Insect health and disease: focusing on the microbiome

Florencia Meyer^*

Mississippi State University, Department of Biochemistry, Nutrition and Health Promotion, Starkville, MS, USA; ^*florencia.meyer@msstate.edu

Insects have emerged as a promising new commodity in agricultural production. Certain insects are particularly efficient at bioconverting a variety of organic waste streams into high-quality protein, lipids and other bioproducts such as chitin. Leveraging these insects could help address critical global issues like food insecurity and environmental sustainability. However, intensive animal farming also presents significant challenges. Housing animals in small spaces, at high densities, and in stressful environments, generates ideal conditions for the development and transmission of infectious diseases. Viral, fungal or bacterial infections can affect a vast fraction of the population in a sometimes very short time frame, which can be devastating to the industry. Studying the collection of microbes (or microbiome) harboured by currently farmed insects is therefore critical to understand what a healthy balance of microbes is and how it differs from the diseased state. Characterizing healthy insect microbiomes could aid producers in monitoring colony fitness and minimise environmental stress in rearing facilities, to ultimately accomplish production objectives. Harnessing the knowledge about a specific harmful virus or bacteria, as well as whole microbial signatures associated with health and positive production traits will better prepare us to minimise stress and disease and to promote more resilient insect production systems.

Insects aren’t failing — they’re just succeeding elsewhere

Daniela A. Peguero^*

Eawag, Department of Sanitation, Water and Solid Waste for Development, Dübendorf, Switzerland; ^*daniela.peguero@eawag.ch

The narrative that the insect industry is failing is increasingly shaped by the recent setback of ventures in Europe and North America. Yet this perspective obscures a more important reality: insects, particularly Black Soldier Fly (BSF), are not failing, they are shifting toward contexts where their value is strongest. This keynote reframes the discussion by centering low- and middle-income countries (LMICs) as the emerging frontier of BSF innovation. Across much of Africa and Asia, the pressures are immediate and structural. Animal feed accounts for up to 60–70% of livestock production costs, driven by reliance on soybean meal and maize. At the same time, waste management systems are under severe strain, with a large portion of waste left uncollected, and organic materials making up 60–70% of municipal solid waste. These challenges are not isolated, they are deeply interconnected. BSF sits precisely at this intersection. Its ability to convert organic waste into high-quality protein for feed and organic fertilizer, positions BSF not only as a feed solution, but as a climate mitigation strategy, reducing methane emissions from decomposing waste while closing nutrient loops. The resulting frass offers a pathway to restore degraded soils, a critical need in Sub-Saharan Africa where soil fertility is declining, and agricultural productivity is under pressure. Year-round tropical climate conditions reduce the need for energy-intensive infrastructure, while urbanisation and agro-industrial growth concentrate reliable waste streams. These conditions fundamentally shift the economics of BSF, enabling lower-cost and more distributed production models. However, realising this potential requires moving beyond inherited assumptions of what scale means. In LMIC contexts, the priority is not maximum technological control, but systems that are cost-efficient, modular, and embedded within local value chains. This places new emphasis on appropriate technology, decentralized supply chains, and enabling policy environments.

Unlike in Europe, where regulatory constraints, high capital intensity, and centralized scaling models have limited viability, LMICs present an opportunity to rethink the sector. Lower legislative barriers and decentralized systems allow BSF to operate closer to both waste streams and feed demand.

This keynote opens a two-part session combining presentations and panel discussions, exploring both opportunity in LMIC and implementation. Rather than asking why insects are failing in Europe and North America, we should ask where they are working, and why.

Insect nutrition, of course

D.G.A.B. Oonincx^*

Animal Nutrition Group, Wageningen University and Research, P.O. Box 383, 6700 AJ Wageningen, The Netherlands;^*dennis.oonincx@wur.nl

This talk on insect nutrition starts with an explanation of why this field is relevant. Then, it deals with the concept of feed suitability, and its underlying factors such as palatability, composition, digestibility and nutritional requirements. Firstly, the classical foundation of these aspects is covered as it was explored in the previous century. Then, it deals with the slowly pivoting approach of experimental studies conducted in the last two decades, which has led up to the GIN-TONIC COST Action. Within this Action questions such as what determines feeding behaviour, how do insect digestive systems function and which nutrients are required in which ratios and forms are addressed. Moreover, it calls for the development of standardized methodologies, as currently knowledge is fragmented, sometimes inconsistent, and has varying levels of detail among the different insect species. The field of insect nutrition should combine knowledge on well-developed animal production systems, with an eye for detail to break through discipline and species boundaries. Understanding the nutritional needs of edible insects will improve the valorisation of underutilized by-products and waste and increase the circularity of our food system. Lastly, we set a point on the horizon: what are the long-term goals to develop his field further? What is the low-hanging fruit, and where do we need to focus our efforts to achieve these goals?

Scientific declaration on insect sentience and welfare

F. Maugère^*

Eurogroup for Animals, Rue Ducale 29, 1000 Brussels, Belgium; ^*f.maugere@eurogroupforanimals.org

In a Scientific Declaration on Insect Sentience and Welfare, coordinated by Eurogroup for Animals, scientists and philosophers express support for increasing research in insect sentience, and for the development and implementation of evidence-based welfare measures in insect farming. This Scientific Declaration aims at synthesising the most recent neurobiological and behavioural evidence on insect sentience and welfare. While scientific consensus on insect sentience remains uncertain, a growing body of evidence suggests that certain insect orders, including those of some farmed species, may experience pain. Moreover, various insect species present behaviours indicative of cognitive complexity, including avoidance learning, risk aversion, site-specific grooming of injuries, and protection from further harm. These behaviours, coupled with the presence of homologous nociceptors akin to those in mammals, suggest pain responses in insects. The prevailing focus of animal sentience and welfare research on vertebrates has overlooked the vast diversity of invertebrates, and notably of insects. There is a gap in understanding the cognitive, neurobiological, and behavioural aspects of insect welfare. As the evidence accumulates, there is a need to extend research efforts into insect welfare to ensure their well-being is considered in the developing insect farming sector. Prioritising the welfare of insects necessitates the development of tailored assessment tools. While some best practices, gathered from evidence on black soldier fly, yellow mealworm, and cricket welfare, can be implemented, there is a pressing need for defining precise, species-specific welfare measures. These include considerations such as humane slaughter methods, appropriate anaesthetics, avoidance of pre-slaughter starvation, safe feeding substrates, nutritional support, genetic manipulation prohibition, optimal densities, light conditions for photophobic species, and disease management protocols. Research is lacking on insect welfare, and thus constitutes one of the limitations of this Declaration. Prioritising this research is crucial in developing updated measures based on the Five Domains of Animal Welfare Model (Nutrition, Environment, Health, Behaviour, and Mental Domains), that are comprehensive and species-tailored. The insect farming sector has an opportunity to heed the latest results in insect welfare research and collaborate in developing efficient and implementable welfare best practices.

Operational drivers and management of a disease event in industrial Hermetia illucens rearing systems

C. Warburton^* and C. Bolduc

Entosystem, 3575 Rue Marie-Curie, Drummondville, QC, Canada J2A0A9; ^*chris@entosystem.com

Entosystem has operated industrial black soldier fly (Hermetia illucens) production systems since 2016 and has valorised recovered retail food waste as larval feedstock since 2019. While biological variability is inherent to large-scale insect production, 2025 marked Entosystem’s first sustained disease event impacting operational performance. This contribution presents an industrial case study focused on process-level drivers, mitigation strategies, and remaining uncertainties. The disease was first observed in autumn 2024 and rapidly contained, but re-emerged in spring 2025 and persisted for approximately 10 weeks. A structured process-mapping approach was used to analyse the full rearing cycle, including larval density, feeding steps, and microclimatic conditions. Environmental parameters (temperature, relative humidity) were monitored alongside operational performance metrics. Preliminary microbiological analyses were conducted in parallel, and the investigation is ongoing in collaboration with veterinary specialists. Postulates of Koch have not yet been fulfilled. Disease expression in our system occurred predominantly during late-stage rearing and was consistently associated with an intermediate feeding step involving high larval densities. This step was characterised by elevated temperatures (approx. 46 °C) and low relative humidity. Affected bins showed reduced harvest performance but did not experience mass mortality, typically retaining approximately 30–80% live larvae. Preliminary analyses identified Paenibacillus thiaminolyticus as a likely causative agent. This bacterium is ubiquitous and has previously been associated with “soft-rot” disease in mass-rearing facilities in China (She et al., 2023). In our system, removal of the intermediate feeding step resulted in operational recovery and continued production. Despite successful mitigation, observations suggest that the pathogen may persist at low levels and re-emerge under stressful conditions, highlighting the difference between disease suppression and eradication in industrial insect systems. Our findings align with previously reported environmental risk factors, while emphasizing the role of process architecture and density management. This case study underscores the need for the insect industry to collectively address pathogenic threats through improved biosafety frameworks, systematic disease reporting, and closer collaboration with veterinary expertise. Advancing colony health will be essential to ensuring the long-term resilience of H. illucens production systems and their contribution to organic waste management and the circular economy.

Feeding adult black soldier flies their preferred foods increases mating activity, egg output, and longevity

M. Barrett^1*, K. Fiocca² and E.A. Waddell³

¹Indiana University Indianapolis, Department of Biology, 723 W Michigan St, Indianapolis, IN 46202, USA; ²Stanford University, 450 Jane Stanford Way, Stanford, CA 94305, USA; ³Holy Family University, 9801 Frankford Avenue, Philadelphia, PA 19114, USA; ^*meghbarr@iu.edu

The black soldier fly (BSF; Hermetia illucens; Diptera: Stratiomyidae) is reared in the trillions annually. Optimising adult breeding systems is an essential part of productivity and welfare. Although much research has focused on optimising larval diet, current industry standard practice generally involves not feeding BSF adults and providing only water, often due to a misconception that BSF adults cannot eat. However, BSF adults have functional mouthparts and digestive systems, gravitate towards and consume food when it is provided, have been found with digestible materials in their guts in the wild, have sugar-specific gustatory receptors, and live longer when provided with food. This suggests there may be untapped potential in optimising adult breeding systems, for both productivity and welfare, in improving adult feed availability and quality. In this study, we determined what carbohydrates and proteins adult BSF preferred, their preferred ratio of these ingredients, and their preferred concentration of these ingredients in water. Then, we assessed mating behaviour, sex-specific longevity, and egg output when flies were fed their preferred diet v. provided only with water. Flies that were provided with the diet were observed to mate more frequently, laid more eggs on days 2–5 post-eclosion, and lived 4–6 days longer compared to flies only provided with water. Gel-based formulations of the preferred diet may be even more practical to use in industry settings than liquid baits. Altogether, these data add to the growing body of literature demonstrating adult BSF can eat both carbohydrates and proteins and that feeding may benefit their reproductive output and welfare.

Breaking the mortality taboo: case report of a pathogen outbreak from an industrial black soldier fly larvae producer

J.B. Guillaume^1*, C. Trublin¹, E. Chardin¹, L. Bridoux², C. Buisson², A. Bolotin², V. Sanchis-Borja², C. Nielsen-Leroux² and P. Lhomme¹

¹Agronutris, R&D Department, 6 rue de la Belle Côte, 08300 Rethel, France; ²Paris-Saclay University, INRAE, AgroParisTech, Micalis Institute, Domaine de Vilvert, 78350 Jouy-en-Josas, France; ^*jeremy.guillaume@agronutris.com

Since the early 2010s, the black soldier fly larva (BSFL; Hermetia illucens) has been increasingly reared as an alternative feed. Numerous studies praised its ability to feed on a wide range of microbe-rich organic substrates and emphasized that no pathogen of BSFL had been identified. However, BSFL mortality outbreaks were reported over the last few years, often with unclear causes. In late 2024, a similar larval mortality event occurred at the industrial BSFL rearing facility of Agronutris in Rethel, France. Symptomatic larvae were 14-day-old or older and showed brown, flaccid, liquified and sometimes swollen bodies. Affected crates were associated with lower activity, moister frass, strong putrid smell and white exuviae. Transmissibility of the symptoms was investigated by inoculating varying amounts of ground symptomatic dead BSFL to the initial substrate and feeding it to healthy starter larvae. Results showed a positive dose response between inoculation rate and both onset of mortality (5 and 10 days with 0.1% and 0.0001% inoculation rates respectively) and mortality rate after 15 days of rearing (100% and 1% with 0.1% and 0.0001% inoculation rates, respectively). Similar mortality patterns were observed when frass from symptomatic crates was used as the inoculum, whereas no mortality was observed when inoculating healthy ground larvae, rotten cold- or heat-killed BSFL, or frass from healthy crates, suggesting that an infectious agent was present in affected crates. Symptoms were also transmitted when reusing incompletely washed affected crates. Moreover, a heat-treated (80 °C, 15 min) inoculum of symptomatic dead BSFL remained infectious, hinting at the involvement of heat-resistant spore-forming bacteria. Isolation of sporulating bacteria, genome sequencing analyses and in vitro feeding bioassays to test per os toxicity of different spore preparations, conducted within the FLYPATH research program, identified the causative agent as Paenibacillus thiaminolyticus. Based on Agronutris’ experience with a successfully identified and contained pathogen, we recommend that insect producers (1) implement a mortality surveillance plan, (2) set-up an investigation task force in case of alerts, including assessment of disease transmissibility, (3) strengthen hygiene practices, particularly crate washing, (4) engage with specialised laboratories capable of identifying insect pathogens, and (5) open up exchanges among insect producers to build a more robust and resilient industry.

Research on the epidemiology and pathogenic mechanism of black soldier fly ‘soft rot’ disease

Q. Xiao^1,2* and L.Y. Zheng^1,2

¹Hubei Hongshan Laboratory, Huazhong Agricultural University, 430070 Wuhan, P.R. China; ²National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, P.R. China; ^*qixiao@webmail.hzau.edu.cn

The black soldier fly (BSF) has emerged as a sustainable agent for converting organic waste into high-value insect protein, lipids, and fertilisers, supporting circular economy. However, industrial-scale BSF production faces significant risks from pathogenic outbreaks, particularly “soft rot” disease, which causes high mortality in BSF larvae (BSFL). This study synthesizes advances of our team in the epidemiology, pathological features, and with the aim of mitigating its impact and ensuring secure BSF production for future food and feed systems. Molecular identification of pathogen bacterium was performed using 16S rRNA gene sequencing and whole-genome analysis. Pathogenic traits were assessed under varying temperatures, feed moisture levels, and larvae ages. RT-qPCR was used to detect the expression levels of immune related genes in BSFL after pathogen infection. Multi-step purification process involving salting out, ultrafiltration, and gel filtration chromatography were conducted to identify virulence proteins. The primary pathogens causing BSF soft rot were identified as Paenibacillus thiaminolyticus. Disease severity increased with higher temperatures (34 °C), lower feed moisture (50–60%), and younger larvae (4–6 days old). The expression of Cecropin-like peptide3 and Hidiptericin-1 genes reached the highest level at 12 h after injection of pathogen bacterium into the abdominal of BSFL, and gradually decreased to the normal level at 24 h and 48 h later. A key virulence protein of the pathogen, Blt-1, was characterised. This is the first report of P. thiaminolyticus as an entomopathogen in BSF. The pathogen virulence depends on environmental conditions and larval developmental stage. The identification of Blt-1 provides insights into the molecular mechanism of pathogenicity. These findings collectively underscore the significant biosecurity challenges posed by pathogenic microorganisms in the scaling up of the BSF industry. The elucidation of their pathogenic mechanisms thus provides a crucial scientific foundation for the strategic development of disease-resistant BSF strains.

Viral and bacterial pathogens associated with black soldier fly larval mortality in a French production facility

C. Wybraniec^1*, V. Wallart¹, J.B. Guillaume², P. Lhomme², C. Savio³, A. Bolotin³, C. Nielsen-Leroux³, V. Sanchis-Borja³, S. Boyer¹ and E. Herniou¹

¹Insect Biology Research Institute, UMR 7261, CNRS, University of Tours, Avenue Monge, Parc Grandmont, 37200 Tours, France; ²Agronutris, R&D Department, 6 Rue de la Belle Côte, 08300 Rethel, France; ³Paris-Saclay University, INRAE, AgroParisTech, Micalis Institute, Domaine de, All. de Vilvert, 78350 Jouy-en-Josas, France; ^*caroline.wybraniec@univ-tours.fr

Black soldier fly (BSF) rearing has gained popularity as a sustainable protein source for animal feed production. However, high-density rearing conditions may facilitate pathogen transmission and disease outbreaks, possibly leading to mortality and economic losses. While BSF-associated pathogens have been increasingly documented in recent years, comprehensive pathogen surveys in commercial facilities remain limited. This study investigated a French industrial BSF production site experiencing an episode of larval mortality. We employed two complementary approaches to characterise the pathogen community associated with larval mortality. First, targeted molecular screening was performed on asymptomatic and symptomatic larvae sampled at two developmental stages to detect and quantify six previously described BSF-associated viruses (HiIfV, HiSgV, HiLbV, HiSvV, HiInV and HiCV) and Paenibacillus 16S rRNA gene. Detection rates varied considerably among pathogens, with HiInV being the most prevalent and HiCV absent from all samples. Second, metatranscriptomic analysis was conducted on the same samples. RNA-seq reads of asymptomatic and symptomatic larvae were mapped against the genomes (WGS) of two bacterial species isolated and sequenced from symptomatic larvae at the same facility: Paenibacillus thiaminolyticus and Bacillus thuringiensis subsp. israelensis}. Differential expression analysis revealed no upregulation at 6 days post-hatching, but significant transcriptional responses at 14 days, with symptomatic larvae showing upregulation of multiple transcripts when mapped against P. thiaminolyticus, including genes potentially associated with virulence, while no differential expression was observed against B. thuringiensis. These findings reveal a complex pathogen landscape in commercial BSF production, with P. thiaminolyticus showing the strongest transcriptional association with larval symptoms, while tested viral pathogens appear unlikely to be primary mortality drivers. The ubiquitous presence of certain viruses regardless of health status, combined with species-specific bacterial responses, underscores the need for integrated pathogen surveillance strategies in the expanding BSF industry.

Green shields for Hermetia illucens: boosting the defense mechanisms of black soldier fly via microalgae supplementation

C. Adamaki-Sotiraki^1*, D. Bruno², M. Marzari², S. Zafeiriadis¹, A. Athanasiou³, D. Roma⁴, I.C. Rumbos⁵, P. Madesis³, M. Casartelli⁴, G. Tettamanti² and G.C. Athanassiou¹

¹University of Thessaly, Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, School of Agricultural Sciences, Phytokou Street, 38333 Volos, Greece; ²University of Insubria, Laboratory of Invertebrate Biology, Department of Biotechnology and Life Sciences, Via Dunant 3, 21100 Varese, Italy; ³University of Thessaly, Laboratory of Molecular Biology of Plants, Department of Agriculture, Crop Production and Rural Environment, School of Agricultural Sciences, Phytokou Street, 38333 Volos, Greece; ⁴University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy; ⁵University of Patras, Department of Agriculture, School of Agricultural Sciences, Messolonghi Campus, 302000 Messolonghi, Greece; ^*cadamaki-s@uth.gr

Feed-induced immunomodulation is an emerging and promising research area that can address the knowledge gaps regarding the nutritional requirements of insect colonies. The black soldier fly larvae (BSFL, Hermetia illucens) can convert low-value organic residues into biomass. Thus, the strengthening of the immunity of the larval stage is currently in the spotlight of research. A promising approach is the supplementation of rearing substrates with natural bioactive ingredients. Thus, in this study we designed diets incorporating the microalga Chlorella vulgaris at three inclusion levels (1.5%, 5% and 10%) to evaluated whether this approach can enhance the immune response of larvae without compromising crucial traits. Hemolymph was collected from last instar larvae, and its antimicrobial activity was tested against Micrococcus luteus and Escherichia coli. The strongest suppression occurred at 1.5% microalgae inclusion against M. luteus, while no inhibitory activity was detected against E. coli. Digestive enzyme activity analysis revealed that the C. vulgaris inclusion did not affect midgut physiology, indicating the feasibility of using microalgae as feed additive without adverse effects on midgut digestion. Finally, larval chemical composition was analysed. Larvae fed diets containing C. vulgaris exhibited higher lipid content compared to larvae reared on control. Overall, C. vulgaris is a promising BSFL feed additive that boosts antimicrobial activity without trade-offs in growth, survival, or digestion, while improving larval nutritional value. This publication is based upon work from COST Action CA23127 — Group on Insect Nutrition: To Open Nutritional Innovative Challenges (GIN-TONIC), supported by COST (European Cooperation in Science and Technology).

Solinvivirus infection induces cellular antiviral response and reduces the lifespan of adult Hermetia illucens

E. Herniou^1*, R. Pienaar^1,2, P. Garcı́a-Castillo², H. Piterois¹, V. Wallart¹, F. Manas¹, C. Bressac¹ and S. Herrero²

¹CNRS — University of Tours, IRBI — UMR 7261, Faculté des Sciences Avenue Monge, Parc Grandmont, 37200 Tours, France; ²Universitat de València, Department of Genetics and University Institute of Biotechnology and Biomedicine (BIOTECMED), 46100 Burjassot (Valencia), Spain; ^*elisabeth.herniou@univ-tours.fr

Viral pathogens pose an emerging threat to the sustainability of insect mass-rearing systems, yet remain understudied in key species like the black soldier fly (BSF, Hermetia illucens). Although multiple viral sequences have been reported in BSF, their role in disease has not been established until now. Here, we provide the first in vivo characterisation of Hermetia illucens solinvivirus (HiSvV), confirming its role as a viral entomopathogen of BSF. Metatranscriptomic analysis of a diseased colony revealed a high viral load attributable to HiSvV. We successfully isolated the virus and developed injection- and oral-based infection assays to investigate replication, tissue tropism, transmission and risk of mortality. HiSvV replicated in inoculated adults, induced premature mortality in flies, and was transmitted both horizontally and vertically. Infected flies also mounted a broad antiviral response, this supported active pathogenesis, even if the small RNA pathways were not activated. These findings establish HiSvV as the first confirmed viral pathogen of BSF and underscore the urgent need for viral surveillance and experimental tools to safeguard industrial insect rearing.

Dietary protein–carbohydrate ratio effects on growth, digestion, gut microbiota and immunity in black soldier fly larvae

I. Armenia^1*, D. Bruno¹, D. Roma², S. Caccia², M. Casartelli^2,3, F. Sandrelli⁴, V. Orlandi¹ and G. Tettamanti^1,3

¹University of Insubria, Biotechnology and Life Science, Via J.H. Dunant 3, 21100 Varese, Italy; ²University of Milano, Department of Biosciences, Via Giovanni Celoria 26, 20133 Milan, Italy; ³Interuniversity Center for Studies on Bioinspired Agro-environmental Technology, Piazza Carlo di Borbone 1, 80055 Portici, Italy; ⁴University of Padua, Department of Biology, Via Ugo Bassi 58b, 35131 Padua, Italy; ^*ilaria.armenia@uninsubria.it

Black soldier fly larvae (BSFL) are a sustainable source of protein and lipids for animal feed due to their ability to convert organic waste and byproducts into high-value biomass. However, rearing on microbially rich substrates exposes larvae to potential pathogens, making immune competence a key factor in ensuring product quality and safety. This study evaluated the effects of diets differing in protein to carbohydrate (P:C) ratio on larval growth, immune response, and physiological parameters, including midgut functionality, gut microbiota composition, and metabolic markers. Larvae were reared on selected diets and challenged with an Escherichia coli (Gram-negative) and Micrococcus luteus (Gram-positive) mix. While larval development was not affected by dietary composition, immune responses—evaluated through cellular (haemocyte count and phagocytic activity) and humoral (lysozyme activity and antimicrobial peptide expression) markers—were strongly modified by diet. Larvae fed a protein-rich diet exhibited enhanced immune competence compared with those reared on carbohydrate-rich substrate. Increased haemocyte number and phagocytic activity, together with higher antimicrobial peptide expression and lysozyme activity, were accompanied by diet-dependent changes in metabolic pathway enzyme expression, lactate production, and ATP levels, indicating coordinated allocation of metabolic resources toward immune functions during bacterial challenge. In contrast, the carbohydrate-rich diet supported adequate larval development but was associated with reduced activation of immune and metabolic markers, suggesting preferential allocation toward growth rather than immune defence. The P:C ratio of the rearing substrate also affected the activity of midgut enzymes involved in nutrient digestion and induced shifts in gut microbiota composition. Overall, these findings highlight a trade-off between growth and immune-related metabolic investment in BSFL and provide insights for optimising dietary formulation to balance growth performance, metabolic efficiency, and immune robustness in BSFL rearing systems. This work was funded by MUR (P2022MZAF8) Finanziamento dell’Unione Europea — NextGenerationEU — PNRR Missione 4, Componente 2, Investimento 1.1

Short-term pupal heat exposure significantly affects black soldier fly life-history traits

T. Klammsteiner^*, E. Gemassmer, P. Lueders, P. Juen, F. Hasler, V. Bangerter, B.C. Schlick-Steiner and F.M. Steiner

Universität Innsbruck, Department of Ecology, Technikerstr. 25, 6020, Austria; ^*thomas.klammsteiner@uibk.ac.at

Mass-rearing subtropical insects in temperate regions requires precise control of abiotic conditions that mimic their natural environment, especially temperature, humidity, and light. In black soldier fly (BSF) larvae, the combination of natural crowding, high densities, and elevated metabolic activity can raise substrate temperatures above 50 °C if not counteracted, inducing irreversible stress responses. Heat stress in BSF larvae has recently gained increased interest, but its impact on the pupal stage remains understudied. Pupae may experience heat stress in dense crates when pupation coincides with peak larval activity. Technical failures of aeration or cooling systems can further intensify fluctuations in temperature. Because pupae remain immobile for approx. ten days during metamorphosis, they cannot actively respond to stress. Consequently, lethal effects of excessive heat may not immediately become apparent, leading to significant delays in compensating for the resulting damages and fluctuations in production. To assess the temperature tolerance of pupae and refine estimates of heat stress risk under production conditions, we conducted controlled heat shock experiments simulating short-term thermal stress as might occur during system failure in industrial facilities. Pupae of two age groups, 2 days (T2) and 5 days (T5) post-pupation, were exposed to temperature levels ranging from 44 to 50 °C for 60 min. Life history traits were subsequently monitored, including eclosion success, male:female ratios, adult survival, egg biomass, egg fertility, and the performance of the next larval generation. Both age groups tolerated 1-h exposure up to 46 °C without major negative effects on eclosion. Higher temperatures significantly decreased eclosion success, with T5 pupae responding more sensitively than T2 pupae. At 48 °C, eclosion dropped to approx. 50% in T2 pupae and below 25% in T5 pupae; temperature stress above this threshold was lethal. Eclosed adults differed in sex ratio, with older pupae (T5) generating proportionally more males. This sex bias, combined with reduced adult fitness caused by heat stress, led to less total egg biomass. Moreover, females from both age groups exposed to 48 °C showed extended oviposition periods and delayed oviposition peaks, however, resulting in lower total egg biomass yields. Our results demonstrate that even short periods of heat stress can have intricate effects on BSF pupal development and subsequent reproductive performance, highlighting the challenges of assessing and mitigating damages in large-scale production systems following incidents with uncontrolled increases in temperatures.

When and where: data insights for identifying temporal patterns in BSF behaviour and biogeographical hotspots

N.B. Lemke^1,2*, B. Chamberlain², C. Li³ and J. De Smet¹

¹KU Leuven Campus Geel, Research Group for Insect Production and Processing, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440, Belgium; ²Companion Intelligence, 8030 La Mesa Boulevard 268, La Mesa, CA 91942, USA; ³Guangzhou Unique Biotechnology Ltd. Co, Room 1717, 101 Science Avenue, Huanpu District, Guangzhou, Guangdong, P.R. China; ^*noahlemke22@gmail.com

This presentation gives data insights for the behavioural ecology of the black soldier fly (BSF), Hermetia illucens. Insights for temporal patterns: Previously it was thought that oviposition behaviour and egg fertility in BSF could not be easily predicted from mating behaviour, due to a complex and nondeterministic mating sequence. However, a cross-correlation analysis of previously collected data revealed that average mating rates were 97.54% correlated with oviposition in traps, if oviposition was lagged by n = 2 days. Similarly, mating was correlated with total egg weights and hatch rates exhibited 60.29% and 75.16% when these were both lagged by n = 3 days. These patterns align with existing knowledge of BSF reproductive dynamics in both captive and wild environments and could be insightful for increasing the predictability of breeding programs. Insights for biogeography: Natural history data on BSF remains scarce, meaning the best source of knowledge about wild distributions currently comes from community science records. An analysis of BSF observation records revealed that 158 out of 254 (62.70%) global regions have more than one BSF sighting. From these records, potential BSF hotspots were identified by correcting for biases such as human population density and community science platform user density. Lastly, the ecological niche hypervolume and the phenology of BSF were both modelled using a dataset comprised of these subsampled observation records paired with weather-climate data gathered via web API. Together, these insights will be important for directing future field-based studies.

Thriving where others fail: the remarkable tolerance of black soldier fly larvae to polluted environments

D. Bruno^1*, M. Marzari¹, J. De Smet², A. De Benedictis¹, N. Sibinga², D. Vandeweyer², M. Van Der Borght², M. Casartelli³ and G. Tettamanti¹

¹University of Insubria, Biotechnology and Life Sciences, Via Dunant 3, 21100 Varese, Italy; ²KU Leuven, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium; ³University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy; ^*daniele.bruno@uninsubria.it

The presence of contaminants in organic waste may compromise the efficiency of black soldier fly-mediated bioconversion, potentially affecting insect physiology, as well as the safety and quality of the resulting insect biomass. In this study, black soldier fly larvae (BSFL, Hermetia illucens) were reared on substrates contaminated with lead, polyethylene terephthalate (PET), and Staphylococcus aureus to assess the effects of heavy metals (HMs), microplastics (MPs), and pathogens, respectively, on insect life-history traits, larval physiology, bioconversion performance, and contaminant accumulation in insect tissues. Among the tested xenobiotics, only HMs induced slight alterations in some physiological parameters, indicating that BSFL responses are strongly dependent on the chemical nature of the contaminant. Notably, lead exposure did not impair larval growth or survival, but affected immune-related markers. Moreover, the complete removal of contaminants by the larvae was observed only for the microbial pathogen, highlighting the recalcitrant nature of HMs and MPs, and raising concerns regarding the use of insect biomass as animal feed or frass as fertiliser. Overall, these findings reveal complex interactions between BSFL and environmental pollutants, providing new insights into their impact on larval biology and bioconversion efficiency. While the ability of BSFL to reduce foodborne pathogens supports their application in waste sanitization, the accumulation of HMs and MPs in larvae and frass underscores critical safety issues. These results emphasize the need for further research to develop effective entomo-remediation strategies and to assess the risks associated with xenobiotic contamination in large-scale BSF production systems. The study was supported by MUR (2020ENH3NZ), FWO (S018524N), and EMBO Short Term Fellowship (8895).

Black soldier fly metabolomics: what can NMR tell us about the dietary flexibility of the larvae?

P.M. Duarte^1,2,3*, O.M.C.C. Ameixa³, M. Palma¹, F. Lourenço³, R. Costa² and I. Viegas¹

¹University of Coimbra, Department of Life Sciences, CC Martim de Freitas, 3000-456 Coimbra, Portugal; ²Polytechnic Institute of Coimbra, Research Centre for Natural Resources, Environment and Society, Coimbra Agriculture School, 3045-601 Bencanta, Portugal; ³University of Aveiro, Centre for Environmental and Marine Studies, Department of Biology, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal; ^*pduarte@student.uc.pt

Black soldier fly (BSF, Hermetia illucens L.) is widely recognized as an effective agent for the bioconversion of organic residues into high-value biomass. While the performance of BSF under different diets is well documented, the metabolic plasticity that enables this adaptability remains poorly understood. Nuclear Magnetic Resonance (NMR) enables simultaneous identification and quantification of metabolites in complex samples and is widely used in metabolomics profiling, with extensive use in insect research spanning ecology, development and stress responses. In this work, BSF larvae were reared on experimental diets consisting of partial replacement of control (chick feed) with unconventional ingredients: spent coffee grounds and hamburger meat at defined inclusion levels. After extraction, polar metabolites were profiled by NMR within an untargeted metabolomics approach. Over 40 metabolites were consistently quantified, defining a core BSF larval metabolome that includes most proteinogenic amino acids and key intermediates of central metabolism. In addition, diet-specific compounds were identified (e.g. caffeine, trigonelline). Multivariate statistics (PLS-DA) revealed clear metabolic differentiation between control and experimental diets. The metabolites contributing most strongly to this separation (variable importance in projection >1.5), including carnitine, acetylcarnitine and β-alanine, indicated a shift towards enhanced amino acid and lipid catabolism. Collectively, these results demonstrate how NMR metabolomics provides a robust framework to interpret larval performance under different diets, informing dietary optimisation for efficient and sustainable bioconversion.

Geography and climate shape genetic diversity in black soldier fly populations across native and introduced ranges

M. Kükrer^1* and C. Sandrock²

¹Kilis 7 Aralık University, Molecular Biology and Genetics, Merkez, 79000 Kilis, Turkey; ²Research Institute of Organic Agriculture, Livestock Sciences, Ackerstrasse 113, 5070 Frick, Switzerland; ^*mertkukrer@gmail.com

We investigated the ecological drivers of differentiation in the black soldier fly (BSF; Hermetia illucens) using 541 samples from 32 populations across the native range via reanalysis of published microsatellite data. Bayesian clustering identified $K=3$ as the optimal number of clusters, yet also revealed substructure within the clusters. Kriging maps across sub-continents point to major breaks around the Isthmus of Panama and the Andes Range. North and Central American populations were monophyletic. Ordination approaches confirmed three major groups, with spatial structure in higher-order axes, suggesting potential temperate adaptations further North, as well as bottlenecks, island adaptations, or recolonization events in Puerto Rico. Gradient Forest analysis highlighted key climatic and geographic predictors, with cumulative importance values of 0.54 compared to 0.38 for Moran’s Eigenvector Maps. A combination of environmental and spatial variables were strong predictors of native population structure. The Southern/Central-South ancestry interface was driven by precipitation variables, while the Northern/Central-South interface was shaped by temperature and seasonality. Applying the model in Africa, where BSF introduced from multiple sources established wild populations, predicted ecological dominance of one ancestry in more humid regions. This prediction aligns with precipitation variables being significant in the native range and also possible resistance to hybridization in West Africa due to ecological adaptation, suggesting success of historic translocations might be non-random. Our results emphasize the combined influence of geography, climate, and history and provide a basis for predicting responses to environmental change across native and introduced ranges. Further research investigating signatures of ecological adaptation at the genome level might be necessary to shed light on specific morphological, physiologic, or behavioural adaptations and their relevance for breeding. Unlike honey bees or silkworms, BSF offers early-stage breeding potential.

Assessing the genomic diversity of black soldier flies (Hermetia illucens) in ten globally distributed strains

F. IJdema^1,2, M. Verschaeren³, L. Frooninckx³, L. Broeckx³, J. Bouhuijzen-Wenger¹, J. De Smet², N. Buys¹, S. Van Miert³ and R. Meyermans^1*

¹KU Leuven, Center for Animal Breeding and Genetics, Department of Biosystems, Kasteelpark A, 3001 Leuven, Belgium; ²KU Leuven, Research group for insect production and processing, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium; ³Thomas More University of Applied Sciences, Centre of Expertise Sustainable Biomass and Chemistry, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*roel.meyermans@kuleuven.be

The black soldier fly (BSF, Hermetia illucens) has become a key species for sustainable bioconversion of nutrients and an insect-based feed industry is arising. Although performance and growth differences between different BSF strains are characterised, insight into their domestication history and global genomic diversity is still limited. In this study, we investigated patterns of genome-wide diversity and population structure across ten BSF populations representing European lineages (4 populations), African lineages (3 populations) and Oceanian lineages (3 populations). For each population, we sequenced 20 individuals using Illumina 150 bp paired-end whole-genome sequencing and conducted a comprehensive set of population genomic analyses. After quality filtering and variant calling against the BSF reference genome (GCF_905115235.1_iHerIll2.2), we examined within-population diversity, between-population differentiation (F_st), potential selection signatures and population relationships using standard diversity metrics such as PCA, admixture inference, and phylogenomic reconstruction. Overall, this study provides an extensive comparative genomic overview of globally distributed BSF populations and offers a foundational resource for breeding, conservation, and bioconversion optimisation efforts.

The effect of genetic homogeneity in farmed insects

N.T. Hamilton^* and C.J. Picard

Indiana University Indianapolis, Department of Biology, 723 W Michigan Street, Indianapolis, IN 46202, USA; ^*nothamil@iu.edu

Climate change has made sustainable agricultural practices increasingly necessary, and as such, mass rearing insects is being investigated as an alternative protein source. Three main species have emerged as leading candidates: the house cricket (Acheta domesticus), the yellow mealworm (Tenebrio molitor), and the black soldier fly (Hermetia illucens). Colony health and productivity are key considerations for mass production. However, genetic diversity loss can increase disease susceptibility and the potential for population reduction and ultimately extinction. The reason is that farming insects likely leads to relaxed selection pressures for some important fitness traits valued in the wild. For example, the selective pressures to maintain a suite of antimicrobial peptides (AMPs), key effectors of insect innate immunity, are metabolically costly, yet maintained through positive selection. This study evaluated genetic diversity among wild, laboratory, and commercial strains in T. molitor, H. illucens, and A. domesticus to understand the correlation between reduced diversity in populations and the role of relaxed selection on genes important for survival in natural environments but less critical in captivity. Analysis of commercial strains revealed a wide range of inbreeding coefficients (F = 0.0–0.38), suggesting different breeding management strategies may minimise inbreeding accumulation. For example, in H. illucens, populations showed elevated and variable inbreeding in farmed strains (F = 0.29–0.53) relative to wild-derived populations (F = 0.23–0.24). In T. molitor, inbreeding varied widely, with two strains having lower inbreeding ($F=0.0$), while other lines derived via experimental manipulations from single-pair matings showed severe inbreeding (F = 0.28–0.36), as expected. Notably, the strain with the greatest inbreeding was a commercial strain ($F=0.38$). To identify gene categories potentially undergoing relaxed selection in highly inbred T. molitor populations, we performed genome-wide variant burden analyses to assess whether genes associated with particular biological processes accumulated excess variation. Genes associated with circadian rhythm regulation were enriched among those with higher variant burdens in highly inbred and farmed individuals compared to low-inbreeding and wild-derived individuals, indicating circadian regulation may be less critical under controlled rearing conditions. These results highlight trait-specific responses to relaxed selection in mass-reared insects and demonstrate the utility of this framework for assessing genomic vulnerability. Future studies should apply this pipeline to larger population samples and additional species, including other farming-relevant insects, to improve inference and generalizability.

High-resolution population genetics of Hermetia illucens across Australia and Asia for sustainable breeding

S. Septriani^1*, K.B. Gowda¹, C. Tedder², N. Thi Thanh Vu¹, D.R. Jerry¹ and K.R. Zenger¹

¹1Centre for Sustainable Tropical Fisheries and Aquaculture, College of Science and Engineering, James Cook University, 1 James Cook Drive, Douglas, QLD 4811, Australia; ²FlyFarm Australia Ltd, Brisbane, Brisbane, QLD, Australia; ^*shafirafira.septriani@my.jcu.edu.au

Commercial production of black soldier fly (BSF), Hermetia illucens, has expanded globally since the early 21st century. However, the increase in BSF productivity to industrial-scale production levels remains limited. To increase productivity through improved farming and breeding practices, key knowledge of genetic structure and diversity of wild and captive BSF populations is required. This information enables design of sustainable breeding that maximises productivity while minimising genetic loss and inbreeding. Furthermore, it provides useful information on colonising history and genetic connectivity of the species throughout its range. In this study, we investigate broad-scale population genetic structure and diversity of wild Australian and captive Asian BSF across multiple geographic regions. A total of 559 wild Australian BSF larvae were collected from 12 regions across Australia (QLD: Cairns, Townsville, Brisbane, Zilzie; NSW: Sydney; VIC: Melbourne, Bendigo; NT: Darwin; WA: Perth, Broome, Bunbury; and SA: Adelaide), along with additional captive Asian population representatives (n = 63, South Korea, Vietnam and Indonesia). BSF samples (n = 622) were genotyped using a custom Allegro array containing 6000 targeted genome-wide SNPs. Amongst the wild Australian populations, genetic diversity parameters were higher in the northern Australian populations (e.g., Cairns; He = 0.32, PPL = 95%) and lower in southern Australian populations (e.g., Melbourne He = 0.27, PPL = 92%), reflecting possible colonisation history effects and/or differences in contemporary effective population sizes. Several of captive Asian sample locations displayed elevated inbreeding coefficients (FIS) and decreased genetic diversity (e.g., Vietnam: FIS = 0.16 and He = 0.25). Low to moderate levels of genetic differentiation were observed across Australian sample locations, with FST values ranging from 0.03 to 0.32, consistent with an isolation-by-distance population genetic structure model ($r=0.44$, $p=0.003$). Principal component analysis (PCA) revealed 4 key genetic clusters, with clear separation among eastern-, western-, northern-Australia, and Asian populations. These patterns reflect colonisation history and current-day genetic connectivity. Overall, these findings not only provide insights into historical and contemporary micro-evolutionary processes shaping Australian BSF populations, but also provide a population-genetic baseline for Australian BSF breeding to ensure long-term sustainability.

Early sexing with a molecular marker unlocks sex-specific phenotyping in BSF larvae

N. Warthmann^1*, A.A. Shah¹, M. Brankatschk^2,3, U. Lutz⁴, M.J. Zorrilla¹ and M. Gold⁵

¹REPLOID Deutschland GmbH, R&D, Löbener Weg 7, 04523 Pegau, Germany; ²Technische Universität Dresden, Applied Zoology, Zellescher Weg 20b, 01217 Dresden, Germany; ³BIOTEC (TU Dresden), Tatzberg 47-49, 01307 Dresden, Germany; ⁴Biogenda, Seelhausgasse 20, 72070 Tübingen, Germany; ⁵Reploid Group AG, R&D, Maria-Theresia-Straße 53, 4600 Wels, Austria; ^*warthmann@reploid.eu

In industrial settings, black soldier fly (Hermetia illucens) breeding programs aim to improve production traits such as growth rate, development time, survival, and conversion efficiency. However, selection and phenotyping are typically performed on mixed-sex larval cohorts because larvae are not sexually dimorphic. This obscures sex-specific trait expression and can reduce selection efficiency, particularly when males and females differ in performance or when sex ratios drift across timepoints due to sex-specific developmental timing. Here we present a sexing assay based on a molecular marker that enables reliable sex assignment in BSF larvae and thereby supports sex-stratified phenotyping in breeding contexts. We developed PCR-based assays targeting male-specific DNA sequences and validated them across dozens of individuals spanning different life stages and genetic backgrounds. Molecular sex calls matched reference sex assignments (based on adult morphology) with 100% accuracy, and the assays performed robustly using genomic DNA and haemolymph as input. This early sex identification reveals sex differences that are otherwise masked in pooled analyses. In a breeding context, this enables (i) estimating sex-specific genetic and phenotypic variation, (ii) identifying whether particular traits are predominantly expressed in males or females, and (iii) designing selection schemes that maintain adequate representation of both sexes — important for mass selection where females may be larger and phenotyping could otherwise inadvertently bias the breeding population. Overall, early molecular sexing provides a practical tool to improve the precision, interpretability and efficiency of BSF phenotyping and selection.

Development and validation of a cost-effective SNP genotyping panel for the black soldier fly

M.J. Davidson, N.T.T. Vu, K.B. Gowda, S. Septriani, D.R. Jerry, D.B. Jones and K.R. Zenger^*

James Cook University, College of Science and Engineering, Townsville, QLD 4818, Australia; ^*kyall.zenger@jcu.edu.au

The black soldier fly (BSF; Hermetia illucens) has emerged as a key commercial production species due to its fast growth, environmental tolerance, and high nutrient conversion efficiency. As the industry expands, managing genetic stocks under domestication is important for maintaining population health, optimising performance, and enabling selective breeding. Although genomic resources for BSF exist (i.e., WGS and GBS), their high unit cost, limited global utility, and low scalability hinder their use in commercial settings. To address this, we developed a custom Allegro 6000 SNP targeted genotyping panel for BSF, which was then evaluated across Australian and Asian BSF stocks. Using ddRAD sequencing, 15 million SNPs were identified from 191 BSF originating from eight geographically distinct Australian populations. These SNPs were then filtered to produce a high-quality candidate set for Allegro SNP panel design. The resulting 6000 SNP panel was independently validated using 250 BSF from six Australian, one South Korean, and a domesticated Australian population. The targeted SNP assay successfully generated 5517 high-quality, polymorphic SNPs across all samples. The average SNP read depth was 161 across individuals. Technical duplicates demonstrated 97.3% concordance across samples, confirming high reproducibility of the Allegro SNP assay. Preliminary population genetic analyses revealed clear genetic differences, with the panel accurately resolving genetic patterns consistent with Australian BSF colonisation history, contemporary genetic connectivity, and potential effects of captive breeding. Linkage disequilibrium (LD) analyses indicated sufficient genome-wide LD among SNPs, highlighting the panel’s utility for quantitative trait loci investigations. Parentage analysis and genomic relationship matrices successfully resolved maternal lineages and sibship, demonstrating applicability in family-based or genomic selection breeding programs. Overall, the 6000 SNP panel provides a scalable, cost-effective, and reproducible platform for routine genotyping in BSF. It can enable population monitoring, support the management of genetic diversity, and inform selective breeding strategies, thereby supporting the expansion of the BSF industry.

Larval weight-based GWAS reveals a sex-specific locus in black soldier fly

K.B. Gowda¹, S. Septriani¹, D.B. Jones^1,2, D.R. Jerry^1,2 and K.R. Zenger^1,2

¹Centre for Sustainable Tropical Fisheries and Aquaculture, College of Science and Engineering, James Cook University, Townsville, QLD 4811, Australia; ²ARC Research Hub for Supercharging Tropical Aquaculture through Genetic Solutions, College of Science and Engineering, James Cook University, Townsville, QLD 4811, Australia; ^*kishorgowda.bommalingegowda@my.jcu.edu.au

Industrial production of black soldier fly (BSF) larvae is expanding globally. Larval body weight (LBW) at the 5th instar is a key production trait, as this stage is nutritionally rich and optimal for animal feed. Sexual dimorphism in BSF is known to influence growth and yield; however, the genetic basis of larval growth and sex determination remains poorly understood. A genome-wide association study (GWAS) using a custom 6K Allegro SNP array was conducted for LBW based on genotyping 2092 BSF larvae. This GWAS identified a significant SNP associated with LBW and a putative SNP linked to sex. The GWAS, conducted in GCTA using the MLMA-LOCO framework, identified a significant SNP on chromosome 4 (NC_051852.1_166367432_G_A; effect (β) = 10.34 ± 2.26; p = 5.07 × 10^−6) along with four SNPs on chromosome 7. Further investigation of the four SNPs on the X chromosome (Chr 7) revealed that SNP NC_051855.1_6352382_A_T exhibited a strictly non-Mendelian inheritance pattern, consistent with a male-heterogametic system (males hemisygous for X) and female-homogametic system. Among the 2092 individuals, 415 were AA, 1677 were AT, and no TT genotypes were observed. Analysis of allele-specific read depth revealed a dosage effect: the AA genotype (putative females) had a mean depth of 272.41x, whereas the AT genotype (putative males) showed reduced depth for allele A (108.89x) and higher depth for allele T (159.62x). Genetic differentiation at this locus was high (F_ST = 0.38). This pattern was validated in an independent subset of individuals with known phenotypic sex (42 males, 57 females), showing complete concordance between genotype and sex. When chromosome 7 was excluded, and putative sex information was included as a fixed effect, the GWAS was rerun for LBW, recovering the same top association on chromosome 4 (NC_051852.1_166367432_G_A; β = 10.32 ± 2.24; p = 2.05 × 10^−6). In addition, a few other loci (p approx. 10^−4) were detected across chromosomes, suggesting a polygenic architecture for LBW. Overall, these results provide evidence for a sex-determining locus on the X chromosome (Chr 7) in BSF and indicate a polygenic genetic architecture underlying LBW. Together, these findings provide a genomic foundation for sex identification and inform breeding strategies aimed at improving LBW in industrial BSF larvae production.

OSDel-BSF: ovary-targeted delivery enables scalable CRISPR editing and RNAi in the black soldier fly

E. Elfer¹, A. Sloutskin², S. Cohen¹, N. Tugentman¹, I. Nevo-Yassaf^2*, I. Alyagor² and I. Khalaila¹

¹Ben-Gurion University, Department of Biotechnology Engineering, Ben-Gurion Blvd 1, Be’er Sheva, 84105, Israel; ²FreezeM, Nachshonim 1, Nachshonim, 7319000, Israel; ^*inbar@freeze-em.com

The black soldier fly (BSF, Hermetia illucens) is a rapidly emerging platform for sustainable protein production, yet functional genetics remains constrained by the low throughput and technical burden of embryo microinjection. We developed OSDel-BSF, an ovary-targeted delivery approach that exploits vitellogenin receptor (VgR) mediated uptake to deliver nucleic-acid cargos into developing oocytes following injection into females. To maximise uptake and reproducibility, we optimised injection timing by mapping VgR expression and introducing a pre-eclosion sex-enrichment protocol (yielding 72% females). We assessed OSDel-BSF for heritable genome editing by delivering CRISPR–Cas9 ribonucleoproteins (RNPs) with multiple sgRNAs and quantifying editing outcomes in egg batches. Across 7 sgRNAs, batch-level editing ranged from 11% to 48%. While per-individual efficiency may be lower than direct embryo injection, OSDel-BSF increased the number of edited eggs per handling effort by approx. 120×, enabling substantially higher-throughput screening. Functional effects were observed for developmental targets, including delayed transition to the prepupal stage following Sema1A targeting, and antennal abnormalities accompanied by reduced eclosion after eyeless targeting. To enable non-transgenic functional testing, we extended OSDel-BSF to dsRNA delivery. dsRNA was detected in ovaries hours after injection, alongside increased expression of canonical RNAi components (Dicer2 and Argonaute2), consistent with activation of RNAi machinery. In a proof-of-concept trehalase assay, OSDel-mediated dsRNA delivery produced measurable knockdown (25% in one experiment and 50% in a second, relative to controls). Ongoing work is focused on calibrating dsRNA dose, timing, and target selection to maximise and standardize knockdown. Overall, OSDel-BSF provides a scalable platform for both heritable CRISPR editing and RNAi-based gene silencing in BSF, supporting higher-throughput target screening and strain-development workflows.

Unveiling host-microbe interactions critical dynamics in black soldier fly: from hatching to bioconversion

Z.L. Li^* and L.Z. Zheng

Huazhong Agricultural University, College of life science and technology, Huazhong Agricultural University, South Luoshi Road, Hongshan District, 430070 Wuhan City, Hubei Province, P.R. China; ^*li-zhe@webmail.hzau.edu.cn

The black soldier fly serves as a cornerstone of the circular bioeconomy, yet its industrial efficacy is fundamentally governed by the insect’s “holobiont” status—an obligate reliance on microbial symbionts. While the role of gut microbiota in waste reduction is acknowledged, the ontogenetic continuity of these interactions from embryonic development to larval bioconversion remains underexplored. This study integrates findings from two distinct phases to elucidate how BSF orchestrates microbial communities to overcome developmental barriers (hatching) and optimise metabolic performance (waste valorisation). We analysed embryonic microbial succession (0–72 h) via sterilization and sequencing to identify hatching promoters. Subsequently, a Germ-free (GF) larval model across three wastes was established to test “host-mediated environmental recruitment.” A synthetic consortium of functional gut symbionts was then constructed to validate its impact on larval development and bioconversion efficiency. Our results demonstrate that BSF performance depends on continuous microbial symbiosis. Hatching proved to be an obligate, microbe-dependent process; surface sterilization reduced hatching rates from 77.1% to 33.7%, whereas re-inoculation with a synergistic Bacillus and Staphylococcus co-culture restored rates to 83.4%. These bacteria putatively secrete chitinases and proteases to degrade the chorion, acting as an “extracorporeal digestive organ” facilitating eclosion. Transitioning to the larval stage, BSF actively recruited specific environmental microbes to compensate for sterile conditions. A host-selected synthetic consortium (Enterococcus, Bacillus, and Lactobacillus) significantly rescued GF larval phenotypes, most notably in chicken manure. Compared to controls, this consortium increased survival by 72.1%, biomass by 193.6%, and feed conversion efficiency by 439.6%, while shortening development time by 62.4%, confirming the efficacy of host-mediated recruitment. The BSF lifecycle is inextricably linked to microbial symbiosis. From the enzymatic “softening” of the eggshell during hatching to the selective enrichment of metabolic partners for waste degradation, the host actively shapes its microbiome to adapt to environmental constraints. These findings underscore the potential of developing stage-specific probiotic formulations — targeting both egg hatching synchronization and larval waste conversion efficiency — to standardize and upscale industrial insect farming.

Phenotypic plasticity and microbial signal: unraveling the Hermetia illucens response to long-term dietary shifts

A. Gligorescu^1*, S.N. Bushi¹, M.B. Lund¹, A. Schramm¹, T.N. Kristensen² and J.G. Sørensen¹

¹Aarhus University, Department of Biology, Ny Munkegade 116, 8000 Aarhus C, Denmark; ²Aalborg University, Department of Chemistry and Bioscience, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark; ^*angl@bio.au.dk

Black soldier fly larvae (BSFL) are increasingly recognized as a promising and sustainable solution for upcycling low quality side streams into high-quality protein, but the underlying genetic and environmental mechanisms influencing their performance remain poorly understood. This study investigated the effects of cross-generational dietary changes on BSF performance and larval gut microbiome composition. We used BSF lines that had been maintained on either low-quality (wheat bran) or high-quality (chicken feed) diets for 30 generations (parental diets) and subjected them to dietary shifts across three generations (F1-F3). Results revealed improvement of growth and reproductive traits when larvae were raised on a high-quality diet, regardless of the parental diet. We also identified subtle effects of parental diet on offspring growth traits when larvae were fed the same diet. However, in most cases this latter effect was transient and disappeared in the F3 generation, suggesting a strong environmental influence on the performance traits. The gut microbiome of BSFL was significantly shaped by dietary changes, with a marked change in composition and generally higher microbial diversity when larvae were reared on wheat bran diets. In addition, a small but significant trans-generational effect of wheat bran parental diet was detected, revealing a potential signal of host/microbiome adaptive response to this diet. However, this potential link between gut microbiome composition and BSF larvae performance was not evident in the traits measured here. These findings provide valuable insight into the effects of diets on fitness components and the microbiome in BSF and offer potential avenues for optimising farming of this species.

Determining the gut microbiota composition of the yellow mealworm Tenebrio molitor in its industrial rearing environment

E. Mollard^1,2*, F. Delbac¹, H. El Alaoui¹, I. Wawrzyniak¹ and V. Ageorges²

¹Laboratoire Microorganismes: Génome et Environnement, Université Clermont Auvergne, CNRS, 1 impasse Amélie Murat, 63178 Aubière Cedex, France; ²INVERS, Champ de la Croix, 63720 Saint-Ignat, France; ^*emilie.mollard@invers.fr

Over the past decade, insects have received increasing interest as an innovative and sustainable source of alternative protein for both human food and animal feed. The yellow mealworm (Tenebrio molitor) is one of the most promising candidates due to its high protein content and ease of breeding and feeding. As in all livestock sectors, understanding and managing the microbiological aspects of mealworm production is crucial to ensuring quality and profitability. To date, the scientific literature is limited regarding mealworm gut microbiota under large-scale rearing conditions, as most studies have been conducted under controlled laboratory conditions. Thus, this study aimed to characterise the microbial communities present in the gut microbiota of T. molitor under industrial rearing conditions. Regular sampling of insects and substrates was performed throughout the life cycle of the insect under large-scale production conditions, including young larvae, fattened larvae, pupae, and beetles. In parallel, welfare indicators and zootechnical performance were rigorously monitored over the insect full cycle. To analyse the gut microbiota of the collected samples, DNA extractions followed by high-throughput sequencing were performed. Two complementary strategies were used: an exploratory shotgun metagenomic approach and a targeted 16S rRNA metabarcoding. The sequencing data are currently under analysis, but the preliminary results provide initial insights into the microbial composition of T. molitor. Among all microorganisms detected in T. molitor larvae, bacteria represent 94%, while eukaryotes, viruses, and archaea account for 3%, 2% and 1%, respectively. Focusing on the bacterial population, the predominant phyla are Bacillota, Pseudomonadota and Bacteroidota. The data generated in this study may offer new perspectives on the interactions between the insect’s gut microbiota, its zootechnical performance and the environmental conditions of industrial rearing.

Beyond linear nutrition: evidence for circular, microbially driven nutrient dynamics in black soldier fly larvae

N.A. Sibinga^* and J. De Smet

KU Leuven, Department of Microbial and Molecular Systems (M2S), Research Group Insect Production and Processing, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*nate.sibinga@kuleuven.be

There is significant interest in defining the nutritional requirements of black soldier fly larvae (Hermetia illucens), yet many foundational principles of classical animal nutrition do not fully translate to this system. Traditional nutrition models assume a linear flow of nutrients from feed to animal biomass, with excreted wastes representing irreversible losses. In contrast, BSF larvae develop within microbially dynamic substrates in which nutrient pools are continuously transformed by resident microbiota. Larval feeding rapidly depletes specific limiting nutrients, imposing selective pressure on the substrate microbiota and favouring taxa capable of synthesizing, mobilizing, or recycling those nutrients. Larval waste products — including exuviae, uric acid, and partially digested feed — can be converted in situ into microbial biomass and subsequently re-enter the larval food supply. Microbial communities thus actively modulate nutrient availability and can partially compensate for nutritionally deficient substrates. Such feedback loops can confound controlled nutritional experiments, as larvae sometimes exhibit robust growth on experimental substrates intentionally formulated to be inadequate. This presentation examines how such microbial dynamics challenge conventional nutritional models and highlights the need for nutritional methods that explicitly account for larva–microbe–substrate interactions. Using an axenic (germ-free) rearing model that does conform to classical nutritional assumptions, we determined the “true” lysine requirement of BSF larvae using substrates with graded supplementation levels (0, 0.05, 0.1, 0.2, 0.5, 1, 2, or 3% lysine added). Isonitrogenous controls were prepared using mixed amino acids from hydrolysed casein at the same levels, and larvae were sampled 20 days after egg collection. Significant dose-dependent differences in growth were observed in axenic larvae ($p=0.007$), whereas lysine depletion/supplementation did not significantly affect larval growth in conventional (i.e. non-axenic) larvae under the same conditions. Together, these results demonstrate that while axenic systems can reveal the absolute biological nutrient requirements of BSF larvae, the relevance of these requirements for practical substrate formulation may be limited in microbially active environments. Accounting for circular, microbially mediated nutrient flows represents the next frontier in developing accurate and useful nutritional models for H. illucens.

Incidence and detection of spore forming bacteria in mass-reared black soldier fly larvae

C. Savio^1*, J.B. Guillaume², P. Lhomme², L. Bridoux¹, C. Buisson¹, V. Sanchis-Borja¹ and C. Nielsen-Leroux¹

¹Paris-Saclay University, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France; ²Agronutris, R&D Department, 08300 Rethel, France; ^*carlottasavio2@gmail.com

The larvae of the black soldier fly (Hermetia illucens) (BSFL) can grow on a wide range of wastes and byproducts, differing in nutritional composition and pH and representing the optimal growing environment for many microorganisms that might represent a risk for human or animal health. BSFL are known for their capacity to reduce some pathogens. However, their efficacy in eliminating spore-forming bacteria is less well documented. In this study we describe Paenibacillus thiaminolyticus, a Gram+ spore-forming bacteria, isolated from symptomatic BSFL in France, and discuss its interactions with the growing media and the host. First, the evaluation of P. thiaminolyticus ecology in diet and its persistence in BSFL was evaluated in order to estimate the risk of contamination and pathogens persistence during insect rearing. Then, challenge tests using pathogen inoculation into the diet were performed to assess the virulence of P. thiamynolitycus on BSFL growth, survival and eventual capacity to reduce P. thiaminolyticus, depending on spores or vegetative form. In laboratory conditions, the infected insects exhibited symptoms such as brownish colour, softening, liquefaction, off flavour, death, decay of the larvae, cannibalistic behaviour, and arrested or delayed development, resulting in reduced larval weight, pupation and adult emergence. However, on the industrial site, symptoms were sometimes more severe, with growth arrest, softening, death, and decay of larvae. During the challenge tests, the presence of the P. thiaminolyticus strain in both BSFL and diet was verified with specific molecular detection probes as well as by using fluorescence and antibiotic labelling genetic techniques. This study describes a new spore-forming BSFL pathogen, a P. thiaminolyticus strain, and underscores the importance of developing good manufacturing practices (GMP) guidelines and detection methods for spore-forming pathogens in rearing equipment, feeds and larvae to preserve insect health in mass rearing environment. Key words: food and feed safety, insect health, Paenibacillus spp., pathogen detection, Hermetia illucens, insect rearing, microbial risk

Histopathology studies in black soldier fly larvae (BSFL) morbidity and mortality cultured in a laboratory sized colony

S. Pyecroft^*

Adelaide University, School of Animal & Veterinary Sciences, College of Science, 1454 Mudla Wirra Road, Roseworthy, SA 5371, Australia; ^*stephen.pyecroft@adelaide.edu.au

Black soldier fly larvae (BSFL, Hermetia illucens) are widely regarded as resilient insects suited to large-scale bioconversion systems; however, their health is influenced by a range of potential pathogens and substrate-dependent risks. Early assessments reported no major natural outbreaks in commercial farms and noted that BSFL show relative resistance to many entomopathogens, despite experimental infections with bacteria, fungi, nematodes, protozoa, and viruses. As global BSFL production expands, the likelihood of pathogen emergence increases, highlighting the need for proactive monitoring and biosecurity. This study assessed histopathology as a diagnostic tool for identifying BSFL disease processes. Histopathology enables detection of structural tissue changes caused by pathogens, toxins, environmental stress, or physiological disruption. Because insects rely primarily on innate immunity, tissue-level alterations often provide early and reliable indicators of disease. During BSFL culture for full-fat insect meal used in fish trials, episodes of tray morbidity and complete mortality occurred. Larvae were fixed in 10% neutral buffered formalin, Davidsons fixative, or Bouin’s variants, processed for wax embedding, sectioned, stained with H&E, and examined under light microscopy. The goal was to document insect-specific causes of morbidity and mortality, including organ pathology and in situ pathogen presence. This approach parallels diagnostic methods used in intensively farmed arthropods such as molluscs and crustaceans. Granulomatous inflammation and coagulative necrosis of tissues such as the gastrointestinal tract and coelomic lining were observed, with bacterial agents commonly identified. BSFL are robust but not immune to disease, capable of experimental infection and susceptible to metabolic disruption under stress. Histopathology provides essential insight into the structural impacts of infection and environmental challenge. As industrial BSFL production accelerates, maintaining colony health will rely on accurate diagnostics, pathogen surveillance, and improved understanding of BSFL responses to infectious and non-infectious stressors.

Profiling the resistome and virulome of the Hermetia illucens midgut microbiota

C.J.R. Scott^1*, D. Roma¹, M. Brilli¹, F. De Filippis², G. Sequino², G. Tettamanti³, S. Caccia¹ and M. Casartelli¹

¹University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy; ²University of Napoli Federico II, Department of Agricultural Sciences, Via Università, 80055 Portici, Italy; ³University of Insubria, Department of Biotechnology and Life Sciences, Via Jean Henry Dunant, 3, 21100 Varese, Italy; ^*conor.scott@unimi.it

The spread of antimicrobial resistance has become a crucial global health threat with agricultural practices and livestock production contributing greatly to the spread of antibiotic resistance genes (ARGs) in the environment. However, the spread of ARGs is not the only challenge facing the agri-food sector which requires urgent innovation for the adoption and development of more sustainable processes. The larvae of the black soldier fly (BSFL) Hermetia illucens are mass-reared to produce animal feed and fertilisers, yet the resistance and virulence encoded within their midgut microbial communities remain underexplored. To elucidate the risk associated with their use, the profiles of antibiotic resistance genes (ARGs), virulence factors (VFs), and plasmids were predicted from the midgut bacterial community of BSFL. Shotgun metagenomic sequencing revealed the presence of genes encoding resistance against 26 classes of antibiotics, and genes encoding virulence via nine mechanisms of which Pseudomonadota phylum taxa were the dominant contributors. Three horizontally-mobile ARG-encoding plasmids were additionally identified. Reconstruction of metagenomes allowed high-resolution taxonomic classification of taxa harbouring multiple genes for resistance and virulence, including a Pseudomonas species with 42 VFs and seven ARGs. Eight unique species of bacteria demonstrated growth on antibiotic containing agar plates inoculated with the BSFL midgut contents. Altogether, this analysis represents the most comprehensive profile to date of the midgut microbiota resistome of BSFL. It will provide both a valuable tool to the research community working on BSF larvae, and for the assessment of the antibiotic resistance risk posed to the environment and to public health through their use.

Black soldier fly immunity shapes the surrounding microbial community via antimicrobial peptides

I. Opatovsky^*, M. Hareb and O. Lemberg

Tel-Hai University, Kiryat Shmona 7918000, Israel; ^*itaio@migal.org

Insects rely on antimicrobial peptides (AMPs) as a central component of their innate immune system. The black soldier fly (BSF), a detritivorous insect that develops in microbe-rich substrates, is known to produce a diverse repertoire of AMPs. Here, we investigated whether AMP production by BSF larvae extends beyond host defence to influence the microbial composition of the surrounding environment. We show that BSF larvae produce AMPs in the gut and release them into the rearing substrate. These secreted peptides selectively modulate the microbial community, promoting the growth of certain fungi such as Candida tropicalis while suppressing others, including Aspergillus niger. This AMP-driven shift in microbial composition is associated with changes in larval performance and may contribute to the exclusion of potentially pathogenic microorganisms from the rearing environment. Our findings suggest that insect immunity can function as an ecological driver shaping microbial communities in insect-rearing systems, with implications for understanding insect–microbe interactions and for improving the stability and safety of industrial BSF production.

Common garden study: stage- and sex-specific trait divergence in black soldier fly lines with contrasting diet histories

Q.-H. Zhang^*, J. Lin, Y. Tang, S. Silvaraju and N. Puniamoorthy

National University of Singapore, 16 Science Drive 4, Block S3, 04-13, 117558, Singapore; ^*zqihui@nus.edu.sg

The black soldier fly (Hermetia illucens, BSF) is widely used in organic waste bioconversion, yet performance varies substantially among strains maintained under different long-term rearing regimes. To examine how long-term dietary history shapes BSF performance across life stages, we conducted a common garden experiment using eight genetically distinct BSF lines maintained on contrasting diets over multiple generations. Each line was reared on both its historical diet and a compositionally distinct novel diet, allowing direct comparison of larval, pupal, and adult traits under standardised conditions. Larval growth and survival differed strongly among lines and were generally highest when individuals were reared on their historical diet. However, these patterns were not consistently reflected at later stages. Pupal development time and eclosion success showed weak or variable correspondence with larval performance, and adult body size and lifespan often contrasted with larval outcomes. Trait divergence was further sex-specific. Females showed marked differences in ovarian investment between diets, whereas males exhibited relatively stable investment in testes and accessory glands across rearing conditions. These results indicate that BSF lines with contrasting dietary histories differ across developmental stages and between sexes, and that patterns observed at the larval stage do not consistently extend to adult reproductive traits.

Stage-specific feeding effects on mealworm growth, survival and reproduction: evidence from two case studies in Uganda

V. Gwokyalya^1*, H. Kelstrup², R. Kayizi¹, R. Schjødt³ and E. Ssebbombo⁴

¹Mothers Against Malnutrition and Hunger, Mityana 256, Uganda; ²6leg solutions, 2440 S 116th Street, Burien, WA 98168-1214, USA; ³Research and Action for Income Security, Lystrup, 8520, Denmark; ⁴Bobo Eco Farm, Mityana 256, Uganda; ^*vgwokyalya@mamah.org.ug

Optimising diets across developmental stages is crucial for improving mealworm farming, particularly in systems relying on agro-byproducts. This study evaluates stage-specific feeding strategies on growth, survival, pupal output, and adult reproduction at two East African farms: Bobo Eco Farm and Kyaka II Refugee Settlement in Uganda. Data from 2024–2025 were analysed, including controlled feeding trials at Bobo Eco Farm and routine KPIs from both sites. Larval diets tested were wheat bran (WB), maize bran (MB), and chicken feed (CF) in different combinations. Growth rate, development time, body weight (ABW), survival, and egg production were assessed. For adults, four diets were tested: MB, WB, 50% MB + 50% WB, and 40% MB + 40% WB + 20% CF. Moisture was applied to some replicates to examine its effect on egg laying. Larval experiments included stocking densities (6000–10 000 M/w), wet vs. dry feeding, and finisher diets (WB + CF). Larval diet had a strong impact on performance. In pre-split larvae, 80% MB + 20% CF reduced development time by approx. 50% and increased daily biomass gain six-fold compared to 100% WB. Finisher diets with 20% CF accelerated pupation and increased pupal size. Adults fed 20% CF laid 4–5 times more eggs and had higher survival than those on other diets. Moisture did not affect egg oviposition. Lower stocking densities (6000 M/w) led to faster development compared to higher densities (10 000 M/w). Routine wet feeding resulted in better survival and faster development than dry feeding or refeeding. At Bobo Eco Farm, pupal ABW stabilized at 125–135 mg, but yields declined (5–12 kg/week), and survival to adulthood was approx. 52%. Egg-to-split larval survival improved from <20% to approx. 53%. At Kyaka II, adults produced 2–5 times more eggs per tray (24 000–35 000/week), with larger pupae (approx. 150 mg ABW), but egg-to-larvae survival was low (2–10%). Stage-specific diets enhance mealworm growth and reproduction. MB and CF diets improve larval growth, while 20% CF in adult diets increases egg production. Lower stocking densities and wet feeding optimise development. Discrepancies between trials and farm outcomes suggest external factors may influence results. Diet optimisation based on developmental stage improves mealworm productivity. These findings have direct relevance to commercial insect farming, showing that targeted feeding strategies enhance performance while reducing feed costs.

From manual measurements to automated precision: a novel predictive phenotyping method for black soldier fly larvae

S. Espinoza-Ulloa^*

NRGene Canada, R&D, 114-111 Research Drive, Saskatoon, SK, Canada S7N 3R2; ^*sebastian.espinoza@nrgenecanada.com

Efficient and accurate phenotyping is essential for optimising black soldier fly (BSF, Hermetia illucens) production, yet traditional manual measurement protocols are time-consuming and limit data collection. Here we present the development and implementation of an automated phenotyping pipeline for BSF larvae, resulting in significant improvements in throughput, data quality, and analytical capabilities. Initial manual protocols, involving random sampling and calliper-based measurement of larval length and width, required approximately one hour per group and restricted phenotyping frequency to two or three times per week. To overcome these limitations, a series of automation solutions were evaluated, including infrared-based counting systems and adapted vibration-detection seed counters, which provided incremental time savings but were constrained by larval size variability. A substantial advancement was achieved through the integration of an image analyser, customised for BSF larvae. This system reduced processing time to under 10 minutes per group and enabled daily phenotyping. Consequently, the dataset expanded from several hundred to over one million entries within a year, facilitating comprehensive developmental analyses. Leveraging this dataset, three-dimensional predictive models were constructed to estimate larval volume and weight based on corrected morphometric parameters. These models demonstrated a very high correlation with actual weights, confirming their robustness and reliability. The automated pipeline now enables high throughput monitoring of larval development, early sex differentiation, and rapid identification of rearing issues. Additionally, the methodology supports advanced applications such as macromolecule concentration estimation, yield-related trait differentiation, and enhanced nutrient profiling. Current efforts focus on further refining predictive models and extending automation to egg-laying, hatching, and reproductive monitoring through image-based analyses in controlled environments.

Tracking and classifying black soldier fly larval group movement using computer vision

M. Hoffmann^1,2*, J. De Smet² and B. Aernouts¹

¹KU Leuven, Department of Biosystems, Animal and Human Health Engineering, Kleinhoefstraat 4, 2440, Geel, Belgium; ²KU Leuven, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440, Geel, Belgium; ^*maximilian.hoffmann@kuleuven.be

Black soldier fly larvae (BSFL) are commonly reared on organic waste streams as a replacement for conventional, less sustainable protein sources in feed for livestock. Organic waste streams can introduce challenging conditions that act as stressors for BSFL, potentially leading to altered movement and group behaviour. Monitoring such behavioural changes may help to identify stressors and address growing concerns regarding insect welfare, while also supporting the optimisation of rearing conditions. However, current BSFL performance metrics are largely limited to end-point measures such as protein yield or feed conversion efficiency and lack the temporal resolution needed to detect behavioural anomalies. This study presents an imaging system, consisting of a camera and a computer vision algorithm, to continuously monitor and classify BSFL movement patterns. The imaging pipeline was applied to ten complete rearing cycles and compared to conventional weight measurements. Subsequently images were analysed using a frame-comparison-based computer vision pipeline to track and classify group-level larval movement. Using this pipeline, we were able to track and compare recurring, development-dependent movement patterns in ten different BSFL life cycles. By comparing high-level movement features with larval growth curves, weight peaks were correlated to activity patterns. This research provides novel insights into movement patterns under standard BSFL rearing conditions and sets a foundation for future work to investigate behavioural changes from suboptimal rearing conditions. Furthermore, it can also give insight into how the growth peaks and weight gain in BSFL rearing can be predicted in a cost-efficient and non-invasive way.

Review of larval performance and composition data of Tenebrio molitor for predictive model development

C. Morand^1,2,3*, C. Constant³ and L. Henault-Ethier^1,2

¹TriCycle Inc., 1401 rue Legendre Ouest, Montréal, QC, Canada H4N 2R9; ²Centre Eau Terre Environnement, Institut National de la Recherche Scientifique, 490 de la Couronne, Quebec City, QC, Canada G1K 9A9; ³Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, 531 boulevard des prairies, Laval, QC, Canada H7V 1B7; ^*charlene.morand@inrs.ca

Tenebrio molitor represents a promising insect species for sustainable protein production to meet increasing protein demand driven by population growth. Its rearing is integrated into circular economy systems that valorise agri-food by-products as feed ingredients. However, feed supply often fluctuates in quantity, composition, and availability over time. This variability creates a need to anticipate the effects of diet composition on larval performance and composition. The objective of this study was to compile and harmonize data from the scientific literature to develop a structured database suitable for meta-analysis and to derive predictive models for key production variables. A data-driven review was conducted to identify studies reporting both dietary composition and relevant response variables. Selected articles included data on larval composition (moisture, protein, fat, carbohydrates, ash) and larval performance metrics (growth, development time, feed conversion efficiency, efficiency of ingested food, nitrogen conversion efficiency). In addition to nutritional variables, non-nutritional factors such as strain, temperature, relative humidity, photoperiod and rearing density were recorded and considered in the analyses. Following screening, a total of 37 articles reporting larval composition and 32 articles reporting larval performance met the inclusion criteria. Articles were primarily excluded because nutritional data of the diet were missing or incomplete, and because data were presented exclusively in graphical form without numerical values provided in the text or supplementary materials. Extracted data were then standardized and transformed to ensure comparability across studies and integrated into a unified database. The resulting dataset enabled the application of meta-analytical approaches and multiple linear regression to quantify relationships between dietary inputs and larval responses. Total nitrogen, ash, fibre and lipids were identified as the main explanatory variables used to model multiple response variables related to larval performance and nutritional composition. This work demonstrates that systematic compilation and harmonization of published data can support the development of predictive models linking diet composition to key production outputs in yellow mealworms. The resulting database provides a foundation for decision-support tools aimed at optimising insect diets.

The carry-over effect of developmental temperature on black soldier fly adult fitness

C. Li^1,2*, H. Zhang¹, Y. Huang¹, N.B. Lemke², D. Salazar², J.K. Tomberlin² and Y. Cao¹

¹Sun Yat-sen University, State Key Laboratory of Biocontrol School of Life Sciences, 135 Xingang West Road, 510275 Guangzhou, Haizhu District, P.R. China; ²Texas A&M University, Department of Entomology, 2475 TAMU, College Station, TX 77843-2475, USA; ^*chujun.li2013@gmail.com

The black soldier fly (BSF, Hermetia illucens) is a vital resource insect for organic waste bioconversion and feed protein. Its minimum developmental temperature is approximately 10 °C, with 30 °C being the optimal temperature, while 20 °C as a suboptimal temperature is in a range where BSF can maintain survival and reproduction. The impacts of thermal acclimation on BSF fitness and the underlying molecular mechanisms remain unclear. This study investigated the effects of two acclimation temperatures (e.g., 20 °C and 30 °C) on BSF fitness-related traits (e.g., growth, thermal tolerance, body composition, and oocyte quantity/quality) and explored regulatory mechanisms via multi-omics analysis. Results showed that 30 °C-acclimated individuals had higher weights at all stages, lower prepupal weight loss, and smaller adult sexual dimorphism than 20 °C-acclimated ones. Compared with 30 °C, BSF reared at 20 °C significantly prolonged median developmental duration and increased developmental asynchrony. For thermal tolerance, 20 °C-acclimated adults exhibited significantly enhanced cold tolerance (e.g., shorter chill coma recovery time) but reduced heat tolerance (e.g., lower heat knock-down temperature). Nutritionally, thermal acclimation affected adult lipid content but not protein content. For potential fecundity, robust linear regression and mediation analysis showed female body weight positively affected oocyte number, and the acclimation temperature effect on fecundity was fully mediated by female body weight (e.g., no direct temperature effect after controlling for weight); however, notably, most 20 °C-developed females failed to achieve normal oocyte vitellogenesis. Multi-omics analysis revealed potential molecular pathways underlying these phenotypic changes. In conclusion, thermal acclimation reshapes BSF adult fitness by regulating development rate, body weight, thermal tolerance, lipid metabolism, and reproductive potential. This study provides critical insights into BSF thermal adaptation strategies with trade-off effects and a theoretical basis for optimising mass rearing conditions.

Fungal contributions to black soldier fly performance: metabolic mechanisms and implications for rearing optimisation

I. Opatovsky^*, Z. Vitenberg, N. Herman and L. Ben-Mordechai

Tel-Hai University, Kiryat Shmona 7918000, Israel; ^*itaio@migal.org.il

Improving the efficiency and stability of black soldier fly (BSF) production systems requires a better understanding of the biological factors shaping larval performance. While bacterial contributions to insect nutrition have been widely explored, the role of fungi in BSF rearing systems remains largely overlooked. Here, we investigated the nutritional and metabolic interactions between BSF larvae and associated fungi. We identified Candida tropicalis as the dominant fungal species in household compost systems colonized by BSF. Supplementation of larval diets with C. tropicalis significantly increased larval weight, indicating a positive contribution to performance. To elucidate the mechanisms underlying this effect, we examined whether fungal contributions occur directly through larval consumption or indirectly via fungal modification of the feeding substrate. Larvae exposed to C. tropicalis showed increased fatty acid biosynthesis, associated with elevated levels of palmitic and myristic acids that are abundant in fungal biomass. In addition, the presence of C. tropicalis in the substrate increased larval levels of essential amino acids (threonine, leucine, and isoleucine), suggesting improved nutrient availability mediated by fungal activity in the substrate. These results support two complementary mechanisms by which fungi enhance BSF performance: direct nutritional contribution and indirect improvement of substrate quality. Our findings highlight fungi as a promising, yet underutilised, lever for optimising BSF rearing systems, with potential applications in improving productivity, robustness, and consistency of insect-based feed production.

Industrial valorisation of dairy, tomato and chili processing by-products using black soldier fly larvae

N. Hermi¹, H. Guebli^1*, O. Nguyen² and V. Steinmetz²

¹nextProtein Tunisia, Km 1 Route de Béni Khalled, 8030 Grombalia, Tunisia; ²nextProtein France, R&D — Engineering, 66 Boulevard Niels Borh, 69100 Villeurbanne, France; ^*h.guebli@nextprotein.co

Converting organic waste into high-protein biomass via the black soldier fly (Hermetia illucens (L.), Diptera: Stratiomyidae) is a key component of circular bioeconomy strategies. However, maintaining consistent industrial performance requires precise diet formulation to manage waste variability. At nextProtein, more than 100 feed ingredients have been screened to bridge the gap between heterogeneous waste streams and production stability. This study evaluates the integration of dairy (yogurt), tomato (concentrate), and chili (harissa) processing residues into BSF larval diets. A stepwise validation protocol was employed, transitioning from lab-scale to industrial pilot tests. Rearing cycles lasted 7 to 10 days. We tested inclusion rates of 15–30% for dairy, 10–21% for tomato, and 4–20% for chili wastes. Performance was measured via larval survival rate, biomass yield, feed conversion ratio (FCR), and residence time, with 30–50 replicates per condition to ensure statistical robustness. Quantitative analysis revealed that dairy wastes significantly boosted efficiency; inclusion rates of 15–30% (fresh matter) resulted in survival rates >94%, a 15–25% increase in biomass yield, and an FCR improvement of up to 20%. Notably, larval residence time decreased from 9 to 7 days. Tomato residues (10–21% inclusion) maintained high survival rates (93–95%) and delivered a 25% increase in yield with 8 day cycles. Finally, chili wastes (4–20%) proved highly compatible, supporting >95% survival and a 15% biomass increase. The results highlight that these wastes are not merely fillers but functional ingredients. The rapid growth observed with dairy by-products suggests a high bioavailability of nutrients that compensates for other waste streams. Tomato residues appear to contribute to stable growth and biomass accumulation, likely due to their balanced carbohydrate and fibre content supporting both feed structure and larval ingestion. The successful integration of chili residues, which contain capsaicinoids, suggests a high metabolic resilience in BSF larvae. This study demonstrates that dairy, tomato, and chili processing wastes can be effectively upcycled at scale. By optimising formulations, we achieved significant gains in FCR and reduced production cycles. These findings support the viability of BSF-based systems to transform diverse agro-industrial side-streams into reliable protein sources, enhancing the overall efficiency of circular waste management.

Ultrasonic processing effects on the structural and nutritional characteristics of black soldier fly protein

B. Zozo^*, M. Wicht and J. Van Wyk

Cape Peninsula University of Technology, Food Science and Technology Department — Agrifood Technology Station, Symphony Way, 7530 Cape Town, South Africa; ^*zozob@cput.ac.za

Ultrasonication has emerged as a viable method for modifying protein structure and improving functionality in food applications. This study examined ultrasound-assisted alkaline extraction to enhance the structure and nutritional features of black soldier fly (BSF) larvae proteins, as edible insects increasingly emerge as sustainable dietary ingredients. BSF samples were sonicated at 300 W for 0, 10, 20, 30 and 40 min. Sonication significantly increased protein recovery (56.14% to 77.07%, $p\mathrm{<}0.05$) and lightness (L*) (27.3 to 35.8, $p\mathrm{<}0.05$). Free sulfhydryl content rose initially but declined with prolonged exposure. Secondary structures shifted, with α-helix decreasing (36.2% to 27.5%) and β-sheet increasing (23.4% to 32.1%, $p\mathrm{<}0.05$). Functional properties, including water and oil binding capacities and solubility, improved significantly ($p\mathrm{<}0.05$). However, excessive sonication reduced these enhancements. These results demonstrate that controlled ultrasonication can effectively tailor BSF protein functionality, depending on protein type, solution, and treatment parameters.

Broodstock: size is fine, diet is better — a production-scale reality

C. Bolduc^*, M. Cooling and C. Warburton

Entosystem, 3575 rue Marie-Curie, Drummondville, QC, Canada J2A 0A9; ^*catherine.bolduc@entosystem.com

Black soldier flies (BSF) are widely recognized as effective agents for organic waste bioconversion. At Entosystem, BSF are reared on grocery store and industrial food-waste streams, including broodstock. While breeder size is known to correlate with reproductive output, the diet used to achieve this size also influences fecundity. During 2025, two broodstock diets were applied sequentially at production scale, supporting the daily production of 1–3 kg of neonates. One diet consisted of a food-waste-derived slurry, while the other was a grain-based formulation excluding food waste. Pupal mass and egg production were monitored continuously under commercial operating conditions. Overall, pupae reared on the grain-based diet were also smaller than those reared on the slurry-based diet (128 mg vs. 140 mg, respectively). However, when comparing multiple periods in which mean pupal mass was equivalent (138 mg), females originating from the slurry-based diet produced approximately 25.3% (about 4 mg) more eggs per female. At production scale, this difference corresponded to an increase of 28%, more than 1 kg of eggs per day. The two diets differed, notably in lipid content, which was higher in the food-waste-derived slurry and may explain the difference in fecundity. These production-scale results indicate that broodstock fecundity in BSF is influenced not only by body size but also by diet composition and this effect can be substantial. We hypothesize that increased dietary lipid availability contributed to enhanced reproductive output. In conclusion enables efficient bioconversion, food waste-based substrates appear to meet the biological needs of BSF breeders and constitute a nutritionally suitable food source for supporting reproductive performance.

Dietary protein level regulates nutrient composition dynamics in black soldier fly larvae across the life cycle

L. Schneider^1,2*, B. Kisinga¹, N.S. Stoehr¹ and G. Dusel¹

¹University of Applied Sciences Bingen, Animal Nutrition, Berlinstreet 109, 55411 Bingen, Germany; ²Justus-Liebig-University Gießen, Animal Nutrition, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany; ^*l.schneider@th-bingen.de

Black soldier fly larvae (BSFL) are an alternative protein source; this study tested whether dietary protein alters nutrient accumulation magnitude without affecting its developmental timing. Larvae were reared under controlled conditions on three isoenergetic diets differing in crude protein content: CPlow (10%), CPmed (15%) and CPhigh (20%), with a standard Gainesville diet as control (n = 6 per diet). From six days post-hatch (days of life, DOL), larvae were maintained at 27 °C and 55% relative humidity. Performance parameters were recorded, and individuals were sampled across developmental stages (6–16 DOL, prepupa, pupa and adult) to quantify protein, lipid, chitin and ash masses. Survival to the prepupal stage was high across all treatments (91.1–94.6%). Total biomass gain in fresh matter was maximised in the CPmed group (2.31 ± 0.2 kg), exceeding the lowest value observed in CPlow larvae (1.66 ± 0.2 kg) by 39% ($P\mathrm{<}0.05$). Total development time from 6 DOL to adulthood was longest under protein-limited conditions (23.3 ± 0.3 days) and shortest in the high-protein group (20.2 ± 0.3 days), representing a reduction of 3.1 days ($P\mathrm{<}0.05$). Prepupal body length was greatest in CPmed larvae (13.3 ± 1.2 mm), exceeding CPlow larvae by 24%, with similar patterns observed in adult females and males. Protein mass increased rapidly during larval growth and peaked at the prepupal stage, reaching a maximum of 26.3 ± 0.9 g dry matter (DM) in CPhigh prepupae compared with 19.8 ± 0.8 g DM in CPlow prepupae (+33%; $P\mathrm{<}0.05$). In contrast, lipid deposition was enhanced under protein limitation, with prepupal fat mass highest in CPlow 19.1 ± 0.9 g DM) relative to CPmed (17.4 ± 0.3 g DM; $P\mathrm{<}0.05$). Structural and mineral components increased disproportionately with higher protein supply: chitin mass peaked at 5.2 ± 0.9 g DM and ash mass at 7.5 ± 0.3 g DM in CPhigh pupae, exceeding CPlow values by 33% and 25%, respectively. Overall, dietary protein level strongly influenced growth rate and nutrient partitioning, while the sequence of nutrient deposition remained conserved. These findings demonstrate that protein supply and harvest timing are key levers for optimising BSFL nutrient composition, performance and the targeted production of physiologically valuable nutrients in sustainable insect-based food and feed systems.

Influence of diet composition on the NPK fertilising value of frass from Tenebrio molitor

C. Morand^1,2,3*, C. Constant³ and L. Hénault-Ethier^1,2

¹TriCycle Inc., 1401 Legendre Ouest, Montréal, QC, Canada H4N 2R9; ²Centre Eau Terre Environnement, Institut National de la Recherche Scientifique, 490 de la Couronne, Québec City, QC, Canada G1K 9A9; ³Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, 531 boulevard des prairies, Laval, QC, Canada H7V 1B7; ^*charlene.morand@inrs.ca

Frass generated during the rearing of Tenebrio molitor can be used as an organic fertiliser due to its NPK composition. As the main by-product of insect production, frass represents a key opportunity for valorisation within circular economy systems. However, variability in feed inputs used in yellow mealworm rearing leads to heterogeneity in the fertilising value of frass. This raises challenges for compliance with guaranteed nutrient regulations and for ensuring agronomic consistency. The objective of this study was to evaluate the impact of diet composition on the NPK fertilising value of frass and to model these relationships. A literature review revealed limited data directly linking diet composition to frass contents (n < 10). A feeding trial was therefore conducted at production scale, in which 10,000 larvae were reared for eleven weeks at 30 °C and 58% relative humidity under natural photoperiod. Ten dietary treatments were formulated from wheat bran, brewer’s spent grain, okara, chicken feed, starch and yeast to generate a wide range of total dietary nitrogen (3.0–6.2%), phosphorus (0.3–1.9%) and potassium (0.1–2.2%) contents. Frass was harvested at the end of the trial and analysed for NPK content. Frass nutrient composition varied across diets, with total nitrogen (N) ranging from 2.0–4.2%, phosphorus (P₂O₅) from 1.0–6.0%, and potassium (K₂O) from 0.5–3.4%. Dietary nitrogen alone was not sufficient to explain variation of total nitrogen in frass. However, multiple linear regression integrating dietary N, P, and fibre was significant ($p\mathrm{<}0.01$; R² adj = 0.75). P₂O₅ and K₂O, contents of frass were explained by simple linear relationships with dietary phosphorus ($p\mathrm{<}0.001$; R²Â = 0.86) and potassium ($p\mathrm{<}0.001$; R²Â = 0.93), respectively. These findings demonstrate that dietary proximate composition directly influences the fertilising NPK values of frass and support the development of predictive models for frass nutrient composition. To date, this study is among the first to quantify these relationships under industrial-scale insect production conditions.

Optimising targeted mating in a black soldier fly breeding program

A.A. Shah^1*, M.J. Zorrilla¹, V. Reda², M. Gold³ and N. Warthmann¹

¹REPLOID Deutschland GmbH, R&D, Löbener Weg 7, 04523 Pegau, Germany; ²REPLOID Deutschland GmbH, Supply Chain and Production, Löbener Weg 7, 04523 Pegau, Germany; ³Reploid Group AG, R&D, Maria-Theresia-Straße 53, 4600 Wels, Austria; ^*shah@reploid.eu

Controlling parentage through targeted matings is a prerequisite for breeding programmes and genetic improvement. Indoor breeding of black soldier fly (BSF, Hermetia illucens) typically relies on unstructured mass-mating swarms, which provide limited control over individual mating events. Practical indoor protocols for efficient targeted matings remain non-trivial because mating behaviour and success depend on a plethora of environmental factors. We identified operational ranges for mating setups and environmental conditions that maximise the likelihood of successful crosses and developed a robust, repeatable targeted-mating protocol for indoor breeding of BSF. We conducted a series of iterative optimisation experiments using different small mating systems (“PP”: 500 ml conical PP plastic cups (5 × 8 × 11 cm), “HD”: PET boxes (9 × 9 × 5 cm), “GF”: 900 ml PP boxes (11 × 8 × 10 cm), “net cage”: 3.4 litre mosquito nets (15 × 15 × 15 cm)). We compared one-on-one pairings (female:male = 1:1) with polygynous setups with female-to-male ratios of 3:1, 4:1, 5:1, 8:1 and 10:1, and systematically evaluated key operational variables including illumination and husbandry factors such as adult feeding. Success was assessed using the proportion of females ovipositing fertile eggs under defined mating and oviposition conditions and the reproducibility of outcomes. Our results show that, across trials, polygynous setups proved more operationally reliable than one-on-one pairings. Oviposition was consistently highest at moderate female densities (4 – 5 females per male), where we regularly achieved 100% of females yielding fertile eggs. Occasional mating/oviposition failures highlighted sensitivity to microenvironmental factors, including differences in light intensity and duration, temperature, humidity and cage geometry, underscoring the need for standardised mating system setups and tightly controlled husbandry. Our study establishes a practical framework for repeatable targeted mating in BSF for breeding applications. The optimised protocol identifies a stable operating window, notably moderate female-to-male ratios, and emphasises controllable environmental drivers, especially illumination and microenvironment. This enables more dependable controlled crosses for breeding programmes.

Mating behaviour and sensory cues in BSF: key factors for optimising reproductive performance in mass rearing

G. Petroni^1*, S. Piersanti¹, G. Salerno² and M. Rebora¹

¹University of Perugia, Chemistry, Biology and Biotechnology, Via dell’ Elce di Sotto 8, 06123 Perugia PG, Italy; ²University of Perugia, Agricultural, Food and Environmental Sciences, Borgo XX Giugno 74, 06121 Perugia PG, Italy; ^*giulia.petroni@dottorandi.unipg.it

The black soldier fly (BSF), Hermetia illucens, is a key species of the circular economy due to its extraordinary larval bioconversion efficiency. While extensive research focuses on larval biomass production, a deeper understanding of adult reproductive biology is essential to further refine mass rearing protocols and ensure consistent performance in industrial facilities. This study provides a detailed ethogram of BSF mating behaviour, adds further information on BSF sexual dimorphism, and highlights sensory cues potentially involved in partner interactions and recognition. Morphological studies reveal marked sexual dimorphism in tarsal adhesive structures, with special discoidal setae, exclusively in males, suggesting their importance during mating. Behavioural observations, conducted through slow-motion video recordings, confirmed that the mating sequence is initiated by the female’s flight, which immediately triggers the male’s pursuit. A peculiar behaviour, “male hindlegs crossing,” was identified for the first time: the male firmly grasps the female mid-air by crossing his hindlegs across the abdomen. After landing, the receptive female remains still while only the male performs vigorous wing fanning. The male simultaneously performs over the female: “tapping” behaviour, a rhythmic touch on the female’s abdomen with the middle and hind legs, and “rubbing,” where the male’s forelegs are used over specific areas of the female’s body. Likewise in Drosophila, these actions may serve as a form of communication between partners, involving (1) tactile stimulation of the female, (2) chemical perception through tarsal gustatory sensilla of cuticular hydrocarbons to assess sex recognition. Wing fanning may represent a signal of mating motivation for females or intraspecific competition with other males. Behavioural observations indicate that agonistic encounters between males exhibit interaction patterns that mirror these courtship attempts, suggesting a possible role in competition. In conclusion, this research offers practical insights into the reproductive biology of BSF, providing a scientific basis for the development of more efficient management strategies for adult populations in mass rearing systems.

Patience pays: a successful multi-year breeding program in black soldier flies

K. Shrestha^1,2*, L.W. Beukeboom² and L. Francuski¹

¹Protix B.V., Research and Development, Van Konijnenburgweg 86, 4612 PL Bergen op Zoom, The Netherlands; ²University of Groningen, Evolutionary Genetics, Nijenborgh 7, 9747 AG Groningen, The Netherlands; ^*kriti.shrestha@protix.com

In 2019, a genetic improvement program was initiated to increase larval body weight in the black soldier fly (Hermetia illucens). Larval body weight increased consistently over 44 generations of directional selection (cumulative 133% gain in individual larval weight) with no indication of a phenotypic plateau, demonstrating potential for further genetic gains. Under automated production conditions, the selected line achieved a +34% increase in wet crate yield, +26% in dry matter yield, +32% in crude protein yield, and +21% in crude fat yield per crate relative to the base population. Throughout the selection program, key fitness components and production-relevant traits were monitored to identify potential correlated responses. There were no reproductive trade-offs associated with increased female body weight. Instead, the selected line showed an 18–49% increase in egg clutch weight per female, a 24–30% increase in the number of eggs per clutch, and a 3–4% increase in egg length. At the adult stage, sexual size dimorphism increased with overall body size, driven by a proportionally greater size increase in females than in males. Seven generations of selection-relaxation resulted in a 2% decline in body weight per generation, indicating that the genetic gains largely remain during several generations of no selection. Periodic refreshment with the selection line is recommended for long-term sustenance of benefits. Overall, this long-term breeding program demonstrates the power of artificial selection to improve biological performance and production efficiency in industrial Black Soldier Fly farming.

Sperm storage in BSF females reveals sexual conflicts: partial storage and digestion

C. Bressac^* and F. Manas

University of Tours — CNRS, Research Institute of Insect Biology, Parc de Grandmont, 37200 Tours, France; ^*christophe.bressac@univ-tours.fr

BSF is not only of great economic interest, but females have highly compartimentalized spermathecae, making it a valuable model for sexual selection investigations. Spatiotemporal dynamics of sperm storage after mating were described with fluorescence and transmission electron microscopy (TEM). Spermatozoa were counted both during and after mating in two successive spermathecae compartments: fishnet canals and reservoirs. In addition to seminal fluids that induce egg-laying and reduce female sexual receptivity, male transfers a sperm plug in the fishnet canals, then only some spermatozoa reach the reservoirs along 2 days. TEM observations of the fishnet canals revealed cells that may have digestive functions, explaining the decline in the number and viability of spermatozoa that have been not stored in the reservoirs. After one mating, females laid up to three fertile clutches, evidencing no constraints on sperm quantity or quality. Both spermatic and ultrastructural investigations strongly suggests that the ejaculate in BSF acts as a sperm plug, which is confirmed by paternity analyses. These results offer valuable perspectives to better understand male/female interactions during and after mating.

Metabolic regulatory networks for targeted nutrient synthesis in black soldier fly

L.Z. Zheng

Huazhong Agricultural University, South Luoshi Road, Hongshan District, 430070 Wuhan City, Hubei Province, P.R. China; ^*ly.zheng@mail.hzau.edu.cn

The black soldier fly (BSF) is a pivotal species for converting organic waste into high-value protein and lipids. However, the industrial application of BSF is currently constrained by unstable nutritional quality and an incomplete understanding of the metabolic mechanisms governing nutrient accumulation. To address the challenge of achieving precise, stable, and predictable production, this study aims to elucidate the key metabolic pathways and regulatory networks controlling lipid and protein synthesis in BSF throughout its life cycle. We constructed a genome-scale metabolic model (iHil3966) integrating life-cycle multi-omics. Dynamic proteomics and WGCNA mapped core larval regulatory networks. Key genes (FAS, ACC, FABP) and transcription factors (IVY, YBOX) were validated using RNAi, EMSA and dual-luciferase assays. Finally, model predictions were verified via nutritional intervention experiments with varying Carbohydrate: Protein ratios. Results confirm BSF nutritional plasticity. A diet with P+C = 70% (P:C 1:2.5) maximized survival (>95%) and biomass. The iHil3966 model successfully predicted that supplementing low-value substrates with valine and isoleucine enhances biomass and fatty acid production. Molecularly, FAS, ACC, ACSBG and DGAT1 were identified as key lipid accumulation genes. RNAi silencing of HiACC and HiFAS significantly reduced triglycerides. We uncovered a mechanism where transcription factors IVY and YBOX negatively regulate lipid synthesis by binding ACC and DGK promoters. Additionally, FABP was identified as a central metabolic regulator downstream of Eip75B. Phosphoproteomics revealed that the TOR pathway (via IRS1 and PDK1) activates rapidly during the egg-to-larva transition. Crucially, we verified a metabolic trade-off where promoting fatty acid oxidation facilitates protein synthesis, providing a mechanism for targeted product quality regulation. This study establishes the first comprehensive multi-omics regulatory map of BSF growth and metabolism. By identifying core regulatory factors (such as FABP, IVY, and YBOX) and optimizing nutritional algorithms, we provide a robust theoretical framework for the “targeted synthesis” of insect nutrients. These findings offer scalable strategies for the insect industry to produce standardized, functional BSF products tailored for specific animal nutrition needs.

Identification of stable reference genes for RT-qPCR normalization in Acheta domesticus

H. Ben-Miled^1*, N. Périard¹, F. Renois¹, M.H. Deschamps², F. Meurens¹ and M.O. Benoit-Biancamano¹

¹Faculté de médecine vétérinaire, Université de Montréal, Pathology and microbiology, 3200 Rue Sicotte, Saint-Hyacinthe, QC, Canada J2S 2M2; ²Faculty of Agricultural and Food Sciences, Department of Animal Sciences, Université Laval, 2425 rue de l’Agriculture, Laval, QC, Canada G1V 0A6; ^*houda.ben-miled@umontreal.ca

Acheta domesticus is attracting increasing interest as a sustainable protein source; however, molecular resources available for this species remain limited. The lack of validated reference genes constrains the reliability of RT-qPCR–based gene expression analyses, which are essential for investigating physiological, immune, and metabolic mechanisms in this insect. To identify and validate stable reference genes for RT-qPCR normalization in A. domesticus, taking into account different tissue types. The expression stability of six candidate reference genes (AdoNEOPT, EF2, 18S rRNA, $\mathit{EF}1\mathrm{Î\pm }$, His H3 and GAPDH) was evaluated across five tissue types (abdomen, legs, wings, head, and whole body). Stability assessment was performed using multiple complementary approaches, including BestKeeper, geNorm, NormFinder, the ΔCt method and the integrated RefFinder platform. Statistical validation using R confirmed the robustness of the results. $\mathit{EF}1\mathrm{Î\pm }$, AdoNEOPT, EF2 and 18S rRNA exhibited high expression stability across all analysed tissues. By contrast, GAPDH and His H3 showed substantial variability and are generally not recommended as reference genes, with the exception of head tissue, where GAPDH displayed stable expression. This study represents the first validation of reference genes in A. domesticus and establishes a robust methodological foundation for accurate, reliable, and reproducible gene expression analyses. It paves the way for future research in functional genomics and for the development of novel qPCR-based diagnostic tools relying on molecular signatures. These advances will support health monitoring in insect farming systems, which remain vulnerable to emerging diseases, and will contribute to the development of more efficient and sustainable insect-based protein production systems.

Investigation and mitigation of a vertically transmissible pathogen in a black soldier fly breeding facility

R.D. Steven^*, A.E. Hamdi, S. Mohammed and L. Wein

Protenga Pte Ltd, Research & Development, 3 Coleman Street, #03-24, 179804, Singapore; ^*rachel@protenga.com

Biosecurity and disease management are major concerns for black soldier fly (BSF) farming operations. The farm investigated in this study exhibited severe larval mortality at the fifth and sixth instar stages (14–18 days old, coinciding with the onset of the prepupal stage), characterised by soft rot, orange-brown colouration, bloating, cessation of feeding activity, and a sludgy, foul-smelling substrate. This study investigated two aspects: (1) verification of suspected biological vertical transmission to enable targeted mitigation, and (2) evaluation of microbial dynamics of healthy and larvae with mortality Verification of vertical transmission was conducted using an inoculant prepared from residual substrate mixed with dead larvae collected from high-mortality trays. Experimental treatments were categorised by feedstock type, inoculum dosage (residual substrate, five dead larvae, or 2% substrate inclusion), microbial suppression strategy (potassium sorbate addition, pH reduction to pH 4, or inoculant sterilisation), tray hygiene (washed and sanitised, sanitised only, or unwashed), larval strain, and rearing environment. All treatments were initiated with 5000 four-day-old larvae and 500 g feedstock, and were refed ad libitum until mortality occurred or pupation was reached. Vertical transmission was confirmed, with faster onset and higher mortality at increased inoculation levels. Inoculation with residual substrate or as few as five dead larvae resulted in >90% mortality by day 10 post-setup. Sterilised inoculant treatments showed no mortality and complete pupation, whereas unsterilised treatments reached 100% mortality within 10 days. Tray hygiene trials demonstrated that washing or sanitising alone was insufficient to disrupt transmission; only the combined washing and sanitising protocol fully prevented mortality. Sanitisation using a disinfectant containing benzalkonium chloride and glutaraldehyde at 1% (v/v) was successfully implemented as mitigation. For microbial analysis, seedling larvae, healthy controls, and mortality-affected larvae were analysed using 16S rRNA gene sequencing. Distinct bacterial community profiles were observed between groups, although no dominant pathogenic taxa were identified. Low-abundance opportunistic bacteria, including Wohlfahrtiimonas, Klebsiella, and anaerobic taxa such as Desulfovibrio spp., were detected. Overall, this study verified the presence of vertically transmissible biological contamination at low inoculation thresholds and identified an effective sanitation-based mitigation measure. The findings highlight the importance of strict tray hygiene, routine colony health monitoring, and further research on BSF-specific pathogens

Flies to feed the flock: a relative welfare exploration

M. Dicke^1*, Y. Kortsmit¹, A. Dörper^1,2, J.J.A. Van Loon¹ and T. Veldkamp²

¹Wageningen University & Research, Laboratory of Entomology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; ²Wageningen University & Research, Wageningen Livestock Research, De Elst 1, 6700 AH Wageningen, The Netherlands; ^*marcel.dicke@wur.nl

Welfare is a topic that gains rapid interest in research on insects as livestock feed. Yet, how this should be assessed remains a topic of exploration. Here we will address the behaviour of an insect as well as that of its predator, a bird, in the context of their behaviour. Video recordings have been made of the behaviour of black soldier fly (BSF) larvae to investigate their aggregation behaviour in dark conditions. Video recordings of the behaviour of poultry (broilers and layers) have been made when provided with BSF larvae, either as live larvae or as larval products. BSF larvae use cues related to conspecifics to aggregate and this behaviour is context dependent. We studied whether pathogen infection influenced this behaviour. Feeding live BSF larvae affected the behaviour of broilers and layers much more than the inclusion of BSF meal in the diet. Black solder fly larvae have adapted to exploit ephemeral food sources. They have evolved to rapidly develop on a diversity of resources. During development they are exposed to a diversity of threats including diseases and predation. This has resulted in behaviour that maximises the use of these resources while minimising the risks. The larvae exhibit dynamic aggregation behaviour which is mediated by cues from conspecifics. Is this information relevant for considerations of BSF welfare? The vast majority of birds on our planet use insects as food source. Wild relatives of poultry birds have evolved as omnivores that readily include insects in their diet and actively forage to find such prey. Insects provide an important source of proteins for adults as well as their chicks. When provided with BSF larvae broilers as well as laying hens readily eat them and this influences their overall behaviour. However, the vast majority of poultry birds do not have access to insects as a food source. Do BSF larvae contribute to poultry welfare? The final element of this presentation will be how to integrate welfare considerations of BSF and poultry when producing animal proteins to feed humans.

Optimising mealworm welfare: results from feeding and environmental trials at Bobo Eco Farm

E. Ssebbombo^1*, R. Kayizzi¹, H. Kelstrup² and V. Gwokyalya¹

¹Mothers Against Malnutrition And Hunger, Mityana, 256, Uganda; ²6-Leg Solutions, 2440 S 116th Street, Burien, WA 98168-1214, USA; ^*essebbombo@gmail.com

Optimising rearing practices is essential for improving efficiency and productivity in mealworm production. This study investigates the impact of diet, environmental conditions, and management strategies on mealworm production. Experiments focused on feeding strategies for egg hatching, larvae development, adult welfare, pupa survival, and post-split larvae feeding. Alternative substrates, such as wheat bran, cassava peels, wet feeding, and protein supplementation with black soldier fly larvae (BSFL), were also tested. At Bobo Eco Farm, larvae were reared in trays with a 50:50 mixture of maize bran (MB) and crushed maize cobs (MC). 100g of split larvae were placed in each tray with 1000 g of substrate. Refeeding and frassing were done biweekly. Trials evaluated diets (MB, wheat bran (WB), and MB + MC) on larvae development and egg hatchability over 6–14 weeks. Adult welfare was assessed in warmer (26–31 °C, 55–70% humidity) and cooler (22–25 °C, 65–80% humidity) rooms. Pupa survival and feeding strategies, including one-time and continuous refeeding, were tested. Wet feeding and alternative substrates were also studied. Larvae fed MB reached split in 8 weeks, while MB + MC took 11 weeks. MC-fed larvae split at 14 weeks. Wheat bran-fed larvae split in 6 weeks, while MB larvae took 9 weeks. WB larvae were larger and easier to separate from frass. Frass as a substrate increased egg yield. Cooler conditions improved adult survival and egg production. Pupa survival dropped from 60% to 30% in warmer conditions. Wet feeding accelerated larvae growth, with wet-fed larvae reaching split in 7–8 weeks, compared to 11–12 weeks for dry-fed larvae. BSFL-fed larvae split in 8 weeks, while wheat bran-fed larvae took 13 weeks. This study highlights the importance of improving welfare conditions to enhance mealworm rearing efficiency. Providing optimal diets, such as WB over MB, accelerated larvae development and minimised stress. Lower temperatures and higher humidity improved adult survival and egg production. Welfare improvements through diet, environmental management, and feeding practices result in healthier and more productive mealworms. Optimising mealworm welfare through diet, environmental conditions, and wet feeding significantly enhanced productivity. By focusing on welfare, mealworms exhibited faster growth, higher survival, and better reproductive success, essential for sustainable mealworm farming.

Genetic effects on larval performance and bioconversion efficiency in Hermetia illucens: validation across generations

L. Frooninckx^1*, L. Broeckx¹, R. Meyermans², S. Berrens¹, A. Wuyts¹, M. Verschaeren¹, C. Sandrock³ and S. Van Miert¹

¹Thomas More University of Applied Sciences, Centre of Expertise Sustainable Biomass and Chemistry, Kleinhoefstraat 4, 2440 Geel, Belgium; ²KU Leuven, Center for Animal Breeding and Genetics, Department of Biosystems, Kasteelpark Arenberg 30 box 2472, 3000 Leuven, Belgium; ³FiBL, Department of Livestock Sciences, Ackerstrasse 113, 5070 Frick, Switzerland; ^*lotte.frooninckx@thomasmore.be

The black soldier fly (Hermetia illucens) is widely studied for its ability to convert low-value organic substrates into high-value insect biomass. In practice, these substrates serve as larval diets and can vary strongly in nutrient composition, fibre content and digestibility, which has a major impact on larval growth, yield and conversion efficiency. While substrate effects on performance are becoming well documented, far less is known about how genetic differences between H. illucens strains interact with nutritionally contrasting diets, and whether such genotype-by-environment (G × E) effects are stable across time. In this study, we evaluated the influence of genetic background on larval performance and bioconversion efficiency on different substrates across time by repeatedly assessing nine genetically distinct captive H. illucens strains, representing European lineages (4 populations), African lineages (2 populations) and Oceanian lineages (3 populations). Strains were genotyped using microsatellites markers and whole-genome sequencing and reared under standardized conditions over the course of 10 generations during this study. Larval performance was assessed on three nutritionally contrasting diets: a nutrient-rich chicken start mash, a cereal-based wheat bran, and a high-fibre horse feed. Following an extensive initial feeding trial, the experiment was repeated in two additional trials conducted over non-consecutive generations. Across all trials, significant effects of strain and substrate were observed for larval yield and bioconversion efficiency. While absolute performance values differed between trials, normalization revealed that relative strain performance remained largely consistent over time. This study demonstrates the importance of genetic background for bioconversion efficiency.

Exploring the hemolymph microbiome: an emerging lever for health management in industrial Tenebrio molitor rearing

R. Tedeschi^1*, V. Candian¹, D. Hentati², C. Savio¹, E. Bigarella², G. Brenna², C. Jucker² and E. Crotti²

¹University of Turin, Department of Agricultural, Forest and Food Sciences (DISAFA), Largo P. Braccini 2, 10095 Grugliasco (TO), Italy; ²University of Milan, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Celoria 2, 20133 Milan (MI), Italy; ^*rosemarie.tedeschi@unito.it

The farming of edible insects represents a promising alternative strategy for the sustainable production of protein-rich food and feed with a low environmental footprint. Rearing substrates strongly influence insect performance and play a key role in shaping insect- associated microbiota, which in turn can affect host physiology and immune competence. While gut microbiota has been extensively investigated, the microbiota associated with insect hemolymph and its potential role in immune responses remain largely unexplored. The HeMiTool project (Hemolymph Microbiome of insects: a promising Tool to develop innovative strategies to control pests and protect beneficials) aims to fill this knowledge gap by providing an in-depth characterisation of hemolymph microbiome–immune system dynamics in mature larvae of Tenebrio molitor L. Larvae were continuously reared on different agro-industrial by-products under controlled conditions, allowing a robust comparison of diet-related effects. Hemolymph and gut microbial communities were analysed using a combined culture- dependent and culture-independent approach, including high-throughput 16S rRNA gene sequencing and quantitative PCR. In parallel, key chemical and biochemical parameters of the hemolymph were assessed, and some immune-related traits were investigated. Methodological optimisation of hemolymph extraction and processing ensured the reliability of downstream microbiological and immunological analyses. Preliminary results indicate that both hemolymph and gut-associated microbial communities are influenced by the rearing substrate, supporting the existence of a previously overlooked, diet-responsive hemolymph microbiome compartment. Observed trends in hemolymph- associated parameters suggest possible interactions between nutrition and host physiological status, although further investigation is required to clarify these relationships. Overall, this study provides first insights into the hemolymph microbiome of an edible insect species and highlights its relevance as an emerging component of insect biology. These findings provide a foundation for future diet-based strategies aimed at improving robustness and health stability in mass-reared edible insects. This work was funded by the PRIN 2022 PNRR project HeMiTool (Grant no. P20228WWB7), European Union – Next Generation EU, Mission 4 Component 1.

Black soldier fly (Hermetia illucens) characterisation of gut microbiome

A. Barca^*

Adelaide University, School of Animal and Veterinary Sciences, Mudla Wirra Road, Roseworthy, SA 5371, Australia; ^*adele.barca@adelaide.edu.au

The exact role and functions of the microbiome of black soldier fly (Hermetia illucens) (BSFL) are not known, however it is essential for them to convert organic waste into high-value biomass, while enhancing said waste into protein and nutrients suitable for other animal consumption. It is important to identify the microbes of the gut in order to characterise possible probiotics or pre-fermentation advantages as both qualitative and quantifying microbial communities informing possible production activities that will maximise BSFL production outcomes. This study examines the taxonomic structure and abundancy of the intestinal bacterial communities of BSFL while fed on selective organic waste, based on the bacterial communities within the full length of BSFL gastrointestinal tract. In addition, the bacterial profiling of the waste substrates samples provides a catalogue of the environmental bacterial taxa. Bacterial assemblages were evaluated using Illumina deep-sequencing of the hypervariable region (V1-V2) of the 16S rRNA gene amplified from each sample. 738 bacteria were identified in 70 samples of larvae and 18 samples of frass, characterising the top phyla among all the communities in terms of relative abundance as Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria. Firmicutes, Actinobacteria and Bacteroidetes were the dominant phyla in the BSFL gut of all organic waste systems with egg substrate BSFL showing additional abundance in Proteobacteria, while Firmicutes dominate the frass, with the addition of Actinobacteria and Pseudomonadales in the egg and mixed substrates. In frass samples the presence of synthesising amino acids, Rhizobiales, Burkholderia and Bacteroidales were found however they were not present in any larvae samples. Key analysis based on genes showed that intestinal microbes’ communities within BSFL differ to their respective frass microbe abundance. These results clarify the biological mechanisms of high-efficiency nutrient conversion in BSFL associated with microbes.

Enhancing insect production with innovative microbial solutions

J.C. Paredes^*

Phileo by Lesaffre, 77 Rue de Menin, 59520 Marcq-en-Barœul, France; ^*j.paredesescobar@phileo.lesaffre.com

The rapid growth of the insect farming industry presents both opportunities and challenges for producers seeking to optimise productivity and sustainability. A key factor in ensuring the success of insect rearing operations is the ability to maintain optimal gut health and nutrient utilisation in the black soldier fly larvae. In this presentation, we will share Phileo by Lesaffre latest findings from our research and industrial trials on the application of innovative microbial solutions to enhance insect performance, create functional products, and reduce GHG emissions. Our trials have demonstrated that the strategic incorporation of probiotics and postbiotics into insect diets can significantly improve feed conversion ratios, increase larval weight gain, and reduce developmental time. By leveraging the natural synergies between insects and microbes, our solutions help to stabilize substrate and intestinal environment and bolster the insect’s immune response. This, in turn, leads to increased productivity, improved product quality and enhanced sustainability for insect farming operations. Drawing on industrial scale studies and data-driven insights, we will explore the mechanisms by which our microbial additives deliver these benefits, as well as provide guidance on the practical implementation of these technologies within commercial insect production systems. Attendees will gain a comprehensive understanding of how innovative microbial solutions can unlock the full potential of insect farming and position the industry for long-term success.

Influence of protein-enriched diets on the larval development of Tenebrio molitor

D.M. Costa-Martı́nez^1,2*, L. Pisa³, J.L. González-Jiménez^2,4 and J. Galian^1,3

¹University of Murcia, Department of Zoology and Physical Anthropology, 30100 Murcia, Spain; ²Protiberia, Villa Insect S.L., 02270 Villamalea, Spain; ³ArthropoTech S.L., University of Murcia, 30100 Murcia, Spain;⁴University of Castilla-La Mancha, Department of Agroforestry Technology and Science and Genetics, 02071 Albacete, Spain; ^*domingomanuel.costam@um.es

Tenebrio molitor (Tm) is a sustainable protein source, and optimising mass rearing requires valorising agri-food byproducts (Sangiorgio et al., 2022). Wheat bran is the standard feed, but balanced macronutrients are essential for profitable yield (Kröncke & Benning, 2022), as high dietary protein alone, in addition to be costly, does not ensure growth when digestibility is low (Montalbán et al., 2022). This study assessed three protein-rich byproducts on Tm larval growth and protein assimilation. Eggs from one cohort were reared on wheat bran until week 6, then assigned to seven dietary treatments (T): 100% wheat bran (control) or wheat bran partially replaced (40 or 30%) with byproduct A (T1–2), B (T3–4), or C (T5–6), yielding approx. 18–23% dietary protein. Five replicates per treatment were maintained at 27 °C and 70% RH with ad libitum feeding. Larval mass was measured weekly (20 larvae/replicate, randomly sampled with replacement) until pupation. Larval and frass crude protein were analysed using the Kjeldahl method (Yu et al., 2021). A Gamma GLMM revealed a strong effect of time on larval mass (χ²(7) = 6876.1, $p\mathrm{<}0.0001$) and a highly significant treatment × week interaction (χ²(42) = 775.5, $p\mathrm{<}0.0001$), while initial treatment effects were absent. This demonstrates that treatment effects emerged as larvae grew. Estimated marginal means indicated that differences appeared in week 7, and were clearer in week 8, where T3–T4 consistently formed the lowest-mass groups. By weeks 10–13, treatments formed three to four distinct statistical clusters per week, with stable rankings: the control and T1–2 had the highest masses, while T3–5 remained the lowest. For the assessment of protein assimilation, preliminary analysis shows higher nitrogen use efficiency in experimental diets, with protein assimilation reaching approx. 20% and feed–protein conversion increasing to 8.5–8.8% versus 7.1% in the control, while body protein content remained stable (approx. 46%). Thus, dietary effects reflected improved protein use rather than altered body protein content. The results demonstrate that clear divergences in growth performance emerge in later instars. Control and byproduct A (T1–T2) showed the highest masses, confirming wheat bran’s efficacy (Bordiean et al., 2022). The poor performance of byproduct B (T3–T4) suggests nutritional imbalance (Morales-Ramos et al., 2013) or lower digestibility (Zhang et al., 2019). This highlights that the entire nutritional matrix, not just protein quantity, must be evaluated for effective diet formulation.

Revealing the reproductive biology of the black soldier fly: from age determination to mating dynamics

Y. Riabtseva^*, B. Grodzki and M. Bolard

Nasekomo AD, Saedinenie Street 299, Lozen village, Sofia 1151, Bulgaria; ^*yulia.ryabceva@nasekomo.life

The black soldier fly (BSF, Hermetia illucens) is a key species for sustainable bioconversion and production of protein, oil and organic fertilisers. Despite its industrial importance, many aspects of its reproductive biology remain poorly understood. To support optimization of mass-rearing systems, a series of studies were conducted to clarify key factors regulating reproductive performance in BSF. Age determination, reproductive morphology and mating performance were examined under controlled environmental conditions. A total of 2500 adults were analysed to identify morphological and physiological age markers, including abdominal “windows,” ovarian development, and mesothoracic phragmata. For reproductive analysis, 700 females were dissected and classified into five ovarian developmental stages. Ovarian length (L), width (W) and index (L/W) were measured, together with wing length (LW) and thorax length (TL) as maturity indicators. Oviposition was studied using 120 egg clutches observed for 5 days to assess fertility, total egg number and the proportion of infertile eggs. To evaluate the influence of body size on reproduction, 720 adults were grouped into four sex – size combinations (large – large, small – small, small male – large female, large male – small female) with a 1:1 sex ratio and 60 flies per cage. Data was analysed using IBM SPSS Statistics. Distinct changes in abdominal “windows,” ovarian morphology, and mesothoracic phragmata correlated strongly with chronological age ($p\mathrm{<}0.001$), enabling accurate identification of sexual maturity and synchronization of breeding cohorts. Ovarian morphometry revealed five developmental stages; L and OI were reliable indicators of reproductive status, while body size alone was insufficient. Infertile eggs (6 – 16% per clutch) appeared consistently and correlated with clutch size ($r=0.97$, $p\mathrm{<}0.001$), possibly indicating a physiological or behavioral function. Reproductive success depended mainly on the size ratio between males and females, not absolute size. The highest mating success (88 – 96%) occurred when the female:male TL ratio ranged from 0.95 to 1.12 (R²Â = 0.91, $p\mathrm{<}0.001$). Small males showed greater agility, while large females produced more eggs, suggesting that optimal size complementarity maximises mating efficiency and reproductive output. Collectively, these findings deepen understanding of BSF reproductive performance by linking morphological age markers, ovarian maturation, mating dynamics and reproductive output. The integration of these parameters provides practical tools for assessing adult readiness, synchronising breeding populations, and enhancing egg production efficiency in industrial-scale BSF rearing.

How larval diet composition and switching shape adult fitness in the black soldier fly

H. Mahdjoub^*, R. Weladji and R. Khelifa

Concordia University, Biology, 7141 Sherbrooke Street W., Montreal, QC, Canada H4B 1R6; ^*hayatmahdjoub@gmail.com

Larval nutrition strongly shapes adult life-history traits and reproductive output in insects. However, most experimental studies use a single diet throughout larval development, an approach that poorly reflects the nutritionally variable conditions insects experience in nature. Understanding how insects respond to such nutritional variability is essential for improving production efficiency under real-world conditions. Here, we examined how larval diet composition and dietary switching influence adult fitness traits in the black soldier fly (Hermetia illucens), a species whose adults do not feed and rely entirely on nutrients accumulated during larval development. Larvae were reared on either a nutritionally complex, balanced diet (Gainesville diet) or a nutritionally simple monodiet (corn). Using a full-factorial design, we tested eight larval diet regimes in which individuals either remained on one diet (low-quality (LLL) or high-quality (HHH)) or experienced one ((HHL, HLL, LHH, LLH)) or two ((HLH, LHL)) switches in diet quality. We measured larval growth rate and assessed adult body size, fecundity, sexual dimorphism, longevity, and reproductive performance. We predict strong, trait-specific plasticity in response to larval nutrition, with individuals reared predominantly on the complex diet exhibiting higher fitness than those reared on the monodiet. We also expect diet variability to produce non-linear outcomes driven by ontogeny-specific sensitivity to nutrition. Overall, our study provides insights into how variable larval nutrition influences performance-relevant traits in H. illucens, with direct implications for optimising feeding strategies, breeding programs, and production stability in insect food and feed systems.

Microbial partners in immunity: probiotic effects on yellow mealworm and black soldier fly physiology

C. Savio^1*, D. Hentati², V. Candian¹, G. Brenna², E. Bigarella², C. Jucker², E. Crotti² and R. Tedeschi¹

¹Università degli Studi di Turin, Department of Agricultural, Forest and Food Sciences (DISAFA), Largo Braccini 22, 10095 Grugliasco (TO), Italy; ²Università degli Studi di Milano, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Celoria 2, 20133 Milan, Italy; ^*carlottasavio2@gmail.com

In these years, the identification of potential probiotic microorganisms within the microbiome of farmed insects, as well as their application in mass-rearing systems, has been increasingly investigated to evaluate their effects on insect growth, reproduction and overall health with the additional aim of mitigating multiple stressors such as entomopathogens, temperature fluctuations and high rearing densities. The PRIN 2022 Project InProFarm (Insect Probiotic-assisted Farming: a promising tool to enhance edible insect health and performances) explored the interactions between beneficial bacterial symbionts and two mass-reared species, Tenebrio molitor (yellow mealworm) and Hermetia illucens (black soldier fly), and their impact on humoral and cellular immune responses. Beneficial bacterial symbionts were screened from larvae of both species, and their probiotic potential was assessed through in vitro and in vivo assays, including feed administration, evaluation of insect performance, and analysis of microbiota composition. In addition to growth performance, genes involved in growth and immune pathways were analysed to uncover the effects of probiotic supplementation on key traits relevant to insects breeding, such as antimicrobial peptides production and reproduction. Overall, the study highlights the importance of promoting healthy and safe insect production systems for feed and food applications. This work was funded by the PRIN 2022 Project InProFarm (Insect Probiotic-assisted Farming: a promising tool to enhance edible insect health and performances), grant number 2022L4NJMK, funded by the European Union — Next Generation EU, Mission 4 Component 1.

Repeat-rich and rapidly evolving: the TE landscape of the black soldier fly

C.J. Picard^*, H. Rosche-Flores and S.A. Fischer

Indiana University Indianapolis, Biology, 723 W Michigan Street, Indianapolis, IN 46202, USA; ^*cpicard@iu.edu

Transposable elements (TEs) are major contributors to genome structure, regulation, and evolutionary change in insects. As Hermetia illucens (BSF) becomes central to global insect agriculture, clarifying its TE landscape is critical for understanding genomic plasticity, domestication processes, and phenotypic variability is relevant to industrial production. We examined whole-genome repeat composition in BSF using curated TE annotations and divergence profiles to characterise TE classes, relative abundance, and signatures of historical versus recent TE activity in four de novo genomes. BSF displays an exceptionally high repeat content (67.6–70.8%), positioning it among the most TE dense insect genomes and reflecting a genomic architecture more complex than traditional insect species. The TE community is composed of substantial LINE, DNA transposon, and unclassified repeat fractions, with evidence for both ancient TE expansions and more recent proliferation events. Class-level TE diversity was nearly identical among genomes, but multiple DNA transposon families showed distinct lineage-specific differences. Patterns of TE diversity may suggest robust mechanisms of genomic variability and provide a framework for understanding lineage specific differences that may influence traits such as development rate, stress tolerance, and reproductive performance. TE driven genomic flexibility likely contributes to the phenotypic variation observed across commercial and laboratory strains, which may make TE associated markers valuable tools for selective breeding and population management. These results highlight the importance of incorporating TE dynamics into ongoing genomic, evolutionary, and applied research efforts. By defining the TE architecture of H. illucens, this study provides foundational genomic insight for a species rapidly becoming indispensable to sustainable insect agriculture.

Water requirements and their effects on growth and gene expression in the yellow mealworm

M. Krzyżaniak^1*, O. Kosewska², S. Przemieniecki², P. Białoskórski¹, A. Aslam¹ and M.J. Stolarski¹

¹University of Warmia and Mazury in Olsztyn, Department of Genetics, Plant Breeding and Bioresource Engineering, Plac Łódzki 3, 10-724 Olsztyn, Poland; ²University of Warmia and Mazury in Olsztyn, Department of Entomology, Phytopathology and Molecular Diagnostics, Prawocheńskiego 17, 10-720 Olsztyn, Poland; ^*michal.krzyzaniak@uwm.edu.pl

Numerous studies have shown that insect growth depends on adequate water supply, including in the yellow mealworm (Tenebrio molitor L.). Although Y. mealworm can develop on dry feed, supplementary water in industrial rearing can accelerate its growth. However, data on the effects of water supply levels and sources remain limited. Therefore, our research team has been conducting studies in this field in recent years. Our preliminary studies indicate that water deficiency significantly reduces the growth rate and biomass production of mealworm larvae, while inducing defence responses manifested by increased expression of water-stress-related genes, such as HSP70 and Coleoptericin B. In addition, analysis of P450 gene expression proved to be a useful indicator for assessing moderate water deficit in insects. Moreover, both the amount of water supplied to the insects and its source affect their growth and development, and larvae without access to an additional water source can attain up to a fourfold lower body mass than larvae provided with an additional source of water. The lack of supplementary water, however, does not significantly affect larval survival, which remains high at approx. 90–98%. When comparing water sources, larvae supplied with carrot as a water source achieved a slightly higher body mass (128 mg per individual) than those provided with agar gel (114 mg per individual), although it should be noted that this difference was not statistically significant. In conclusion, the water source and its rate can significantly affect the development of mealworm larvae in industrial rearing. Therefore, determining the optimal rate of supplementary water and its source is of key importance, as this can substantially influence the profitability of insect production as well as its environmental impact (e.g. carbon or water footprint). For this reason, our research team will continue investigations in this field. This research was funded by the University of Warmia and Mazury in Olsztyn, Faculty of Agriculture and Forestry, through the Department of Genetics, Plant Breeding, and Bioresource Engineering (topic number 30.610.007-110) and the Department of Entomology, Phytopathology, and Molecular Diagnostics (topic number 30.610.011-110). Moreover, it was funded by the Minister of Science under “the Regional Initiative of Excellence Program.”

Digestibility of cereal-based nutritional substrates in Tenebrio molitor

M. Petrović^*, M. Vukadinović, S. Krstović, N. Laćarac, I. Jajić, A. Popović and M. Polovinski-Horvatović

Faculty of Agriculture, University of Novi Sad, Department of environmental and plant protection and Department for Animal Husbandry, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia; ^*milos.petrovic@polj.edu.rs

In the context of sustainable insect production and circular bioeconomy, increasing attention has been given to the use of agricultural residues and agro-industrial byproducts as alternative substrates for rearing Tenebrio molitor larvae. Tenebrio molitor exhibits a high capacity to adapt to diverse substrates, which is associated with its ability to degrade complex carbohydrates such as cellulose. However, the specific digestive efficiency and metabolic responses of this species to low-quality feed remain insufficiently understood, highlighting the importance of further research on food digestibility in this insect. Some of the production parameters and digestibility were evaluated during the rearing of T. molitor. The experiment was initiated with 25 g of 30-day-old Tenebrio molitor larvae and lasted for six weeks, during which weekly weight gain, feed intake, frass production, and feed conversion were recorded. For the experiment it was used eight different feedstuffs. All parameters were measured per tray. In terms of total weight gain for the whole period, the highest gain was observed in the group fed with wheat bran (average 39.97 g/tray), while the lowest weight gain was observed in the group fed with breadcrumbs (only 9.85 g/tray). The digestibility coefficient for each feed was calculated based on the feed consumption and the frass production. The highest digestibility was observed in oat flakes (0.78) and the lowest in sunflower meal (0.30). While the weight gain was very low in the group with breadcrumbs, the digestibility was high (average 0.77). The feed conversion ratios for wheat bran, sunflower meal, breadcrumbs, oat, oat flakes, barley, barley flakes, and DDGs were 2.11, 3.8, 8.6, 4.02, 3.49, 7.05, 3.80 and 1.95, respectively. Overall, T. molitor larvae showed the best growth performance and feed utilisation when reared on wheat bran, as indicated by the highest total weight gain and the lowest feed conversion index compared with other used feed. These results confirm the strong adaptability of this species to different cereal-based substrates and underline the importance of substrate quality for optimising digestibility and production efficiency in sustainable insect rearing systems.

Safeguarding insect mass-rearing: molecular approaches to pathogen identification and surveillance

L. Hernández Pelegrı́n^1,2*, P. Garcı́a-Castillo¹ and S. Herrero¹

¹Research Institute of Biotechnology and Biomedicine, University of Valencia, Department of Genetics, Dr Moliner, 50, 46100 Burjassot, Valencia, Spain; ²Cucare Diagnostics, Cl Catedrático Agustı́n, 9, 46980 Paterna, Valencia, Spain; ^*luis.hernandez.pelegrin@gmail.com

Insect mass-rearing is a rapidly growing field with applications ranging from sustainable protein production to biocontrol. While most research has focused on improving production efficiency, the health of reared insects has received comparatively less attention, despite its critical impact on industry sustainability. Our work aims to bridge this gap by applying advanced scientific methods to detect and monitor pathogens threatening insect-rearing operations. By combining specialized databases of insect pathogens with high-throughput sequencing, we have identified novel pathogens associated with impaired production in mass-reared insects, such as the black soldier fly, the mealworm, and biocontrol agents of the Nesidiocoris genus. In parallel, we have developed targeted molecular techniques to deliver cost-effective and tailored approaches for the detection and quantification of known insect pathogens. Overall, these case studies highlight the importance of integrating pathogen discovery and health monitoring into standard rearing practices to ensure the resilience and safety of this emerging industry.

Assessment of physiological status and antimicrobial potential in larvae of H. illucens reared on aquaculture sludge

D. Santori^*, F. Di Donato, G. Grifoni, E. Sezzi and S. Cucci

Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, via Appia Nuova, 1411, 00178 Roma, Italy; ^*davide.santori@izslt.it

The bioconversion of aquaculture sludge by Hermetia illucens larvae (BSFL) represents a key strategy for the circular economy. It is well established that the nature of the growth substrate can modulate the insect’s physiology and immune response. However, the energetic cost of such adaptation remains less explored. The present study aims to verify the substrate-induced antibacterial properties of the hemolymph against Aeromonas salmonicida, a major fish pathogen, and to evaluate the temporal dynamics of the stress response (Heat Shock Protein — HSP70) and metabolic vigor (Vitellogenin — VTG). Fourth instar larvae were subjected to three different diets: Control (Gainesville diet), 50% sludge + 50% Gainesville diet, and 100% aquaculture sludge. Sampling was performed at T0 (baseline), T1 (4 hours), and T2 (24 hours). Gene expression analysis for VTG and HSP70 was conducted via qPCR on cDNA, normalizing data against the 16S gene. Concurrently, hemolymph bactericidal activity was evaluated using in vitro antibiogram assays. Molecular tests showed at T1 (4h) a significant up-regulation of VTG in the 100% sludge group (Fold Change 3.38). At T2 (24h), VTG expression underwent a marked reduction (FC 0.07 in the 50% sludge group). HSP70 expression showed a significant increase exclusively at 24h in the 100% group (FC 3.36), suggesting the late activation of cellular protection mechanisms. Microbiological analyses against A. salmonicida revealed distinct patterns: in the Control group (T1 and T2), hemolymph produced an inhibition zone of approx. 6.6 mm with the presence of internal colonies. The 50% sludge group generated a wider zone (6.9 mm), albeit with colonies present within. In the 100% sludge group, despite a reduced zone (approx. 5.8 mm), a total absence of colonies within the zone was observed. Data integration highlights an energetic trade-off. Although defensive efficacy remains maximal in the 100% sludge group, VTG analysis reveals the sustained metabolic effort: the initial overexpression (T1) suggests energetic mobilization, while the subsequent drop (T2) indicates potential transcriptional exhaustion, with the larva downregulating growth functions to prioritize survival. The delayed activation of HSP70 suggests the intervention of chaperones to counteract proteotoxic damage. In conclusion, pure sludge acts as a potent immunostimulant but imposes a significant physiological cost, as evidenced by the molecular pattern.

Potential host-parasite relationships between the parasitoid hymenoptera Trichopria drosophilae and Hermetia illucens

E. Sezzi^1*, S. Cucci¹, D. Santori¹, R. Fochetti² and G. Polgar²

¹Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, via Appia Nuova 1411, 00178 Roma, Italy; ²Università della Tuscia, Dipartimento per la Innovazione nei sistemi Biologici, Agroalimentari e Forestali, Largo dell’Università snc, 01100 Viterbo, Italy; ^*erminia.sezzi@izslt.it

Hermetia illucens (Linnaeus), the black soldier fly (BSF), is undoubtedly the most studied species among those recently authorised by European regulations for use in insect meal as a protein source in feed for poultry, swine, and fish. However, despite the extensive volume of studies available on this species, knowledge regarding its potential pathogens remains scarce. An analysis of specific literature indicates that the pupal stages of the black soldier fly could be parasitized by certain species of the genus Trichopria (Hymenoptera, Diapriidae). The aim of the present study was to verify experimentally whether the parasitoid Trichopria could have an actual parasitic incidence on H. illucens rearing systems. This is particularly important considering that T. drosophilae is currently used for the biological control of Drosophila suzukii Matsumura, which was recently accidentally introduced into the country. T. drosophilae has been commercially available in Italy since 2017, is commonly on sale, and has been widely and regularly released across the national territory. The possible parasitic action exerted by the parasitoid T. drosophilae towards the pupal stages of H. illucens was considered, with the aim of potentially suggesting the implementation of containment measures in facilities intended for rearing the latter, ranging from large centralized plants to smaller, locally-based structures typical of circular economy models. To verify the type and degree of interspecific interactions, an experimental laboratory setup was created to house the larval stages of H. illucens and D. melanogaster together with adults of T. drosophilae. Three series of experiments were carried out by exposing T. drosophilae females, previously kept for 24 hours with males, to D. melanogaster pupae and the different larval stages of H. illucens for 48 hours under different experimental conditions. T. drosophilae was shown to parasitize D. melanogaster in all trials but never parasitized H. illucens. When placed with D. melanogaster, Trichopria exhibited parasitisation rates varying between 40% and 80%. D. melanogaster placed in the absence of Trichopria (negative control) showed very high success and emergence rates (75%). H. illucens in the absence of Trichopria (negative control) had equally high success rates (80%); when placed together, no larval stage or pupa was parasitized, and the emergence rate remained high.

Abundance of selected amino acid transporter mRNAs in the gut of starved and fed black soldier fly larvae

M. Mielenz^*, A.-K. Möller, G. Daş and C.C. Metges

Research Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; ^*mielenz@fbn-dummerstorf.de

The growth of black soldier fly larvae (BSFL) depends on availability and uptake of nutrients such as amino acids (AA), which occurs via intestinal AA transporters (AAT). The capacity and affinity of these AAT are responsible for the efficiency of AA uptake, with their abundance depending on the insect’s developmental status and feed supply. In this initial work, we quantified the expression of several putative AAT transcripts in the mid- and hindgut of fed and starved BSFL. The BSFL (12 vessels each with 150 larvae) were kept at 27.5 °C and 65% relative humidity until harvest on day (d) 16 and fed chicken feed (CF). On d 15, larvae from 6 vessels were isolated and kept separately without feeding substrate for 24 h (starved; n = 6) while larvae in remaining 6 vessels continued on CF until d 16 (fed; n = 6). At harvest, all larvae were isolated from the substrate, washed, dried and frozen in liquid nitrogen. The hindgut and the posterior midgut were isolated from 5 starved and 5 fed frozen larvae/vessel and analysed as pooled samples. After extraction, the reverse transcribed RNA was quantified by qPCR. Primers were selected based on predicted sequences (NIH GenBank). The data were analysed by PROC NPAR1WAY (SAS 9.4) with the Wilcoxon test (significance: $P\mathrm{<}0.05$, trend: 0.05<$P\mathrm{<}0.1$). Four putative neutral AAT (XR_005249910.1 (N1), XM_038061550.1 (N2), XM_038062991 (N3), XM_038064401.1 (N4)) and two putative cationic AAT (XM_038052742.1 (C1), XM_038052605.1 (C2)) were quantified in mid- and hindgut of BSFL. In the midgut of starved larvae, N4 abundance was 1.8 times higher than in fed larvae ($P=0.054$). In the hindgut, abundance of N2 was 4 times higher ($P=0.017$) and N4 ($P=0.093$) was 2 times higher in starved larvae, while C1 ($P=0.055$) was 3 times higher in fed larvae. When comparing midgut vs. hindgut of starved larvae, N1 was 6 times higher in the hindgut ($P=0.017$) and C2 was 13 times higher in the midgut ($P=0.017$). In fed BSFL, N1 was 12 times higher in the hindgut ($P=0.017$) and C2 was 3 times higher in the midgut ($P=0.04$). All AATs analysed were expressed in midgut and hindgut of BSFL. Based on their abundance, the neutral AAT N1 and N2 and the cationic AAT C2 seem to be the most relevant in the current experimental setting. Judging by their abundance, C2 appears to respond primarily to starvation in midgut, while N1 and N2 in hindgut. The AAT N1 seems to be relevant for absorption of AA from the gut regardless of feeding status. The expression of N1 and N2 in the hindgut could be important for the recovery of AA from primary urine. In conclusion, under starvation, most of the AAT mRNAs analysed in the mid- and hindgut responded differently to differences in AA availability.

Hybridisation among geographically distinct Tenebrio molitor strains: multigenerational effects on key biological traits

P. Soulioti^1*, E. Skreti¹, C.I. Rumbos², K. Mathiopoulos³ and C.G. Athanassiou¹

¹University of Thessaly, Department of Agriculture, Crop Production and Rural Environment, Phytokou Street, 38446 Volos, Greece; ²University of Patras, Department of Agriculture, 30200 Messolonghi, Greece; ³University of Thessaly, Department of Biochemistry and Biotechnology, Biopolis, 41500 Larissa, Greece; ^*pasoulioti@uth.gr

The yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), has been widely studied and mass-reared for decades, yet many aspects of its rearing optimisation remain insufficiently explored. Crossbreeding geographically distinct T. molitor strains offers a promising path for improving key biological traits, but this approach has not been thoroughly examined. This study investigated the effects of reciprocal hybridisation on reproductive performance, adult longevity, and larval growth across multiple generations, using four purebred T. molitor strains originating from Greece, Italy, Germany and the USA. Adult reproductive output and survival were monitored in both inbred and outbred lines by weekly recording of egg production, hatchability and adult survival in the F0 and F1 generations. Larval performance was assessed in the F3 generation by measuring larval weight and survival every 14 days until the appearance of the first pupae. Performance differed significantly among crosses for all traits across all generations. Several outbred lines showed clear reciprocal effects, while two hybrids, i.e., German (߻) × USA (߼) and USA (߻) × German (߼), exhibited heterotic effects in both fecundity and fertility. Adult survival also varied significantly among strains in both generations. Although the inbred Italian strain outperformed all other inbred and outbred crosses, the German (߻) × USA (߼) hybrid consistently showed the strongest performance across all life-history traits and generations among the outbred crosses. In the F3 generation, the Greek (߻) × German (߼) hybrid displayed the shortest development time (61 days). Overall, these results underscore the potential of targeted crossbreeding strategies to enhance T. molitor mass-rearing efficiency and support the development of strains better suited for industrial-scale production. Acknowledgement: This study was supported by project “Mealworm Innovations” (MIS: 7591), carried out within the framework of the National Recovery and Resilience Plan Greece 2.0, funded by the European Union – NextGenerationEU (Implementation body: HFRI).

High-throughput computer vision pipeline to study mating behavior across 10 strains of black soldier fly

N.B. Lemke^1,2*, M. Hoffman^1,2, B. Aernouts² and J. De Smet¹

¹KU Leuven Campus Geel, Research Group for Insect Production and Processing, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium; ²KU Leuven Campus Geel, Division of Animal and Human Health Engineering, Department of Biosystems, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*noahlemke22@gmail.com

The black soldier fly, Hermetia illucens, is mass-reared throughout the globe to valorise organic waste into many products including animal feed. For the success of industrial mass-rearing programs, efficient breeding is needed. While studies have begun to reveal a complex genomic landscape in wild and industrial BSF strains, practically nothing is known about how BSF strains of different genetic provenances may exhibit different reproductive phenotypes, nor potentially contribute to reproductive incompatibility or hybrid vigor. Of course, part of the reason is that behavioural assays of adult BSF are time consuming, labor intensive, and low throughput. Here, we present on a computer-vision apparatus and machine-learning pipeline that directly address this need; a retrofitted 30 x 30 x 30 cm cage automatically records images of live BSF breeding populations, after which a pipeline extracts meaningful features from these images (e.g., number of mating pairs, motion of males and females, etc.). Preliminary experimentation using the verified system will focus on detecting observable differences in mating behaviour and fitness during 6-day experiments (replicated n = 3 times) in response to shifts in cohort demography (i.e., ages), sex ratio, population density, and abiotic conditions. Thus, this system is well-poised to quickly document the BSF reproductive behavioural phenome, which will be informative for ongoing and future industrial breeding programs.

Developing a mutagenesis breeding protocol for black soldier fly: evaluating UV, EMS, and colchicine on eggs

Y. Xing^*, Y. Ma and Z.X. Qi

Guangdong Hairui Biotech Co., Ltd., No.5, 8th Street, Fuping Road, Xiaoping Industrial Zone, Shatou Street, Panyu District, 511430 Guangdong, P.R. China; ^*xingy1@haid.com.cn

The genetic improvement of the black soldier fly (BSF), a key resource insect for waste bioconversion, is constrained by limited germplasm diversity domestically. Introducing foreign strains is challenging. Mutagenesis breeding, proven in plants and Drosophila, offers a solution to create novel genetic variation. However, its efficacy in BSF, particularly at the egg stage, remains unexplored. This study systematically compared common mutagens to establish a foundational breeding protocol for BSF. Eggs from a laboratory population were treated with ultraviolet radiation (UVA/UVC at various durations), the alkylating agent EMS (0.5–2%), or colchicine (0.5–5%). A negative control was included. Treatments were applied consecutively over five generations (P1-P5). Hatching rates were recorded. Genetic divergence between treated and control groups was assessed by genotyping adults from the P5 generation using a custom-designed 40K SNP liquid chip and analysing genetic distances. Hatching rate analysis showed UVC caused the highest lethality, while colchicine had minimal effect. Genetic analysis revealed a critical divergence: UV treatments induced significant genetic differentiation from the control group after five generations. By contrast, EMS treatments, despite causing dose-dependent lethality, showed no significant genetic divergence from the control. Colchicine was ineffective and not pursued beyond initial tests. The results demonstrate that UV irradiation, not EMS, is effective for inducing heritable genetic variation in BSF eggs under our protocol. This suggests different mutagens have distinct effect modes in BSF–UV causes genomic changes viable for selection, while EMS’s primary effect is physiological toxicity. We have successfully developed a practical mutagenesis workflow using UV treatment on eggs and high-density SNP chip verification. This protocol provides a powerful new tool to expand genetic diversity for BSF breeding programs, mitigating reliance on limited native germplasm.

Does size matter? Allometry in adult black soldier flies under body-weight selection

K. Shrestha^1,2*, S. Karanjit¹, R. Jacobse¹, A. Shoureh¹ and L. Francuski¹

¹Protix. B.V., Research and Development, Van Konijnenburgweg 86, 4612 PL Bergen op Zoom, The Netherlands; ²University of Groningen, Evolutionary Genetics, Nijenborgh 7, 9747 AG Groningen, The Netherlands; ^*kriti.shrestha@protix.com

Directional selection for increased body size is expected to generate coordinated morphological responses through changes in cell size and number, potentially altering trait proportionality, sexual dimorphism and functional performance. Quantifying these allometric responses is therefore essential for predicting outcomes and trade-offs in long-term breeding programs. We investigated allometric patterns across 39 generations of artificial selection for increased body size in the black soldier fly (Hermetia illucens). Specifically, we tested whether trait enlargement is proportional, allometric relationships change over generations and differential responses between sexes. We analysed 331 adult flies from the parental populations of body-weight line under selection for generations 2, 20 and 39 using a combination of traditional linear morphometrics (on head width and tibia length) and landmark-based geometric morphometrics (on wings). Linear traits were evaluated relative to wing centroid size (CS), whereas wing shape variation was assessed following Generalized Procrustes Analysis and multivariate regression of shape on size. All three traits showed that sexual size dimorphism (SSD) increased with selected generations, with a proportionally greater size increase in females than in males. Strong proportional scaling was detected between log values of head width, tibia length and wing CS (r = 0.77–0.92), with comparable patterns in males and females, indicating largely isometric growth of major body components under selection. Larger adults exhibited proportionately larger wings, heads, and legs across all generations. Although wing shape varied significantly between sexes, the contribution of allometry to shape variation remained low throughout the selection program, ranging between approx. 1% and 3.3%. Overall, our results demonstrate that long-term selection for increased body size in H. illucens largely preserves proportional morphology while maintaining subtle allometric effects on wing shape. We discuss these findings as a quantitative basis for incorporating allometric constraints into industrial breeding strategies aimed at optimising black soldier fly performance for sustainable mass-rearing systems.

Evidence of frequency-dependent stress responses in Tenebrio molitor larvae: implications for welfare in rearing systems

M. Marson^1*, C. Uboni², F. Tulli² and C. Chiandetti¹

¹University of Trieste, Department of Life Sciences, Via A. Valerio 28/1, I-34127 Trieste, Italy; ²University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, viale delle Scienze 206, 33100 Udine, Italy; ^*matteo.marson@phd.units.it

Recent studies indicate that Tenebrio molitor larvae exhibit behavioural flexibility and context-dependent responses that go beyond simple reflexive patterns, supporting the hypothesis that this species may possess basic intentional-like capacities even at the larval stage. These findings raise important questions regarding how standard husbandry practices may affect larval welfare, particularly in large-scale and semi-intensive rearing systems. Mechanical vibrations are routinely generated during common management procedures, including substrate replacement, container handling, mechanical feeding systems, and environmental maintenance. Despite their widespread use, the potential role of vibrational stimuli as stressors for insect larvae has received little empirical attention. In this study, we investigated the behavioural responses of T. molitor larvae to controlled vibrational stimuli, with a focus on frequencies relevant to those produced by typical rearing equipment. Larvae were exposed to mechanical vibrations across a range of frequencies, and freezing behaviour, a stress-related behavioural indicator, was quantified. Results suggest that vibrations already at around 25 Hz induce a significant increase in freezing responses, consistent with a heightened state of alertness or stress sensitization. This pattern indicates that low-frequency vibrations may carry a negative valence for larvae and are not behaviourally neutral. Importantly, these frequencies overlap with those generated by everyday rearing activities. Although the present data are exploratory, they highlight the need to consider vibrational exposure as a potentially relevant factor in insect welfare. Further investigation is required to clarify frequency-specific thresholds, long-term effects, and possible mitigation strategies. Incorporating sensory ecology and behavioural responsiveness into rearing protocols may contribute to more welfare-oriented and sustainable insect production systems.

In vitro detection of antiprotease activity in the digestive tract of black soldier fly larvae (Hermetia illucens)

L. Lapierre¹, E. Soler-Warin², M.P. Rivera Rodrı́guez^1*, M. Lapierre³, G.W. Vandenberg¹ and M.H. Deschamps¹

¹Université Laval, Département des sciences animales, 2425 Rue de l’Agriculture, Laval, QC, Canada G1V 0A6; ²Saliège Campus, 3 Rue Georges Bernanos, 31130 Balma, France; ³La Crevette du Nord Atlantique, 553 Boulevard du Griffon, Gaspé, QC, Canada G4X 6B6; ^*martha.rivera-rodriguez.1@ulaval.ca

Amid global population growth and environmental concerns, the valorization of agricultural waste has become increasingly important. Black soldier fly larvae (BSFL) represent a promising solution, converting organic residues into protein-rich biomass and fertilizer. However, substrate variability might influence larval growth and bioconversion efficiency, highlighting the need for prior substrate screening, such as in vitro assays. While proteolytic activity can be measured by in vitro protocols to assess protein digestibility in other animal species, no standardised method currently exists for BSFL. Therefore, this study aims to develop a protocol to determine the in vitro antiprotease activity in BSFL, using enzyme extracts derived from the digestive tract (DT) of the larvae and exposing them to inhibitor extracts from different substrates. Different larval fasting conditions were tested (3, 6, 9, 12 and 24 h at 25, 27 °C). Potential inhibitory extracts (IE; Gainesville diet and ovalbumin) were obtained by mixing with a 50 mM Tris-HCl (pH 7.4) buffer. Enzyme extract (EE) was prepared using empty and full digestive tracts to assess the potential impact of the food bolus on protein concentration. Subsequently, the DT were extracted and stored in two different buffers: Tris-HCl, frequently used in proteolytic assays in fish, and PBS, which is widely used in insect samples. The protein content of the EE was determined by Bradford method at different EE: BSA ratios (1:1, 1:2, 1:5, 1:10). Proteolytic activity in alkaline medium of the EE was measured by spectrometry in both tested buffers with 0.5% casein. The addition of salt (CaCl₂ and CaCO₃) was also evaluated, as Ca is an important enzyme cofactor. Finally, the effect of IE on proteolytic activity was measured at 8 concentrations of IE. Results showed that 12 h was the optimal fasting period at 27 °C, though fasting had no significant effect on protein concentration nor alkaline proteolytic activity. The highest proteolytic activity in alkaline medium was achieved at a dilution of 1:5 of EE in 50 mM Tris-HCl (pH 7.5) buffer. During inhibition assays, optimal conditions were obtained using 50 mM Tris-HCl supplemented with 0.5% casein without added salts. At these conditions, ovalbumin inhibition reached 99.9% in full DT at 75 μg/U act. Further trials with additional substrates are still required. The proposed method provides a novel tool for identifying limitations in the development of new BSFL diets.

Nanoparticle-mediated CRISPR/Cas9 delivery in Hermetia illucens for enzymatic PET degradation

I. Armenia^1*, N. Esteban-Pérez^2,3, E. Catelan-Carphio⁴, A. Grapputo⁴, F. Sandrelli⁴, V. Grazù^2,3, J.M. De La Fuente^2,3 and G. Tettamanti¹

¹University of Insubria, Biotechnology and Life Science, Via J.H. Dunant 3, 21100 Varese, Italy; ²Centro de Investigación Biomédica en Red de Bioingenierı́a, Biomateriales y Nanomedicina, Av. Monforte de Lemos 3–5, 28029 Madrid, Spain; ³Aragon Nanoscience and Materials Institute (INMA-CSIC-UNIZAR), Department of Bionanomedicine, calle Mariano Esquillor s/n, 50018 Zaragoza, Spain; ⁴University of Padua, Department of Biosciences, Via U. Bassi 58b, 35131 Padua, Italy; ^*ilaria.armenia@uninsubria.it

Polyethylene terephthalate (PET) is among the most extensively used synthetic polymers and represents a major source of persistent plastic pollution due to its high chemical stability and resistance to natural degradation. The accumulation of PET-derived micro- and nanoplastics in organic waste streams highlights the need for innovative and sustainable degradation strategies. PET-hydrolysing enzymes, such as PETases, provide a promising biological approach for polymer depolymerization under mild conditions. In this study, we explore a nanotechnology-based gene delivery strategy to enable PETase expression in the larvae of Hermetia illucens, an insect species widely used for organic waste bioconversion. Biodegradable chitosan-based nanocarriers were developed for the encapsulation and delivery of CRISPR/Cas9 components. Nanocapsules were produced using ionic gelation or water-in-oil emulsification methods, and formulation parameters were optimised to enhance nucleic acid encapsulation efficiency. The resulting nanocarriers were characterised in terms of hydrodynamic size, morphology, colloidal stability, and controlled release behaviour. Ongoing and future investigations will assess transfection efficiency in insect cell cultures through fluorescence-based uptake studies, molecular analyses to detect CRISPR-induced genome modifications, and enzymatic assays to evaluate PETase activity. This work aims to establish a versatile and biocompatible platform for gene delivery in insects and to assess the feasibility of engineering H. illucens as a biological system capable of contributing to PET degradation. By integrating nanomaterials science, molecular biotechnology, and insect physiology, this approach lays the groundwork for innovative solutions in sustainable plastic waste bioprocessing. This project has received funding from the European Union’s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101204748

Biochar for animal feed – can we use it for insects?

A. Nycz^1*, M. Kaczor¹, M. Krzyżaniak², S. Przemieniecki³, K. Szewczuk-Karpisz¹ and P. Bulak¹

¹Institute of Agrophysics Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland; ²University of Warmia and Mazury in Olsztyn, Department of Genetics, Plant Breeding and Bioresource Engineering, Plac Łódzki 3, 10-724 Olsztyn, Poland; ³University of Warmia and Mazury in Olsztyn, Department of Entomology, Phytopathology and Molecular Diagnostics, Prawocheńskiego 17, 10-720 Olsztyn, Poland; ^*ola141b@gmail.com

Biochar, a carbon-rich material produced by pyrolysing biomass, is increasingly explored as a feed additive in animal husbandry. Across ruminants, pigs, poultry and fish, inclusion rates of roughly 0.1–4% of daily feed intake have been associated with improved growth performance, feed conversion, product quality (meat, milk, eggs), and enhanced resistance to gut pathogens (Nair et al., 2023). Adding biochar to the feed of conventional farm animals has been practiced for a very long time. Cato the Elder mentioned this use in the context of treating oxen, a 1906 Animal Husbandry Manual also noted that pigs naturally tend to consume small pieces of coal. Similar behavior has been observed in some wild animals, which voluntarily ingest charcoal fragments (Schmidt et al., 2019). Studies on animals raised with biochar supplementation (including cattle, goats, pigs, chickens, ducks, and fish) report increased body weight and reduced feed conversion ratio (FCR), indicating greater biomass gains and more efficient feed utilisation (Man et al., 2020). In relation to insects, research focuses less on biochar as a nutrient and more on its biological effects. When mixed directly into artificial diets or pupation media, biochar generally suppresses growth, survival, and successful metamorphosis in lepidopteran pests such as Manduca sexta and Spodoptera frugiperda (Wagle et al., 2025). However, is there a possibility to use it in insect reared for feed and food, to improve its development? This work will try to summarize what we know so far about the use of biochar for insect feeding. This work was funded by the National Science Centre, Poland, Opus 28 number 2024/55/B/NZ9/02474. References: Man, K.Y., Chow, K.L., Man, Y.B., Mo, W.Y. and Wong, M.H., 2021. Use of biochar as feed supplements for animal farming. Critical Reviews in Environmental Science and Technology 51: 187–217. https://doi.org/10.1080/10643389.2020.1721980. Nair, P.S., P S, S.M., Suresh, S., A J, S., K, S., S, A.K., P R, A., Krishnan, N., S, P., Chakraborty, S., Chopra, H., Akash, S., Amin, R., Dey, A., Alagawany, M., Chandran, D. and Dhama, K. (2023). Beneficial impacts of biochar as a potential feed additive in animal husbandry. Journal of Experimental Biology and Agricultural Sciences 11: 479–499. https://doi.org/10.18006/2023.11(3).479.499. Schmidt H.-P., Hagemann, N., Draper, K. and Kammann, C., 2019. The use of biochar in animal feeding. PeerJ 7: e7373. Wagle N., Unnikrishnan, S., Kaur, S., Pereira, E. and Kariyat, R., 2025. Biochar suppresses growth, pupation and eclosion success of a specialist (Manduca sexta) and a generalist (Spodoptera frugiperda) insect herbivore. Journal of Sustainable Agriculture and Environment 4: e70069.

The effect of black soldier fly larvae inclusion on hepatic gene expression in Bianca di Saluzzo chicken breed

F. Shirjandi^1*, N. Stoppani¹, F. Hossein Zadeh Behrouz², E. Fiorilla¹, C. Forte¹, A. Schiavone¹ and D. Soglia¹

¹University of Turin, Department of Veterinary Sciences, Largo Paolo Braccini n. 2, 10095 Grugliasco (TO), Italy; ²University of Padua, Department of Agricultural Sciences and Veterinary Medicine, Agripolis — Viale dell’Università, 16, 35020 Legnaro (PD), Italy; ^*faraz.shirjandi@edu.unito.it

The chicken industry is significantly dependent on soybean meal as it is a crucial protein source. Alternative feeding ingredients, like soy-free vegetal protein sources and black soldier fly larvae (BSFL), are in high demand due to their favourable nutritional profile (1). This study evaluated the effects of BSFL and soy-free vegetal meal replacements on hepatic gene expression in the slow growing Bianca di Saluzzo chicken breed. A total of 192 male chickens were randomly allocated to 24 pens (8 birds each) and four dietary groups (6 replicates/diet): Control (C), Soy-Free (SF): replace soybean with vegetal protein, dried larvae (DL): SF with 5% dried BSFL supplemented, and live larvae (LL): SF with 5% live BSFL supplemented (2). 96 liver samples were collected at 147 and 174 days (each time 48 birds, 2 birds per each replicate) for RNA extraction. Targeted RNA sequencing focused on genes related to stress response (CASP6, CAT, HSPA2, SOD1) and lipid metabolism (ACOX1, FABP1, LPL, SREBF2, FADS2). Substituting soybean meal did not negatively affect the lipid metabolism gene expression. DL and LL increased HSPA2 and CASP6 with no differences between them. Expression of these two genes was upregulated at older age in all dietary groups, indicating physiological changes related to sexual maturity and meat composition rather than stress caused by feed (3). In older insect-fed birds, CAT, SOD1 and SREBF2 were upregulated, while SOD1 was uniquely upregulated in LL, suggesting increased antioxidant resilience and lipid metabolism associated with weight gain in elderly birds (4). In conclusion, replacing soybean meal with alternative protein sources yielded comparable molecular outcomes to conventional diets. Insect-larvae supplemented diets mildly activated cellular stress and apoptotic pathways in young chickens, likely reflecting early physiological development. Both DL and LL demonstrated equivalent molecular effects, confirming their similar functional potential as sustainable protein ingredients in poultry nutrition.

Selection of bacterial probiotics to enhance bioconversion ability and growth performance of mass-reared insects

E. Crotti^1*, E. Bigarella¹, D. Hentati¹, V. Candian², C. Savio², G. Brenna¹, C. Jucker¹ and R. Tedeschi²

¹University of Milan, Department of Food, Environmental and Nutritional Sciences, via Celoria 2, 20133 Milan, Italy; ²University of Turin, Department of Agricultural, Forest and Food Sciences, Largo Paolo Braccini 2, 10095 Grugliasco (TO), Italy; ^*elena.crotti@unimi.it

In recent years insect farming has gained great attention to bioconvert agro-industrial by-products into valuable biomass in the context of circular economy and to partially or fully substitute conventional protein sources used in feed and food systems. Insects have established intricate associations with diverse microbial partners which play crucial roles in host physiological processes, defence, and overall fitness. Gut eubiosis is critical for host health and physiological homeostasis, as alterations of the gut microbial structure may negatively affect host health and physiological functions. Thus, supplementation of beneficial bacteria may help insects improve their growth, bioconversion ability and health in general. Aim of this work was to characterise the cultivable microbiota associated with the yellow mealworm (YM) Tenebrio molitor L. (Coleoptera: Tenebrionidae) to select candidate probiotics that can promote host growth and health, particularly when insects are reared on different agro-industrial by-products. To obtain a detailed description of the gut bacterial community associated to the insects, 16S rRNA gene amplicon sequencing was performed. Guts were dissected from surface-sterilized insects and used for bacterial isolation on selective culture media. The resulting bacterial collections were dereplicated by ITS-PCR fingerprinting and taxonomically identified through 16S rRNA gene sequencing. The isolates were further evaluated for their ability to hydrolyse key dietary polysaccharides, including cellulose, starch, and pectin, which can be found in agro-industrial by-products, as well as for their antimicrobial activity against entomopathogenic microorganisms. Overall, this study provides a comprehensive characterisation of the gut microbiota of an edible insect species, with particular emphasis on its cultivable fraction. This approach enables the identification of potentially beneficial bacterial strains that may be exploited in microbially assisted rearing strategies to enhance insect production and promote the bioconversion of organic wastes into high-quality products. Work funded by PRIN 2022 Project InProFarm (Insect Probiotic-assisted Farming: a promising tool to enhance edible insect health and performances), grant number 2022L4NJMK, European Union — Next Generation EU, Mission 4, Component 1.

Macronutrient profile shapes mealworm life history, but not its microbiome

R.A. Stoner¹, H.K. Walt¹, H.R. Jordan², C.J. Picard³, J.K. Tomberlin⁴, S.T. Behmer⁴, E. Harris⁴, S. McPeek⁴, E. Oedii³, F.G. Hoffmann^1,5* and F. Meyer¹

¹Mississippi State University, Department of Biochemistry, Nutrition, and Health Promotion, Mississippi State, MS 39762, USA; ²Mississippi State University, Department of Biological Sciences, Mississippi State, MS 39762, USA; ³Indiana University Indianapolis, Department of Biology, Indianapolis, IN 46202, USA; ⁴Texas A&M University, Department of Entomology, College Station, TX 77845, USA; ⁵Mississippi State University, Institute for Genomics, Biotechnology and Biocomputing, Mississippi State, MS 39762, USA; ^*fgh19@msstate.edu

The mealworm (Tenebrio molitor) is an important industrial insect that is commonly reared as a high-quality protein source for pet food and livestock. Additionally, there is a growing interest in their capability to digest plastics like polystyrene. Therefore, efforts to understand the effect that diet has on the mealworm and its gut microbiome are important to determine its performance under varying nutritional contexts and bolster production systems. In this study, we measured survival and weight of mealworms that were reared on three different synthetic diets: a carbohydrate-rich diet (1P:3C), a balanced diet (1P:1C), and a protein-rich diet (3P:1C). We found that mealworm survival and weight were highest when reared on the protein-rich diet. To understand the impact of these diets on the mealworm microbiome, we conducted metatranscriptomic sequencing on mealworm gut tissues. We found that these diets did not have large impacts on the microbial community, since differences in alpha and beta diversity were not significant. However, we found 40 differentially expressed microbial transcripts that were upregulated in the balanced diet but downregulated in the carbohydrate and protein-rich diets. Interestingly, we detected Zophobas morio black wasting virus in five of our 12 samples, which was responsible for a recent epidemic in superworms (Zophobas morio) and could have implications for safe rearing practices in facilities with both mealworms and superworms. Overall, our results suggest that protein-rich diets can increase mealworm performance, but these improvements are not necessarily related to the gut microbiota.

Bacterial microbiota analysis of Hermetia illucens larvae feed with meat-based former foodstuffs

G. Spatola^1*, E. Copelotti^1,2, S. Mancini¹, A. Zanzot¹, A. Giusti¹, A. Trocino^3,4, R. Nuvoloni¹ and A. Armani¹

¹University of Pisa, Department of Veterinary Sciences, Viale delle Piagge n. 2, 56124 Pisa, Italy; ²IRTA, Animal Nutrition, Mas Bové, 43120 Costanti, Catalonia, Spain; ³University of Padua, Dipartimento di Biomedicina comparata e Alimentazione (BCA), Viale dell’Università, 16, Legnaro, 35122 Padua, Italy; ⁴University of Padua, Department of Agronomy, Animals, Food, Natural Resources and Environment, Viale dell’Università, 16, Legnaro, 35020 Padua, Italy; ^*gabriele.spatola@phd.unipi.it

Edible insects are recognised as a sustainable source of animal protein for food and feed, and several formulations have been authorised within the European Union. Among edible insects, black soldier fly (Hermetia illucens) larvae (BSFL) can efficiently convert organic waste and former foodstuffs (FFs) into protein-rich biomass, representing sustainable and economically viable feed and food. However, EU food and feed legislation currently prohibits the use of meat and fish as components of rearing substrates in insect farming. Indeed, safety concerns persist, and additional data are needed to evaluate the risks associated with the use of meat and fish based FFs as BSFL substrates. In this study, the bacterial microbiota of BSFL reared on substrates containing raw and processed meat based FFs was investigated, together with the effects of post-harvest treatments including fasting, rinsing with distilled water, and washing with 70% ethanol (EtOH) solution. Three substrates were tested: a 100% plant-based control (C), and two substrates containing 25% of FFs raw meat (R) or 25% FFs processed meat (P). BSFL were sampled at the end of the rearing period as untreated larvae, after 24 h fasting, rinsing, and EtOH washing. Bacterial DNA was extracted and analysed using 16S rRNA metabarcoding. The bacterial community was dominated by Pseudomonas spp., Providencia spp., Myroides and Sphingobacteriaceae. Major foodborne pathogens, including Listeria spp., Staphylococcus spp., Salmonella spp. and Campylobacter spp., were not detected. As expected, both alpha and beta diversity analysis revealed that substrate type significantly affected the BSFL bacterial microbiota. Specifically, Shannon and Simpson diversity indices were significantly higher in BSFL reared on C and R substrates, suggesting that the use of processed FFs as part of the rearing substrate may reduce the BSFL bacterial microbiota biodiversity and potentially the level of microbial contamination. On the contrary. fasting, rinsing, and EtOH washing had limited impact on the bacterial microbiota. These results support the potential use of BSFL in circular economy strategies. However, further investigations are needed before meat based FFs can be considered safe substrates for food or feed production.

Sublethal effects of cyromazine on life-history characters and reproductive performances of Hermetia illucens

A. Zanzot^1,2*, T.N. McNeil², S. Mancini¹ and J.K. Tomberlin²

¹University of Pisa, Department of Veterinary Sciences, Viale delle Piagge 2, 56124 Pisa, Italy; ²Texas A&M University, Department of Entomology, 160 Agronomy Road, College Staiton, TX 77843, USA; ^*asia.zanzot@phd.unipi.it

The circular economy is increasingly emphasized in food and feed systems due to the 1.3 billion tonnes of agricultural by-products generated annually. Black soldier fly (Hermetia illucens) farming represents a promising bioconversion strategy; however, widespread pesticide use and presence in residue for larval substrates, may compromise immature and adult performances, while transferring the active compounds through the food chain. Cyromazine, an insect growth regulator, was assessed on larval weights, growth rates, feed conversion ratio frass corrected (FCR frass corrected), efficiency on conversion of ingested (ECI) and digested (ECD) feed and reproductive performances of Hermetia illucens. Larvae were fed, in triplicates, 1 kg of Gainesville diet (i.e., standard feedstock) at 70% moisture (control) or four Gainesville diets contaminated with cyromazine (0.0625, 0.125, 0.25, 0.5 ppm). Ten largest larvae were weighed daily and sieved three days after reaching a maximal stable weight, then transferred to plastic boxes for emergence. Sixty adults (40 males, 20 females) were sexed and placed in a cage for each replicate, and mating and oviposition events were recorded daily for 12 h. Only the highest treatment (0.5 ppm) significantly affected larval weights, whereas no differences were observed between the other treatments and the control (0.170 g ± 0.004, 0.166 ± 0.006, 0.162 ± 0.000, 0.162 ± 0.003, and 0.136 ± 0.004, from control to 0.5 ppm, $P\mathrm{<}0.05$). Similarly, bioconversion indexes FCR frass corrected, ECI and ECD were not negatively impacted by the presence of the contaminant, except for 0.5 ppm (2.81 ± 0.17, 2.95 ± 0.08, 3.00 ± 0.17, 3.04 ± 0.15, 5.18 ± 0.71 for FCR frass corrected, 0.36 ± 0.02, 0.34 ± 0.01, 0.33 ± 0.02, 0.33 ± 0.02, 0.20 ± 0.03 for ECI and 0.39 ± 0.02, 0.37 ± 0.01, 0.37 ± 0.02, 0.36 ± 0.02, 0.23 ± 0.03 for ECD, from control to 0.5 ppm, $P\mathrm{<}0.05$). Adult reproductive performance differed among treatments. Cyromazine affected negatively the number of mating events. From 0.25 ppm, cyromazine presence increased oviposition events; however, the amounts of eggs laid per oviposition event were significantly lower – by up to 40% – than in 0.0625 ppm and the control. Total egg production did not differ significantly among treatments. These results show that sublethal cyromazine exposure may negatively affect growth performances, conversion and efficiency indexes, as well as the reproduction performances of the adults. Results highlight the need of close monitoring for the presence of chemical and pesticides residues in agricultural by-products when used as substrates in the insects farming sector.

Effects of zinc on life-history traits including heat resistance of Hermetia illucens larvae

A. Zanzot^1,2*, T.N. McNeil², S. Mancini¹ and J.K. Tomberlin²

¹University of Pisa, Department of Veterinary Sciences, Viale delle Piagge 2, 56124, Italy; ²Texas A&M University, Department of Entomology, 160 Agronomy Road, 77843, USA; ^*asia.zanzot@phd.unipi.it

Zinc is an essential trace element involved in physiological processes in all living organisms, including heat resistance; however, excessive levels can be toxic. Heavy metal contamination in byproducts-derived substrates is a major concern in insect farming. Larvae of the black soldier fly (Hermetia illucens) are widely used for bioconversion of low-quality substrates, yet heavy metals may impair larval performance, highlighting the need for careful monitoring of substrate contamination. In this study, approx. 1000 7-day-old Hermetia illucens larvae were fed diets (1 kg) containing double (D, 6 mg) and triple (T, 9 mg) the zinc concentration normally present in their standard basal diet Gainesville (G, 3 mg), which served as the control. Treatments were performed in triplicate. Larvae were reared until reaching 19 days of age. To evaluate whether increased zinc intake affected larval resistance to elevated temperatures, heat resistance was assessed at completion of 75% of development of both larval stage and prepupal stage, calculated based on accumulated degree days (ADD). Accordingly, ten larvae per treatment were weighted and subjected to the experimental procedure at 11 and 19 days of age. Larvae were placed in glass vials and immerged in a water bath (reverse osmosis water). Water temperature (27 °C) was gradually increased, and heat knockdown was recorded when each larva ceased movement. Conversion and efficiency indexes (Feed Conversion Ratio frass corrected, Efficiency of Conversion of Ingested Food ECI, and Efficiency of Conversion of Digested Food ECD) were assessed. Results show that growth performances were not impacted by the zinc concentration of the diets, as average final weights did not significantly differ between treatments (G: 56.47 g; D: 61.73 g; T: 74.20 g;). Similarly, performance indexes did not show statistical differences, therefore zinc at the levels examined did not exert toxic effects (G: 5.818, D: 5.555, T: 4.663 for FCR frass corrected; G: 0.174, D: 0.182, T: 0.214 for ECI; G: 0.195, D: 0.202, T: 0.233 for ECD). Regarding the heat treatments, average knockdown temperatures were similar between treatments both at 11 (G: 50.5 °C; D: 50.3 °C; T: 50.1 °C) and at 19 days of age (G: 50.9 °C; D: 50.7 °C; T: 50.3 °C). These data indicate that zinc concentrations up to three times the baseline level present in a standard diet do not exert toxic effects on Hermetia illucens larvae and, at the same time, did not improve larval resistance to heat stress.

Transfer and accumulation of arsenic, cadmium and lead in farmed insects from endogenous substrate contaminants

N.A. Sibinga^1*, L. Vogels², L. Frooninckx³, J. Claes¹, E. Smolders⁴ and M. Van Der Borght¹

¹KU Leuven, Department of Microbial and Molecular Systems (M2S), Research Group Insect Production and Processing, Kleinhoefstraat 4, 2440 Geel, Belgium; ²Thomas More University of Applied Sciences, Department of Agro- and Biotechnology, Kleinhoefstraat 4, 2440 Geel, Belgium; ³Thomas More University of Applied Sciences, Centre of Expertise Sustainable Biomass and Chemistry, Kleinhoefstraat 4, 2440 Geel, Belgium; ⁴KU Leuven, Soil and Water Management, Kasteelpark Arenberg 20, 3001 Leuven, Belgium; ^*nate.sibinga@kuleuven.be

Uptake and accumulation of undesirable trace elements is a complex process governed by both physico-chemical and biological variables. Many studies on trace element contamination in farmed insects rely on spiked substrates, producing unrealistically high contaminant levels and potentially overestimating bioavailability. This study examined transfer of endogenous arsenic (As), cadmium (Cd) and lead (Pb) under lab and production-scale rearing conditions using 27 practical feed substrates and five insect species: Hermetia illucens, Tenebrio molitor, Alphitobius diaperinus, Acheta domesticus and Locusta migratoria. Element concentrations were measured via ICP-MS, and bioaccumulation factors (BAF) determined on a dry-matter (DM) basis. Substrate concentrations varied widely, with maxima of 1.15 mg As/kg DM, 1.43 mg Cd/kg DM and 13.60 mg Pb/kg DM. In contrast, insects showed narrower ranges, with highest observed levels of 0.386 mg As/kg DM, 1.066 mg Cd/kg DM and 2.085 mg Pb/kg DM. Bioaccumulation of As and Pb was not observed in any species (all BAF<1). Cd bioaccumulation occurred in H. illucens (mean BAF 3.74) and T. molitor (mean BAF 3.07), whereas L. migratoria showed low mean BAF values across all elements (As 0.17, Cd 0.36, Pb 0.18). All insects were below EU feed ingredient limits for As, Cd, and Pb. In the context of novel food legislation, A. diaperinus and A. domesticus met all regulatory thresholds, while L. migratoria exceeded Cd and Pb limits when reared on highly contaminated grass. One T. molitor sample slightly exceeded the Cd limit for frozen larvae (0.051 mg/kg WM vs. 0.050 mg/kg WM). Importantly, Pb levels in all T. molitor samples were below the analytical LOD of this study (0.023 mg/kg WM), preventing confirmation of compliance with the extremely low permitted Pb levels (0.01–0.02 mg/kg). This study provides the most comprehensive evaluation to date of endogenous trace element transfer in farmed insects under realistic production conditions. Results confirm Cd bioaccumulation in H. illucens and T. molitor, reveal strong element exclusion in L. migratoria, demonstrate broad compliance with feed safety standards, and highlight regulatory challenges when legal limits fall below achievable analytical sensitivity.

Influence of agro-industrial by-product diets on the nutritional values of Tenebrio molitor

A. Angioni and F. Corrias^*

University of Cagliari, Life and Environmental Science, Cittadella Universitaria di Monserrato SS 554, Bivio Monserrato, SP 8 Monserrato-Sestu Km 0,700, 09042 Monserrato, Italy; ^*francesco.corrias@unica.it

Intensive livestock and aquaculture systems require high-quality feed that is nutritionally balanced. The decrease in wild fish protein has led to demands within the feed supply chain for new protein sources to fulfil the growing demand. In this context, edible insects such as the yellow mealworm (Tenebrio molitor) are the most promising alternative protein source. This study evaluated the nutritional profile of yellow mealworm larvae reared in a laboratory on eight different agro-industrial by-products: wheat middlings, durum wheat bran, rice bran, hemp cake, thistle cake, dried brewer’s spent grains, dried tomato pomace, and dried distillery grape marc. The quantitative and qualitative impacts of the rearing substrates on the larvae were compared. The results showed that the larvae adapted well to different substrates with different nutritional compositions, including the fibrous fraction. However, substrates affect larval macronutrient composition. Hemp cake stood out for its superior nutritional value, as reflected by its high protein content and moderate NDF levels. Conversely, imbalanced lipid or carbohydrate content in the substrate (rice bran) and the presence of potential antinutritional compounds (thistle cake) appeared to negatively affect Tenebrio molitor. The larvae of T. molitor are a stable protein source but variable for lipids and moisture. The substrates recommended are hemp cake, wheat bran, and wheat middling. Substrates that should be avoided include RB, TC, and DGM. Analysis performed on residual feed and insect frass supported the selective feeding behaviour and high plasticity of mealworm larvae and their potential to adapt to different substrates. Indeed, larvae try to maximise the intake of sugars, fats and amino acids while minimising the ingestion of fibre.

The efficient degradation mechanisms of aflatoxin B1 by Hermetia illucens combined with gut microbe S. geniculata ZJA10

A.R. Henawy^* and Z. Jibin

Huazhong Agriculture University, National Key Laboratory of Agricultural Microbiology, National Engineering Research Center of Microb, Hongshan, Wuhan, Hubei 430070, P.R. China; ^*ahmed_henawy@mail.hzau.edu.cn

Aflatoxin contamination is a widespread and persistent problem in agriculture sector, posing serious risks to food safety, public health, economy and international trade. Consequently, the development of environmentally friendly and efficient approaches for aflatoxin removal has become a global priority highlighting microbial biodegradation. Recently, Black soldier fly larvae (BSFL; Hermetia illucens) has gained attention for its remarkable ability to bio-convert organic waste and bioremediation in general. This ability has largely attributed to its diverse and active gut microbiota. In this study, natural consortium was derived from BSFL gut and evaluated their ability to degrade AFB1 over 16 days. It was revealed that the AFB1 concentration decreased to 50% then 99% in the day 4 and 8 respectively and reached complete degradation on the sixteen^th day. Metagenomic analysis was used to understand microbial community composition during these days. It was revealed that Pseudomonadata taxa was the predominant taxa participating in AFB1 biodegradation in these enriched natural consortia. Additionally, there are significant shifts in microbial diversity composition which reveal the most adapted genera and species. Subsequently, isolation of efficient AFB1 degrading bacterial strains was performed and inoculated sterile BSFL with them. The inoculated sterile BSFL showed a remarkable decline in AFB1 concentration particularly the BSFL inoculated by Stenotrophomonas geneiculata ZJA10. So ZJA 10 strain was selected as the most efficient strain for whole genome sequencing to investigate the genetic determinants behind AFB1 degradation. This study integrates multi-omics and culturomics approaches to elucidate the role of BSFL gut microbiota in AFB1 biodegradation, highlighting their potential as valuable bioresources for the remediation of aflatoxin contamination.

Nutritional quality and oxidative status of house crickets (A. domesticus) in iron-enriched rearing conditions

J.M. Malek¹, M. Maria², O.A. Sylvester³, O.O. Godwin³, R. Charlotte⁴, K. Claudia⁵ and G. Rossi^6*

¹Benue State University, Department of Chemistry, 102119 Makurdi, Nigeria; ²Universität Potsdam, Department of Food Chemistry, 14476 Potsdam, Germany; ³Benue State University, Department of Chemistry, 102119 Makurdi, Nigeria; ⁴Freie Universität Berlin, Institute for Biology, 14195, Berlin, Germany; ⁵Technische Universität Berlin, Institut für Lebensmitteltechnologie und Lebensmittelchemie, 13355 Berlin, Germany; ⁶Leibniz-Institut für Agrartechnik und Bioökonomie e.V. (ATB), Department of Bioenergy, 14469 Potsdam, Germany; ^*Grossi@atb-potsdam.de

Dietary iron deficiency remains a public health challenge in low-income countries. Insects are a source of highly bioavailable iron; iron enrichment of A. domesticus might mitigate the low iron bioavailability. Iron is an essential micronutrient for metabolic processes, but it has toxic effects when in excess. This study investigated the effects of dietary iron supplementation on rearing performance and oxidative status of house crickets (Acheta domesticus). Neonate crickets (20 days old) were reared under controlled conditions (31 ± 1 °C temperature, 50 ± 5% RH, 12:12 (L:D) photoperiod), and fed with commercial chicken feed supplemented with increasing FeSO₄ concentrations as the iron source (1, 5, 10, 25 and 100 mmol Fe/kg diet). Five replicates/treatment were prepared. The first adult emerged after 21 days in every treatment. Growth parameters (survival, weight gain, and rearing efficiency) and lipid peroxidation (TBARs) were evaluated. The survival was significantly higher in the 1 (91.2 ± 3.43%), 5 (96.42 ± 1.90%) and 10 (91.65 ± 4.31%) mmol Fe groups compared to the CTR (88.46 ± 1.22%), while it was decreased in the 25 (88 ± 2.71%) and 100 (86.88 ± 3.15%) mmol Fe groups. The total feed intake was higher in the 1-25 mmol Fe groups (20.09 ± 0.64, 17.24 ± 0.76, 17.07 ± 1.34, 15.82 ± 0.79 g) compared to the CTR (15.78 ± 0.73 g) and lower in the 100 mmol Fe treatment (12.59 ± 0.43 g). The Feed Conversion Ratio (FCR) was lower in the 100 mmol Fe group (0.98 ± 0.04) compared to all the other treatments, indicating altered feeding behavior and metabolic stress. Oxidative stress was higher in the 100 mmol Fe group (199 ± 29.02 μg MDA/kg) compared to the CTR (120 ± 6.23 μg MDA/kg). No significant differences were observed between CTR and the other groups (values ranged between 135.00 ± 29.64 and 136.00 ± 19.86 μg MDA/kg). The results highlight that optimised dietary iron enrichment improves A. domesticus growth while limiting lipid peroxidation. In conclusion, iron supplementation of 5-10 mmol Fe/kg diet is recommended for optimising cricket rearing, thereby guaranteeing low oxidative damage and ensuring the adequate nutritional quality of edible crickets.

Experimental model for evaluating rearing conditions and bio-conversion by BSF

A. Desaegher and M. Radek^*

Le Gouessant, rue des frères Pieto, 22400 Lamballe-Armor, France; ^*mathilde.radek@legouessant.fr

The MicroDEMi experimental model was designed to evaluate rearing conditions, feeding diets, and genetics of black soldier fly (BSF) using controlled experimental units. Experimental conditions, including ambient parameters and larval rearing duration, are predefined to align with industrial realities. The model allows for twenty-five replicates per experimental condition and up to 4 conditions + control per trial, enabling robust statistical evaluation. This framework provides a valuable tool for assessing raw materials, genetic performance and optimising growth time in BSF farming.

Water spinach as a green feed supplement: impacts on survival and feed utilization in two-spotted crickets

D.T. Pham^* and P.T.H. Bui

An Giang University, Vietnam National University Ho Chi Minh City (VNU-HCM), Department of Animal Sciences and Veterinary Medicine, 18 Ung Van Khiem Street, Long Xuyen 90000, An Giang, Viet Nam; ^*phamduytan03041997@gmail.com

Insect farming may support more sustainable protein systems by using locally available feed resources. Water spinach (Ipomoea aquatica) is widely available in Vietnam, but experimental evidence for its use in cricket rearing is limited. We tested whether adding water spinach leaves to commercial broiler feed affected growth performance and survival of the two-spotted cricket (Gryllus bimaculatus) under captive conditions. Two diets were compared: (T1) commercial broiler feed (control) and (T2) broiler feed plus fresh water spinach leaves. Each diet had 8 replicates; each replicate was a 15-litre plastic box stocked with 200 one-day-old crickets (n = 3200). After one week of adaptation, data were recorded for four weeks. Outcomes included average daily gain (ADG), feed conversion ratio (FCR), and survival at different stages. Data were analysed using a general linear model ($P\mathrm{<}0.05$). ADG was similar between diets (T2: 41.85 vs T1: 39.08 mg/individual/day; $P=0.71$). FCR tended to be higher in T2 than T1 (2.08 vs 1.42; $P=0.06$). Survival differed by stage: in the first two weeks, survival was lower in T2 than T1 (47.88% vs 60.00%; $P\mathrm{<}0.01$), while harvest survival was numerically higher in T2 (73.22% vs 58.36%; $P=0.20$). Water spinach can be used as a low-cost local green supplement for G. bimaculatus, but it involves trade-offs. Supplementation did not increase growth significantly and tended to increase FCR, and early survival was lower. Future work should optimise supplementation strategy and early-stage management to improve overall performance.

“Micro” nutrients, major impacts: effects on growth and nutritional value of Tenebrio molitor larvae

T.I. Gogou^1*, S. Zafeiriadis¹, G.V. Baliota¹, C.I. Rumbos², M. Psarianos³, O. Schlüter^3,4 and C.G. Athanassiou¹

¹University of Thessaly, Phytokou, 38446 Volos, Greece; ²University of Patras, 30200 Messolonghi, Greece; ³Leibniz institute for agricultural engineering and bioeconomy (ATB), Max Eyth Allee 100, 14469 Potsdam, Germany; ⁴University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy; ^*theo.gog9@gmail.com

Global food demand continues to rise, while micronutrient deficiencies remain widespread. Micronutrients occur naturally in soil and plants and are essential for human and animal health. Insects, such as Tenebrio molitor, can efficiently convert various substrates into high-quality feed and food products and their nutritional value can be influenced by the feeding substrate. This study evaluated the effects of dietary supplementation with four micronutrients (Mn, Zn, Ca, P) at three inclusion levels on the growth and nutritional composition of T. molitor larvae. Manganese was added at 10, 20 and 200 ppm, zinc at 15, 30 and 300 ppm, and calcium and phosphorus at 0.5, 1 and 2%. All micronutrients were incorporated into wheat bran, which served as both the experimental base and the control diet. Larval growth and survival were monitored weekly and the experiment concluded with the appearance of the first pupa. Feed utilisation parameters, including Feed Conversion Ratio (FCR) and Specific Growth Rate (SGR), were calculated and chemical analyses were performed to determine larval mineral composition. Significant dietary effects were observed for most parameters, while development time was less affected. Diets supplemented with manganese consistently produced the highest larval weights (107–113 mg), survival rates of approx. 97%, and improved feed efficiency. By contrast, zinc-supplemented diets, particularly at medium and high levels, resulted in lower larval growth (98–103 mg) and survival of 83–88%. Calcium- and phosphorus-enriched diets supported intermediate to high growth and survival, with larval weights of 103–106 mg and 106–109 mg, respectively, and survival up to approx. 90%. Zinc at 30 ppm yielded the largest FCR (approx. 2), while the maximum phosphorus inclusion led to the lowest SGR (7.72%). Mineral analyses revealed similar concentration ranges across treatments, with some variability. Overall, these results demonstrate that targeted micronutrient supplementation can enhance T. molitor growth, survival, and feed efficiency, providing valuable insights for the optimisation of insect-based feed and food production.

Linking biological performance to economic outcomes: a sensitivity analysis of Acheta domesticus rearing

R. Zorrilla Gonzalez*¹ and E. Neff²

¹Bravo Farm and Laboratories, Carretera los Saucos Valle de Bravo Km3, Los Saucos, Estado de Mexico 51200, Mexico; ²Fluker’s Cricket Farm Inc., 1333 Plantation Avenue, Port Allen, LA 70767, USA; ^*rozg@bravofl.com

Despite the industry’s promise, economic viability remains a persistent hurdle for ventures of all scales. This challenge is exacerbated by a lack of unified quantitative frameworks linking biological metrics (growth, survival) with financial outcomes. This disconnect limits researchers’ ability to determine if biological gains observed in empirical trials translate into actual economic returns, and prevents farmers from identifying which biological traits to prioritize for profitability. We developed an open-source bioeconomic model to bridge these gaps for Acheta domesticus. Calibrated using empirical datasets, the model integrates functions for survivorship, logistic growth, and age-dependent feed conversion. This framework simulates batch production dynamics to determine the harvest timing that maximises annualized profit per square area. Our sensitivity analyses reveal that profitability drivers are not universal. Instead, economic outcomes are sensitive to specific parameters contingent on a farmed population’s specific life history traits. Furthermore, we demonstrate that the optimal harvest window is dynamic, shifting significantly in response to biological performance and local cost structures. This work proposes a standardized baseline for bioeconomic benchmarking. By quantifying the relative economic weight of different biological traits, the framework allows researchers to prioritize high-leverage targets for selective breeding and management. Crucially, it provides a flexible tool for producers to generate personalized insights aligned with their unique biological performance and cost constraints. The model is designed to accommodate future extensions of relevant covariates such as density, temperature, and feed compositions, or stochastic risk factors such as pathogen-driven colony collapse to reflect the complexity and volatility of industry-scale production.

Techno-economic model of insect farming — viable scenarios and trade-offs

L. Wein^* and J. Lean

Protenga Pte Ltd, 3 Coleman Street, #03-24, 179804 Singapore, Singapore; ^*leo@protenga.com

As the global demand for sustainable protein alternatives intensifies, insect farming — specifically the production of black soldier fly larvae (BSFL) — has emerged as a frontrunner in the circular bioeconomy. However, the industry faces significant hurdles regarding high capital expenditure (CAPEX) and volatile operational costs. A comprehensive Framework for Techno-Economic Modelling (TEM) designed to evaluate the financial viability and industrial scalability of insect protein production systems is proposed. The framework integrates mass-balance equations, CAPEX and OPEX assumptions with discounted cash flow (DCF) analysis to quantify the relationship between biological performance (e.g., feed conversion ratios, larval growth rates) and economic indicators such as Net Present Value (NPV). Central to this model is a multi-objective optimisation layer that identifies critical trade-offs between automation levels and labour costs. While high-tech environments may optimise yields, the initial investment often extends the payback period beyond viable commercial thresholds. Furthermore, the study conducts a Viable Scenario Analysis across distinct market price assumptions and facility capacities. By utilising sensitivity analysis, we assess how fluctuations in cost factors such as energy prices and substrate (organic waste) consistency impact long-term profitability. Results indicate that the “decentralized-hybrid” model—combining low-tech waste processing with centralized refining—yields the most robust economic resilience. This framework provides stakeholders, from agri-investors, operators to policy makers, with a standardized tool to evaluate investments and optimise the resource-efficiency of insect-based value chains.

Effects of adult density on egg production and neonatal output in industrial-scale black soldier fly rearing

T.B. Barber^1*, M.B. Ala Eddine^1,2, L.I. MacAvei³, S. Dabbou², L. Maistrello⁴ and G. Benassi¹

¹Kinsect S.r.l. SB, Via Mazzacurati, 7/D, 42124 Reggio Emilia, Italy; ²University of Trento, C3A, Via E. Mach, 1, 38098 San Michele All’Adige (TN), Italy; ³USAMV Cluj, Calea Mănăştur 3–5, 400372 Cluj-Napoca, Romania; ⁴University of Modena and Reggio Emilia, Piazzale Europa, 1, 42124 Modena (RE), Italy; ^*trent@kinsect.eu

Optimising adult rearing conditions is critical for maximising egg production and neonatal output in industrial-scale black soldier fly (BSF, Hermetia illucens L.) systems. While larval rearing has been extensively studied, adult reproductive performance remains a key bottleneck strongly influenced by adult density. This study evaluated the effects of adult density on reproductive output in a proprietary industrial batch-rearing system. Six adult densities (15 000, 25 000, 30 000, 35 000, 45 000 and 48 000 adults per cage) were tested in 1.42 m³ cage under controlled conditions (27 °C, 70% RH, 12:12 h LED photoperiod). Automated hydration (three times per day) and a proprietary oviposition attractant were provided; Pupae were introduced 1–2 days prior to adult emergence, and each density was replicated three times over a six-month period. Eggs were collected daily over a 10–12-day production cycle corresponding to peak oviposition. Performance indicators included total egg production per cage and batch, eggs per female (assuming a 1:1 sex ratio and supported by preliminary observations), adult emergence rate, and cumulative neonatal yield per batch; total egg production and eggs per female were analysed using one-way ANOVA. Total egg production peaked at intermediate densities, with cages stocked at 25 000–30 000 adults yielding significantly higher egg output per cage (199 ± 4 g at 30 000 and 184.33 ± 2.30 g at 25 000) than both lower densities (100.67 ± 9.71 g at 15 000) and higher densities (170.67 ± 9.45 g at 35 000, 157.33 ± 5.50 g at 45 000 and 125.67 ± 3.79 g at 48 000). Eggs per female were highest at 25 000 adults (3.97 ± 0.04 mg) indicating reduced reproductive efficiency at both lower and higher densities. Adult emergence exceeded 90% across all treatments, and cumulative neonatal yield remained consistent at approx. 31% across all densities, indicating stable hatching performance regardless of adult density. Overall, intermediate adult densities (25 000–30 000) maximised both total egg output and per-female efficiency, while lower densities limited mating opportunities and higher densities reduced efficiency due to high-density constraints. Consistent neonatal yields suggest density-dependent effects primarily act on adult reproductive performance rather than egg viability. Defining optimal adult density thresholds is thus essential for improving efficiency and scalability in industrial BSF production.

Black soldier fly H. illucens farming in rural Greece under variable temperature regimes and local agrifood residues

A. Yarali Paisios^*, A. Anthousi and E. Paisios

MAGMA Agro S.A., Research & Development, Kathiana, Akrotiri, 73100 Chania, Greece; ^*yarali.paisios@magma-agro.gr

Insects will only feed the world if their farming can be implemented cost-effectively and sustainably across the globe. Thus, rural small/ medium-scale applications are at least as important as industrial ones. In Europe, the mild climate and unvalorised agrifood residues of the Mediterranean region can allow insects to be integrated into existing farm structures with minimal active climate control and feedstock-costs. To demonstrate the potential of such context, we took as a case, black soldier fly H. illucens (BSF) farming in rural Greece. We focused on two aspects that are key to cost-effectiveness and sustainability: climatisation and feedstock. Energy-efficient BSF farming under variable temperature regimes in a Mediterranean greenhouse: We conducted BSF larval growth experiments in industry-type rearing trays using brewer’s spent grain (BSG) as feedstock. The experiments took place July 2024-December 2025 in an experimental greenhouse in southern Greece, with minimal climatisation, mimicking commercial horticulture greenhouses of the region. Larval growth and bioconversion performance as well as the effects of rearing parameters on these were in line with findings under more tightly controlled conditions: Individual larval growth improved but the efficiency of bioconversion decreased with increasing feed availability per larva, while the feed dry matter content was a key source of variation. The unstable ambient temperature enabled us to pinpoint the boundary conditions for bioconversion under variable temperature regimes, which, to our knowledge has not been previously studied. Based on this, we estimated the energy demand for maintaining permissive variable temperature regimes for year-round BSF farming in a Mediterranean greenhouse, in comparison to the respective energy demands in other settings with constant temperatures. Unvalorised Mediterranean agrifood residues as BSF larva feedstock: In the same greenhouse setting, we incorporated local agrifood residues as feedstock, focusing on materials which, due to physicochemical properties or logistics were otherwise unvalorised. Particular combinations of olive mill residue, cheese whey, raw dough, bakery rejects, greenhouse crop residues and BSG were efficiently bioconverted. We present an outlook on how these partially seasonal materials can be stored and pre-processed with minimal energy use to be integrated into year-round operation at a meaningful scale. Overall, these results concerning climatisation and feedstock highlight rural Greece in particular, and the Mediterranean region in general as a promising context for cost-effective, sustainable BSF farming.

Beyond biomass: microbial stabilization of host homeostasis in Hermetia illucens

H.K. Walt¹, E. Harris², S. McPeek², E. Odii³, J. Smink^4*, F. Meyer¹, S. Behmer², F.G. Hoffmann¹, J.K. Tomberlin², C. Picard³ and H.R. Jordan⁴

¹Mississippi State University, Department of Biochemistry, Nutrition, and Health Promotion, 945 Stone Boulevard, Mississippi State, MS 39762, USA; ²Texas A&M, Entomology, 370 Olsen Boulevard, College Station, TX 77843, USA; ³Indiana University at Indianapolis, Department of Biology, 723 W Michigan Street, Indianapolis, IN 46202, USA; ⁴Mississippi State University, Biological Sciences, P.O. Box GY, Mississippi State, MS 39762, USA; ^*jordan@biology.msstate.edu

Understanding the tripartite interactions among host, diet, and microbiome is fundamental to optimising black soldier fly (Hermetia illucens, BSF) production at scale. While research often prioritizes microbial drivers of absolute biomass, this study utilises the BSF as a model of physiological resilience. We demonstrate how specific microbes act as “biotic filters” to stabilize host development, ensuring predictable outcomes even when a biological ceiling prevents further increases in mass. By enriching gut microbes from high-performing larvae, we identified K. aerogenes as a microbial “landmark” associated with peak performance. We then probed this landmark by supplementing high-dose K. aerogenes into an already balanced rearing substrate. Because the control larvae were performing at peak physiological efficiency, the supplement did not significantly increase absolute mass. However, the addition of K. aerogenes triggered a profound stabilization of the phenotype, resulting in an 80% decrease in weight variation (Coefficient of Variation) compared to the control group. Mechanistically, K. aerogenes establishes niche dominance within the gut, acting as a biotic filter that causes a functional crash of the resident fungal community. This bacterial-fungal competition governs the stability of the internal environment, which we further validated through host transcriptomics. We identified 52 differentially expressed host transcripts pointing to a process of “molecular housekeeping,” suggesting that the host “sensed” the stabilized microbial environment and synchronized its metabolism toward internal homeostasis. The significance of this work lies in its shift from the “bigger is better” narrative toward a focus on physiological stability. In industrial bioconversion, achieving population uniformity is a primary hurdle for predictable scaling and economic efficiency. This study provides a mechanistic blueprint for standardizing larval output through microbial niche gatekeeping, offering a new ecological lens for probiotic interventions in sustainable protein production.

Dietary sugars influence substrate temperature and larval growth in black soldier fly larvae bioconversion

A. Fuhrmann^1,2, M. Gold^1,2, Y. Bi², P. Azzari¹, M.J. Zorrilla³, N. Puniamoorthy⁴ and A. Mathys^1,2

¹ETH Zurich, Sustainable Food Processing Laboratory, Schmelzbergstrasse 9, Zurich, Switzerland; ²Singapore-ETH Centre, 1 Create Way, Singapore, Singapore; ³REPLOID Deutschland GmbH, Löbener Weg 7, 04523 Pegau, Germany; ⁴National University of Singapore, Department of Biological Science, 14 Science Drive 4, Singapore, Singapore; ^*adrianfu@ethz.ch

Substrate temperature is a key factor for climate control and larval growth in black soldier fly larvae (BSFL) bioconversion. Excessive heat interrupts operation, yet its drivers are understudied. Digestible carbohydrates, often a highly variable and abundant component of organic waste substrates in BSFL, could be a critical driver of temperature variation. Therefore, this study investigated whether different digestible carbohydrates in organic waste affect temperature and larval growth. In a 7-day controlled feeding trial with industrial crates (60 × 40 cm crates, 8,640 larvae each), we added carbohydrates to the substrate (20% based on dry matter) to determine whether the sugar type (isomaltulose versus sucrose, i.e. high versus low digestion rate) and availability (day 0, day 3) affected larval dry mass, substrate temperature, metabolites, and pH, CO₂, and NH₃ emissions. We found that sugars strongly altered substrate temperature in a time-dependent manner. Sucrose addition on day 0 produced higher temperatures (mean increase: 2.1 °C, max.: 7.9 °C) and resulted in a lower final larval mass (approx. 20%) compared to sucrose addition on day 3. Isomaltulose addition resulted in a lower mean (0.4 °C versus sucrose) but highest peak (51.3 °C) temperature. pH and NH₃ concentration dropped after sucrose addition. Trends in metabolites suggested O₂-dependent metabolic shifts during the early bioconversion. These results showed that initial amounts of digestible sugars in organic wastes can lead to elevated temperature peaks early in the bioconversion process and lower larval yield. The study improves the understanding of heat generation in food waste recycling with BSFL and suggests that tailored, sequential feeding strategies could optimise substrate temperatures, larval yield and environmental process performance.

Effects of early larval weight on survival of Hermetia illucens across waste substrates

V. Wiklicky^*, I. Lopes, M. Kullberg and C. Lalander

SLU (Swedish University of Agricultural Sciences), Energy and Technology, Almas Allé 8, 75651 Uppsala, Sweden; ^*viktoria.wiklicky@slu.se

BSF farming has become increasingly optimised, yet key uncertainties remain regarding management of the nursery stage. In particular, there is no consensus on when larvae should be transferred from nursery substrates to the waste feedstock. Current recommendations are typically based on larval age, despite substantial variation in early growth rates under different rearing conditions. To improve the comparability of studies and farming practices, evaluating the effect of early larval weight on survival is crucial. This study was conducted at SLU to evaluate whether early larval weight provides a robust criteria for the successful transfer of BSF larvae to waste substrates. Prior to the start of the experiment, approx. 100 000 neonate larvae originating from a single egg cohort, all hatched within an 8-hour window, were reared on chicken feed (CF) provided ad libitum until they reached a size that permitted manual handling and counting. Throughout the experiment, the cohort was maintained parallel under the same controlled laboratory conditions (29 ± 1.5 °C, RH 40%) as the following waste treatment. The experiment began when the individual average weight of the seed larvae was 0.13 mg. On the first day, the first waste treatments were established, using a set amount of hand counted larvae per replicate. New groups of progressively heavier larvae were collected from the same cohort and introduced into the same waste source in triplicates every 24 h. This approach resulted in waste treatments containing larvae of increasing initial weight over 5 consecutive days, allowing assessment of early-stage mortality across a range of seed larval sizes. Each waste stream triplicate started on different larva sizes was terminated after seven days, and the survival of the larvae was checked. Using this approach, we evaluated larval survival across commonly used substrates (CF and food waste (FW)) as well as more challenging wastes, including fermented FW with/without insect frass, orange and cabbage cuttings, and mussel meat (MM). The results of this experiment identified a minimum weight threshold for successful establishment and results show that across most waste types, larvae weighing approximately 0.4 mg could be transferred without negatively affecting survival. Reduced survival was observed only on particularly tough substrates, such as high-protein MM but the use of heavier larvae (10 mg) increased the survival significantly. These findings demonstrate that larval weight, rather than age, is a key determinant of early survival and provide a practical, weight-based criteria for optimising nursery duration and larval transfer in BSF production systems

Achieving thermogenesis in bench-scale black soldier fly larvae experiments

M.J. Zorrilla^1*, R.F. Calderon¹, M. Tayyab¹, T. Saleem¹, N. Warthmann¹, F. Weihnhold¹ and M. Gold²

¹REPLOID Deutschland GmbH, R&D, Löbener Weg 7, 04523 Pegau, Germany; ²REPLOID Group AG, R&D, Maria-Theresia-Straße 53, 4600 Wels, Austria; ^*zorrilla@reploid.eu

Reproducibility of black soldier fly larvae (BSFL) rearing results within and among locations, and especially when scaling from bench scale to industrial scale, remains a challenge. One factor that has so far been insufficiently considered is the accurate mimicking of substrate temperature. Substrate temperature can rise above ambient due to accumulated metabolic heat from larvae and microbes, a phenomenon termed thermogenesis. Among others, this effect depends on substrate and larval mass and the corresponding surface area-to-volume ratio. However, BSFL research rarely accounts for scale-dependent thermogenesis in experimental designs, complicating interpretation and replication of results. Specifically, thermogenesis in small-scale laboratory experiments (low substrate/larval mass, higher surface area-to-volume ratio) rarely mimics thermogenesis in large-scale industrial settings (high substrate/larval mass, lower surface area-to-volume ratio). We hypothesized that lower temperatures in small-scale experiments are caused by elevated heat loss, and that this can be overcome using insulated rearing containers. We tested this hypothesis in controlled bench-scale rearing experiments. Insulated and uninsulated stainless steel vessels (12 cm diameter, 1100 larvae each, 700 g chicken feed-based diet at 28% dry matter) were compared in a 7 day experiment including six replicates per treatment. During the experiment substrate temperature was measured every 5 minutes using Testo 175 T3 loggers (Testo, Germany) with external thermocouple probes. After harvest the individual larval weight, total larvae weight, frass total weight, and dry matter content for both biomass fractions were measured. Data was analysed with Anova and post hoc testing. Results demonstrated that insulated rearing containers effectively minimised biological heat loss in small-scale experiments. Insulated vessels showed greater temperature increase above ambient compared to uninsulated vessels (mean increase 15.7 °C vs. 5.4 °C), with higher peak temperature (50.5 °C vs. 37 °C). This suggests that insulation alters the energy balance, and heat accumulates when loss rate to the environment is lower than production rate. High temperatures in the insulated containers seemed to mirror the phenomenon of overheating, which is common with large substrate mass (9–40 kg) in BSFL rearing, where the substrate insulates itself. This approach could improve consistency and replicability of BSFL rearing experiments, while increasing the predictability of bench-scale to industrial-scale results.

Biowaste treatment using black soldier fly larvae: effect of substrate macronutrients on process performance

M. Henjak^1*, V. Grossule¹ and J.K. Tomberlin²

¹University of Padova, DICEA, Department of Civil, Architectural and Environmental Engineering, via Marzolo 9, 35131 Padova, Italy; ²Texas A&M University, Department of Entomology, 370 Olsen Boulevard, College Station, TX 77843, USA; ^*mia.henjak@phd.unipd.it

Black soldier fly larvae (BSFL) are widely used for the treatment of solid organic waste by converting biowaste into a protein- and lipid-rich larval biomass. Process performance, evaluated with survival rate (SR, %) and waste reduction efficiency (RE, % dry matter), depends on substrate nutritional quality. Substrate quality can be described by the overall macronutrient concentration (∑PCL), defined as the sum of proteins (P), lipids (L) and non-fibre carbohydrates (NFC), and by the relative abundance of a single macronutrient (XP, XNFC, XL). This study quantitatively combines published literature to assess how substrate quality affects BSFL process performance. A systematic literature review was conducted following the PRISMA methodology. Data on substrate macronutrient composition and process performance were extracted from 35 peer-reviewed studies (83% Q1), covering 177 substrates, including biowaste and artificial diets. Substrates were classified into six categories: meat and fish waste, manure, grain waste, food waste, vegetables and fruits, and artificial substrates. Most substrates showed high nutritional quality, with 133 substrates exhibiting ∑PCL > 50% and 124 substrates exhibiting balanced macronutrient ratios (XNFC > XP > XL). These substrates were mainly food waste, vegetables and fruits, and artificial diets. In contrast, data on low-quality and unbalanced substrates, such as meat and fish waste, were limited. SR is independent of ∑PCL, as most substrates achieved SR ≥ 80%. However, SR is influenced by macronutrient ratios and decreases under lipid excess (XL > 0.6), protein deficiency (XP ≤ 0.05), or protein excess (XP > 0.5) combined with low NFC (XNFC < 0.15). RE, in contrast, depends on ∑PCL. On low-concentrated substrates (∑PCL < 60%), RE decreases below 40%, especially in the case of lipid excess (XL > 0.65) and/or protein imbalance (XP ≤ 0.05 or XP > 0.5 with XNFC < 0.20). Highly concentrated substrates with ∑PCL > 60% generally achieve RE ≥ 40%, which further increases when macronutrients are balanced, with XP > 0.6 and XNFC > XL. By integrating both SR and RE data, good-quality substrates for BSFL treatment were identified (when SR ≥ 80% and RE ≥ 40%) based on the relative macronutrient abundances. The results can be used as a simple tool for rapid evaluation of individual substrate suitability for the BSFL process and for identifying the optimal substrate mixture to improve process performance.

Batch-invariant identification of edible insects from NIR spectra with explainable AI

M. Babor^*, G. Rossi, A. Altavilla, O. Schlüter and M.M.-C. Höhne

Leibniz Institute for Agricultural Engineering and Bioeconomy e.V. (ATB), Max-Eyth-Allee 100, 14469 Potsdam, Germany; ^*MBabor@atb-potsdam.de

Reliable authentication of edible-insect species is essential for the safety, traceability, and regulatory compliance of emerging insect-based food and feed supply chains to prevent food fraud and process inefficiencies. Near-infrared (NIR) spectroscopy offers a fast, non-destructive way for automated species verification, but its reliability is often undermined by data shift that occurs when spectra are collected under different real-world conditions. Strong batch effects due to variation between production runs and insect processing-induced spectral distortions can alter signals, scattering behaviour, and the intensities of biochemical absorption bands. To address this, we introduce the Batch-Invariant Spectral Transformer (BIST), a batch-generalizing model that jointly learns a spectral preprocessing module and a species classifier while using an adversarial branch to suppress batch-discriminative information. We use a full-factorial dataset of 2700 spectra, spanning three insect species (Acheta domesticus, Hermetia illucens, Tenebrio molitor), three batches, three processing treatments (tap water, blanched with tap water, plasma-activated water), and two ultrasound conditions (with and without ultrasound). This dataset enables comprehensive evaluation of BIST under leave-one-batch-out (LOBO) protocol. BIST achieves 0.93 ± 0.04 accuracy, outperforming classical chemometric models by 9–39% and recent deep spectral models by 5–41%, while producing batch-invariant, biochemically meaningful representations. We integrate a gradient-based counterfactual framework to BIST that quantifies the minimal spectral adjustments required for a sample to be reassigned to its true insect class. Spectral region-restricted counterfactuals, defined across twelve chemically curated NIR bands, show that lipid- and protein-dominated regions provide the causal leverage necessary for true species recovery. These counterfactual signatures align with established biochemical differences among the insects and clarify the mechanisms underlying systematic misclassification under specific processing conditions. Together, these analyses demonstrate that BIST achieves robust cross-batch generalization while presenting interpretable biochemical structure and spectral region-level insight, establishing a reliable foundation for NIR-based species authentication in industrial settings.

Controlling black soldier fly adult emergence via low-temperature storage

M.B. Ala Eddine^1,2*, L.I. MacAvei^2,3, T.B. Barber², G. Benassi² and S. Dabbou¹

¹University of Trento, Center Agriculture Food Environment (C3A), Via E. Mach 1, 38098 San Michele All’Adige (TN), Italy; ²Kinsect S.r.l. SB, Via Mazzacurati, 7/D, 42124 Reggio Emilia, Italy; ³University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Calea Mănăştur 3–5, 400372 Cluj-Napoca, Romania; ^*mohamadbaker.alaeddine@unitn.it

Industrial black soldier fly production requires precise synchronization of life cycles to ensure efficient breeding and egg collection. Variability in pupal emergence can cause overlapping generations and reduce process control. This study evaluated low-temperature storage to delay pupal emergence, enabling batch alignment and improved workflow in scalable industrial production. Approximately 6000 individuals (>75% pupae) from a standard growth cycle at 27 °C and 70% relative humidity (RH) were used. Larvae were fed a wet Gainesville housefly diet (70% water) at 0.114 g larva^−1 day^−1. One week after harvest, at the onset of adult emergence, individuals were equally divided into six containers (1000 individuals per container). Three replicates were assigned to an “immediate emergence (IE)” treatment, in which containers were transferred directly to adult cages (30 × 30 × 30 cm) maintained at 27 °C, 70% RH and a 12:12 h LED light-dark cycle. The remaining three replicates were assigned to a “delayed emergence (DE)” treatment, in which containers were stored at 17 °C and 70% RH for 12 days; based on preliminary observations indicating the onset of emergence at this temperature before transfer to cages under the same conditions as the IE group. Eggs were collected every 2–3 days over a 10-day reproductive cycle period following full adult emergence, corresponding to the peak oviposition phase. Total egg production and adult emergence rates were recorded and compared between treatments using independent samples t-tests. Total egg yield did not differ between treatments (IE: 6.14 ± 0.69 g; DE: 5.70 ± 0.52 g), and adult emergence rates remained high and comparable in both groups (>90%). No differences were observed in oviposition timing or daily egg collection counts. These results indicate that DE did not compromise reproductive performance under these conditions, as total egg production remained comparable to that of the IE group. Similarly, adult emergence rates were unaffected by the delay, suggesting that temporary low-temperature storage did not negatively impact pupal-to-adult survival. Under the tested conditions, storage at 17 °C and 70% RH enabled a delay of up to 12 days while maintaining reproductive potential. This approach represents a practical strategy for improving batch synchronization and operational flexibility in industrial BSF production.

Optimising larval density in black soldier fly (BSF) production using a dry hatching system

R. Selmane¹, A. Kamoun Rebai^1*, N. Hermi¹, G. El Mabrouk¹, L. Voutey² and N. Kinet²

¹nextProtein Tunisia, Km 1 Route de Béni Khalled, 8030 Grombalia, Tunisia; ²nextProtein France, Engineering — Performances, 66 Boulevard Niels Borh, 69100 Villeurbanne, France; ^*a.kamoun@nextprotein.co

Efficient and scalable techniques for black soldier fly (Hermetia illucens (L.), Diptera: Stratiomyidae) larval production are essential for advancing sustainable waste management and providing alternative protein sources. This study presents the implementation and qualification of a dry hatching system for BSF neonate collection and incubation at nextProtein’s industrial production facility. The case study shares nextProtein’s experience transitioning to a new production process while maintaining consistent performance at scale. Since its founding in 2015, nextProtein has relied on a simple reproduction process: collecting fresh eggs daily and directly dosing them into crates supplied with fresh feed. While effective and low-cost in early operations, this method introduced major challenges in later stages due to high heterogeneity in larval weight and density both within and between crates. Based on preliminary tests, results and consulting recommendations, a dry hatching process was designed and all related equipment and devices were developed or sourced to ensure its industrialisation following a strict phasing plan. First, the weighing of the egg collectors (“eggies”) is done before they are placed into the cages. Then, the following day, these eggies are weighed again to determine the mass of the eggs. After 24 h, these same egg collectors are transferred to specially designed equipment aiming to regularly collect the newly hatched larvae for 5 days within a precisely controlled environment (temperature & humidity). The system demonstrated high efficiency in guiding newly hatched larvae toward targeted collection areas, reducing losses from hatching through early larval stages and reducing the total growth time before harvest. The process allowed an important hatching rate of 92%, an average of neonates weight of 0.0159 mg an average weight after incubation (5 DOLS) of 4.9 mg with an overall survival rate of 94%. Following implementation, larval yield doubled for an equivalent egg input, while reducing the fattening phase duration by 20%. The findings highlight the potential for scalable applications of dry hatching systems in BSF production, particularly for operations aiming to improve biosecurity, resource efficiency and productivity. Further investigations will focus on refining incubation conditions and scaling the system for larger industrial setups.

Effect of high-intensity ultrasound on quality of proteins from three different insect species

E. Gousgoula^1,2, A. Poccia^1,3, L. Rossi Ribeiro¹, A. Altavilla¹, O.K. Schlüter¹ and G. Rossi^1*

¹Leibniz-Institut für Agrartechnik und Bioökonomie e.V. (ATB), Max-Eyth-Allee 100, 14469 Potsdam, Germany; ²National and Kapodistrian University of Athens, Panepistimiou Street 30, 10679 Athens, Greece; ³University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy; ^*grossi@atb-potsdam.de

The low acceptance of insects among consumers suggests that enriching food products with extracted insect proteins could be a useful approach. Ultrasound processing (US) can improve the protein extraction yield. However, little is known on the impact of US on the quality of the proteins. In the current study, three insect species (Tenebrio molitor (Tm), Hermetia illucens (Hi), Acheta domesticus (Ad)) and three independent batches were investigated. Frozen insects were mixed with water and reduced into paste by applying three different treatments (A, tap water; B, blanching (70 °C, 5 min) + tap water; C, plasma water). Obtained pastes were treated with US (700 W, 30 min, 5 s ON–55 s OFF) and the proteins were extracted. Techno-functional properties (water holding capacity (WHC); oil holding capacity (OHC)), oxidation status (number of thiol, disulphide and carbonyl groups) and biological activities (antimicrobial and antioxidant capacity) were investigated. Results showed that Ad had the highest WHC (3.35 ± 0.19%), regardless of the treatment and the US process. No significant differences were observed in OHC. Number of thiols, disulphides and carbonyls were significantly affected by species and treatment. The highest thiols were always detected in Hi (39.19 ± 1.70 μmol SH/g protein), followed by Tm (20.26 ± 1.61 μmol SH/g protein) and Ad (12.10 ± 0.92 μmol SH/g protein). US led to an increase of thiols in Tm-A (+2.83 μmol SH/g protein), while they decreased in Tm-B (−2.61 μmol SH/g protein) and Hi-C (−4.22 μmol SH/g protein). US reduced the number of disulphides in Tm (−84.96 μmol SS/g protein), while increased them in Ad (+40.44 μmol SH/g protein) and Hi (+44.45 μmol SH/g protein). Number of carbonyls increased in Tm-C (+0.36 μmol COO/g protein) and Ad-C (+0.82 μmol COO/g protein), while decreased in Tm-B (−0.42 μmol COO/g protein) and Ad-B (−0.16 μmol COO/g protein) after US. Application of US always led to an increase of antioxidant activity (FRAP: +1.09 μmol TE/mg protein, ABTS: +8.60 μmol TE/mg protein). Antimicrobial activity against Escherichia coli was only noticed in Hi-A and disappeared after US. Activity against Salmonella enterica was recorded in Ad and Hi and boosted by US. Antimicrobial activity toward Staphylococcus aureus was observed in Tm. In summary, this study shows that protein extracts from different insect species have significantly different biological and techno-functional properties and that these activities can be influenced by the processing method used.

From insect proteins to food systems: a stepwise processing and mimicking strategy for edible insect-based foods

E.Y. Oh^1* and Y.Y. Kim²

¹Korea University, Human Ecology Research Center, Seoul 136-701, South Korea; ²Korea University, Department of Human Ecology (Food Science and Nutrition), Seoul 136-701, South Korea; ^*oheuy0725@korea.ac.kr

Edible insects are increasingly recognized as sustainable alternative protein sources; however, their application in food systems remains limited by poor solubility, restricted techno-functionality, and low compatibility with conventional food matrices. This study investigates a stepwise processing and mimicking strategy to systematically transform mealworm (Tenebrio molitor larvae) proteins into food-ready ingredients by sequentially aligning their functional and structural properties with those of conventional food proteins. First, the effects of extraction strategies on health-related functionality were evaluated using conventional (alkali and salt) and non-conventional (enzyme and screw-press) methods. Enzyme- and salt-extracted protein concentrates significantly enhanced insulin signalling in cell-based models, increasing p-Akt/Akt ratios by approximately 2.3–2.7-fold and 1.3–1.6-fold, respectively, compared with untreated controls. In addition, bioactive peptides derived from mealworm protein hydrolysates (FDKY and FDRL) significantly suppressed myostatin expression and downregulated muscle atrophy markers under dexamethasone-induced conditions, indicating tunable anti-diabetic and anti-muscle atrophy potential through extraction and hydrolysis design. To improve techno-functional performance, physicochemical processing combining heat treatment, pH-shifting, and high-pressure homogenization (HP + HT + PS) was applied to mealworm flour. This integrated treatment increased protein solubility from 0.23 to 7.50 mg/ml and enhanced surface hydrophobicity by up to 85-fold, resulting in improved emulsifying activity, stability, and reduced droplet size. These improvements are critical for incorporating insect proteins into emulsion-based and liquid food systems. For further functional mimicry of conventional protein powders, mealworm protein isolate (MPI) was co-spray dried with gum Arabic (GA) or maltodextrin (MD). Co-spray-dried powders showed enhanced in vitro digestibility, with higher degrees of hydrolysis (MPI + GA: 55.8%; MPI + MD: 47.6%) compared with freeze-dried or spray-dried MPI alone. Finally, mealworm-derived proteins were incorporated into a tofu system as a representative soy-protein matrix. Partial substitution of soybean protein enabled the development of protein-enriched tofu with tailored structural and functional characteristics. Overall, these results demonstrate that edible insect proteins can be effectively converted into food-compatible ingredients through a processing-driven, stepwise mimicking strategy, supporting their practical application in sustainable and functional food systems.

Diet effects on development, nutrition, safety, and gut microbiota of Protaetia brevitarsis larvae

S.Y. Kim^*, M.J. Park, K.M. Park, B. Koo, S. Ji and K.-H. Park

National Institute of Agricultural Sciences, Development of Agricultural Biology, 166 Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeonbuk state 55365, South Korea; ^*carp0120@korea.kr

Protaetia brevitarsis larvae are traditionally consumed in Korea and are gaining global attention as a sustainable protein source. However, large-scale utilisation requires scientific validation of how feed substrates influence larval development, nutritional quality, safety, and gut microbiota. This study evaluated the effects of fermented oak sawdust (FOS) and fermented oyster mushroom cultivation by-products (OMCB) on larval performance and quality. Larvae were reared under controlled conditions and fed either FOS or OMCB throughout the larval stage. Developmental traits, including survival rate, body weight gain, and development period, were recorded. Proximate composition, amino acid profiles, fatty acids, minerals, and hazardous substances were analysed using standard AOAC and food code methods. Microbiological safety was assessed by detecting foodborne pathogens and heavy metals. Gut microbiota composition was analysed using 16S rRNA gene sequencing. Larvae fed OMCB showed significantly higher weight gain and shorter development periods compared to those fed FOS, while maintaining high survival rates. OMCB-fed larvae exhibited higher crude protein content and comparable essential amino acid profiles, with elevated levels of proline and unsaturated fatty acids. Heavy metals and pathogenic bacteria were below detection limits in both groups. Gut microbiota analysis revealed distinct community structures depending on feed source, with mushroom-based diets promoting higher bacterial diversity and enrichment of host-associated taxa involved in carbohydrate degradation. Feed substrate strongly influenced larval growth performance and gut microbial composition without compromising nutritional value or safety. Fermented mushroom by-products provided improved nutrient availability and supported beneficial gut microbiota, demonstrating advantages for sustainable insect production. Fermented oyster mushroom cultivation by-products are a safe and effective alternative feed for Protaetia brevitarsis larvae, enhancing growth efficiency, nutritional quality and gut microbiota profiles. These findings support the use of agro-industrial by-products in edible insect production and contribute to sustainable circular bioeconomy strategies.

Impact of membrane separation on functional and bioactive properties of insect proteins

M.K. Ranasinghe^1,2, P. Aguadé¹, R. Mazzei³, F. Bazzarelli³, L. Giorno³, A. Groso⁴, G. Collet⁴, R. Daniellou⁴, M. Ferrando¹ and C. Güell^1*

¹Universitat Rovira i Virgili, Departament d’Enginyeria Quı́mica, Tarragona, Spain; ²Uva Wellassa University, Dept. of Food Science and Technology, Badulla 90000, Sri Lanka; ³Institute on Membrane Technology, CNR-ITM, University of Calabria, Rende, Italy; ⁴Chaire de Cosmétologie, AgroParisTech, Orléans, France; ^*carme.guell@urv.cat

Membrane based fractionation offers a low impact route to tailor protein functionality. We studied how mild alkalinization of protein solutions before ultrafiltration (UF) and cascade membrane systems modulate the techno functional/bioactive properties of A. domesticus (cricket) and H. illucens (black soldier fly; BSF) proteins. Cricket and BSF proteins were solubilized at pH 7 or 9.5 and processed in a single 30 kDa UF step. Cricket protein was also processed in two cascade systems: Config. A (0.2 μm MF, 500 kDa, 100 kDa, 30 kDa) and Config. B (500 kDa, 100 kDa, 30 kDa). Permeate flux, protein rejection, emulsifying activity, and foaming capacity were quantified for all permeates and retentates. Bioactivity of low molecular weight (<30 kDa) fractions from the single 30 kDa was assessed by collagenase inhibition and in vitro cell proliferation/cytotoxicity up to 48 h. Adjusting the solubilization pH from 7 to 9.5 altered the UF fractions obtained with the 30 kDa membrane for both cricket and BSF. While these pH shifts strongly affected techno functional properties, the most notable effects concerned bioactivity. Cricket permeate at pH 7 showed clear bioactive potential, combining collagenase inhibition with enhanced cell proliferation after 48 h, without any cytotoxicity — indicating suitability for cosmetic applications. By contrast, BSF protein solutions at both pH levels stimulated cell proliferation at 24 and 48 h without cytotoxic effects, revealing previously unreported bioactivity in low molecular weight (<30 kDa) BSF fractions. These findings highlight that both insect species can yield biologically active protein fractions only applying membrane separation without requiring hydrolysis. Cascade membrane processing showed that membrane sequence strongly affects efficiency and protein functionality. Configuration A (starting with microfiltration) maintained a higher permeate flux (approx. 40 l ⋅ h^−1 ⋅ m^−2) and yielded a more balanced protein distribution with lower rejection, producing an MF retentate with strong emulsifying properties. Configuration B, starting with 500 kDa UF, resulted in a lower steady state flux (approx. 30 l ⋅ h^−1 ⋅ m^−2) and earlier protein concentration, with >80% rejection at the 500 kDa step. Overall, both set-ups show that cascade membrane separation can tailor insect protein fractions, but membrane choice and sequence are critical for optimal performance and protein functionality

Insect proteins as sustainable multifunctional emulsifiers for diverse colloidal food architectures

M. Ferrando^1*, C. Güell¹, A. Ballon¹, S. De Lamo-Castellvı́^1,2, J. Jayakumar¹, C. Camelo-Silva³ and M. Ortiz¹

¹Universitat Rovira i Virgili, Departament d’Enginyeria Quı́mica, Tarragona, Spain; ²The Ohio State University, Department of Food Science and Tech, Columbus, OH, USA; ³Universidade Federal de Santa Catarina, Departamento de Engenharia Quı́mica e Engenharia de Alimentos, Florianopolis, Brazil; ^*montse.ferrando@urv.cat

Insect proteins offer a versatile platform for structuring a wide spectrum of food colloids with contrasting techno-functional demands. Microfluidic observations, used to map how formulation variables influence early droplet stability, showed that lesser mealworm protein concentrate (LMPC) performs comparably to whey protein, with monodisperse droplets (55–118 μm) and coalescence suppressed at 0.02% protein, while pH and oil fraction modulated dimer formation. These insights help define stability windows across different emulsion formats (Jayakumar et al., 2023). Chemical modification further broadens functionality: covalent conjugation of LMPC with tannic or chlorogenic acid markedly increased antioxidant capacity (up to approx. 845 μmol TE/g) without impairing emulsifying activity (approx. 53–55%). In PUFA rich O/W emulsions, these conjugates substantially reduced lipid oxidation during accelerated storage (Ballon et al., 2024). Such antioxidant emulsifiers also enable multicomponent systems. Double emulsions with plant derived polyphenols and linseed oil achieved high encapsulation efficiencies (≥67.5%) and small droplets (≤8.35 μm), while exhibiting enhanced oxidative stability when stabilized with LMPC tannic acid. These systems illustrate how insect protein emulsifiers can support bioactive protection and controlled release in functional foods (Camelo-Silva et al., 2025). Insect proteins (native or heat-treated) also stabilize high-internal-phase emulsions, HIPEs, (80% oil; 2–3% protein) with solid like rheology and yield stresses of 255–399 Pa. These HIPEs show adequate viscosity, elasticity and self-supporting capacity for extrusion-based 3D printing, producing fat analogues that retain shape for at least nine days under refrigeration. Thermal treatment increases viscosity and structure stiffness, expanding the textural palette accessible to formulators (Ballon, et al., 2025). Altogether, native, thermally treated and polyphenol-conjugated insect proteins provide a sustainable toolbox for designing antioxidant emulsifiers, bioactive-loaded double emulsions and printable fat-rich HIPEs, demonstrating their relevance for food applications requiring tailored interfacial and rheological functionalities. References: Ballon, A., Romero, M.-P., Rodriguez-Saona, L.E., de Lamo-Castellvı́, S., Güell, C. and Ferrando, M., 2024. Conjugation of lesser mealworm (Alphitobius diaperinus) larvae protein with polyphenols for the development of innovative antioxidant emulsifiers. Food Chemistry 434: 137494. Ballon, A., Sessa, S., Cito, S., de Lamo-Castellvı́, S., Güell, C. and Ferrando, M., 2025. High internal phase emulsions stabilized by insect proteins: a path to 3D printable fat analogues. Food Hydrocolloids 166: 111330. Camelo-Silva, C., Ballon, A., Ranasinghe, M.K., Verruck, S., Ambrosi, A., Di Luccio, M., Güell, C. and Ferrando, M., 2025. Lesser mealworm (Alphitobius diaperinus) protein concentrate conjugated with tannic acid improves the oxidative stability of W1/O/W2 emulsions loaded with beet by-product extract and linseed oil. Food Chemistry 463: 141542. Jayakumar, J., Ballon, A., Pallarès, J., Vernet, A., de Lamo-Castellvı́, S., Güell, C., Ferrando, M., 2023. Lesser mealworm (A. diaperinus) protein as a replacement for dairy proteins in the production of O/W emulsions: droplet coalescence studies using microfluidics under controlled conditions. Food Research International 172: 113100.

High-pressure–assisted protein extraction to modulate functionality in black soldier fly

M. Ferrando^1*, S. De Lamo-Castellvi^1,2, T. Kaothaisong², M.C. Alvarado¹, M. Ortiz¹, S.S. Guduru³, C.T. Simons², C. Güell¹ and V.M. Balasubramaniam^2,3

¹Universitat Rovira i Virgili, Departament d’Enginyeria Quı́mica, Avda. Països Catalans, 26, 43007 Tarragona, Spain; ²The Ohio State University, Department of Food Science and Technology, 2015 Fyffe Road, Columbus, OH 43210-1007, USA; ³University of Georgia, Department of Food Science & Technology, Griffin, GA 30223-1797, USA; ^*montse.ferrando@urv.cat

High-pressure-assisted extraction (HPAE) of edible insects remains underexplored. In this study, the potential of HPAE to produce black soldier fly (Hermetia illucens) protein extracts with tailored technological properties was investigated. High-pressure treatments at 100, 200, 300 and 400 MPa were applied for 5 min at 30 °C using a bench-scale high-pressure processor to organic solvent–defatted BSF fractions (0.8 g) under alkaline conditions (0.05, 0.10, 0.20, and 0.25 M NaOH) in the presence of 0.05 M NaCl. The alkaline condition, combined with low ion concentration (<0.05 M NaCl), was selected to enhance protein extraction. Protein extraction yield, protein molecular weight distribution, emulsifying activity, and emulsion zeta potential were analysed to assess the combined influence of pressure and alkaline extraction conditions on the functional properties of BSF protein extracts. HPAE significantly increased protein recovery ($p\mathrm{<}0.05$) compared with the control. With NaOH at 0.20 and 0.25 M, protein yield was significantly higher ($p\mathrm{<}0.05$), between 250 and 390 times that of control samples with 0.05 and 0.10 M NaOH being particularly enhanced at 200 and 300 MPa. Molecular weight distribution analysis indicated that HPAE increased the intensity of protein bands, though the distribution remained predominantly at 10–15 kDa, suggesting denaturation under these conditions. Nonetheless, samples suspended in 0.05 and 0.10 M NaOH and treated at 200 and 300 MPa showed a significant increase in protein bands at 100–150 kDa ($p\mathrm{<}0.05$), which were not found in the control samples. Emulsifying activity was higher in HPAE-treated samples compared with the control, and was particularly higher in samples suspended in 0.10 M NaOH and treated at 300 MPa. Zeta potential values of the emulsions were below –30 mV in all samples, including the control and HPAE-treated groups, showing the potential for producing stable emulsions from these protein extracts. This study demonstrates that HPAE can be effectively used to modulate the yield, molecular weight distribution, and functional properties of BSF protein extracts.

Bringing order to complexity: performance monitoring at black soldier fly reproduction units

A. Walker^* and C. Zurbrügg

Eawag: Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf, Switzerland; ^*walker.alvi@gmail.com

Biowaste processing in low- and middle-income countries using black soldier fly (BSF) bioconversion has recently gained significant traction. To process the daily waste received, facilities ensure a steady supply of hatchlings in their reproduction units (RU). However, operators often struggle to maintain a stable and reliable RU. The complexity of the BSF life cycle combined with varying or sub-optimal operating procedures highly impacts their efficiency. As inefficiencies and disruptions in the RU impact the whole system, it is crucial to monitor key parameters and react with measures in a timely manner. Despite the importance of RUs, there is currently no consensus on monitoring methods nor on target performances among practitioners. We propose a BSF RU monitoring framework using four Reproductive Performance Indicators (RPI). The RPI-G contains information about the performance of the whole RU. The other three indicators: The RPI-P, RPI-E and RPI-N provide information about the larval, adult, and neonatal stages respectively. Using this framework, we analyse and compare nine existing RUs of bioconversion facilities around the world with different types of monitoring datasets. Subsequently, based on interviews with operators, we hypothesize possible associations between operating procedures and performance. The RPI framework was compatible with all 9 different datasets and data structures. Results show that there are large differences in performance among facilities, with variations in specific RPIs up to a factor of 4. Operational measures leading to increases in performance (by a factor of 2–3) were identified during interviews and are presented. We show that the RPI framework is near universal and enables comparison of a variety of RUs from different BSF facilities, independent of scale, operational protocol, and data collection methods. As RPIs are expressed in ratios, they can be calculated over multiple days, weeks or months, filtering out the day-to-day variability and offering an unbiased estimation of performance. This framework can be applied and implemented in RU monitoring, to identify a declining performance and simultaneously identify the source of the decline. The RPIs offer straightforward and interpretable performance measures for BSF RUs. They are robust to variability and can be used on historic data. This enables BSF practitioners to efficiently monitor their RU, to assess their RU quality, and to compare it with their peers’.

Integration of emerging technologies for insect processing in a biorefinery framework

H. Mhemdi^*, R. El Hajj and E. Vorobiev

Université de technologie de Compiègne, Ecole Supérieure de Chimie Organique et Minérale, TIMR (Integrated Transformations of Renewable Matter), Centre de recherche Royallieu, CS 60319, 60203 Compiègne Cedex, France; ^*h.mhemdi@escom.fr

This study assesses the integration of emerging processing technologies into an insect biorefinery framework, using yellow mealworm (Tenebrio molitor) larvae as a sustainable protein source. Effective upstream pretreatments are essential to ensure microbial safety, promote efficient fractionation, and preserve product quality. Instant Controlled Pressure Drop (DIC) and Pulsed Electric Fields (PEF) were therefore evaluated and compared with conventional hot-water blanching. Larvae were subjected to pretreatment followed by killing/decontamination, hydraulic pressing for simultaneous defatting and dewatering, and convective drying. Performance was assessed through microbiological analyses (total viable counts, yeasts and moulds), juice colour and browning, pressing yield, protein recovery, cake composition (dry matter and lipid content), protein structural changes (fluorescence spectroscopy), drying behaviour (shrinkage, effective water diffusivity, surface area, particle size distribution), and energy consumption. DIC pretreatment at 0.3 MPa for 15 s achieved rapid microbial reductions below European regulatory limits within seconds, outperforming blanching, which required several minutes, while PEF effectively killed larvae but showed limited microbial inactivation. DIC pretreatment also resulted in lighter juice colour due to enzymatic inactivation, whereas blanching and PEF promoted browning. Conventional blanching yielded 68% press juice, 48% protein recovery, and press cakes containing 45% dry matter and 14.7% lipids (dry basis). In contrast, DIC reduced juice yield to 55% but significantly enhanced protein retention in the press cake (78% recovery), with higher dry matter content (55%) and lower lipid levels (12% dry basis), likely due to protein aggregation. PEF increased juice yield (78%) but resulted in greater protein losses to the liquid fraction (43% recovery), with 54% dry matter and 14% lipids (dry basis) in the cake. DIC pretreatment substantially improved drying performance by reducing shrinkage, increasing effective water diffusivity, expanding surface area by 125%, and producing more uniform particle sizes. PEF also accelerated drying, though to a lesser extent than DIC. Energy consumption followed the order: PEF (21.7 kWh ⋅ t^−1)<blanching (61.7 kWh ⋅ t^−1)<DIC (111 kWh ⋅ t^−1). Overall, DIC and PEF offer complementary advantages within insect biorefinery systems, with DIC excelling in decontamination and drying enhancement, and PEF improving process efficiency and protein quality. Strategic integration of these technologies enables more sustainable and efficient insect processing pathways.

An AI-based framework for feed optimisation in black soldier fly rearing under substrate variability

S. Mysore Guruprasad^*, M. Hasan and U. Liebau

August-Wilhelm Scheer Institut GmbH, Uni Campus Nord D 5 1, 66123 Braunschweig, Germany; ^*shreyas.mysoreguruprasad@aws-institut.de

Feed formulation is a key determinant of growth performance and biomass composition in black soldier fly (Hermetia illucens) rearing. Industrial optimisation is challenged by strong variability in organic waste substrates and the lack of structured methods to link feed composition, processing and biological response. This study presents an AI-based framework to systematically address feed optimisation under substrate heterogeneity. The feed formulation was modelled as a constrained, multi-objective mixture optimisation problem. The experimental feed classes included household vegetable waste, supermarket vegetable waste, agro-industrial residues (cassava pulp, palm oil decanter cake, and brewery spent grains), and fermented variants using Lactobacillus spp. A Material–Process–Biological response ontology was introduced to semantically structure substrate origin, pre-treatment, compositional descriptors, and larval response variables. Supervised machine learning models act as surrogate predictors, whereas Bayesian and evolutionary optimisation algorithms explore feasible feed combinations under cost, availability, and sustainability constraints. Simulation-based case studies using synthetic and literature-derived datasets showed that ontology-guided optimisation reduced the effective feed search space by approximately 40–60% compared to unstructured mixture exploration. Surrogate model benchmarking indicates predictive errors below 10–15% RMSE for growth-related target variables under realistic noise assumptions. Multi-objective optimisation yields Pareto fronts that explicitly quantify the trade-offs between growth rate, protein–lipid allocation, and feed cost, thereby enabling transparent decision support. The results demonstrate that combining formal knowledge representation with AI-driven optimisation improves robustness and interpretability when dealing with heterogeneous and seasonally variable substrates. The fermentation state and substrate class emerged as dominant explanatory factors in the optimisation landscape, highlighting the importance of structured data integration. The proposed framework provides a reproducible and extensible methodology for feed optimisation in BSF farming. By combining ontology-based knowledge modelling with AI optimisation, this study supports the scalable valorisation of heterogeneous organic residues and establishes a foundation for future experimental validation in industrial insect production systems.

What’s good enough? Bioconversion heuristics model based on mass balance and cost-benefit analysis

L. Wein^* and R. Steven

Protenga Pte Ltd, 03-24, 3 Coleman Street, 179804 Singapore, Singapore; ^*leo@protenga.com

Black Soldier Fly insect farming is a frontrunner among circular bio-economy solutions. To deliver on its positive impact potential, insect farms need to be profitable investments and deliver competitively priced products. This can be achieved only, when the bioconversion process, whereby seedling larvae and feed as input turn into mature larvae and frass as outputs, delivers a positive economic value. This study proposes a mass-balance based cost-benefit analysis model, including as-is mass balance and dry-matter mass balance, that enables a simple, practical and comparable assessment for research and industry with harmonised parameters and definitions. The key parameters proposed for the model are feedstock (weight kg, moisture%, unit cost as is $/kg), seedling larvae (weight kg, moisture%, larvae average weight g/unit, count and cost, whereby cost is normalised to $/g neonates at 50 000 larvae/g), mature larvae (weight kg, moisture%, average weight g/count, count and value/price $/kg) and frass (weight kg, moisture% and value/price $/kg). Additional parameters considered are growth area (m2), density (larvae count/m²) and cycle time (days). Key process ratios used are bioconversion efficiency (BCE, also larvae/feed ratio), frass/feed ratio, frass/larvae ratio, larvae-out/larvae-in (LOLI) and bioconversion gross margin (BGM). The BGM is calculated by multiplying weight with unit value of the outputs and subtracting the sumproduct of the inputs; in addition unit values should be normalised to moisture content, for example if frass at 30% moisture has a value of 0.1$/kg and the process yields 40% frass moisture, the unit value is discounted accordingly to 0.86$/kg. The model was explored by analysing a selection of experimental and production data. Findings included anti-proportional relationship between average weight and density balancing a relatively stable total weight of larvae, higher BCE for higher larvae densities, trade-offs between feed weight, larvae density and dry matter losses and dry matter losses typically ranging between 30-60%. Consequently, trade-offs can be quantitatively evaluated, for example higher larvae density resulting in higher larvae yields and feed efficiencies (lower feed costs), but higher seedling costs. More feed resulting in higher larvae yields per growth unit, but also in higher dry matter losses and therefore lower frass value. The model offers assessment of multiple parameters and identification of heuristics ranges to evaluate ‘what’s good enough’ for profitable bioconversion and demonstrates its value as a simple, comprehensive and standardised framework enabling bioconversion process evaluation and comparison for both research and industrial contexts.

Thermal effects on growth and development of Duolandrevus (Eulandrevus) ivani

S.M. Ji^*

National Institute of Agricultural Sciences, 166, Nongsaengmyeong-ro, Deokjin-gu, Jeonju-si, Jeonbuk-do 55365, South Korea; ^*jeee3ang@korea.kr

In 2024, a densovirus outbreak caused mass mortality in the two-spotted field cricket (Gryllus bimaculatus), resulting in substantial disruption to the supply chain of cricket farms in South Korea. This event highlighted the need to explore additional indigenous genetic resources that could potentially contribute to the stability of insect-based feed production systems. In this context, the present study examines the life-history traits of Duolandrevus (Eulandrevus) ivani, a large cricket species native to Jeju Island, under different thermal conditions, with the aim of assessing its potential applicability for industrial rearing. Growth, adult emergence, and survival of D. ivani were monitored at constant temperatures of 20, 25 and 30 °C. Developmental performance was quantitatively evaluated using area under the curve (AUC) analysis derived from weekly body weight measurements. At 20 °C, development was markedly delayed, resulting in a low AUC value (2.92) and a relatively small final body weight (approximately 0.30 g). In contrast, individuals reared at 25 °C and 30 °C exhibited substantially higher AUC values (10.98 and 12.32, respectively), and reached comparable maximum body weights of around 0.63 g. Adult emergence patterns varied with temperature. At 30 °C, emergence occurred earlier and within a narrower time window, whereas at 25 °C, emergence was distributed over a longer period. Adult survival appeared to be relatively consistent across temperature treatments, with mean survival times of approximately 26–27 weeks. In addition, body weight differed significantly between sexes, with females tending to be heavier than males. Overall, these results suggest that D. ivani can maintain stable growth and survival within the temperature range of 25–30 °C. The more synchronized emergence observed at higher temperatures may offer practical advantages for harvest management in mass-rearing systems. Although further studies on nutrition, density, and large-scale rearing performance are required, the present findings indicate that D. ivani could be considered a promising candidate as a supplementary or alternative feeder insect, and provide baseline information for the development of future rearing protocols.

Industry shakeout: risk signal or normal maturation phase?

B. Holtermans^*

Insect Engineers, Nijverheidsstraat 2a, 5961 PJ Horst, The Netherlands; ^*bob@insectengineers.com

The insect industry has recently experienced increasing reports of financial distress and company failures following a period of strong capital inflow. While often interpreted as negative signals for sector viability, similar patterns have historically occurred in emerging manufacturing industries during early industrialization phases. In this work, we analysed scale-up performance, commissioning timelines, and operational stability indicators across multiple commercial insect production facilities. The objective was to evaluate whether recent financial failures reflect sector-specific weaknesses or typical industry lifecycle dynamics observed in emerging manufacturing sectors. Comparable developments were identified in other emerging technology sectors. The cultivated meat sector demonstrates that even well-funded companies may fail when technological scale-up, regulatory development, and capital market confidence do not progress at similar rates. Early solar photovoltaic manufacturing similarly experienced failures of highly funded companies despite long-term sector growth and eventual industrial maturity. These patterns are consistent with industry lifecycle models describing early experimentation phases followed by consolidation and emergence of dominant production designs. Our analysis indicates that commercial insect farming is entering a comparable phase typical for capital-intensive industries transitioning from pilot to industrial scale. Several structural drivers were identified. Capital was raised during a period characterised by low interest rates and strong investor appetite for sustainability-driven technologies, incentivizing rapid scale-up. In multiple cases, large-scale production facilities were constructed while biological and process technologies were still under development. Analysis of implemented facilities showed that scaling feedstock handling capacity from pilot levels (≈10 t/day) to industrial levels (>300–500 t/day) introduces non-linear complexity in logistics, process integration, climate control, and biological performance. Results indicate that early operational output typically reaches approx. 40–70% of designed capacity during the first operational year, while time to stable production varies significantly between sites. Furthermore, strong IP-driven strategies resulted in parallel development of proprietary technologies, limiting cross-sector knowledge sharing during early industry formation. Early market positioning strongly focused on alternative protein markets, while waste management and nutrient recovery applications were comparatively underexplored. Overall, the results demonstrate that current developments are consistent with established industry lifecycle models describing early experimentation phases followed by industry shakeout and consolidation.

Drying technologies for black soldier fly larvae: process efficiency, energy demand, and product quality

M. Lehmad¹, B. Nomeir², N. Hidra¹, P. Lhomme³, N. Abdenouri¹ and Y. El Hachimi^1*

¹Cadi Ayyad University, B.P 549 Av. Abdelkarim Elkhattabi, 40000 Guéliz, Morocco; ²Moroccan Foundation for Advanced Science Innovation and Research (MAScIR), Rue Mohamed Al Jazouli, 10000 Rabat, Morocco; ³Mons University, 7000 Mons, Belgium; ^*yelhachimi@gmail.com

Black soldier fly larvae (Hermetia illucens) (BSFL) are increasingly recognized as a sustainable source of protein and micronutrients for food and feed. Owing to their high initial moisture content, drying is a critical post-harvest step affecting product quality, energy use, and industrial feasibility. This study compares five drying technologies — sun drying (SD), hybrid solar–electric drying (HSED), vacuum freeze drying (VFD), greenhouse drying (GD) and microwave drying (MD) — to identify efficient and sustainable processing options for BSFL. Fresh larvae were dried to a final moisture content below 8% using each method. Drying performance was assessed through drying time, yield, specific electrical energy consumption (SEEC), and average drying rate (ADR). Dried larvae were further characterised for proximate composition, physicochemical and techno-functional properties, microstructure, and thermal stability. Drying time ranged from 120 h for SD to 0.33 h for MD, with intermediate durations of 6 h for HSED, 5 h for GD, and 72 h for VFD. MD achieved the highest ADR (5.16 kg water/kg dm ⋅ h), followed by GD (0.34 kg water/kg dm ⋅ h) and HSED (0.29 kg water/kg dm ⋅ h). VFD exhibited the lowest ADR (0.02 kg water/kg dm ⋅ h) but resulted in the lowest residual moisture content (3.61%). Energy demand varied substantially, with VFD requiring 206.86 kWh/kg, compared to 3.24 kWh/kg for MD and 1.62 kWh/kg for HSED, while SD and GD operated without electrical energy input. Drying method significantly influenced larval composition and functionality ($P\mathrm{<}0.05$). Protein content ranged from 33.36% (GD) to 44.63% (VFD), and crude fat from 24.71% to 34.80%. HSED yielded a balanced nutritional profile (42.03% protein, 32.05% fat) with a high energy value (497.57 kcal/100 g). Functional properties were highest in VFD and HSED, whereas GD showed the lowest performance. Structural analyses revealed marked tissue damage in GD and MD samples, while VFD best preserved microstructure. Overall, clear trade-offs exist between drying efficiency, energy demand, and product quality. Although VFD ensures superior quality preservation, its high energy requirement limits scalability. HSED represents a favourable compromise, combining low energy consumption, short drying time, and acceptable product quality, making it a promising option for sustainable large-scale BSFL processing.

Design of an industrial modular Tenebrio molitor farm for the food industry

A.L. Nava Sedano^1*, D. Ocampo Nava², C. Osorio Cano¹, L.J. Muñiz Rascado¹ and A. Olarte Paredes¹

¹TECNM/ Iinstituto Tecnológico de Zacatepec, Calzada Tecnológico No. 27, Zacatepec, Morelos 62780, Mexico; ²Universidad Gestalt de Diseño, Av. 1o. de Mayo 113, Xalapa-Enrı́quez, Veracruz 91020, Mexico; ^*lilly.nava@gmail.com

Eleven years after the 2015 approval of the UN Sustainable Development Goal “Zero Hunger” and the 2021 European Union (EU) legislation on insect-based food products, the commercial insect farming sector faces challenges related to innovation in systematized breeding equipment. These challenges focus on maintaining food safety standards, 24/7 operability, and ergonomic conditions for tools and machinery. This article, describe the process for designing a modular Tenebrio molitor breeding facility, addressing the aforementioned challenges. Based on opinions, the needs and frustrations of insect producers were identified. Additionally, through an operational breakdown, postures involving high physical effort and repetitive movements were analysed. Similarly, the optimal growth parameters for Tenebrio molitor were considered. With this information, a Value Proposition Canvas was integrated to define the initial design characteristics of the breeding facility. The design process was approached using the Design Thinking methodology, covering the first step, Empathize, with the conclusions from the Value Proposition Canvas. The Define problem step was complemented by a technological screening of the Mexican Institute of Industrial Property and WIPO’s databases to establish design constraints and avoid including intellectually protected elements. The Ideate step was materialized through proposal sketches, selecting one for the final design in CAD software. The Prototype and Testing stages are in process and the application of the first three stages of Design Thinking led to the conceptualization of a digital prototype with an innovative design. The finalized design of the modular industrial breeding facility is characterised by:

1. (1) Dimensions: 50 × 40 × 160 cm.
2. (2) Stackable design for optimised vertical space utilisation.
3. (3) Mechanical system for feeding, growth and sifting featuring 8 trays.
4. (4) Compatible with remote visual, temperature and humidity monitoring systems.

Its primary innovation is the tray sifting system, which facilitates cleaning, separation, and feeding during larval growth, resulting in significantly reduced human effort. Discussion. The Prototyping phase is being developed with the certainty of integrating a design that meets the sector’s requirements through an innovative, scalable, and industrial-grade proposal. Identified areas for improvement will be evaluated during the Testing phase to determine their inclusion or dismissal.

Friend, foe or feint? Metagenome-guided insights into Scrofimicrobium in black soldier fly larvae

F. IJdema^*, D. Lachi, N. Sibinga and J. De Smet

KU Leuven, Research Group for Insect Production and Processing, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*freek.ijdema@kuleuven.be

Larvae of the black soldier fly (Hermetia illucens, BSFL) are viewed as a sustainable alternative protein source due to their capacity to convert low value waste streams, such as food waste, manure, and agricultural side streams, into high value insect biomass. This broad substrate flexibility is thought to be partially mediated by the BSFL gut microbiome, which comprises diverse bacteria, fungi, and viruses that may contribute metabolic and protective functions. Previous studies have shown that the composition of the BSFL gut microbiome is shaped by environmental factors, including diet and rearing conditions, as well as host related factors, resulting in considerable variability in the insect’s gut bacterial composition between studies. Nevertheless, several bacterial taxa have been repeatedly detected across independent investigations, suggesting that they may be intrinsically associated with BSFL and play a role in supporting its niche as a generalist detritivore. Our recent metagenomic analyses have identified a putative novel strain of Scrofimicrobium, a genus often described in association with BSFL. In the present study, we investigate the potential functional role of Scrofimicrobium_sp_concoct.67, a metagenome assembled genome (MAG) recovered from whole genome sequencing of BSFL reared on two distinct diets. As an initial step, over 600 KEGG annotated genes were examined to identify metabolic pathways that could facilitate the conversion of nutrients commonly present in BSFL substrates, as well as functions that may enhance microbial survival in the gut environment. In addition, several genes associated with antimicrobial resistance were detected, indicating a possible protective role for the bacterium within the gut microbial community. To assess whether the predicted genomic functions are biologically relevant and actively expressed, ongoing work aims to isolate the Scrofimicrobium strain using Actinomycetota selective agar media. Candidate isolates are being identified using two sets of primers specifically designed from the Scrofimicrobium_sp_concoct.67 MAG. Successful isolation would enable downstream phenotypic testing, including the evaluation of metabolic capabilities and antimicrobial resistance profiles using high throughput, plate based substrate utilisation assays. Collectively, this work illustrates how metagenome mining can guide the identification of potentially beneficial gut associated bacteria and outlines a workflow for linking genomic predictions to laboratory validation. In the longer term, such microorganisms may be explored as probiotic candidates to support BSFL performance on specific waste substrates.

Limits of enzymatic and chemical chitin degradation in Tenebrio molitor larvae

P. Sudwischer^1,2*, M. Rickers¹, J. Heidhues³, D. Sindermann³, S. Wildförster⁴, F. Tometten⁵, M. Kölln⁶, A. Grümpel-Schlüter⁶, J. Kluess⁶, D. Dänicke⁶, W. Sitzmann^1,5 and M. Hellwig²

¹Internationale Forschungsgemeinschaft Futtermitteltechnik e.V. (IFF), Frickenmuehle 1A, 38110 Braunschweig, Germany; ²Technische Universität Dresden, Chair of Special Food Chemistry, Bergstraße 66, 01062 Dresden, Germany; ³GEA Westfalia Separator Group GmbH, Werner-Habig-Straße 1, 59302 Oelde, Germany; ⁴DEVEX Verfahrenstechnik GmbH, Splieterstraße 70, 48231 Warendorf, Germany; ⁵Amandus Kahl GmbH & Co. KG, Dieselstraße 5-9, 21465 Reinbek, Germany; ⁶Friedrich-Loeffler-Institut, Bundesallee 37, 38116 Braunschweig, Germany; ^*iff@iff-braunschweig.de

The increasing use of insects as a sustainable protein source in livestock nutrition is constrained by the presence of chitin in the exoskeleton, which potentially impairs digestibility and nutrient availability. Efficient strategies to reduce or remove chitin are therefore essential to improve the nutritional value of insect-derived feed ingredients. This study compared different chemical, enzymatic and mechanical approaches for chitin reduction in Tenebrio molitor larvae. Chitin degradation was investigated using 6 approaches: enzymatic hydrolysis with chitinase, enzymatic pre-treatments with lysozyme, ultrasound or hydrogen peroxide, as well as partial and total hydrochloric acid hydrolysis. All treatments included an initial protease pre-treatment to disrupt the protein-chitin matrix. In addition, a mechanical separation process was evaluated to physically remove chitin-rich fractions during protein meal production. Chitin contents were quantified, with a limit of quantification (LOQ) of 1 g/100 g dry matter. The pre-treatments resulted in only minor or negligible chitin reduction. Partial acid hydrolysis achieved approximately 30% chitin reduction, while total acid hydrolysis exceeded 80%. In contrast, mechanical separation proved to be the most effective strategy, yielding an almost complete removal of chitin from the protein meal. Residual chitin levels were only slightly above the LOQ. The mechanically separated chitin fraction was obtained as a distinct side stream. The results confirm the high crystallinity and recalcitrance of insect chitin, which strongly limits enzymatic degradation without prior structural disruption. While acid hydrolysis was effective, its practical application is restricted by safety, environmental, and economic concerns. Mechanical separation emerged as the most efficient and scalable approach, enabling near-complete chitin removal while simultaneously generating a chitin-rich side stream suitable for further valorisation. This strategy offers a promising pathway for improving protein quality in insect-based feed ingredients and supports integrated concepts for insect biomass.

Effect of water-to-larvae ratio during boiling on processing performance and quality of black soldier fly larvae

R. Sorayani Bafqi^1*, Z. Loiotine¹, I. Biasato¹, V. Bongiorno², L. Gasco¹ and S. Bellezza Oddon¹

¹University of Turin, Department of Agricultural, Forest and Food Sciences, Largo Paolo Braccini 2, 10095 Grugliasco, Italy; ²Michigan State University, Department of Animal Science, Michigan State University, Room 1290, 474 S. Shaw Lane, East Lansing, MI 48824, USA; ^*reyhane.sorayanibafqi@unito.it

Boiling is widely recognized as an effective post-harvest processing step in black soldier fly management. Rapid larval deactivation is a key factor influencing final product quality as it immediately stops metabolic and enzymatic activity. By quickly inactivating enzymes and reducing microbial activity, it prevents protein degradation, lipid oxidation, and off-flavour formation. Prompt larval deactivation also helps preserve nutritional value, colour, and of the final product and improves stability and process consistency. Despite extensive research on drying methods, the influence of boiling conditions remains poorly explored, although it may strongly affect heat transfer efficiency and product quality. This study investigated the effect of different water-to-larvae ratios during boiling on the quality of dried larvae, with emphasis on colour attributes. Five treatments were tested (6 replicates/treatment) using constant water volume (0.425 L) and larval loads of 0.3 (T1), 0.6 (T2), 0.9 (T3), 1.0 (T4), and 1.2 (T5) kg/l. The time required for most larvae to be deactivated (MD) and for full deactivation (FD) was assessed by two operators, showing excellent agreement (ICC>0.99). The minimum temperature (MT) reached after larval addition and the temperature at larval removal (RT) were recorded. Boiling was continued until all larvae observed at the surface were fully deactivated. Larvae were then dried at 70 °C for 18h, ground, and analysed for colour (L*, a*, b*). Data were analysed by One-way ANOVA (SPSS 20.0, $p\mathrm{≤}$0.05). The shortest MD and FD durations were observed in T1 and T2, while the longest in T5 ($p\mathrm{<}0.05$). MT was highest in T1 and T2 and decreased with increasing larval biomass ($p\mathrm{<}0.05$) while T3, T4, and T5 showed comparable MT values ($p\mathrm{>}0.05$). RT followed a similar pattern, with higher values in T1 and T2 compared to T3 and T4 ($p\mathrm{<}0.05$), while T5 showed intermediate values. All treatments exhibited short MD times (<40 s), although FD extended to several minutes at higher larval loads. Colour analysis indicated higher L* values in T1 compared with T3 and T4 ($p\mathrm{<}0.05$), while T2 and T5 showed intermediate lightness. Only T1 exhibited lower a* values ($p\mathrm{<}0.05$), whereas b* values were higher in T1 and T5 compared with the remaining treatments. Overall, the larvae-to-water ratio significantly influenced processing temperature, deactivation dynamics, and colour attributes, highlighting the importance of ratio optimisation to preserve product quality.

Optimised bioprocessing of Tenebrio molitor larvae for protein and oil valorisation

P. Navarro^1,2,3, J.C. Ribeiro^1*, M.E. Brassesco³, M. Pintado³, G. Pereira² and L.M. Cunha¹

¹GreenUPorto/Inov4Agro, Faculty of Sciences, University of Porto, DGAOT, Rua da Agrária 747, 4485-646 Porto, Portugal; ²Corial Foods – Sustainable Food Products, Portugal, R. Eng. Adelino Amaro da Costa 35 C, 4740-472 Esposende, Portugal; ³CBQF, Catholic University of Portugal, Rua Diogo de Botelho 1327, 4169-005 Porto, Portugal; ^*joribeiro@fc.up.pt

Edible insect fractionation has the potential to valorise major insect fractions while improving their quality and applicability as food ingredients. Protein concentrates/isolates with high functionality may enhance food quality and consumer acceptance. Nonetheless, developing scalable and efficient processing strategies remains a key challenge for industrial implementation. Membrane filtration, a rarely explored yet scalable and sustainable method, was first reported by our research group to recover alkali-solubilised protein from T. molitor larvae using a 50 kDa cut-off membrane; however, despite yielding highly pure protein fractions (>80%), the approach was limited by modest extraction yields (33%) and protein recovery rates (30%) on a defatted basis. This study aimed to optimise the complete fractionation process (defatting, solubilisation and filtration) with emphasis on process efficiency and scalability. Defatting was evaluated using mechanical pressing (screw press) and Soxhlet extraction with various solvents and their combinations. Pressing resulted in a recovered defatted flour (60.50 ± 0.00 g/100 g) with high residual fat (29.96 g/100 g) and excessive temperatures (>100 °C), which can promote protein degradation, whereas the oil (32.08 g/100 g) showed high impurity levels. Soxhlet extraction achieved effective lipid removal, showing no significant differences among solvents; still, ethanol provided the highest fat extraction efficiency (40.93 ± 1.16 g/100), yielding oils with favourable fatty acid profiles, characterised by higher polyunsaturated and lower saturated fatty acid contents. Protein recovery was subsequently optimised through alkaline solubilisation followed by ultrafiltration (UF). A Box–Behnken design (3 levels) was used to test pH (10–13), temperature (25–50 °C), time (2–4 h), and solute/solvent ratio (1/25–1/50). The pH and solvent ratio were identified as key factors. Optimal conditions were 44.0 °C, pH 12.98, ratio 1/44 and 2 h 55 min. Centrifugation conditions showed no significant effect. UF using 10- and 50-kDa membranes resulted in similar extraction yields and protein recovery rates (>10 kDa: 48%, 43%; >50 kDa: 49%, 40%, respectively). Overall, the optimisation enabled the efficient recovery of both protein and oil fractions from T. molitor, resulting in improved yields and product quality. Ongoing characterisation and semi-industrial-scale-up further support the potential of this approach as a sustainable, scalable strategy.

Grill or chill: preliminary results on how does temperature affect the performance of crickets?

J. Folke^*, M. Kulma and P. Kouřimský

Czech University of Life Sciences Prague, Zoology and fisheries, Kamýcká 129, 16500 Praha 6, Czech Republic; ^*folke@af.czu.cz

Microclimate temperature is among the critical factors influencing the performance of farmed insects. This study examined the effect of temperature on growth, feed conversion ratio (FCR), and nutritional value of two cricket species: the house cricket (Acheta domesticus) and the Jamaican field cricket (Gryllus assimilis). The experiment was conducted in climate chambers under controlled conditions (65% relative humidity and a 12:12 photoperiod) at four temperatures (25, 28, 31 and 34 °C), with chicken feed used as the diet. The trial started with 0.25 g of one-day-old pinheads and was terminated after 40 days. Regardless of temperature, house crickets achieved higher yields with similar FCR as Jamaican field crickets. Temperature significantly influenced most of the monitored parameters. In house cricket, harvested biomass increased with increasing temperature, whereas differences in yields among Jamaican field cricket groups were not significant. FCR and frass production increased with temperature in both species. Significant differences in chemical composition were also observed. In Jamaican field cricket, protein content decreased while lipid content increased with rising temperature. In AD, protein and lipid contents did not differ significantly among groups reared at 25–31 °C; however, crickets reared at 34 °C contained more protein and less fat than those reared at lower temperatures. This study highlights the importance of temperature in cricket farming and emphasizes the need to balance input costs with output gains. The two species exhibited distinct responses to temperature. Overall, the house crickets appears more suitable for insect farming, as higher rearing temperatures increased biomass yield, albeit at the cost of higher feed conversion rates. Further research should focus on a more detailed analysis of nutrient quality, verification at a larger scale, and evaluation using different diets. The study was funded by INTER-COST project titled “A comprehensive strategy for the safe production of nutritious edible insects” nr. LUC25019.

From substrates to safety: larval production and HACCP-based risk analysis of Hermetia illucens meal

K.C. Torres Monroy^*, E.M. Menjura Rojas, H.A. Arevalo Arevalo, S.M. Vásquez Mejı́ and K.B. Barragán Fonseca

Universidad Nacional de Colombia, Facultad de Medicina Veterinaria y de Zootecnia, Carrera 30 # 45-01, 111321 Bogota, Colombia; ^*kctorresm@unal.edu.co

This study examined how alternative feeding substrates, including pig manure, influence the productive performance of black soldier fly (Hermetia illucens) larvae and the associated risks during meal production under a Hazard Analysis and Critical Control Points (HACCP) approach. Larvae were reared on two experimental substrates containing 20% and 25% pig manure and a control diet. Development time, biomass production, dry matter yield, feed conversion efficiency, nitrogen-adjusted and corrected feed conversion efficiencies, bioconversion rate and residue reduction were measured. The diet containing 25% pig manure produced the best overall performance, reaching harvest at 19 days and yielding 1446 ± 53.4 g biomass and 428 ± 25 g dry matter. Larvae from all treatments were processed into meal using a laboratory-developed protocol, informed by the literature and optimised through pilot trials to preserve product quality. Proximate composition was broadly similar among treatments, except for moisture, ash and nitrogen-free extract. Protein content ranged from 40.5 ± 1.79 to 41.0 ± 0.57%, while ether extract ranged from 26.7 ± 0.9 to 32.6 ± 0.4%. Amino acid profiles were stable, whereas fatty acid composition showed high lauric acid levels (32.5 ± 9.69 to 45.0 ± 0.46%). Calcium, phosphorus and iron were prominent in mineral profiles, and functional properties such as water-holding capacity and oil absorption supported technological performance in feed formulations. Microbiological analyses met safety criteria, except for the aerobic mesophilic count, which exceeded values reported for processed BSF larvae in previous studies. This count slightly surpassed the reference hygiene threshold (<1 × 10⁴ CFU g^−1) recommended by the IPIFF for insect-derived feed ingredients. This result highlights the need to reinforce Good Manufacturing Practices (GMP) during handling and processing. A HACCP-based analysis of the production process identified biological, chemical and physical hazards, including microorganisms, contaminants, allergens and particulate matter. Two Critical Control Points were defined: raw material reception to manage chemical risks and drying to control biological risks. Additional hazards were addressed through process controls, with monitoring variables, critical limits and corrective actions established. Taken together, these results show that substrate composition shapes larval performance and that HACCP provides a robust framework for identifying hazards and strengthening process control in H. illucens meal production for animal feed.

Why dry matter is not enough: comprehensive physical property characterisation of black soldier fly larvae substrates

M.L. Schøn^1,2*, M. Gold¹, C. Zurbrügg² and A. Mathys¹

¹ETH Zurich, Sustainable Food Processing Laboratory, Schmelzbergstrasse 9, 8092 Zürich, Switzerland; ²Eawag, Sandec, Ueberlandstrasse 133, 8600 Dübendorf, Switzerland; ^*maja.schon@hest.ethz.ch

Black soldier fly larvae (BSFL) feed and develop in biowaste substrates whose physical properties can be as critical as nutrient content for stable and efficient production. Across diverse biowastes, key properties such as bulk density, water-holding capacity (WHC), and particle size vary widely, influencing aeration, microbial activity, heat generation, and overall bioconversion efficiency. Despite their importance, these parameters remain poorly characterised. To close this knowledge gap, we performed the first comprehensive physical property characterisation study of BSFL substrates to establish reference values, identify correlations, and enable substrate comparison. We analysed 16 commercially relevant BSFL substrates (i.e. mixtures of individual biowaste streams) with differing compositions provided by nine companies. Analyses included bulk density, viscosity, penetration force, WHC, water release by filtration (WRF), free water, and dry matter (DM). Relationships among structural, mechanical, and water-related properties were examined using Pearson correlations and principal component analysis (PCA) in Rstudio. Although all substrates were optimised for efficient bioconversion, their physical properties differed substantially. For instance, bulk density varied over a twofold range (0.5–1.1 g cm^−3), viscosity spanned more than two orders of magnitude (8–2000 Pa ⋅ s at shear rate 1), and penetration force differed by over 300-fold (0.01–3.30 N). Likewise, WHC ranged from 2–5 g water g^−1 DM (a 150% difference), WRF from 0–16%, free water from 0–10% of total water, and DM content from 24–33%. Strong statistically significant internal coupling was observed within groups of similar properties: mechanical properties (viscosity and penetration force) showed a strong positive correlation (r = 0.95), and water-release metrics (WRF and free water) were likewise closely linked (r = 0.85). In contrast, bulk density showed weak relationships with other properties, indicating that substrate structure represents an independent physical characteristic. PCA confirmed that BSFL substrates vary across multiple, partially independent physical dimensions, demonstrating that DM — or any single factor alone — is insufficient to capture the physical complexity of BSFL feeding substrates. This study supports the need to consider physical substrate properties as design and control parameters in BSFL production and, while controlled rearing trials were not feasible due to company restrictions and multidimensional substrate variability, it provides a foundation for future work linking substrate physical properties to bioconversion performance.

Antioxidant activity of edible insects: effect of method of extraction, bioactivity assay and diet

E. Rodrı́guez-González^1,2*, V. Da Cunha-Borges^1,2, D. Martin^1,2 and M.R. Garcı́a-Risco^1,2

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto. de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ^*esther.rodriguez01@uam.es

Edible insects are increasingly studied for their antioxidant activity, which is influenced by species and diet. However, the subsequent production of insect-based ingredients for health, such as antioxidant extracts, will also depend on the extraction method. Therefore, this study compared the antioxidant activity of extracts from Hermetia illucens (HI) and Tenebrio molitor (TM) larvae obtained by two extraction methods typically used to produce bioactive extracts from natural sources, such as ultrasound-assisted (UAE) and pressurized liquid extraction (PLE). For both methods, the effect of diet was also tested by comparing antioxidant extracts obtained from larvae fed conventional diet or the by-product olive pomace. Both species were reared on their conventional diet (wheat bran) or wheat bran including 50 % of olive pomace (OP). Reared larvae were slaughtered, dried, ground, and defatted, and extracted by UAE (with EtOH 70%, ratio sample to solvent 1:20, for 25 min at 40 °C) and PLE (with EtOH 70%, ratio sample to solvent 1:20, 10 min, at 80 and 120 °C). Antioxidant activity was evaluated by the two popular DPPH and ABTS radical scavenging assays. Results showed that extraction method and diet markedly influenced antioxidant activity. It was also evidenced that the antioxidant assay used was relevant. In general, UAE extracts showed higher antioxidant activity than PLE extracts for DPPH (46% vs 44.9%), whereas ABTS results were similar between both extraction methods. TM extracts exhibited greater DPPH inhibition (53.7%), whereas HI extracts showed higher ABTS inhibition (55.9%). OP feeding enhanced DPPH inhibition in both species (up to 30%), while ABTS activity appeared similar between diets. For PLE, higher temperatures (120 °C) increased DPPH inhibition in both species and diets, reaching the highest for TM-OP (44.5%), whereas ABTS inhibition was favoured at 80 °C, mainly for HI-OP (55.9%). In conclusion, antioxidant activity of insect extracts is influenced by species, diet, extraction method, and antioxidant assay used. These findings evidence the potential selective production of antioxidant insect extracts through specific extraction strategies, together with the valorisation of agri-food by-products, to produce ingredients for health-related applications. Acknowledgements: ENTOMOTIVE project, PID2022-136238OB-I00 and grant PREP2022-000828, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU.

The influence of disturbance of Hermetia illucens pupae on egg production

C.L. Coudron^*, J. Claeys, L. De Praetere and D. Deruytter

Inagro vzw, Insect Research Centre, Ieperseweg 87, 8800 Roeselare, Belgium; ^*carl.coudron@inagro.be

Egg production is a key indicator in black soldier fly (Hermetia illucens, BSF) breeding systems, as it determines the supply of progeny and the proportion of larvae that can be allocated to biomass production. Although adult reproductive performance is often optimised through cage design and environmental control, the influence of pre-imaginal handling on subsequent egg yield remains insufficiently understood. Producers may remove frass from (pre)pupae by sieving, an action that consists of high-frequency micro-impacts. This intervention improves the accuracy of estimating the desired number of flies per cage, but it may also impose mechanical disturbance that affects metamorphosis, adult fitness, and ultimately reproductive output. Therefore, 3 trials were performed to evaluate the impact of vibrations (sieving) on egg production. Each trial consisted of 24 reproduction cages (60 × 60 × 90 cm), maintained under uniform conditions (30 °C, 80% RH) with controlled lighting, water access, lures and egg traps. Eggs were collected at fixed two-day intervals until production ceased, and total egg yield (g) per cage was used as the response variable. In the first trial, pupae ( ± 250 g) were subjected to a harsh treatment of daily sieving (1 minute) or no sieving at all. This resulted in a 97% reduction in egg production, which prompted a closer examination of the intensity of the mechanical disturbance. A second trial was therefore designed in which 24 batches of late prepupae were prepared. Each day, 3 batches were randomly selected for a single one-minute, maximum-intensity sieving treatment, ensuring that each batch was disturbed only once and allowing the identification of the most sensitive developmental days prior to adult emergence. The results indicate that sieving when prepupae or young pupae (1–3 days old) are present has no significant negative effect on egg production. Nevertheless, a single one-minute sieving event 4 days into pupation (or older) decreased egg output by 37%. A final trial quantified the relationship between sieving intensity and reproductive performance using an accelerometer to measure the amplitude of both hand-sieving and mechanical-sieving motions. Mechanical sieving within a certain low-amplitude range did not negatively affect egg yield, whereas exceeding this safe threshold led to a linear decrease in egg production with increasing amplitude. No effect on pupae eclosion was observed. In our setup, this threshold corresponded to accelerometer readings averaging 100 G. This leads us to conclude that BSF pupae are more fragile than expected. Handling pupae is therefore best avoided and, when necessary, should be performed as early as possible and with great care to prevent reductions in egg production.

Slaughtering methods outcomes on the oxidative stability, antioxidant response, safety and functionality of T. molitor

P. Navarro^1,2,3*, J.C. Ribeiro¹, C. Brollo¹, T.R. Fernandes¹, S.M.P. Carvalho¹, M. Pintado³, G. Pereira² and L.M. Cunha¹

¹GreenUPorto — University of Porto, Faculty of Sciences, Rua da Agrária, 747, 4485-646 Vila do Conde, Portugal; ²Corial Foods — SFP, Rua Engenheiro Adelino Amaro da Costa, 35 Fracção C, 4740-472 Esposende, Portugal; ³Catholic University of Portugal, Centre for Biotechnology and Fine Chemistry, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal; ^*up202310452@up.pt

A strong focus has been placed on safety, pre-processing, functionality, and the fractionation of insects as food ingredients to enhance product quality and consumer acceptance. However, the impact of insect-slaughtering processes on various properties remains poorly understood. To address this, reared T. molitor larvae, collected at 8-9 weeks old, were subjected to 3 slaughter methods, each in 2 different conditions: Freezing at –24 °C for 3 h (FZ-1) and 24 h (FZ-2); Blast-Chilling at –45 °C, for 3 h (BC-1) and 1.5 h (BC-2); and Blanching at 100 °C for 5 min without (BL-1) and with a 0 °C (5 min) pre-chilling step (BL-2). The techno-functional properties, oil oxidative stability, antioxidant enzyme activity, and microbiological safety were evaluated. Foaming (8–12%) and emulsifying (2–4%) capacities and oil (8.5–9.9%) and water (7.6-12.5%) binding capacities showed no significant differences among treatments due to high lipid and chitin content in the whole insect flour. In contrast, the processing methods exerted a pronounced impact on colour. The BC-1 yields the highest lightness value (L* = 35.20 ± 0.76), whereas BC-2 results in the most severe browning index (47.78 ± 4.89). Regarding microbial load, evaluated upon sample processing and after 6 months of storage in sealed bags (c.a. 25 °C), slaughtering methods had a significant effect on TVC and Enterobacteriaceae, with lower loads for BC methods. However, all samples stayed within EU safety limits (EFSA). FZ-2 presented the lowest lipid oxidation markers in oil samples (3.76 ± 0.71 mg KOH/g, 0.24 ± 0.15 mg MDA/kg, and 1.00 ± 0.00 meq O₂/kg oil) and minimal antioxidant enzyme activity (SOD 0.01 ± 0.01 U/mg, CAT 2.73 ± 0.01 U/mg and GPx 0.97 ± 0.14 U/mg), indicating preserved oxidative stability. By contrast, BL-2 induced pronounced lipid oxidation, reflected by elevated peroxide value (71.21 ± 1.21 meq O₂/kg oil) and TBARS (2.57 ± 0.20 mg MDA/kg), accompanied by increased CAT (15.21 ± 0.28 U/mg) and GPx (3.59 ± 0.00 U/mg) enzyme activities. BC samples showed intermediate levels of oxidative damage, suggesting that rapid cooling may promote oxidative stress by disrupting cells. Overall, these results show that slaughtering strategies modulate oxidative stress in T. molitor larvae, thereby influencing lipid stability and antioxidant enzyme activity, without affecting the techno-functional properties of the flours.

Induced growth suspension for enhanced logistics in black soldier fly production: industrial validation of the StayPrime

A. Gligorescu^*

EntoPrime, Universitetsbyen 14, 8000 Aarhus, Denmark; ^*angl@entoprime.com

The insect protein is affected by several bottlenecks, such as high production costs, high complexity and natural fluctuations in egg output. These affect the entire value chain, from full-life cycle producers to specialized breeders, to small farms and R&D labs. This study evaluates StayPrime, a patented technology that reduces production costs and logistical complexity by inducing suspended animation to extend the shelf life, storage, and transportability of black soldier fly (BSF) neonates and juveniles. StayPrime was first evaluated through trying out sample tests at different producers, successfully suspending the young larvae for 7–14 days. These tests were followed by a one-year validation phase, during which suspended BSF juveniles were shipped and stored across multiple insect farms. This external validation ensures that the technology is robust and transferable across different operational environments. In the validation phase, batches of juveniles were packaged with StayPrime and shipped to regional rearing partners for 7-day storage before being reared in commercial settings. Performance was benchmarked against non-arrested controls to evaluate survival, weight and Feed Conversion Ratios (FCR) in production. Findings confirm that StayPrime successfully arrests development with minimal weight flux and high post-suspension survival (>80%). During the trying out tests, suspended neonates exhibited a significant compensatory growth effect, reducing overall production time from 17 to 14 days and increasing final larval weight from 130 mg to 151 mg compared to the control. The one-year validation confirmed that StayPrime-treated juveniles consistently averaged high harvest survival (>90%), with mean weights of 128–136 mg and mean FCRs of 1.35–1.39 (calculated as kg dry matter feed per kg fresh larval weight), matching standard production benchmarks throughout the period. Economically, the technology reduced shipping costs by more than 50% by replacing multiple express deliveries to different farms with a single weekly shipment under standard condition. StayPrime provides a robust applicable solution for stabilizing the insect supply chain and lowering the barrier of production costs. By creating “buffer capacity” for neonates or juveniles, the technology offers immediate value to researchers, pilot-scale innovators, and full-scale industrial producers alike. To date, StayPrime has facilitated the production of over 100 tonnes of BSF biomass across diverse partner facilities. A standardized physical product is currently under development and is expected to be available on the market soon. This commercial solution offers a plug-and-play tool to streamline production schedules, reduce overhead, and enhance efficiency across the global insect farming industry.

Hatching methods black soldier fly eggs: implications for industrial rearing efficiency

M.B. Ala Eddine^1,2*, L.I. MacAvei^2,3, T.B. Barber², G. Benassi² and S. Dabbou¹

¹University of Trento, Center Agriculture Food Environment (C3A), Via E. Mach, 1, 38098 San Michele All’Adige (TN), Italy; ²Kinsect S.r.l. SB, Via Mazzacurati, 7/D, 42124 Reggio Emilia, Italy; ³University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania; ^*mohamad.alaeddine@unitn.it

The black soldier fly (BSF) is a key species in circular economy models, where early developmental stages strongly influence production efficiency. Although most rearing protocols rely on moist substrates for egg hatching, BSF eggs can hatch without nutritive support. This study compared two hatching methods under controlled conditions to optimise egg-to-larvae conversion at industrial scale. Eggs (<24 h old) from an in-house colony were collected using standardized 3D-printed oviposition devices (Kinsect S.r.l), weighed, and incubated in plastic containers (20 × 30 cm) using either “wet hatching” (WH; Gainesville diet, 70% water) or “dry hatching” (DH; non-nutritive support). Experiments were conducted at 27 °C and 70% relative humidity with ten replicates per treatment. In WH, containers with fresh wet substrate beneath the oviposition platforms were replaced daily from first hatch to day 5, and larvae dropping into the substrate were counted daily. In DH, neonates dropping onto the container surface were collected daily, weighed, and transferred to WH substrate to estimate larval counts. Day-specific counts reflected larval recovery integrating hatching timing with early survival and handling effects rather than representing pure hatching rates. Larval output was expressed per gram of eggs, and survival was calculated using a literature-based egg weight (0.028 mg). Total larval output, survival and day-specific counts were compared between treatments using independent samples t-tests. Total larval output and survival did not differ significantly (WH: 20 075 ± 4531 larvae (g eggs)^−1, 56 ± 13%; DH: 18 053 ± 4301 larvae (g eggs)^−1, 51 ± 11%). Temporal patterns differed: WH showed earlier larval recovery on day 3 (7783 ± 3289 larvae), whereas DH had no larvae. By day 4, DH surpassed WH in larval recovery (16 844 ± 4356 vs. 11 920 ± 5710 larvae), and on day 5, DH still yielded higher counts (1208 ± 583 vs. 372 ± 316 larvae), with no larvae observed thereafter. Although cumulative output was similar, dry hatching concentrated larval emergence, reduced contamination risk, and allowed flexible handling. These results suggest that dry hatching can be implemented in industrial BSF production to synchronize larval availability and improve operational efficiency, while minimizing challenges related to early larval mortality, uneven distribution, and substrate contamination.

Effects of dietary energy and protein levels on growth performance and chemical composition of Tenebrio molitor larvae

M. Vukadinović^*, M. Polovinski Horvatović, M. Petrović, M. Ivković, B. Iličić, K. Nikolić, D. Beuković and S. Krstović

Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia; ^*marko.vukadinovic@stocarstvo.edu.rs

To optimise protein production from insect larvae, a comprehensive understanding of their nutritional requirements, production performance, and the chemical composition of the produced biomass is required. In this study, nine experimental diets based on wheat bran were formulated, with corn, soybean meal, and sugar beet pulp incorporated to modulate dietary energy and protein levels. Three proteins and three energy levels were combined. Each of nine treatments had 4 replicates, and each replicate contained 2 g of Tenebrio molitor larvae. The results showed that larval growth performance was significantly affected by protein level in diets, whereas energy level had no significant effect on weight gain expressed as grams per day per tray. Weight gain ranged from 0.57 g/day per tray for larvae fed low-energy, low-protein diets to 0.71 g/day per tray for those receiving high-protein, medium-energy diets. FCR varied between 1.83 and 2.34 and was significantly influenced by both energy and protein levels in the diet. The protein effect on FCR was significant in four of the five weeks of the duration of the trial and in the whole period. Lipid content ranged from 38.5% in larvae fed low-protein diets to 26.9% in those fed high-protein diets. These results indicate that dietary protein level is a key driver of growth performance, while both energy and protein levels strongly influence feed efficiency and body composition. From the standpoint of feed cost per unit of gain, medium levels performed best. Low protein resulted in reduced gains, while high protein did not lead to sufficient improvement compared to medium levels to justify the increased cost. Gain was only 3% higher, with price 7% higher for the high protein feed compared to the medium protein feed. Feed cost per unit of protein gain was again affected by feed protein level, rather than energy level. The difference between medium and high protein feeds was moderate, while the low protein group performed worse. Despite better protein conversion when low protein feed is used, the cheapest protein production is achieved with higher protein levels. The findings highlight the importance of diet formulation as a practical tool for steering both production efficiency and the nutritional quality of insect biomass intended for use as a sustainable protein source. To obtain sustainable insect farming, this emerging industry will have to focus on the economic parameters of production. This research shows that finding an economically optimal solution might be a challenging task involving integrating multiple important parameters.

DIC technology on the BSF larvae preservation: microbiological decontamination, quality characteristics and COVs profile

C. Jablaoui^1*, J. Dhaouafi¹, V. Lefrançois², J. Hauchecorne¹, H. Romdhana¹ and S. Mezdour¹

¹UMR Sayfood — Food and Bioproduct Engineering Research Unit, Paris-Saclay University, AgroParisTech, 91120 Palaiseau, France; ²ABCAR DIC PROCESS, 11 Rue du Four Saint-Jacques, 60200 Compiègne, France; ^*cherif.jablaoui@agroparistech.fr

The global population is steadily increasing, resulting in a growing demand for protein for both human and animal consumption. Edible insects represent a promising and environmentally sustainable alternative for future food systems. To make insects suitable for food or feed formulations, various processing techniques must be employed while ensuring the safety, nutritional value, and sensory quality of the final product. These techniques include conventional methods such as aqueous processing, salt treatments, solvent, detergent, alkali, and thermo-mechanical processing, as well as innovative techniques considered as green extraction or intensification methods, including enzyme, ultrasound, High Pressure (HP), pulsed electric field, and instant controlled pressure drop (DIC). Among this emerging process, DIC emerges as a relevant technology for insect processing, as it allows simultaneous microbial decontamination and modification of the physical structure of insect matrices. This study aimed to assess the effect of DIC treatment on the microbial decontamination of black soldier fly larvae, while also evaluating its role in preserving product quality and improving the sensory attributes of the resulting larvae flour. Using HS-SPME/GC–MS quantification, the volatile profile of raw dried larvae was quantitatively characterised. A total of 44 major volatile organic compounds (VOCs) were identified, including 5 acids, 6 alcohols, 7 aldehydes, 8 alkanes, 7 esters, 3 pyrazines, 5 ketones and 3 other compounds. Compared with raw BSF larvae, DIC-treated larvae flour exhibited a significant reduction in the overall volatile compound content. A short DIC treatment of 55 sec at 0.6 MPa resulted in reductions of 87.52, 64.62, 34.18 and 74.57% in the concentrations of 1,4-dimethylcyclooctane, hexanal, dodecanoic acid and acetic acid, respectively. In addition to preserving insect biomass quality (Protein and Oil), the effect of DIC processing can be attributed to the inactivation of oxidative enzymes in the larvae flour. In the other hand, some conditions of this treatment result in a markedly reduced microbial load, with low viable counts compared to untreated larvae. The application of rapid (55 s) DIC treatment at 0.6 MPa allowed the microbial reduction of 2.69 ± 0.09 log cfu/g “total aerobic count”, highlighting the effectiveness of DIC as green process. Moreover, DIC treatment was proved to be much more effective than conventional thermal processes, including water blanching, conventional steam treatment, and HP processing.

Hatching methods of black soldier fly eggs: implications for industrial rearing efficiency

M.B. Ala Eddine^1,2*, L.I. MacAvei^2,3, T.B. Barber², G. Benassi² and S. Dabbou¹

¹University of Trento, Center Agriculture Food Environment (C3A), Via E. Mach 1, 38098 San Michele All’Adige (TN), Italy; ²Kinsect S.r.l. SB, Via Mazzacurati 7/D, 42124 Reggio Emilia, Italy; ³University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania; ^*mohamad.alaeddine@unitn.it

The black soldier fly (BSF) is a key species in circular economy models, where early developmental stages strongly influence production efficiency. Although most rearing protocols rely on moist substrates for egg hatching, BSF eggs can hatch without nutritive support. This study compared two hatching methods under controlled conditions to optimise egg-to-larvae conversion at industrial scale. Eggs (<24 h old) from an in-house colony were collected using standardized 3D-printed oviposition devices (Kinsect S.r.l), weighed, and incubated in plastic containers (20 × 30 cm) using either “wet hatching” (WH; Gainesville diet, 70% water) or “dry hatching” (DH; non-nutritive support). Experiments were conducted at 27 °C and 70% relative humidity with ten replicates per treatment. In WH, containers with fresh wet substrate beneath the oviposition platforms were replaced daily from first hatch to day 5, and larvae dropping into the substrate were counted daily. In DH, neonates dropping onto the container surface were collected daily, weighed, and transferred to WH substrate to estimate larval counts. Day-specific counts reflected larval recovery integrating hatching timing with early survival and handling effects rather than representing pure hatching rates. Larval output was expressed per gram of eggs, and survival was calculated using a literature-based egg weight (0.028 mg). Total larval output, survival and day-specific counts were compared between treatments using independent samples t-tests Total larval output and survival did not differ significantly (WH: 20 075 ± 4 531 larvae (g eggs)^−1, 56 ± 13%; DH: 18 053 ± 4301 larvae (g eggs)^−1, 51 ± 11%). Temporal patterns differed: WH showed earlier larval recovery on day 3 (7783 ± 3289 larvae), whereas DH had no larvae. By day 4, DH surpassed WH in larval recovery (16 844 ± 4356 vs. 11 920 ± 5710 larvae), and on day 5, DH still yielded higher counts (1208 ± 583 vs. 372 ± 316 larvae), with no larvae observed thereafter. Although cumulative output was similar, dry hatching concentrated larval emergence, reduced contamination risk, and allowed flexible handling. These results suggest that dry hatching can be implemented in industrial BSF production to synchronize larval availability and improve operational efficiency, while minimising challenges related to early larval mortality, uneven distribution, and substrate contamination.

Effect of hybrid solar–electric drying temperature on the quality and functional properties of black soldier fly larvae

M. Lehmad¹, B. Nomeir², P. Lhomme^3*, N. Hidra⁴, Y. El Hachimi¹ and N. Abdenouri⁴

¹Laboratory of Bioresources and Food Safety, Cadi Ayyad University, Avenue Abdelkrim Khattabi, 4000 Marrakech, Morocco; ²Moroccan Foundation for Advanced Science Innovation and Research, rue Mohamed Al Jazouli, 10000 Rabat, Morocco; ³Laboratory of Zoology, Mons University, 20 Place du Parc, 7000 Mons, Belgium; ⁴Laboratory of Control and Computing for Intelligent Systems and Green Energy, Cadi Ayyad University, Avenue Abdelkarim Elkhattabi, 40000 Marrakech, Morocco; ^*patrick.lhomme@umons.ac.be

Black soldier fly (Hermetia illucens) larvae (BSFL) represent a promising sustainable nutrient source for food and feed. Drying is essential to ensure microbial safety and shelf stability, but conventional technologies are often energy-intensive and poorly suited to small-scale or resource-limited systems. Hybrid solar–electric drying (HSED) offers a low-energy alternative, particularly in regions with high solar availability, by reducing reliance on electricity or fuel. However, its effects on BSFL quality remain poorly documented. Whole larvae were killed by freezing (−20 °C for 24 h) and dried using HSED at 40, 50, 60 and 70 °C until constant weight was reached, corresponding to drying times of approx. 25, 19, 8 and 3 h, respectively. Dried larvae were analysed for proximate composition, mineral content, pH, colour, bulk density, techno-functional properties (water and oil binding, emulsifying and foaming capacities), microstructure, thermal stability, and molecular structure. Drying temperature significantly influenced larval quality. Protein content decreased from 45.8% at 40 °C to 37.7% at 70 °C, while crude fat declined from 31.8% to 24.3%. By contrast, carbohydrate content increased from 7.3 to 21.2%, and ash from 7.1 to 9.5%. Dried larvae showed high mineral levels, notably calcium (3208–3238 mg/100 g), iron (52–53 mg/100 g), and zinc (20–27 mg/100 g). Higher temperatures resulted in darker colouration (ΔE up to 12.2), increased bulk density (0.5–0.7 g/ml), and reduced techno-functional properties, including water-binding capacity (88.2 to 74.3%) and oil-binding capacity (98.6 to 75.4%). Scanning electron microscopy revealed greater tissue damage and porosity at higher temperatures, while thermal analysis indicated high thermal stability across treatments. Overall, HSED can produce nutritionally valuable BSFL ingredients. Lower temperatures (40–50 °C) better preserve protein and functional properties, whereas higher temperatures (60–70 °C) may provide practical drying efficiency depending on the intended application.

Identification of process phases in black soldier fly larvae bioconversion of food waste

A. Fuhrmann^1,2*, M. Gold^1,2, M. Bours^2,3, E. Turrisi^2,3, A. Spaccasassi⁴, C. Dawid^4,5, T. Furuhashi⁶, L.E. Yu⁶, I. Haberkorn², N. Puniamoorthy⁷ and A. Mathys^1,2

¹ETH Zurich, Sustainable Food Processing Laboratory, Schmelzbergstrasse 9, Zurich, Switzerland; ²Singapore-ETH Centre, 1 Create Way, Singapore, Singapore; ³Wageningen University and Research, Droevendaalsesteeg 4, Wageningen, The Netherlands; ⁴TUMCREATE, 1 Create Way, Singapore, Singapore; ⁵Technical University of Munich, TUM School of Life Science, Lise-Meitner-Strasse 34, Freising, Germany; ⁶National University of Singapore, Dept. of Civil and Environmental Engineering, Engineering Drive 2, Singapore, Singapore; ⁷National University of Singapore, Department of Biological Science, 14 Science Drive 4, Singapore, Singapore; ^*adrianfu@ethz.ch

Industrial black soldier fly larvae (BSFL) bioconversion with food waste can be hampered by challenges such as extensive heat production, reduced larval growth and variable gaseous emissions. These stem from the complex biology of the system, lacking an established succession framework. This study performed a systematic multi-omic bioconversion description via delineating process phases through the integration of macro-variables (larval mass, temperature, moisture, pH), microbial actors (fungi, bacteria) and metabolites. For industrial relevance, feeding trials were conducted in large-scale crates (40 × 60 cm) with 3 distinct food waste batches under high and low temperature regimes, with and without larvae. Throughout bioconversion, macro-variables, microbes (amplicon sequencing), metabolites (LC-MS), and VOCs (GC-MS) were tracked. We identified four consecutive core process phases via hierarchical clustering of microbes, metabolites and macro-variables. Phase characterisation employed linear mixed models, alongside ecological-role annotations of microbes. Strikingly, in the first two phases, bacteria and metabolites did not differentiate based on larval presence indicating that early metabolic shifts were driven primarily by microbes. While fermenters, constituted ≥60% relative abundance throughout the first three phases they dropped ≤40% within phase 4, alongside reductions in fermentation indicators (e.g. Clostridium, succinate). This drop suggested an anaerobic–aerobic shift in the substrate related to reduced moisture (−6%) and higher temperatures (+8 °C). Additionally, significant increases in antimicrobial metabolites (e.g. macrolactams, aminoglycosides) indicated intense microbial competition. Finally, PLS-DA based phase prediction via VOC patterns showed high accuracy (>80%) for phases 1,3 and 4, demonstrating the potential for real-time phase monitoring via exhaust gases. This research emphasizes the heavy influence of metabolic shifts in the substrate related to thermal peaks and emissions and suggests a phase-based framework for systematic optimisation of the process.

Microbiological food safety hazards related to Bacillus cereus group in black soldier fly (Hermetia illucens) production

D. Vandeweyer^*, J. Maeriën, J. Vermeulen, D. Lachi and J. De Smet

KU Leuven, Research Group for Insect Production and Processing, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*dries.vandeweyer@kuleuven.be

The production of black soldier fly larvae (BSFL, Hermetia illucens) commonly relies on agrifood by-products and organic waste streams, enabling their valorisation into high-value biomass for food and feed applications. However, these substrates may harbour microbiological hazards like foodborne pathogens. Among these, the Bacillus cereus group represents a major concern in insect production systems due to its endospore-forming capacity and relevance to food safety. This study evaluated the occurrence and dynamics of B. cereus in BSFL production by addressing two key aspects. First, the prevalence of B. cereus was assessed in an industrial organic waste stream (swill; i.e. supermarket food leftovers) obtained from a local waste processor and sampled at multiple time points. The effects of substrate heat treatment (60 °C, 30 min) and 7-day storage, either refrigerated or at room temperature, on B. cereus persistence were investigated as well. Second, pathogen survival and transmission during BSFL production on swill were assessed via challenge tests. BSFL were reared on artificial swill (a controlled substrate mimicking industrial swill, consisting of fruits, vegetables and dairy) inoculated with B. cereus and both vegetative cell and endospore counts were monitored. Results indicate that B. cereus can frequently be detected in industrial swill, with counts ranging from 6.3 × 10³ to 2.5 × 10⁵ cfu/g, occasionally reaching levels of food safety concern. Heat treatment reduced B. cereus counts by approx. 1.5 log cycles, leaving predominantly endospores at levels of up to 5.0 × 10³ cfu/g. During storage, only vegetative B. cereus counts were slightly reduced in amount under both refrigerated and ambient conditions. In the challenge tests, B. cereus was successfully introduced into the artificial swill, mainly in its vegetative form. Preliminary results indicate a decrease in pathogen levels over time during BSFL rearing. The persistence of B. cereus, particularly in its endospore form, highlights the resilience of this pathogen in (potential) BSF substrates. The observed reduction during larval rearing suggests that rearing conditions may limit pathogen survival or transmission. Further experiments will assess the risks associated with inoculation of B. cereus in its endospore form. In conclusion, this study confirms that B. cereus is a relevant microbiological hazard in BSF substrates such as swill, occurring both as vegetative cells and endospores. While BSF rearing conditions may reduce pathogen levels, further research is needed to fully understand the food safety implications and to optimise risk mitigation strategies in insect production systems.

Effects of long-term storage on Hermetia illucens larval performance

L. Broeckx¹, L. Frooninckx¹, M. Verschaeren^1*, R. Meyermans², F. IJdema³ and S. Van Miert¹

¹Thomas More University of Applied Sciences, Centre of Expertise Sustainable Biomass and Chemistry, Kleinhoefstraat 4, 2440 Geel, Belgium; ²Center for Animal Breeding and Genetics, Department of Biosystems, Kasteelpark Arenberg 30, box 2472, 2472 Leuven, Belgium; ³Research group for insect production and processing, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*Maaike.verschaeren@thomasmore.be

The production of black soldier fly larvae (BSFL, Hermetia illucens) is widely used for organic waste conversion and as a sustainable biomass source, particularly for animal feed. As production systems scale up, maintaining reliable larval stocks becomes essential. Larval storage is often applied to manage fluctuations in production and demand, yet its long-term biological and genetic effects remain poorly understood. In this study, 7-day-old larvae (7 DOL) were subjected to repeated storage cycles at 19 °C using hydrated straw pellets sealed with aluminium foil to maintain moisture. Each cycle consisted of two months of storage followed by one generation of rearing before returning larvae to storage. By the time of this study, the stock population had undergone 32 consecutive storage cycles. Both the stock line and a continuously reared control line originated from the same initial population. To assess the impact of repeated storage, both lines were reared under identical conditions on three nutritionally distinct diets, with 100 larvae per replicate in triplicate. Survival, development time, growth curves, final larval weight, feed conversion ratio, and waste conversion efficiency were recorded. Preliminary observations from previous experiments indicate that larval survival during storage ranges from 77 to 99%, while pupal hatching ratios remain high (84–100 %), suggesting that storage does not critically impair pupal development. Slower growth has been observed in stored populations compared to continuously reared larvae. Genetic divergence between the lines will be assessed using Illumina paired-end whole-genome sequencing to determine whether repeated storage induces measurable genetic changes. By combining phenotypic and genomic analyses, this study clarifies whether prolonged storage leads to measurable biological or genetic divergence, providing insights for more robust stock management in BSFL production.

Exploring the impact of dietary changes during black soldier fly rearing on their microbiome composition and performance

K. Cerckel^1*, F. IJdema¹, S. Berrens², L. Frooninckx² and J. De Smet¹

¹KU Leuven, Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium; ²Thomas More University of Applied Sciences, Centre of Expertise Sustainable Biomass and Chemistry, Kleinhoefstraat 4, 2440 Geel, Belgium; ^*kato.cerckel@kuleuven.be

Black soldier flies are key organisms for large-scale insect rearing and bioconversion, owing to their ability to convert diverse organic substrates into high-quality biomass. Achieving true circularity, requires optimising rearing practices when nutritionally poor, low-cost substrates are used. In many systems, larvae are first raised on a nutrient-rich nursery diet before being transferred to a fattening diet. A key question is whether this two-phase feeding strategy improves performance or whether similar results can be obtained when larvae are reared directly on the fattening diet. This directly concerns dietary flexibility and how it can be used most effectively in practice. To explore the effects of dietary shifts on larval performance and the microbiome, this study investigated the impact of a diet change between the nursery stage (0 – 7 days after egg harvest; DAH) and the fattening stage (8 – 15 DAH), using chicken feed (CF) and artificial supermarket waste (SW). At the end of the nursery stage, CF-nursed larvae were more than twice as heavy as SW-nursed larvae (0.0185 ± 0.0028 g vs. 0.0074 ± 0.0009 g). After diet shift, however this effect diminished by the end of the fattening phase, though the rate at which was variable between diets. This indicates that a diet transition might results in a temporary slowdown, but not necessarily a lasting reduction in performance. Microbiome analyses based on 16S rRNA gene sequencing revealed distinct bacterial communities associated with CF-based and SW-based rearing. The CF-to-CF group consistently maintained a diverse community, whereas the SW-to-SW group showed persistently low diversity and, from DAH 9 onward, microbiome was dominated by five zero-radius operational taxonomic units. Three of these belonged to the family Lactobacillaceae, and two were identified as Lactobacillus. Following the diet change, the microbiome gradually shifted from a nursery-driven towards a fattening-driven profile, although full convergence was not achieved. This indicates that early-life diet leaves a lasting imprint on microbial assembly and limits later microbiome adjustment. Together, these findings show that diet – microbiome – insect interactions shape larval performance and highlight the need to consider these dynamics in multi-diet rearing protocols, as early-life nutrition can constrain later microbiome adjustment and affect responses to subsequent feeds.

Moisture optimisation for adult beetles: assessing hydration needs in Tenebrio molitor

D. Deruytter^*, J. Claeys and C.L. Coudron

Inagro vzw, Insects and aquaculture, Ieperseweg 87, 8800 Rumbeke-Beitem, Belgium; ^*david.deruytter@inagro.be

Previous studies have shown that giving a source of moisture to mealworm larvae is necessary to ensure efficient and rapid growth. In contrast, the hydration requirements of adult beetles remain poorly documented, despite indications from pilot scale production that moisture availability may strongly influence adult health and reproductive output. However, supplying moisture increases labour demands and may involve work on holidays/weekends. A better understanding of how long beetles can be deprived of moisture without compromising performance is therefore essential for efficient mealworm farming. Two dehydration–rehydration experiments were conducted to quantify the effects of moisture deprivation on adult survival, body weight and fertility (larvae/female per day). In the first experiment, five groups were created where beetles were deprived of moisture for 0, 1, 2, 3 or 4 days. Each group was replicated five times with a 100 beetles per replicate (14 days, 50/50 sex ratio assumption). Mortality and weight change of beetles was assessed over the course of 10 days to cover both the de- and rehydration period. In a second experiment, a similar set-up was used but with 0, 1, 2, 4 and 6 days without moisture. Besides the weight and survival, the daily fertility was also determined. Both experiments were performed at 27 °C and a relative humidity of 60%, wheatbran as dry feed and an agar cube as moisture source if appropriate. Across all treatments, mortality remained unaffected, even after six days without moisture. The animals did lose a consistent 5 mg (or 4%) of bodyweight per day without access to moisture for up to at least six days. The beetles fully regained their initial weight within 24 hours once moisture was reintroduced. Fertility did however does not follow this pattern. A single day without access to a moisture source did not affect fertility compared to the control. Starting from two days a reduction is visible (20-30%) with severe effects after 4 (75% reduction) and 6 days (90% reduction). More importantly, there is no immediate recovery. With 2, 3 or 4 days needed to return to control fertility levels after respectively 2, 4 or 6 days without moisture. These findings demonstrate that while adult beetles tolerate short term dehydration, their reproductive performance is sensitive to moisture availability. For example, omitting moisture provision during the weekend can reduce fertility by 20% during the weekend and, crucially, also on Monday due to the lag in recovery. Hence avoid more than two consecutive days without moisture in the breeding units.

Bodybuilding babies – an optimised Sofia Diet for black solider fly neonates

W. Grindle^1,2*, S. Popova¹, M. Partalozova¹, D.G.A.B. Oonincx², M. Bolard^1,3 and M. Tejeda^1,3

¹NASEKOMO EAD, Saedinenie Street 299, 1151 Lozen Village, Sofia, Bulgaria; ²Wageningen University & Research, Animal Nutrition Group, Droevendaalsesteeg 4, 6708 PB Wageningen, The Netherlands; ³Flygenetics AD, Saedinenie Street 299, 1151 Lozen Village, Sofia, Bulgaria; ^*grindleewayne@gmail.com

The larvae of black soldier flies (Hermetia illucens; BSFL) show great promise as a high-quality source of protein. Until recently, comparing studies on BSFL on a dietary basis has been difficult. There is a need for the development of an artificial, globally available, and consistent diet that will enhance comparison effectively between studies. This study expanded the use of the artificial Sofia Diet by optimising the composition for neonates during the 0 to 5 day growing period. A series of experiments using mixture design models was used to narrow the region for optimisation. Quadratic models were used to evaluate the effect of ingredient inclusions on life history traits, with fitness (percent survival to adulthood*fecundity) and larval weight at 5 days used as key performance indicators of a successful diet. The optimal diet for neonates consisted of 0.25% xanthan gum, 75% water, 21% hydrolysed yeast protein, 3.65% sucrose and 0.1% premix, which differed from the Sofia Diet for the 5–12 day growing period. With this ingredient mixture, the model predicted 5-day-old larval weight of 22.9 mg with a biological fitness of 1098. Known nutrient requirements for BSFL were exceeded by the optimal diet and with a protein to sucrose ratio of 5.75:1, this suggested that nutritional requirements differ between neonates and fattening larvae. Analysis of ingredient effects revealed that physical properties of the diet were critical in determining larval survival. Now that an artificial and standardized Sofia Diet has been established across the larval life cycle and can sustain multiple generations, the Sofia Diet can reduce variation between studies under controlled conditions. This allows for standardisation and shared practice, enabling more reliable comparisons and enhancing collaboration among the research, academia and industry sectors.

A framework for assessing and tracking digital maturity in the insect industry

U. Liebau^*, M. Hasan, S. Mysore Guruprasad and A. Flore

August-Wilhelm Scheer Institut, Uni Campus D 5 1, 66123 Saarbrücken, Germany; ^*Urs.Liebau@aws-institut.de

The insect industry is under increasing economic pressure while simultaneously being expected to improve efficiency, scalability, and sustainability. Although digital technologies and artificial intelligence are frequently discussed as potential solutions, there is currently no structured framework for systematically assessing the digital maturity of insect production systems. This study develops and applies a research framework to evaluate the level of digitalization in insect farming, with a focus on black soldier fly (BSF) production systems. The framework combines (i) industry and market analysis, (ii) an empirical survey among insect production stakeholders, and (iii) an analytical synthesis including digital maturity assessment and cross-industry benchmarking with digitalization approaches from other agri-food sectors. For the empirical component, a structured survey was distributed to insect industry stakeholders across the value chain. The results of the pilot survey indicate that digitalization within insect production is unevenly distributed across operational processes. Respondents identified insect counting and biomass monitoring, feeding optimisation, and process monitoring as key operational challenges where digital solutions could significantly improve efficiency and scalability. At the same time, environmental monitoring and climate control systems were reported as comparatively more digitalized, often using sensor-based monitoring and partial automation. The findings suggest that current digital adoption in insect farming is largely technology-driven and process-specific rather than strategically integrated across production systems. Based on the empirical results, the study proposes a digital maturity assessment approach for insect farming that adapts digitalization metrics used in other agricultural and industrial sectors to the specific characteristics of insect production. The proposed framework provides a methodological basis for future empirical studies and supports policymakers, researchers, and industry stakeholders in identifying priority areas where digital technologies and AI applications could realistically contribute to improved operational efficiency and economic viability in insect farming.

Pilot-scale drying of black soldier fly adults: a preliminary study for compliance to veterinary feed requirements

L. Rossi Ribeiro¹, O. Schlüter¹, F. Schindler² and G. Rossi^1*

¹Leibniz Institute for Agricultural Engineering and Bioeconomy e.V. (ATB), Max-Eyth Alee 100, 14469 Potsdam, Germany; ²Hermetia Baruth GmbH, An der Birkenpfuhlheide 10, 15837 Baruth, Germany; ^*Grossi@atb-potsdam.de

End-life black soldier fly adults are an important side-stream of the black soldier fly rearing process. Valorisation of this product allows higher production sustainability and economic viability for the producers. The current EU legislation allows to feed chicken with live insects belonging to the Hermetia illucens species (Reg. EC 767/2009). On the other hand, when killed, insects need to be opportunely processed (Reg. EU 2017/893). This study aims to evaluate different drying processes for obtaining microbiologically safe black soldier fly adults. A pilot-scale drier consisting in 9 layers was used. Three drying operations consisting in (A) 70 to 80 °C for 4.5 h, (B) 50 to 60 °C for 24 h, (C) 40 to 50 °C for 24 h and 90 °C for 1 h, were conducted. Samples were collected every 30 min during the first 4.5 h of drying and at the end of each drying. Drying kinetics, microbial and chemical analysis were considered. Results showed that the moisture of fresh flies (50.78 ± 0.90% DM) decreased fast during the first hours of drying when 70-80 °C were applied (sample A), reaching 93.38 ± 2.26% DM after 4 hours. On the other hand, when the samples were dried at lower temperature (B), the moisture decreased slowly during the first hours (69.73 ± 0.57% DM after 4 h), and faster in the following 30 min (91.20 ± 0.48% DM). Longer treatments at 50-60 °C only showed a limited effect on the total moisture content. Application of two-steps drying (sample C) did not significantly improve the dry matter of the final samples (92.02 ± 0.36% DM) but resulted in samples with different microbial counts with aerobic mesophilic bacteria, Enterobacteriaceae and yeasts and moulds showing reduction of 1.8 log, 3.0 log, and 4.3 log, respectively. Limited microbial reductions were instead observed when A (aerobic mesophilic bacteria: –1.4 log, Enterobacteriaceae: –2.6 log; yeasts and moulds: –3.3 log) and B (aerobic mesophilic bacteria: –1.3 log, Enterobacteriaceae: –2.3 log; yeasts and moulds: –3.9 log) were applied. Application of B and C also resulted in insects with higher protein content, amounting for approx. 40% DM, against the 25% of fresh insects and the 31% of treatment A insects. No significant differences were detected on the amino-acid composition. In conclusion, a drying process for 4 h at 80 °C or for 24 h at lower temperatures (40–50 °C) followed by 1 h at 90 °C was advised for obtaining dry black soldier fly adults able to be stored for long time at room temperature and complaining the safety requirements for feed use.

Microwave-assisted extraction of insect proteins: comparative analysis of alkaline and saline conditions

M. Alvarado^*, M. Keshani, M. Ortiz, C. Güell and M. Ferrando

Universitat Rovira i Virgili, Departament d’Enginyeria Quı́mica, Avda Països Catalans 26, 43007 Tarragona, Spain; ^*michelecristina.alvarado@urv.cat

Insect-derived proteins are sustainable alternatives to animal proteins but are conventionally isolated by energy-intensive alkaline extraction with isoelectric precipitation (AEIP), while microwave-assisted and saline extraction offer intensified and milder alternatives, respectively. This study compared alkaline MAE and saline MAE in terms of protein yield, molecular weight distribution and emulsifying capacity. Alkaline MAE (0.25 M NaOH) was applied to grasshopper (GH) (Locusta migratoria) defatted meal during 7 minutes at absorbed energy densities (AED) from 33 to 129 J ml^−1 and compared with AEIP (1 h) (Laroche et al., 2022). Under alkaline conditions, MAE improved protein yield (from 30 to 51%), while reducing drastically reducing extracting time. SDS-PAGE showed progressively more intense protein bands from 10–180 kDa with increasing AED, consistent with enhanced solubilization of proteins that are not efficiently released under conventional AEIP. GH extracts exhibited consistently high emulsifying activity (EA ≈60%) across AED values, indicating that, microwave exposure did not compromise the hydrophobic–hydrophilic balance required for interfacial functionality. Saline MAE (0.15 M NaCl) was evaluated under comparable conditions and, then applied to GH, black soldier fly (BSF) (Hermetia illucens) and lesser mealworm (LMW) (Alphitobius diaperinus) at AED 87, 98 and 178 J ml^−1. Protein yields saline conditions, remained around 20%, markedly lower than alkaline MAE and consistent with limited protein solubilization at near-neutral ionic conditions (Aniyam et al., 2024). SDS-PAGE revealed the appearance of higher-molecular-weight proteins (up to approx. 130 kDa) as AED increased, indicating improved extraction of protein fractions that remain embedded in the solid matrix at low energy inputs. This pattern, observed in GH, BSF and LMW, suggests that microwave-driven intensification enhances mass transfer even under moderate ionic conditions. Nonetheless, additional mechanisms, such as microwave-induced structural disruption or partial unfolding, cannot be excluded. Functionally, saline MAE extracts showed EA increasing from ≈10% to ≈40% with higher AED, reflecting the broader range of proteins mobilized during intensified extraction. Overall, alkaline MAE enhanced protein yield and functionality while reducing extraction time, whereas saline extraction highlighted complementary ionic and energy-density effects. These results support microwave-based technologies and ongoing comparisons with other intensification approaches, such as high-pressure-assisted extraction. References: Anyiam, P.N., Nwuke, C.P., Uhuo, E.N., Ajah, O., Uche, C.P., Dike, O.D. and Onyemuchara, T.C., 2024. Influence of pH and salt conditions on extraction efficiency and functional properties of Macrotermes nigeriensis protein concentrate for food applications. Discover Food 4: 100. Laroche, M., Perreault, V., Marciniak, A., Mikhaylin, S. and Doyen, A., 2022. Eco-Efficiency of Mealworm (Tenebrio molitor) Protein Extracts. ACS Food Science & Technology 2: 1077-1085.

Evaluating the impact of Grow Detect AI, an AI-driven monitoring tool on BSF production efficiency

M. Hasan^*, U. Liebau and S. Mysore Guruprasad

August-Wilhelm Scheer Institut GmbH, Center for Digital Greentech, Uni Campus D 51, 66123 Saarbrücken, Germany; ^*moynul.hasan@aws-institut.de

Large-scale insect production faces challenges such as process variability, high labor demand, and inefficient monitoring. While the sector is gradually adopting digital solutions, the use of AI-driven monitoring in black soldier fly (BSF) production remains limited. Tools such as Grow Detect AI could improve production efficiency and enhance process stability. Grow Detect AI is a modular AI-based monitoring system for black soldier fly (BSF) production that uses image analysis to count larvae, estimate larval size at different developmental stages, and assess pupal hatching patterns from representative image samples. The system operates with standard smartphone images and does not require specialized hardware when recommended image-capture protocols are followed. It can analyse images containing up to 500 larvae with detection accuracy exceeding 95%, even in the presence of small amounts of frass. However, accurate size measurements are currently limited to larvae with body lengths greater than 1 mm. This study evaluated the impacts of Grow Detect AI use for 5-day-old larvae (5 DOL) counting and size estimation on a pilot BSF farm, in a trial conducted over three production cycles. The measured KPIs included larvae estimation accuracy, labour hours for monitoring, feed conversion ratio (FCR), growth uniformity (coefficient of variation, CV), and batch-to-batch production variability. Operator feedback was collected via structured interviews to assess the usability, adoption barriers, and perceived value. The use of Grow Detect AI reduced 5 DOL larvae counting error to 3.28 ± 0.71% from 19.11 ± 0.83% in manual counting (92% counting accuracy improvement). The labour hours required for larvae counting decreases from 203 ± 6.32 s to 91.1 ± 3.47 s to count 500 larvae (45% improvement), feed application was improved through the homogenous size larvae use, resulting in a 15% improvement in FCR from 1.38 ± 0.02 to 1.20 ± 0.02. Growth uniformity improved, with the CV of adult larval weight, decreasing from 17% to 6%, and batch-to-batch production variability (total yield) decreased by 27% (variability decreased from 10.4% to 7.5%). Operators reported higher confidence in production planning and decision-making processes. Adoption barriers were adequate personnel training requirement to support the transition from manual counting procedures to a software-based workflow. Grow Detect AI improves the efficiency and productivity of Black Soldier Fly (BSF) production. Improvements in key KPIs indicate strong potential for broader adoption. These results highlight digital tools as important enablers of scalable and sustainable insect farming.

Automated image-based assessment of black soldier fly larval quality at batch level

D.T. Tippani¹, H.T. Tödtmann¹, D.E. Ewald² and B.K. Kubsch^1*

¹Fraunhofer Institute for Graphical Data Processing, Bioeconomy Competence Center, Joachim-Jungius-Strasse 11, 18059 Rostock, Germany; ²Digitales Innovationszentrum Rostock, Albert-Einstein-Strasse 21, 18059 Rostock, Germany; ^*bastian.kubsch@igd-r.fraunhofer.de

The larvae of the black soldier fly (Hermetia illucens) are increasingly recognised as a sustainable biological resource for protein and lipid production. With the scale-up of insect-based bioconversion, there is a need for robust, objective assessment of larval quality in terms of traits directly indicative of harvestable biomass (size), metabolic vitality (activity) and physiological health (colour), which are critical for predicting yield, optimising feeding regimes and ensuring suitability for downstream processing. This work presents a proof-of-concept, fully automated image- and video-based framework for low-cost, scalable batch-level quality monitoring. Low-cost RGB images and videos were acquired using a consumer-grade smartphone camera positioned above random samples of larvae drawn directly from the production process and processed with Canny edge detection and a watershed algorithm for larval segmentation, achieving about 99% agreement with manually annotated ground truth on a small, preliminary dataset without the need for high-end GPU hardware. From the segmented larvae, morphometric, colour and motion features are extracted and aggregated into interpretable indicators. For size, non-parametric, quartile-based thresholds define area-based classes (e.g. <3.3, 3.3–7.2, >7.2 mm²). Colour scores are computed as CIEDE2000 distances to a reference colour representing visually healthy, well-fed larvae, and activity is quantified via a normalised motion history image score. The framework was evaluated on data from a laboratory imaging setup designed to be transferable to industrial practice and from pilot-scale industrial conditions during rearing and pre-harvest inspection. It performed well under standardised illumination, while larval clumping at high densities and variable ambient lighting were identified as key challenges, particularly for colour-based indicators. The resulting operator-independent indicators provide early warning signals that can support data-driven decision-making in insect factories and inform potential corrective actions such as adjusting climate or feed regimes. Systematic validation under a broader range of process conditions and substrates is still pending, and ongoing work focuses on extending the labelled data basis and refining the thresholds, including for substrate-specific colour variations.

A low-cost larvae counter for black soldier fly systems

J. Biner¹, S. Diener^2*, B. Dortmans², C. Zurbruegg³, J. Tkaczuk¹ and E. Tilley¹

¹ETH Zurich, Global Health Engineering, Clausiusstrasse 37, 8092 Zurich, Switzerland; ²Eclose GmbH, Im Gumpersloo 1391, 9602 Bazenheid, Switzerland; ³Eawag: Swiss Federal Institute of Aquatic Science and Technology, Sandec, Ueberlandstrasse 133, 8600 Duebendorf, Switzerland; ^*stefan.diener@eclose.info

Accurate dosing of black soldier fly larvae is a critical control point in both grow-out and reproduction systems. The number of larvae used as stocking material in relation to the amount of substrate fed directly determines growth performance, survival rates, and the predictability of work processes. Despite its importance, reliable quantification of young larvae remains difficult in practice. Larvae are small, highly mobile, and typically mixed with heterogeneous feed residues and frass. Manual counting of samples is therefore time-consuming and unpopular with workers. As a result, this step is often bypassed and operators rely on rough weight-based estimates, introducing substantial uncertainty into process control. This contribution presents the development of a larvae counting system designed to automate the quantification of representative samples of stocking larvae while remaining simple to operate under field conditions. The system combines a mechanical pre-processing step based on passive sieving with an automated counting unit using controlled funneling and machine-vision-based detection implemented on a compact camera and single-board computer platform within a closed, illuminated counting chamber. Experimental validation was conducted by comparing automated counts with manual reference counts across a total of ten trials covering different operating conditions and larval size ranges. Tested larvae covered an average individual weight range of 0.85–5.3 mg. Initial experiments resulted in substantial overcounting (up to 147%), caused by the misclassification of non-larval particles such as frass. Following adjustment of detection thresholds to supress noise, repeated trials (n = 3) with sample sizes ranging from 613 to 762 larvae (0.84–0.87 mg per larva) demonstrated a counting accuracy of 96–100%. The observed counting duration ranged between 21 and 25 min/sample, with most larvae being counted within the first 10 min. Pre-processing required approx. 20–25 s/g mixed sample. Ongoing development focuses on improving the feeding mechanism to increase larval flow and reduce total counting time. Application areas include routine sampling in reproduction and grow-out operations, where improved quantification of stocking larvae enables more accurate dosing, higher process reliability, and contributes to the professionalisation of the black soldier fly technology.

Integrating bioacoustics into multi-sensor monitoring systems for precision insect farming

S. Mysore Guruprasad^*, M. Hasan and U. Liebau

August-Wilhelm Scheer Institut GmbH, Uni Campus Nord D 5 1, 66123 Saarbrücken, Germany; ^*shreyas.mysoreguruprasad@aws-institut.de

Monitoring larval behaviour and health in black soldier fly (Hermetia illucens) production systems is challenging because larvae develop within opaque substrates and at high stocking densities, limiting direct observation. While visual monitoring systems can measure the larval size, number, and growth, they provide limited insight into behavioural activity. This study proposes a hypothesis-driven monitoring framework to investigate whether acoustic emissions produced by larval movement and feeding can serve as indirect indicators of stress, overcrowding, or suboptimal rearing environments. Larval movement and feeding produce low-amplitude vibrations through cuticle friction and particle movement within the substrate. Similar bioacoustic approaches have been used to study behavioural dynamics in insects such as bees, termites, and crickets, indicating that acoustic activity may provide measurable proxies for colony behaviour. We present a methodological framework that integrates passive acoustic sensing into a multi-sensor monitoring system for insect farming. Acoustic sensors pick up sounds when the larvae move around in the substrate. These signals are synchronised with environmental measurements and process metadata. Signal analysis extracts activity-related acoustic features such as signal energy, dominant frequency bands, and temporal activity rates. These acoustic descriptors are combined with environmental variables through data fusion to support behavioural inference. Conceptual signal analyses demonstrate that acoustic feature extraction can reduce monitoring data volumes by roughly 70–80% compared to image based analysis. Activity-related acoustic indicators are expected to capture behavioural changes associated with feeding intensity, larval density, and environmental stress. For example, a drop in acoustic activity may indicate decreased feeding due to substrate degradation, while an increased activity patterns may signal overcrowding or agitation under suboptimal environmental conditions. These behavioural indicators could support operational decisions such as adjusting feed moisture, replacing degraded substrates, or modifying stocking density before measurable declines in larval growth occur. However, acoustic monitoring provides indirect behavioural proxies rather than direct biomass measurements. The proposed framework positions bioacoustic sensing as a complementary modality for precision insect farming. Continuous, non-invasive monitoring of larval activity may improve early detection of suboptimal rearing conditions and support data-driven operational decisions in BSF production, providing a basis for future experimental validation.

Prophylactic efficacy of bacteriophages against Salmonella spp. in black soldier fly larvae rearing systems

J. Maeriën^1*, D. Vandeweyer¹, J.A. Wouters², B. Marcelli² and J. De Smet¹

¹KU Leuven, Department of Microbial and Molecular Systems (M²S), Kleinhoefstraat 4, 2440 Geel, Belgium; ²Micreos Food Safety B.V., Nieuwe Kanaal 7P, 6709 PA Wageningen, The Netherlands; ^*jasmine.maerien@kuleuven.be

Various organic waste streams can be utilised as feed for black soldier fly larvae (BSFL) but potentially harbour pathogenic Salmonella. Bacteriophages, which are natural enemies of bacteria, have arisen as a potential biocontrol strategy for Salmonella contaminations in the meat processing industry, e.g., sprayed on poultry carcasses. Yet, their application and efficacy in insect-rearing environments is largely unexplored. This study aimed to provide a proof-of-concept for using bacteriophages as a prophylactic administration strategy for targeted mitigation of Salmonella in BSFL rearing. In a challenge experiment, the following four conditions were tested, with each replicated n = 3 times: (i) 200 g of standard BSFL diet (1:1 chicken feed to water); (ii) the same amount of diet to which 500 7-day-old larvae were added; (iii) feed-only inoculated with a mixture of two pathogenic Salmonella (7 log CFU/g, 1:1 S. typhimurium and S. infantis); and (iv) diet with 500 larvae and the pathogen mixture. These four conditions were duplicated, with one set treated with a commercially available bacteriophage cocktail, Phageguard S. (9 log PFU/g), and the other set being untreated. The presence of both Salmonella and bacteriophages within the substrates and the tissues of surface-sterilized BSFL were destructively sampled on days 0, 2 and 6 of the experiment. Results indicate that treatment with the bacteriophage cocktail led to a substantial reduction in Salmonella counts (3 log CFU/g) within as little as 1h post-treatment. For treatments without larvae present in the substrate, pathogen counts continued to decline over the experimental period, reaching the detection threshold (<3.0 log CFU/g). However, in the presence of larvae, the counts recovered by day 6 of the experiment (6.4 ± 0.3 log CFU/g). The decline of phage numbers over time in the substrate, on the other hand, was slower in the presence of larvae. The results also confirmed that functional Salmonella phages could be retrieved from the larvae after day 2 and 6 of the experiment. Interestingly, these findings suggest that larval presence may contribute to phage persistence over time, but counter-intuitively this is not associated with a more efficient reduction of Salmonella. As such, this proof-of-concept study showcases the potential of phage therapy as a control strategy to reduce the concentration of unwanted microbes in insect production systems, but also highlights several research questions that need to be addressed (e.g. how larvae impact both phage infectivity and pathogen dynamics).

Black soldier fly adults spatial distribution by age, weight, and love-cage environmental conditions

R. Garsia¹, A. Doretto¹, M. Meneguz² and F. Grosso^2*

¹University of Eastern Piedmont, Department for Sustainable Development and Ecological Transition, Piazza Sant’Eusebio 5, 13100 Vercelli, Italy; ²BEF Biosystems^†, Strada di Settimo 224/15, 10156 Turin, Italy; ^*frgrs@hotmail.com

Industrial breeding cages for black soldier flies (BSF) are designed to increase and ensure stable, high-quality egg production. To achieve this goal, several factors must be considered (environment, population dynamics, physiological fly parameters, and artificial habitat structure), but they are often analysed separately rather than together. This research aims to evaluate how the spatial distribution of flies in the love-cage was affected by environment (temperature (T) and humidity (RH)) and physiological fly parameters (age, weight, and sex). Inside a 24 m³ (6 × 4 × 1 L × W × H m) climatic chamber with constant T (27 ± 1 °C), RH (65 ± 5%), a 1.5 × 2 m resting area was used to assess fly spatial behaviour. For data collection, the area was divided into three zones (0.73 × 1.5 m each) at heights of 36,106, and 176 cm from the ground. To verify the behavioural differences during the day, sampling was done twice: at 9.30 am and at 2.30 pm. The experiment was repeated for two consecutive weeks during the spring months of 2024. During sampling from each zone of the resting area, T and RH of were measured, and 10 specimens were taken, individually sexed, weighted, and estimated their age by quantifying the fat reserves present in the abdominal ‘window’. Window fullness is an indicator of age: value close to 1 indicate young flies, while values closer to 0 indicate older ones. To understand the distribution of individuals on the resting surface, all parameters of adult BSF (sex, weight and age) were correlated with T and RH of each zone. Considering the entire resting area, 60% of the individuals sampled were female. Regardless of the sampling zone, the weight of adults was strongly and positively correlated with the age. Indeed, females were heavier (41.93 ± 18.79 mg) and with more fat reserves (so younger) than males (36.95 ± 13.45 mg) ($p\mathrm{<}0.005$). Negligible correlations were found among the weight and age of adults and the environmental parameters (T and RH). Nevertheless, regression models revealed a significant increment in the fat resource with increased RH was found in BSF females. In conclusion, in industrial system young BSF female use resting areas more frequently than males’ ones, and RH influences their spatial distribution. Therefore, it can be inferred that the female specimen requires energy derived from fat reserves and a proper RH environment for oocyte maturation. This could be a good starting point for future research.

Economic performances of small-scale insect dry larvae production: a comparison between control and waste-based diets

L. Baima^*, V.M. Merlino, S. Bellezza Oddon, L. Gasco, I. Biasato, Z. Loiotine, S. Blanc and F. Brun

University of Turin, Department of Agriculture, Forest and Food Sciences, Largo Paolo Braccini 2, 10095 Grugliasco (TO), Italy; ^*lorenzo.baima@unito.it

The high market price of insect-based proteins remains a key barrier to large-scale adoption in animal feed, despite their recognised environmental and nutritional potential. In this context, improving the economic sustainability of insect farming is crucial, especially for small- and medium-scale production systems typical of Europe. This study evaluates the economic performance of black soldier fly (Hermetia illucens) meal production under two feeding strategies: a diet based on commercially available feed -Gainesville diet- (case study 1), and a waste-based diet based on selected agri-food by-products (case study 2). Data were collected from a pilot-scale insect rearing and low-tech processing facility and analysed through full cost accounting, separating fixed costs (infrastructure, machinery, financial charges) from variable costs (labour, feed inputs, energy use, water, consumables, process control and quality assurance). Costs were expressed as € per kg of dried larvae at pilot scale. Results show that labour represents the dominant cost component in both scenarios, accounting for more than 60% of total production costs and increasing to over 70% under the waste-based diet. In the conventional scenario, feed inputs represented around 15% of total costs; when by-products were used as feed, their contribution became marginal (approx. 1–2%). Fixed costs -including infrastructure, machinery, and financial charges- remained relatively stable across scenarios, indicating limited short-term flexibility in small-scale systems. Overall, the waste-based diet substantially reduced variable costs and improved cost efficiency. However, the high incidence of labour and capital-related expenses suggests that further competitiveness will depend on increased automation and scale expansion. In addition, heterogeneous by-products may require extra management to maintain nutritional consistency and process stability. Integrating waste recovery into insect diets can enhance economic sustainability and support local circular feed supply chains; nevertheless, at pilot and small-scale, production costs remain higher than those of conventional protein sources. These systems should therefore be regarded as a promising pathway towards more resilient and environmentally sustainable feed supply chains. Overall, the waste-based scenario (2) outperforms the conventional feeding strategy in terms of cost efficiency, but its competitiveness ultimately depends on structural changes in production systems rather than feed substitution alone.

Defatting of Acheta domesticus by supercritical fluid technology

L. Vázquez^1,2*, D. Martı́n-Hernández^1,2, D. Villanueva-Bermejo^1,2, T. Fornari^1,2 and D. Martin^1,2

¹Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ²Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ^*luis.vazquez@uam.es

Despite the high protein content of insects such as Acheta domesticus, defatting processes can be performed to obtain products with an even higher protein concentration because of their high lipid content. This process generates a substantial volume of lipid co-product, which is increasingly attracting interest owing to its valuable nutritional composition, particularly its high degree of unsaturation, such as A. domesticus. Traditionally, defatting is performed using organic solvent extraction or mechanical pressing; however, alternative methods such as supercritical fluid extraction (SFE) are emerging as cleaner and more selective options. This study focused on developing a sustainable SFE method for the defatting of A. domesticus and on the characterisation of the two main products obtained: fat and defatted powder. A traditional method of defatting using hexane was compared to SFE. Different combinations of pressure (350 or 450 bar) and temperature (40 or 60 °C) at fixed CO₂ flow of 80 g/min were tested. Kinetics of defatting were obtained under the 4 tested conditions during 60 min of extraction. Proximate composition of the defatted powder and the fatty acid profile and minor lipid compounds of the lipid fraction was performed. The condition of 450 bar-60 °C was the most efficient method of defatting, producing the highest oil yield (22%) at the shortest time of extraction (40 min). After these conditions, the derived defatted powder consisted of 74% protein, 13% carbohydrates, 5% lipids and 5% ashes. The notable concentration of proteins due to the SFE defatting process was evidenced, since the starting material (cricket powder) consisted of 63% protein, 19% lipids, 7% carbohydrates and 4% ashes. With respect to the lipid obtained co-product, this showed the typical fatty acid profile for A. domesticus, with a similar content of saturated (35%) and unsaturated fatty acids (43%), with linoleic acid (40%), palmitic (26%), oleic (21%) and stearic (9%) acids as the most abundant. Additionally, minor compounds such as cholesterol, desmosterol, and stigmasterol were detected, offering valuable insights into the lipid composition. Therefore, this study demonstrates that SFE can be a sustainable and efficient alternative for defatting A. domesticus without compromising the conventional nutritional composition of the derived products. Acknowledgement: Funded by the European Union under grant agreement No 101162517. Call: HORIZON-EIC-2023-PATHFINDERCHALLENGES-01.

Supercritical CO₂ for sequential defatting and production of bioactive extracts from Acheta domesticus

D. Villanueva-Bermejo^1,2*, D. Martı́n-Hernández^1,2, T. Fornari^1,2, L. Vázquez^1,2 and D. Martı́n^1,2

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Producción y Caracterisación de Nuevos Alimentos, c/ Nicolás Cabrera 9, 28049 Madrid, Spain; ^*david.villanueva@uam.es

Acheta domesticus (AD) is an edible insect approved for food and feed rich in proteins, lipids and micronutrients. Beyond its nutritional value, AD has also been reported as a source of minor compounds associated with bioactive effects, such as antioxidant, anti-inflammatory and hypolipidemic properties. The production of insect extracts with high concentration of bioactive compounds and enhanced biological activity often requires a prior defatting step. In addition, the appropriate selection of extraction techniques is crucial to maximising extract bioactivity. In this context, supercritical CO₂ (SCCO₂) is a sustainable alternative to conventional defatting methods based on the use of toxic and polluting solvents. Additionally, SCCO₂ allows the extraction of bioactive compounds from the defatted material. The aim of this study was to evaluate the potential of SCCO₂ for sequential defatting and production of bioactive extracts from AD. Extractions were carried out sequentially in two stages. First, neat SCCO₂ (350 bar, 40 °C, 80 g/min, 60 min) was applied for the defatting of AD powder. Subsequently, a second extraction was performed on the defatted material using two different SCCO₂ flow rates (60 and 100 g/min) and EtOH concentrations (10% and 20%) as co-solvent. Total phenolic content (TPC. Folin-Ciocalteu method, as g of gallic acid equivalent (GAE)/100 g extract) and the antioxidant (DPPH and ABTS assays) and anti-inflammatory (lipoxygenase -LOX- inhibition) activity of the extracts were determined and expressed as IC₅₀ values. The defatting step resulted in a lipid removal of 84.7%. The second extraction step for obtaining bioactive extracts showed that the extraction yield increased with both CO₂ flow rate and EtOH concentration, reaching a maximum of 4% at 100 g/min and 20% EtOH. A similar trend was observed for TPC, antioxidant and anti-inflammatory activities. Thus, the highest TPC value (15.8 mg GAE/g extract) was obtained at 100 g/min and 20% EtOH, together with the lowest IC₅₀ values for ABTS (405.5 μg/ml), DPPH (690.3 μg/ml) and LOX inhibition (433.1 μg/ml). These results highlight the potential of SCCO₂ as a sustainable and efficient method for sequential defatting and production of bioactive extracts from AD that could be used for the development of insect-based ingredients for health. Acknowledgement: Funded by the European Union under grant agreement No 101162517. Call: HORIZON-EIC-2023-PATHFINDERCHALLENGES-01.

Mislabelled insect-based products: use of Near Infrared Spectroscopy techniques to ensure quality control

A. Anselmo^1*, A. Pissard¹, A. Marien¹, S. Gofflot¹, V. Baeten¹ and F. Debode²

¹Walloon Agricultural Research Centre, Knowledge and valorisation of agricultural products Department, Chaussée de Namur 24, 5030 Gembloux, Belgium; ²Walloon Agricultural Research Centre, Life Sciences Department, Chaussée de Charleroi 234, 5030 Gembloux, Belgium; ^*a.anselmo@cra.wallonie.be

Over the past few years, a wide range of insect-based products has emerged on the international and European markets, promoted for their nutritional benefits and low environmental impact. Although these products represent a major economic opportunity, they are currently subject to very limited quality control. Consequently, a recent study revealed that, among one hundred insect-based products analysed using genomic methods, the majority exhibited labelling inaccuracies. However, genomic methods require the use of expensive products and involve time-consuming sample preparation. To develop rapid and non-invasive approaches for assessing the quality of insect-based products available on the market, two Near Infrared Spectroscopy (NIRS) techniques were applied on commercially feedstuffs containing one or more insect species. First, a Near Infrared Microscope (NIRM) was used to authenticate insect species present in the samples. Second, NIRS was employed to evaluate protein, lipid and moisture contents of feedstuffs. The same samples were also analysed using real-time PCR, metabarcoding and conventional wet chemistry analyses to accurately assess the performance of NIRM and NIRS, respectively. The results obtained using NIRM and NIRS enabled the detection, authentication and compositional evaluation of the analysed insect-based products and were consistent with those obtained by real-time PCR and wet chemistry analyses. By comparing the different analytical results with the declared compositions on product labels, several labelling discrepancies were identified. NIRM analyses revealed errors related to the insect species, including the presence of species different from those declared or the complete absence of certain species despite being listed on the labels. Furthermore, NIRS analyses highlighted substantial discrepancies in protein content for several samples, with measured values around 9% compared to the 55% indicated on the labels. Overall, these findings underscore the importance of establishing robust quality control analysis for insect-based products, not only to prevent fraud but also to support the sustainable development of this emerging sector.

Supercritical fluid extraction of Tenebrio molitor larvae fed on rice crop byproducts rearing substrates

L. Tarraran¹, B. Aiuto¹, F. Romaniello¹, A. Rossi², C. Lamberti^1*, S. Fraterrigo Garofalo³, S. Bellezza Oddon⁴, I. Biasato⁴, F. Gai¹ and S. Cirrincione¹

¹National Research Council, ISPA, L.go P. Braccini 2, 10095 Grugliasco, Italy; ²National Institute of Metrological Research, S.da delle Cacce 91, 10135 Torino, Italy; ³Polytechnic of Turin, DISAT, C.so Duca degli Abruzzi 24, 10129 Torino, Italy; ⁴University of Turin, DISAFA, L.go P. Braccini 2, 10095 Grugliasco, Italy; ^*cristina.lamberti@cnr.it

The Sustainable Development Goal 2 aims to achieve food security, improve nutrition and develop sustainable and resilient food production systems. Edible insects (EIs) were recognised as alternative sources of nutrition with lower carbon and water footprints than traditional livestock. Their consumption could even be more beneficial to sustainability if they are reared on agricultural byproducts. Among EIs, Tenebrio molitor larvae (TML) are abundant in nutrients like proteins, fatty acids, minerals, and vitamin C. EFSA approved TML for use as a food and feed in European countries. Nevertheless, consumers’ acceptance remains low. Studies suggest it will increase if EIs are incorporated into food, such as bakery products. This study aims to obtain a rich protein flour from TML grown on rice crop by-products for use in baked products, including its polyunsaturated fatty acid-rich fraction. TML were grown on crop rice byproduct rearing substrates containing 10.5% white rice byproducts. TML were freeze-dried and finely minced. To separate the protein from the oil fraction, CO₂ supercritical fluid (SC-CO₂) extraction was performed. This extraction technique efficiently produced food-grade solvent-free extracts. In this study, two different methods were applied. In method I, the chosen process parameters were 400 bar and 65 °C. A 50-ml extraction vessel was filled with minced TML flour, and SC-CO₂ was provided. A first extraction phase was conducted in batch mode for 50 minutes, followed by continuous SC-CO₂ flow for 40 minutes to collect the extracted oil. In method II, the parameters chosen were 300 bar, 65 °C in a 500-ml extraction vessel. Flours with two degrees of defatting were obtained: 90% defatted (fl10) and 45% defatted (fl65). Based on dry matter, results indicate that they contained 59% and 47% crude protein, respectively. The results obtained are within the range reported in the literature (47 – 60%) for SC-CO₂ extraction. According to the data, method I allows the production of a flour with a higher protein content. Nevertheless, fl65 could also offer advantages for the preparation of baked products from a technological standpoint. Thus, the oil and protein fractions were characterised and used to prepare preliminary baked products. Research supported by Fondazione Cariplo and Compagnia San Paolo (newRIFF and TOWINBAKE projects).

Support for the implementation and development of NIRS technology for the insect sector

A. Desaegher, M. Radek^* and L. Le Picard

Le Gouessant, rue des frères Pieto, 22400 Lamballe-Armor, France; ^*mathilde.radek@legouessant.fr

Near-infrared spectroscopy (NIRS) is an analytical technique that relies on infrared light absorption to generate an electromagnetic spectrum, producing a unique “spectral fingerprint” associated with chemical composition. By establishing correlations between spectral data and chemical properties, NIRS technology enables predictive modelling of organic materials. After calibration, this approach allows accurate estimation of key nutritional components such as proteins, dry matter, lipids, and sugars in substrates, frass, and insect larvae at different processing stages. This technology offers significant advantages for industrial insect production. On the one hand, in R&D applications involving highly variable samples of substrates, larvae, and frass, we obtained robust equations that enable real-time substrate control, as well as high-throughput analysis of frass and larvae within short time frames. For example, for protein content in substrate (ranging from 8 to 30%), using 130 samples with chemical reference analyses and four years of R&D improvements (now 325 samples), we achieved very satisfactory results: n = 325; slope = 1.01; SEC = 1.55; R²Â = 0.91; RPD = 2.44. The chemical uncertainty is 1.2, and the maximum target for NIRS is an SEC of 1.4. On the other hand, in industrial applications—where variability is lower but real-time measurements are required on production lines with high throughput and the ability to scan a large share of the output—we also achieved strong performance. For example, for fat content in live larvae (ranging from 7% to 15%), using 35 samples and 10 months of development, the results were satisfactory: n = 35; slope = 1.06; SEC = 0.8; R²Â = 0.96; RPD = 3.85. The chemical uncertainty is 0.8, and the maximum target for NIRS is an SEC of 1.0. In routine use, ongoing improvements have increased the calibration set to 110 samples. NIRS enables reduced costs compared with conventional methods. Its implementation supports precise monitoring of production cycles and improves process control, ultimately leading to better product characterisation and enhanced operational efficiency. Supported by Le Gouessant, NIRS can be deployed at both laboratory scale and online production environments, contributing to the optimisation of insect-based bioprocesses.

Oil extraction from black soldier fly larvae: impact of dehydration method and extraction process on yield and recovery

J. Torrão¹, A. Carreira^2*, A. Gramacho², J. Regedor², M. Lima¹, V. Andrade¹ and O. Moreira²

¹Escola Superior Agrária de Santarém — ESAS, Quinta do Galinheiro; S. Pedro, 2001-904, Santarém, Portugal; ²Instituto Nacional de Investigação Agrária e Veterinária — INIAV, I.P., Av. Professor Vaz Portugal, 2005-424, Vale de Santarém, Portugal; ^*ana.carreira@iniav.pt

Black soldier fly (BSF, Hermetia illucens) larvae represent a promising lipid source for feed and non-feed biorefinery applications. This study evaluated the influence of dehydration methods and extraction strategies on oil recovery from BSF larvae, comparing aqueous extraction with the green solvent 2-methyltetrahydrofuran (2-MeTHF) as an alternative to conventional hexane. Larvae were dehydrated by microwave (MW; 100 °C, 20 min), oven (OV; 65 °C, 24 h), or freeze-drying (FD; 48–72 h). The resulting samples showed dry matter contents between 95.9% (OV and FD) and 96.9% (MW), while crude fat contents ranged from 32.6% (FD) to 34.3% (MW). Oil extraction results showed a clear difference between methods. Extraction with 2-MeTHF achieved about 35% oil yield corresponding to nearly complete lipid recovery (≈100%), regardless of dehydration method. In contrast, aqueous extraction showed strong dependence on pre-treatment. FD samples formed stable emulsions preventing phase separation and oil recovery, while OV- and MW-dried larvae allowed partial oil recovery with significantly lower performance (6% yield and 17% lipid recovery for OV; 16% yield and 45% lipid recovery for MW). These results demonstrate that dehydration method critically affects aqueous extraction efficiency due to emulsion formation, whereas 2-MeTHF enables consistently high lipid recovery. The performance obtained with 2-MeTHF was comparable to oil yields reported for hexane extraction in the literature, while offering improved safety and environmental performance. Further work will focus on improving aqueous extraction by reducing emulsion formation to enhance oil recovery and enable its integration as a complementary low-cost strategy in BSF biorefineries.

Valorisation of mealworm exuviae as a sustainable source of chitosan

T. Sedlar^1*, D. Dragojlović¹, Ž. Radonić², M. Polić Pasković³, J. Vujetić¹, N. Spasevski¹ and S. Rakita¹

¹Institute od Food Technology Novi Sad, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; ²Faculty of Technology Novi Sad, University of Novi Sad, Bul Cara Lazara 1, 21000, Serbia; ³Department of Agriculture and Nutrition, Institute of Agriculture and Tourism, K. Huguesa 8, 52440 Poreč, Croatia; ^*tea.sedlar@fins.uns.ac.rs

The growing need for sustainable food systems has intensified interest in insect farming as an efficient source of protein for human food and animal feed. In addition to their nutritional value, insects generate by-products that offer further opportunities for resource valorisation. In this context, the larval exuviae of Tenebrio molitor, a by-product of mealworm production, represent an underutilised biomass with high potential for conversion into value-added materials. In this study, chitosan was extracted from T. molitor larval exuviae using sequential demineralisation, deproteinisation, and deacetylation. The chitin yield from exuviae reached 12.86%, while the chitosan yield was 7.41% relative to the initial dry biomass, corresponding to 57.76% of the extracted chitin. The obtained chitosan exhibited a medium degree of deacetylation (72.27%) and a viscosity-average molecular weight of 612 kDa. High product purity was confirmed by low residual ash (0.33%) and amino acid content (0.14%). Morphological analysis revealed a fibrous and porous microstructure, particularly after purification. Chitosan films prepared from a 1% solution were uniform and transparent, with low thickness (0.0197 mm), high swelling capacity (335.07%), moderate water solubility (46.99%), and moisture content of 32.39%, indicating good film-forming ability and functional performance. These properties support the applicability of insect-derived chitosan in biodegradable films and other advanced applications. The results demonstrate that by-products of insect farming can be effectively integrated into circular bioeconomy models. Valorisation of T. molitor exuviae not only reduces waste from insect-based food and feed production but also enhances the overall sustainability of insects as a resource for feeding the world. Acknowledgements: This research was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Project Nos. 451-03-136/2025-03/200222 and 451-03-136/2025-03/200134) and by the Croatian Science Foundation (CSF(HRZZ)) and Slovenian Research Agency (ARIS) under the Weave initiative project nos. HRZZ IP-2022-10-8305 and ARIS N4-0346 (PROGRESS) and under CSF project HRZZ-DOK-2021-02-5517.

Frass or feed? Analytical challenges in insect meal quality control

Y. Khatabi¹, M.C. Lecrenier^2*, A. Anselmo², S. Bellezza Oddon³, Z. Loiotine³, L. Gasco³ and V. Baeten^1,2

¹University of Louvain-la-Neuve (UCLouvain), Croix du Sud, 2, 1348 Louvain-la-Neuve, Belgium; ²Walloon Agricultural Research Centre (CRA-W), Chaussée de Namur, 24, 5030 Gembloux, Belgium; ³University of Turin (UniTo), Largo P. Braccini 2, 10095 Grugliasco (TO), Italy; ^*m.lecrenier@cra.wallonie.be

Within the framework of the EU Green Deal and the Circular Economy Action Plan, reducing dependence on critical feed materials and limiting food waste are key objectives. In this context, insect meal has emerged as a promising alternative protein source for livestock feed. However, due to higher production cost compared to conventional proteins, its use in animal nutrition remains limited. Frass, a co-product of insect farming composed of faeces, residual substrate, dead insects and cuticular fragments, is produced in large quantities. Under economic pressure, some producers may be tempted to adulterate insect meal with frass to increase volume and reduce costs. Once mixed, frass is visually indistinguishable from insect meal, and no official analytical method currently allows its detection. This study investigates how proteomic analysis could identify and quantify frass adulteration in insect meal? The reliability of proteomics depends on efficient and representative protein extraction, yet no optimised protocol exists for frass. Developing a robust extraction procedure is therefore essential for achieving sensitive and reproducible analyses. This work focuses on optimising protein extraction from Hermetia illucens frass. Insects were reared on plant-based substrates and harvested at three developmental stages (6-DOL, 13-DOL and pupae). Insects and frass were collected, dried and ground. Four extraction protocols were tested, combining two urea concentrations (low/high) with or without a cold-acetone defatting step. Protein yields were then quantified. High urea concentration consistently improved extraction efficiency for both insect meal and frass. In insect meals, defatting further increased protein yields, whereas in frass it had the opposite effect, reducing extracted protein quantities. These trends were identical across developmental stages, confirming their robustness. These results show that extraction conditions are a critical source of variability in proteomic workflows. However, protein quantification alone cannot predict whether these conditions will also allow the detection of specific peptide markers distinguishing frass from insect meal. Therefore, all samples will now be analysed by UHPLC-MS/MS to verify whether the protocols maximising protein yield also optimize marker identification. This preliminary work lays the methodological basis for developing a reliable proteomic method to detect frass adulteration in insect meal.

Insect counting system on production line

A. Desaegher¹, M. Radek^1*, J. Costil² and J. Ferro²

¹Le Gouessant, rue des frères Pieto, 22400 Lamballe-Armor, France; ²MUTATEC, 1998 Chem. du Mitan, 84300 Cavaillon, France; ^*mathilde.radek@legouessant.fr

The insect industry is facing many challenges to improve its operational performance. One of the them is to measure the number of animals used per tray of production as it was mainly evaluated by a mean weight bringing to much variability and uncertainty. Moreover quality control was performed manually by operators and this step was time consuming and with a questioning reliability. Ideally the industry was looking for a system to be implemented directly on the production line without compromising the productivity rate. The insect counting system was designed to perform an exhaustive (95%) count of insect larvae of few millimetres in farming, directly on a production conveyor at 25m/min without separation from their feeding substrate and the full population of each tray. The development relied on a thorough selection of waves lengths (infrared and visible lighting), image acquisition with a specific camera (resolution and speed) and paired with deep learning-based analysis (trained on an important dataset labelled by experts). The device enables the distinction between all type of substrate, live and dead larvae and adapts to various industrial conditions (conveyor speed, width, and substrate type). The only constraint is to implement the equipment on a part of the process delivering a thin layer of substrate to avoid larvae to be totally hidden by the substrate particles while counting. Results demonstrate a high counting accuracy (>95%) with a variation coefficient below 1% (assessed on 10 samples per 10 repetitions). These results are reaching the performance targeted by the industrial as it is above both the accuracy and consistency delivered by the former system on place. Continuous learning ensures long-term consistency, paving the way for further applications in automated insect farming. The technology is patented and is also implemented for R&D operations.

Comprehensive characterisation of adult insects to assess their potential for sustainable biorefinery processes

J.M. Salgado^*, N. Muñoz-Seijas, H. Fernandes, B. Fernández-Alonso, F. Soto-Beltran and J.M. Dominguez

University of Vigo, Chemical Engineering, Edificio Campus da Auga Universidad de Vigo (Campus Ourense), 32004 Ourense, Spain; ^*jmsalgado@uvigo.es

A large part of current research focuses on the larval stage of insects. However, the adult stage can also be valorised. Through insect bioprocessing, insects can be separated into several fractions: protein, lipids, and chitin. This work focused on carrying out a comprehensive characterisation of adult insects: Acheta domesticus (AD), Locusta migratoria (LM) and Hermetia illucens (HI) to evaluate their potential in biorefinery processes. The three adult insects were characterised in triplicate as follows: protein (after subtracting the nitrogen of chitin origin), lipids, chitin, ash, and moisture content. Also, amino acids, fatty acids and minerals profile were assessed, as well as the in vitro digestibility of protein mimicking human digestion conditions. AD exhibited higher protein (73.5% dry weight, DW) content, which is 15% and 25% more than HI (49.4% DW) and LM (45.3% DW), respectively. Contrarily, higher lipids content was observed in LM (31.4% DW), followed by HI (20.1% DW) and AD (6.89% DW). Chitin content was similar among AD and HI, ranging between 9.3% (HI) and 10% DW (AD), with lower amount found in LM (4.4% DW). Higher essential amino acids were found in AD (19.9% DW), followed by HI (13.9% DW) and LM (9.57% DW). Among the three species, AD exhibited lower saturated fatty acids (36% DW; SFA) and higher polyunsaturated fatty acids (46.2% DW; PUFA) content, while HI has a higher content in SFA (76.4% DW) and LM showed the lowest PUFA content (11.9% DW). The in vitro digestibility of protein was higher in AD (63.8%), followed by HI (58.0%) and LM (53.3%). Regarding minerals, high sodium (6.13 g/kg DW), calcium (1.45 g/kg DW) and potassium (12.9 g/kg DW) levels were found in AD, while HI was an important source of potassium (10.6 g/kg DW) and magnesium (4.03 g/kg DW). It is not uncommon to observe a wide range of protein values in insects reported in literature. In this work, nitrogen of protein and chitin origin was quantified, permitting not overestimate the protein content. Also, in vitro digestibility results highlight the promising applicability of each insect species, highlighting the importance of AD for foods formulations. These results highlight the strong potential of these insect species, particularly as versatile and sustainable feedstocks for biorefinery processes, enabling the efficient production of multiple high-value products such as proteins, lipids and chitin.

Effect of larvae inoculation weight and substrate type on development and bio-conversion rate of black soldier fly

D. Nakimbugwe^1*, G. Ssepuuya², A. Kabuye¹, C. Ndagire³ and C. Tanga⁴

¹Makerere University, Food Technology & Nutrition, College of Agricultural & Environmental Services, 7062 Kampala, Uganda; ²Kyambogo University, Food Technology, Kyambogo 1, Uganda; ³Mountains of the Moon University, Food and Innovation Nutrition, Fort Portal 837, Uganda; ⁴International Center for Insect Ecology and Physiology, Insects for Food, Feed and Other Uses, Nairobi 30772-00100, Kenya; ^*dnakimbugwe@gmail.com

Productivity of black soldier fly larvae (BSFL) is strongly influenced by larval inoculation density and substrate characteristics. Low larval densities may lead to inefficient nutrient use, whereas high densities increase competition and reduce performance. This study therefore assessed the effects of substrate type, substrate depth, and larval inoculation density on BSFL growth and yield. In a completely randomised design with triplicates, 2.5 g of BSFL eggs were inoculated into six substrates to evaluate hatchability and development during the first six days. Substrates were placed in plastic basins (30 × 27 × 12 cm) at depths of 0.75, 1.50 and 3.00 inches at an average temperature of 27 °C and relative humidity of 54%. Six-day-old larvae (6DOL) were transferred into nine grower substrates, of plant- and animal-based origin, at inoculation levels of 20.8, 26.0, 31.2, 36.4 and 41.6 g. Growth, yield, and substrate bioconversion were assessed. Maize bran produced the highest larval weight among starter substrates, followed by fermented soy residue. A substrate depth of 3 inches produced comparable larval yields to other depths while generating the highest compost output. Brewery waste, poultry manure, Irish potato peels, and banana peels resulted in the greatest larval biomass. Inoculation rates above 31.2 g per 3 kg of substrate significantly reduced larval weight and overall yield. Larval growth at day 21 did not differ significantly from day 18. Irish potato waste, pineapple waste, and brewery waste recorded the highest bioconversion rates and orange peels and cow dung the lowest. Moderate larval densities (31.2 g) enhanced growth through improved aggregation and feed utilisation, while excessive densities caused competition and reduced nutrient availability. Growth peaked between days 9 and 12 due to fresh substrates with higher nutrient and moisture content and improved feeding capacity. Reduced growth after day 15 was associated with nutrient depletion and the onset of pupation. Inoculation of 2.5 eggs in maize bran starter feed, followed by inoculation of 31.2 g of 6 DOL in 3 kg of a ‘3 inch high’ plant-animal substrate mixture, and harvesting after 12 days results in the best performance.

Impact of post-harvest treatment on the yellow mealworm (T. molitor) metabolite profile

O. Pospı́šil^*, K. Šebelová, F. Beneš, M. Benešová and J. Hajšlová

University of Chemistry and Technology Prague, Technická 5, 160 00 Prague 6, Czech Republic; ^*pospisaf@vscht.cz

The quality of the produced edible insect is influenced by farming practices, including the choice of feeding substrate. However, post-harvest treatment has a significant impact on final product composition. In our study, three different post-harvest treatments were investigated. yellow mealworm (T. molitor) larvae were euthanised by (i) boiling in water (100 °C, 5 min), (ii) freezing (–20 °C, 24 h) and (iii) microwave treatment (800 W, 1.5 min). Subsequently, each variant was dried to constant weight using microwave drying (800 W), conventional hot-air drying (60 °C) and freeze-drying (–55 °C, vacuum). All samples, including lyophilised bath (water) obtained by boiling larvae, were examined by untargeted fingerprinting using ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (U-HPLC-HRMS/MS). Advanced statistical methods, including multivariate statistical analyses (PCA, PLS-DA), were used to evaluate the data. The results showed that insect samples euthanised by freezing and microwave contained a higher abundance of polar metabolites than those killed by boiling. The reason might be the transfer of some water-soluble compounds, such as amino acids or short peptides, into the boiling bath. Analysis of the amino acid profile confirmed the differences among the three post-harvest treatments of yellow mealworm (T. molitor) larvae. The results of the study may contribute to the optimisation of post-harvest treatment of edible insects for food purposes.

Intelligent feeding method for automated insect rearing

V. Klapper^1*, T. Röthig², E. Werner³ and C. Henze¹

¹Fraunhofer IVV, Department for Cyber-Physical Processing and Cleaning Systems, Heidelberger Strasse 20, 01189 Dresden, Germany; ²Fraunhofer IME, Biodiversity research, Ohlebergsweg 12, 35392 Giessen, Germany; ³Alpha-Protein GmbH, Werner-von-Siemens-Straße 2-6, 76646 Bruchsal, Germany; ^*vinzenz.klapper@ivv-dd.fraunhofer.de

Edible insects such as Tenebrio molitor, Hermetia illucens and Acheta domesticus can provide protein with relatively low environmental impacts (European Parliament, 2024; van Huis, 2013). Global protein demand for food and feed is projected to rise by about 70% by 2050 (FAO, 2017), and the insect protein market is expected to grow strongly (SkyQuest, 2026). In this context, the InFueSekt sub-project develops a T. molitor reproduction module with an integrated, barrier-based feeding concept. In standard crate-based reproduction, beetles and feed, as well as eggs and frass accumulate in a single layer, causing high manual effort for separation, egg losses and limited hygiene control. InFueSekt replaces this with a four-layer barrier design: (i) a substrate layer, (ii) a beetle layer with minimal substrate, (iii) an egg collection layer and (iv) a frass collection layer. Dry feed is sieved from above onto the beetle layer as particles with defined size, large enough not to pass through egg and frass sieves, but small and rounded enough not to injure beetles and to ensure access. Sieves between the layers allow eggs and frass to fall into separate zones while retaining beetles and feed above. In replicated trials, the barrier-based module is benchmarked against a conventional crate baseline. Beetle survival, oviposition, egg recovery, egg damage, hatch rate and early larval performance are quantified, and offspring quality is characterised via selected nutrient and microbiological parameters. Handling time and suitability for decentralised on-farm use are recorded alongside technical indicators. As eggs have the highest value density in the mealworm chain, the module targets improved egg yield, recovery and hygiene as a basis for scalable, decentralised breeding of T. molitor. The project is funded by the German Federal Ministry of Food and Agriculture (BMEL) within the innovation programme “Nachhaltigere menschliche Ernährung”. References: (1) European Parliament, 2024. Alternative protein sources for food and feed. Available at https://www.europarl.europa.eu/RegData/etudes/STUD/2024/757806/EPRS_STU(2024)757806_EN.pdf. FAO, 2017. The future of food and agriculture. DFAO, Rome. SkyQuest, 2026. Insect protein market report. Available at https://www.skyquestt.com/report/insect-protein-market. van Huis, A., 2013. Potential of insects as food and feed. Annual Reviews of Entomology 58: 563-583.

Calcium fortification of Tenebrio molitor larvae: powder mixing versus spray application

M.L. Cocato^*, G.P. Sant’anna, M.A. Hortellani, P. Ragozzino-Paulino and J.E.S. Sarkis

Nuclear and Energy Research Institute (IPEN), Lasers and Applications Center, Av. Prof. Lineu Prestes, 2242, São Paulo, SP 05508-000, Brazil; ^*mlcocato@hotmail.com

Low calcium (Ca) content and inverted Ca:P ratios are major nutritional limitations of Tenebrio molitor larvae when used as feeder insects or as ingredients in compound feeds, since they may contribute to mineral imbalance in animal diets. This study evaluated two Ca fortification strategies by comparing wheat-bran diets nominally supplemented with calcium carbonate (CaCO₃) at 4, 8 and 12% (w/w): dry powder mixing (POW) and spraying an aqueous CaCO₃ suspension onto the substrate followed by drying (SPR). Larvae were fed the experimental diets for 48 h (ad libitum), a short-term feeding interval selected based on previous biofortification studies to enable comparison with the literature, and then fasted for 48 h. Calcium concentrations in diets and larvae were quantified by flame atomic absorption spectrophotometry. Dietary Ca increased strongly with CaCO₃ inclusion in both series, with all fortified diets higher than the control ($p\mathrm{<}0.001$). At 4 and 8% CaCO₃, SPR diets contained more Ca than the corresponding POW diets, whereas no difference was observed at 12%. Larval Ca also increased significantly with inclusion level ($p\mathrm{<}0.001$): relative to control larvae, POW diets produced approximately 1.7 – 2.7-fold enrichment and SPR diets produced approximately 2.5 – 3.4-fold enrichment. Two-way ANOVA confirmed significant effects of fortification strategy and inclusion level, with SPR consistently yielding higher larval Ca than POW at the same CaCO₃ level. These results demonstrate pronounced mineral plasticity in T. molitor and identify spray-based CaCO₃ application as an effective short-term approach to produce calcium-fortified larvae with improved mineral balance, supporting their use in animal nutrition.

Optimisation and modelling of protein extraction from Tenebrio molitor larvae

H. Mhemdi^*, N. Boussetta, N. Lebovka and E. Vorobiev

Université de technologie de Compiègne, Ecole Supérieure de Chimie Organique et Minérale, TIMR (Integrated Transformations of Renewable Matter), Centre de recherche Royallieu, 60203 Compiègne Cedex, France; ^*h.mhemdi@escom.fr

This study examines the kinetics and efficiency of protein extraction from Tenebrio molitor larvae using an integrated processing sequence comprising blanching, drying, hydraulic pressing and grinding, followed by aqueous extraction under controlled operating conditions. Extractions were conducted at temperatures of 20, 40 and 60 °C and alkaline pH values of 7, 10 and 12. Kinetic analysis revealed a biphasic extraction mechanism consisting of an initial rapid washing stage, with characteristic times of ≤4 min, dominated by convective mass transfer, followed by a slower diffusion-controlled stage (characteristic times ≥30 min) governed by intraparticle transport within the larval matrix. Protein extraction yield was strongly influenced by both temperature and pH. At neutral to moderately alkaline conditions (pH 7 – 10), increasing temperature enhanced extraction efficiency due to improved protein solubility and diffusivity. In contrast, at pH 12, extraction yields declined above 40 °C, likely as a result of thermally induced protein aggregation and partial denaturation. Overall, the results define a scalable and environmentally benign extraction strategy that enables efficient protein recovery from insect biomass. This approach demonstrates strong potential for sustainable food ingredient production and the valorisation of insect-based agro-resources.

Practical case studies of black soldier fly production

K. Unger^* and J. Wixler

LIVIN farms AgriFood GmbH, 2. Haidequerstraße 1/Halle 42, 1110 Vienna, Austria; ^*contact@livinfarms.com

Insect-based feed systems offer significant potential to upcycle organic by-products into locally produced protein for animal nutrition. This study presents an analysis of six black soldier fly (BSF) production systems implemented across European agricultural value chains using modular and scalable factory concepts. The systems range from rearing-only units to fully integrated facilities combining breeding, rearing, and processing, operated under both manual and automated conditions. These factories are currently converting over 100 000 Tons of organic material into insect protein. A comparative framework was applied considering customer type, substrate streams, production scale, integration level, end-product form, and regulatory context. Two dominant implementation models were identified: (1) industrial-scale production of processed insect protein by agro-industrial actors, and (2) decentralized, smaller-scale systems enabling on-farm use of live larvae in livestock feeding. The results demonstrate that system configuration such as integration level, automation, and output form determine how BSF production contributes to circular feed strategies. Modular system design enables adaptation to regional conditions and supports the conversion of local organic side streams into feed resources. These findings provide practical insights into the deployment of BSF systems within European agri-food value chains.

Learnings from a decade of insect processing: processing as one key driver for profitability

A. Kühl*

Reinartz GmbH & Co. KG, Industriestrasse 14, 41460 Neuss, Germany; ^*a.kuehl@reinartz.de

Over the past decade, industrial insect farming has developed from experimental pilot systems to commercial-scale production facilities. However, many projects still face challenges in achieving stable economic performance. Industrial experience indicates that biological production efficiency alone is insufficient to ensure profitability. Downstream processing design increasingly emerges as a decisive factor influencing plant economics. This contribution analyses observations from several industrial insect production projects implemented between 2015 and 2025 involving commercial-scale production facilities. Experiences from plant design, commissioning, and early operational phases were evaluated with respect to processing configuration, product strategy, and integration of processing units. Particular attention was given to differences between facilities designed primarily for the sale of unprocessed insect biomass and plants integrating processing steps such as drying, oil extraction, fractionation, and product specification. Across the analysed projects, facilities focused on the sale of unprocessed insect biomass showed significantly higher exposure to market price volatility and margin pressure. In several cases, attempts to introduce processing capacity after plant start-up increased capital expenditure by more than 25% due to retrofitting constraints and suboptimal process integration. In contrast, facilities where processing concepts were defined during early plant design achieved improved process integration, multiple product streams, and more stable product specifications, enabling access to higher-value market segments. The observations indicate that delayed integration of processing systems can lead to structural inefficiencies that are difficult to correct during later plant operation. Conversely, early integration of processing enables better alignment of production systems with downstream product requirements and market demands. In conclusion, the observations confirm that downstream processing is not merely an auxiliary step in insect production but a key driver of profitability. Industrial insect production facilities therefore benefit from being conceptualized as integrated protein processing systems from the earliest project stages in order to improve long-term economic performance.

Fermentation metabolite dynamics during black soldier fly larval rearing

R.F. Calderon Barrientos^1*, M. Gold² and M.J. Zorrilla¹

¹REPLOID Deutschland GmbH, R&D, Löbener Weg 7, 04523 Pegau, Germany; ²REPLOID Group AG, R&D, Maria-Theresia-Straße 53/2 OG, 4600 Wels, Austria; ^*calderon@reploid.eu

Fermentation of substrates is a common pre-treatment in black soldier fly larvae (BSFL) bioconversion. However, little is known about substrate fermentation occurring during larval rearing, despite the ideal conditions for microbial growth during BSFL bioconversion. This study characterised temporal changes in fermentation metabolites in a high-moisture substrate (73% moisture, 27% dry matter) during BSFL rearing and evaluated impacts on substrate nutrient availability and microbial activity under larval influence. Larvae were reared on an experimental substrate (whey, wheat bran) under controlled rearing conditions in stainless steel trays (2.68 × 1.12 m). Each tray contained 260 000 larvae and 180kg feed. Ambient temperature was 30 °C, relative humidity 70%, and initial substrate temperature 28 °C. Feed samples were collected daily at 14:00 on days 0, 2, 5 and 6 and analysed for key fermentation metabolites using including lactic acid, ethanol, 1-propanol, and 1,2-propanediol. Total solids were determined gravimetrically (VDLUFA III, 3.1, 1976), while organic acids and alcohols were quantified using internal chromatographic and gas-chromatographic methods (LKS FMUAA 166:2024-11 and 181:2024-11). Metabolite concentrations were normalized to dry mass to assess temporal dynamics during active larval feeding. Fermentation metabolite profiles changed markedly over the rearing cycle. Data were analysed using ANOVA with post-hoc testing ($p\mathrm{<}0.05$). Lactic acid dominated the metabolite spectrum and increased substantially during the first 3 days of rearing with an observed increase of approximately 20% between day 2 and day 3. Alcohols such as ethanol and 1-propanol were present at lower concentrations (<15% of total fermentation metabolites). Ethanol reached its highest concentrations during early rearing (day 2) and decreased thereafter, while 1-propanol remained at consistently low levels (<2%) across all sampling days. These dynamics indicate shifts in the substrate microbial metabolism during rearing, consistent with reported changing redox conditions driven combined larval and microbial respiration, substrate turnover and larval movement. Thus, temporal patterns in fermentation metabolites, particularly lactic acid, may therefore serve as indicators of microbial activity and metabolic state during insect rearing. The results further demonstrate that nutrient concentrations in the rearing substrate change dynamically during bioconversion, such that initial substrate values are unlikely to represent nutrient availability at peak larval demand. Nutrient availability during BSFL rearing should thus be considered a dynamic process rather than a static substrate property.

Insects can bioaccumulate phytochemicals from olive oil by-products

V. Da Cunha-Borges^1,2, E. Rodrı́guez-González^1,2, E. Cantero-Bahillo^1,2, D. Villanueva-Bermejo^1,2, M.R. Garcı́a-Risco^1,2 and D. Martin^1,2*

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Departamento de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ^*diana.martin@uam.es

Research on the functional properties of insects, such as Hermetia illucens (HI) or Tenebrio molitor (TM) is growing, evidencing their potential as novel health-related foods. Their bioactive composition and derived bioactivity seem to be influenced by diet, particularly when insects are fed vegetable ingredients rich in bioactive phytochemicals. In this context, feeding bioactive-rich agri-food by-products could valorise side streams while enriching insects with phytochemicals, although evidence on the effective bioaccumulation of specific compounds and related bioactivities remains limited. This study aimed to evaluate the effect of feeding HI and TM larvae with different levels (15–90%) of bioactive-rich by-products well-known from the olive oil industry (olive leaves or olive pomace) on the phytochemical bioaccumulation and antioxidant activity of insect meals. Reared larvae were slaughtered, dried, ground, and defatted. Meals were characterised for target olive phytochemicals (GC-MS previous silylation or HPLC-DAD) following 70% ethanol ultrasound-assisted extraction. The efficiency of bioaccumulation (EB,%) of phytochemicals was estimated as the proportion of the ingested compound accumulated relative to the theoretical maximum. Antioxidant activity was evaluated (DPPH method). Insects accumulated most targeted dietary phytochemicals, which were not detected in control larvae. However, despite diets being richer in phenolic compounds such as oleuropein (olive leaves) and hydroxytyrosol (olive pomace), together with minor levels of pentacyclic triterpenes, the insect meals mainly accumulated pentacyclic triterpenes, particularly oleanolic and maslinic acid (HI, TM) and ursolic acid (TM). EB values (0.6-3.5% for HI; 0.4-1.8% for TM) suggested limited retention of the phytochemicals. Nevertheless, the achieved enrichment was relevant enough considering the absolute accumulated content of phytochemicals. In fact, the total amount of phytochemicals in the meals strongly correlated with antioxidant activity. This study evidences the possibilities of dietary strategies to enrich insects with bioactive phytochemicals, enabling the use of insects as bioactive carriers and enhancing their health-related properties, while supporting circular economy approaches through by-products valorisation. Acknowledgment: ENTOMOTIVE project, PID2022-136238OB-I00 and grant PREP2022-000828, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU.

Refining protein quantification in mealworm and cricket meals: impact of processing on K_p and K_A factors

G. Pellerin^* and A. Doyen

Institute of Nutrition and Functional Foods (INAF), Université Laval, Department of Food Sciences, 2440 Boulevard Hochelaga, Quebec City, QC, Canada G1V 0A6; ^*genevieve.pellerin.3@ulaval.ca

Conflicting values have been reported for the nitrogen-to-protein conversion factor (K_p) used to estimate protein content from total nitrogen (N_total) in edible insects, complicating accurate protein estimation. This work aims to demonstrate that protein concentration in insect meals can be determined more reliably by applying the K_A conversion factor to protein-bound nitrogen (N_P). Cricket (A. domesticus) and mealworm (T. molitor) meals were prepared from insects treated by various killing (freezing, blanching) and drying (freeze-drying, oven-drying, roasting) methods. Amino acid profiles of insect meals were obtained by UPLC, and K_p and K_A factors were calculated. Non-protein and non-chitin nitrogen (N_NP,NC) was quantified through protein precipitation using tricholoracetic acid and acetone. Chemical deproteinisation (1 M NaOH, 75 °C, 60 min) was then performed, followed by filtration to recover N_P. Nitrogen content in all nitrogenous fractions (N_total, N_NP,NC, N_P, N_other) were measured using the micro-Kjeldahl method. Crickets killed by freezing exhibited significantly lower K_p factors (3.11–3.69) than those subjected to blanching (4.15–4.75), whereas K_A factors remained unaffected by processing (5.63 ± 0.03). Similarly, K_p factors of mealworm meals varied with processing (4.53–5.50), while K_A values remained constant (5.72 ± 0.02). In both crickets and mealworms killed by freezing, the drying method strongly impacted N_NP,NC fractions as well as meal colour, with N_NP,NC accounting for 19% and 37% of N_total in oven-dried crickets and mealworms, respectively. In contrast, blanched insects had similar N_NP,NC concentrations (8–10% of N_total). These results indicate that non-protein nitrogenous compounds were generated through proteolysis and enzymatic browning during drying. Protein contents calculated using N_P and K_A ranged from 33–37% for cricket meals and 19–42% for mealworm meals, compared to 49–50% for both insects when using N_total and a K_p factor of 4.76. Thermal processing significantly affects the K_p value of insect meals, limiting its reliability. Therefore, protein quantification based on protein-bound nitrogen and K_A factors of 5.61 (A. domesticus) and 5.69 (T. molitor) provides a more suitable approach for determining protein content in insect ingredients.

Effect of storage temperature and duration on lipid oxidation of black soldier fly larvae meal

G. Kanbur^*

Selcuk University, Animal Science, Alaaddin Keykubat Kampüsü, Selçuklu, 42100 Konya, Turkey; ^*gkanbur@selcuk.edu.tr

Black soldier fly larvae meal (BSFLM) is considered a promising alternative protein source and a valuable lipid source for poultry. However, the elevated lipid fraction may increase susceptibility to oxidative deterioration during storage and under environmental temperatures typical of poultry housing conditions. This study evaluated the effects of storage temperature and storage duration on lipid stability of BSFLM. Samples were stored at +4 °C, 25 °C, and 35 °C for 14 and 32 days. Free acidity (%) and thiobarbituric acid reactive substances (TBARS, mg MDA/kg) were determined. Data were analysed using two-way ANOVA to evaluate the main effects of temperature and storage duration, as well as their interaction. Free acidity was significantly affected by both temperature ($P=0.022$) and storage duration ($P\mathrm{<}0.001$), while their interaction was not significant ($P=0.163$). Mean free acidity increased from 11.41% on day 14 to 12.69% on day 32. TBARS values were significantly influenced by storage duration ($P\mathrm{<}0.001$) and showed a strong temperature × duration interaction ($P\mathrm{<}0.001$). The highest TBARS value was observed at 25 °C after 32 days (4.93 mg MDA/kg), whereas the lowest value occurred at 25 °C after 14 days (3.67 mg MDA/kg). Temperature-related differences were limited at shorter storage periods but became more pronounced with prolonged storage. These findings indicate that storage duration is a key driver of lipid oxidation in BSFLM, while temperature effects emerge mainly during extended storage. Appropriate storage strategies, particularly under poultry house temperature conditions, are therefore essential to preserve oxidative stability and nutritional quality of insect-based feed ingredients.

Host-dominant degradation of sulfonamides in black soldier fly larvae mediated by CYP4d14

C. Sun^*, M. Cai and J. Zhang

Huazhong Agriculture University, Life science collage, 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei Province, P.R. China; ^*sunc1998@webmail.hzau.edu.cn

Antibiotic residues in livestock manure, particularly sulfonamides (SAs), pose environmental risks. Black soldier fly larvae (BSFL) are effective in waste valorisation. However, the relative contributions of the larval host versus the gut microbiome to this degradation process remain unclear. This study systematically elucidates the degradation mechanism of Sulfadiazine (SD) and Sulfamethazine (SMZ) from phenotypic, physiological, and molecular perspectives. We established conventional, sterile, and isolated gut microbiome systems to determine degradation contributions. Comparative transcriptomics and molecular docking were used to screen candidate genes. Finally, mechanism validation was conducted using the P450 inhibitor 1-aminobenzotriazole (1-ABT) and functional verification via RNA interference. Phenotypic assays showed that BSFL possesses robust degradation capacity in conventional environments, with removal rates reaching 88.11% for SD and 80.79% for SMZ. Crucially, the sterile BSFL system maintained high degradation efficiency of 72.18% for SD and 85.72% for SMZ, whereas the isolated gut microbiome showed minimal activity ranging from 3.32% to 17.57%, confirming that the larval host rather than the microbiota is the primary driver. Transcriptomic analysis identified the upregulation of Cytochrome P450 genes. This was physiologically validated by adding the inhibitor 1-ABT, which suppressed SD and SMZ degradation rates to 63.17% and 46.66%, respectively. Further molecular docking and RNAi assays pinpointed CYP4d14 as the key gene. Silencing CYP4d14 significantly reduced the degradation efficiency of SD and SMZ to 53.64% and 51.42%, respectively, compared to the control group, which maintained higher rates of 86.67% and 94.38%. This study establishes a comprehensive evidence chain: BSFL degrades sulfonamides primarily through its intrinsic metabolic system, specifically mediated by the CYP4d14 enzyme, rather than gut bacteria. These findings provide a theoretical basis for genetic screening or inducing specific P450 activities to enhance the safety and efficiency of insect-based waste management.

CaCO₃ in shrimp residue inhibits bioconversion and development of black soldier fly (Hermetia illucens, Linnaeus, 1758)

A. Thompson De La Chenelière^1*, M.P. Rivera Rodriguez¹, L. Lapierre¹, V. Banville², M. Lapierre³, G.W. Vandenberg¹ and M.-H. Deschamps¹

¹Université Laval, Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation, Pavillon Paul-Comtois, 2425 rue de l’Agriculture, Laval, QC, Canada G1V OA6; ²Centre de Développement Bioalimentaire du Québec, 1660 Rue de la Ferme, La Pocatière, QC, Canada G0R 1Z0; ³La Crevette du Nord Atlantique, 553 Boulevard du Griffon, Gaspé, QC, Canada G4X 6B6; ^*arthd@ulaval.ca

Worldwide population growth has increased the need for sustainable waste management within food systems. Black soldier fly larvae (BSFL) can upcycle various organic residues by converting them into fertiliser (frass) and protein-rich animal feed (larvae). Shrimp processing co-products could be valorised in this way. However, preliminary trials suggested that their high CaCO₃ contents could inhibit BSFL growth and bioconversion. This project thus aimed to evaluate the impacts of shrimp co-products and CaCO₃ on bioconversion and lifecycle indicators through feeding trials (3 trials; 2 repetitions/diet) using BSFL (4 days post-hatching; n = 1000 larvae/container; 30 mg/ind. per day). We hypothesized that increasing CaCO₃ concentration, with or without shrimp products, would diminish growth and bioconversion, and delay development. A control diet (Gainesville, 70% RH) was compared to 9 experimental diets supplemented with raw and hydrolysed shrimp powder (10, 20 and 40%, dry basis) or with CaCO₃ (3.3, 4.3 and 8.3% Ca^{2 +}, dry basis). Calcium did not accumulate in frass as levels were found to be similar to initial diet levels. In contrast, higher levels of Ca^{2 +} were measured in BSFL reared on diets containing 8.3% calcium (CaCO₃ supplementation), 40% raw shrimp powder and 40% hydrolysed shrimp powder (from 5.8 ± 0.4% to 15.2 ± 1.0%, 18.7 ± 1.8% and 17.2 ± 0.1%, respectively). Lower bioconversion rates were observed in CaCO₃-enriched diets containing 4.3 and 8.3% calcium (from 11.6 ± 0.3% to 5.0 ± 0.5% and 2.8 ± 0.5%, respectively) and 40% raw and hydrolysed shrimp powder (from 11.6 ± 0.3% to 3.7 ± 1.7% and 4.5 ± 0.3% respectively). The CaCO3-enriched diet containing 8.3% calcium also delayed adult emergence (from 29.8 ± 0.3 to 36.5 ± 2.0 days post-hatching). These results indicate that diets containing more than 8% calcium exceed the regulation mechanisms of BSFL, resulting in limited bioconversion and delayed development. Though physiological mechanisms of disturbance (e.g., osmotic stress, ion transport disorders, limiting sequestration capacity of the midgut and Malpighian tubules) require further studies in BSFL, the use of chelating agents could be a promising avenue to enable the upcycling of residues with high calcium content (e.g., marine residues, digestate, milk processing coproducts) through BSFL bioconversion.

Carbon balance and emission reduction in BSF organics conversion significantly enhanced by microbial supplementation

M.Z. Li^*, M.M. Cai and J.B. Zhang

Huazhong Agricultural University, National Key Laboratory of Agricultural Microbiology, 1 Lion Mountain Street, Hongshan District, 430070 Wuhan City, Hubei Province, P.R. China; ^*mzli@webmail.hzau.edu.cn

Global organic waste generation, projected to exceed 6 million tons daily by 2050, drives severe pollution and greenhouse gas emissions. Conventional waste management methods, such as landfilling and composting, contribute significantly to these issues through nitrogen loss and methane release. Black soldier fly larvae bioconversion has emerged as a sustainable alternative that converts waste into valuable protein and biofertiliser. This study employed ¹³C-labelled sodium carbonate (Na₂¹³CO₃) to trace carbon dynamics during BSFL bioconversion of artificial feed, with and without supplementation of the functional microorganism Stenotrophomonas acidaminiphila (CF-5). Carbon emissions were quantified using absorption and multi-gas systems, and microbial community dynamics were profiled via 16S rRNA sequencing and ¹³C-DNA-based stable isotope probing. The results showed that BSFL conversion alone reduced cumulative CO₂ emissions by 23.37% compared to natural fermentation over 51 days. CF-5 supplementation further reduced emissions by 13–13.38% and suppressed system carbon loss by approximately 75%. Isotopic tracing revealed that CF-5 enhanced ¹³C assimilation into larval biomass (increasing carbon content by 11–28%), with significant enrichment in fat bodies and exuviae, while concurrently decreasing ¹³CO₂ release. Microbial analysis indicated that CF-5 enriched Actinobacteria in BSFL guts and upregulated key carbon fixation pathways — including the Calvin–Benson–Bassham cycle in the substrate and the Wood-Ljungdahl, rTCA, and 3HB pathways in the guts — without altering overall microbial diversity. The results indicated that functional microorganisms such as CF-5 optimise carbon flux in BSFL systems, augmenting carbon sequestration by redirecting waste-derived carbon into larval biomass. This approach establishes an efficient strategy for carbon-neutral organic waste management.

Valorisation of challenging waste streams using Hermetia illucens for animal feed and human food

S.A. Mallard^1,2*, L. Swinden¹, L. Donnellan², K. Wilkinson² and P. Deo²

¹ASC, 467 Vulture Street, East Brisbane, QLD 4169, Australia; ²Adelaide University, North Terrace, Adelaide, SA 5005, Australia; ^*smallard@asc.co

Black soldier fly larvae (BSFL) are increasingly recognised as an efficient and sustainable tool for converting organic waste streams into high-quality protein for animal feed and potential human food. However, many of the abundant food-chain wastes used as feeding substrates (e.g., grape marc (GM), olive pomace (OP), paunch/offal, pre-consumer food waste) are considered “challenging” due to high lipid, phenolic, fibre, or salt content, which can impair larval growth and survival. These substrates may also contain contaminant residues that implicate the final BSFL safety. This study investigated the performance and safety of BSFL reared on 11 real-world, complex substrates that are currently under-investigated due to these technical challenges. Neonates from commercial Australian colonies were reared to maturity on the following substrates: mixed fruit/vegetable, GM, OP, fermented banana skins, creamed corn, dog food or cattle paunch/offal mixture. Larval growth, substrate reduction, bioconversion rate, and final biomass yield were recorded. Proximate composition and fatty acid profiles of BSFL were determined, with >300 pesticide residues (including glyphosate, cyprodinil, and metalaxyl) monitored in substrate, larvae and frass. BSFL achieved good performance on most substrates: growth 49–178 mg/larvae; waste reduction 52.9–78.5% (w/w); and feed conversion ratio 14.7–22.4 dry matter (DM). Grape marc and olive pomace at 50% inclusion, traditionally considered difficult, supported larval final weights of 106 ± 6 mg and 109 ± 6 mg (fresh), respectively, with protein content 38–43% and fat 26–35% DM. Larval biomass contained favourable ω-6/ω-3 ratios <8:1 in several treatments. Pesticide carry-over was compound-specific: metalaxyl was eliminated, cyprodinil was quantitatively transferred to frass, and glyphosate showed partial uptake (10–43% of substrate concentration as dry matter) with significant excretion. Highly toxic compounds such as paraquat accumulated in some BSFL protein fractions, highlighting the need for substrate screening. BSFL demonstrated remarkable adaptability to high-phenolic GM, high-lipid OP, and high-sodium (paunch and dog food) substrates, producing nutritionally valuable protein meals with quantitative performance comparable to fruit and vegetable substrates. Pesticide fate was predominantly favourable, with strong reduction or excretion for most compounds, confirming the bioremediation potential of BSFL. These results support the safe upscale of BSFL technology for valorising challenging food-chain side streams that are currently landfilled, contributing to circular economy and sustainable feed and food production.

Integrating sustainability assessment and advanced analytics in insect farming

A. Francis¹, V. Khieya¹ and S. Smetana^1,2*

¹German Institute of Food Technologies — DIL e.V., Prof.-Von-Klitzing Strasse 7, 49610 Quakenbrück, Germany; ²Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany; ^*s.smetana@dil-ev.de

Insect farming is increasingly recognised as a promising pathway for sustainable food and feed production, yet its effective scaling is constrained by fragmented analytical approaches and limited integration of sustainability assessment with advanced data analytics. While environmental, economic, and social impacts are routinely discussed, there is a lack of unified methodological frameworks capable of supporting robust evaluation, optimisation, and data-driven decision-making in research, industrial and policy contexts. This gap reduces comparability between studies, limits reproducibility, and weakens the evidence base required for large-scale deployment and regulatory acceptance. This study synthesises current analytical practices in insect farming, with a specific focus on the integration of sustainability assessment tools and advanced data analysis methods. Sustainability evaluations based on life cycle assessment (LCA), life cycle costing (LCC), and social life cycle assessment (S-LCA) are reviewed as key instruments for capturing environmental performance, economic viability, and social implications of insect-based systems. In parallel, there is an obvious development of advanced analytics, including decision support systems, bioeconomic models, machine learning techniques, and automated monitoring tools, to optimise production efficiency, resource use, behaviour analysis, and selective breeding. Even though there are advances in both fields, their cross-application is limited. Advanced analytics has a strong potential to improve not only operational efficiency and predictive capacity but also automate and standardise sustainability assessment if assured with good data quality and interoperability. It can solve the issues of methodologic heterogeneity and inconsistency in reporting practices of sustainability assessment. Furthermore, emerging digital technologies, including blockchain and predictive analytics, offer additional opportunities to enhance traceability, regulatory compliance, and food safety assurance in automated regime. Overall, the integration of harmonised sustainability assessment frameworks with advanced analytical tools is critical for advancing insect farming from experimental systems to scalable, resilient, and compliant production models. Establishing unified guidelines, improving data infrastructures, and aligning analytical methods with regulatory and industry needs will be essential to unlock the full sustainability potential of insect-based food and feed systems.

Ingestion of polyurethane foam by Tenebrio molitor larvae: effects of nutritional and probiotic supplementation

G. Fondevila^1*, V.C. Sein-Echaluce² and M. Fondevila¹

¹Universidad de Zaragoza, Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Miguel Servet 177, 50013 Zaragoza, Spain; ²AITIIP Centro Tecnológico, Polı́gono Industrial Empresarium, Romero 12, 50720 La Cartuja Baja, Zaragoza, Spain; ^*guillermofondevila@gmail.com

Insect species such as Tenebrio molitor have been proposed as an alternative to mitigate the environmental impact of plastic waste. A 28-day experiment was conducted to evaluate the ingestion of polyurethane foam by T. molitor larvae and to assess their ability to use it as an energy source when supplemented with a nutritional substrate and a bacterial probiotic mixture. Eighteen trays containing 60 larvae (average initial weight: 25.3 mg) were assigned to three treatments, in which polyurethane foam was offered ad libitum as the sole substrate (PU) or supplemented with wheat bran (0.4 g per week) either without (PUB) or with a mixed bacterial culture (Kocuria rhizophila, Priestia megaterium and Cupriavidus respiraculi) administered as a probiotic (PUBP). Fresh carrot was provided twice a week as a source of water. Despite the limited nutritional value of polyurethane, larval mortality was low (average 7.2%) and did not differ among treatments. Larval growth and total mass produced were greater in PUB and PUBP than in PU ($P\mathrm{<}0.001$), although numerical improvements observed in PUBP compared to PUB were not statistically significant. Polyurethane intake reached 9.85 mg/larva and did not differ among treatments, whereas frass production was higher in PUBP than in PU, with intermediate values observed in PUB ($P\mathrm{<}0.001$). Microscopic examination of frass revealed the presence of numerous polyurethane particles across all treatments, indicating incomplete polymer degradation. In conclusion, T. molitor larvae were able to ingest polyurethane foam independently of additional nutrient supplementation. The provision of small amounts of wheat bran improved larval performance without affecting plastic intake. Although larval depolymerising capacity and the potential contribution of probiotic supplementation could not be conclusively demonstrated under the present experimental conditions, the numerical improvements in productive performance observed in probiotic-supplemented larvae suggest that a potential enhancement of plastic degradation cannot be completely discarded.

Monitoring and manipulating the microbiota of yellow mealworm larvae (YML) to improve their bioconversion performance

M. Sajid^1*, A.-A. Durand¹, E. Yergeau¹, J. Laur², G. Vandenberg³ and P. Constant¹

¹Armand-Frappier Santé Biotechnologie Research Centre-Institut national de la recherche scientifique, Laval, QC, Canada H7V 1B7; ²Université de Montréal, Montreal, QC, Canada H3C 3J7; ³Université Laval, QC, Canada G1V 0A6; ^*menailsajidhussain@gmail.com

Tenebrio molitor (YML) offers promising solutions for organic waste upcycling into protein and biofertiliser. However, upcycling waste is complicated by its instability, variability and digestibility. Therefore, this project seeks to implement a microbiota-centric approach to improve bioconversion performance and reduce the environmental footprint of waste upcycling bioprocess. Considering the central role of microbes for insect host and feedstock stability, we hypothesized that bioconversion performance is related to microbial community structure and functioning in feedstock and YML. The hypothesis is tested by longitudinal monitoring of YML farm and laboratory-scale experiments. The farm-scale experiment began in June 2025. YML conversion rate, mortality, farm temperature and microbiome of feedstock, larvae and frass are monitored to relate microbiome and environmental conditions to farm productivity. Microbial taxa whose distribution is beneficial or deleterious to YML productivity will be identified and directed isolation procedures will be deployed to demonstrate how their introduction in the YML affects biomass production yield. While farm monitoring efforts continue until June 2026, preliminary data assess variation of microbiome composition, potential functions and diversity in YML, frass and feedstock. These observations also support the second, lab-scale experiment seeking to manipulate the microbiota of wet feedstock and YML for better yield. The lab-scale experiment aimed to improve the shelf-life of wet diet by lactic-acid producing bacteria (LAB) fermentation. Besides feedstock stabilization, few LAB strains were shown to act as probiotics in YML. A collection of LAB associated with YML was assembled, and isolates were subjected to DNA extraction and 16S rRNA gene PCR for molecular identification. Isolates were screened for their fermentation performance using fruit-vegetable mixture from waste management industry. Around 50 colonies were isolated from YML under anaerobic conditions, mainly representing the genera Lactococcus, Enterococcus, Staphylococcus, Weissella and Brevibacterium. Preliminary results reporting the feedstock fermentation impact for 1,7 or 14 days on YML growth performance and feedstock quality will be presented. In conclusion, this communication is expected to give rise to inspiring exchanges about the challenges and promises of microbiological applications in ento-technologies to improve organic waste upcycling.

A modular decision support system (DSS) for cost and environmental optimisation in insect production

D. Rastogi^*, V. Khieya, J.I. Petrusan and S. Smetana

DIL Deutsches Institut für Lebensmitteltechnik e.V., Food Data Group, Prof.-von-Klitzing-Strasse 7, 49610 Quakenbrück, Germany; ^*d.rastogi@gmail.com

Insect-based protein production offers a potential for reducing the environmental footprint of food and feed systems while providing protein-rich outputs. However, large-scale production is often constrained by high feed and processing costs, inconsistent nutritional contents, and processing-related environmental impacts. This study presents the development of a data-driven decision support system (DSS) to assist insect producers in minimising production costs and environmental impacts while maintaining higher protein content and potential financial gains. The DSS integrates experimental performance data, feed costs and process-level resource use into a modular optimisation framework. Feed requirements and costs per unit of dried larval biomass are estimated using feed conversion ratios and biomass yields, including mixtures of multiple feed types to identify cost-efficient combinations with high protein output. Environmental impacts are modelled across the production chain, distinguishing feed-related effects from processing inputs (electricity, heat, water and land) in relation to country and insect species. Cost and environmental indicators are combined using multi-criteria optimisation, including constraint-based filtering and Pareto-front analysis, supported by linear programming and heuristic optimisation methods depending on data availability. In a preliminary analysis of five feed types from Greece, the cost and environmental impacts of producing 1 kg of yellow mealworm dry larval biomass were evaluated using blanching and oven drying as the standard processing route. A feed mixture of oats by-products (65%) and maize by-products (35%) resulted in the lowest carbon emissions (0.122 kg CO₂-eq), representing a 75% reduction compared to the mean emission value (0.474 kg CO₂-eq). The estimated feed cost for this mixture was approximately €0.61 per kg of dry larvae, excluding processing costs. DSS demonstrates how heterogeneous experimental data can be translated into an actionable tool for sustainable insect production. By integrating cost modelling, environmental assessment, and optimisation logic within a software-driven framework, the DSS provides a practical foundation for informed decision making in insect-based production systems and is being implemented as a web-based application for stakeholder use.

Field evaluation of black soldier fly frass as an organic fertiliser for maize production in southeastern Madagascar

C.L. Solofondranohatra^1*, C. Andrianorosoa Ony¹, S. Burza² and B.L. Fisher^1,3

¹Madagascar Biodiversity Center, Parc Botanique et Zoologique Tsimbazaza, Antananarivo 101, Madagascar; ²Health In Harmony, Portland, OR, USA; ³California Academy of Sciences, Department of Entomology, San Francisco, CA, USA; ^*lovacedrique@gmail.com

Food insecurity remains a major challenge for smallholder farming communities in Madagascar, underscoring the need for locally available and sustainable soil fertility inputs. Black soldier fly frass (BSFF), a by-product of insect-based organic waste conversion systems, is an emerging organic fertiliser; however, field-based evidence of its agronomic performance under smallholder conditions remains limited. This study evaluated the effects of composted BSFF (CBSFF) and fresh BSFF (FBSFF) on maize (Zea mays L.) growth and yield in comparison with cattle manure (CM) and an unfertilised control in acidic sandy clay soils of southeastern Madagascar. A field experiment was conducted using a nitrogen application rate of 43 kg N ha^−1, corresponding to approximately one-third of the national recommendation and reflecting prevailing farmer practice in the region. Germination rate, plant height, grain yield, and agronomic efficiency of nitrogen (AE_N) were assessed. Maize treated with FBSFF exhibited significantly lower germination rates than all other treatments, indicating sensitivity to application timing. No statistically significant differences in final plant height or grain yield were observed between CBSFF and CM; however, plants fertilised with CBSFF were on average 6% taller than those receiving CM and 13% taller than those receiving FBSFF. Grain yield under CBSFF was 38% higher than under CM. All fertilised treatments produced significantly greater yields than the unfertilised control, which resulted in no harvestable maize. CBSFF showed the highest agronomic efficiency of nitrogen (46 kg grain per kg N), although differences among fertiliser treatments were not statistically significant. Overall, results indicate that BSFF, particularly in composted form, can supply sufficient nitrogen to support maize production under smallholder conditions. Composted BSFF appears more suitable for pre-planting application, while fresh BSFF may be better applied after crop establishment. Even at sub-recommended fertiliser rates, BSFF demonstrates potential to enhance maize productivity within circular, insect-based waste management systems and contribute to local food security. Further research across multiple agroecological zones, including economic analyses of cost, labor, and transport, is recommended to support broader adoption.

A review on insects and urban circularity: future pathways for food loss and waste interventions

O. Sanusi^1*, K. Kantamaneni¹, P. Tiwasing², A. Andongma³, U. Ravindran¹, F. Baker⁴ and D. Mwai¹

¹University of Lancashire, University of Lancashire Business School, Fylde Road, Preston, PR1 2HE, UK; ²University of Nottingham, University Park, Nottingham NG7 2RD, UK; ³Lancaster University, Lancaster, LA1 4YW, UK; ⁴University of Gloucestershire, The Park, Cheltenham GL50 2RH, UK; ^*OSanusi@lancashire.ac.uk

Over one-third of food produced for consumption is either lost or wasted in the United Kingdom (UK), which is 25% of edible food costing the economy over ¿20 billion. Meanwhile, a significant percentage of the population experiences food insecurity and hunger across the globe. To avoid these circumstances, as policy attention gradually shifted towards circular economy explanations, such as simpler recycling legislation in the UK, voices advocating for alternative interventions for organic waste valorisation and sustainable food system transitions likewise amplified. Therefore, this study investigates the role of insects as a circular bio-solution in the city value chain and design, examining their potential impact on organic waste interventions in the UK. Drawing on food system theory and city-region perspectives, this study assesses the insect-based interventions as an integrated unit for managing FLW, where organic waste from metropolitan areas is considered a resource and transformed into valuable produce for both urban and rural adoption needs. All these steps are carried out by reviewing literature on FLW, unpacking policy, practice, Life Cycle Assessment, circularity, insect bio-conversion and valorisation, towards determining key enablers, players, barriers, pathways and opportunities for adoption. Findings highlight the potential of insect-based approaches in rethinking urban organic waste design and propose a new “insect city approach” to decentralising FLW and organic waste, often mismanaged and a missed opportunity for sustainable food systems, while also revealing key policy drawbacks and green investment prospects. This research argues that green spaces in cities are vital spaces for insect bio-solution take-off and integration into FLW interventions, hence the need for a robust policy framework and implementation. By reframing insects as enablers of city circularity, this study contributes to debates on urban design and circular food systems and provides insights for various stakeholders exploring innovative pathways to integrate FLW management into urban food systems and shift the burden of FLW from waste redistribution companies.

Recirculating BSF frass for an optimised waste bioconversion and high-quality fertiliser production

I. Guidini Lopes^1* and C. Lalander²

¹Swedish University of Agricultural Sciences, Department of Biosystems and Technology, Box 190, 23422 Lomma, Sweden; ²Swedish University of Agricultural Sciences, Department of Energy and Technology, Box 7032, 75007 Uppsala, Sweden; ^*iva.guidini.lopes@slu.se

The use of drying agents when treating waste with Hermetia illucens larvae is a common industrial practice that increases costs and affects the activity’s environmental performance. Frass recirculation, in which frass is used as a dietary component while simultaneously serving as a drying agent, has been proposed as an alternative. In this study, frass recirculation (15-25% inclusion level) was studied for various organic wastes — food waste, spent grains, pig manure, fruits and expired chicken feed — with initial DM contents of 29, 24, 22, 13 and 46%, respectively, which after a first cycle of bioconversion generated frass with DM contents ranging between 46 and 75%. Frass obtained from recirculation cycles were analysed for their maturity (pH, electrical conductivity, seed germination) and composition (plant nutrients, microorganisms and phytohormones). Frass collected from a bioconversion with four recirculation cycles were used as fertilisers for cultivating pak choi (Brassica rapa), and its effects on plant growth and photosynthetic performance were assessed. Recirculation appeared viable for most of the tested substrates, achieving good process performance in relation to larval yield (comparable with the controls, without recirculation). The exception was spent grains, likely due to the high bulkiness of the generated frass, which increased substrate depth. Recirculating frass at least once resulted in increased maturity of the fertiliser in comparison to fresh (non-recirculated) frass, with seed germination indexes being up to 10-fold higher in recirculated frass. Recirculation was found to shift the frass’ microbial community. Firmicutes and Proteobacteria were more prominent in recirculated frass, while Actinobacteria was more prominent in fresh frass. Specific phytohormones were screened in frass samples, such as cytokinins (up to 70 pmol (g DM)^−1) and auxins (up to 1050 nmol (g DM)^−1), positively correlated with humic substances. Specific bacteria (e.g. Oceanobacillus, Cerasibacillus and Morganella) positively correlated with phytohormones, suggesting that the biostimulant effects of frass may be derived from its microbial composition. Pak choi plants fertilised with recirculated frass reached similar fresh weight (241 ± 19 g fresh weight) in comparison to control plants (232 ± 44 g) cultivated with a chemical fertiliser, while achieving 5-15% higher photosynthetic efficiency. Frass recirculation is a promising alternative to reduce the need for drying agents, resulting in positive effects on process efficiency and frass fertilising quality.

Impact of predigestion of organic residues on black soldier fly protein and its use in organic post-weaning pigs

R. Caparros Megido^1*, H. Luttenschlager¹, J. Carpentier¹, Y. Beckers¹, S. Finet², J. Wavreille³ and F. Francis¹

¹Gembloux Agro-Bio Tech, Passage des déportés 2, 5030 Gembloux, Belgium; ²Biowaste Upcycling, Rue Culée 77, 1410 Waterloo, Belgium; ³CRAW, Rue de Liroux 8, 5030 Gembloux, Belgium; ^*r.caparros@uliege.be

Black soldier fly (BSF) Hermetia illucens larvae offer a robust option to upcycle food loss and waste into high-quality proteins for animal nutrition in circular and organic systems. Within the ASTIPPOR project, we established in Wallonia a pipeline that predigests local organic food surpluses and brewers’ spent grains using a thermophilic aerobic digester, rears BSF larvae on the stabilized substrate, and valorises defatted BSF meal in post-weaning piglet diets under organic conditions. We first assessed five physical and microbial pretreatments (grinding, heating at 50 °C, and thermophilic microbial inoculation, alone or combined) of a mixture of unsold fruits and vegetables, bread, and brewers’ spent grains on BSF performance and composition. All larval groups reached high final weights (0.278–0.307 g at day 12; 0.326 g on chicken feed) with survival rates above 92%, while predigestion significantly altered larval lipid content and fatty acid profile but not protein or ash content (≈30–31% crude protein DM, ≈32% lipids DM, ≈3.5% ash DM). Predigested substrates produced larvae with lower total lipid and saturated fatty acid proportions and slightly higher unsaturated fatty acids, improving the protein-to-lipid ratio for monogastric diets. Defatted BSF meal was then used to partially replace the protein core (soybean meal, pea meal, potato protein) in organic post-weaning piglet diets. In a five-week trial with 72 female piglets, four isoenergetic and isonitrogenous diets were tested: a control without insects and three diets where 15, 25, or 35% of the protein core was replaced by defatted BSF meal. At 15% replacement, growth performance, weekly gain, body-weight trajectories and feed conversion ratio did not differ from the control, whereas higher inclusion levels caused modest but significant increases in FCR and slightly lower final body weights. Cumulative FCR remained within typical organic ranges, but economic modelling showed that the break-even price of insect meal rapidly declines when inclusion exceeds 15% of the protein core, constraining economic viability at current market prices. Overall, ASTIPPOR demonstrates that thermophilic microbial and physical predigestion of local organic residues can generate BSF larvae with a nutritionally favourable profile, and that defatted BSF meal can safely replace up to 15% of the protein core in organic post-weaning piglet diets without impairing performance.

Upcycling regional fruit and vegetable by-products into eco-feed using black soldier fly (Hermetia illucens)

B.W. Koo^*, K.W. Park, S.M. Ji, S.Y. Kim and J.H. Song

National Institute of Agricultural Sciences, South Korea, 166 Nongseonmyeongro, Iseo-myeon, 55365 Wanju, South Korea; ^*bonwoo9@korea.kr

Globally, the black soldier fly (Hermetia illucens, BSF) has emerged as a major insect species for organic waste bioconversion, supported by increasing industrial deployment in feed and waste-processing sectors. In South Korea, the first-tier sales value of BSF increased from KRW 0.8 billion in 2017 to KRW 1.09 billion in 2021, accompanied by an increase in the number of BSF farms from 166 to 224, indicating rapid expansion of on-farm production capacity based on national insect industry statistics. This study quantitatively evaluated the bioconversion performance of BSF larvae fed regional fruit and vegetable by-products, focusing on waste reduction efficiency, biomass conversion, and key biological parameters directly relevant to industrial eco-feed utilisation. BSF larvae were reared on apple pomace, mandarin waste, and oriental melon waste under controlled conditions (26 °C, 60% RH), and larval growth, developmental time, survival, fecundity, waste reduction ratio, and bioconversion efficiency were measured. Larvae decomposed 48.0% of apple pomace and 61.5% of mandarin waste, yielding bioconversion efficiencies of 9.1% and 12.1%, respectively, with no significant differences in larval survival (≥88%) or adult fecundity (≥99%) compared with a commercial control diet. When fed oriental melon waste, BSF larvae achieved a substantially higher waste reduction rate of 80.5% and a bioconversion efficiency of 11.8%, although larval development was prolonged and individual larval biomass was reduced relative to nutrient-balanced feeds. Across substrates, waste reduction efficiency was strongly associated with substrate physical properties (semi-liquid texture, low fibre content), whereas larval biomass accumulation was primarily constrained by protein and lipid availability. These results demonstrate that region-specific horticultural by-products can be reliably processed by BSF larvae with predictable trade-offs between waste reduction efficiency and larval biomass yield. The quantitative outcomes provide a technical basis for (i) substrate-specific precision rearing strategies, (ii) selective breeding targeting improved conversion efficiency under low-protein substrates, and (iii) optimisation of reproductive performance for large-scale BSF eco-feed system integration.

Treatment of food processing wastewaters by using black soldier fly larvae

V. Grossule^* and M. Henjak

University of Padova, DICEA, Department of Civil, Architectural and Environmental Engineering, via Marzolo 9, 35131 Padova, Italy; ^*valentina.grossule@unipd.it

The use of black soldier fly larvae (BSFL) is a promising alternative for treating high-organic-content (HOC) wastewaters (WWs), with high efficiency and the production of valuable biomass. Although widely applied to solid biowaste, their use in wastewater is limited by drowning. This was overcome by providing inert solid support (plastic granules) saturated with wastewater, allowing larvae to dive to feed and resurface to breathe. This process is termed LarWaR (Larvae for Wastewater treatment and Resource recovery). This study evaluates LarWaR on six food-processing WWs: bakery (BA), brewery (BR), dairy (D), juice (J), slaughterhouse (S) and winery (W). Suitability was assessed based on organic content and macronutrient composition (protein, non-fibre carbohydrates (NFC), lipids). Performance was evaluated in batch tests by monitoring larval growth, mortality, and removal of organic carbon, nitrogen, and phosphorus. Each reactor (13.5 × 13.5 × 5.5 cm) contained 80 six-day-old larvae (8.4 mg wet weight, SE = 1.08; density 0.4 larvae/cm²) and a plastic granular bed (VALOX, 2 – 3 mm) saturated with 150 ml WW. Reactors were covered with permeable fabric and perforated lids to allow aeration and prevent escape or oviposition. WW was replaced weekly for 49 days. WWs differed in organic concentrations, resulting in different applied loads. Each WW was tested in triplicate (21 reactors total) at 25 – 30 °C, 18/6 h light/dark. All WWs achieved TOC removal of 76 – 93%, BOD 86 – 97% and COD 75 – 85%. Concentrations halved within 2 weeks and stabilized by 3 weeks, indicating a short start-up compared to conventional treatments. The specific consumption rate (mg C/larva per day) increased proportionally with organic load, following a Michaelis – Menten relationship, while removal efficiency decreased with increasing load. This relationship can be applied to design LarWaR systems and predict performance across HOC WWs. Considering both treatment and larval development, BA, BR and W (TOC > 1500 mg C/l; load 0.4 – 0.55 mg C/larva per day) performed the best, with the lowest mortality (<20%) and the highest larval weight (109 mg, SE = 4.5). These WWs had higher levels of volatile fatty acids (VFAs) and ethanol than NFC, suggesting that larvae used them as additional carbon sources, thereby improving macronutrient balance and performance. Although LarWaR effectively removes organics, nitrogen and phosphorus were only partially removed, indicating the need for additional treatment steps to address these contaminants. Results highlight the importance of organic concentration and macronutrient quality, which can be optimized by mixing WWs to enhance performance and applicability.

Gut immune responses in black soldier fly larvae: the role of the gut microbiota to prime pathogen resistance

M. Marzari^1*, N. Sibinga², D. Bruno¹, M. Van Der Borght², G. Tettamanti¹ and J. De Smet²

¹University of Insubria, Biotechnology and Life Science, Via J. H. Dunant 3, 21100 Varese, Italy; ²KU Leuven, Department of Microbial and Molecular Systems (M²S), Kleinhoefstraat 4, 2440 Geel, Belgium; ^*mmarzari@uninsubria.it

Organic substrates used for rearing black soldier fly larvae (BSFL) may harbour pathogenic bacteria, such as Staphylococcus aureus, Salmonella spp., Escherichia coli and Bacillus cereus, posing potential risks to both human health and animal welfare. Nevertheless, BSFL thrive in pathogen-rich environments and can significantly reduce bacterial loads in the rearing substrate, suggesting the presence of a highly effective immune system. While the cellular and molecular mechanisms underlying systemic immune responses to infections have been partially characterised, the contribution of gut microbiota and gut physiology to pathogen defence remains poorly understood. To investigate the role of the gut microbiota as a factor for immune stimulation in the defence mechanisms of BSF, we adopted two complementary challenges to assess changes in host immunity within an axenic BSFL rearing system: (i) S. aureus and E. coli were inoculated in the rearing substrate (7 log CFU/g), to assess pathogen resistance in the gut environment and (ii) the same microbes were injected into the hemocoel (7 log CFU/ml) to assess pathogen resistance within the larvae. Challenged axenic larvae exhibited higher survival rates following the direct injection than control, while gut microbial loads were comparable to those of controls after the dietary challenge, indicating that gut microbiota is not essential for the activation of BSFL immune defences. Larvae remain, indeed, immunologically primed despite the absence of a gut microbial community. However, while control larvae significantly reduced bacterial loads in the substrate after the dietary challenge, pathogen levels remained high under axenic conditions, highlighting the importance of interactions between BSFL and the environmental microbial community in pathogen suppression. The gut immune response will be further analysed to verify that no underlying shifts in the activity of immune effectors have occurred. This study provides new insights into the complex interplay between host immunity, gut microbiota, and environmental microbes in BSFL. Such insights offer a foundation for future research that aims to develop strategies that enhance larval immune competence, with potential applications in improving biosafety, resilience and sustainability in large-scale waste bioconversion systems.

Well-timed provision of digestible carbohydrates improves manure utilisation by housefly larvae

D.G.A.B. Oonincx^*, L. Pisa, W.H. Hendriks and G. Bosch

Wageningen University and Research, Animal Nutrition Group, department of animal sciences, De Elst 1, 6708 WD Wageningen, The Netherlands; ^*dennis.oonincx@wur.nl

Rearing housefly larvae (Musca domestica L.) on livestock manure, such as chicken excreta, can contribute to sustainable feed production and waste reduction. As chicken excreta contains low amounts of digestible carbohydrates, adding these could enhance larval biomass and nitrogen retention. However, ontogenetic changes in morphology and enzymology indicate that larval use of carbohydrates may differ over larval stages. This study tested how carbohydrate type (glucose, starch, cellulose, fresh excreta as control) and timing of supplementation (0 h or 72 h) affects larval growth, bioconversion and residue characteristics. Glucose and starch improved larval performance compared to fresh manure and cellulose. Late (72 h) supplementation was most effective, with late glucose increasing individual larval mass by 12%, total biomass by 33% and survival by 19%. Late starch increased total biomass by 30% and survival by 24%, while effects on individual mass were small (2% increase). Dry matter bioconversion efficiency increased by 27% with late glucose and 20% with late starch, whereas late excreta addition caused an 18% decline. Nitrogen bioconversion efficiency rose by 36% with late glucose and by 37% with late starch. Residue ammonia increased with all supplementations but less for with late additions than with early additions, especially for starch. Residue pH increased with all supplementations, with excreta and cellulose showing the largest early-late increases (0.3-0.8), early and late glucose numerically decreasing and late starch producing the overall lowest pH. This study shows that adding starch to fresh chicken manure later in the larval development period improves survival, bioconversion and yield.

Enabling waste-based insect feed systems for circular agri-food transformation: governance lessons from Uganda

F. Lwiza^* and A.J. Komakech

Makerere University, PO Box 7062, Kampala, Uganda; ^*lwizaflorence@gmail.com

Insects reared on organic waste streams offer a promising pathway for advancing circular agri-food systems by converting waste into high-quality animal feed while reducing environmental pressures. In low- and middle-income countries, waste-based insect feed systems are particularly relevant due to high feed costs, widespread organic waste mismanagement, and growing demand for sustainable livestock production. Despite strong technical and agronomic evidence, adoption and scaling of insect-based feed systems remain limited. This paper examines how policy, legal, regulatory, and institutional frameworks shape the emergence and scaling of waste-based insect feed systems in Uganda, with complementary insights from waste-derived organic fertiliser pathways. The study combines a systematic review of national policies, laws, regulations, and standards governing waste management, animal feed, and fertilisers with a stakeholder power-influence analysis to explore the political economy of implementation. Findings reveal strong alignment between insect-based circular economy principles and national objectives on waste reduction, livestock productivity, and soil health. However, regulatory ambiguity around the classification of organic waste and waste-derived insect feed, fragmented institutional mandates, and centralized compliance systems constrain operationalization. High compliance costs and procedural complexity disproportionately exclude micro, small and medium enterprises, which dominate insect production and innovation. Power-influence asymmetries further reinforce regulatory inertia, as actors with practical expertise in insect rearing and waste valorisation have limited influence over rule-making processes. The paper demonstrates that regulatory clarity, institutional coordination, and inclusive governance are critical for unlocking the scaling potential of waste-based insect feed systems and advancing circular agri-food transformation.

Synthesising the evidence on Salmonella spp. inactivation in black soldier fly larvae bioconversion of biowastes

C. Lalander^*, E. Were and I. Guidini Lopes

Swedish University of Agricultural Sciences, Box 7032, 750 07 Uppsala, Sweden; ^*cecilia.lalander@slu.se

Insect bioconversion has been recognised as a circular waste management method and protein production strategy. Life cycle assessments show that environmental benefits require the use of “true” waste substrates. Post-consumer food waste may contain pathogenic microorganisms, including zoonotic Salmonella spp. At the Environmental Engineering group at SLU, we have studied Salmonella inactivation during black soldier fly larvae (BSFL) bioconversion since 2013 and consistently observed inactivation (Lalander et al., 2013, 2015). The frequency of contaminated feed influenced reduction (Lopes et al., 2020), with lower total reduction (3.3 log₁₀) under multiple feedings compared to a single feeding (6.2 log₁₀). Frass alone also inactivated Salmonella when reinoculated without larvae, suggesting antimicrobial activity. In a later study (Lalander et al., 2025), neither treatment scale (2400 vs 231 cm²) nor 20% frass inclusion affected inactivation, despite the hypothesis that frass would enhance it. In our most recent work, we examined larval density (1, 3, 6 larvae/cm²; 0.6, 0.2, 0.1 g VS/larva) and substrate composition (carbohydrate-rich (CHO) vs protein-rich (Pr) diets). In the CHO diet, reduction appeared mainly chemical, as similar reductions occurred in controls; this was not observed in the Pr diet. Under repeated contamination, inactivation occurred only in the Pr diet and decreased at higher densities. At high densities, larval weights were <150 mg/larva compared to 165–200 mg/larva at lower densities, indicating suboptimal growth. The Pr diet had similar crude protein (approx. 30% DM) but lower crude fat (12% vs approx. 30% DM; Lopes et al., 2024). It could be speculated that reduced inactivation at higher densities may be linked to nutrient or energy deficiency. Lalander, C., Diener, S., Magri, M.E., Zurbrügg, C., Lindström, A., and Vinnerås, B., 2013. Faecal sludge management with the larvae of the black soldier fly (Hermetia illucens) – from a hygiene aspect. Science of the Total Environment 458-460: 312-318. Lalander, C., Fidjeland, J., Diener, S., Eriksson, S. and Vinnerås, B., 2015. High waste-to-biomass conversion and efficient Salmonella spp. reduction using black soldier fly for waste recycling. Agronomy for Sustainable Development 35: 261-271. Lalander, C., Lopes, I.G., Gyftopoulos, N. and Vinnerås, B., 2025. The impact of scale and frass recirculation on pathogen inactivation dynamics in black soldier fly larvae bioconversion. Frontiers in Microbiology 16: 153948. Lopes, I.G., Lalander, C., Vidotti, R.M. and Vinnerås, B., 2020. Reduction of bacteria in relation to feeding regimes when treating aquaculture waste in fly larvae composting. Frontiers in Microbiology 11: 1616. Lopes, I.G., Wiklicky, V., Vinnerås, B., Yong, J.W.H. and Lalander, C. 2024. Recirculating frass from food waste bioconversion using black soldier fly larvae: Impacts on process efficiency and product quality. Journal of Environmentl Management 366: 121869.

Valorisation of school canteen food waste with black soldier fly larvae in Central Europe via decentralised insect farms

J. Vasickova^1*, R.F. Calderon Barrientos², G. Obersteiner³, E. Schmied³ and M. Gold¹

¹REPLOID Group AG, Durisolstraße 6, 4600 Wels, Austria; ²REPLOID Deutschland GmbH, Löbener Weg 7, 04523 Pegau, Germany; ³BOKU, Gregor-Mendel-Straße 33, 1180 Vienna, Austria; ^*vasickova@reploid.eu

Food waste (FW) from school canteens is an underutilised resource that can be valorised through insect bioconversion, support circular food systems and local education. Because school FW production is decentralized, we investigated how school leftovers could be valorised by decentralized insect bioconversion using black soldier fly larvae (BSFL) within the EU Interreg FoodCIRCUS project. As EU regulations prohibit the use of postconsumer catering waste as insect feed, practical recommendations were formulated. The case study combined three methods. First, FW generation was quantitatively assessed in school canteen across five Central Europe countries. Monitoring surveys covered 23 schools in Austria, 17 in Italy, 6 in Poland, 9 in the Czech Republic and 10 in Hungary. Second, survey data were used to calculate mass flow for integrating FW into a decentralized insect bioconversion system (9500 t substrate/year). Third, representative FW samples collected over four days in schools in Vienna and Leipzig were used as substrates for BSFL. Wheat bran and water were used as a control diet. FW was minced (< 5 mm), adjusted to 73–75% moisture, and portioned into 9 kg batches in 40 x 60 cm boxes. Each batch reared 13 000 7-day-old BSFL for 8 days. Process performance indicators, such as larval yield, bioconversion efficiency, substrate reduction, frass output and characterisation of the chemical and microbiological properties of both larval biomass and frass were assessed. Monitoring in the five FoodCIRCUS partner countries provided a baseline data: Hungary showed the highest share of discarded food (48%) followed by Poland (46%), Austria (44%), the Czech Republic (35.5%), and Italy (29.3%). Austrian schools generated on average 12.6 kg of FW per canteen per day. Extrapolated to 250 Viennese schools, this equals about 3.1 t per day (≈ 567 t per school year). Scenario analysis showed that school FW from larger cities could supply 6% of substrate demand for a decentralized insect farm. The study confirmed that school FW is an efficient BSF substrate. Bioconversion efficiency of FW reached 18%, substrate reduction rate was up to 91%, frass output was lower on FW diets (0.83–0.97 kg vs 1.24 kg in the control), and no Salmonella was detected in the frass. The technical findings were linked to practical constraints such as collection frequency, pre-treatment and hygienisation options, and storage capacity. Despite current legal barriers, integrating school canteen food waste as a substrate material in decentralized insect farming is technically feasible.

Valorisation of olive pomace and other Algerian agro industrial by products in black soldier fly rearing

B. Hamdani^1*, Z. Cherifi¹, W. Merrouche¹, H. Hammoudi¹, N. Badis¹, N. Mabrek¹, M.T. Capucchio² and A. Abdelli¹

¹University of Bouira, Department of Agricultural Sciences, Bouira, Algeria; ²University of Turin, Department of Veterinary Sciences, Largo Braccini 2, 10095 Grugliasco (Turin), Italy; ^*bacem.hamdani@univ-bouira.dz

Hermetia illucens larvae are increasingly recognized as a sustainable biological agent to convert organic waste into protein-rich biomass and frass within a circular bioeconomy framework. In Mediterranean regions, agro-industrial activities produce substantial by products each year, notably olive pomace, constitutes a significant fraction of these residues. Their use remains limited, underscoring the importance of exploring alternative valorisation strategies. This study evaluated the efficiency of a mixed substrate formulated with 33% olive pomace, 34% brewery spent grain and 33% recycled bread on larval growth, substrate reduction, bioconversion efficiency, and nutritional quality of larval biomass and frass, compared to a standard Gainesville diet as a control. A total of 27 000 5-day-old larvae were allocated into two treatments with three replicates each. Larval growth parameters (weight, length, width) were recorded every two days. At the end of the trial, substrate reduction rate (WR), bioconversion efficiency (BRE), mortality rate (MR), meal yield, and the chemical composition of larval biomass and frass were determined. Larval growth was significantly influenced by substrate type ($p\mathrm{<}0.05$). On day 15, larvae fed the mixed substrate reached a higher average weight (0.233 ± 0.009 g), length (24.23 ± 0.31 mm), and width (5.30 ± 0.06 mm) than those fed the Gainesville diet (0.184 ± 0.006 g; 22.16 ± 0.61 mm; 4.52 ± 0.19 mm). Substrate reduction was significantly higher with the mixed diet (85.40%) than the control (66.40%, $p\mathrm{≤}$0.01), and bioconversion efficiency was more than twice as high (21.69 ± 0.63% vs 10.05 ± 0.20%). Mortality rates remained very low in both treatments. The chemical composition of larval meal was similar between treatments, with crude protein around 44% and ether extract between 41 and 44%. In summary, incorporating olive pomace even at moderate levels into BSFL diets allows efficient valorisation of local agro-industrial waste enhancing BSFL growth, substrate reduction, and bioconversion efficiency. It produces protein-rich biomass and nutritionally valuable frass, contributing to a sustainable circular bioeconomy. Acknowledgement: This research is supported by the EU-PRIMA program — project OLIWA Repurposing OLIve Waste in circular economy solutions for feeds, additives, packaging, and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus.

Regulatory latitude and black soldier fly rearing in North America

H.A. Hobbs^*

North American Coalition for Insect Agriculture (NACIA), 3531 N Dinwiddie Street, Arlington, VA 22207, USA; ^*aaron.hobbs@gmail.com

Insect, and more specifically black soldier fly (Hermetia illucens), rearing is increasingly promoted as a circular solution for organic waste valorisation. However, sustainability performance and economic viability differ substantially across regions, often reflecting regulatory context rather than biological or technical constraints. This study examines how feedstock regulations shape outcomes in insect-based waste valorisation systems. A comparative review of regulatory frameworks was conducted across Europe, the United States, Australia and China, focusing on feedstock eligibility, waste classification, and sustainability objectives. International trade data for insect-derived products were reviewed as an indicator of global production capacity and demand. Substantial regulatory divergence was identified. In the United States, Australia and China, black soldier fly systems are commonly integrated into waste management strategies and may utilise a broad range of organic inputs such as recovered food waste, post-consumer substrates, or manure. In contrast, other regions apply more restrictive feedstock rules, emphasizing precaution and feed safety. Despite these differences, global production continues to expand, with North America importing more than 10 000 tonnes of insect-derived products annually. These results demonstrate that sustainability outcomes are closely linked to regulatory design. Broader access to organic waste streams generally supports higher waste diversion and enhanced circularity, while more restrictive frameworks prioritize traceability and risk management. Both approaches reflect valid policy goals but lead to different operational and environmental results. We conclude that black soldier fly rearing is a flexible technology whose contribution to sustainable waste valorisation depends strongly on regulatory context. Evaluations of insect agriculture should therefore be region-specific. Aligning science-based risk management with circular economy objectives will be critical to maximising environmental benefits.

EcoeFISHent: Circular valorization of fish by-products through insect bioconversion

M. Tejeda^1,2*, S. Belleza-Oddon³, M. Aleksandrova¹, I. Mincheva¹, A.A. Melis³, L. Gasco³ and M. Bolard^1,2

¹Nasekomo EAD, Research and Development, Saedinenie Street 299, Lozen, Sofia 1151, Bulgaria; ²FlyGenetics AD, Research and Development, Saedinenie Street 299, Lozen, Sofia 1151, Bulgaria; ³University of Torino, Department of Agricultural, Forest and Food Sciences, Largo P. Braccini 2, Grugliasco, Turin, 10095, Italy; ^*marco.tejeda@nasekomo.life

The EcoeFISHent project, funded under Horizon Europe, addresses the challenge of underutilised fish processing by-products by developing and put in action innovative circular solutions. The project explores insect bioconversion as a sustainable pathway to transform these streams into valuable outputs. A dedicated pilot unit running by an Industry-Academia partnership is assessing the technical feasibility of using fish by-products as substrates for insect rearing. While current EU regulations currently restrict the use of certain fish-derived side-streams as insect substrate, EcoeFISHent focuses on research and demonstrate how high-quality fish side-streams can be safely bio converted, paving the way for more direct circular value chains and open the dialogue for future frameworks. Furthermore, the 10 tons annual biomass capacity of the pilot aims to produce frass, chitin/chitosan, and insect oil, unlocking applications beyond feed markets: frass as an organic fertilizer, chitosan for agriculture and pharmaceuticals, and oil for cosmetics. These outputs provide a qualitative demonstration of how insect technologies can complement other circular strategies, creating new value chains aligned with the European Green Deal, EU Bioeconomy Strategy, EU Circular Economy Action Plan and other sectoral or regional sustainability strategic frameworks. EcoeFISHent unites industry, research and technology partners in the first regionally defined cluster approach to deliver scalable solutions for the blue economy, emphasising sustainability, innovation and systemic change. The project is supported by EcoeFISHent project 101036428/01.10.2021.

Life cycle assessment of mealworm production in closed-loop systems enhancing crop production

A.F. Francis^1,2*, A.T. Thomsen³, J.R. Rukov⁴ and S.S. Smetana^1,5

¹German Institute of Food Technologies – DIL e.V., Food Data Group, Professor-von-Klitzing-Straße 7, 49610 Quakenbrück, Germany; ²Technische Universität Braunschweig, Universitätsplatz 2, 38106 Braunschweig, Germany; ³Nordicflexhouse, ApS Lautruphøj 1–3, 2750 Ballerup, Denmark; ⁴Bugging Denmark, Otto Busses Vej 63 2450 København V, Denmark; ⁵Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany; ^*a.francis@dil-ev.de

Circular economy strategies and advanced agricultural technologies can reduce environmental impacts of food production; therefore, this study evaluates the life cycle assessment (LCA) of mealworm (Tenebrio molitor) production in refurbished containers integrated into a closed-loop system with lettuce cultivation in biopod units. The primary focus of the assessment is mealworm production, while lettuce is treated as a co-product system utilising recovered waste streams. Within this system, mealworm frass and nutrient-rich fish waster are reused as inputs for lettuce cultivation, reducing the need for external fertilisers and improving resource efficiency. Using a combination of primary and secondary data, the study assesses environmental impacts within the Biocircular project, which aims to valorise organic side streams through the integrated production of insects, fish, and vegetables. Seperate LCAs were conducted for insect and fish production to quantify their individual contributions before integrating them into a circular model that includes lettuce cultivation using recovered biofertilisers, oxygen, and carbon dioxide from insect and fish waste streams. The system boundary covers the full life cycle from raw material extraction to end-of-life, with a functional unit of 1 kg of lettuce produced per biocontainer and 27 impact categories evaluated using Environmental Footprint (EF) 3.1 methodology in SimaPro 9.6.0.1. Results show that standalone mealworm production has a climate change impact of 1.64 kgCO₂eq per kg; however, when integrated with lettuce as a co-product system, the impact is reduced to 0.948 kgCO₂eq per kg of mealworm. Additionally, lettuce production achieves a low impact of 0.0303 kgCO₂eq per kg due to the utilisation of recovered nutrients. These findings demonstrate that coupling mealworm production with lettuce cultivation in a circular system substantially improves environmental performance by converting waste streams into valuable inputs.

Milk for beetles: alternative wet feed sources for insect rearing

A. Resconi^1*, G. Baliota², S. Zafeiriadis², L. Gasco¹ and C. Athanassiou²

¹University of Turin, Department of Agriculture, Forest and Food Sciences, Largo Paolo Braccini 2, 10095 Grugliasco, TO, Italy; ²University of Thessaly, Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and R, Fytokou Street, 384 46 Nea Ionia, Volos, Greece; ^*andrea.resconi@unito.it

In the rearing of beetle (Coleoptera) species for food and feed, the main source of feed is constituted by dry substrates, while a high-water containing substrate is used as a moisture source. Although commonly used moisture sources are agar, carrots and potatoes, these substrates pose certain limitations. Potatoes and carrots are food grade items and their use in insect rearing may not be viable from an economic standpoint, while agar is very expensive. The dairy industry stands out for producing several waste streams and byproducts that are nutritionally valuable. This trial explored these substrates as wet feed for the lesser mealworm, Alphitobius diaperinus. The substrates tested were expired food grade products: yogurt (Y), milk (M), kefir (K), plant-based milk (PM) and byproducts from the dairy industry: sour lactose (SoL), sweet lactose (SwL), both from yogurt production, whey (W) and sour whey (SW), both originating from ricotta production. Starch was added to each dairy byproduct (7% g/vol) to jellify it and render its administration possible to the larvae. For the control, a mixture of starch and water (7% g/vol) was used. Each treatment consisted a plastic vial containing 50 7-day-old larvae and 2 g of wheat bran. The wet feed was administered three times/week to each replicate, and larvae were sampled at weekly intervals for survival and average weight. The experiment was considered over for a specific replicate when the average weight difference between two consecutive samplings was<50%. The experimental data was analysed with RStudio software. Larval survival was always above 70%, with no significant differences among treatments ($P\mathrm{>}0.05$). The final weight of the larvae was also not significantly affected by the wet feed, although the highest value was obtained by the K treatment (0.018 g), higher than the control (0.014 g). The development time of the larvae was comparable for Sol, SwL, PM (42 days), K, SW, W (43.2 days) and the control (44.2 days), while longer for Y (55.3 days). Regarding bioconversion values, K performed very well, with a feed conversion ratio (FCR) value of 2.71, one of the most efficient recorded, more efficient than the control (4.03; $P\mathrm{<}0.05$). This treatment also allowed for one of the highest specific growth rate (SGR) values (10.58), statistically similar with the control (10.17, $P\mathrm{>}0.05$). This experiment explores novel sustainable substrates that could be used as wet feed sources for mass rearing protocols of A. diaperinus.

Using an insect for sustainable waste management of a superabundant bird

C. López-Manzano^*, H. Mahdjoub, L.R. Arce-Valdés and R. Khelifa

Concordia University, Biology, 7141 Sherbrooke Street W., Montreal, QC, Canada H4B 1R6; ^*lopezmanzano3494@gmail.com

Urban environments accumulate large volumes of faecal waste from superabundant wildlife, yet few circular strategies exist to convert this biomass into useful products. We evaluated the potential of black soldier fly (BSF; Hermetia illucens) larvae to bioconvert faeces from the Canada goose (Branta canadensis), a common bird that creates significant faecal pollution in parks and green spaces. Our objectives were to (i) test whether BSF larvae can develop on goose faeces and reduce waste, and (iii) assess whether resulting frass can serve as an effective fertiliser for duckweed (Lemna minor). Fresh faeces were manually collected from urban parks (Southern Quebec, Canada) using gloves, transported within 2 h, and stored at 5 °C prior to experiments. In a field survey across 11 sites, flock size strongly predicted faecal abundance (R²Â = 0.88), confirming high spatially concentrated waste inputs. In laboratory trials, 6-day-old larvae (n = 100 per replicate; 200 g substrate; three replicates per treatment) were reared on diets containing 0, 50, or 100% goose faeces, under controlled conditions (30 °C, 80% RH). Both raw and autoclaved substrates were tested to distinguish between nutritional and microbial (including pathogen-related) effects of faeces. BSF larvae successfully completed development on all diets. In raw substrates, larvae reduced dry matter by 64.0% (control), 63.5% (50% faeces), and 56.9% (100% faeces), with highest waste-reduction rates in mixed diets (3.77 g DM day^−1). Autoclaving reduced consumption ($P=0.007$) and larval performance in faeces-only diets, indicating a beneficial role of native microbiota. Survival remained high in control (96.4%) and mixed diets (95.4%) but declined in 100% faeces (80.6%). Increasing faeces content slowed development and reduced adult body mass and lifespan (9.0 days in control vs 5.4 days in 100% faeces), linking waste quality to insect performance and biomass production. Frass derived from BSF processing was then tested as fertiliser using duckweed as a plant model. Frass significantly outperformed fresh faeces and nutrient-rich Hoagland medium: at 10 g l^−1, frass increased duckweed yield by 32% relative to Hoagland solution and produced higher frond numbers (6.0 vs 4.6). In contrast, fresh faeces yielded lower growth (4.7–5.0 fronds). Root length was reduced by up to 83% in frass treatments, consistent with high nutrient availability. Overall, BSF larvae efficiently converted Canada goose faeces into insect biomass and a nutrient-rich fertiliser that outperformed untreated faeces. These results demonstrate a practical circular strategy for managing wildlife-derived faecal waste in urban environments through collection, insect-based bioconversion and reuse in plant production systems.

Synthetic microbial community enhances the protein bioconversion from organic wastes by black soldier fly larvae

Y.Q. Yu^*, C. Huang, J. Zhang, B.B. Wang, Z.Z. Zhao, F. Huang, M.M. Cai, L.Y. Zheng and J.B. Zhang

Huazhong Agricultural University, College of life science and technology, Huazhong Agricultural University, 1 Shizishan Street, Hongshan District, 430070 Wuhan, Hubei Province, P.R. China; ^*vvvyyq@yeah.net

Black soldier fly larvae are promising agents for converting organic waste into high-value biomass as sustainable alternative protein resources. However, the interactive mechanisms between the core gut microbiota and its host that govern efficient protein degradation and synthesis remain elusive. This study employed a multi-omics approach—integrating gut microbiomes, transcriptomics and metabolomics—within germ-free and gnotobiotic BSFL models to decode host-microbe interactions. Also, a defined synthetic microbial community (SynCom), rationally constructed from core taxa (Bacillus, Lactobacillus, Issatchenkia), was used for functional dissection. The tripartite SynCom established a stable division of labor along the gut pH gradient, maximising protein degradation and increasing final larval protein content by 63% in controlled feeding experiments. Mechanistically, the host aldehyde dehydrogenase (ALDH) was identified as a key hub, integrating microbial signals from branched-chain amino acid and tryptophan metabolism into the host’s TOR pathway to boost protein synthesis. When applied to chicken manure and kitchen waste, the SynCom significantly enhanced system performance, increasing total larval protein yield (17.4 and 8.3%, respectively), feed conversion ratio (17.1 and 11.3%), and dry matter reduction (28.1 and 18.7%). Our findings provide a scalable framework for engineering insect-microbe symbiosis, enhancing the efficiency of organic waste valorisation and sustainable protein production.

Functional expression of PET-degrading enzymes in the BSF: a platform for insect-mediated plastic bioconversion

M. Marzari¹, C. Fontana¹, D. Bruno¹, I. Nevo², I. Alyagor², D. Roma³, S. Caccia³, M. Casartelli³, E. Caruso¹, A. Grapputo⁴, G. Molla¹, F. Sandrelli⁴ and G. Tettamanti^1*

¹University of Insubria, Department of Biotechnology and Life Sciences, via Dunant 3, 21100 Varese, Italy; ²FreezeM, Nachshonim 1, 7319000 Nachshonim, Israel; ³University of Milano, Department of Biosciences, via Celoria 26, 20133 Milan, Italy; ⁴University of Padua, Department of Biology, via Ugo Bassi 58/B, 35121 Padua, Italy; ^*gianluca.tettamanti@uninsubria.it

Despite the implementation of the EU Strategy for Plastics in the Circular Economy in 2018, plastic waste management remains a major challenge, with less than 30% of the 25.8 million tonnes of plastic waste generated annually in Europe being recycled. Recycling rates increase slowly, and biological recycling approaches are limited by the scarcity of efficient enzymes and scalable systems, underscoring the need for alternative and innovative plastic valorisation strategies. This trend is particularly relevant for polyethylene terephthalate (PET), a dominant packaging plastic and major contaminant of the organic fraction of municipal solid waste, which can contain up to 5% of plastic debris. The presence of these polymers, resulting from the improper disposal of plastics, makes the management procedures of this organic waste more problematic. Black soldier fly (Hermetia illucens) larvae (BSFL) are widely used for organic waste bioconversion into protein-rich biomass thanks to their unparalleled feeding plasticity, but lack intrinsic plastic-degrading ability. Here, we present an innovative biotechnological platform that exploits BSFL as living bioreactors for PET biodegradation that could help address the management of plastic-contaminated waste. The approach is based on the genetic modification of BSFL to express high-performance microbial PET-degrading enzymes (PETases) in their gut. To this purpose, we selected an improved variant of a leaf-branch compost cutinase (TA-ΔLCC). Using PiggyBac-mediated transgenesis, a stable homozygous BSF line carrying a $\mathit{Hi}\mathrm{−}\mathit{TA}\mathrm{−}\mathrm{Δ}\mathit{LCC}$ construct was generated. Transgenic larvae showed the expression of LCC in the gut epithelium. Moreover, the enzyme was secreted in the gut lumen, displayed activity, and demonstrated the capability to bioconvert PET, without detectable morphological and functional alterations of the alimentary canal. Further analyses are in progress to quantify the extend of PET degradation by the larvae. These findings demonstrate the feasibility of using transgenic BSFL for integrated management of organic waste streams contaminated with PET. This approach opens new perspectives for coupling insect-based bioconversion with plastic biodegradation, contributing to circular economy strategies and offering scalable solutions for sustainable waste management.

Feeding Tenebrio molitor larvae (TML) with aquaponics waste for circular and sustainable production

S. Errico^*, A. Verardi, S. Moliterni, R. Rebuzzi, P. Sangiorgio and S. Dimatteo

ENEA — Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Sustainability, Trisaia Research Centre, SS Jonica 106, Km 419+500, I-75026 Rotondella (Matera), Italy; ^*simona.errico@enea.it

The DEMETRA project, “An integrated approach based on aquaponics and waste reuse for a green and smart prototype food system” (https://sostenibilita.enea.it/en/node/12802), aims to develop a sustainable food system capable of producing low-impact, high-nutritional-value food in extreme environments (e.g., underground cavities, abandoned industrial structures, large urban buildings) through aquaponics, integrating aquaculture and hydroponics. The system also incorporates waste valorisation processes, including recovery of organic matter via bioconversion (by microalgae and insects), and production of bioactive molecules and biopolymers. Within DEMETRA, Tenebrio molitor (TM) was selected as a model species for insect-based bioconversion. Two laboratory-scale trials were conducted. In the first, T. molitor larvae (TML) were reared on aquaponic dried waste (ADW) to assess its suitability as a substrate for protein production. ADW was provided as partial or total replacement of the standard diet at 50% and 100% inclusion levels, named ADW-50 and ADW-100, respectively. Growth performance was evaluated in terms of TML weight gain (as a percentage ratio between TML initial and final weight) and survival rate. In the second trial, three aquaponic crops — savoy cabbage (Brassica oleracea var. sabauda), broccoli (Brassica oleracea var. italica) and cauliflower (Brassica oleracea var. botrytis) — were tested as wet supplements (WS) for larvae fed a standard diet. Growth performance, shelf life under rearing conditions, and larval preference (choice tests) were assessed. The best performance was observed with ADW-50, yielding a mean weight gain of 221.43%, compared to 72.41% and 79.10% for ADW-100 and the standard diet, respectively. Among WS treatments, broccoli resulted in the highest weight gain (50.19%), followed by cauliflower (38.25%) and savoy cabbage (24.66%). However, cauliflower showed higher palatability in choice tests and more consistent overall performance. These findings demonstrate the feasibility of using aquaponic by-products as feed for TML, supporting the integration of TM rearing within circular food systems. Further research will address performance under extreme conditions and the application of TML-derived products in aquafeed.

Tomato leaf waste handling affects growth performance and glycoalkaloids in desert locusts

Y. Luo^1*, N. Förster¹, M. Bogdanova², S. Schulz¹, I. Mewis¹, C. Ulrichs¹ and W. Yakti¹

¹Humboldt University of Berlin, Lentzeallee 55, 14195 Berlin-Dahlem, Germany; ²Institut für Lebensmittel- und Umweltforschung e. V. (ILU), Papendorfer Weg 3, 14806 Bad Belzig, Germany; ^*luoyanhuilove@gmail.com

Tomato leaves represent a large but underutilised agricultural waste stream, yet their direct use as feed is limited by high fibre content and the presence of toxic glycoalkaloids. Feeding tomato leaves to desert locusts (Schistocerca gregaria) may provide a sustainable pathway for converting plant residues into high-value insect protein for food and feed applications. In this study, desert locusts were reared on tomato leaves subjected to four handling strategies: (i) freshly harvested leaves (FL), collected and fed daily; (ii) leaves stored at 8 °C for 3 days and fed daily (LS); (iii) leaves stored at 8 °C for up to 6 days with daily removal for feeding (LSD); and (iv) a leaf-cutting method (LCM), in which freshly cut stems were placed in water to maintain hydration and physiological freshness. Leaves treated with LCM remained usable for up to 7 days, thereby reducing the need for daily harvesting and feeding. Despite higher glycoalkaloid concentrations in LCM-treated leaves, accumulation of α-tomatine and dehydrotomatine in locust biomass was lower, and no significant effects on growth performance or survival were observed. The chemical composition of the produced locusts varied across treatments, with the leaf cutting method resulting in higher accumulation of Cu, Zn and Pb compared to feeding with fresh leaves. The crude protein content of the locusts remained stable across treatments (59.37–60.51%), so was their fatty acid composition. These findings suggest that the LCM strategy represents a promising approach for the development of efficient, low-labour insect bioconversion systems for agricultural waste valorisation.

Black solider fly: a potential insect for managing kitchen bio-waste

A. Abdullah^* and M. Ali

Bahauddin Zakariya University, Department of Entomology, Dept of Entomology, FAS&T, BZU, 60000 Multan, Punjab, Pakistan; ^*abdullah_ento@outlook.com

The management of organic waste material is becoming a challenge due to the increase in global population which leads to greenhouse gas emissions and environmental loss. Our project was to evaluate the potential of larvae of black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae) in the management of kitchen bio-waste by evaluating their bioconversion rate and potential for sustainable waste management. Kitchen bio-waste (15 kg per house having vegetables and fruit peels and food residues) was collected from seven volunteer houses (H1-H7) for fifteen days. Waste was processed and divided into three 5 ± 0.1 kg replicates per household. The H1 and H4 kitchen bio-waste gave the highest larval growth compared to H2, H3, H5, H6 and H7, showing that waste composition affects larval development. Moreover, in comparison to the waste residues from the other houses, H1, H2, H3, H4, H6 and H7, the kitchen bio-waste residues from house H5 showed the highest weight. This suggests that even though each bin contained 600 first-instar larvae and was divided into three equal pieces, the quality or content of the 15 kg of kitchen bio-waste collected in H5 did not favour larval development more than that of the wastes in H1, H2, H3, H4, H6 and H7. Black soldier fly larvae expressed as an effective waste manager, which is also cost-effective and sustainable method of waste management, with also production of useful products of protein-enriched larvae biomass (used in animal feed) and frass (used as fertiliser for plants).

Dairy sludge as a rearing diet for black soldier fly larvae (Hermetia illucens L.): Successful? Efficient?

S. Naser El Deen^1*, T. Veldkamp¹, P. Van Wikselaar¹, K. Van Rozen², W.C.W. Tao³, H. Elissen² and K.C.W. Van Dongen³

¹Wageningen University and Research, Livestock Research, De Elst 1, 6700 AH Wageningen, The Netherlands; ²Wageningen University and Research, Plant Research, Edelhertweg 1, 8219 PH Lelystad, The Netherlands; ³Wageningen University and Research, Food Safety Research, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands; ^*somaya.nasereldeen@wur.nl

Black soldier fly larvae (BSFL) are known for their capacity to valorise diverse residual streams by converting them into valuable resources. This study evaluated BSFL rearing efficiency on industrial dairy sludge (DS), alone or mixed with different materials to improve the sludge’s nutritional and physical properties. BSFL were reared for 7 days on wheat bran, dairy sludge, DS mixed with wheat bran (80:20 weight-based), and DS mixed with sawdust (80:20 weight-based), and for 14 days on DS alone (DS-14). For each diet, four crates (replicates) were prepared, each with 10 kg fresh diet and 18 500 5-day-old BSFL. Dietary crude protein ranged from 18.1% in wheat bran to 46.6% in DS, while crude fat ranged from 1.6% DS-sawdust mix to 4.9% in the DS-wheat bran mix. Assessed parameters included larval growth (harvested total mass, individual weight, growth rate and survival), waste reduction index (WRI), efficiency of conversion of ingested feed (ECI), and biochemical composition of larvae and frass. Larvae reared on DS for 7 days had the highest harvested total mass (1,720 g), individual weight (63.6 mg), growth rate (8.3 mg per larva/day), and ECI (0.3). DS-14 larvae had the highest WRI (11.7 g/day) but the lowest ECI (0.1). Wheat bran-fed larvae showed poor growth, with the lowest harvested total mass (1,073 g) and growth rate (6.1 mg per larva/day). DS mixed with sawdust resulted in the lowest WRI. Larval crude protein ranged from 42.2–48.3% (DM basis), and did not directly reflect dietary protein. Larval crude fat was highest on wheat bran (9.0% DM basis) and lowest (2.9% DM basis) on DS-14. Frass from DS-7 had the highest nitrogen (6.8% DM basis) and phosphorus (4.6% DM basis) contents, while frass from DS mixed with sawdust had the lowest (2.2% and 1.3% DM basis, respectively). In conclusion, these findings indicate that DS can solely support BSFL rearing under the tested conditions, and adding 20% wheat bran or sawdust did not improve performance. This highlights the potential of dairy sludge as a sustainable substrate for insect-based bioconversion, contributing to circular economy strategies and sustainable nutrient recycling in agro-industrial systems.

Efficacy of thermophilic aerobic fermentation of pig carcasses and subsequent bioconversion by black soldier fly larvae

L. Broeckx^1*, L. Saucier¹, M.H. Deschamps¹, M. Dallaire-Lamontagne¹, M.P. Rivera Rodrı́guez¹, C.S. Osorio Gonzalez², J. Deslauriers³, J.M. Allard Prus⁴, M.O. Benoit-Biancamano⁵ and G.W. Vandenberg¹

¹Université Laval, Département des sciences animales, 2425 rue de l’Agriculture, Laval, QC, Canada G1V 0A6; ²Université Laval, Département des sols et de génie agroalimentaire, 2425 rue de l’Agriculture, Laval, QC, Canada G1V 0A6; ³WAKAN Environnement, 3065 boulevard Losch, Saint-Hubert, QC, Canada J3Y 3V6; ⁴Couvoir Scott Inc., 1798 Route du Président-Kennedy, Scott, QC, Canada G0S 3G0; ⁵Université de Montréal, Département de pathologie et microbiologie, Faculté de médecine vétérinaire, 1500 av. des Vétérinaires, Saint-Hyacinthe, QC, Canada J2S 2M2; ^*laurens.broeckx.1@ulaval.ca

The management of animal carcasses remains a critical environmental and logistical challenge for livestock production systems. Conventional disposal methods present major limitations: burial poses long-term risks to groundwater contamination; composting is time-consuming and associated with substantial greenhouse gas emissions; incineration and rendering are highly energy-intensive; and, except for rendering, most approaches result in a near-complete loss of recoverable nutrients. Solid-state aerobic digestion represents a potential alternative, in which thermophilic bacteria drive rapid microbial activity and heat generation. We hypothesize that a thermophilic fermentation of 24 h may allow biological stabilization by inactivating pathogenic micro-organisms, while producing a microbiologically stable digestate rich in nutrients for subsequent valorisation. In this study, two fermentation approaches of pig carcasses were tested using Bacillus smithii as the thermophilic bacterium, at pork carcass:sawdust ratios of 2:1 and 3:1 (w/w). A standard Gainesville diet and a non-fermented 3:1 pork carcass:sawdust mixture were used as controls, and a 50:50 (w/w) mixture of Gainesville diet and fermented 3:1 substrate was also evaluated. Five-day-old black soldier fly larvae (BSFL) were reared on each substrate for 7 days (500 larvae per treatment, 0.2 g DM/larva, three replicates). Bioconversion efficiency differed significantly among diets (one-way ANOVA, $p\mathrm{<}0.0001$). The fermented 3:1 pork carcass:sawdust substrate outperformed its non-fermented counterpart by 26.3%, while supplementation of the fermented 3:1 substrate with the Gainesville diet at a 50:50 ratio resulted in a 30.8% higher bioconversion efficiency compared to the Gainesville diet alone. In contrast, the fermented 2:1 treatment showed significantly lower performance. Overall, thermophilic aerobic fermentation has the potential to biologically stabilize carcass waste while producing nutrient-rich digestates that support efficient BSFL bioconversion, both as a substrate and as a supplementation.

When apple meets olive: nutrient balanced pomace diets driving black soldier fly density and performance

P. Soulioti^1*, T.I. Gogou¹, R.H. Nakirya², E. Valli³, C.I. Rumbos⁴, M. Sakka¹, M.T. Capucchio⁵ and C.G. Athanassiou¹

¹University of Thessaly, Department of Agriculture, Crop Production and Rural Environment, Phytokou Street, 38446 Volos, Greece; ²Università di Bologna, Department of Biological, Geological and Environmental Sciences, Alma Mater Studiorum, Via Irnerio 42, 40126 Bologna, Italy; ³Università di Bologna, Department of Agricultural and Food Sciences, Alma Mater Studiorum, piazza Goidanich, 60, 47521 Cesena, Italy; ⁴University of Patras, Department of Agriculture, 30200 Messolonghi, Greece; ⁵University of Turin, Department of Veterinary Sciences, Via Verdi, 8 10124, Turin, Italy; ^*pasoulioti@uth.gr

Agricultural processing generates large volumes of byproducts every year, and in Mediterranean regions, olive and apple pomaces account for a large portion of these residues. Finding effective ways to repurpose these materials remains a challenge, making the search for alternative valorisation pathways increasingly relevant. Black soldier fly larvae (BSFL) are known for their ability to consume a wide range of organic byproducts and are considered efficient bioconverters. This study evaluated the impact of isonitrogenous and isoenergetic diets on larval density and overall performance. Three diets were formulated using apple pomace, olive pomace, or a combination of both, each supplemented with wheat bran and brewer’s spent grains. These diets were tested at three larval densities: 1, 2.5 and 5 larvae/cm². Larval weight and survival were recorded every three days, and the experiment ended once 10% of the larvae reached the prepupal stage. Larval protein content was also analysed. The results revealed significant differences across all measured parameters except survival, which remained higher than 90% for all treatments. Individual larval weight varied among treatments, with the olive pomace diet at the highest density producing the heaviest larvae (96 mg), followed by the apple pomace diets across all densities (82-90 mg). The lowest weights were recorded in the olive pomace diet at the lowest density (60 mg). Development time also differed, with the apple pomace diet at the highest density reaching prepupal stage in just 6 days, while all olive pomace diets required around 12 days. Larval crude protein ranged from 48 to 56%, with the highest value recorded in the apple pomace diet at 5 larvae/cm² density and the lowest in the olive pomace diet at the highest larval density. Collectively, these findings highlight the strong influence of substrate type and larval density on BSFL growth and efficiency. Acknowledgement: This research is supported by the EU-PRIMA program — project OLIWA- Repurposing OLIve WAste in circular economy solutions for feeds, additives, packaging, and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus.

From Mediterranean waste to insect protein: larval performance of Hermetia illucens on two-phase olive Pomace diets

P. Soulioti^1*, G. Baliota¹, C.I. Rumbos², M.T. Capucchio³ and C.G. Athanassiou¹

¹University of Thessaly, Department of Agriculture, Crop Production and Rural Environment, Phytokou Street, 38446 Volos, Greece; ²University of Patras, Department of Agriculture, 30200 Messolonghi, Greece; ³University of Turin, Department of Veterinary Sciences, Via Verdi 8, 10124, Turin, Italy; ^*pasoulioti@uth.gr

Olive pomace is one of the most abundant byproducts in Mediterranean regions, generated during olive oil extraction. The valorisation of two phase olive pomace (TPOP) remains challenging, mainly because of its high moisture content (>60%). The black soldier fly (BSF) is widely recognized as an efficient bioconverter capable of utilising a broad range of organic byproducts. Considering the species’ preference for moist substrates, this study examined the partial inclusion of TPOP in BSF larval diets alongside chicken feed. Both dried TPOP (74 °C for 5 h) and wet TPOP were tested at inclusion levels of 10, 15, 25, 50 and 75% for the dried form, and 5, 10, 15, 25, 50 and 75% for the wet form. A 100% chicken feed diet served as the control, and all diets were adjusted to 70% moisture. Larval survival and weight were recorded every three days until 10% of the larvae reached the prepupal stage, marking the end of the trial. Protein and lipid content were subsequently analysed, and feed conversion ratio (FCR) and bioconversion rate (BCR) were calculated. Significant differences were observed across nearly all measured parameters except survival, which remained above 94% in all treatments. The highest larval weights were recorded in the control diet (146.4 mg), with no significant differences compared to both TPOP forms up to the 25% inclusion level. Diets containing more than 50% TPOP resulted in reduced individual larval weights. The 25% wet TPOP diet showed the best FCR and BCR values (7.6 and 13.2%, respectively), whereas the 75% dried TPOP diet led to the poorest performance and the highest crude lipid content (around 34%). Protein content increased with higher inclusion levels of dried TPOP, reaching 47% at the 50% level, but dropped to 39.6% at 75% dried TPOP diet. Taken together, these results demonstrate the potential of incorporating TPOP into BSF larval diets and emphasize how drying and inclusion level shape both nutritional outcomes and larval growth performance. Acknowledgement: This research is supported by the EU-PRIMA program — project OLIWA- Repurposing OLIve WAste in circular economy solutions for feeds, additives, packaging, and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus.

Safety assessment of black soldier fly larvae reared on non-permitted substrates

K. Niermans^1*, N. Meijer¹, S. Naser El Deen², T. Veldkamp², C.J. De Vos-De Jong³, M.J. Appel¹ and E.F. Hoek-Van Den Hil¹

¹WUR, Wageningen Food Safety Research, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands; ²WUR, Wageningen Livestock Research, De Elst 1, 6700 AH Wageningen, The Netherlands; ³WUR, Wageningen Bioveterinary Research, Houtribweg 39, 8221 RA Lelystad, The Netherlands; ^*kelly.niermans@wur.nl

Black soldier fly larvae (BSFL; Hermetia illucens) can convert diverse organic residual streams into valuable biomass, including materials currently excluded under EU feed legislation, which offers opportunities for circularity. Within the Safe Insects 1 project, we assessed BSFL performance and food safety outcomes on several non-authorised substrates.

Feeding trials used a control diet, kitchen waste, supermarket waste, chicken manure and category 2 meat meal. Pre studies showed that category 2 meat meal was unsuitable for BSFL and was excluded. All substrates and larvae were analysed for chemical contaminants, heavy metals, pesticides, PFAS, dioxins, PCBs, veterinary drugs and microbiological hazards. To study pathogen behaviour, chicken manure was spiked with Salmonella enterica, Clostridium perfringens, Bacillus cereus, Klebsiella pneumoniae, Eimeria oocysts and five viruses. A quantitative risk assessment evaluated the likelihood of prion transfer.

Across all substrates, dioxins, PCBs and PFAS were detected only at low levels. Kitchen waste and supermarket waste had high microbial loads, including C. perfringens and B. cereus, while Salmonella was found only in supermarket waste. Kitchen waste and supermarket waste contained pesticides, and supermarket waste also showed unexpected high nicotine levels. Heavy metals were present in both substrates. Cadmium accumulated in BSFL reared on kitchen waste but remained below legal limits. Feeding on supermarket waste resulted in bioaccumulation of several elements, although BSFL concentrations stayed within regulatory thresholds. Chicken manure contained elevated metals and coccidiostats, which did not accumulate in BSFL but exceeded carry over limits in the substrate.

Some pathogens transferred from the spiked substrate to the BSFL, while others were not detected. Two viruses appeared only on day 1, whereas hepatitis E virus and Eimeria persisted in the BSFL. B. cereus, K. pneumoniae and S. abaetutuba were found in both substrate and BSFL, though pre-existing bacteria complicate interpretation. C. perfringens appeared only in the substrate. Lasty, the quantitative risk assessment concluded that prion transfer from insects reared on these substrates is very unlikely. A follow up project, Safe Insects 2.0, is examining animal by product residual streams, focusing on processing effects on the presence of animal DNA and microbiological risks, and the legal conditions for safely using currently unauthorised substrates.

Progress on research and application of black soldier fly and microbes in China

J. Zhang^*, M. Cai, L. Zheng, F. Huang and Z. Yu

College of Life Science and Technology, Huazhong Agricultural University, No1 Shizhishan street, Hongshan District, 430070, Wuhan, P.R. China; ^*zhangjb@mail.hzau.edu.cn

Over the past twenty years, investigators in China have made great progress on basic research and industry application of black soldier fly (BSF) and microbes. We bred Chinese local black soldier fly Wuhan strain, sequencing its genome and discovered a large amount of active substances in black soldier flies, such as antimicrobial peptides and active enzymes. We first found that the gut microbiota is an essential organ of the black soldier fly. Subsequent studies have found that key microorganisms in the gut of black soldier flies play different roles in the conversion of organic waste, such as promoting protein degradation in organic solid waste by black soldier flies, promoting egg laying by adult black soldier flies, degrading cellulose in materials, promoting the growth and development of black soldier flies, assisting black soldier fly larvae in inhibiting zoonotic pathogens in substrates (such as livestock manure), degrading antibiotic residues in livestock manure and reducing antibiotic resistance genes and their mechanism. We are on the way to explore exactly low cost and high efficiency conversion process and equipment of organic solid waste, and plays an important role in alleviating the shortage of soybean and fish meal in China. We have established safe conversion and detection technologies, and the conversion products of black soldier flies have been included in the Chinese feed catalogue. We have also discovered some BSF pathogens and find the green control methods, and established national, group and local standards in BSF breeding, conversion process and its production (BSF larvae powder) for black soldier flies industrialization.

Olive pomace and goat manure valorisation by anaerobic digestion and bioconversion with Hermetia illucens

A. Dolce^1*, G. Lomonaco¹, F. Iannielli¹, N. Rubiejo Pérez², R. Salvia^1,3, C. Scieuzo^1,3, J.D. Fernandez-Bayo² and P. Falabella^1,3

¹University of Basilicata, Department of Basic and Applied Sciences, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy; ²University of Granada, Department of Soil Science and Agricultural Chemistry, Faculty of Pharmacy, Campus de Cartuja, s/n, 18071 Granada, Spain; ³Spinoff XFlies s.r.l, University of Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy; ^*antonio.dolce@unibas.it

The increasing demand for sustainable management of agro-industrial residues has intensified interest in circular bioeconomy strategies integrating energy recovery and material valorisation. Olive pomace (OP) and goat manure (GM) are abundant organic wastes with high potential for biological conversion, although OP recalcitrance often limits bioconversion efficiency. This study investigated an integrated two-stage pathway combining anaerobic digestion (AD) and bioconversion using Hermetia illucens (black soldier fly larvae, BSFL). In the first stage, the effect of OP thermal pre-treatments (90, 120 and 150 °C) on biogas production performance was assessed. Pre-treated OP-GM (2:1, dry weight) were anaerobically digested under mesophilic conditions (37 °C, 17 days) and compared with an untreated OP-GM and GM. In the second stage, solid digestates, untreated OP-GM, raw OP and GM were used as rearing substrates for BSFL to compare performance on digested and non-digested substrates. Survival, growth and bioconversion efficiency, using a standard chicken feed diet as control. Phytotoxicity of digestates obtained from anaerobic digestion and frass produced by larvae reared on a raw OP-GM was evaluated using a germination index (GI) on alfalfa seeds. The 120 °C pre-treatment yielded the highest cumulative biogas pressure (1400 hPa), whereas GM reached about 500 hPa. However, untreated OP-GM showed comparable yields, indicating limited benefits of thermal pre-treatment. BSFL reared on digestates exhibited high survival rates (up to 98%) but reduced growth, with final larval weights of 0.09–0.11 g on digestates, compared to 0.083 g on untreated OP-GM, 0.08 g on standard diet, 0.03 g on raw OP and 0.04 g on GM. Bioconversion efficiency decreased from 18.7% (standard diet) to below 12% on digestates. Phytotoxicity tests revealed strong inhibitory effects for digestates (GI 7–10%), whereas frass showed no phytotoxicity, with GI exceeding 130%. Overall, thermal pre-treatment does not significantly improve AD performance and may compromise digestate suitability for insect bioconversion. Conversely, the AD-BSFL approach without thermal pre-treatment represents a promising strategy for renewable energy recovery, waste reduction and the production of stable, agronomically valuable frass and insect protein.

BioInsectonomy: a trinational collaborative project for circular insect-based feed production from agricultural residues

C. Garino^1*, S. Broll¹, A.P. Muñoz Ramı́rez², O. Pötz³, T. Meisinger³, I. Belghit⁴, H. Broll¹ and K. Barragán-Fonseca⁵

¹German Federal Institute for Risk Assessment (BfR), Department of Food Safety, Max-Dohrn-Strasse 8–10, 10589 Berlin, Germany; ²Universidad Nacional de Colombia, UN-ACUICTIO research group, Universidad Nacional de Colombia, 111321 Bogota, Colombia; ³SIGNATOPE GmbH, Markwiesenstraße 55, 72770 Reutlingen, Germany; ⁴Norwegian Institute of Marine Research, Nordnesgaten 50, 5005 Bergen, Norway; ⁵Universidad Nacional de Colombia, Centro de Investigación de Artrópodos Terrestres (CINAT), Universidad Nacional de Colombia, 11001 Bogota, Colombia; ^*Cristiano.Garino@bfr.bund.de

BioInsectonomy is a trinational collaborative research project involving institutions from Germany, Colombia, and Norway, aimed at advancing circular solutions based on insect farming for sustainable feed production. The project was developed by integrating complementary expertise while conducting experiments in different regions. The European members addressed analytical validation by developing four real-time PCR assays for the species-specific identification of insects in feed, as well as mass spectrometry-based methods for the detection of insect and non-insect peptide markers. In Colombia, researchers analysed the production chain from the use of agricultural residues as substrates for insect, larval rearing, processing, to the formulation of insect meals, evaluating the performance of four diets in two insect species, Hermetia illucens and Tenebrio molitor. Insect meals were produced using harmonized processing protocols and analysed to assess their safety. Feeding trials conducted in Colombia evaluated four aquaculture diets formulated with H. illucens meal in the tropical fish Piaractus brachypomus, indicating that this insect-based ingredient could be incorporated into aquaculture feeds without adverse effects under the experimental conditions. In parallel, guidelines and standard operating procedures for the establishment and operation of insect farms producing processed animal proteins were jointly developed in accordance with both Colombian and EU regulatory frameworks. These activities were embedded in a shared vision to strengthen long-term scientific and technical cooperation between the EU and Colombia. More than 8 scientific papers and a review authored by all unit heads were drafted for international peer-reviewed journals. Overall, the results demonstrate the feasibility of integrating insect production, analytical control, and regulatory alignment within a coordinated international research framework, illustrating how sustained trinational collaboration can support the development of safe, traceable and context-adapted insect-based feed systems aligned with bioeconomy and sustainability strategies.

Black soldier fly frass as a multifunctional input for sustainable crop protection and productivity

L. Shaltiel Harpaz^1,2*, A. Jonas-Levi¹, M. Gezonterman¹, R. Cohen-Hayat^1,2, M. Dafni Yelin², A. Shaviv³, H. Castel⁴, S. Gal⁴, Y. Cohen² and E. Palevsky⁴

¹Tel-Hai University of Kiryat Shmona, Kiryat Shmona 12210, Israel; ²MIGAL, Nothern R&D, South Industrial Zone, Kiryat Shmona 11016, Israel; ³Technion – Israel Institute of Technology, 3200003 Haifa, Israel; ⁴Agricultural Research Center (ARO), Newe Ya’ar, Ramat Yishay, Ramat Yishay 30095, Israel; ^*liorash@telhai.ac.il

Agricultural waste management represents a major environmental and economic challenge worldwide. The use of black soldier fly larvae (Hermetia illucens) for bioconversion of agricultural residues into insect biomass is increasingly recognized as a key component of circular agri-food systems. This process generates frass as a secondary product composed of larval excreta, exoskeleton residues, and partially digested organic matter. Despite its increasing availability, the biological and agronomic properties of BSF frass remain insufficiently characterised. In this study, we evaluated BSF frass as a multifunctional input for sustainable crop production, focusing on its effects on plant performance, pest and disease incidence, and soil microbial communities. Frass samples derived from different production systems underwent thermal stabilization prior to application. Chemical properties were characterised, and laboratory and field experiments were conducted using tomato and cucumber as model crops. Frass application consistently enhanced plant vigor and yield compared to untreated controls and was associated with reduced pest and disease incidence. Analysis of soil bacterial communities revealed that frass treatments did not alter overall taxonomic richness or evenness but significantly reshaped community composition and assembly patterns. Incorporation of frass as a treatment variable contributed to explaining between-group differences in microbial profiles, primarily through shifts in the relative abundance of low-abundance but agriculturally relevant bacterial groups. Notably, frass-amended soils tended to be enriched in nitrogen-fixing bacterial groups and depleted in groups associated with reduced nutrient availability. Overall, frass inputs favoured specific functional groups without causing broad losses in bacterial diversity. Our results demonstrate that BSF frass functions not only as a nutrient source but also as a biologically active input capable of shaping soil microbial communities in ways relevant to crop productivity. These findings highlight the potential of BSF frass as a value-added product within insect-based waste management systems and support the need for classification and standardization of frass products based on both chemical and microbial characteristics to enable reliable and scalable application in sustainable agriculture.

Advances in the production and application of insect frass fertilisers

D. Beesigamukama^1*, J.G. Njagi¹, R. Malingumu^1,2, L.O. Onyango¹, N. Kagehi¹, S.Y. Chia¹, S. Subramanian¹ and C.M. Tanga¹

¹International Centre of Insect Physiology and Ecology, P.O. Box 30772, 00100 Nairobi, Kenya; ²Kenyatta University, Department of Agricultural Science and Technology, P.O. Box 43844, 00100 Nairobi, Kenya; ^*dbeesigamukama@icipe.org

Insect frass fertiliser is gaining global recognition as high-value product for enhancing soil health and crop productivity. There is increased diversification of frass fertiliser in addressing soil health and crop production challenges. This study presents recent efforts in frass fertiliser optimization and application for soil fertility management, yield enhancement and pest management. We evaluated the effect of pit and heap composting and influence of phosphate rock (PR) amendments (5 – 25%) on the quality, yield and profitability of black soldier fly frass fertiliser (BSFFF) production. We used agronomic studies to assess of different BSFFF products (solid, liquid, chitin-fortified solid, chitin-fortified liquid on broccoli yield and nutritional profiles in comparison with conventional organic fertiliser (Safi) and unfertilised soil (control). Lastly, we assessed the pest control effects of insect oil/neem oil-BSFFF biorationals against tomato leaf miner and fall armyworm using application rates of (25, 50 and 100 μl/ml). Findings showed that composting methods did not significantly affect nutrient levels, but the heap method produced BSFFF with high-level of maturity, indicated by lower carbon to nitrogen ratio (< 12) and higher germination index (> 80%). Heap composting also yielded 7% more frass and higher net income. We noted that amendment of substrates with PR yielded 1.1 – 1.4-fold more BSFFF. The BSFFF with 20% PR inclusion had 2.9 – 7.8-fold and 1.1 – 1.7-fold higher concentrations of phosphorus and macronutrients, respectively, compared to unamended fertilizer. Field studies showed that soils amended with BSFFF products produced broccoli with 26 – 138% more yield and 4.6-fold higher agronomic use efficiency compared to Safi and control treatments. Liquid BSFFF produced broccoli with higher levels of crude fat (61%), carbohydrates (16%), and calcium (38%) compared to other BSFFF products while the highest crude protein, potassium, crush ash and phosphorus were achieved using chitin-fortified BSFFF. Bioassays showed no significant differences in pest mortality between insect oil- and neem-oil-based biorationals. Pest mortalities of 34 – 93% and 11 – 94% were achieved for tomato leaf miner and fall armyworm, respectively, with efficacy increasing at higher concentrations. Our findings provide insights for optimising frass fertiliser production and diversification to address biotic and abiotic challenges to crop production, thus contributing to improved soil health, food security and climate resilience through circular economy.

Research on the efficiency and application prospects of black soldier fly cascading degradation of high-fibre waste

R. Shang^*, L. Zheng, J. Zhang and Z. Yu

Huazhong Agricultural University, 1 Shizishan Street, Hongshan District, 430070 Wuhan City, Hubei Province, P.R. China; ^*1421303955@qq.com

With global population growth and changes in consumption patterns, the production of high-fibre organic waste (such as agricultural straw, distiller’s grains, and fruit and vegetable residues) continues to rise. Due to their high cellulose, hemicellulose, and lignin content, these wastes are difficult to efficiently degrade using conventional biological methods, making their treatment a major challenge in the field of solid waste management. The development of efficient and environmentally friendly resource utilisation technologies is urgently needed. Black soldier fly larvae, as resource insects, have shown great potential in organic waste treatment. Current research has mainly focused on their ability to process food waste, livestock manure, and other similar materials, while systematic studies on their degradation of high-fibre waste remain insufficient. This study aims to thoroughly investigate the degradation efficiency of black soldier fly larvae on high-fibre waste and the feasibility of large-scale application. This study evaluated the growth and waste reduction effects (weight reduction rate, fibre component degradation rate) of black soldier fly larvae and cascading transformation processes on high-fibre substrates primarily composed of distiller’s grains and corn straw. The results showed that using black soldier fly larvae (which are more adaptable to high-moisture substrates) to decompose organic matter in fresh distiller’s grains, followed by the introduction of yellow mealworm larvae (which have stronger cellulose decomposition capabilities) for subsequent degradation, proved effective. This insect cascading biotransformation technology can efficiently convert distiller’s grains into insect biomass rich in protein and balanced in amino acid composition, providing high-quality raw materials to address the shortage of feed protein in China. This solution not only effectively tackles the challenges of treating high-fibre waste such as distiller’s grains but also contributes to promoting the sustainable development of agricultural production and ecosystems. Future research should focus on optimising pretreatment processes, screening efficient synergistic degradation strains, and promoting the engineering demonstration and application of this technology.

Enhancing the nutritional value of olive pomace via microbial fermentation for Hermetia illucens larvae production

J.D. Fernandez-Bayo^1*, S. Ortega², N. Rubiejo Pérez¹, B. Brown³, F. Carvalho Muzzi³ and D. Altmajer Vaz²

¹Universidad de Granada, Department of Soil Science and Agricultura Chemistry, Faculty of Pharmacy, University of Granada, Campus de Cartuja, s/n, 18071 Granada, Spain; ²Universidad de Granada, Department of Chemical Engineering Department, Faculty of Sciences, Faculty of Sciences, University of Granada, Av Fuentenueva, s/n, 18071 Granada, Spain; ³InsectBiotech, Calle Cardenal Cisneros 4, 41002 Sevilla, Spain; ^*jdfbayo@ugr.es

The escalating global demand for protein-rich feed necessitates the urgent development of sustainable alternatives. Black soldier fly larvae (BSFL) represent a promising solution due to their high nutritional value, including proteins, minerals, and beneficial fatty acids. BSFL can be reared on agricultural by-products such as olive pomace (OP), a by-product of olive oil production that poses an environmental challenge due to its large volumes and high toxicity. This study evaluated the potential of olive pomace as feedstock for BSFL rearing following different biological pretreatments. Experiments were conducted by inoculating OP with bacterial strains (Bacillus subtilis, B. pumilus, and B. megaterium) or fungal strains (Trametes versicolour, Pleurotus eryngii and Phanerochaete chrysosporium) under controlled temperature and moisture conditions. The effects of inoculum proportion, medium composition, and substrate sterilisation were assessed under both solid-state fermentation (SSF) and submerged fermentation (SF) conditions. The efficacy of the biologically pretreated OP was assessed through laboratory-scale BSFL growth trials by evaluating survival and larvae biomass and compared to a control feed (chicken feed) and non-treated OP. Larvae grow on non-treated OP for nine days showed a biomass that was 20% of the biomass obtained in the control feed (chicken feed). Biological treatment of OP with T. versicolour under SSF conditions significantly increased BSFL biomass to 40–60% of the control biomass. In contrast, bacterial pretreatment with B. pumilus and B. subtilis under SF conditions improved BSFL growth, achieving approx. 80% of the biomass observed in the control. The highest BSFL biomass was obtained when OP was mixed with wheat bran (WB) and pre-digested with T. versicolour, P. eryngii or P. chrysosporium under SSF, or with B. pumilus and B. megaterium under SF conditions, exceeding the control biomass by 20–40%. In conclusion, fungal pretreatment of OP under solid-state fermentation alone showed limited effectiveness in improving its digestibility for BSFL. Submerged fermentation with bacterial strains yielded more promising results but requires further optimisation. The best overall performance was achieved through co-digestion of OP with wheat bran under both SSF and SF conditions, confirming co-digestion as one of the most effective strategies for BSFL rearing.

Fish side-streams to high-value resources: environmental and economic sustainability of black soldier fly biorefinery

A. Aldaghi^1*, M. Costamagna¹, S. Truffa¹, M. Perucca¹, I. Mincheva², M. Tejeda² and M. Aleksandrova²

¹Project HUB-360, Corso Laghi 22, 10051 Avigliana (TO), Italy; ²Nasekomo EAD, Saedinenie Street 299, 1151 Lozen village, Sofia, Bulgaria; ^*ahmad.aldaghi@projecthub360.com

The transition towards a circular Blue Economy requires internalizing industrial residues through “zero-waste” systems. In this framework, the EcoeFISHent project utilises the black soldier fly (BSF), which consumes fish side-streams, as a biological engine to upcycle them into high-demand resources: chitin, oil, and organic fertilisers. Despite growing interest, integrated environmental and economic assessments of multi-output insect production systems remain limited, especially at industrial scale. This study presents a combined life cycle assessment (LCA) and life cycle costing (LCC) of an industrial BSF process rearing on fish side-streams to co-produce oil, chitin, and frass fertiliser. A “cradle-to-gate” approach was applied, covering the entire value chain from feed preparation — fish side-streams (approx. 20% mass) and agricultural by-products (wheat and rice bran) — to the extraction of final co-products. Primary data from the Nasekomo facility demonstrate high bioconversion efficiency, with biomass scaling from 959 g of 5-DOLS larvae to 39 235 g of 12-DOLS larvae, and will be corroborated on the EcoeFISHent larva bioconversion pilot facility in UNITO and integrated with secondary data from reference databases. Results indicate environmental impacts consistent with, and in some categories lower than, literature benchmarks. Global Warming Potential (GWP) was 26.6 kg CO₂ eq/kg chitin and 27.8 kg CO₂ eq/kg oil, representing a 75–97% reduction compared to conventional crustacean systems due to avoided chemical demineralization. The protein-rich pulp is used for oil extraction, while the remaining fraction is reused as larvae feed, enhancing circularity. Electricity is the dominant contributor (approx. 95% of GWP and Fossil Resource Depletion), mainly in extraction, thermal deactivation, and deskinning. Wheat bran drives approx. 97% of Land Use and Freshwater Eutrophication due to upstream agricultural burdens. The valorisation of BSF frass plays a key role in nutrient loop closure and significantly improves economic performance. Economic modelling estimates production costs of approximately €2.80–5.20 per kg of output, aligning with current market price ranges. Its value lies in returning organic matter and nutrients to soil and displacing synthetic fertilisers associated with high environmental burdens. Overall, this study demonstrates the environmental and economic viability of BSF bioconversion as a zero-waste solution for the fishing sector. Future efforts should prioritize renewable energy integration and replacing agricultural substrates with fishery side-streams to further enhance sustainability.

Olive it or leave it: valorising olive fragments in black soldier fly larvae diets

T.I. Gogou^1*, R.H. Nakirya², P. Soulioti¹, E. Valli³, C.I. Rumbos⁴, M.T. Capucchio⁵ and C.G. Athanassiou¹

¹University of Thessaly, Fytokou, 38446 Volos, Greece; ²University of Bologna, Via Irnerio 42, 40126 Bologna, Italy; ³University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy; ⁴University of Patras, 30200 Messolonghi, Greece; ⁵University of Turin, Via Verdi 8, 10124 Turin, Italy; ^*theo.gog9@gmail.com

Olive oil production generates a wide range of by-products, including olive fragments, mainly in Mediterranean countries. These fragments, mostly olive leaves and branches, are often discarded after harvesting, although they retain nutritional potential. Black soldier fly larvae (BSFL) can efficiently utilise such agricultural residues and convert them into high-quality protein. Therefore, this study investigated the impact of incorporating different levels of olive fragments into BSFL diets on larval growth and performance. Diets with three inclusion levels of olive fragments (5, 12 and 25%) were combined with whey, brewers’ spent grains and wheat bran, while the control diet contained the same by-products without olive fragments. All diets were formulated to meet the larvae’s protein and lipid requirements, except for the 25% olive fragments diet, which had a slightly lower protein content; three of the diets were isoenergetic. Larval performance was evaluated every three days by recording weight and survival until 10% of the larvae reached the prepupal stage. Feed utilisation parameters, specifically Feed Conversion Ratio (FCR) and Bioconversion Ratio (BCR), were calculated, and nutritional analyses of diets and larvae were performed. Survival remained higher than 90% for all diets, with no significant differences. The control diet and the 5% inclusion diet consistently showed the best performance across all the evaluated parameters. The 12% olive fragments diet demonstrated intermediate results, while the 25% diet performed the least favourably. Chemical analyses revealed that larvae contained 50–56% protein and 2–9% lipids, with the highest values being recorded for the 5% and control diet, respectively. In conclusion, these findings indicate that moderate inclusion of olive fragments in BSFL diets can enhance feed efficiency and growth, whereas higher levels may reduce performance. Acknowledgement: This research is supported by the EU-PRIMA program — project OLIWA-Repurposing OLIve WAste in circular economy solutions for feeds, additives, packaging, and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus.

Closing the loop: evaluation of tomato by-products as feed for Tenebrio molitor larvae

R.H. Nakirya^1*, T.I. Gogou², P. Soulioti², E. Valli³, M. Guida³, C.I. Rumbos⁴ and C.G. Athanassiou²

¹University of Bologna, Via Irnerio 42, 40126 Bologna, Italy; ²University of Thessaly, Phytokou, 38446 Volos, Greece; ³University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy; ⁴University of Patras, 30200 Messolonghi, Italy; ^*racheal.nakirya@studio.unibo.it

During industrial tomato processing, large amounts of by-products are generated and often remain underutilised. Within a circular economy framework, these by-products could be used as alternative feeding substrates for Tenebrio molitor larvae, contributing to sustainable insect production. The aim of the present study was to evaluate the suitability of tomato by-products, mixed at different ratios with wheat bran, as diets for T. molitor larvae. The diets were formulated with tomato by-products at inclusion levels of 10, 25 and 50%, while two controls were used: 100% wheat bran and 100% tomato by-product. Larval performance was evaluated on a weekly basis by recording larval weight and survival, and the experimental period ended with the appearance of the first pupa. In addition, feed utilisation parameters, such as Feed Conversion Ratio (FCR) and Specific Growth Rate (SGR), were calculated. Analyses for proteins and lipids were performed in larvae and diets, while tomato by-products were tested for lycopene. Statistical analysis revealed significant differences among most evaluated parameters, while survival rate remained above 83% in all cases. Wheat bran showed the highest overall performance, followed by tomato inclusion levels of 10% and 25%. The 50% tomato diet presented intermediate to lower performance, whereas the 100% tomato diet was less efficient across all parameters. Regarding larval chemical composition, protein content was approximately 52% across most diets, whereas larvae fed exclusively on tomato by-products showed notably higher results (approx. 69%). Lipid content was around 30% in larvae from all diets, but was markedly reduced (approx. 3%) in the 100% tomato treatment. The analyses of tomato by-products indicated the presence of lycopene that may act as a beneficial factor for T. molitor larvae. These findings suggest that tomato by-products can be effectively used when mixed with other substrates, contributing to the optimisation of T. molitor larval production.

Mitigating antimicrobial resistance transmission in manure via black soldier fly larvae-microbe synergistic conversion

M. Cai^*

Huazhong Agriculture University, Life science collage, No. 1, Shizishan Street, Hongshan District, 430070 Wuhan, Hubei Province, P.R. China; ^*cmm114@mail.hzau.edu.cn

The dissemination of antibiotic resistance genes (ARGs) is a critical factor driving the global increase in environmental antimicrobial resistance (AMR), with animal manure being a primary reservoir posing significant public health risks. Black soldier fly larvae (BSFL) treatment is an emerging agricultural waste valorisation technology that simultaneously addresses pollution and produces insect biomass for feed protein. This study focuses on controlling the environmental risks of ARGs during BSFL conversion and elucidating the underlying microbial mechanisms. Our results demonstrate that BSFL-microbe synergistic conversion effectively reduced the total absolute abundance of ARGs in pig manure by 93.2%, significantly mitigating the risk of multidrug-resistant pathogens. Field experiments using insect frass as biofertiliser revealed a specific microecological mechanism: the application of frass activated the Bacillaceae family in the soil. This bacterial family, known for inhibiting antibiotic-resistant bacteria (ARB), showed enhanced carbohydrate metabolism pathways and promoted biofilm formation, which antagonized ARB and consequently reduced ARG proliferation. Furthermore, tracking experiments in radish (Raphanus sativus) cultivation showed that frass application significantly decreased the richness and abundance of ARGs, mobile genetic elements (MGEs), and virulence factors (VFs) in the edible roots compared to conventional fertilisation, thereby reducing potential pathogenic resistant bacteria. This study provides comprehensive technical and theoretical support for mitigating the transmission of antibiotic resistance in agricultural environments, highlighting the safety and ecological benefits of BSFL-derived biofertilisers.

Integrating dairy side-streams into insect rearing: performance of Hermetia illucens and Tenebrio molitor larvae

G.V. Baliota^1*, S. Zafeiriadis¹, G. Vrontakis¹, T. Gogou¹, C. Adamaki-Sotiraki¹, C.I. Rumbos² and C.G. Athanassiou¹

¹Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, Phytokou Street, 38446 Nea Ionia, Greece; ²University of Patras, Department of Agriculture, Messolonghi Campus, 30200 Messolonghi, Greece; ^*mpaliota@uth.gr

Dairy side-streams are generated in large volumes and may pose environmental risks due to their high organic load, odours and microbiological hazards when inadequately managed. Although these materials are often redirected to biogas production, insect bioconversion provides an additional circular pathway for nutrient recovery. Here, we evaluated expired dairy products and side-streams from cheese production as rearing inputs for two commercially relevant insect species: the black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), and the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). Across all dairy-based treatments, H. illucens larvae showed rapid development (10–13 days) and high survival (>90%), with expired yogurt consistently yielding the best performance, including higher larval weight and improved feed conversion ratio. For T. molitor, dairy side-streams were provided as moisture sources with agar or starch as gelling agents. All supported successful development, with high final larval weights and survival generally >80%, while development time was shortened (<49 days) in some treatments. Proximate larval composition analyses indicated no treatment effects on larval nitrogen content in either species, but this was not true in the case of lipids. This research is supported by PRIMA, a program supported by the European Union, under grant agreement No 2231, project CIPROMED (PRIMA Call 2022 Section 1 Agri-food IA).

From soil to water: agronomic performance and environmental safety of black soldier fly frass as a sustainable fertiliser

D.N. Cardoso^1*, A. Rodrigues¹, I. Cruz¹, J. Pinto¹, A. Mostafaie¹, C. Malheiro¹, A.R. Silva¹, I.G. Lopes², G. Pinto¹, P. Monteiro¹, C. Souza^1,3,4 and S. Loureiro¹

¹CESAM – Centre for Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal; ²Swedish University of Agricultural Sciences, Department of Biosystems and Technology, Alnarp, Sweden; ³Ingredient Odyssey SA – Entogreen, Santarém, Portugal; ⁴Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Caparica, Portugal; ^*dfilipe@ua.pt

The transition towards sustainable fertilisation strategies requires alternatives to synthetic mineral fertilisers that maintain crop productivity while reducing environmental impacts. Insect frass derived from Hermetia illucens larvae represents a circular, bio-based fertiliser with potential to improve soil functioning and nutrient retention. However, integrated assessments across soil–plant systems and aquatic compartments remain limited. Two entomofertilisers produced from H. illucens frass (olive pomace-based and cereal-based) were evaluated against a conventional mineral NPK fertiliser using integrated experimental systems. Soil–plant assays with Brassica rapa were conducted over two full life cycles under equivalent nitrogen inputs to assess plant performance, soil enzymatic activity, nutrient dynamics, and soil ecotoxicology. In parallel, soil column experiments simulated intense rainfall to quantify nutrient leaching (NH₄⁺, NO₃^−, PO₄^3−), and leachate ecotoxicity to freshwater organisms was assessed. Frass-based fertilisation supported plant development comparably to 100% NPK, particularly when combined with mineral fertiliser, while exclusive frass applications showed slightly lower early-stage biomass, consistent with the slow-release dynamics of frass. Plant physiological indicators remained stable, indicating no metabolic stress. Frass amendments enhanced soil enzymatic activities related to C, N, and P cycling and promoted balanced ammonium–nitrate dynamics. Soils amended with frass sustained plant growth in a second cultivation cycle without re-fertilisation, demonstrating residual fertility. Leaching experiments showed lower N and P losses from frass-amended soils than from NPK-treated soils. Soil ecotoxicological assays revealed no adverse effects on soil invertebrates, and frass leachates induced no toxicity in aquatic organisms, whereas NPK leachates inhibited algal growth. H. illucens frass is an environmentally safe and effective bio-based fertiliser that supports plant performance, improves soil health, reduces nutrient losses, and limits ecological risks, making it a viable alternative or complement to synthetic fertilisers in circular and climate-smart agriculture.

Colonisation of the black soldier fly larval midgut by Escherichia coli for improved biotransformation strategies

D. Roma*¹, M. Vaghi¹, D. Bruno², C.J.R. Scott¹, M. Attafi³, M. Vanoni³, A. Pessina³, L. Brambilla³, L. Pollegioni², G. Tettamanti², M. Casartelli¹ and S. Caccia¹

¹University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy; ²University of Insubria, Department of Biotechnology and Life Sciences, Via Dunant 3, 21100 Varese, Italy; ³University of Milano-Bicocca, Department of Biotechnology and Biosciences, Piazza della Scienza 2, 20126 Milan, Italy; ^*davis.roma@unimi.it

Insect-mediated bioconversion represents a promising approach for the valorisation of organic wastes and by-products of the agri-food chain, enabling the production of high-value insect biomass. Black soldier fly larvae (BSFL) are among the most effective bioconversion agents, industrially exploited to obtain protein for animal feed, and extensively studied for a wide range of biotechnological applications, including sustainable waste management strategies. Bioconversion of organic waste may be challenging due to the presence of recalcitrant, hardly digestible, polymers, such as plastics or lignocellulose. Their breakdown during the bioconversion process could be facilitated by conditioning BSFL midgut microbiota with engineered bacteria producing specific hydrolytic enzymes and making available degradation products to boost BSFL growth. To this purpose, protein engineering and synthetic biology made Escherichia coli a valuable biotechnological tool for the expression of single or multiple genes of interest. However, E. coli is not a natural inhabitant of BSFL gut. For this reason, it is important to assess whether this bacterium can colonize the BSFL gut. In the present work we developed an effective and simple protocol to monitor the colonization dynamics of BSFL midgut by E. coli. In particular, we: (i) determined the factors affecting E. coli survival in different BSFL rearing substrates and in the larval midgut; (ii) identified simple, cost-effective strategies to improve the persistence of E. coli in the BSFL midgut; and (iii) verified the expression of a newly introduced gene in midgut-colonizing E. coli by RT-qPCR. The results showed that the engineered E. coli can colonise the BSFL midgut and proved to be metabolically active. However, the outcome of the colonisation largely depends on the rearing substrate used. In particular, evidence suggested an antagonistic role of substrate and midgut microbiota, both driven by chemical composition of the diet, against E. coli. Importantly, the metabolically active bacterium can produce the protein of interest after several days post-inoculation in the substrate. Overall, this study demonstrated that recombinant E. coli can colonize BSFL midgut and could be used as a tool for engineering midgut microbiota to support larvae digestive functions.

Feeding Alphitobius diaperinus larvae with adults of Aedes albopictus and Bactrocera dorsalis

G.V. Baliota^1*, X. Gkargkavouzi¹, A. Michaelakis², C.I. Rumbos³, A. Puggioli⁴, R. Bellini⁴, T. Ozeri⁵, S. Thammasart⁵ and C.G. Athanassiou¹

¹University of Thessaly, Department of Agriculture, Crop Production and Rural Environment, Phytokou Street, 38446 Nea Ionia, Greece; ²Benaki Phytopathological Institute, Stefanou Delta Street, 14561 Kifissia, Athens, Greece; ³University of Patras, Department of Agriculture, Messolonghi Campus, 30200 Messolonghi, Greece; ⁴Centro Agricoltura Ambiente “G. Nicoli”, Via Sant’agata 835, 40014 Crevalcore, Italy; ⁵Flying Spark (Agritech) Co. Ltd., 88/8 Moo 3, Huai Rong Subdistrict, Khao Yoi District, Phetchaburi Province 76140, Thailand; ^*mpaliota@uth.gr

Insects are mass-reared for pest-control programs (e.g., Sterile Insect Technique (SIT)) and for food and feed applications. A common yet underutilised byproduct of these systems are the parental adults that remain after mating, having no further operational value. Developing valorisation pathways for this “adult” side-stream could improve the sustainability and profitability of mass-rearing facilities. One promising approach is their conversion through secondary insect production systems, such as the one of the lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), which can efficiently convert organic substrates into high-nutritional biomass and more. In this study, we assessed adult side-streams from the Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae) from a mass rearing lab, and the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), as dietary ingredients for A. diaperinus larvae. Wheat bran-based diets were formulated with 0, 25, 50, 75, or 100% (w/w) adult side-stream from each species. The results indicated that inclusion of 25 to 75% adults of both species in the A. diaperinus larval diet improved multiple developmental traits in comparison to the wheat-bran control treatments, whereas performance at 100% inclusion was less favourable. These findings support the concept that adult Diptera can function as a valuable feed ingredient for A. diaperinus production and highlight an opportunity for integrated, circular use of this biomass across insect-rearing facilities. Further work should characterise the nutritional drivers of the positive responses observed, and particularly which nutrients are complemented by the dipteran adults that wheat bran is lacking, in order to refine A. diaperinus diet formulations. Acknowledgement: This research is supported by PRIMA, a program supported by the European Union, under grant agreement No 2231, project CIPROMED (PRIMA Call 2022 Section 1 Agri-food IA).

Agro-industrial by-products for black soldier fly larvae: gut microbiota, performance and probiotic application

C. Jucker^1*, G. Brenna¹, E. Bigarella¹, D. Hentati¹, R. Tedeschi² and E. Crotti¹

¹University of Milan, Italy, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Celoria 2, 20133 Milan, Italy; ²University of Turin, Department of Agricultural, Forest and Food Sciences, Largo P. Braccini 22, 10095 Grugliasco (TO), Italy; ^*costanza.jucker@unimi.it

Larvae of black soldier fly (Hermetia illucens L.) efficiently bioconvert low value organic mass into high quality biomass enabling the valorisation of agri-food side-streams within circular bioeconomy paradigms. Scaling up black soldier fly production in a sustainable and cost efficient manner, requires the identification of feedstocks economically accessible and available at industrial volumes. Diet composition is known to modulate not also larval performance, but also gut microbiota, a key determinant of host physiology including nutrition and development. Larvae were reared on agri food by products (okara, brewer’s spent grain, potato peels, and potato sorting residues) alongside a standard diet. Growth performance was quantified, and gut microbial communities were profiled in terms of taxonomic composition and total bacterial load. Moreover, to enhance larval performance on potato wastes, four bacterial strains were administered via the substrate with two inoculations; heat inactivated counterparts were also provided to evaluate their contribution as a source of nutrients. Okara supported the highest bioconversion efficiency and larval performance, followed by brewer’s spent grain and potato sorting residues, whereas potato peels induced substantial mortality (57%). Substrate reduction ranged from 34 to 55%, with okara again yielding the highest rate. Different substrate dependent gut microbial community were detected, yet Enterococcaceae consistently dominated, albeit with reduced relative abundance in larvae fed with potato peels and potato sorting waste. These to latter substrates promoted also higher bacterial loads compared with the standard diet. Bacterial supplementation did not influence larval survival or final biomass yield, although significant differences in larval weight emerged after the second inoculation, particularly with active strains. Overall, the exploitation of agri-food by products represents a valuable strategy to enhance the sustainability of black soldier fly production. Further insights are required to elucidate how substrate influence the gut microbiota and, in turn, insect–microorganism interactions, ultimately supporting the development of probiotic assisted rearing strategies to optimise insect rearing and its health. Acknowledgement: Work funded by PRIN 2022 Project InProFarm (Insect Probiotic-assisted Farming: a promising tool to enhance edible insect health and performances), grant number 2022L4NJMK-Next Generation EU, Mission 4, Component 2.

Evaluating the effect of chemical pretreatment of lignocellulosic biomass through black soldier fly larvae performance

M. Sultana^1,2*, A. Rath², C. Schwennen², A. Wahab², S. Lipinski^1,3, C. Visscher², N. Grabowski³, M. Plötz³ and K. Rehman^1,4

¹German Institute of Food Technologies (DIL e.V.), Insect Research Group, Advanced Technology, Prof.-v.-Klitzing-Strasse 7, 49610 Quakenbrück, Germany; ²University of Veterinary Medicine Hannover, Institute for Animal Nutrition, Bünteweg 2, 30559 Hannover, Germany; ³University of Veterinary Medicine Hannover, Institute of Food Quality and Food Safety, Bünteweg 2, 30559 Hannover, Germany; ⁴The Islamia University of Bahawalpur, Faculty of Veterinary and Animal Sciences, Bahawalpur 6314, Pakistan; ^*m.sultana@dil-ev.de

Lignocellulosic biomass (LCB), composed mainly of cellulose, hemicellulose and lignin, is the most abundant renewable resource with potential applications for bioconversion. Recalcitrant structure limits LCB’s biological utilisation. Chemical pretreatment (C-PT) reduces covalent and hydrogen bonds, crystallinity and the degree of polymerization, thereby increasing cellulose accessibility, disrupting lignin-cellulose and lignin-hemicellulose complexes. This study investigated the effect of C-PT of LCB on black soldier fly larvae (BSFL) performance, including survival rate (SR), larval growth (LG), bioconversion rate (BCR), efficiency of conversion of ingested feed (ECI), and feed conversion ratio (FCR). LCB substrates were pretreated with aqueous solutions of 1.0% and 2.0% NaOH and 1.0% and 2.0% citric acid (CA), separately, defined as A, B, C and D, and untreated substrate as control diet (CRL). One hundred BSFL were raised on untreated and treated substrates in plastic containers (diameter 10–14 cm, height 8 cm) for 14 days with three replicates. Rearing conditions: 28.5 ± 0.2 °C, RH 56.2 ± 3.2%, feeding rate 50 mg/larva/day, feed moisture 85%. SR exceeded 85% in all trials. The highest SR (98.67 ± 0.88) and LG (78.04 ± 2.48 mg larva-1) were determined for BSFL reared on A compared to CRL (95.67 ± 1.76% and 23.48 ± 0.38 mg larva-1, respectively). ECI (%) = 12.23 ± 1.20 (CRL); 44.89 ± 2.99 (A); 54.84 ± 5.61 (B); 35.36 ± 7.41 (C); and 32.52 ± 1.58 (D) as well as BCR (%) = 3.31 ± 0.06; 11.25 ± 0.36; 5.63 ± 1.03, 8.21 ± 0.69; and 9.32 ± 0.17 for BSFL, while FCR (mg larva-1) = 8.35 ± 0.88; 2.25 ± 0.15; 1.87 ± 0.21; 3.07 ± 0.60 and 3.09 ± 0.16, respectively. Increased LG, ECI, and BCR as well as reduced FCR compared to CRL indicate substrates become more convertible into BSFL-biomass upon C-PT. LG and BCR (except CRL vs 2.0% NaOH (B)), ECI (except CRL vs 2.0% CA (D)) and FCR are statistically significant ($p\mathrm{<}0.05$) compared to CRL. In conclusion, C-PT improves LCB quality for BSFL cultivation, supporting sustainable waste valorisation and circular bioeconomy strategies. Acknowledgement: Funded by Volkswagen Foundation.

The effects of fish-faeces diets on the growth and elemental composition of Hermetia illucens and Tenebrio molitor

W. Yakti^1*, E.W. Poley¹, C. Shaw², I. Mewis¹, W. Kloas², D. Deruytter³ and C. Ulrichs¹

¹Humboldt University of Berlin, Lentzeallee 55/57, 14195 Berlin, Germany; ²Leibniz Institute of Freshwater Ecology, and Inland Fisheries, Müggelseedamm 310, 12587 Berlin, Germany; ³Inagro, Ieperseweg 87, 8800 Beitem, Belgium; ^*Wael.yakti@hu-berlin.de

The potential use of aquaculture sludge as a substrate for the black soldier fly (BSFL) has been investigated in various studies, and this use is usually associated with the risk of accumulating heavy metals in the larval biomass produced. In the current study, pure fish faeces, not contaminated with fish feed or fish carcass, was included in the substrates of BSF and the yellow mealworm. The substrates contained chicken feed and straw, and the chicken feed was replaced with fish faeces. Growth performance, fresh and dry yield, crude protein content, feed conversion ratio (FCR), survival rate, and elemental composition were measured. The results showed that even a 19% inclusion of fish faeces did not impair BSFL performance indicators, while dry yield and FCR were optimal at 15%, inclusion and survival rates remained high across all treatments. Elemental analysis revealed increasing concentrations of various elements (e.g., Fe, Zn, Cu, Pb, Cd) the higher the inclusion of fish faeces is; however, the levels of As, Cd, Pb, and Hg remained at or below current EU maximum limits for feed materials, with only Cd nearly reaching the limit for complete feed at the highest inclusion rate. The yellow mealworms (MW) were fed the same diet and the same parameters were assessed. Growth performance did not differ among treatments regardless of fish faeces inclusion level, and the concentration of most measured elements increased with the inclusion rate. However, accumulation of all examined elements was markedly higher in BSFL than in mealworms. Overall, the results indicate that both insect species can be fed diets containing up to 19% or moderate levels of fish faeces without impairing larval growth. MW biomass appears to be “safer” if the goal is to produce insect biomass with lower concentrations of the undesirable heavy metals.

Developing cost-effective enzymatic aqueous oil extraction for industrial BSF biorefineries: preliminary results

A. Carreira^1*, R. Fantatto², D. Murta² and O. Moreira¹

¹Instituto Nacional de Investigação Agrária e Veterinária — INIAV, I.P., Av. Professor Vaz Portugal, 2005-424 Vale de Santarém, Portugal; ²EntoGreen — Ingredient Odyssey, S.A., Casais do Mocho, 2005-002 Várzea, Portugal; ^*ana.carreira@iniav.pt

The black soldier fly (Hermetia illucens, BSF) is a key species for emerging insect biorefineries due to its capacity to convert low-value organic substrates into high-value lipids, proteins, and bioactive compounds. To enhance sustainability and industrial scalability, alternatives to conventional solvent-based oil extraction are required. Aqueous enzymatic extraction (AEE) represents a promising solvent-free method with lower environmental impact and improved product quality, suitable for food, feed, and other refined applications. This study evaluated the use of Alcalase (ALC) from Bacillus licheniformis and Protease (PROT) from Bacillus sp. at 0, 0.4 and 2% (enzyme:dry larval biomass) for aqueous oil extraction from BSF larvae reared on olive pomace, a common agro-industrial by-product in Portugal. Following pH adjustment, incubation at 55 °C for 1h45min, centrifugation, and oil recovery, oil yield (%) and extraction efficiency (%) were determined. Both parameters increased consistently with enzyme concentration. Yield ranged from 17.70 to 19.79% (mean ± SD = 18.95 ± 1.02%, and extraction efficiency from 51.56 to 56.95% (mean ± SD = 54.34 ± 2.94%). For ALC, yield increased from 17.70 ± 0.71% to 19.55 ± 1.40% and efficiency from 51.56 ± 2.07% to 56.95 ± 4.09%, whereas for PROT yield rose from 18.35 ± 0.33% to 19.79 ± 0.68% and efficiency from 51.80 ± 0.92% to 55.88 ± 1.93%. No significant differences were observed between enzymes, and no enzyme–concentration interaction was detected, although enzyme concentration showed a trend toward higher yield ($p=0.056$) and efficiency ($p=0.058$). While oil yields remain lower than those typically achieved by solvent extraction (30–40%), AEE offers advantages in sustainability, safety, and product quality. Future work will focus on process optimisation (enzyme dosage and reaction conditions), enzyme recycling across multiple extraction cycles, and full valorisation of residual fractions (oil, proteins, chitin, and other bioactive compounds) to enhance economic and environmental feasibility. Process robustness will also be assessed using BSF larvae reared on diverse biowaste substrates. This integrated, solvent-free approach supports circular bioeconomy principles by coupling biowaste valorisation with high-value biorefinery outputs and cost-efficient processing.

Spent coffee grounds and their mode of drying as feed ingredient for Tenebrio molitor

V. Da Cunha Borges^1,2*, R. Rodrı́guez Gonzalez^1,2 and D. Martin^1,2

¹Universidad Autónoma de Madrid, Sección Departamental de Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Producción y Caracterisación de Nuevos Alimentos, C/ Nicolás Cabrera 9, 28049 Madrid, Spain; ^*virginia.dacunha@uam.es

Spent coffee grounds (SCG) are a major by-product of the coffee industry that remains underutilised despite being rich in interesting bioactive compounds with health properties. This study evaluated the potential valorisation of SCG through their inclusion in the diets of Tenebrio molitor larvae, assessing growth performance, survival, and larval nutritional composition under different inclusion levels and drying processes of SCG. Experimental diets were formulated using wheat bran as a control (C) or partially replaced with SCG. Freeze-dried SCG were included at 15, 30 and 50% (FD15, FD30, FD50) to evaluate larval tolerance to increasing inclusion levels, while oven-dried SCG were tested at 15% (OD15) to compare drying methods at the same inclusion rate. The trial was performed in triplicate (845 larvae, 4 weeks-old, 8.6 mg/larva, 210 mg feed/larva) at 27 °C, 60% RH, and water supplied as agar, until 12 weeks-old. Mortality, weight gain (WG) and mass reduction (MR) were estimated. Larvae were blanched (100 °C, 1 min), oven-dried (60 °C, 18 h), and analysed. Larvae fed the control diet exhibited WG of 103.9 mg/larva. SCG inclusion induced a dose-dependent decline in larval growth, influenced by the drying process of SCG. Thus, inclusion of freeze-dried SCG above 15% led to severe growth impairment (WG <10 mg/larva) and mortality (72–97%). At the same inclusion level (15%), oven-dried SCG resulted in WG value around 40% higher than those observed for freeze-dried SCG. Nevertheless, oven-dried SCG still resulted in around 40% lower larval growth than control (62.5 mg/larva). At 15% inclusion, MR also decreased with SCG treatments, particularly for freeze-dried SCG. Despite reduced growth, SCG increased protein content in larvae (36, 41 and 51% for C, OD15 and FD15, respectively), and reduced lipid levels (36, 20 and 11 for C, FD15 and OD15, respectively). This preliminary assay demonstrates that SCG, its drying method and inclusion level affects T. molitor rearing performance and composition. While reduced growth and survival suggest the need for caution, the notable larval nutritional improvement, by protein enrichment and lipid reduction, supports further research on optimised SCG processing and inclusion strategies, as well as further exploration on the potential changes in the functional properties of insect biomass. Acknowledgment: ENTOMOTIVE project, PID2022-136238OB-I00 and grant PREP2022-000828, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU.

Conservation of organic solid waste and agro-industrial by-products for bioremediation with black soldier fly larvae

J. Regedor^*, I. Rehan, J. Costa and O. Moreira

Insituto Nacional de Investigação Agrária e Veterinária, Pólo de Inovação da Fonte Boa, 2005-424 Vale de Santarém, Portugal; ^*jose.regedor@iniav.pt

The inadequate management of organic solid waste (OSW) and agro-industrial by-products is an environmental and economic challenge. Their valorisation through bioremediation with black soldier fly Hermetia illucens (BSF) larvae is a sustainable alternative, but its efficiency depends on the preservation of the substrate. This study aimed to investigate the conservation of OSW and by-products zucchini (CUR) and broccoli stalks (TB) over 45 days, combining packaging type (plastic and vacuum), temperature (ambient and refrigeration at 4 °C), and the use of a preservative (0.5% citric acid). For each substrate, the following treatments were applied: (1) plastic bags at ambient temperature (SPAmb); (2) plastic bags under refrigeration at 4 °C (SPRef); (3) vacuum bags at ambient temperature (SVAmb); (4) vacuum bags under refrigeration at 4 °C (SVRef); (5) preservative in bag at ambient temperature (CVAmb) and (6) preservative in bag under refrigeration at 4 °C (CVRef). Chemical composition and nutritional losses were analysed, and statistical analysis was performed using PROC MIXED procedure of SAS (Statistical Analysis System). The CV treatments showed better stability of dry matter (DM) and non-nitrogen extractives (NNE) (values above 480 and 670 g/kg DM in OSW and by-products, respectively), indicating effective inhibition of microbial degradation, although with higher losses of crude protein (CP) (up to 38.8 and 25.4% in CUR and TB, respectively) and crude fat (CF) (83.4 and 57.5% in CUR and TB, respectively). In contrast, the SP and SV methods better preserved protein and fibre but resulted in higher losses of NNE (up to 24.4% in OSW and 27.4% in by-products). Refrigeration reduced some losses, but the preservation method was the key factor. Overall, preservation with citric acid optimises substrate stabilization for BSF larvae, while plastic and vacuum methods may be preferable when the primary goal is protein-rich larval biomass.

Composting of Tenebrio molitor frass to reduce phytotoxicity: implications for agronomic use as an organic fertiliser

G. Sabbatini, C. Uboni, E. Pellegrini, F. Tulli, M. Contin and G. Fellet^*

University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Via delle Scienze 208, 33100 Udine, Italy; ^*guido.fellet@uniud.it

The growing interest for insect farming for food and feed production is generating increasing amounts of insect frass, a by-product with considerable potential as an organic fertiliser. Previous studies by the authors have shown that the soil application of frass can stimulate soil biological activity, increasing microbial biomass C and respiration by 4 and 20 times compared to the control, respectively, as well as double the availability of mineral N. Such effects have been attributed both to the stimulation of native soil microorganisms and to the direct input of exogenous microbial biomass contained in the frass itself. Anyway, the agronomic use of frass is often constrained by phytotoxic effects. This study evaluates composting as a practical post-treatment to reduce the phytotoxicity of Tenebrio molitor (TM) frass and to improve its suitability for agricultural applications. TM colony was reared in plastic trays (60 × 40 × 13 cm) at 1 larva/cm² rearing density in a climate-controlled chamber (T = 26 ± 1 °C, RH = 55 ± 3%, 10L:14D) at the University of Udine. Wheat bran was used as substrate, and potato slices were added every 4 days as a water source. TM frass was composted for 30 days under controlled moisture (60%) and aeration conditions. A germination trial was conducted using maize (Zea mays L.) and sunflower (Helianthus annuus L.) grown on soil amended with composted frass (CF) or untreated frass (UF) at two application rates (4% and 8%, w/w), alongside an unamended control. Plant responses were assessed after 15 days by measuring fresh weight (FW), dry weight (DW) and germination index (GI), followed by two-way ANOVA. UF significantly reduced plant performance, particularly at the 8% rate, with GI values indicating phytotoxic conditions. Composting improved frass performance, increasing GI values to non-phytotoxic or stimulating classes depending on crop and dose. In maize, CF applied at 4% resulted in GI values above 80% and significantly higher FW and DW compared to UF, with whole-plant FW exceeding the control, in some cases. In sunflower, composting reduced phytotoxicity at both application rates, although biomass responses were more variable. Overall, composting reduced without eliminating frass phytotoxicity, with the 4% application rate consistently outperforming the 8% rate. When combined with the stimulation of soil microbial activity and nutrient availability, these results support composted TM frass as a promising organic fertiliser once appropriate post-treatment is adopted to safely integrate insect farming by-products into sustainable agricultural systems.

Investigating black soldier fly Hermetia illucens frass fertility: from crop residues to insect feed and bio-fertilisers

I.M. Scott^*, R. Murray, S. Deelen and F. Longpre

Agriculture and Agri-Food Canada, London Research and Development Centre, 1391 Sandford Street, London, ON, Canada N5V4T3; ^*ian.scott2@agr.gc.ca

Edible insects are increasingly grown in Canada for pet and livestock protein supplements, and to digest and manage food waste streams. Mass-reared insects like black soldier fly (BSF) are used in the bioconversion of food waste, including fruits and vegetables, to protein and frass. Other available food grade plant residues include field crops (edible bean, soybean, corn) and cover crops (buckwheat, mustard, peas) that have minimal pesticide use. Trials with various crop residues and seeds from cover crops indicated that BSF had 100% survival up to the pre-pupal stage (11 days) and larval weights were similar to those fed the control diet. Typically, the materials that produced the higher BSF weights, for example buckwheat (BW) seed, had higher carbohydrates (20%), but less crude protein (20%) than the control feed, Gainesville diet (GVD). Promising crop wastes were corn red dog (kernel chaff/dust), soybean hulls, and edible bean husks and stems. A combination of BW seed with less nutritious residues, such as dried distillers grain (DDG) at 40:60 BW:DDG, increased the weight of BSF to that of the GVD-fed larvae by the prepupal stage. Insect frass produced by BSF from the crop residue diet can also provide a source of fertiliser for growing vegetables and field crops. Field trials with frass produced by a Canadian commercial BSF supplier determined that recommended chemical fertiliser rates could be reduced if 500 to 1000 kg/Ha frass contributed part of the N-P-K. The growth (biomass, plant density, canopy area) of cover crops, BW and brown mustard (BM), was no different between fertiliser alone and frass-fertiliser combined. For example, 48.2% less N, and 100% less P and K were needed to grow BW when 1000 kg/Ha frass was applied. With BM, 28.4% less N, 60.9% less P, and 75.9% less K were required. The benefit of harvesting the BW seed for use as BSF feed was that the soil nutrients (N-P-K) were not significantly different between the time when cover crops are terminated (before seed set) and the time required for seed harvest (2-4 weeks later). Measurement of contaminants in the frass from these promising feed sources indicated that heavy metals met the standards for use as a fertiliser. These findings suggest that selected field crop residues and cover crops, singly or in combination, can: (1) provide nutritious feed for BSF; (2) reduce crop wastes; (3) provide a rich fertiliser for cover crop growth; (4) reduce chemical fertiliser use and (5) constitute key components in a novel circular economy. Future studies will examine the application of frass produced from mixtures of the field crop wastes as an amendment for cover crop growth and seed yield.

Valorisation of agricultural by-products as nutritional substrates for Tenebrio molitor larvae

C. Uboni, G. Pascon, G. Cardinaletti and F. Tulli^*

University of Udine, Department Agriculture, Food, Environment and Animal Science, Viale delle Scienze 206, 33100 Udine, Italy; ^*francesca.tulli@uniud.it

The utilisation of locally produced agricultural by-products as a feed source for Tenebrio molitor larvae offers numerous advantages and contributes to the development of sustainable circular economy models. The study aimed to evaluate the impact of various by-product-based diets on larval growth, survival, Efficiency of Conversion of Ingested Food (ECI) and on the nutritional profile of yellow mealworm (MW) larvae. Homogeneous larval populations were obtained by collecting eggs from peak-reproduction adults of the stable colony of the University of Udine. Experimental substrates were formulated using pea (P), buckwheat (BW), barley (B) and soyabean (S) as 50% replacement to the high-value wheat bran (WB) rearing substrate (CTRL). Groups of 50 28-day larvae (1.99 ± 0.05 mg) per replicate (5/substrate) were transferred to a plastic box (17.5 × 9.3 × 6.3 cm) with aeration slits on the top according to a completely randomised design and maintained at 27 °C and 60% RH. Larvae were bulk weighed at the beginning and during the experimental period. The MW grew in all the substrates and MANOVA revealed a significant effect of the substrate on the following combined production variables: single-larva weight and their survival at the day of first pupal appearance, fresh yield, and pupation time ($p\mathrm{<}0.05$), but not on the pupal weight (119 ± 19 mg). The mean single-larva weight resulted the highest in CTRL (122 ± 13.7 mg) and BW followed by S, PP and the lowest in B (47 ± 8.9 mg). The highest larvae survival rate was observed in the CTRL (98%), followed by S, BW, PP (70%). The barley-based diet (B50) showed significantly lower survival (47%), with most mortality occurring in the first 20 days. Time of pupation differed with values ranging from 84 days (CTRL) to 154 days (B). Substrate significantly affected ECI (% DM): the highest value was registered in BW (58 ± 8.5) and lowest values in B50 (15.4 ± 4.6) ($p\mathrm{<}0.05$). Rearing substrate significantly affected larvae DM, protein (Kjeldhal N*4.76), lipid, ash and chitin content (ADF-ADL) ($p\mathrm{<}0.05$) as well as the fatty acids and mineral composition. The research shows that specific agri-food by-products like buckwheat, soy and pea can be effectively integrated into T. molitor diets, thereby supporting the transition to more sustainable and circular food systems while suggesting the possibility to tailor products for specific targets of consumers. Acknowledgement: This work was supported by the University of Udine in the framework of the Strategic Plan 2022-25 Interdepartmental Research Project CibiAmo.

Bioaccumulation and fate of per- and polyfluoroalkyl substances (PFAS) in black soldier fly (Hermetia illucens) larvae

D. Gomez¹, S. Sébastien², V. Céline¹, B. Simon³ and G. Vandenberg^1*

¹Universitè Laval, Sciences animales, 2425, rue de l’Agriculture, Pavillon Paul-Comtois, Local 4211, Quebec City, QC, Canada G1V 0A6; ²Université de Montréal, 1375 Avenue Thérèse-Lavoie-Roux, Complexe des sciences, B-5017, Montréal, QC, Canada H2V 0B3; ³Université de Québec a Trois Rivières, 3351 boulevard des Forges, Trois-Rivières, QC, Canada G8Z 4M3; ^*Grant.Vandenberg@fsaa.ulaval.ca

The black soldier fly (Hermetia illucens) industry is an evolving and expanding sector globally, driven by its capacity to upcycle diverse organic matter into valuable products like larval biomass and frass. Previous work in our lab has demonstrated that digestates from anaerobic digestion originating from industrial plants can be used as a feeding substrate for black soldier fly larvae when an energy source is supplemented, thereby enabling the valorisation of the other nutrients present. However, using waste substrates, such as anaerobic digestates, poses contamination risks from trace elements, agrochemicals, antibiotics, microplastics and persistent toxins, including per- and polyfluoroalkyl substances (PFAS): synthetic “forever chemicals” known for environmental persistence, bioaccumulation, and health impacts at low concentrations. This study assessed PFAS dynamics during larval bioconversion of solid digestates from an urban organic waste biomethanisation plant, primarily processing wastewater treatment sludges mixed with household organic residuals. Up to 80 PFAS compounds were analysed in digestates (feed), larvae, and frass using LC-MS/MS at two independent laboratories (Université de Montréal and SGS-Axys), finding the same dynamics in both. Total PFAS concentrations in digestates were 36.5 and 47.9 μg/kg dry weight (dw), dominated by perfluorooctane sulfonate (PFOS; 33.2 and 32.4 μg/kg dw). Post-bioconversion, PFAS distributed to larvae (179 and 200 μg/kg dw) and frass (25.9 and 40.4 μg/kg dw), indicating bioaccumulation in larvae (e.g., PFOS up to 189 μg/kg dw) and a reduction of 16-67% in frass for key compounds such as PFOS and PFHxA. In absolute terms, input PFAS (6 and 7.9 μg per unit) partitioned to larvae (1 and 1.1 μg) and frass (4.4 and 7.1 μg), suggesting minor losses, transformations or external inputs according to specific PFAS (e.g., reductions in PFHxA and NEtFOSE, increases in PFNA and PFDoA). This dynamic depends on several factors but shows that some of the PFAS are being transformed during the process. These findings demonstrate PFAS bioaccumulation in larvae, with potential frass decontamination, highlighting opportunities for defining bioremediation strategies in waste management.

Circular use of olive waste (OLIWA) via black soldier fly rearing for feed, biopackaging and biogas

M. Jimenez Serrano^1*, R. Sorayani Bafgi², L. Gasco², A. Athanassiou³, P. Barattini⁴, S.L. Bavaro⁵, E.M. Lopez Hernandez⁶, Z. Cherifi⁷, K. Titouh⁸, H. Meziani⁹, C. Garces Narro¹⁰, J. De La Osada Garcia¹¹, E. Ivanova Stojcheva¹², A. Naderpour Peñalver¹³, F. Alegre Sasian¹⁴, C. Athanassiu¹⁵, A. Paraskevopoulou¹⁶, F. Trabelsi¹⁷, E. Ben Boubaker¹⁸, E.A. Hayouni¹⁹, B. Yurdakok-Dikmen²⁰, S.M. Ceylan²¹ and M.T. Capucchio¹

¹University of Turin, Department of Veterinary Sciences, Grugliasco (Turin), Italy; ²University of Turin, Department of Agricultural, Forest and Food Sciences, Grugliasco (Turin), Italy; ³Italian Institute of Technology, Genoa, Italy; ⁴Kontor 46 S.a.s., Turin, Italy; ⁵Institute of Science-Food Production, CNR, Turin, Italy; ⁶Consorzio Italiano Compostatori, Rome, Italy; ⁷University of Bouira, Department of Agronomy Sciences, Bouira, Algeria; ⁸National Institute of Agronomic Research of Algeria, Oued Ghir, Algeria; ⁹Specialised Technological Institute for Agricultural Training, Tizi Ouzou, Algeria; ¹⁰Universidad Cardenal Herrera, Valencia, Spain; ¹¹Universidad de Zaragoza, Veterinary School, Zaragoza, Spain; ¹²NATAC Biotech SLU, Madrid, Spain; ¹³AIMPLAS, Valencia, Spain; ¹⁴Metanogenia SL, Badajoz, Spain; ¹⁵University of Thessaly, Volos, Greece; ¹⁶Aristotle University of Thessaloniki, Thessaloniki, Greece; ¹⁷Association for Development-Preservation of the Environment and Heritage, Béja, Tunisia; ¹⁸Hay-Green Sarl, Tunis, Tunisia; ¹⁹Center of Biotechnology of Borj-Cédria, Hammam-Lif, Tunisia; ²⁰Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey; ²¹Esa Group Energy Co Inc, Ankara, Turkey; ^*maria.jimenezserrano@unito.it

Olive oil production results in waste and by-products (OBs) containing bioactive compounds. The OLIWA project aims to create a circular economy model applicable at the local level and to reduce food loss and waste across six Mediterranean countries (Italy, Spain, Greece, Turkey, Algeria and Tunisia). More specifically, the project will explore OB’s use in insect rearing, investigate the effects of olive natural extracts (NE), insect meals and their association on the intestinal barrier by means of in-vitro testing, investigate the effects of NE and insect meals on animal models, poultry and fish, trying to enhance gut health and final product quality, investigate OB’s potential in food packaging and use insect frass, poultry manure and olive waste for biogas production to be used in local industries. By proposing insects as an alternative source of proteins for animal feed and utilising OBs for insect meal production, OLIWA addresses the EU’s need for sustainable food security and reduced reliance on imported feeds. Furthermore, producing new active bio packaging material will prolong the shelf life of animal products reducing food waste and improving the product quality. Acknowledgement: This research is supported by the EU-PRIMA program — project OLIWA- Repurposing OLIve WAste in circular economy solutions for feeds, additives, packaging and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus.

From waste to wealth: engineering black soldier fly bioconversion for a circular waste economy in Saudi Arabia

D. Kigen^1*, S. Giri¹, M. Sarathy¹ and H. Almotairy²

¹King Abdullah University of Science and Technology, Clean Energy Research Platform, 23955-6900 Thuwal, Saudi Arabia; ²Saudi Food and Drug Authority, Feed Risk Assessment, 4904 Hittin, Saudi Arabia; ^*derrick.barissa@kaust.edu.sa

Saudi Arabia faces a dual challenge of increasing organic waste generation and dependence on imported feed and fertilisers. Approximately 33.1% of available food is lost or wasted across the supply chain, while per-capita municipal solid waste generation average 1.72 kg person a day between 2010 and 2018. Municipal solid waste management costs are projected to exceed USD 10 billion annually, and food waste is estimated at 4.07 Mt yearly, intensifying pressure on food security and waste management systems. Addressing these challenges aligns with the Kingdom’s Vision 2030 priorities for sustainability, circular resource use, and local value creation. At KAUST, we developed a modular and easily deployed black soldier fly (Hermetia illucens) bioconversion system designed to valorise heterogeneous organic waste streams under arid climatic conditions. The modular architecture enable scalable, rapid deployment across municipal and agro-industrial sectors. A pilot facility processing up to 5 t/day of mixed municipal and agro-industrial waste in the Thuwal region demonstrates the scalability of this waste-to-resource approach. Substrate specific feeding trials quantified feed conversion ratio (FCR), biomass yield, nutrient recovery, and environmental performance. Larval biomass achieved up to 47% crude protein (dry weight), with FCR values ranging from 1.6 to 1.9, depending on substrate composition. Amino acid profiling and the vitro digestibility confirmed suitability for aquaculture and poultry feed formulations. Organic waste mass was reduced by 62–71% within a 10–12-day larval growth cycle. Residual frass was thermochemically upgraded into a biochar-enriched soil amendment, increasing soil water holding capacity by 28–35% and improving controlled nutrient release in sandy soils. Extracted larval lipids (up to 32% dry weight) exhibited favourable fatty acid profiles for biodiesel and feed applications, while purified chitin yielded chitosan with up to 85% deacetylation efficiency. Anaerobically digested sewage sludge evaluated as a substrate resulted in substantial reductions of Escherichia coli and Salmonella spp., producing an organic fertiliser compliant with national regulatory thresholds. These results demonstrate the technical feasibility and regulatory readiness of BSF-based bioconversion into Saudi’s municipal waste management. The modular platform delivers co-benefits in sustainable feed production, soil rehabilitation, biosolid stabilization, and localized resource recovery, supporting a scalable circular bioeconomy for arid environments.

Productivity of Tenebrio molitor adults on different by-products

S. Malabusini, S. Viganò, S. Savoldelli and C. Jucker^*

University of Milan, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Celoria 2, 20133 Milan, Italy; ^*costanza.jucker@unimi.it

Thanks to their ability to convert organic matter into nutritious biomass, insects can help solve the issue of food waste and loss. Tenebrio molitor, the yellow mealworm, is one of the most studied species for the bioconversion of agro-industrial by-products. According to most current research, our primary focus is the evaluation of various byproducts (tomato peels, potato waste, brewer’s grain, okara, hemp waste, and wheat bran as control diet) for their ability to support the growth of yellow mealworm larvae. However, diet also has a profound impact on the lifespan, growth and productivity of adults. The reproductive phase and tray-based production are fundamental aspects of mass rearing. This study aims to evaluate the effectiveness of by-products, previously tested for their influence on larval development, on the oviposition. Newly emerged adults, sexed during the pupal stage and emerged from different substrates, were used for the experiments. Two different tests were set up: I) one adult couple per replicate; II) four adults couples per replicate. After ten days, adults were removed from each replicate for seven times (total of 70 days), and moved to another box with the same substrate. The tests were observed weekly, and once the new larvae in containers were visible, they were counted. Potato waste was the worst substrate, allowing the emergence of very few adults. Similar trends were observed in both experiments with one or four couple of yellow mealworm. Overall, higher number of larvae were observed on wheat, hemp waste and tomato peels (wheat: 29.33 ± 2.63; hemp waste: 22.46 ± 1.59 and tomato peels: 21.70 ± 0.77). A significantly lower number of larvae was observed on okara and brewer’s grain (respectively, okara: 5.23 ± 0.96 and brewer’s grain: 6.03 ± 1.09). When observing the egg-laying time, significant differences were only observed between the wheat and hemp waste, with a significantly higher presence of larvae developing between 30 and 40 days for the test with single couple. With four couples, no differences were found. Tests on okara and brewer’s grain revealed a large number of dead small larvae, indicating that oviposition was possible but probably not favourable for larval growth. In fact, number of offspring or productivity may not always be related to female fecundity, but rather to larval mortality after egg hatching. Conversely, hemp waste and tomato peels proved suitable for both larval growth and oviposition, which is an important requirement for efficient mass rearing. Acknowledgement: Research funded by the EU project “ON Foods – Research and Innovation Network on Food and Nutrition Sustainability, Safety and Security — Working ON Foods” (NRRP, No. 1550).

Antimicrobial extracts from edible insects: modulation through olive oil by-product-enriched diets

E. Rodrı́guez-González^1,2*, J.M. Silván^1,2,3, A.T. Varga⁴, C. Vargas⁴, A.J. Martinez-Rodriguez³ and D. Martin^1,2

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ³Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Biotecnologı́a y Microbiologı́a de Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ⁴Universidad de Almerı́a, Dpto de Biologı́a Aplicada, CEI3, 04120 Almerı́a, Spain; ^*esther.rodriguez01@uam.es

Supplying insects with by-products rich in antimicrobial compounds may enable to valorise side streams, while enhancing the antimicrobial activity of insect-derived products through bioaccumulation of such dietary compounds. The aim of this study was to assess the impact of dietary supplementation of Hermetia illucens (HI) and Tenebrio molitor (TM) larvae with by-products rich in antimicrobial compounds, such as olive by-products (olive leaves and olive pomace) on the subsequent antibacterial activity of extracts obtained from the fed larvae. Larvae of both species were reared a control diet (wheat bran), or wheat bran replaced with 30% olive leaves or 30% olive pomace. After rearing, larvae were slaughtered, dried, ground, and defatted. Bioactive extracts were obtained by ultrasound-assisted extraction with 70% ethanol from defatted flours. Antibacterial activity of the insect extracts was evaluated by broth microdilution against six bacterial species, including three Gram-positive (Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis) and three Gram-negative (Escherichia coli, Salmonella spp. and Campylobacter jejuni) bacteria. TM extracts lacked significant antibacterial activity against most tested bacteria, except for C. jejuni, for which larvae fed olive pomace showed a 1.22 log reduction in growth. In contrast, HI extracts exhibited strong antibacterial effects against the Gram-positive strains (1.56–5.82 log reduction), regardless of the diet. It was also remarkable that extracts obtained from larvae fed olive oil by-products showed a strong antibacterial effect (>6 log reduction) against S. aureus and B. subtilis, compared to extracts from control larvae. These findings indicate that both insect species and diets modulate the antibacterial activity of extracts obtained from insect flours. Overall, the study highlights the potential to produce tailored antibacterial extracts from H. illucens larvae reared on specific olive by-products as promising ingredients for health- and safety-related applications within a circular economy framework. Acknowledgment: ENTOMOTIVE project, PID2022-136238OB-I00 and grant PREP2022-000828, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU

Effects of heavy metal exposure on Hermetia illucens larvae

A. De Benedictis¹, D. Bruno¹, I. Armenia¹, M. Marzari¹, D. Panu², V. Prina¹, M. Casartelli^3,4, S. Caccia⁴, G. Tettamanti^1,3 and O. Santoro¹

¹University of Insubria, J.H. Dunant 3, 21100 Varese, Italy; ²University of Insubria, Valleggio 11, 22100 Como, Italy; ³Interuniversity Center for Studies on Bioinspired Agro-environmental Technology, Università 100, 80055 Portici (NA), Italy; ⁴University of Milan, Giovanni Celoria 26, 20133 Milan, Italy; ^*adebenedictis@studenti.uninsubria.it

The larvae of the black soldier fly (BSFL), Hermetia illucens, are highly efficient bioconversion agents of a wide range of organic waste. The possibility to tap into new rearing substrates for BSFL not yet authorised by EU legislation could allow increasing production volumes and reducing the price of BSF meal. In this setting, the organic fraction of municipal solid waste (OFMSW) is one of the most promising wastes to be used due to its high nutritional value and high quantities. However, the use of these substrates, that are considered unsafe due to the possible presence of chemical and microbiological hazards, may expose BSFL to various pollutants, including heavy metals (HMs), that could potentially compromise larval growth and health. Moreover, the possible accumulation of these xenobiotics in larval organs and tissues may pose a risk for BSFL-derived products. In this study we reared BSFL on OFMSW contaminated with two concentrations of PbCl₂ (i.e., 30 mg/kg and 120 mg/kg, d.m.). Growth performance, survival rate, and bioconversion efficiency were monitored and compared with control larvae fed on OFMSW devoid of lead. In addition, to evaluate the effects of this HM on larval physiology, the activation of the immune system was monitored through cellular (i.e., haemocyte counts) and humoral (i.e., antimicrobial activity) markers. Finally, lead accumulation was quantified in whole larvae and in different body tissues, as well as in the frass. Our results demonstrate that, although no effects on life cycle and development were observed — as confirmed by comparable survival rate and maximum weight reached by BSFL across different treatments — exposure to high lead concentrations induced the activation of both cellular and humoral immune components. In addition, we detected the accumulation of lead in various larval tissues (including the midgut epithelium, fat body, and Malpighian tubules) and in frass and whole larvae, underlining the potential risk associated with the use of these larvae for the preparation of BSFL-based products. This study provides important insights into the effects of HM contamination on BSFL, highlighting the need for rigorous safety evaluations when using BSFL to bioconvert contaminated substrates for production purposes.

What is the phytohormone content of Hermetia illucens frass?

M. Kaczor¹, A. Nycz¹, A. Bieganowski¹, K. Szewczuk-Karpisz¹, P. Waligórski² and P. Bulak^1*

¹Institute of Agrophysics Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland; ²The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland; ^*p.bulak@ipan.lublin.pl

Organic fertilisers produced via bioconversion are an important element of sustainable, cleaner agricultural systems. In this context, insect frass has gained attention as a promising biofertiliser because it is nutrient-dense, harbours beneficial microbiota, and can promote plant growth. Nevertheless, the phytohormone profile of frass generated by Hermetia illucens (black soldier fly, BSF) larvae is still poorly described (Green 2023; Lopes et al., 2025). Here, we present a comprehensive evaluation of the three major phytohormone groups in BSF frass and associated leachates obtained during the bioconversion of pea and bean seed waste. These plants, belonging to legumes, are important annual agronomic crops grown primarily for their protein-rich seeds. According to FAO, legume production reached about 9.6 billion tons worldwide in 2022, and projections suggest it will rise to roughly 125 million tons by 2032 (FAO,2023). Yet industrial processing of the seeds produces substantial residues, typically accounting for approx. 5–25% of the initial raw material mass (Karaca and Nickerson, 2022). Using H. illucens larvae we bioconverted very low-quality pea and bean seed waste, which cannot be use for feeding others animals and then determined phytohormones in it. Both substrates enabled efficient larval development. Frass from bean waste contained higher amounts of indole-3-acetic acid, stress-associated hormones, and the total cytokinin pool, whereas leachate from pea frass showed increased levels of trans-zeatin, cis-zeatin, and IP. IAA and selected cytokinins were also identified in the larvae. Overall, our results indicate that BSF-driven bioconversion improves the phytohormonal profile of frass more effectively than microbiota-only decomposition, thereby strengthening its prospects as a multifunctional, environmentally friendly biofertiliser. This work highlights how insect-based bioconversion can upgrade agri-food residues by adding functional value within sustainable production frameworks. References: FAO, 2023. Agricultural production statistics 2000–2022. FAOSTAT Analytical Briefs, No. 79. FAO, Rome. Green, T., 2023. A biochemical analysis of black soldier fly (Hermetia illucens) larval frass plant growth promoting activity. PLoS One 18: e0288913. Karaca A.C. and Nickerson M.T., 2022. Developing value-added protein ingredients from wastes and byproducts of pulses: challenges and opportunities. ACS Omega 23: 18192-18196. Lopes I.G., Gómez-Brandón, M., Praeg, M., Claeys, J., Yakti, W., Bitterlich, M., Jones, J.J., Geilfus, C.M. and T. Klammsteiner, T., 2025. BugBook: Critical considerations for evaluating and applying insect frass. Journal of Insects as Food and Feed 11: S507-S532.

Valorisation of olive oil by-products in Algeria using the black soldier fly

Z. Cherifi^1*, A. Abdelli¹, T. Maza², B. Hamdani¹, N. Mabrek¹, T. Titouh³, Z. Meniche⁴, H. Meziani⁵, L. Ibaliden⁶ and M.T. Capucchio¹

¹University of Bouira, Department of Agricultural Sciences, Bouira, Algeria; ²University Ahmed Zabana of Relizane, Relizane, Algeria; ³National Institute of Agronomic Research of Algeria, Oued Ghir, Algeria; ⁴SOLSTAR, Dély Brahim, Algeria; ⁵Specialised Technological Institute for Agricultural Training, Tizi Ouzou, Algeria; ⁶Oleocoop Soummam, Tisserifine Maakel, El Flaye, Algeria; ^*z.cherifi@univ-bouira.dz

In recent years, Algeria’s olive oil sector has grown significantly due to national programs. Olive groves now cover over 440 000 ha, producing 70 000–100 000 tonnes annually, making olive cultivation a key driver of rural development and the agri-food sector. The increase in production generated substantial quantities of organic by-products, including liquid effluents and solid residues such as olive pomace and olive mill wastewater, characterised by a significant content of phenolic compounds with phytotoxic activity, which pose serious environmental challenges in the absence of appropriate valorisation pathways. In this context, the black soldier fly (BSF) emerges as an innovative biotechnological solution for the bioconversion of low-value organic substrates. Its larvae exhibit a strong capacity to transform complex matrices into a protein- and lipid-rich biomass, which can be used as a sustainable source of alternative proteins for animal feed. Beyond larval biomass, BSF by-products, such as larval cuticle and exuviae, represent a promising source of chitin. This biopolymer has attracted increasing interest in pharmaceutical, biomedical, and biomaterials applications due to its biocompatibility and biodegradability. In addition, the frass generated during larval rearing constitutes a valuable organic amendment that can improve soil fertility and biological activity. This research highlights the strategic potential of integrating BSF into the management of olive oil by-products in Algeria, in line with the principles of circular bioeconomy and zero-waste valorisation. Although BSF farming remains poorly structured in Algeria, it shows strong development potential and deserves further attention. The PRIMA OLIWA project aligns with this objective by aiming to demonstrate the technical and environmental feasibility of integrating BSF into circular valorisation systems for olive oil co-products in the Mediterranean context. Acknowledgements: This research is supported by the EU-PRIMA program — project OLIWA- Repurposing OLIve WAste in circular economy solutions for feeds, additives, packaging, and biogas — GA 2432, Call 2024 Section 1 Food Value Chain in Nexus

From waste to resource: agri-food by-products as substrates for Tenebrio molitor rearing

G. Brenna^1*, C. Savio², V. Candian², R. Tedeschi², E. Crotti¹ and C. Jucker¹

¹University of Milan, Department of Food, Environmental and Nutritional Sciences, via Celoria 2, 20133 Milan, Italy; ²University of Turin, Department of Agricultural, Forest and Food Sciences, largo Paolo Braccini 2, 10095 Grugliasco, Italy; ^*gabriele.brenna@unimi.it

Steady population growth and the increasing global demand for protein require the identification of sustainable alternative sources. Insect farming is widely recognized as a promising solution, particularly when integrated into a circular economy framework. Among major agrobusiness, tomato processing and brewery production generate by-products accounting for approx. 10–30% of the total raw material processed, which contain valuable nutritional components that make them attractive substrates for insect rearing. This study evaluated the suitability of two by-products as feed source for the yellow mealworm Tenebrio molitor L. (Coleoptera, Tenebrionidae). Experiments were conducted separately on two genetically distinct yellow mealworm populations, originating from Milan and Turin. Dried tomato peels and brewer’s spent grain were mixed with wheat bran (1:1, v/v) as growth substrates and were provided along with wheat bran as control treatment. Development time, survival, larval biomass, individual weight and individual length were assessed. For the Milan population, no significant differences were observed between diets in terms of survival (≈95%), total larval biomass, or development time (34 days to first pupation). However, larvae reared on the tomato peel mixture showed a significantly higher mean individual weight and length (0.144 ± 0.004 g; 27.01 ± 0.23 mm) compared to the control (0.096 ± 0.003 g; 22.88 ± 0.28 mm). For the Turin population no significant differences were observed between diets in terms of survival (≈89%), and total larval biomass. Differences were found in development time between brewer’s (118 days) and control diet (50 days). Larvae reared on brewery’s mixture showed a significantly lesser mean individual weight and length (0.075 ± 0.002 g; 19.41 ± 0.22 mm) compared to the control (0.085 ± 0.002 g; 21.48 ± 0.19 mm). Overall, these findings support the potential of tomato peels and brewer’s spent grain as sustainable substrates for yellow mealworm mass rearing. Nevertheless, deformities observed at late larval and pupal stages with tomato peels highlight the need for further investigations on adult reproductive performance and on the possible presence of pesticide residues or anti-nutritional factors, emphasizing the importance of careful selection and quality control of by-products as insect feeding. Acknowledgement: Work funded by PRIN 2022 Project InProFarm (Insect Probiotic-assisted Farming: a promising tool to enhance edible insect health and performances), grant number 2022L4NJMK — Next Generation EU, Mission 4, Component 2.

Growth performance and microbial composition of black soldier fly larvae fed different food waste substrates

C. Liu^1*, J. Zhao¹, J. Love¹, D. Schneider² and L. Flores Renteria¹

¹San Diego State University, San Diego, CA 92182, USA; ²Centro de Investigación Cientı́fica y de Educación Superior de Ensenada, Ensenada, Baja California 22860, Mexico; ^*changqi.liu@sdsu.edu

Despite increasing food insecurity, >30% of the global food supply is wasted, contributing substantially to greenhouse gas emissions. Black soldier fly larvae (BSFL) composting offers a circular solution by converting food waste into insect biomass for animal feed and nutrient-rich frass for biofertilisation. However, diet-specific impacts on larval performance, nutrient transformation, and microbial composition remain insufficiently characterised. This study evaluated BSFL growth, feed conversion, nutrient composition, and associated microbial community dynamics across four food-waste substrates: cooked chicken, fruits and vegetables, starch-rich foods, and a mixed diet combining all categories. Food waste was fed to BSFL over a 14-day period. Larval growth was assessed by changes in length, width, and mass on days 1, 7 and 14, and feed conversion ratios (FCR) were calculated. Proximate composition of feedstocks and larvae was determined. Microbial abundance and community structure in food waste and frass were analysed using 16S rRNA amplicon sequencing. Statistical differences were evaluated using ANOVA with Tukey’s HSD (α = 0.05). Larvae fed exclusively on chicken exhibited significantly reduced growth, failing to reach the prepupal stage, with final mass 33% lower than those reared on fruit/vegetable waste and 56–62% lower than starch-rich and mixed diets. Starch-rich and mixed diets achieved the highest biomass gain (6.8–7.9 mg/larva) and most efficient feed conversion (FCR = 2.9–3.2). Although chicken-fed larvae accumulated the highest protein content (26.7 ± 0.5%), inadequate carbohydrate availability limited development. Balanced macronutrient profiles were observed in larvae reared on starch-rich and mixed substrates. Microbial analyses revealed strong diet-driven effects. Chicken waste exhibited the highest bacterial abundance (8 × 10¹² gene copies/g) but lowest alpha diversity (Shannon index), dominated by lactic acid bacteria. Fruit/vegetable and mixed wastes showed 38–40% higher alpha diversity. Following digestion, starch-derived frass exhibited the highest bacterial load, while mixed-waste frass showed marked selective enrichment dominated by Atopostipes suicloacalis (65.3%) and Vagococcus lutrae (21.1%). Beta-diversity analyses confirmed distinct, diet-specific microbial communities that persisted post-digestion. Overall, diversified, carbohydrate-rich food waste optimised BSFL growth, bioconversion efficiency, and microbial restructuring, supporting the scalability of BSFL-based systems for sustainable food waste management and tailored biofertiliser production.

The giving gut: valorisation of insect residues via insect gut microbiome fermentation for postbiotic production

I.D. Okehie^* and R. Ovissipour

Texas A&M University, Food Science and Technology, 370 Olsen Boulevard, 2474 TAMU, College Station, TX 77843, 77843, USA; ^*ikennaokehie@tamu.edu

Postbiotics, defined as preparations of inactivated microorganisms and/or their components that confer functional benefits in biological systems, are emerging as safe and stable alternatives to live probiotics for applications in food, feed, and biomanufacturing. Insect gut microbiomes represent a largely untapped reservoir of enzymatic and metabolic diversity capable of converting low-value agro-industrial residues into functional postbiotic preparations. In this study, gut microbial consortia from selected insect species were used as biocatalysts to valorise lignocellulosic and insect-derived substrates, including corn stover and insect frass. Shotgun metagenomic analysis revealed diverse microbial communities enriched in genes encoding carbohydrate-active enzymes, including cellulases, chitinases, and proteases, as well as pathways associated with short-chain fatty acid and amino acid metabolism. Electron-beam irradiation was applied as a non-chemical pretreatment to enhance substrate accessibility while maintaining a clean-label process. Subsequent anaerobic fermentation yielded metabolite-rich fractions, including short-chain fatty acids, organic acids, and bioactive peptides. Post-fermentation inactivation yielded sterile postbiotic preparations, which were fractionated into cell-free supernatants and microbial lysates. Functional evaluation demonstrated that selected postbiotic fractions exhibited significant antioxidant activity, ACE-inhibitory potential, and antimicrobial effects against model pathogens, with minimal cytotoxicity and controlled endotoxin levels. Notably, substrate type and insect source influenced both metabolic output and functional activity, highlighting the role of microbial ecology in tailoring postbiotic functionality. Collectively, this study establishes a scalable and clean-label framework for agro-waste valorisation using insect gut microbiomes and positions insect-derived postbiotics as multifunctional ingredients for applications in food systems, animal feed, and cell-culture platforms.

Detoxification of Fusarium toxins in black soldier fly larvae (DetoxBug)

L. Ivanova¹, F.-L. Tukun¹, N. Dhaoui², E. Byhrø² and C.K. Fæste^1*

¹Norwegian Veterinary Institute, Pb 64, 1431 Ås, Norway; ²Pronofa ASA, Øraveien 15B, 1630 Fredrikstad, Norway; ^*christiane.faste@vetinst.no

Black soldier fly larvae (BSFL, Hermetia illuscens) are of growing interest as sustainable ingredient in agri- and aquafeeds. Their capability to tolerate elevated levels of dietary contaminants can make them essential tools for the revalorisation of grain spoilt with mycotoxins. Fusarium spp. infection in grain is common in temperate climate and leads to harvest losses because of high contents of mycotoxins such as deoxynivalenol, T2/HT2-toxin, enniatins and zearalenone. BSFL have been shown to grow on feed containing the Aspergillus mycotoxin aflatoxin B1 (Heuel et al., 2023); however, the exploitation of their potential for recirculating resources into the feed chain is still prohibited due to safety concerns. Thus, the established maximum levels for mycotoxins in feed are also applicable to BSFL until it has been confirmed that they do not accumulate contaminants. Studying the detoxifying abilities of BSFL with regard to Fusarium mycotoxins, we have exposed BSFL to chicken feed incurred with high levels of DON or T2, or to fungi-inoculated oat grains. The larvae received the contaminated feeds between days 7 and 15 of their lifecycle. BSFL mortality and growth performance were determined. Larvae and frass were collected at four timepoints, extracted and analysed with a validated multi-mycotoxin liquid-chromatography high-resolution mass spectrometry (LC-HRMS) method (Ivanova et al., 2026). Larval growth remained unaffected even at substantial dietary mycotoxin levels, which were comparable to or above the highest detected levels in harvested grains. Mortality was low. The different mycotoxins were determined in feed, frass and larvae by LC-HRMS, showing that the concentrations in the larvae were low and decreasing at the different sampling timepoints. In contrast, mycotoxin levels were substantial in the frass and increasing throughout the study period. These findings indicated that the BSFL were apparently able to eliminate feed-borne mycotoxins from their organism by detoxification and excretion after they had adapted to the contaminated diet. In conclusion, the DetoxBug study showed that BSFL fed with elevated levels of different Fusarium mycotoxins could be safely used as protein and lipid sources in animal and fish feeds, whereas the frass is unsuitable for applications in feed. References: Heuel M., Kreuzer, M., Gangnat, I.D.M., Frossard, E., Zurbrügg, C., Egger, J., Dortmans, B., Gold, M., Mathys, A., Jaster-Keller, J., Weigel, S., Sandrock, C. and Terranova, M., 2023. Low transfer of cadmium, lead and aflatoxin B1 to eggs and meat of laying hens receiving diets with black soldier fly larvae reared on contaminated substrates. Animal Feed Science and Technology 304: 115733. Ivanova L., Dhaoui, N., Tukun, F.-L., Byhrø, E. and Fæste, C.K., 2026. Multi-mycotoxin method applicable for the safety assessment of waste-fed black soldier fly (Hermetia illuscens) larvae on the way to circular bioeconomics. Talanta 302: 129432.

Boosting the health properties of edible insects while valorising by-products

E. Cantero-Bahillo^1,2, E. Rodrı́guez-González^1,2, J.M. Silván³, V. Da Cunha-Borges^1,2, A.J. Martinez-Rodriguez³, D. Villanueva-Bermejo^1,2* and D. Martin^1,2

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ³Instituto de Investigación en Ciencias de la Alimentación (CIAL), Dpto de Biotecnologı́a y Microbiologı́a de Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ^*david.villanueva@uam.es

Edible insects are a sustainable source of nutrients and bioactive compounds, whose health properties depend on insect species and diet. In this study, we approached that feeding insects with bioactive-rich by-products is an innovative strategy to valorise side streams while enhancing the bioactivity of insect-derived products. To illustrate this idea, we evaluated the incorporation of well-known bioactive-rich by-products, such as olive leaves (OL) and olive pomace (OP), into the diets of Tenebrio molitor (TM) and Hermetia illucens (HI) larvae and its effect on multi-bioactive properties. Larvae were reared on wheat bran–based diets or on diets partially substituted with OL (15, 30, 50%) or OP (30, 50, 70, 90%). Reared larvae were slaughtered, dried, ground, and defatted. In vitro bioactivities were assessed after ultrasound-assisted extraction with 70% ethanol, including antioxidant (DPPH assay), anti-inflammatory (lipoxygenase -LOX- inhibition) and hypolipidemic (pancreatic lipase inhibition) activities, expressed as IC₅₀. Selected OL30 and OP30 samples were further tested in cellular models (RAW264 macrophages for reactive oxygen species -ROS- as antioxidant method, and for nitric oxide -NO- production as anti-inflammatory activity). In vitro antioxidant activity was higher for TM than HI, regardless of diet. However, TM showed no detectable antioxidant activity in cell models, while HI showed the same pattern both in vitro and in cells. For both species, feeding olive oil by-products increased their antioxidant capacity. HI showed a higher overall anti-inflammatory activity than TM, both by in vitro and cell models. The feeding of olive oil by-products enhanced this bioactivity, with the improvement being more evident for HI. Similarly, hypolipidemic activity was higher for HI than TM, with the inclusion of olive oil by-products improving such activity, more markedly for TM. These findings highlight the potential of using olive oil industry by-products into insect diets as an effective and sustainable strategy to simultaneously boost the health properties of edible insects while valorising agri-food side streams. Acknowledgment: ENTOMOTIVE project, PID2022-136238OB-I00 and grant PREP2022-000828, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU.

Grassland biomass as a substrate for black soldier fly larvae production

N.S. Stoehr^*, L. Schneider and G. Dusel

University of Applied Sciences Bingen, Department of Agriculture, Berlinstraße 109, 55411 Bingen, Germany; ^*n.stoehr@th-bingen.de

In many regions in Germany, structural changes in livestock production have reduced the demand for grass feed. Consequently, grasslands are either underutilised, with biomass no longer fully harvested or economically valorised, or extensively managed, with reduced inputs, low grazing pressure, and infrequent mowing. The decline in livestock as primary biomass consumers leads to reduced management intensity and the accumulation of low-valuable biomass. These conditions favour the spread plant species, that are toxic to ruminants or monogastric animals. Alternative uses for grassland biomass are needed and rearing black soldier fly larvae (BSFL) representing a promising yet unexplored option. However, insect meal production commonly relies on substrates also suitable as feed for pigs and poultry, such as the Gainesville diet (GD). To improve sustainability and minimise competition for feed resources, alternative substrates to the GD are needed. Five-day old (DOL) BSF-larvae (initial weight of 1.07mg/larvae) were allocated equally to 7 treatments. In each case, 14 000 BSFL were housed in one of 21 identical plastic boxes (40 × 60 cm) and reared in a controlled climate chamber (28–30 °C, 55-65%RH). The experimental setup consisted of GD (50% wheat bran, 30% corn, 20% Alfalfa) as control group (CON). The following 6 treatments consisted of fresh grass, hay, and silage, 25% and 50% respectively in GD, resulting in the treatment groups Grass25, Grass50, Hay25, Hay50, Silage25 and Silage50. Grassland biomass was harvested from a single grassland site. The grass, hay, and silage were processed to the finest possible particle size to optimise their availability for the larvae. Each substrate had an initial dry matter of 20–25%. To determine BSFL growth, 50 larvae were removed from each box every 2 days and weighed to calculate the average weight per larvae and treatment. PH value and temperature of rearing substrates were measured every 2 days. Data were analysed using one-way ANOVA ($p\mathrm{<}0.05$), and post-hoc-Tukey HSD. After 9 days of trial, no significant differences among treatments were observed ($p\mathrm{>}0.05$). Individual larvae weight was highest in CON, and Silage50 (107mg/larvae), whereas the remaining treatments averaged 91-101mg/larvae, with Gras50 showing the lowest mean value (91mg/larvae). Rearing substrates containing grassland biomass had higher pH values and lower temperatures compared to CON. However, no correlation between the pH value or temperature and larvae growth performance was observed. In conclusion, the utilisation of grassland biomass in combination with GD represents a suitable substrate for BSFL rearing. Further research is needed to investigate different inclusion levels, processing strategies and larvae characteristics.

Effects of agricultural by-products and surplus food-based diets on Hermetia illucens larval biomass and frass quality

B. Ufitinema^*, S. Mansbridge, S. Jeffery, J. Eastham and L. Vickers

Harper Adams University, Agriculture and Environment, Edgmond, Newport, Shropshire TF10 8NB, UK; ^*bufitinema@live.harper.ac.uk

Food waste is a global concern, significantly contributing to the growing challenges of waste management. The use of insects such as black soldier fly larvae (BSFL, Hermetia illucens), in organic waste management has emerged as a promising sustainable solution for the revalorisation of such waste through conversion to insect protein. This bioconversion process reduces waste volume and generates protein-rich larval biomass for animal nutrition and nutrient-dense frass for soil and plant nutrient supply. This study examined how different, seasonally available agricultural by-products and surplus food-based diets affect BSFL composition and frass nutrient profiles. Three diets namely: Summer (tomato, French beans, wheat bran, whey protein), Winter (potato, wheat bran, whey protein), and Control (wheat bran, layer’s grain) were tested at a commercial insect farm facility over 13 days. Approximately 10 000 eight-day-old BSFL were added to 10 kg of each diet with 10 replicates per diet. Larvae fed summer and winter diets exhibited higher gross energy (24 MJ/kg) and protein content (46%) compared to the control (22 MJ/kg and 39%, respectively), likely due to the greater nitrogen and crude protein content of the respective substrates. Across diets, BSFL biomass contained 29 – 30% fat, and essential macro- and micro-elements consistent with literature. Moreover, during the first 10 days of the experiment, larvae reared on summer and winter diets were larger (summer: 1.6 cm, 126 mg/larva; winter: 1.4 cm, 91 mg/larva) than those in the control group (1.2 cm, 40 mg/larva). Frass from all treatments was rich in essential nutrients (N, P, K, Ca, Mg), with NPK ratios of 1:1.8:1.5 (Control), 1:1.9:2.4 (Summer) and 1:1.2:2.3 (Winter), and C/N ratios of 14.4 (Control), 17.4 (Summer) and 18.8 (Winter) comparable to solid manure and bio-compost, indicating suitability as an organic soil amendment. Trace elements and heavy metals (Cu, Zn, As, Cd, Pb, Ni) fell within EU regulatory limits, except for chromium (Cr) in the control diet frass, which exceeded the permissible level (3.15 mg/kg vs. the 2 mg/kg limit). These findings highlight the impact of different diets on the growth rate and nutrient content of BSFL and show that diet composition should be considered when formulating optimum diets for BSFL production systems.

Bugs in the system: have the economic and environmental benefits of insect farming been overstated?

C. Biteau^*

Observatoire national de l’élevage d’insectes — National Observatory on Insect Farming, 20B rue de l’avenir, 78400 Chatou, France; ^*corentin.biteau@onei-insectes.org

Insect farming has attracted over $2 billion in investment, positioned as a sustainable route to reducing dependence on fishmeal and soy in animal feed. The sector’s value proposition rests on key assumptions: that insects can be fed on food waste, that production costs will decrease with scale, and that insect-based products offer environmental benefits over conventional alternatives. However, much of the supporting evidence comes from small-scale or pilot studies, often under conditions not representative of Western industrial production. This presentation examines whether these assumptions hold in light of recent developments.

This work draws on a series of peer-reviewed critical reviews by the author, synthesising life cycle assessment data, market and cost data, and industry evidence. Results indicate that insect meal currently costs $3800–6000 per ton, compared to $1400–1800 for fishmeal and approx. $500 for soybean meal, a gap that available modelling suggests is unlikely to close under realistic near-term conditions. Investment in the sector has declined sharply since 2022, and several leading companies, which collectively received over €900 million in investment, have gone bankrupt or entered insolvency proceedings, raising questions about commercial viability at scale.

From an environmental perspective, the circular economy narrative central to the sector remains largely aspirational, as large-scale farms rarely utilise genuine food waste due to regulatory, logistical, and nutritional barriers. Our review of life cycle assessments found that when food waste is not used, greenhouse gas emissions for insect meal range from 3.0 to 30.2 kg CO₂e per kg of protein. These findings are consistent with a UK government-commissioned LCA, which reported 12.9 to 30.1 kg CO₂e per kg of protein, approximately 1.8 to 13.5 times the impact of fishmeal and soybean meal, respectively. In the pet food market, the comparison is similarly unfavourable, as conventional pet foods primarily rely on low-impact slaughterhouse by-products. In the human food market, a global market audit found that only 7% of commercially available insect-based food products are marketed as meat alternatives, meaning insect-based foods mostly displace plant-based foods, which have low environmental footprints, further undermining the sustainability case for entomophagy.

These findings suggest that the gap between theoretical promises and industrial realities is substantial. For the sector to build a credible future, research and investment decisions should be grounded in evidence that reflects actual operating conditions rather than optimistic projections.

Production of black soldier flies (Hermetia illucens) from fish residues for sustainable aquaculture

I. Zaouite^1,2,3, I. Eeckhaut³, H. Chairi¹ and M. Gosselin²

¹Polydisciplinary Faculty of Larache, Abdelmalek Essaâdi University, Research Team in Agricultural and Aquacultural Engineering, Department of Biology, 93030 Tetouan, Morocco; ²Haute Ecole Provinciale de Hainaut-Condorcet, Laboratory of Entomology, Rue de la sucrerie 10, 7800 Ath, Belgium; ³University of Mons (UMONS), Research Institute for Biosciences, Biology of Marine Organisms and Biomimetics Unit, 23 Place du Parc, 7000 Mons, Belgium; ^*imad.zaouite@student.umons.ac.be

Aquaculture feeds are still heavily dependent on fishmeal and fish oil, contributing to overfishing and ecosystem degradation. Black soldier fly (Hermetia illucens) larvae offer a sustainable alternative by valorising organic waste, including fish residues, for circular economy applications. This study aimed to optimize a BSF larval rearing substrate using fermented fish waste, with larvae intended for inclusion in formulated feeds for European seabass (Dicentrarchus labrax). Two successive rearing cycles with the same diets were conducted (4 diets in triplicate/cycle): control 100% chicken feed and three experimental diets containing 25% fermented fish waste following the fermentation protocol of Dallaire-Lamontagne et al. (2025), blended with variable bread and vegetable waste proportions; BR25 (25% bread-50% vegetable), BR50 (50% bread-25% vegetable), BR37.5 (37.5% bread-37.5% vegetable). In each rearing cycle approximately 4 grams of newly hatched larvae (1-day) were collected and reared for 5 days in chicken feed. Plastic crates measuring 48 × 38 × 15 cm were each stocked with approximately 7000 larvae (5 days old) with an initial individual weight of (Cycle 1: 18.1 mg; Cycle 2: 11.3 mg). Larvae were fed at 0.5 g feed per larva under controlled conditions (27 °C, 60 – 70% RH), with improved temperature stability in Cycle 2. Daily monitoring of 50 larvae per crate set cycle end (<10% daily weight gain). Growth (final weight, biomass, GI), survival (SR), and bioconversion (FCR, ECI, FR) parameters were assessed. Diet composition significantly affected performance ($p\mathrm{<}0.05$), with Cycle 2 showing superior results. BR50 exhibited the highest performance: final weight 172.0 ± 6.1 mg, FCR = 3.52 ± 0.06, ECI = 34.24 ± 0.28%, comparable to the control (170.7 ± 1.5 mg, FCR = 3.33 ± 0.14, ECI = 37.27 ± 1.53%) and superior to other studies reporting 25 and 20% fish waste. BR37.5 showed intermediate performance, BR25 was consistently lowest (final weight 126.3 mg, FCR = 5.61). These results demonstrate that the BR50 diet can effectively replace conventional feed and supports the sustainable valorisation of organic wastes through black soldier fly larval bioconversion. Reference: Dallaire-Lamontagne, M., Lebeuf, Y., Saucier, L., Vandenberg, G.W., Lavoie, J., Allard, J.-M. and Deschamps, M.-H., 2025. Optimization of a hatchery residue fermentation process for potential recovery by black soldier fly larvae. Poultry Science 104: 104946.

Social Life Cycle Assessment of insect-based protein supply chains

J.I. Petrusan^1*, V. Khieya¹ and S. Smetana^1,2

¹German Institute of Food Technologies (DIL e.V.), Food Data Group, Prof.-von-Klitzing-Strasse 7, 49610 Quakenbrück, Germany; ²University of Veterinary Medicine Hannover, Institute of Food Quality and Food Safety, Bünteweg 2, 30559 Hannover, Germany; ^*j.petrusan@dil-ev.de

Insect-based proteins are increasingly promoted as sustainable protein alternatives to conventional protein feed ingredients. Yet, existing studies rarely assess Social Life Cycle Assessment (SLCA), predominantly evaluating the environmental or economic dimension, which limits the ability to identify sustainability across all dimensions. This study applies a reference scale following ISO 14075, prototyping the Social Hotspot Database (SHDB) to evaluate five insect-based protein production systems across different regions. Social risk scores for the insect-protein production system were calculated by converting the inventory data and were expressed in monetary terms (Medium Risk Hours and Labour Hour Risk). The Functional Unit for the study is 1 kg of insect-based protein. In total, five countries have been assessed (Italy, Greece, Germany, Tunisia, Morocco), from which only two countries/products are herewith addressed (Tunisia and Morocco) due to their significance. At the national level, Tunisia and Morocco exhibited comparatively higher composite risks, indicating weaker baseline social protection conditions. However, our results illustrate that among all products assessed, Product 3 (black soldier fly produced in Tunisia) showed a lower final social risk score than expected based on its national composite risk, whereas Product 4 (BSF Morocco) demonstrated the highest risk among all cases. Tunisia had a composite risk of 0.42 and a final social risk of 0.66, whereas Morocco had a composite risk of 0.40 but a final social risk score of 0.93. Conversely, the high final risk of Product 4 shows that favourable baseline conditions alone do not guarantee strong social performance; product-specific efficiencies within the sector can either mitigate or exacerbate social outcomes, particularly in regions characterised by higher social risk factors. The underlying social indicators in the SLCA conducted were manually extracted and represent sector-level averages; they might not capture the full heterogeneity of labour conditions between producers and the insect industry, which inherently limits granularity. Overall, the study demonstrates that sustainable insect-protein production, as assessed through SLCA, is highly dependent on product-specific characteristics along with regional conditions.

Growth, performance and larval composition of Hermetia illucens reared on industrially processed former foodstuffs

T. Ratajc¹, L. Bonin¹, L. Lavrič² and A. Kuhar¹

¹University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; ²KOTO d.o.o., Agrokombinatska cesta 80, 1000 Ljubljana, Slovenia; ^*tim.ratajc@bf.uni-lj.si

Rising global populations and limited arable land require innovative, sustainable methods for food and feed production. Local insect production can be economically and environmentally sustainable if insects are reared on low-opportunity-cost substrates that would otherwise have a higher environmental impact. While current research mainly focuses on optimising black soldier fly larvae (BSFL; Hermetia illucens) growth using predefined diets, studies on real-life former foodstuffs are lacking. These organic streams have significant potential, as their use in animal feed can prevent their classification as organic waste, thereby avoiding the costs and environmental burdens associated with waste legislation. In this study, we tested real-world pre- and post-consumer waste-derived substrates, with and without packaging, sourced from a large-scale organic waste processor handling heterogeneous agri-food sector waste. We evaluated five diets in a standardised insect bioconversion assay with BSFL: (1) broiler starter feed (control), (2) pasteurised canteen waste, (3) retail food waste (without meat, with packaging), (4) retail food waste (without meat, without packaging) and (5) retail food waste (including meat, with packaging). We calculated biomass gain (BG), feed conversion ratio (FCR), specific growth rate (SGR), waste reduction efficiency (WRE), bioconversion efficiency (BCE), nitrogen bioconversion efficiency (NBCE) and survival rate (SR). Nondigested residues in frass were quantified, and proximate composition (Weende analysis) and heavy metal content (Mn, Zn, Cd, Pb) were analysed for both substrates and harvested BSFL. Pre-consumer expired foods with and without packaging supported growth (SGR up to 35.7% per day; BG ≈ 25% of initial larval mass) comparable to the control, with SR of 66 – 72% (control: 70%) and WRE of 68 – 71%. These substrate streams can be efficiently converted by BSFL into protein- and lipid-rich biomass with high waste reduction potential. In contrast, the post-consumer canteen waste performed poorly across all metrics, indicating that untreated kitchen waste requires structural bulking and improved aeration. Manual depackaging after grinding reduced packaging residues in frass from 6.6 to 1.8% of the initial substrate and increased NBCE from approximately 69% to 80%. Proximate analysis revealed similar BSFL nutritional profiles despite varying substrate compositions. Heavy metal content was low in all substrates and harvested BSFL, supporting compliance with EU feed safety standards when legally permitted substrate categories are used.

Impact of adding goat cheese whey to the substrate and the slaughter method on Hermetia illucens larvae powder quality

S. Valente^1*, M.T. Santos^1,2, A. Almeida^1,3,4, L. Madeira^1,4, E. Mabécua¹, F. Carvalho^1,2,4, D. Murta^5,6 and A. Macedo^1,2

¹Instituto Politécnico de Beja, Ap.158, 7801-902 Beja, Portugal; ²MED, Universidade de Évora, Pólo da Mitra Apartado 94, 7006-554 Évora, Portugal; ³CREATE, Pole of Polytechnic Institute of Beja, 7800-295 Beja, Portugal; ⁴CENSE, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal; ⁵CiiEM-Egas Moniz Center for Interdisciplinary Research (CiEM), Egas Moniz School of Health & Science, University Campus, Quinta da Granja, 2829-511 Caparica, Portugal; ⁶Ingredient Odyssey SA-EntoGreen, 2005-002 Várzea, Portugal; ^*susana.silva.valente@gmail.com

The increase in global demand for sustainable food has driven interest in edible insects due to their high nutritional value and production efficiency. Hermetia illucens (BSF) has high potential and used in animal feed. This study aimed to evaluate the effect of supplementing the substrate with goat cheese whey (GCW) and slaughter methods on the quality of BSF larvae powders. The larvae reared on a standard chick feed based substrate, moistened with distilled water or goat cheese whey (GCW). After harvesting, the larvae killed by deep freezing or blanching, dehydrated, ground, and analysed for nutritional and microbiological quality. The powders obtained had average protein 29 ± 2.18% and ash 8.3 ± 0.6% contents, with no significant differences between diets or slaughter methods. Total fat content was 31.2 ± 2.2% with significantly higher values in larvae from the whey diet compared to the control ($p=0.0146$, n = 12). Total fibre content was 9.5 ± 0.9%, being significantly higher in the whey diet ($p\mathrm{<}0.001$, n = 12). The mesophilic aerobic count ranged from 3.43 to 6.08 log CFU/g, with half of the samples exceeding the legal limit (≤ 10⁵ CFU/g). In the mould (<1–3.54 log CFU/g) and yeast (<1–3.97 log CFU/g) counts, half of the samples exceeded the legal limit (≤100 CFU/g). Related to sulphite-reducing anaerobes and B. cereus, 4 samples exceeded legal limits. Non-conformities are possibly associated with fungal contamination of the substrate supplied to the larvae or recontamination of the final product. Pathogens and indicators of faecal contamination were within legal limits in all samples. Supplementing the substrate with GCW improved the nutritional profile of BSF, increasing fat and fibre content without affecting protein and mineral content. Slaughter methods did not significantly influence microbiological quality. Acknowledgements: Work carried out within the scope of the project InsectERA (C644917393- 00000032), funded by the Recovery and Resilience Plan (RRP), Portuguese Republic, Measure RE-C05 — Mobilizing Agendas for Business Innovation and European Union, NextGenerationEU.

Olive pomace application of feedstock for Hermetia illucens after different chemical and thermal pretreatments

N. Rubiejo Pérez^1*, J.D. Fernández Bayo¹, R. Martı́n Aguilera², B. Brown³, F. Carvalho Muzzi³, M.C. Almécija² and A. Fernández-Arteaga²

¹Universidad de Granada, Department of Soil Science and Agricultura Chemistry, Faculty of Pharmacy, University of Granada, Campus de Cartuja, s/n, 18071 Granada, Spain; ²Universidad de Granada, Department of Chemical Engineering Department, Faculty of Sciences, Av Fuentenueva, s/n., 18071 Granada, Spain; ³InsectBiotech, Calle Cardenal Cisneros 4, 41002 Sevilla, Spain; ^*nazaretrubiejo@correo.ugr.es

Spain is the world’s largest producer of olive pomace (OP). Olive pomace is a semi-solid by-product of the olive oil industry composed of pulp, skin, olive pit fragments, and water. It presents significant environmental challenges due to its complex chemical composition. Among the alternative strategies proposed for olive pomace valorisation, its use as feedstock for black soldier fly larvae (BSFL, Hermetia illucens) has emerged as a promising option. However, OP’s high lignin content, organic load, and phytotoxicity make its direct use for BSFL rearing challenging. This study evaluated the impact of different pretreatments on olive pomace properties and its suitability as feedstock for BSFL. Acidic pretreatments using hydrochloric acid (1M), hydrothermal pretreatments at 160, 175 and 190 °C, and thermal pretreatments at 28, 40, 50, 60, 70 and 80 °C were applied to the olive pomace. Changes in reducing sugar and polyphenol content in OP following pretreatment were monitored. The efficacy of each pretreatment was assessed through BSFL growth trials, with performance compared to larvae reared on chicken feed and non-treated olive pomace. Acid pretreatments removed more polyphenols (>200 ppm) than hydrothermal pretreatments (19–30 ppm). Soft thermal pretreatments did not significantly affect polyphenol or reducing sugar availability. Larvae fed with olive pomace non-pretreated or pretreated with hydrochloric acid pretreatment showed similar values of biomass (0.013 g dw/larva). This was significantly lower than the larvae reared on chicken feed (0.070 g dw/larva). Hydrothermal pretreatments also showed low yields with larva biomass values of 0.012, 0.007 and 0.001 g dw/larva at 160, 175 and 190 °C. Finally, soft thermal treatments also showed limited effectiveness, with BSFL biomass across all temperatures reaching approximately 0.015–0.011 g dw/larva). These preliminary tests showed that the tested acidic and hydrothermal pretreatments conditions removed polyphenols and reducing sugars. However, these effects were not translated in higher BSFL growth. This, combined with high environmental risks and costs, suggests that future pretreatment research should focus on alternative approaches such as basic pretreatments or co-digestion strategies.

Trade-offs in insect-based waste valorisation of spent coffee

R. Khelifa^* and H. Mahdjoub

Concordia University, Biology Department, 7141 Sherbrooke Street W., Montreal, QC, Canada H4B1R6; ^*rassim.khelifa@concordia.ca

Global coffee consumption generates a large stream of organic waste that could be valorised through insect-based bioconversion. This study examined how incorporating spent coffee grounds (SCG) into a high-quality black soldier fly (BSF, Hermetia illucens) diet affects insect performance and the fertilization potential of the resulting frass. BSF larvae were reared on a Gainesville diet supplemented with increasing proportions of SCG (0, 10, 25 and 50%), and larval growth dynamics, development time, and adult traits were quantified. Subsequently, basil (Ocimum basilicum) and duckweed (Lemna minor) were grown in substrates amended with frass derived from selected diets (0, 25 and 50% SCG) at varying application rates (0, 10 and 20% for basil and 0, 10 and 20 g/l for duckweed). Increasing SCG content reduced larval growth rate, peak weight and life history traits, indicating costs to insect performance. By contrast, frass derived from coffee-supplemented diets enhanced plant growth in both plants when applied at moderate rates (10% for basil or 10 g/l for duckweed). These results reveal a trade-off between BSF performance and frass quality and highlight the potential of BSF bioconversion to transform spent coffee grounds into value-added agricultural inputs when application rates are optimised.

Effects of feeding on Salix gordejevii on the growth and development of Protaetia brevitarsis larvae

B. Lei¹, K. Zhang¹, Y. Ding¹, J. Hao², Y. Xie², W. Tang², Y. Wu², Y. Xu², K. Ni¹, X. Wang¹, X. Yang¹, Z. Xu³ and F. Yang^1,2*

¹College of Grassland Science and Technology, China Agricultural University, Beijing 100193, P.R. China; ²College of Animal Science, Guizhou University, Guiyang, Guizhou Province 550025, P.R. China; ³Ulanqab Agricultural and Forestry Sciences Institute, Ulanqab 012000, China; ^*yfuyu@cau.edu.cn

As a saprophagous resource insect, Protaetia brevitarsis larvae have the potential to efficiently transform organic agricultural and forestry wastes. With high protein content and abundant amino acid composition, the larvae are high-quality animal protein sources. Salix gordejevii is one of the key tree species for windbreak and sand fixation in the sandy areas of northern China. Due to its high fibre content, poor palatability, and rich secondary metabolites such as phenols, its utilisation methods are relatively single, mostly used for sand barriers and weaving, with insufficient resource utilisation. To efficiently utilise Salix gordejevii resources, fermented Salix gordejevii feed was prepared by adding lactic acid bacteria and Bacillus subtilis to Salix gordejevii in this study. A feeding experiment on Protaetia brevitarsis larvae was conducted with commercial insect feed as the blank control. The results showed that Protaetia brevitarsis larvae could efficiently transform Salix gordejevii waste, with the highest feed utilisation rate of 64.64% and larval conversion rate of 89.03%, which were not significantly different from those of the commercial insect feed ($P\mathrm{>}0.05$). The protein content of larvae fed with fermented Salix gordejevii was above 50%, with the highest value of 57.97%, and the fat content of each treatment group ranged from 2% to 4%, indicating that they are high-protein and low-fat protein sources. This study provides theoretical guidance for the resource utilisation of Salix gordejevii branches, an agricultural and forestry waste, and also a reference for new-type feed for the low-cost large-scale breeding of Protaetia brevitarsis larvae. Funding: Guizhou Provincial Major Scientific and Technological Program (2025) 004.

Valorising unsold supermarket products through black soldier fly larvae rearing

E. Copelotti^1,2*, S. Mattioli³, M. Vispi³, N. Tous¹, A. Zanzot², N. Chiasso², L. Fihurska^4,5, K. Campbell⁴ and S. Mancini²

¹IRTA, Animal Nutrition, Mas Bové, 43120 Constantı́, Catalonia, Spain; ²University of Pisa, Department of Veterinary Sciences, Viale delle Piagge 2, 56124 Pisa, Italy; ³University of Perugia, Department of Agricultural, Food and Environmental Sciences, Borgo XX Giugno 74, 06123 Perugia, Italy; ⁴Queen’s University, Institute for Global Food Security, School of Biological Sciences, Chlorine Gardens 19, Belfast BT9 5DL, United Kingdom; ⁵Odesa National University of Technology, Department of Grain and Compound Feeds Technologies, Kanatnaya 112, 65039 Odesa, Ukraine; ^*emma.copelotti@irta.cat

Recently, the European Parliament adopted new measures to prevent and reduce food waste across the EU by 2030. To date, several former foodstuff products could be used as feed ingredients if they do not contain meat or fish. The aim of this research was to test the effect of substrates made with different unsold supermarket products on the growth performance and chemical composition of BSF larvae. Products from a local supermarket removed from sale for nearing expiration date or quality issues were collected. Three substrates were formulated: (P), composed of a mix of fruits and vegetables; (C), with 75% P and 25% of a cheese mix (on a wet basis, WB); (Y), with 75% P and 25% yoghurt mix (on a WB). Growth rate (GR), feed conversion ratio (FCR), efficiency of conversion of ingested feed (ECI), substrate reduction (SR) and waste reduction index (WRI) were calculated. Furthermore, the proximate composition, fatty acid profile, antioxidants and mineral concentrations were analysed in substrates and larvae. The results showed that C larvae had the highest average larval weight. Accordingly, the C larvae had the highest GR, SR, WRI and the lowest FCR in comparison to P and Y larvae. The CP% (DM) content does not differ significantly among P, C, and Y larvae. However, the highest ether extract content (EE%, DM) was in the C substrate, while the highest EE% content was in Y larvae, consistent with the presence of soluble sugars in the yoghurt. The fatty acid profile of larvae (%FA on total FA) changed in the total amount of saturated and monounsaturated FA, resulting in higher concentrations in C and Y than in P larvae. The levels of tocopherols and carotenoids (μg/g) in larvae varied with the different proportions of plant-based substrate. P larvae resulted in the highest concentrations of Ca, K, P, and Mg (mg/kg DM). These results support the growing body of evidence on the significant role of BSF larvae in reducing food losses and increasing the sustainability of the agrifood system. Acknowledgement: The work done at IRTA is financed by the European Union’s European Regional Development Fund, within the framework of the Catalonia FEDER Programme 2021-2027 and the STEP initiative.

Nutritional, antioxidant and sensory acceptability in wheat biscuits fortified with black soldier fly and mopane worm

V.V. Mshayisa^1*, N. Vanqa¹ and J. Kinyuru²

¹Cape Peninsula University of Technology, Department of Food Science and Technology, Symphony Way, 7535 Cape Town, South Africa; ²Jomo Kenyatta University of Agriculture and Technology, Department of Food Science and Technology, Juja, Nairobi, Kenya; ^*mshayisav@cput.ac.za

Edible insect flours represent a promising strategy for enhancing the nutritional quality of cereal-based snacks, although the inclusion level and insect species may impact product quality and consumer acceptance. This study evaluated the effects of fortifying wheat biscuits with flours from Gonimbrasia belina and Hermetia illucens on their nutritional composition, antioxidant capacity, and sensory acceptability. Biscuits were produced by replacing wheat flour with H. illucens or G. belina flour at 5, 10, 15 and 20% (w/w). Proximate composition, colour parameters, antioxidant indices including total phenolic content, DPPH, and ABTS, and sensory acceptability were determined. Protein content increased significantly with insect inclusion compared to the control ($p\mathrm{<}0.05$), ranging from 14.09 to 17.80% for H. illucens and 15.04 to 19.91% for G. belina. The fat content was significantly higher in H. illucens biscuits compared to the control ($p\mathrm{<}0.05$), while differences among inclusion levels of 5 to 15% were not significant ($p\mathrm{>}0.05$). Moisture and ash contents increased significantly in all insect-fortified biscuits ($p\mathrm{<}0.05$). Antioxidant capacity increased significantly with inclusion level ($p\mathrm{<}0.05$), with total phenolic content rising from 6.05 mg GAE per 100 g in the control to 251.14 mg GAE per 100 g for H. illucens and 77.43 mg GAE per 100 g for G. belina. Sensory acceptability declined significantly at higher substitution levels ($p\mathrm{<}0.05$). PCA separated low and high inclusion treatments, with PC1 and PC2 explaining 45.93 and 23.04% of total variance. Insect flour fortification significantly enhanced nutritional and antioxidant properties, while sensory constraints limited acceptable inclusion levels. Lower substitution levels represent the most viable balance between functionality and consumer acceptance

Nutritional quality of edible insects: impact of species, developmental stage, sex and fermentation

A. Vehar^1,2*, M. Mencin¹, D. Potočnik¹, L. Strojnik¹, M. Korošec³, B. Ferjančič³, M. Jagodic Hudobivnik¹, T. Zuliani^1,2, P. Jamnik³, U. Vrhovšek⁴, P. Škvorová⁵, A. Ota⁶, L. Kouřimská⁵, M. Kulma⁵, D. Heath¹ and N. Ogrinc^1,2

¹Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; ²Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia; ³Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; ⁴Fondazione Edmund Mach, Via Edmund Mach 1, 38098 San Michele all’Adige, Italy; ⁵Czech University of Life Sciences, Kamýcká 129, 165 00 Praha-Suchdol, Czech Republic; ⁶Jata Emona, Kavčičeva ulica 72, 1000 Ljubljana, Slovenia; ^*anja.vehar@ijs.si

As the insect farming industry grows, understanding how species, developmental stage, sex, and processing methods affect nutritional composition is crucial, as variations in fat, protein, carbohydrate and mineral content arise from these factors. Additionally, fermentation, a traditional processing method, can enhance sensory qualities, improve nutrient bioavailability, reduce anti-nutritional factors, increase bioactive compounds, support gut health and extend shelf life. This study assessed how species, developmental stage, sex and fermentation influence the nutritional quality of farmed insects. Seven species (desert locust, migratory locust, yellow mealworm, house cricket, Jamaican field cricket, lesser mealworm and discoid cockroach) of different sex and developmental stage were analysed for nitrogen, fatty and amino acids, elements and polyphenols. After that, yellow mealworm, house cricket and migratory locust were fermented with Lactobacillus plantarum and a commercial starter culture for 48 hours and then analysed for proximate composition, fatty and amino acids, elements and oxidation stability. Results showed that species, developmental stage, sex, and fermentation significantly affected nutritional quality. Insects were rich in essential fatty acids, amino acids, and minerals. Polyphenol content ranged from 7.6 to 511 mg/kg dry weight, with the highest levels in female desert locust (511 mg/kg, 19 compounds), adult migratory locust (288 mg/kg, 34 compounds), and discoid cockroaches (male 449 mg/kg, 21 compounds; female 357 mg/kg, 20 compounds). Fermentation reduced dietary fibre in yellow mealworm (33%) and house cricket (12%) but increased non-protein nitrogen by 38% and 16%, respectively. Fatty acid profiles were largely unchanged, while amino acid composition varied by species and fermentation culture. Mineral concentrations (Fe, K, Mg, Mn, Na, P) increased, whereas Se and Cu decreased. Zinc levels showed inconsistent trends. Oxidative stability decreased in yellow mealworm (42%) and migratory locust (25%), but improved in house cricket (160%). Overall, fermentation enhanced insect nutritional value, with variation by species and fermentation culture.

Comparative analysis of amino acids, minerals, thermal decomposition, and colour in six full-fat edible insect species

V.V. Mshayisa¹, W.G. Lindeque^1*, P. Netsianda¹, S.Y. Chia², B.O. OcHieng², J. Kinyuru³, M. Kababu² and C.M. Tanga²

¹Cape Peninsula University of Technology, Department of Food Science and Technology, Bellville, South Africa, 7535 Cape Town, South Africa; ²International Centre of Insect Physiology and Ecology, Icipe Road, Kasarani, P.O. Box 30772, 00100 Nairobi, Kenya; ³Jomo Kenyatta University of Agriculture and Technology, Department of Food Science and Technology, JKUAT Towers (former ICEA), Kenyatta Avenue, P.O. Box 62000, 00200 Nairobi, Kenya; ^*220201862@mycput.ac.za

Understanding species-level variability in edible insects is fundamental for their use as food ingredients. However, comparative datasets integrating protein composition, micronutrient content, thermal behaviour and colour across multiple insect species remain limited. Six edible insect species, T. obscura, T. molitor, H. illucens, G. bimaculatus, R. differens and S. gregaria, were analysed in the full-fat state using triplicate samples. Amino acid composition, mineral content, thermal stability, and colour attributes were quantified and evaluated using multivariate analysis of variance (MANOVA) to determine overall species effects. Amino acid profiles differed significantly among species ($p\mathrm{<}0.001$), with clear separation observed for total, essential, hydrophobic, and charged amino acid fractions. Mineral composition was likewise species dependent ($p\mathrm{<}0.001$), revealing distinct macro- and trace-mineral signatures across species. Thermogravimetric analysis further demonstrated species-specific thermal decomposition behaviour, indicating differences in thermal stability and compositional architecture among insects. Colour attributes also varied markedly, with S. gregaria exhibiting higher lightness and chroma, while H. illucens and T. molitor displayed darker, less saturated profiles. Within-species variability was low across all measurements. The pronounced inter-species differences observed across nutritional composition, thermal behaviour, and colour attributes demonstrate that edible insects represent a diverse ingredient space rather than a uniform protein category. From an industrial perspective, these baseline differences have direct implications for ingredient specification, formulation strategy, nutritional positioning, and consumer acceptance.

Multivariate evaluation of nutritional, techno-functional, and structural properties of wheat-mopane worm composites

V.V. Mshayisa^*, T.A. Nkosi, N. Dlelanga, K.S. Matladi and L. Mayekiso

Cape Peninsula University of Technology, Department of Food Science and Technology, Symphony Way, 7535 Cape Town, South Africa; ^*mshayisav@cput.ac.za

Edible insects are increasingly recognised as sustainable protein sources, yet their interaction with cereal matrices during processing remains poorly understood. This study investigated how mopane worm (Gonimbrasia belina) powder influences structure–function relationships in wheat-based composite flours. Methods Composite flours were produced by substituting wheat flour with mopane worm powder at 10, 20 and 30% (w/w). Nutritional composition, techno-functional, pasting behaviour, thermal stability, and molecular structure were characterised using standard analytical techniques, including Rapid Visco Analysis, thermogravimetric analysis, and FTIR spectroscopy. Results Mopane worm inclusion significantly increased protein and mineral content while reducing carbohydrate proportion ($p\mathrm{<}0.05$). Hydration behaviour was altered, with 100% mopane powder exhibiting a significantly higher water binding capacity than wheat flour, while composite blends showed moderated responses. Pasting profiles were markedly affected, with peak viscosity decreasing from approximately 1920 cP in wheat flour to below 900 cP at 30% inclusion, alongside significant reductions in breakdown and setback viscosities. Thermal analysis showed delayed degradation in insect-enriched flours, indicating improved thermal resilience. FT-IR spectra demonstrated progressive intensification of amide I and II bands and aliphatic stretching vibrations, confirming a transition from starch-dominated to protein-dominated matrices. PCA explained over 80% of the total variance and revealed distinct clustering driven by protein content, colour metrics, and functional properties. Discussion and conclusion Mopane worm powder induces coordinated structural and functional transitions within wheat flour systems. These findings provide mechanistic insight into insect–cereal interactions and support the rational design of protein-enriched composite flours for processed food applications.

Protein/peptide bioaccessibility and bioactivity of five traditional edible insects from Mexico after INFOGEST digestion

O.A. Sanchez-Velazquez^1*, L.C. Acevez-Mares^1,2 and A.J. Hernandez-Alvarez^1,2

¹University of Leeds, School of Food Science & Nutrition, Woodhouse Lane, Leeds LS3 9JT, United Kingdom; ²National Alternative Protein Innovation Centre, Woodhouse Lane, Leeds LS3 9JT, United Kingdom; ^*o.sanchez@leeds.ac.uk

Edible insects are increasingly recognized as sustainable sources of high-quality nutrients and bioactive compounds. This study evaluated the protein digestibility, nutritional quality, and antioxidant capacity of five traditionally consumed edible insect species from Mexico: mesquite bug (Thasus gygas), maguey white worm (Aegiale hesperiaris), maguey red worm (Comadia redtenbacheri), milpa grasshopper (Sphenarium purpurascens) and leaf-cutter ant (Atta mexicana). Samples were subjected to standardized in vitro gastrointestinal digestion following the INFOGEST 2.0 protocol. Protein solubility, soluble peptide content, amino acid profile, mineral composition, in vitro antioxidant activity (ABTS, reducing power, iron, and copper chelation), cellular antioxidant activity (CAA), and reactive oxygen species (ROS) inhibition in Caco-2 cells were assessed before and after digestion. Protein solubility increased between 3- and 12-fold ($p\mathrm{<}0.05$) depending on the species, with T. gygas showing the highest increase. Soluble peptide content increased significantly after digestion, reaching up to 70-fold ($p\mathrm{<}0.05$) in some species (C. redtenbacheri and A. mexicana), although the highest absolute peptide concentrations were observed in other insects (A. hesperiaris and T. gygas). In vitro antioxidant assays showed markedly enhanced activity after digestion, with values up to four times higher than undigested samples ($p\mathrm{<}0.05$). CAA increased by 14.23–43.67% ($p\mathrm{<}0.05$) compared to untreated Caco-2 cells, and ROS inhibition reached up to 85.26% ($p\mathrm{<}0.05$) in digested T. gygas (after intestinal phase 120 min). Amino acid profiles indicated enhanced protein quality in all five species, even before digestion (≥1.0 EAAI, ≥100 AAS, ≥100% BV). Additionally, mineral profiling revealed a wide diversity of macro- and microelements across all species, while heavy metals and medically relevant elements remained below hazardous levels. These findings demonstrate that gastrointestinal digestion substantially improves the bioaccessibility and bioactivity of proteins and antioxidant compounds from traditional Mexican edible insects. The high nutritional quality and improved antioxidant effects support their potential as functional and sustainable food ingredients for human nutrition.

Structure and gelling properties of house cricket (Acheta domesticus) myofibrillar proteins

G. Pellerin^*, F. Martel and A. Doyen

Institute of Nutrition and Functional Foods (INAF), Université Laval, Department of Food Sciences, 2440 Boulevard Hochelaga, Quebec City, QC, Canada G1V 0A6; ^*genevieve.pellerin.3@ulaval.ca

Incorporation of edible insect meals into meat formulations has been reported to negatively affect texture and colour at inclusion levels above 10% (w/w), thereby limiting their use as meat alternatives. The textural properties of meat products are largely governed by the gelling behavior of myosin. House crickets (A. domesticus) contain a high proportion of muscle proteins, including myosin. Thus, this study investigates the structural characteristics and gelling properties of isolated cricket myofibrillar proteins. Crickets (A. domesticus) were killed by flash-freezing and subsequently freeze-dried. Myofibrillar proteins were extracted by ammonium sulfate precipitation (50% saturation), followed by dialysis and desalting by ultracentrifugation. Protein profile was analysed by SDS-PAGE, while protein structural properties were characterised through surface hydrophobicity (H₀), free and total sulfhydryl groups and differential scanning calorimetry. Modifications in protein secondary structures were visualized by FTIR. Protein suspensions (1–5% (w/v) proteins, 0.6 M KCl, pH 7.0) were heated at 80 °C for 1 h to determine the least gelation concentration and water holding capacity. Gel strength was assessed by rheological measurements. Protein interactions involved in gel network formation were investigated using destabilising agents (1 M urea, 8 M urea and 0.1 M DTT) combined with Bradford method and SDS-PAGE analysis. Protein concentration of the myofibrillar protein-enriched extract was 63 ± 2%, with myosin heavy chain (25%), paramyosin (16%) and actin (17%) as the predominant proteins. Cricket myofibrillar proteins formed gels at 25 °C and 80 °C at a minimum concentration 3% m/v, exhibiting a water holding capacity of 98%. Differential scanning calorimetry revealed two denaturation peaks at 51 °C and 83 °C. H₀ increased markedly at 70 °C, whereas free sulfhydryl group content decreased at temperature exceeding 50 °C. Rheological analysis showed that the solid-like behaviour of a 3% protein suspension observed at 25 °C was thermo-reversible above 50 °C. Gelation initiated at 65 °C and strengthened upon cooling, resulting in a weak gel ($G^{\mathrm{′}}$ = 25 Pa) with characteristics more similar to polysaccharide gels. SDS-PAGE analyses suggest that myofibrillar gel network formation is predominantly formed through hydrophobic interactions. Cricket myofibrillar proteins exhibited structural characteristics comparable to those of animal muscle proteins but formed weaker gels. Further work should investigate cricket-animal myofibrillar protein systems to better model the substitution of animal-derived ingredients by insect-based alternatives in processed meat products.

Characterisation and application of Acheta domesticus in sustainable food systems

A. Brena-Melendez and V. Tejada-Ortigoza^*

Tecnológico de Monterrey, Bioengineering Department, Av. Eugenio Garza Sada 2501, 64700 Monterrey, Mexico; ^*viri.tejada@tec.mx

This work presents an integrated framework encompassing farming, processing, and formulation of cricket (Acheta domesticus L.) ingredients to support sustainable food systems. First, diet optimisation through cricket self-selection of regional agro-industrial by-products under an industrial farming system was evaluated. This was followed by an assessment of how commercial processing conditions affect the nutritional composition and techno-functional properties of edible crickets. Subsequently, the protein profile and techno-functional potential of cricket protein concentrates were characterised, concluding with an evaluation of insect-plant protein blending using a machine-learning approach. In the farming stage, stage-specific self-selection diets incorporating 13 Mexican agro-industrial by-products improved biomass yield (+59%), survival (+35%), and feed conversion efficiency (+14%) compared to conventional broiler feed. Ingredient preferences varied across developmental stages, with fibre-rich corn bran favoured during initiation, carbohydrate-rich corn flour during growth, and lipid-dense toasted sesame paste during reproduction, highlighting dynamic nutritional requirements. Regarding processing, the techno-functional properties and composition of different cricket fractions revealed that underutilised materials, particularly legs, may represent valuable food ingredients. Legs, head-and-torso, and whole-body crickets were systematically evaluated. Legs exhibited the highest protein content (75.2 g/100 g dw) and distinct cuticular protein bands (approx. 20 kDa, <15 kDa). Protein digestibility was highest in head-and-torso fractions (85.5%, PDCAAS = 0.86) and lowest in legs (PDCAAS = 0.73). ATR-FTIR analysis indicated predominant β-sheet structures, especially in legs (89.6%), suggesting a higher degree of protein aggregation. Finally, ingredient application was explored using a dataset of 124 formulations (66 single ingredients and 58 binary blends spanning insect-insect, insect-plant, and plant-plant combinations). Machine-learning models linked compositional inputs to five key techno-functional outputs. Spline regression performed best overall (R²Â = 0.763, MAE ≈ 0.23 for multi-output prediction), with strong performance in oil-in-water emulsifying capacity and oil-holding capacity predictions. Overall, this work demonstrates an integrated strategy to enhance crickets as a versatile source of food ingredients by addressing bottlenecks in production, processing, and formulation. Together, these findings provide a foundation for expanding the application of cricket-based ingredients in sustainable food systems.

The edible insects of Tanzania: current potential and future opportunities

M.M. Mmari^1*, J.N. Kinyuru² and S. Rashid¹

¹Sokoine University of Agriculture, Department of Food Science and Agro-processing, P.O. Box 3006, Morogoro 255, Tanzania; ²Jomo Kenyatta University of Agriculture and Technology, Department of Food Science and Technology, P.O. Box 62000-00200, Nairobi 254, Kenya; ^*mmarimercy@gmail.com

Edible insects are considered the fastest-growing alternative protein market in the world. The practice of eating insects by humans is known as human entomophagy, and people throughout the world have been eating edible insects as a regular part of their diets for millennia. It is considered safe as there are no reported cases of disease transmission, parasitoids, or heavy metals contamination to humans from the consumption of insects. The field of Entomophagy is under- researched in Tanzania, and there are no specific policy issues for it. Despite the fact that edible insects are reported to contain more nutrients like protein, fats, vitamins and minerals of high value compared to other conventional sources, bioactive compounds, and medicinal traits, there is limited data. The current study is looking at the occurrence, habitats, indigenous processing methods, cultures, beliefs, nutritional composition and value addition of the edible insects of Tanzania. This is being done through ethnography, key informant interviews, observations, literature, the internet, chronological reports and experimental laboratory analysis. Several tribes in Tanzania are reported to have edible insects as part of their traditional diets with cultural significance, and some treat them as respectable delicacies. Commercialisation and consumption of processed edible insects such as longhorn grasshopper (senene) is increasing countrywide. There are several farms identified in Tanzania that are practising insect rearing primarily for animal feed. There are different value-addition possibilities with scaling up potential, focusing on nutrition improvement in traditional diets and livelihood improvement in low-income societies. Innovations in the value addition of edible insects have the potential to fight malnutrition and poverty following increased commercialisation. Regulatory bodies and policy frameworks need to include the adoption of edible insect aspects, like food safety standards covering production to consumption.

Nutritional characterisation of 30 insect species for their potential as food or feed

B. Gautam¹, S. Tiwari¹ and P. Khanal^2*

¹Agriculture and Forestry University, Rampur Chitwan, 063, Nepal; ²Nord University, Skolegata 22, 7713, Norway; ^*prabhat.khanal@nord.no

The rising global demand for sustainable protein sources has intensified interest in alternative food and feed systems, with edible insects gaining increasing attention. We evaluated 30 insect species from Nepal for their nutritional potential as future food or feed. Species identification was initially based on morphological traits and subsequently confirmed using DNA barcoding. After sample collection, they were freeze-dried, ground, and analysed for proximate composition, amino acid, and fatty acid profiles. Preliminary results revealed substantial interspecific variation in nutritional composition. Crude protein content ranged from 30 to 60%, with orthopterans exhibiting the highest levels. Mulberry silkworms, scorpions, wasps and water beetles also exceeded 50% crude protein. Crude fat content varied widely (5–45%). Amino acid profiles were generally balanced and met recommendations for children and adults, as well as requirements for poultry and pigs, except for methionine. Among insect species, scavenger species, including mole crickets, black soldier flies, blowflies and certain lepidopteran larvae, were especially rich in minerals. Overall, multiple insect species exhibit strong nutritional potential, with high-quality protein, balanced amino acids, and valuable lipid and mineral profiles, underscoring their suitability as future food and feed resources. Strategic identification, domestication, and management of locally available nutrient-rich species can substantially enhance sustainable protein supply and strengthen global food and feed security.

Nutritional and physicochemical characterisation of Tenebrio molitor and Hermetia illucens larvae and flours

J. Dhaouafi^1*, C. Turchiuli¹, R. Karoui², C. Jablaoui¹, H. Romdhana¹ and S. Mezdour¹

¹Agroparistech, Université Paris Saclay, INRAE, Agroparistech, UMR 782 SayFood, Palaiseau 91120, France; ²Université d’Artois, Université d’Artois, UMRT BioEcoAgro INRAe 1158, Lens 62300, France; ^*Jihen.dhaouafi@agroparistech.fr

Global food security and sustainability challenges have driven interest in edible insects as low-impact protein sources. In particular, black soldier fly (Hermetia illucens, BSFL) and yellow mealworms (Tenebrio molitor, TNL) larvae stand out for their rich nutritional and functional properties. This study aimed to comparatively assess the nutritional composition, structural features, and techno-functional properties of BSFL and TNL, alongside their industrially processed flours (BSFF and TNF). Significant differences ($P\mathrm{<}0.05$) were observed among the powders in terms of composition, and physico-chemical and thermal properties in comparison with their larvae. For instance, the total lipid content decreased by 62.28 and 59.50% db in BSFF and TNF, measuring 14.56 and 10.57% db, respectively, while BSFL and TNL showed higher values of 38.60 and 26.10% DW. In contrast, the total protein content increased by 68 and 22% db in BSFF and TNF, reaching 60.84 and 72.08% db, respectively, compared with BSFL and TNL, yielding values of 36.22 and 59.04% db. Thermal gravimetry and differential scanning calorimeter analysis, highlight the conformational physical changes induced by the partial removal of fats which has an influence on protein denaturation. Fourier transform infrared spectroscopy further revealed that the industrial processing induced structural modifications on the amide I and amide II (1500-1700 cm^−1) regions. At the powder scale, morpho-granulometric analysis revealed reduced and more homogeneous particle size distributions in BSFF and TNF than in BSFL and TNL, reflecting the strong impact of industrial processing on powder structure and surface properties. These compositional and structural modifications collectively contributed to the enhanced techno-functional performance of the flours. Accordingly, BSFF and TNF showed superior techno-functional properties, with highest oil (increased by 22 and 31,4% reaching 3.33 and 3.47 g/g wb) and water holding capacity (increased by 17.7 and 11.2% reaching 4.58 and 3.47 g/g wb) than their corresponding larvae (BSFL and TNL). In addition, the mineral composition further supported the nutritional relevance of the flours. Overall, industrial processing had a different impact on BSFL and TNL, but in the two cases, it increased protein content and improved techno-functional properties, supporting insect flours as nutrient-dense ingredients for sustainable food formulations.

Molecular characterisation and assessment of potential allergens in Hermetia illucens

T. Tedeschi^1*, B. Prandi¹, K. Bugatti¹, S. Cutroneo¹, L. Calcinai¹, D. Delfino¹, L. Dellafiora¹, E. Ridolo² and C. Folli¹

¹Department of Food and Drug Sciences, University of Parma, University of Parma, Parco Area delle Scienze 27/A, 431233 Parma, Italy; ²Medicine and Surgery Department, University of Parma, 43126 Parma, Italy; ^*tullia.tedeschi@unipr.it

According to the UN Food and Agriculture Organization (FAO), insects have promising potential as an alternative food source, with both environmental and health benefits. In parallel with development of regulations surrounding the use of insects for human consumption, the associated risks of food allergies should be explored. Allergic reactions to edible insects have been reported, as well as the possibility of cross-reactivity with crustaceans and house dust mites. Tropomyosin (TPM) and Arginine Kinase (AK) are, indeed, the primary allergenic proteins in crustaceans and house dust mites. Hermetia illucens, black soldier fly (BSF), is one of the most interesting insects for use in the human diet and, in this perspective, it is essential to assess possible cross-reactivity with known Arthropoda allergens. In this work, we investigated the allergenic potential of TPM and AK identified in BSF genome by bioinformatic analysis. Both proteins were expressed as recombinant proteins in E. coli. They were fully characterised by LC-MS analysis and CD spectroscopy. Bioinformatics tools were used to predict possible linear and conformational epitopes, and in vitro simulated digestion associated to high-resolution mass-spectrometry allowed the identification of peptides resistant to gastro-intestinal conditions, which were compared with epitopes of Arthropoda and Mollusca allergens to predict the persistence of allergenicity upon digestion. In the context of food allergy, sequential IgE binding epitopes are particularly relevant as conformational epitopes are easily degraded in the gastrointestinal tract. The identified sequences were synthesized using solid phase peptide synthesis. Preliminary NMR Conformational analyses were performed, with the aim to assess the correspondence between the epitope’s solution conformation and the bioinformatically predicted structure. Finally, IgE-immunoblotting assays conducted with sera from patients with food allergies to shrimps and/or airway allergy to mites showed reactivity to both recombinant proteins and synthesized epitopes, suggesting a cross-reactivity of both H. illucens TPM and AK with shrimps and mites. These results highlight that the allergenicity assessment of Novel Foods is crucial to correctly inform the potentially allergic consumer. The availability of recombinant TPM from BSF will also allow the inclusion of these allergens in molecular screening aimed to specifically identify reacting allergenic molecules in view of the use of this insect and its derivatives in the human diet.

Development of corn-based extruded snack enriched with lesser mealworm flour

A. Kozlu^1*, D.K. Baigts-Allende¹, T. Kudera¹ and L. Seguı́ Gil²

¹Czech University of Life Sciences Prague (CZU), DRIFT-FOOD Research Center, Kamycka 129, 16500 Prague, Czech Republic; ²Instituto Universitario de Ingenierı́a de Alimentos—FoodUPV, Universitat Politècnica de València, Camino de Vera, s/n, 46022 Valencia, Spain, Spain; ^*kozlu@af.czu.cz

The use of insect-derived ingredients in extruded snack products offers a versatile way to provide sustainable, safe, and nutritious insect-based alternatives that consumers are willing to accept. This study involved the production of corn extruded snacks enriched with lesser mealworm (Alphitobius diaperinus) flour, as well as the physicochemical (moisture content, water activity, water absorption index (WAI), water solubility index (WSI), swelling index, expansion index, hygroscopicity, colour) and microbiological (anaerobic mesophiles, enterobacteria, yeast and moulds) characterisation of the final products. Corn flour was partially substituted with lesser mealworm flour at varying levels: 2.5, 5, 7.5, 10 and 12.5%. The extrusion process utilised a single-screw extruder operating at 150 rpm, with a screw length-to-diameter ratio of 25:1, a compression ratio of 3:1, a die diameter of 3 mm, and a feed rate of approximately 1.58 kg/h. A control sample consisting of 100% corn flour was also included in the study. A significant reduction in moisture content was observed, decreasing from 6.54 to 5.24 g/100 g, along with a decrease in water activity from 0.450 to 0.369 as the levels of insect flour increased. All samples remained within the low-moisture range. The WAI increased (4.56–5.59 g/g), while the WSI decreased (16.81–5.35%), accompanied by reduced swelling and expansion indices, reflecting denser, less expanded structures at higher levels of insect flour incorporation. Protein content increased at all levels of replacement, peaking at 12.47 g/100 g at the highest substitution level, compared with 6.10 g/100 g in the control. At the same time, colour darkening (L* decreased from 73.93 to 65.3) and reduced chroma (29.92 to 26.0). These changes were attributed to protein-induced browning reactions during extrusion. Texture measurements demonstrated reduced hardness and crispness in samples with higher substitution levels, aligning with limited expansion and altered starch–protein interactions. Microbiological analysis showed no detectable growth in any culture media, confirming that all extruded products were microbiologically safe for consumption. Overall, lesser mealworm flour can be successfully incorporated into corn-based extruded snacks without compromising physicochemical stability or microbiological safety, with moderate inclusion levels (≤7.5%) providing optimal protein content and structural properties.

Bioassay-guided evaluation of chitin–protein fractions from Hermetia illucens as inhibitors of AGEs

N.J. Wilden¹, T.H.D. Le¹, C. Malcolm², J. Malcolm², S.A. Mallard¹ and P. Deo^1*

¹Adelaide University, City Campus, North Terrace, Adelaide, SA 5000, Australia; ²Mobius Farm Pty. Ltd, Nuriootpa, SA 5355, Australia; ^*permal.deo@adelaide.edu.au

Advanced glycation endproducts (AGEs) are implicated in the development of chronic metabolic and age-related diseases, and there is growing interest in identifying natural inhibitors of AGE formation from sustainable bioresources. Hermetia illucens larvae represent a promising source of functional biopolymers, particularly chitin-based fractions, with potential health-related applications. This study investigated the antiglycation potential of fractions derived from H. illucens larvae using a bioassay-guided fractionation approach. Chitin–protein mix, chitin-rich fractions with minerals, chitin-rich fractions without minerals, and fat fractions were evaluated for their inhibitory activity against protein-bound fluorescent AGEs. Larvae were reared on fruit and vegetable substrates at 50 and 85% inclusion levels, as well as on food industry by-products consisting of fruits and vegetables, olive pomace, and grape marc. For fruit and vegetable substrates, fractions derived from larvae fed 85% FV showed a decreasing trend in IC₅₀ values compared to 50% FV; however, these differences were not statistically significant. In larvae reared on food industry substrates, the chitin–protein mix exhibited IC₅₀ values ranging from 106.96 to 210.77 μg/ml, with no significant differences among substrate types. Notably, the chitin-rich fraction without minerals derived from grape marc-fed larvae (282.40 ± 50.74 μg/ml) showed a significantly lower IC₅₀ value compared to those obtained from fruits and vegetables (435.93 ± 24.70 μg/ml) and olive pomace (464.75 ± 56.84 μg/ml). Multiple comparison analysis indicated that chitin-rich fractions without minerals were consistently the least effective inhibitors compared with chitin–protein mix and chitin-rich fractions containing minerals, irrespective of substrate. Fraction type was the primary determinant of antiglycation activity, explaining over 80% ($p\mathrm{<}0.0001$) of the variance for fruit and vegetable substrates and nearly 69% ($p\mathrm{<}0.0001$) for food industry substrates, while substrate type had little to no effect. Principal component analysis supported these findings, with the first two components explaining more than 80% of total variance. Overall, the results demonstrate that inhibition of protein-bound fluorescent AGEs is driven primarily by the fraction derived from H. illucens rather than the rearing substrate. Chitin–protein mix and chitin-rich fractions with minerals show promise as natural AGE inhibitors; however, further studies are needed to elucidate the synergistic physicochemical properties underlying chitin’s enhanced antiglycation activity.

The effect of yellow mealworm-enriched maize porridge on growth of school-going children in Western Uganda

T. Boit^1*, V. Gwokyalya², D. Nakimbugwe³, B. Grenov¹, E. Mupere⁴ and N. Roos¹

¹University of Copenhagen, Nutrition, Exercise and Sports, Rolighedsvej 26, 1958 Frederiksberg, Denmark; ²Mothers Against Malnutrition And Hunger, Lulagala, Mityana, Uganda; ³University of Makerere, Department of Food technology & Nutrition, University Road, 7062 Kampala, Uganda; ⁴University of Makerere, Department of Pediatrics and Child health, University Road, 7062 Kampala, Uganda; ^*tcb@nexs.ku.dk

Children living in food-insecure environments consume diets that are nutritionally inadequate, characterised by limited intake of animal-sourced foods (ASFs). Malnutrition in school-aged children adversely affects their physical growth, cognitive development, and academic performance. Increased consumption of ASF’s can improve children’s growth, nutrition and health. Edible insects are nutrient-dense and can be produced sustainably as a novel protein, alternative to conventional ASF. However, their impact on children’s nutrition and health is not well established. This study investigated the effects of a yellow mealworm-enriched porridge on growth of 6–9-year-old school children living in and around Kyaka II refugee settlement in Uganda. A randomised, double blinded, two-arm parallel trial was conducted. Children were individually randomised to receive either yellow mealworm enriched maize porridge (YMP) or standard maize porridge (MP) for 14 weeks. The primary outcome was height gain and secondary outcomes were other anthropometric measurements. Dietary data was assessed by 24-hour recall. The intervention involved inclusion of 25 g/100 g (dry weight) yellow mealworm powder. A portion of 400 ml of porridge was served daily, providing 15.8 g of protein in the YMP group and 5.7 g in the MP group. Children’s household diet was low in protein. Consumption of YMP significantly increased protein energy%. The YMP group showed greater height gain and lower accumulation of subcutaneous fat mass compared to the MP group. Changes in weight remained similar between both groups. Detailed results on the effect of the intervention on growth will be presented at the conference. The consumption of YMP improved linear growth in school going children with low habitual consumption of ASF’s. The growth effects elicited by the yellow mealworm in this study show similarities to those associated with dairy. Therefore, the yellow mealworm may serve as an alternative and sustainable protein source for children with limited access to ASF’s. This study highlights the nutritional potential of edible insects in the diets of children.

School children acceptance of ready-to-cook pottages fortified with African palm weevil larvae as school lunch in Ghana

A.A. Okyere^1*, K.K. Ganyo², G.E. Kpadonou², C. Tortoe³ and F.B. Zotor¹

¹University of Health & Allied Sciences, Family & Community Health, PMB 31, Ho, Ghana; ²Conseil Ouest and Centre Africain pour la Recherche et le Développement Agricoles, 7 Avenue Bourguiba, B.P. 48, 18523 Dakar, Senegal; ³Council for Scientific & Industrial Research, Food Research Institute, P.O. Box M 20, Accra, Ghana; ^*aokyerepg@sph.uhas.edu.gh

This pilot study investigated nutritional content, acceptability and cost of ready-to-cook (RTC) pottages fortified with African palm weevil larvae (APW). It aimed to provide evidence as a potential public health nutrition intervention strategy in Ghana’s school feeding programme (GSFP). The RTC pottages were produced from white yam or cocoyam or sweet potato and APW flours. Mixture design was used to generate 5 composites. The 5 composites obtained were processed into ready-to-cook pottages for each root and tuber crop. 50 school children aged 8 to 12 years served as sensory evaluation panellists for each root and tuber crop pottage. A modified WHO One Health Tool ingredients-based approach of intervention costing was used in estimating the cost of serving per child. Protein, fat, iron and zinc content increased with the increase in APW content of the composite. Sensory evaluation of the products showed no significant differences ($p\mathrm{<}0.05$) in terms of taste and overall acceptability of the RTC products. The products were highly acceptable to the panellists. 68, 84 and 92% of the panellists were willing to consume the RTC cocoyam, white yam and sweet potato pottages respectively as school lunch. The cost ranged GHC| 7.33 to GHC| 8.15 per 150 g serving for a child. The cost estimation provides a higher cost comparable to GHC| 2.00, which is the current GSFP government approved budget of food per child. However, policymakers would be able to lower the improved meals cost by making available less expensive APW production and processing technologies to farmers and processors.

Edible cricket supplementation and nutrition education modulate gut microbiota of infants aged 6–14 months in Kenya

J.K. Ng’ang’a^1*, N. Okeyo², H. Kihuga³, S.M. Kiiru¹, B. Kanoi³, S. Konyole², N. Roos⁴ and J.N. Kinyuru¹

¹Jomo Kenyatta University of Agriculture and Technology, Department of Food Science and Technology, 00200 Nairobi, Kenya; ²Masinde Muliro University of Science and Technology, Department of Nutritional Sciences, 50100 Kakamega, Kenya; ³Mount Kenya University, 00100 Nairobi, Kenya; ⁴University of Copenhagen, Department of Nutrition, Exercise and Sports, 1017 Copenhagen, Denmark; ^*Kandojeremies@yahoo.com

Edible crickets can potentially replace common animal-source proteins in complementary food (CF); however, their effects on the emerging gut microbiome are not fully described. We analysed secondary output measures from a 2 × 2 factorial randomised control trial (clinicaltrials.gov: NCT06002620) to evaluate the effects of edible cricket-enriched CF on the gut microbiome based on Cricket treatment (+/−CR) and Nutrition Education treatment (+/−ED). Eligible mother-infant dyads with infants aged ≥ 6 months were enrolled at Rwambwa Sub-County Hospital in Siaya County, Kenya. Participants were randomised into treatment arms, received intervention foods +/−CR monthly as take-home rations, and were offered monthly +ED sessions delivered as audiovisual content with education messages. The +CR rations were age-adjusted (29–80 g/day) and contained 24% cricket powder. Stool samples collected at baseline and after eight-month intervention were characterised using 16S rRNA gene sequencing. Statistically significant changes in microbial diversity were observed post-intervention (Shannon $p=0.016$; Chao1 and Observed species $p=0.27$; Simpson $p=0.12$). Cricket intervention increased Faecalibacterium prausnitzii, Bacteroides uniformis, Lactobacillus reuteri and Akkermansia muciniphila, which are considered beneficial taxa, and decreased potential pathogens Escherichia coli and Clostridium innocuum. The CR + ED treatment resulted in microbiome modification with significant increases in short-chain fatty acid producing species (F. prausnitzii, Roseburia intestinalis, Coprococcus eutactus and Bifidobacterium adolescentis), as well as inhibition of Escherichia coli and Clostridium difficile. These findings suggest that edible cricket supplementation can beneficially modulate the gut microbiome of infants. The additional impact of caregiver nutrition education points to complex pathways that warrant further investigation. Integrating insect-based complementary foods represents a safe and biologically meaningful strategy for supporting early-life gut health and improving child nutrition in resource-limited settings.

Nixtamalization of Sphenarium purpurascens and its application in maize tortillas

B.A. Acosta-Estrada¹, E. Perez-Carrillo^1*, M.T. Ramirez-Arriaga¹, C.M. Rosell² and S.O. Serna-Saldivar¹

¹Tecnologico de Monterrey, Bioingeneria, Ave. Eugenio Garza Sada 2501 Sur, 64700 Monterrey, N.L., Mexico; ²University of Manitoba, Department of Food and Human Nutritional Sciences, 144 Dafoe Library, Winnipeg MB, Canada R3T 2N2; ^*perez.carrillo@tec.mx

The growing demand for sustainable protein sources has increased interest in edible insects; however, most studies focus on thermal or fermentation treatments, while alkaline processing remains poorly explored. This study evaluated the application of nixtamalization (alkaline treatment) to adult Sphenarium purpurascens (SP) and its incorporation into nixtamalized maize tortillas. Grasshoppers were cooked in 1% Ca(OH)₂ at 98–100 °C for 0–40 min and steeped for 14 h. Physicochemical properties, protein digestibility, chitin content, calcium uptake, and protein structural changes (ATR-FTIR) were evaluated. Tortillas were produced using a 90:10 maize nixtamal:SP ratio and characterised for colour, texture, proximate composition, in vitro protein digestibility, and PDCAAS. Alkaline treatment of SP increased dry matter loss from 25.6% at 0 min to 31.5% after 40 min of cooking, accompanied by a pH increase from 4.8 to 9.2, while moisture content changed (8.5–79.5%). Crude protein content remained relatively stable during treatment (52.7–58.7% db up to 30 min), whereas crude fat decreased significantly by 27.2% after 40 min of cooking. Chitin content increased by up to 47.4% at short treatment times (0–10 min), while calcium concentration increased progressively with cooking time, reaching 514 mg/100 g at 40 min. FTIR analysis revealed an increase in β-sheet structures from 57.1% (control) to 68.8% (40 min) and a simultaneous reduction in random coil content from 14.3% to 6.3%, which correlated with reductions in free amino nitrogen (48.8 to 8.9 mg/g) and in vitro protein digestibility (88.7% to 74.4%). Tortillas supplemented with alkali-treated S. purpurascens (T0) showed higher protein content (10.4 vs 7.2% db), increased lysine contribution (56.3 vs 45.6% of FAO requirement), and a higher PDCAAS (67.1 vs 51.1%) compared to control tortillas. Textural analysis indicated softer but more fragile tortillas, with lower breaking force after 24 h of storage (0.252 vs 0.414 kg), suggesting delayed structural stiffening. These results demonstrate that alkaline treatment modifies the functional and nutritional properties of Sphenarium purpurascens, enabling its successful incorporation into maize tortillas. Applying nixtamalization to an insect-based protein matrix represents a novel processing strategy to improve protein quality and diversify edible insect applications in staple foods.

Processing the black soldier fly larvae as ingredient in extruded corn snacks

A. Culetu^1*, J. Wagner², F. Schindler³, H. Wedwitschka⁴ and M. Kreische¹

¹Institut für Getreideverarbeitung GmbH, Arthur-Scheunert-Allee 40-41, 14558 Nuthetal, Germany; ²Frankenförder Forschungsgesellschaft mbH, Potsdamer Straße 18a, 14943 Luckenwalde, Germany; ³Hermetia Baruth GmbH, An der Birkenpfuhlheide 10, 15837 Baruth/Mark, Germany; ⁴Deutsches Biomasseforschungszentrum gemeinnützige GmbH, Torgauer Strasse 116, 04347 Leipzig, Germany; ^*alina.culetu@igv-gmbh.de

Edible insects are increasingly being studied and promoted as a valuable nutritional resource. Hermetia illucens (black soldier fly, BSF) is a promising candidate for commercial insect-derived protein production owing to its high protein content and a favourable essential amino-acid profile. While BSF biomass is currently deployed mainly in animal feed, growing nutritional and processing evidence indicates substantial potential for its sustainable incorporation into food systems. The aim of this study was to obtain protein-enriched flour from BSF larvae by supercritical carbon dioxide extraction and to evaluate its inclusion in extruded products. The obtained BSF flour contained 60% protein, 2.7% fat, 12.6% ash, 8.9% fibre and 9.2% carbohydrates. Glutamic and aspartic acids were the most abundant amino acids (6.9 and 6.2%, respectively) followed by leucine (4.5%) and lysine (4.2%). BSF flour was incorporated into corn grits at 5, 10 and 20% substitution level, and extrudates were produced using a twin-screw extruder. Snacks without BSF flour served as the control. Addition of BSF flour increased protein content up to 2.2-fold and mineral content up to 3.5-fold compared with the control. With BSF flour addition, the bulk density and hardness of extrudates increased. Colour changes were observed: higher BSF flour levels produced darker products, and the colour parameters a* and b* shifted toward red and blue, respectively. Sensory evaluation indicated that formulations containing 5 and 10% BSF flour showed slight reductions in overall score (29 and 27 points, respectively) compared with the control (30 points), while the sample with 20% addition recorded the lowest overall score (24 points). The results of this study may contribute to optimising the acceptability of BSF in food products. The project OptiFood is supported by funds of the Federal Ministry of Agriculture, Food and Regional Identity (BMLEH) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support programme.

Safety assessment and characterisation of silkworm pupae as a potential novel food in Europe

L. Tassoni^*, A. Saviane and S. Cappellozza

Council for Agricultural Research and Economics, Research Centre for Agriculture and Environment, Sericulture Laboratory of Padua, Via Eulero 6/a, 35143 Padua, Italy; ^*tassoni.luca@gmail.com

Silkworm pupae are a common by-product of sericulture and provide a source of proteins, polyunsaturated lipids and bioactive compounds. In Asia, silkworm pupae have a long tradition of consumption as food; however, in Europe — despite a long tradition of silkworm rearing — they have not been widely used. For this reason, any potential authorisation of silkworm pupae as a novel food in Europe must follow the procedure established by the EU Novel Food Regulation. In accordance with the technical and safety requirements set by European legislation, a dedicated processing method and a full characterisation of a potential novel food product were carried out. The suitable pupae came from second-grade cocoons not intended for reeling. The pupae were removed alive from the cocoons and euthanised in accordance with principles of animal welfare applicable to insects. The final product consisted of dehydrated silkworm pupae that were fully characterised in terms of composition and potential hazards. All chemical and biological safety parameters remained well below the limits established for other insect-based products authorised in the European Union. Regarding composition, proteins were the main nutrient, accounting for 45.9–50.4 g/100 g, while fats represented the second most abundant macronutrient, with values ranging from 32.6 to 42.7 g/100 g. With respect to contaminants, cadmium and mercury levels were below the limits of quantification, whereas lead concentrations ranged from 0.013 to 0.04 mg/kg and arsenic from 0.039 to 0.275 mg/kg. Dioxins and dioxin-like PCBs ranged from 0.185 to 0.679 pg WHO-PCDD/F-PCB-TEQ/g. All microbiological indicators investigated in the novel food were below the limits of quantification. The only exception was Bacillus cereus, which reached 230 CFU/g in a single batch. Shelf-life analyses covered eight months and showed no significant changes in microbial load. Regarding oxidative stability, peroxide values ranged from 0.6 to 2.9 mEq O₂/kg at time zero, increasing to 1.7–7.3 mEq O₂/kg at the eighth month. Apart from possible allergenicity, the main potential hazards regard the presence of heavy metals, whose levels strongly depend on soil contamination affecting mulberry leaves, and which therefore require future monitoring. Overall, our results demonstrate that the developed product represents a nutritious and safe alternative food source, with the added benefit of deriving from an environmentally sustainable agricultural practice. Moreover, its use could increase the value of sericulture by further enhancing traditional agricultural activities in Europe.

Fate of chitin from different insect fractions during in vitro human gut fermentation

C. Pedrazzani^1*, M. Mishyna², E. Capuano², V. Fogliano² and A. Caligiani¹

¹University of Parma, Food and Drug Department, Parco Area Delle Scienze 17/A, 43124 Parma, Italy; ²Wageningen University, Department of Agrotechnology and Food Sciences, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands; ^*clara.pedrazzani@unipr.it

This study explores the behaviour of insect-derived ingredients in the human gastrointestinal tract, focusing on chitin, a structural polysaccharide of the exoskeleton of arthropods. As insects are increasingly recognised as a sustainable food source, understanding chitin digestion and its impact on gut microbiota is essential for developing health-promoting food products. In vitro digestion and 48-h fermentation experiments were conducted on several insect species (Acheta domesticus, Tenebrio molitor, and Hermetia illucens) and their chitin-rich fractions (defatted insects, cuticle, and chitin). Additionally, a 15-day experiment employing the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) was performed with a basal diet supplemented with 30% previously digested insect ingredients. Chitin was resistant to digestion and not rapidly fermented under short-term fermentation, as reflected by negligible short and branched fatty acids (SCFAs, BCFAs), and ammonia production. However, relevant metabolic and microbial changes were observed in the insect-based diet during the long-term SHIME® experiment. These changes became evident after 5 days of treatment, indicating gradual microbial adaptation. Notably, the insect-supplemented diet led to shifts in SCFA profiles, with reduced acetate (24 mM and 38 mM in the proximal (PC) and distal colon (DC) compared to 42 mM and 53 mM in the control diet) and increased butyrate (9 mM both in PC and DC compared to 1 mM and 4 mM). In addition, higher ammonia levels and BCFAs (3 mM both in PC and DC compared to<1 mM in the control diet), reflected enhanced proteolytic fermentation, likely related to the progressive degradation of chitin-protein complexes. Furthermore, the microbial community composition evolved over time, with an increase in beneficial bacteria, particularly members of the family Bifidobacteriaceae, and the emergence of new bacterial populations (e.g. Synergistaceae family), suggesting adaptation and possible selective stimulation of gut bacteria. Insect chitin represents a novel dietary fibre with possible prebiotic properties that can modulate gut microbiota over extended periods. These findings contribute to a better understanding of insects as a sustainable and health-promoting diet, encouraging further research into the functional applications of insect-based foods.

Functional peptide fractions from black soldier fly protein: bioactivity, selectivity and gut microbial metabolism

I.D. Okehie^*, J.K. Tomberlin, W. Thilakarathna and R. Ovissipour

Texas A&M University, Food Science and Technology, 370 Olsen Boulevard, 2474 TAMU, College Station, TX 77843, USA; ^*ikennaokehie@tamu.edu

Black soldier fly larvae (BSFL) protein is an emerging sustainable ingredient with relevance in food and feed systems. Beyond its nutritional value, enzymatic processing and gastrointestinal digestion can release bioactive peptides with functional and gut-relevant properties; however, how different processing routes influence biological and microbial responses remains unclear. This study evaluated how enzymatic hydrolysis and simulated digestion affect the functional activity, cellular selectivity, and gut fermentation behaviour of BSFL protein–derived peptide fractions. BSFL protein was hydrolysed using Alcalase, bromelain, trypsin, and a standardized in vitro gastrointestinal digestion model (INFOGEST), followed by molecular weight fractionation. Hydrolysates were assessed for antioxidant capacity, angiotensin-converting enzyme (ACE) inhibitory activity, and selective cellular responses using human colon cancer and healthy intestinal cell models. Selected fractions were further evaluated using an in vitro human gut fermentation system to quantify short-chain fatty acid (SCFA) production. Simulated gastrointestinal digestion produced the highest extraction yield (approx. 65%) and degree of hydrolysis (approx. 43%), compared with enzymatic treatments (30–40% yield; 20–27% hydrolysis). Hydrolysis significantly enhanced antioxidant activity, with low-molecular-weight peptide fractions exhibiting 1.5–1.8-fold higher activity than native protein. Several fractions demonstrated notable ACE-inhibitory activity, reaching 50–60% inhibition. Bromelain-derived peptide fractions reduced colon cancer cell viability to approximately 45–55% at 1 mg/ml while maintaining healthy cell viability above 95%, accompanied by increased activation of intrinsic apoptosis markers. In vitro gut fermentation of selected peptide fractions resulted in substantial SCFA production, with acetate, propionate and butyrate concentrations ranging from approx. 165–695 μM, 568–791 μM and 401–691 μM, respectively. INFOGEST and enzyme-derived fractions promoted higher total SCFA production than raffinose and control substrates while maintaining moderate levels of branched-chain fatty acids, indicating active yet controlled microbial protein fermentation. Overall, these findings demonstrate that BSFL-derived peptide fractions are functionally distinct and strongly dependent on processing strategy. By linking enzymatic processing, peptide bioactivity, and gut microbial metabolite production, this work highlights the potential of insect-derived proteins as multifunctional ingredients for sustainable food and feed applications.

Extrusion of insect-plant protein blends: from insect ingredients to structural diversity

M. Mishyna^*, M. Verhoeven and C. Lakemond

Wageningen University and Research, Food Quality and Design Group, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands; ^*maryia.mishyna@wur.nl

To support development of insect food products with high added value, innovative processing using insect fractions as ingredients needs to be explored. Combining insect ingredients with other alternative protein sources in extrusion processes is one way to diversify of insect food applications. This work integrates studies on incorporation of insect-derived ingredients into extruded protein matrices to demonstrate how ingredient composition and moisture conditions determine extrudate structure and quality. High-moisture extrusion studies focused on fibrous structure formation through the partial substitution of soy or pea protein with different yellow mealworm fractions, including whole meal and soluble and insoluble protein fractions, at inclusion levels up to 50%. Increasing insect inclusion level altered extrudate composition and led to reduced texturization, fibre strength, and structural anisotropy while increasing extrudate darkening. Distinct fraction-dependent effects were observed, with chitin-rich fractions constraining the development of continuous fibrous networks. Across experiments, ingredient composition showed a stronger influence on structural outcomes than processing conditions and screw configuration. Insect-plant protein high moisture extrudates can serve as structured protein matrices for applications such as meat analogues and other fibrous protein foods. Low-moisture extrusion tests investigated the partial replacement of lentil flours with black soldier fly larvae ingredients at inclusion levels between 10 and 25%. Across formulations, changes in hardness, water absorption capacity, expansion ratio, and colour were primarily associated with the inclusion of insect ingredients. These extrudates are suitable for applications where compact, shelf-stable products with controlled hydration properties are required, such as dry protein ingredients, snacks, or feed formulations. The results demonstrate how systematic variation in insect ingredient composition and extrusion moisture conditions enables a wide range of extrusion outcomes, from compact low-moisture products to anisotropic fibrous structures.

Production and characterisation of extruded snacks with incorporation of mixtures of house crickets and lupin beans

R.N. Isaı́as^1,2,3*, J.C. Ribeiro¹, L. Loureiro^3,4, C. Rocha^1,2, A.A. Vicente^3,4, L.M. Cunha¹, M. Igual⁵, P. Garcı́a-Segovia⁵ and J. Martı́nez-Monzó⁵

¹GreenUPorto/INOV4Agro, DGAOT — Faculty of Sciences, University of Porto, Campus de Vairão, Rua da Agrária 747, 4485-646 Vairão, Portugal; ²Sense Test, Lda., Rua Zeferino Costa 341, 4400-345 Vila Nova de Gaia, Portugal; ³CEB — Centre of Biological Engineering, Department of Biological Engineering — University of Minho, University of Minho — Campus of Gualtar, 4710-057 Braga, Portugal; ⁴LABBELS — Associate Laboratory, Braga/Guimarães, 4710-057 Braga, Portugal; ⁵i-Food Group, IIA-FoodUPV, Universitat Politècnica de València, FoodUPV, Camino de Vera s/n, 46022 València, Spain; ^*id10818@alunos.uminho.pt

Food products containing alternative proteins are an expanding market in the agri-food sector, but their functionality and sensory properties still affect their success. This study aimed to develop and characterise extruded snacks incorporating controlled blends of whole house cricket (Acheta domesticus) flour (WC) and lupin bean protein concentrate (L), and to subsequently fit an appropriate statistical model to the resulting data to support product optimisation. A 13-point experimental mixture design was used, with corn flour as the main component (minimum 85%) and L and WC added to a maximum of 15%. Extruded samples were characterised for their physical and compositional properties (e.g., sectional expansion index (SEI), porosity), colour, and texture parameters. These data were processed and adjusted to optimal response surface models. All samples expanded acceptably; however, protein incorporation significantly decreased SEI, particularly in samples with 15% WC or 10% WC + 5% L (7.28 and 6.86, respectively; control 10.22). Most water-related properties were similar to control, especially the swelling index (ca. 6-7 ml/g; control 6 ml/g), and the water content (between 0.030–0.040 g/g; control 0.041 g/g), while porosity increased, particularly in samples with higher protein content (e.g., 2.5% WC + 10% L: 0.94; 5% WC + 10% L: 0.94; control: 0.91). Regarding colour parameters, samples with increased protein content became darker (lower L* and b* values), a known effect of adding dry crickets, with no impact of lupin incorporation. Texture parameters were largely unaltered by the different blends, resulting in products like the control, suggesting an overall positive outcome. Comparing different statistical models and their quality of adjustment, a cubic model was chosen as an overall optimal response surface, which can be useful in further product development. All in all, the study demonstrates that snacks enriched with cricket and lupin proteins can be produced with desirable structural, optical, and textural attributes, within feasible and permitted protein inclusion levels.

Mono- and disaccharide composition of insects and their suitability for individuals with disaccharide intolerance

L. Kourimska^1*, E. Vaclavikova², F. Beno², P. Skvorova¹, M. Kulma³, P. Kourimsky¹ and F. Kvasnicka²

¹Czech University of Life Sciences Prague, Department of Microbiology and Nutrition, Kamycka 129, 165 00 Praha-Suchdol, Czech Republic; ²University of Chemistry and Technology in Prague, Department of Food Preservation, Technicka 5, 160 00 Praha 6-Dejvice, Czech Republic; ³Czech University of Life Sciences Prague, Department of Zoology and Fisheries, Kamycka 129, 165 00 Praha-Suchdol, Czech Republic; ^*kourimska@af.czu.cz

Insects are gaining attention as a sustainable and nutritionally valuable food source, yet information on their carbohydrate fraction, especially simple sugars, is still limited. The present study focused on the determination of selected mono- and disaccharides in a diverse set of insect species and on evaluating their suitability for consumption by individuals with disaccharidase deficiencies. A total of nineteen insect samples covering five taxonomic orders, various developmental stages and both sexes were analysed. Concentrations of trehalose, glucose, fructose, lactose, sucrose and maltose were quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), while total soluble saccharides were assessed by the anthrone method. The combined content of quantified mono- and disaccharides ranged from 49 to 1675 mg/100 g of lyophilised insect material, whereas total soluble saccharides reached 253–5403 mg/100 g. Trehalose represented the dominant disaccharide in most species, with maximum levels of 495 mg/100 g in Hermetia illucens larvae and 215 mg/100 g in Tenebrio molitor larvae. Glucose concentrations varied markedly among species, peaking at 1328 mg/100 g in subadult Locusta migratoria. Lactose was absent or detected only at trace levels (<5 mg/100 g) in most samples; an exception was observed in Musca domestica larvae, where 84 mg/100 g was found, likely reflecting residual dietary sources. Statistical analysis confirmed a significant influence of species, developmental stage and sex on saccharide composition ($p\mathrm{<}0.001$). In general, the low levels of lactose and sucrose observed in most insect species indicate favourable suitability for individuals affected by common disaccharide intolerances. In contrast, the highly variable trehalose content suggests that certain species may pose a risk for individuals with trehalase deficiency. These findings contribute to a better understanding of carbohydrate composition in insects and support informed dietary recommendations regarding insect consumption for people with disaccharidase-related intolerances. Acknowledgments: The study was supported by the METROFOOD-CZ research infrastructure project (MEYS grant number: LM2023064), including access to its facilities.

Chitin-rich black soldier fly larvae fractions as functional ingredients for hyperglycaemia management

T.H.D. Le¹, N.J. Wilden¹, C. Malcolm², J. Malcolm², S.A. Mallard¹ and P. Deo^1*

¹Adelaide University, City Campus, North Terrace, Adelaide, SA 5000, Australia; ²Mobius Farm Pty. Ltd, Nuriootpa, SA 5355, Australia; ^*permal.deo@adelaide.edu.au

Black soldier fly larva (Hermetia illucens, BSFL) represents a sustainable and underutilised bioresource with growing relevance for functional foods and nutraceutical applications. Chitin extracted from BSFL has previously demonstrated antioxidant and anti-inflammatory properties, but its potential in managing hyperglycaemia and type 2 diabetes mellitus through carbohydrate-digesting enzyme inhibition and antiglycation activities remains underexplored. This study compared the inhibitory effects of chitin-rich BSFL fractions, obtained through chemical and microwave-assisted extraction methods, on α-amylase, α-glucosidase, and protein-bound fluorescent advanced glycation end-products (AGEs) inhibition. Defatted BSFL samples were prepared as three fractions: untreated protein/chitin mix, chemically extracted fractions and microwave-assisted extracts. α-amylase and α-glucosidase inhibitory activities were assessed in vitro using the 2-chloro-4-nitrophenyl α-d-maltotrioside reagent and the p-nitrophenyl-α-d-glucopyranoside substrates, respectively. Antiglycation activity was evaluated by measuring the formation of protein-bound fluorescent AGEs levels in an in vitro model system. The untreated protein/chitin mix showed significantly stronger α-amylase inhibition (IC₅₀ = 3780.00 ± 58.59 μg/ml) compared with microwave-assisted extracts (IC₅₀ = 6423.00 ± 380.10 μg/ml, $p\mathrm{<}0.05$). Chemically extracted fractions (IC50 = 5190.00 ± 1690.00 μg/ml) showed no significant difference when compared with protein/chitin mix and microwave-assisted extracts, respectively. In contrast, α-glucosidase inhibition showed limited activities. The chemically extracted fraction demonstrated the strongest antiglycation activity (IC50 = 239.40 ± 19.45 μg/ml), which was significantly ($p\mathrm{<}0.05$) lower than both protein/chitin mix (945.70 ± 49.70 μg/ml) and microwave-assisted extracts (1065.70 ± 31.64 μg/ml), and notably lower than the positive control aminoguanidine (IC₅₀ = 558.40 ± 23.70 μg/ml). Overall, chitin-rich fractions derived from BSFL exhibited notable α-amylase inhibitory and antiglycation activities, supporting their potential role in hyperglycaemia management. While extraction process reduced the α-amylase inhibition when compared to untreated samples, chemical extraction significantly enhanced the antiglycation activity. These findings highlight BSFL as a sustainable ingredient for future functional food and health-oriented applications.

Papain-mediated hydrolysis of yellow mealworm (Tenebrio molitor) proteins for hypoallergenic ingredient development

C.S.S. Teixeira^*, C. Villa, R. Biltes, C. Dias, B. Carriço-Sá, J. Costa and I. Mafra

REQUIMTE/LAQV, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; ^*cteixeira@ff.up.pt

Edible insects are emerging as sustainable alternative protein sources due to their high nutritional value, reduced environmental footprint and versatility in diverse food applications. However, their incorporation as ingredients in conventional food matrices where their presence may go unnoticed, raises safety concerns, primarily related to allergenic potential. In particular, yellow mealworm (Tenebrio molitor) contains pan-allergenic proteins (e.g., tropomyosin) which are homologous to major crustacean allergens and may induce IgE-mediated reactions in sensitized individuals due to cross-reactivity. This work proposes a novel papain-mediated hydrolysis strategy to mitigate the cross-reactivity of yellow mealworm flour. Papain, a cysteine endopeptidase extensively used in the food industry, was applied in a simple and cost-effective process involving a 3-hour incubation at 65 °C of insect flour suspended in water, using enzyme concentrations ranging from 1.25 to 2.50 U/ml. Under these conditions, extensive proteolysis was achieved, resulting in the disruption of conformational and linear IgE-binding epitopes. Immunoblotting analysis demonstrated complete elimination of IgE reactivity when tested with sera from crustacean-allergic patients. In silico hydrolysis simulation further corroborated the degradation of known allergenic epitopes within allergenic proteins. Collectively, these results demonstrate that the developed method offers a technologically feasible strategy for producing hypoallergenic, protein-rich, insect-derived ingredients suitable for incorporation into a wide range of food products. The proposed methodology has been protected as an innovation through a provisional patent application (PT120590) filed with the National Institute of Industrial Property (INPI) (Texeira et al., 2025). Reference: Teixeira, C.S.S., Villa, C., Carriço-Sá, B., Biltes, R., Dias, C., Costa, J. and Mafra, I., 2025.750 Method for producing an edible insect-based product. Provisional patent application PT120590 filled in 15/09/25. Portuguese Institute of Industrial Property. Patent pending. Funding: This work received financial support from European Union, FEDER, through the project EMPOWER (COMPETE2030-FEDER-00861800). Acknowledgments: This work was also supported by the national PT funds (FCT/MECI) through the projects INSPIRE (2024.14868.PEX), ALLEVIATE (2023.12193.PEX), UID/50006/2025, and IMMUNOGATE (COMPETE2030-FEDER-00848700).

Biocomputing analysis of allergenic glutathione transferases in edible insects

N.E. Labrou¹ and K.G. Athanasiadis^1,2*

¹Agricultural University of Athens, Biotechnology, Iera Odos 75, 11855, Athens, Greece; ²TUV Austria Labs SA, Laboratories, Kapodistriou 6, 15343, Agia paraskevi, Greece; ^*Kiriakos.athanasiadis@tuvaustria.com

Insect proteins are increasingly explored for food, feed and biotechnological applications, raising the need for systematic allergenicity assessment. This study employed advanced bioinformatic tools to investigate the allergenic potential of Glutathione Transferases (GSTs) from selected insect species. Protein sequences from Tenebrio molitor, Locusta migratoria, Acheta domesticus and Alphitobius diaperinus were analysed against curated databases of known allergens to identify sequence similarity patterns indicative of IgE-binding capacity. GST sequences were evaluated using established similarity thresholds, with values ≥90% considered indicative of a high likelihood of allergenicity. The analysis revealed that T. molitor GSTs showed the most pronounced allergenic signatures, with two sequences exceeding the 93% threshold relative to GST allergens from Blattella germanica. This high level of homology strongly suggests structural conservation of IgE-reactive epitopes and underscores the potential of T. molitor GSTs to elicit allergic responses. Although L. migratoria did not present sequences surpassing the cutoff, several GSTs exhibited high similarity values, indicating partial conservation of allergenic domains and possible immunological relevance. Phylogenetic reconstruction supported these findings by demonstrating evolutionary relatedness among insect GSTs, further implying epitope preservation across species. In contrast, GSTs from A. domesticus and A. diaperinus showed weaker homology, suggesting comparatively lower allergenic potential based on current computational evidence. Collectively, these findings indicate that insect-derived GSTs, particularly two isoenzymes from Tenebrio molitor and Locusta migratoria, display strong molecular signatures consistent with potential allergenic activity. Based on these results, the corresponding GST-encoding genes from T. molitor and L. migratoria were successfully cloned into T7 promoter–driven expression plasmids optimised for heterologous expression in Escherichia coli. Both enzymes were subsequently expressed as soluble recombinant proteins, enabling their isolation in quantities suitable for downstream analyses. Ongoing and future work will focus on the comprehensive biochemical and immunological characterisation of the recombinant GSTs, with particular emphasis on IgE epitope mapping and the evaluation of immunoreactivity using sera from sensitized human donors. These investigations are expected to experimentally validate the predicted allergenic potential inferred from sequence-based analyses and to provide critical data for allergenicity risk assessment.

Evaluation of the allergenic potential of novel insect proteins by IgE cross-reactivity

M.R. Carillo¹, S. Cirrincione¹, L. Tarraran¹, B. Aiuto¹, A. Quinternetto², F. Romaniello³, C. Rumbos⁴, C. Athanassiou⁵, E. Gronich⁶, F. Gai¹ and C. Lamberti^1*

¹National Research Council, ISPA, L.go P. Braccini 2, 10095 Grugliasco, Italy; ²Mauriziano Hospital, Via Magellano 1, 10128 Turin, Italy; ³National Institute of Metrological Research, S.da delle Cacce 91, 10135 Turin, Italy; ⁴University of Patras, Messolonghi Campus, 30200 Messolonghi, Greece; ⁵University of Thessaly, Phytokou Street, 38446 Volos, Greece; ⁶Flyng Spark Ltd, 2 Yizhak Modai Street, 7608804 Rehovot, Israel; ^*cristina.lamberti@cnr.it

The increasing adoption of sustainable alternative protein sources, such as edible insects in both human and animal diets raises important questions regarding their risk-assessment. One of the most relevant issues is the potential to elicit allergic reactions in individuals sensitized to phylogenetically related protein sources. This study aimed to assess the cross-reactive potential of insect-derived proteins using a combined immunological and proteomic approach. Sera from 48 patients with documented allergies to shrimp and/or house dust mites (HDM) were used to evaluate IgE cross-reactivity against protein extracts from two insect species, i.e., the yellow mealworm, Tenebrio molitor L., and the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Proteins present in immunoreactive bands were identified by high-resolution mass spectrometry (HPLC-HRMS) and their amino acid sequences were retrieved and compared against the AllergenOnline database using the dedicated search tool (www.allergenonline.org). Sequence alignments were evaluated according to internationally accepted criteria for allergenicity assessment, with significant matches defined as a minimum of 35% sequence identity over at least 80 contiguous amino acids (FAO/WHO recommendations). Our results showed that insect proteins exhibited pronounced IgE cross-reactivity with common invertebrate allergens, such as tropomyosin, paramyosin, troponine, myosin and arginine kinase. These findings suggest that exposure to these novel food sources could potentially trigger allergic reactions in patients with established sensitisation patterns, highlighting the need of integrating allergenic risk assessment into the safety evaluation of novel protein sources. Overall, this work supports the development of evidence-based strategies to ensure a safe introduction of sustainable alternative proteins into the animal and human diets. Acknowledgement: Financial support for this research has been provided by PRIMA under grant agreement No 2231, project CIPROMED (PRIMA Call 2022 Section 1 Agri-food IA).

Food allergen carryover in the edible insect supply chain: investigation in a pilot plant

C. Tramuta^*, S. Morello, A. Provera, R. Rosi, S. Lupi and D.M. Bianchi

Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta — CReNaRiA, Via Bologna 148, 10154 Turin, Italy; ^*clara.tramuta@izsplv.it

The use of insects as source of food has an important environmental and economic benefit: for these reasons all insect-based foods are included among the categories of food constituting novel foods. Insects may be an alternative food source but consider insects as food requires scrutiny due to risk of allergens. The aim of this work was to evaluate if food allergens could be vehiculated by edible insects from the substrate on which they are grown to the final product. Hermetia illucens larvae were grown on substrates containing 10% of each of the following allergens: peanuts, celery, soy, almonds and gluten. At the end of the feeding period, larvae were sampled at T0 (end of feeding), T1 (24 h fasting), T2 (48 h fasting) and subsequently frozen, dried and ground. Additionally, for each of the three time points, a negative control group was tested, consisting of larvae reared on substrates free of food allergens. Larva samples reared on substrates with and without allergens were analysed to evaluate the presence of allergen residues using: i) real-time PCRs (SPECIALfinder MC, Generon) and ii) enzyme-linked immunosorbent assays (ELISA RIDASCREEN® Gliadin, r-Biopharm). Real-time PCR for the detection of the soy allergen showed positive results in all analysed samples, (mean Ct: 28.84 at T0, 29.4 at T1, 30.95 at T2). Similarly, the celery target was detected in 7/9 replicates tested (mean Ct: 26.74 at T0, 26.90 at T1, 29.77 at T2), while the almond target was identified in one replicate at T0 (Ct 33.96) and in two replicates at T1 (mean Ct 34.01). Negative results were obtained for larva samples treated with the peanut allergen. As expected, the negative controls showed no fluorescence signals for the targets investigated. Values<5 mg/kg were obtained at T0, T1, and T2 for the gluten allergen as well, using the ELISA test. Insects may represent an alternative food source; however, their use requires careful evaluation due to the potential presence of allergens. The results obtained in this study highlight that if insects are fed on substrates containing allergens, these allergens can be transferred to the insects and, consequently, to the food products derived from them. To reduce the ’allergy risk’ for final consumers, it is essential to consider using allergen-free growth substrates or to include appropriate precautionary labelling that refers to allergens potentially present even in trace amounts. Acknowledgement: Project funded by the Italian Ministry of Health: IZSPRC, CUP J19I23001630001

Edible insect-derived ingredients for type 2 diabetes: the DiBaN project

D. Martin^1,2*, V. Da Cunha-Borges^1,2, E. Cantero-Bahillo^1,2, E. Rodrı́guez-González^1,2, D. Martı́n-Hernández^1,2, D. Villanueva-Bermejo^1,2, M.R. Garcı́a-Risco^1,2, L. Vázquez^1,2 and T. Fornari^1,2

¹Universidad Autónoma de Madrid, Sección Departamental Ciencias de la Alimentación, Facultad de Ciencias, 28049 Madrid, Spain; ²Instituto de Investigación en Ciencias de la Alimentación (CIAL), Departamento de Producción y Caracterisación de Nuevos Alimentos, Nicolás Cabrera 9, 28049 Madrid, Spain; ^*diana.martin@uam.es

Type 2 diabetes (T2D) is a chronic metabolic disorder characterised by insulin resistance and hyperglycaemia, with intestinal dysbiosis emerging as a key contributing factor, often linked to poor dietary habits. In this context, edible insects are gaining attention as sustainable and innovative sources of nutrients and health-promoting bioactive compounds. The DiBaN project aims to develop novel ingredients from bioactive-enriched edible insects to counteract dysbiosis and improve metabolic health related to T2D. Acheta domesticus has been selected as the model species for this project due to its high nutritional value and its approval for human foods. Specifically, DiBaN investigates how the nutritional and metabolic status of insects influences the subsequent bioactivity of insect-derived products, and how this can be boosted through dietary supplementation with specific bioactive and sustainable diets, such as microalgae. As general approaches of DiBaN, insects reared on conventional or microalgae-enriched diets are being processed into conventional products (full-fat powder, defatted powder or oil). Furthermore, advanced extraction techniques are being applied to develop innovative insect-based bioactive extracts. The nutritional composition and bioactivity of the products are being evaluated for vitro antioxidant, anti-inflammatory, anti-diabetic and hypolipidemic properties. The most promising insect-derived products are being assessed in ex vivo models (organoids and organ-on-a-chip). In parallel, two in vivo intervention studies (mice and human) are investigating the personalized metabolic and microbiome responses to dietary supplementation with A. domesticus powder. Finally, all generated data will be integrated into the development of a prototype of an AI-based predictive tool capable of forecasting personalized responses to nutritional interventions based on insect-derived ingredients. Therefore, the DiBaN project pioneers a new strategy to tackle T2D by exploiting the potential of edible insects as tailored sources of bioactive compounds capable of modulating the gut microbiome–host metabolic axis through targeted feeding strategies of the insects. Acknowledgement: This project is funded by the European Union under grant agreement No 101162517, call HORIZON-EIC-2023-PATHFINDERCHALLENGES-01.

Identification of bioactive peptides with potential human health applications from black soldier fly larvae

M. Hoffmann^1*, S. Karnaneedi¹, R. Nugraha², F. Kurniawan², K.R. Zenger¹ and A.L. Lopata¹

¹James Cook University, College of Science and Engineering, 1 James Cook Drive, Townsville, QLD 4814, Australia; ²Bogor Agricultural University, Department of Aquatic Product Technology, Jl. Raya Darmaga Kampus IPB, Babakan, Kec. Dramaga, Kabupaten Bogor, Jawa Barat, 16680 Bogor, Indonesia; ^*maximilian-h2@web.de

Black soldier fly larvae (BSFL) are increasingly being recognised as an alternative source of food protein among other edible insects. However, little research has been conducted into the potential beneficial effects of consumption of BSFL proteins. This study investigated the potential of BSFL as a source of bioactive peptides with antidiabetic, antihypertensive, and antioxidant properties combining in silico and experimental processes. The in vitro approach involved collecting, washing and starving the BSFL for 24 h before producing protein extracts. Two sample types were generated: one dried by microwave and the control which was not treated. Both samples were stored frozen at –80 °C before analyses with in vitro bioassays and advanced mass spectrometric analysis to identify proteins via UniProt databases. The in silico approach involved using several bioinformatic tools to simulate in vivo digestion of BSFL proteins by pepsin and trypsin, as well as determining the bioactivity of the resulting peptides. Finally, molecular docking experiments were performed to establish the extend of possible bioactivity. A total of 73 bioactive peptides were identified during the in silico process, focusing only on the ten most abundant proteins identified in BSFL by mass spectrometry. Most of these peptides are derived from hexamerin/hemocyanin and possess antihypertensive properties (n = 64). Other identified properties include antidiabetic, DPP-IV inhibition and antioxidant activity. Antioxidant potential was evaluated using the DPPH assay, with the non-treated BSFL protein extract exhibiting 71.2% radical scavenging capacity at a concentration of 30 μg/ml, decreasing to 14.3% at 3.75 μg/ml. Computational molecular docking experiments using the HADDOCK 2.4 cloud server produced HADDOCK scores ranging from –16.4 to –70.2, indicating high to very high binding affinities to suitable targets. These finding are supported through calculation of intermolecular interactions and the prediction of protein-peptide binding energies, resulting in $\mathrm{Δ}G$ values ranging from –9.9 to –6.4 kcal/mol, crucial for high affinity binding to target proteins. This study demonstrates that the BSFL are not only rich in proteins, but digestion potentially generates a promising panel of bioactive peptides, with antidiabetic, antihypertensive and antioxidant properties. More detailed in-vitro assay will support these finding.

Bioactive peptides from Acheta domesticus: in silico, in vitro, and cellular cardiometabolic assessment

C.S.S. Teixeira^1*, C. Villa¹, R. Biltes¹, S.F. Sousa¹, J. Costa¹, I.M.P.L.V.O. Ferreira¹, T.G. Tavares², M.A. Faria¹ and I. Mafra¹

¹REQUIMTE/LAQV, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; ²LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr Roberto Frias, 4200-465 Porto, Portugal; ^*cteixeira@ff.up.pt

Entomophagy is emerging as a sustainable and nutritious alternative protein source. Beyond their nutritional value, insect-derived peptides released during gastrointestinal (GI) digestion may confer health benefits, with antihypertensive activity, mediated through inhibition of somatic angiotensin-converting enzyme (sACE), being among the most widely reported. This study began with an in silico approach to simulate the GI digestion of house cricket (Acheta domesticus) proteins and identify peptides with potential sACE-inhibitory activity. Molecular docking was then performed to evaluate binding interactions between the resulting peptides and sACE catalytic domains. Peptides AVQPCF, CAIAW, IIIGW, QIVW, PIVCF and DVW exhibited high docking scores, suggesting the potential to inhibit sACE activity. Those peptides were subsequently validated in vitro. LC-MS/MS confirmed their presence in A. domesticus digesta, and enzymatic assays demonstrated potent sACE-inhibitory activity for AVQPCF, PIVCF and CAIAW, with IC₅₀ values of 3.69 ± 0.25, 4.63 ± 0.16 and 6.55 ± 0.52 μM, respectively. The digesta also exhibited significant sACE inhibition (IC₅₀ = 77.1 ± 11.8 μg protein/ml extract). Finally, the peptides were assessed in Caco-2 cells to evaluate intestinal absorption, cytotoxicity, barrier integrity, and modulation of gene expression markers associated with diabetes (DPP-4, SGLT1) and hypertension (sACE, ACE2). All peptides were non-cytotoxic up to 2 mM; however, DVW and PIVCF disrupted epithelial integrity. Only DVW was transported across the epithelium intact. Peptides DVW and PIVCF significantly downregulated SGLT1 expression (0.42- and 0.52-fold, respectively), suggesting a potential antidiabetic mechanism mediated by reduced intestinal glucose absorption. Taken together, this work demonstrates the bioactivity of A. domesticus-derived peptides, highlighting the potential of this insect as a multifunctional ingredient for promoting cardiometabolic health. Funding: This work received financial support from European Union, FEDER, through the project EMPOWER (COMPETE2030-FEDER-00861800). Acknowledgments: This work was also supported by the national PT funds (FCT/MECI) through the projects INSPIRE (2024.14868.PEX), ALLEVIATE (2023.12193.PEX), UID/50006/2025, and IMMUNOGATE (COMPETE2030-FEDER-00848700).

Development and quality evaluation of a novel protein bar enriched with food-grade roasted Tenebrio molitor powder

S.S.K. Daluwatta^1,2*, E.S.L.H. Ranathunga³, E.R.J. Samarakoon^2,4, L. Gasco¹ and P. Weththasinghe^2,3

¹University of Turin, Department of Agricultural, Forestry and Food Sciences, Largo Paolo Braccini, 2, 10095 Grugliasco, Turin, Italy; ²Postgraduate Institute of Agriculture, P.O. Box 55 Old Galaha Road, 20400 Peradeniya, Sri Lanka; ³University of Peradeniya, Department of Animal Science, Faculty of Agriculture, 20400 Peradeniya, Sri Lanka; ⁴University of Peradeniya, Department of Food Science and Technology, Faculty of Agriculture, 20400 Peradeniya, Sri Lanka; ^*sujivisamangakumari.daluwatta@unito.it

Insect-based food consumption is a rising global trend, driven by sustainability, circular economy, nutrition and food security. This study aimed to develop protein bars incorporating roasted Tenebrio molitor (TM) larvae flour and evaluate their physicochemical, nutritional and sensory properties. Initially, the effect of three roasting temperatures (90,110, 130 °C) on colour, water-holding capacity (WHC), oil-holding capacity (OHC), swelling capacity (SWC), Thiobarbituric acid reactive substances (TBARS), and total phenolic content (TPC) of the flour was examined. Protein bars were then developed with 12% (12MB) and 15% (15MB) roasted TM flour (roasted at 130 °C), blended with oats, chickpea, peanuts, coconut treacle, salt, vanilla and spices without added sugar. The bars were analysed for proximate composition, TBARS, TPC, and consumer sensory acceptance compared to control bar without TM (CB). Colour, WHC, OHC, SWC and TBARS of roasted flours were not significantly different ($p\mathrm{>}0.05$), but TPC was statistically different ($p\mathrm{<}0.05$) among treatments remaining within the acceptable range (5–20 mg GAE/g). Considering the pleasant aroma, 130 °C was selected for the preparation of protein bars. The crude protein content was 12.9% in the CB, while in 12MB and 15MB it was 18.9 and 21.5%, respectively, significantly higher ($p\mathrm{<}0.05$) compared to CB. Crude fat contents were 10.5, 12.5 and 12.6% for CB, 12MB and 15MB, respectively. Crude fibre contents were 2.9% (CB), 5.5% (12MB) and 4.0% (15MB). Both crude fat and fibre contents showed significantly higher values ($p\mathrm{<}0.05$) in TM contained bars. TBARS of 12MB showed a significantly low ($p\mathrm{<}0.05$) value, but both 12MB and 15MB were in the acceptable level of ≤2.0 mg MDA/kg. TPC values did not differ significantly among three formulations ($p\mathrm{>}0.05$). In the sensory evaluation, a considerable consumer segment (20/31) was interested in TM included protein bars in terms of all sensory attributes, in particular 15MB was the most preferred ($p\mathrm{<}0.05$), showing strong consumer acceptance. These findings demonstrate that appropriate thermal processing, such as roasting, allows TM flour to be successfully included in protein bars, supporting the development of sustainable, high-protein foods with favourable consumer acceptance.

Integration of Tenebrio molitor flour in protein bars: from formulation to industrial scale-up and market comparison

A. Vinhas^1*, A. Guimarães¹, A. Mota¹, H. Pinheiro¹, M. Teixeira¹, B. Barbosa², C. Rocha² and R. Lima²

¹Colab4Food, I&D, Vairão, 4485-655, Vila do Conde, Portugal; ²SenseTest, Lda, R. Zeferino Costa 341, 4400-345 Vila Nova de Gaia, Portugal; ^*ana.vinhas@colab4food.com

Consumer demand is shifting toward protein-rich snacks that combine health appeal with improved sustainability accelerating interest in novel protein ingredients such as insects. These ingredients provide nutrient-dense alternatives with low environmental impact. This study aimed to develop protein bars incorporating Tenebrio molitor larvae flour (TM) at different concentrations levels and to evaluate and compare performance across successive stages of product development. Four variants of protein bars were formulated, containing 0, 5, 10 or 15% (w/w) TM. Sensory screening enabled the down-selection of the 10 and 15% (w/w) formulations, which progressed to further technological optimisation. This was supported by sensory mapping that clarified the drivers of liking, indicating that consumer acceptance was associated with attributes such as a brownish appearance, intense odour, crunchy texture and earthy-bitter flavour. The formulation containing 10% (w/w) TM aligned with these attributes and was positioned close to the acceptance vector. Although the 15% (w/w) formulation also presented a positive acceptability score, it was characterised by less favourable textural attributes, particularly a pronounced grainy and powdery texture. Therefore, these formulations were subsequently subjected to texture-focused reformulation. An alternative processing technique (NP) was evaluated, applied exclusively to the insect flour (freezing and grinding), in comparison with the flour as originally supplied. Texture analysis showed that the NP consistently improved firmness and toughness compared with the old method, although at the cost of markedly increasing adhesiveness, effect that aligned with sensory results, which identified the 10% (w/w) NP formulation as the preferred sample and the 15% (w/w) NP formulation as the least appreciated. Based on that the 10% (w/w) TM formulation was selected for industrial Scale-up. Following scale-up, this bar was benchmarked against tree commercial bars, including one containing insect-derived ingredients and two conventional bars without insect components. This final sensory test validates that the formulation obtained achieves sensory performance comparable to commercial products. Overall, this work highlights the importance of an integrated approach combining formulation design, sensory evaluation, process optimisation, and market benchmarking in the development insect-based functional foods. Acknowledgement: This work was funded by InsectERA (No. C644917393-00000032), WP InFood, supported by Next Generation EU and Portugal’s PRR under “Agendas para a Inovação Empresarial” (C5).

Acheta domesticus or Tenebrio molitor? Species-dependent effects of insect powders on wheat bread quality

A. Orkusz^1*, J. Harasym¹, M. Mozzon² and R. Foligni³