Whole-genome sequencing of two captive black soldier fly populations: Implications for commercial production.

Black soldier fly (BSF; Hermetia illucens) is a promising insect species for food and feed production as its larvae can convert different organic waste to high-value protein. Selective breeding is one way to optimize production, but the potential of breeding is only starting to be explored and not yet utilized for BSF. To assist in monitoring a captive population and implementing a breeding program, genomics tools are imperative. We conducted whole genome sequencing of two captive populations separated by geographical distance - Denmark (DK) and Texas, USA (TX). Various population genetics analyses revealed a moderate genetic differentiation between two populations. Moreover, we observed higher inbreeding in the DK population, and the detection of a subpopulation within DK population aligned well with the recent foundation of the DK population from two captive populations. Additionally, we generated gene ontology annotation and variants annotation for wider potential applications. Our findings establish a robust marker set for research in population genetics, facilitating the monitoring of inbreeding and laying the groundwork for practical breeding programs for BSF.

Impact of age on the reproductive output of Hermetia illucens (Diptera: Stratiomyidae).

The current study examined the impact of adult black soldier fly (Hermetia illucens (L.)), age on reproductive output. Four treatments consisting of an equal sex ratio of the following combinations were used: 2-day-old males with 2-day-old females (YMYF); 6-day-old males with 2-day-old females (OMYF); 2-day-old males with 6-day-old females (YMOF); 6-day-old males with 6-day-old females (OMOF). Adult age was a significant factor for the overall number of mating events observed, with YMYF having 2.2× more than YMOF and 2.7× more than OMOF. Time was a significant factor in the number of mating events observed, with 97.8% observed on days 2-6. Age was not a significant factor for the total number of oviposition events, but the inclusion of time as a factor was significant, with 91.5% of all events occurring on days 2-6. Treatment and trial significantly interacted with egg production. This response can be attributed to treatments containing old females, which had a 50% variation in egg output across trials. Treatments containing young females were consistent in terms of egg production. As anticipated, due to oviposition events, 94.8% of total eggs were oviposited on days 2-6. Fecundity and egg hatch were also significantly impacted by female age, with a 50% reduction in both cases across trials occurring in old females. Young females paired with old males had a 20% reduction in egg hatch, but this difference was not statistically significant.

Evaluation of lauric acid enhancement of black soldier fly larvae from coconut.

The current study evaluated the potential enhancement of lauric acid (LA) in black soldier fly, Hermetia illucens, (L.) (Diptera: Stratiomyidae) larvae (BSFL), a source of this short-chain fatty acid which has antimicrobial and immunostimulatory properties. Replicate groups of BSFL were reared on either the coconut or Gainesville diet for 7 days. After the rearing period, BSFL were harvested, purged, dried, and subjected to proximate, fatty acid and amino acid compositions, and pepsin digestibility analyses. Results demonstrate changes in proximate composition. BSFL reared on the coconut had significantly (P = 0.002) higher lipid content (47.3% vs. 25.2%) on a dry-matter basis. The LA concentration in BSFL produced on the coconut was 31% greater than those reared on Gainesville, resulting in almost 150% more LA. Furthermore, BSFL-fed coconut had reduced crude protein (29.7% of dry weight) and ash (3.7% of dry weight) relative to those fed Gainesville (43.4% and 7.5% for crude protein and ash, respectively) but higher pepsin digestibility (91.0% vs. 87.0%). The relative amounts of various amino acids in the 2 BSFL meals did not differ extensively, with statistically lower concentrations of only phenylalanine and tryptophan and higher concentrations of alanine, arginine, isoleucine, leucine, and serine in BSFL reared on coconut. Results demonstrate that the nutritional composition of BSFL can be manipulated, and an enhancement of LA concentrations of 150% was achieved with coconut, which has value for BSFL as a feed for various livestock, including aquaculture. Lower protein content is a tradeoff in terms of BSFL value as a feed additive.

Reproductive output and other adult life-history traits of black soldier flies grown on different organic waste and by-products.

The interest in mass-rearing black soldier fly (Hermetia illucens) larvae for food and feed is rapidly increasing. This is partly sparked by the ability of the larvae to efficiently valorise a wide range of organic waste and by-products. Primarily, research has focused on the larval stage, hence underprioritizing aspects of the adult biology, and knowledge on reproduction-related traits such as egg production is needed. We investigated the impact of different organic waste and by-products as larval diets on various life-history traits of adult black soldier flies in a large-scale experimental setup. We reared larvae on four different diets: spent Brewer's grain, ground carrots, Gainesville diet, and ground oranges. Traits assessed were development time to pupa and adult life-stages, adult body mass, female lifespan, egg production, and egg hatch. Larval diet significantly impacted development time to pupa and adult, lifespan, body size, and egg production. In general, flies reared on Brewer's grain developed up to 4.7 d faster, lived up to 2.3 d longer, and produced up to 57% more eggs compared to flies reared on oranges on which they performed worst for these traits. There was no effect of diet type on egg hatch, suggesting that low-nutritious diets, i.e. carrots and oranges, do not reduce the quality but merely the quantity of eggs. Our results demonstrate the importance of larval diet on reproductive output and other adult traits, all important for an efficient valorisation of organic waste and by-products, which is important for a sustainable insect-based food and feed production.

Rearing methods of four insect species intended as feed, food, and food ingredients: a review.

Over the past 2 decades, the potential of insects as food and feed has been recognized globally. Insects as feed ingredients can improve sustainability because of their lower greenhouse gas emissions and their potential to transform organic wastes into high-quality feed rich in nutrients. However, currently, the practical use of insects as food or feed is limited by the high costs of insect production. A great deal of effort is required to improve the rearing technology necessary to establish the principles of insect farming. Several insect species have become industrialized using existing methods of production. The most common industrialized insect species intended as feed and food include the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae); the house cricket, Acheta domesticus L. (Orthoptera: Gryllidae); the black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae); and the house fly, Musca domestica L. (Diptera: Muscidae). This review focuses on describing the existing rearing methods for these 4 insect species, which may provide a basis for future research to enhance insect production capabilities.

Grinding as a slaughter method for farmed black soldier fly (Hermetia illucens) larvae: Empirical recommendations to achieve instantaneous killing.

At least 200 billion black soldier fly (Hermetia illucens) larvae (BSFL) are reared each year as food and feed, and the insect farming industry is projected to grow rapidly. Despite interest by consumers, producers, and legislators, no empirical evidence exists to guide producers in practicing humane - or instantaneous - slaughter for these novel mini-livestock. BSFL may be slaughtered via freezing, boiling, grinding, or other methods; however standard operating procedures (SOPs) and equipment design may affect the likelihood of instantaneous death using these methods. We tested how larval body size and particle size plate hole diameter affect the likelihood of instantaneous death for black soldier fly larvae that are slaughtered using a standard meat grinder. Larval body size did not affect the likelihood of instantaneous death for larvae that are 106-175 mg in mass. However, particle size plate hole diameter had a significant effect on the likelihood of instantaneous death, with only 54% of larvae experiencing an instant death when using the largest particle size plate (12-mm hole diameter) compared to 84% using the smallest particle size plate (2.55 mm). However, a higher percentage of instantaneous death (up to 99%) could be achieved by reducing the proportion of larvae that become stuck in the machine. We conclude by outlining specific recommendations to support producers in achieving a 99% instantaneous death rate through specific SOPs to be used with similarly designed machines. We also develop a protocol for producers that wish to test their own grinding SOPs.

Flight toward Sustainability in Poultry Nutrition with Black Soldier Fly Larvae.

Black soldier fly larvae (BSFL), Hermetia illucens (L.) (Diptera: Stratiomyidae), have emerged as a promising feed ingredient in broiler chicken diets, known for their high protein content, nutritional richness, and environmental sustainability. This review examines the effects of integrating BSFL into broiler feeds, focusing on aspects such as growth performance, nutrient digestibility, physiological responses, and immune health. The ability of BSFL to transform waste into valuable biomass rich in proteins and lipids underscores their efficiency and ecological benefits. Protein levels in BSFL can range from 32% to 53%, varying with growth stage and diet, offering a robust source of amino acids essential for muscle development and growth in broilers. While the chitin in BSFL poses questions regarding digestibility, the overall impact on nutrient utilization is generally favorable. The inclusion of BSFL in diets has been shown to enhance growth rates, feed efficiency, and carcass quality in broilers, with the larvae's balanced amino acid profile being particularly advantageous for muscle development. BSFL may also support gut health and immunity in broilers due to its bioactive components, potentially influencing the gut's microbial composition and enhancing nutrient absorption and overall health. Moreover, the capacity of BSFL to efficiently convert organic waste into protein highlights their role as an environmentally sustainable protein source for broiler nutrition. Nonetheless, further research is necessary to fully understand the long-term effects of BSFL, ideal inclusion rates, and the impact of varying larval diets and rearing conditions. It is crucial for poultry producers to consult nutritionists and comply with local regulations when incorporating new feed ingredients like BSFL into poultry diets.

Bokashi fermentation of brewery's spent grains positively affects larval performance of the black soldier fly Hermetia illucens while reducing gaseous nitrogen losses.

Digestion of waste feedstocks by larvae of the black soldier fly Hermetia illucens (Diptera: Stratiomyidae) (BSF) results in proteins for animal feed and organic fertilizer with a reduced environmental footprint, but it can still have negative environmental effects through greenhouse gas (GHG) and ammonia (NH3) emissions. Both biomass conversion by BSF larvae and associated GHG and NH3 emissions can depend on substrate properties that may be optimized through microbial inoculation pre-treatments, such as bokashi fermentation. Here, we quantified the effects of bokashi fermentation of brewery's spent grains on BSF rearing metrics and associated GHG and NH3 emissions at benchtop scale. We found that bokashi fermentation increased larval biomass by 40% and shortened development time by over two days on average, compared with unfermented spent grains. In line with increased larval growth, CO2 emissions in BSF larvae treatments were 31.0 and 79.0% higher in the bokashi fermented spent grains and Gainesville substrates, respectively, compared to the unfermented spent grains. Adding BSF larvae to the spent grains increased cumulative N2O emissions up to 64.0 mg N2O kg substratedry-1 but there were essentially no N2O emissions when larvae were added to fermented spent grains. Bokashi fermentation also reduced NH3 fluxes from the volatilization of substrate nitrogen in the BSF larvae treatment by 83.7-85.8% during days 7 and 9, possibly by increasing N assimilation by larvae or by reducing the transformation of substrate NH4+ to NH3. Therefore, bokashi fermentation may be applied to improve performance of BSF larvae on a common industrial waste stream and reduce associated emissions.

Insights into the reduction of antibiotic-resistant bacteria and mobile antibiotic resistance genes by black soldier fly larvae in chicken manure.

The increasing prevalence of antibiotic-resistant bacteria (ARB) from animal manure has raised concerns about the potential threats to public health. The bioconversion of animal manure with insect larvae, such as the black soldier fly larvae (BSFL, Hermetia illucens [L.]), is a promising technology for quickly attenuating ARB while also recycling waste. In this study, we investigated BSFL conversion systems for chicken manure. Using metagenomic analysis, we tracked ARB and evaluated the resistome dissemination risk by investigating the co-occurrence of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial taxa in a genetic context. Our results indicated that BSFL treatment effectively mitigated the relative abundance of ARB, ARGs, and MGEs by 34.9%, 53.3%, and 37.9%, respectively, within 28 days. Notably, the transferable ARGs decreased by 30.9%, indicating that BSFL treatment could mitigate the likelihood of ARG horizontal transfer and thus reduce the risk of ARB occurrence. In addition, the significantly positive correlation links between antimicrobial concentration and relative abundance of ARB reduced by 44.4%. Moreover, using variance partition analysis (VPA), we identified other bacteria as the most important factor influencing ARB, explaining 20.6% of the ARB patterns. Further analysis suggested that antagonism of other bacteria on ARB increased by 1.4 times, while nutrient competition on both total nitrogen and crude fat increased by 2.8 times. Overall, these findings provide insight into the mechanistic understanding of ARB reduction during BSFL treatment of chicken manure and provide a strategy for rapidly mitigating ARB in animal manure.

A gut commensal bacterium promotes black soldier fly larval growth and development partly via modulation of intestinal protein metabolism.

IMPORTANCE: Black solider fly larvae and the gut microbiota can recycle nutrients from various organic wastes into valuable insect biomass. We found that Citrobacter amalonaticus, a gut commensal bacterium of the insect, exerts beneficial effects on larval growth and development and that the expression of many metabolic larval genes was significantly impacted by the symbiont. To identify the larval genes involved in the host-symbiont interaction, we engineered the symbiont to produce double-strand RNA and enabled the strain to silence host genes in the larval gut environment where the interaction takes place. With this approach, we confirmed that two intestinal protease families are involved in the interaction and provided further evidence that intestinal protein metabolism plays a role in the interaction. This work expands the genetic toolkits available to study the insect functional genomics and host-symbiont interaction and provide the prospective for the future application of gut microbiota on the large-scale bioconversion.

Bioinformatic Surveillance Leads to Discovery of Two Novel Putative Bunyaviruses Associated with Black Soldier Fly.

The black soldier fly (Hermetia illucens, BSF) has emerged as an industrial insect of high promise because of its ability to convert organic waste into nutritious feedstock, making it an environmentally sustainable alternative protein source. As global interest rises, rearing efforts have also been upscaled, which is highly conducive to pathogen transmission. Viral epidemics have stifled mass-rearing efforts of other insects of economic importance, such as crickets, silkworms, and honeybees, but little is known about the viruses that associate with BSF. Although BSFs are thought to be unusually resistant to pathogens because of their expansive antimicrobial gene repertoire, surveillance techniques could be useful in identifying emerging pathogens and common BSF microbes. In this study, we used high-throughput sequencing data to survey BSF larvae and frass samples, and we identified two novel bunyavirus-like sequences. Our phylogenetic analysis grouped one in the family Nairoviridae and the other with two unclassified bunyaviruses. We describe these putative novel viruses as BSF Nairovirus-like 1 and BSF uncharacterized bunyavirus-like 1. We identified candidate segments for the full BSF Nairovirus-like 1 genome using a technique based on transcript co-occurrence and only a partial genome for BSF uncharacterized bunyavirus-like 1. These results emphasize the value of routine BSF colony surveillance and add to the number of viruses associated with BSF.

Behavioral interplay between mosquito and mycolactone produced by Mycobacterium ulcerans and bacterial gene expression induced by mosquito proximity.

Mycolactone is a cytotoxic lipid metabolite produced by Mycobacterium ulcerans, the environmental pathogen responsible for Buruli ulcer, a neglected tropical disease. Mycobacterium ulcerans is prevalent in West Africa, particularly found in lentic environments, where mosquitoes also occur. Researchers hypothesize mosquitoes could serve as a transmission mechanism resulting in infection by M. ulcerans when mosquitoes pierce skin contaminated with M. ulcerans. The interplay between the pathogen, mycolactone, and mosquito is only just beginning to be explored. A triple-choice assay was conducted to determine the host-seeking preference of Aedes aegypti between M. ulcerans wildtype (MU, mycolactone active) and mutant (MUlac-, mycolactone inactive). Both qualitative and quantitative differences in volatile organic compounds' (VOCs) profiles of MU and MUlac- were determined by GC-MS. Additionally, we evaluated the interplay between Ae. aegypti proximity and M. ulcerans mRNA expression. The results showed that mosquito attraction was significantly greater (126.0%) to an artificial host treated with MU than MUlac-. We found that MU and MUlac produced differential profiles of VOCs associated with a wide range of biological importance from quorum sensing (QS) to human odor components. RT-qPCR assays showed that mycolactone upregulation was 24-fold greater for MU exposed to Ae. aegypti in direct proximity. Transcriptome data indicated significant induction of ten chromosomal genes of MU involved in stress responses and membrane protein, compared to MUlac- when directly having access to or in near mosquito proximity. Our study provides evidence of possible interkingdom interactions between unicellular and multicellular species that MU present on human skin is capable of interreacting with unrelated species (i.e., mosquitoes), altering its gene expression when mosquitoes are in direct contact or proximity, potentially impacting the production of its VOCs, and consequently leading to the stronger attraction of mosquitoes toward human hosts. This study elucidates interkingdom interactions between viable M. ulcerans bacteria and Ae. aegypti mosquitoes, which rarely have been explored in the past. Our finding opens new doors for future research in terms of disease ecology, prevalence, and pathogen dispersal outside of the M. ulcerans system.

Quorum sensing gene regulation in Staphylococcus epidermidis reduces the attraction of Aedes aegypti (L.) (Diptera: Culicidae).

Introduction: Identifying mechanisms regulating mosquito attraction to hosts is key to suppressing pathogen transmission. Historically, the ecology of the host microbial community and its influence on mosquito attraction, specifically, whether bacterial communication through quorum sensing (QS) modulates VOC production that affects mosquito behavior have not been extensively considered. Methods: Behavioral choice assays were applied along with volatile collection, followed by GC-MS and RNA transcriptome analyses of bacteria with and without a quorum-sensing inhibitor, furanone C-30. Results: Utilizing the quorum-sensing inhibitor on a skin-inhabiting bacterium, Staphylococcus epidermidis, we disrupted its interkingdom communication with adult Aedes aegypti and mitigated their attraction to a blood-meal by 55.1%. Discussion: One potential mechanism suppressing mosquito attraction could be the reduction (31.6% in our study) of bacterial volatiles and their associated concentrations by shifting S. epidermidis metabolic (12 of 29 up regulated genes) and stress (5 of 36 down regulated genes) responses. Manipulating the quorum-sensing pathways could serve as a mechanism to reduce mosquito attraction to a host. Such manipulations could be developed into novel control methods for pathogen-transmitting mosquitoes and other arthropods.

Impacts of black soldier fly, (Diptera: Stratiomyidae) larval frass on tomato production.

The "insects as food and feed" movement is gaining considerable momentum as a novel means to provide protein to people (i.e., food) and other animals (i.e., feed). Insects require significantly fewer resources, such as water and land, to produce, process, and distribute as a food or feed source. While the production of insect biomass has received considerable attention for use as food or feed, little is known about the value of the residual materials remaining after digestion. One insect, the black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), can generate large quantities of residual (i.e., frass) that is high in nitrogen, phosphorus, and potassium. These materials could serve as a partial replacement for fertilizer or peat, thus creating added value to the insects as food and feed sector. Greenhouse studies were designed to investigate the use of frass in vegetable production. In pot studies with tomatoes, different ratios of peat:vermicompost and peat:insect frass were compared to a 100% peat control. Across all other parameters, tomato fruits and vegetative biomass did not produce significant differences across treatments, indicating results were comparable to the control (i.e., 100% peat). Thus, replacing peat with black soldier fly frass is a viable option and could allow for the peat industry to become more sustainable and regenerative. However, it should be noted that average individual tomato fruit weight was significantly (P < 0.05) higher (by 19%) in the vermicompost 10% treatment compared to the control, which did not differ from treatments including black soldier fly frass.

Large-scale production of house fly, Musca domestica (Diptera: Muscidae), larvae fed 3 manure types.

House flies, Musca domestica, L., (Diptera: Muscidae) are well-known pests at animal facilities; however, they can be used for manure biodegradation. Utilizing house flies to process animal manure offers a means to recycle nutrients and reduce contaminants (e.g., pathogens and heavy metals), while also producing multiple revenue streams (e.g., protein for feed, fat for biodiesel, frass as a soil amendment). This study determined house fly larval performance on a larger scale (kilogram of wastes; thousands of larvae; single feeding) as a follow-up to a previous experiment performed at a bench-top scale (g of wastes; hundreds of larvae; incremental feeding). Four thousand larvae were fed 1 kg of swine, dairy, or poultry manure, or a control (Gainesville diet: 50% wheat bran, 30% alfalfa meal, and 20% corn meal). Peak larval weight occurred 4 days after inoculation and no significant difference in development time to first pupariation occurred across diets. However, percent survivorship to pupariation varied, with the highest occurring in Gainesville (74%), swine (73%), and poultry (67%) manure, whereas 50% survived when fed dairy manure. The highest pupal weight was found for those fed Gainesville (27 mg), and similar weights were found for those fed swine (21 mg), dairy (24 mg), and poultry (25 mg) manure. Although using house flies to manage manure has received little consideration in Western countries, other regions have this practice in place. Results may provide insight on differences between small- and large-scale studies, which is valuable for industrialization of this species for waste management and creating a more circular economy.

Black soldier fly (Diptera: Stratiomyidae) larval heat generation and management.

Mass production of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae results in massive heat generation, which impacts facility management, waste conversion, and larval production. We tested daily substrate temperatures with different population densities (i.e., 0, 500, 1000, 5000, and 10 000 larvae/pan), different population sizes (i.e., 166, 1000, and 10 000 larvae at a fixed feed ratio) and air temperatures (i.e., 20 and 30 °C) on various production parameters. Impacts of shifting larvae from 30 to 20 °C on either day 9 or 11 were also determined. Larval activity increased substrate temperatures significantly (i.e., at least 10 °C above air temperatures). Low air temperature favored growth with the higher population sizes while high temperature favored growth with low population sizes. The greatest average individual larval weights (e.g., 0.126 and 0.124 g) and feed conversion ratios (e.g., 1.92 and 2.08 g/g) were recorded for either 10 000 larvae reared at 20 °C or 100 larvae reared at 30 °C. Shifting temperatures from high (30 °C) to low (20 °C) in between (∼10-d-old larvae) impacted larval production weights (16% increases) and feed conversion ratios (increased 14%). Facilities should consider the impact of larval density, population size, and air temperature during black soldier fly mass production as these factors impact overall larval production.

Mitigation of antibiotic resistome in swine manure by black soldier fly larval conversion combined with composting.

The increasing prevalence of antibiotic resistance genes (ARGs) in animal manure has attracted considerable attention because of their potential contribution to the development of multidrug resistance worldwide. Insect technology may be a promising alternative for the rapid attenuation of ARGs in manure; however, the underlying mechanism remains unclear. This study aimed to evaluate the effects of black soldier fly (BSF, Hermetia illucens [L.]) larvae conversion combined with composting on ARGs dynamics in swine manure and to uncover the mechanisms through metagenomic analysis. Compared to natural composting (i.e. without BSF), BSFL conversion combined with composting reduced the absolute abundance of ARGs by 93.2 % within 28 days. The rapid degradation of antibiotics and nutrient reformulation during BSFL conversion combined with composting indirectly altered manure bacterial communities, resulting in a lower abundance and richness of ARGs. The number of main antibiotic-resistant bacteria (e.g., Prevotella, Ruminococcus) decreased by 74.9 %, while their potential antagonistic bacteria (e.g., Bacillus, Pseudomonas) increased by 128.7 %. The number of antibiotic-resistant pathogenic bacteria (e.g., Selenomonas, Paenalcaligenes) decreased by 88.3 %, and the average number of ARGs carried by each human pathogenic bacterial genus declined by 55.8 %. BSF larvae gut microbiota (e.g., Clostridium butyricum, C. bornimense) could help reduce the risk of multidrug-resistant pathogens. These results provide insight into a novel approach to mitigate multidrug resistance from the animal industry in the environment by using insect technology combined with composting, in particular in light of the global "One Health" requirements.

Establishment of highly efficient transgenic system for black soldier fly (Hermetia illucens).

The black soldier fly (BSF), Hermetia illucens, is a promising insect for mitigating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass. We recently reported a high-quality genome assembly of the BSF; analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion. To enable genetic manipulation of the BSF, we have established the first transgenic methods for this economically important insect. We cloned and identified the ubiquitous actin5C promoter (Hiactin5C-p3k) and 3 endogenous U6 promoters (HiU6:1, HiU6:2, and HiU6:3). The Hiactin5C promoter was used to drive expression of a hyperactive variant of the piggyBac transposase, which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase. Furthermore, we evaluated the 3 HiU6 promoters using this transgenic system. HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) development. Overall, our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.