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Larvae of the black soldier fly Hermetia illucens (Diptera: Stratiomyidae) (BSFL) are increasingly used in a circular economy context for industrial production of protein, oil, and frass, while serving as a sustainable method for managing numerous types of organic waste. On both fronts, there are ongoing efforts to optimise feedstocks for increased larval performance, yields of protein and/or oil, and efficiency of volumetric reduction of waste. Fermentation of organic waste prior to providing it to BSFL can help accomplish both goals. A few studies have individually evaluated fermenting agents such as lactic acid bacteria, yeasts, and fungi, showing that they can improve BSFL digestion of biowaste. However, the potential of co-fermentation by multiple microbes to improve waste digestion by BSFL has not been well explored. Here we tested a type of anaerobic fermentation, known as bokashi, that simultaneously uses lactic acid bacteria, yeasts, and fungi, on a common nutritious industrial feedstock (brewery’s spent grains) and on a nutritionally poor agricultural waste (unharvested oranges) on resulting life-history traits of BSFL. We show that bokashi-fermented substrates increased BSFL biomass and growth rate on both feeding substrates and dramatically reduced BSFL development duration on the nutritionally challenging oranges. Besides this, BSFL reared on fermented industrial feedstock reached the peak weight a day earlier, on average, than those feeding on the same unfermented substrate. Collectively, these effects would be beneficial for industrial BSF farming. We also highlight research areas to be tackled before bokashi fermentation can become widely adopted by the BSF farming sector.

Black soldier fly larvae (BSFL) can convert organic waste into high-quality biomass. In this study, we tested the potential of sugarcane by-products as a food source for BSFL and compared larval development and nutritional value with some other organic substrates. Seven different substrates were used, including carrot pomace (C), carrot pomace and leftover bread (CB) (50/50), bagasse and vinasse (BV), bagasse and molasses (BM), bagasse, vinasse, and molasses (BVM), a mixture of all the above treatments (MX), and university canteen leftovers (UCLs). The larval weight and length were measured for two weeks from day 5 to 19. Then, the BSFL were harvested and analyzed for dry matter, crude protein, oil, ash, mineral, and fatty acid composition. Larval weight and length varied depending on the feeding substrate provided. University canteen leftovers resulted in the BSFL having at least 18% greater length (17.00 mm) and 56% greater weight (3.15 g) compared to other treatments. The highest amounts of protein (38.9%) and oil (39.06%) were observed in the UCL treatment, while the BV treatment larvae had the highest quantities of ash (28.9%) and dry matter (28.0%). The fatty acid profile of the BSFL revealed three-times-higher levels of saturated fatty acids than unsaturated fatty acids in the UCL treatment and was at least twice as high in other treatments. Overall, the BSFL had the best growth on the UCL substrate, and the combination of bagasse and vinasse (BV) was the most appropriate substrate for BSFL development among the sugarcane by-products.

Artificial perches are implemented by many companies that mass-rear the black soldier fly (BSF), to emulate a natural breeding environment or provide additional surface area for flies to rest; however, basic information about perching behavior is lacking. This experiment tested the effect of adding 0.00, 0.04, 0.26, or 0.34 m 2 of surface area to 0.93 m 3 cages, each supplied with 90 male and 90 female adults. Female thoraxes marked with acrylic paint, and the number of perching flies of each sex were recorded over 6 d. A time-series analysis revealed the following: (a) females utilized perches 1.42 times more often than males across two trials; (b) especially in the morning where the difference could be as high as 2.56 times as great; (c) this decreased to 0.20-1.57 times more females than males by 1600 h; and (d) this cyclical pattern repeated each day throughout the week with a decreasing female-bias, starting from 2.41-times more females on day 1, which fell to 0.88-1.98-times more females than males on day 6. These dynamics are likely due to the presence of male flies engaging in aerial contests near ultraviolet lamps required for mating, especially during the early hours and early adulthood, aligning with and expanding prior knowledge of black soldier fly mating behavior.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Dr. Tomberlin has a significant financial interest in EVO Conversion Systems LLC. This conflict of interest is managed by a plan submitted to and approved by Texas A&M University and Texas A&M AgriLife., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

Thermal tolerance and preference are key parameters impacting agricultural production systems. In this study, the impact of larval diet on black soldier fly thermal tolerance and preference across life-stages and sexes was examined. Larvae were fed either a low-protein high-carbohydrate synthetic diet (i.e., P 7 C 35 ), a high-protein low-carbohydrate synthetic diet (i.e., P 35 C 7 ), or the Gainesville diet (i.e., C) as a control and reference. Our results demonstrate that the impacts of larval diet on black soldier fly thermal tolerance and preference could be stage and sex specific. The mean heat knockdown temperatures (HKT) ranged between 46.6 and 47.9 °C. Synthetic diets resulted in greater HKT and the difference decreased form larvae (e.g., ∼1 °C) to adults (e.g., ∼0.2 °C). The mean chill-coma recovery time (CCRT) ranged between 8.3 and 21.6 min. Not much differences were detected between diets, but CCRT became longer from larvae to adults. The mean thermal preference ranged between 13.6 and 29.5 °C. Larvae fed synthetic diets preferred much lower temperatures than the control diet. A bimodal distribution was observed for adults regardless of sex. Differences on body mass, lipid, and protein contents were detected among diets; however, more research should be done before any conclusions can be linked to their thermal traits. These findings highlight the importance of considering the ingredients and nutritional makeup of larval diets when optimizing temperature management protocols for mass production of black soldier flies. Conversely, specific diets can be developed to promote survival under extreme rearing temperatures., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Material was purchased for use in this project from EVO Conversion Systems, LLC, a company with which Dr. Tomberlin has a significant financial interest. This conflict of interest is managed by a plan approved by Texas A&M AgriLife., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: MB and BF report a relationship with the Insect Welfare Research Society that includes: board of advisors (unpaid). CP reports a relationship with the Insect Welfare Research Society that includes: employment. JKT reports a relationship with EVO Conversion Systems, LLC. Regarding funding, MB was an NSF postdoctoral research fellow (2109399) at the time this work was conducted. CP was supported by the Insect Welfare Research Society. JKT and CF appreciate financial support from the Texas A & M AgriLife Research State Air Quality Initiative (SAQI). JKT and CM used resources from the Texas A&M AgriLife Institute for Advancing Health Through Agriculture., (© The Author(s) 2024.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 (NH 3 ) emissions. Both biomass conversion by BSF larvae and associated GHG and NH 3 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 NH 3 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, CO 2 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 N 2 O emissions up to 64.0 mg N 2 O kg substrate dry -1 but there were essentially no N 2 O emissions when larvae were added to fermented spent grains. Bokashi fermentation also reduced NH 3 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 NH 4 + to NH 3 . Therefore, bokashi fermentation may be applied to improve performance of BSF larvae on a common industrial waste stream and reduce associated emissions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

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., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kim, Crippen, Jordan and Tomberlin.)

With the potential to process the world's agricultural and food waste, provide sustainable fodder for livestock, aquaculture, and pet animals, as well as act as a source of novel biomolecules, the black soldier fly, Hermetia illucens, has been launched into the leading position within the insects as feed industry. Fulfilment of these goals, however, requires mass-rearing facilities to have a steady supply of neonate larvae, which in-turn requires an efficient mating process to yield fertile eggs; yet, little is known about adult reproductive behavior, nor what physiological factors lead to its emergence. Moreover, fertile egg production tends to be highly variable in colony. Therefore, this review brings together what is currently known of the organismal biology of H. illucens, compiling information on adult morphology, physiology, biogeography, genomics, and behavioral ecology. As a holistic synthesis, it highlights several directions of interest for research to follow., (© 2022 Wiley Periodicals LLC.)

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut-associated microorganisms., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Flies and other arthropods mechanically transmit multiple pathogens and a recent experimental study demonstrated house flies, Musca domestica L. (Diptera: Muscidae), can mechanically transmit SARS-CoV-2. The purpose of this study was to explore the possibility of mechanical transmission of SARS-CoV-2 by domestic insects and their potential as a xenosurveillance tool for detection of the virus. Flies were trapped in homes where at least one confirmed human COVID-19 case(s) resided using sticky and liquid-baited fly traps placed inside and outside the home in the Texas counties of Brazos, Bell, and Montgomery, from June to September 2020. Flies from sticky traps were identified, pooled by taxa, homogenized, and tested for the presence of SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR). Liquid traps were drained, and the collected fluid similarly tested after RNA concentration. We processed the contents of 133 insect traps from 40 homes, which contained over 1,345 individual insects of 11 different Diptera families and Blattodea. These individuals were grouped into 243 pools, and all tested negative for SARS-CoV-2 RNA. Fourteen traps in seven homes were deployed on the day that cat or dog samples tested positive for SARS-CoV-2 RNA by nasal, oral, body, or rectal samples. This study presents evidence that biting and nonbiting flies and cockroaches (Blattodea) are not likely to contribute to mechanical transmission of SARS-CoV-2 or be useful in xenosurveillance for SARS-CoV-2., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Biochar is known to accelerate composting process and improve the quality of end-products. However, its effects on bioconversion of organic waste by black soldier fly larvae (BSFL) remains largely unexamined. To investigate the effects of corn straw biochar (CS-BC) on bioconversion of soybean dregs (SD) by BSFL, SD was amended with four different dosages of CS-BC [0%, 2%, 5%, and 8% (w/w)] and digested by BSFL for ten days. The results indicated that the peak values of single larva wet weight in the treatments amended with CS-BC were advanced by 2-3 days and the reduction rate of SD increased from 72.09% to 85.37% with the increasing dosage of CS-BC. Meanwhile, SD mixed with 2%, 5% and 8% of CS-BC decreased ammonia (NH 3 ) emission by 2.7%, 3.6% and 18.0%, respectively. The nitrous oxide (N 2 O) emissions reduced (-23.6%, -29.1% and -49.2%) with 2%, 5% and 8% CS-BC additions, respectively. In addition, the residual nitrogen of SD‑nitrogen proportionally increased with CS-BC application (28.3%, 28.6%, 30.1% and 35.0% for application at the dosage of 0%, 2%, 5% and 8%, respectively). Based on the comprehensive evaluation of bioconversion performance, alleviation of pollutant gas emission, and nitrogen conservation, we recommend the introduction of 8% (w/w) CS-BC during bioconversion of SD by BSFL. This study confirmed the feasibility of CS-BC as an amendment for the BSFL-based bioconversion system., Competing Interests: Declaration of competing interest The authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The feeding of vertebrate scavengers on animal remains has been noted for at least 30 years in relation to the creation of postmortem artifacts. However, the subsequent impact on other necrophagous arthropods, which interact with these remains has not been well documented. Herein, we report a rodent (Rattus spp.) feeding event that altered a perimortem wound beneath the jaw of a decomposing swine carcass. Point trauma such as this has been cited as insufficient for insect colonization; however, the resulting enlargement of the wound due to scavenger feeding has the potential to serve as an oviposition site where colonization would typically not be expected otherwise (i.e., in the absence of a wound or the presence of a small wound such as point trauma). In fact, colonization was observed surrounding the site of scavenger alteration. If scavenger artifacts (e.g., postmortem alteration of remains by feeding) are not identified appropriately and recognized for the effects they may have on necrophagous arthropods then associated assessments in forensic investigations could be affected., (© 2022 American Academy of Forensic Sciences.)

Lincomycin fermentation residues (LFR) are the byproducts from the pharmaceutical industry, and contain high concentrations of antibiotics that could pose a threat to the environment. Here, we report that black soldier fly larvae (BSFL) and associated microbiota can effectively degrade LFR and accelerate the degradation of lincomycin in LFR. The degradation rate of lincomycin in LFR can reach 84.9% after 12 days of BSFL-mediated bioconversion, which is 3-fold greater than that accomplished with natural composting. The rapid degradation was partially carried out by the BSFL-associated microbiota, contributing 22.0% of the degradation in the final composts. Based on microbiome analysis, we found that the structure of microbiota from both BSFL guts and BSFL composts changed significantly during the bioconversion, and that several bacterial genera were correlated with lincomycin degradation. The roles of the associated microbiota in the degradation were further verified by the ability of two larval intestinal bacterial isolates and one bacterial isolate from BSFL composts to lincomycin degradation. The synergy between BSFL and the isolated strains resulted in a 2-fold increase in degradation compared to that achieved by microbial degradation alone. Furthermore, we determined that the degradation was correlated with the induction of several antibiotic resistant genes (ARGs) associated with lincomycin degradation in larval guts and BSFL composts. Moreover, the environmental conditions in the BSFL composts were found to be conducive to the degradation. In conclusion, these findings demonstrate that the BSFL-mediated bioconversion of LFR could effectively reduce residual lincomycin and that the associated microbiota play crucial roles in the process., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Antibiotics are challenging to degrade and are excreted by livestock which results in environmental pollution. In this paper, we demonstrated that environmentally friendly manure bioremediation performed by black soldier fly larvae (BSFL) is a wise alternative, which could effectively degrade ciprofloxacin (CIP) by approached 85.48% in artificial diet and 84.22% in poultry manure within 12 days. They are up to 2.5-4.0 fold more than that achieved by natural fermentation. The five CIP-degrading strains were isolated from the larval gut, two of which, named by Klebsiella pneumoniae BSFLG-CIP1 and Proteus mirabilis BSFLG-CIP5, could degraded CIP by nearly 98.22% and 97.83% in vitro, respectively. When the intestinal isolates were re-inoculated to sterile BSFL system, the degradation level significantly increased up to 82.38%, comparing with the sterile BSFL system (21.76%). It is proved that the larvae intestinal microbiota might carry out this highly-efficient CIP-degradation. Furthermore, seven possible metabolites were identified for CIP-degradation in vitro, and they were referring three main potential degrading mechanisms of hydroxylize, piperazine ring substitute and cleavage, and quinoline ring cleavage. In conclusion, the present study may provide a strategy to reduce antibiotics pollution in animal waste through bioremediation with BSFL and adjusted intestinal microbes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Organic decomposition processes, involving the breakdown of complex molecules such as carbohydrates, proteins and fats, release small chemicals known as volatile organic compounds (VOCs), smelly even at very low concentrations, but not all readily detectable by vertebrates. Many of these compounds are instead detected by insects, mostly by saprophytic species, for which long-range orientation towards organic decomposition matter is crucial. In the present work the detection of aldehydes, as an important measure of lipid oxidation, has been possible exploiting the molecular machinery underlying odour recognition in Hermetia illucens (Diptera: Stratiomyidae). This voracious scavenger insect is of interest due to its outstanding capacity in bioconversion of organic waste, colonizing very diverse environments due to the ability of sensing a wide range of chemical compounds that influence the choice of substrates for ovideposition. A variety of soluble odorant binding proteins (OBPs) that may function as carriers of hydrophobic molecules from the air-water interface in the antenna of the insect to the receptors were identified, characterised and expressed. An OBP-based nanobiosensor prototype was realized using selected OBPs as sensing layers for the development of an array of quartz crystal microbalances (QCMs) for vapour phase detection of selected compounds at room temperature. QCMs coated with four recombinant H. illucens OBPs (HillOBPs) were exposed to a wide range of VOCs indicative of organic decomposition, showing a high sensitivity for the detection of three chemical compounds belonging to the class of aldehydes and one short-chain fatty acid. The possibility of using biomolecules capable of binding small ligands as reversible gas sensors has been confirmed, greatly expanding the state-of the-art in gas sensing technology., (Creative Commons Attribution license.)

Antibiotic resistance is a continuing challenge in medicine. There are various strategies for expanding antibiotic therapeutic repertoires, including the use of blow flies. Their larvae exhibit strong antibiotic and antibiofilm properties that alter microbiome communities. One species, Lucilia sericata, is used to treat problematic wounds due to its debridement capabilities and its excretions and secretions that kill some pathogenic bacteria. There is much to be learned about how L. sericata interacts with microbiomes at the molecular level. To address this deficiency, gene expression was assessed after feeding exposure (1 h or 4 h) to two clinically problematic pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii. The results identified immunity-related genes that were differentially expressed when exposed to these pathogens, as well as non-immune genes possibly involved in gut responses to bacterial infection. There was a greater response to P. aeruginosa that increased over time, while few genes responded to A. baumannii exposure, and expression was not time-dependent. The response to feeding on pathogens indicates a few common responses and features distinct to each pathogen, which is useful in improving the wound debridement therapy and helps to develop biomimetic alternatives., (© 2021 The Royal Entomological Society.)

Black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae) larvae (BSF larvae or BSFL) offer an environmental-friendly method for degrading antibiotics, such as tylosin (TYL) and enrofloxacin (EF), in swine manure. This study examined the impact of temperature on this process, role of associated microbes, dynamics of resistant genes, and a description of the microbial community associated with the BSF larval gut, how microbes isolated from the BSF larval gut as inoculants impact the process as well as enhance antibiotic digestion, and finally a quantification of antibiotics in BSF larvae fed manure with TYL or EF. Antibiotic degradation in manure was optimized at 28 °C with at least 10% greater than 23 °C and 37 °C. More than 40% reduction in TYL and EF concentrations in the manure occurred when BSF larval gut associated microbes were present. Furthermore, DNA extracted from the gut of non-sterile BSF larvae fed manure with TYL or EF indicated at least two 2 -△△Ct fold increase in antibiotic resistance genes for TYL and EF. We identified 250, 4, and 16 unique operational taxa for larvae fed control manure and manure with either TYL or EF. Intestinal microbes isolated from non-sterile larvae fed manure with TYL or EF, were identified, cultured, and examined for their ability to degrade TYL and EF in Luria-Bertani (LB) medium. Three strains (two strains of Enterococcus faecalis and one strain of Proteus mirabilis) resulted in at least 50% TYL or EF degradation within 96 h. Sterile BSF larvae inoculated with P. mirabilis recovered >60% of the degradation ability exhibited by non-sterile larvae. Finally, no TYL residuals were found in 14-d-old larvae, prepupae, or pupae of BSF immatures fed manure containing these antibiotics. While ∼65 μg/g and ∼20 μg/g of EF were found in larval contents and pupal exoskeleton, respectively., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Black soldier fly (BSF) larvae are often exposed to organic waste which harbors abundant zoonotic pathogens. We investigated the ability of BSF larvae to inhibit the zoonotic pathogens naturally found in pig manure. The zoonotic pathogens populations were detected by using selective medium during the conversion. Results showed that the viability of the zoonotic pathogens in pig manure was significantly affected. After eight days of conversion, the Coliform populations were undetected, and Staphylococcus aureus and Salmonella spp. decreased significantly on the eighth day. Antimicrobial assays of the purified recombinant defensin-like peptide 4 (DLP4) showed that this peptide exhibits inhibitory activity against S. aureus , Salmonella enterica serovar typhimurium, and Escherichia coli in vitro. Bacteria BSF-CL and BSF-F were isolated from the larvae gut, and both inhibited the growth of S. aureus and E. coli , but Salmonella spp. was sensitive to the BSF-CL strain (but not to the BSF-F strain). The results from our experiments indicate that BSF larvae are capable of functionally inhibiting potential zoonotic pathogens in pig manure through a variety of mechanisms including antimicrobial peptides expression and the gut associate microorganisms. This study provides a theoretical basis for further study on the combined mechanism of BSF larvae immunity and its gut microbes against the zoonotic pathogens in pig manure.

Flesh flies are received greater attention due to their use as evidence in forensic investigations of decomposing human remains. Investigators will often use age of immature insects associated with such remains as the minimal postmortem interval (PMI min ) given certain assumptions. Boettcherisca peregrina is a significant flesh fly species of forensic importance in several countries, whereas B. nathani is potentially a species of forensic importance. Distinguishing these two species is difficult and relies on close examination of the male terminalia. For genus Boettcherisca belonging to subfamily Sarcophaginae, identification is primarily based on the morphological structures associated with male terminalia. Using light microscopy and scanning electron microscopy, similarities and differences were determined for these two species. In this study, B. peregrina was collected from an urban area of Mueang District, while B. nathani was collected from the highland forest area of Doi Saket District, Chiang Mai province, northern Thailand. The morphological observations indicated similarities in the morphology of vesica (large, rounded anterior margin, bi-lobed, half upper-outer margins serrated), harpes (well-developed, elongated, broad basally with bifurcated apically) and lateral styli (two parallel tube-like with open ends with microserration apically) in both species. However, differences were found with the cercus and juxta. With regards to the cercus, B. peregrina has bristles or setae only along the posterior surface and the cercal tip is pointed, while B. nathani has bristles or setae on both the anterior and posterior surfaces, and the cercal tip is hook-shaped. Morphometric measurements of three criteria of the cerci (length from top to middle, internal distance at narrow part and distance between apex of cercal prong) revealed that the average length of each criteria of B. nathani cerci (n = 30) were significantly greater than B. peregrina cerci (n = 30). As for the juxta, the juxtal lateral plate was different: primarily bifurcated in B. peregrina and non-bifurcated in B. nathani. The medial part of juxta was different: V-shaped 95.8% (46/48) and, to a lesser extent, W-shaped 4.2% (2/48) in B. peregrina, only W-shaped 100% (52/52) in B. nathani. Such information is useful for entomologists to distinguish between adult males of these two species, which could be valuable when determining colonization patterns in association with decomposing human remains., (Copyright © 2021. Published by Elsevier B.V.)

One important feature of insect rearing is its apparent, and sometimes non-apparent, reliance on the bacterial ecosystem. Indeed, microbes contribute to insect nutrition, protection against natural enemies, and detoxification of dietary compounds, antibiotics, and insecticides. Further, microbes have been implicated as the source of signals and cues important to insect communication. But the incidence and general significance of these functions is only just being explored in the context of mass production of insects. Knowledge of the diversity and functional distribution of these microorganisms in mass-rearing systems is key to understanding microbial dynamics and to enhance system performance. Therefore, this brief review is a synthesis of literature surrounding insect rearing systems for the primary insects reared as food and feed (i.e. black soldier fly, Hermetia illucens (Diptera: Stratiomyidae), mealworms (Coleoptera: Tenebrionidae), and cricket (Orthoptera: Grylloidea) with a focus on recent advances pertaining to microbial contribution to reproduction, growth, and waste conversion., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acari community structure and function associated with delayed pig carrion decomposition has not been examined. In this study, 18 swine carcasses were studied in central Texas, USA, during two consecutive summers (2013 and 2014). Samples of ca. 400 g soil were collected from beneath, aside, and 5 m away from each pig carcass over 180 days. Mites from soil samples were extracted using Berlese funnels and identified to order and family levels and classified according to ecological function. In total 1565 and 1740 mites were identified from the 2013 and 2014 soil samples, respectively. Significant differences were determined for mite community structure at order and family levels temporally on carrion (e.g., day 0 × day 14) regardless of treatments and between soil regions where mites were collected (e.g., soil beneath vs. soil 5 m away from carrion). However, no significant differences were found in mite community structure at the order level between pig carrion with and without delayed Diptera colonization (i.e., treatments). Analysis at the family level determined a significant difference across treatments for both summers. Ecological function of mites did not change significantly following the delayed decomposition of pig carcasses. However, detritivores and fungivores were significant indicator groups during the pig carrion decomposition process. Furthermore, 13 phoretic mite species associated with eight forensically important beetle species were documented. Data from this study indicated that the rate of nutrient flow into the soil impacted associated arthropod communities; however, detecting such shifts depends on the taxonomic resolution being applied., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), has considerable global interest due to its outstanding capacity in bioconverting organic waste to insect biomass, which can be used for livestock, poultry, and aquaculture feed. Mass production of this insect in colonies requires the development of methods concentrating oviposition in specific collection devices, while the mass production of larvae and disposing of waste may require substrates that are more palatable and more attractive to the insects. In insects, chemoreception plays an essential role throughout their life cycle, responding to an array of chemical, biological and environmental signals to locate and select food, mates, oviposition sites and avoid predators. To interpret these signals, insects use an arsenal of molecular components, including small proteins called odorant binding proteins (OBPs). Next generation sequencing was used to identify genes involved in chemoreception during the larval and adult stage of BSF, with particular attention to OBPs. The analysis of the de novo adult and larval transcriptome led to the identification of 27 and 31 OBPs for adults and larvae, respectively. Among these OBPs, 15 were common in larval and adult transcriptomes and the tertiary structures of 8 selected OBPs were modelled. In silico docking of ligands confirms the potential interaction with VOCs of interest. Starting from the information about the growth performance of H. illucens on different organic substrates from the agri-food sector, the present work demonstrates a possible correlation between a pool of selected VOCs, emitted by those substrates that are attractive for H. illucens females when searching for oviposition sites, as well as phagostimulants for larvae. The binding affinities between OBPs and selected ligands calculated by in silico modelling may indicate a correlation among OBPs, VOCs and behavioural preferences that will be the basis for further analysis.

Avian infectious bronchitis virus (IBV), belonging to Gammacoronavirus, is an economically important respiratory virus affecting poultry industry worldwide. The virus can infect chickens at all ages, whereas young chickens (less than 15 day old) are more susceptible to it. The present study was conducted to investigate effects of dietary supplementation of black soldier fly (Hermetia illucens L.) larvae (BSFL) on immune responses in IBV infected 10-day-old chickens. BSFL were ground to powder and mixed with commercial fodder (1%, 5%, and 10 % [mass] BSFL powder) to feed 1-day-old yellow broilers for ten days and then challenged with IBV. Our results indicated that commercial fodder supplemented with 10 % BSFL [mass] reduced mortalities (20 %) and morbidities (80 %), as well as IBV viral loads in tracheas (65.8 %) and kidneys (20.4 %) from 3-day post challenge (dpc), comparing to that of IBV-infected chickens fed with non-additive commercial fodder. Furthermore, at 3-day post challenge (dpc), 10 % BSFL [mass] supplemented chickens presented more CD8 + T lymphocytes in peripheral blood and a rise in interferon-g (IFN-γ) at both mRNA and protein levels in spleens, comparing with chickens fed with commercial fodder. Furthermore, the mRNA abundance of MHC-I, Fas, LITAF, and IL-2 in the spleens of 10 % BSFL [mass] supplemented chickens increased at different time points after challenge. The present results suggest that supplemental BSFL could improve CD8 + T lymphocytes proliferation, thus benefit young chickens to defend against IBV infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The industrial production of insects for waste management or as a protein source is becoming vital to our society. Large volumes of manure are produced by concentrated animal facilities around the globe that must be managed, utilized, and disposed of properly. Flies offer a partial solution with their abilities to reduce these wastes and heavy metal pollutants. Meat and crop proteins are being supplemented by insect proteins for many feeds across the globe, yet science-based studies behind the mass-rearing of insects are still in their infancy. In the current study, the percent change in the composition of nutrients, heavy metals, and fiber, in dairy, poultry, and swine manure degraded by either black soldier fly (BSF) or house fly (HF) larvae was explored. Pre-digested and post-digested manure samples were collected from four independent studies that differed in production scale (number of larvae and feeding regimen): 1) BSF small-scale (100 larvae fed incrementally), 2) HF small-scale (100 larvae fed incrementally), 3) BSF large-scale (10,000 larvae fed a single time), and 4) HF large-scale (4,000 larvae fed a single time). Results indicate that nitrogen is a key nutrient impacted by larval digestion of manure by both species, regardless of scale. However, scale significantly impacted reductions of other nutrients, as did the type of manure in which the insects were reared. Ultimately, this study demonstrated that manure type and rearing scale impact the ability of BSF and HF larvae to reduce nutrients and heavy metals in manure, and thus insect management procedures need to be congruent with production emphases of the insects for waste management or protein products. Failure to take scale into consideration could lead to inaccurate assumptions related to industrialized efforts on this topic., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A basic tenet of forensic entomology is development data of an insect can be used to predict the time of colonization (TOC) by insect specimens collected from remains, and this prediction is related to the time of death and/or time of placement (TOP). However, few datasets have been evaluated to determine their accuracy or precision. The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae) is recognized as an insect of forensic importance. This study examined the accuracy and precision of several development datasets for the black soldier fly by estimating the TOP of five sets of human and three sets of swine remains in San Marcos and College Station, TX, respectively. Data generated from this study indicate only one of these datasets consistently (time-to-prepupae 52%; time-to-eclosion 75%) produced TOP estimations that occurred within a day of the actual TOP of the remains. It is unknown if the precolonization interval (PreCI) of this species is long, but it has been observed that the species can colonize within 6 d after death. This assumption remains untested by validation studies. Accounting for this PreCI improved accuracy for the time-to-prepupae group, but reduced accuracy in the time-to-eclosion group. The findings presented here highlight a need for detailed, forensic-based development data for the black soldier fly that can reliably and accurately be used in casework. Finally, this study outlines the need for a basic understanding of the timing of resource utilization (i.e., duration of the PreCI) for forensically relevant taxa so that reasonable corrections may be made to TOC as related to minimum postmortem interval (mPMI) estimates., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chrysomya rufifacies (Macquart), the hairy maggot blow fly, is of great importance for the field of forensic entomology due to its habit as an early colonizer of decomposing vertebrate remains and myiasis producer. Development studies on this species have been conducted in scattered regions of the world, using types of tissue from several species of animals as a rearing medium. Despite the commonality of C. rufifacies in Sri Lanka, developmental studies have never been performed in this region. As well, the effects of diet on development have not been tested. In the current study, C. rufifacies immatures were reared on skeletal muscle, liver, and heart from domestic swine, with flies from colonies maintained at 25 and 28°C. The minimum time needed to complete each stage at 25°C on liver (224.14 h) was fastest followed by skeletal muscle (249.33 h) and heart (251.64 h) respectively, whereas at 28°C, fly development was quickest on heart muscle (178.27 h) followed by liver (178.50 h) and skeletal muscle (186.17 h) respectively. A significant difference in total development time was determined for temperature, while the rearing medium was not significant. Temperature also showed a significant effect on the length and the width of the larvae, while the type of tissue statistically impacted only the width., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Detecting and locating a carrion resource is critical for the reproduction of necrophagous insects and initiating forensically important timelines. Blow flies (Diptera: Calliphoridae) primarily use olfactory cues in the form of volatile organic compounds to locate a suitable resource. Factors governing detecting and locating a resource have been studied using various behavior assays with modifications to suit the experiment design, such as the dual-choice cube olfactometer, which was examined in the current study. Systems optimization ensures biologically relevant and consistent results across replicates. In this study, two responses were measured: 1) leaving cube for either control or treatment and 2) choice between control and treatment. Phenotype (e.g., male, non-gravid, gravid) and total blow fly, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), number to respond were measured. Four parameters were assessed for their impact on response: 1) adjustment time in cube before trial, 2) trial length, 3) sugar/water presence, and 4) screening type in arms. Approximately, 70% of all phenotypes responded to liver with the 30-min adjustment period where only 50% responded with other adjustment periods. Trial length had a significant impact on response (35% increase in the 8 h trial compared to shorter durations); however, significant response to treatment was lost by increasing trial length. The presence of sugar/water decreased gravid and non-gravid response by 35% but did not impact males. Screening had no influence on overall or treatment response. Data indicate experiment design impacts fly response. Future studies should optimize parameters for their given fly population prior to initiating experiments., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Background: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents., Results: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions., Conclusions: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector.

Under normal circumstances, insects such as blow flies will oviposit and larvae will colonize a carcass as soon as possible. However, insect colonization on a carcass may be delayed due to the effects of wrapping, shallow burial, addition of lime derivatives to mitigate scavenging and odor, or extreme weather. The impacts of delayed insect colonization on carcass decomposition and its subsequent effect on soil chemistry profiles have not been examined to date. The objectives of this study were to determine soil chemistry dynamics associated with porcine carcasses experiencing delayed insect colonization for 7-day or 14-day. Soil chemistry profiles such as ammonium-N (NH 4 -N), orthophosphate-P (PO 4 -P), and dissolved organic carbon (DOC) were significantly different among treatments: insect inclusion (immediate access of blow fly colonization on porcine carcasses), 7-day insect exclusion and 14-day insect exclusion (blow fly access was delayed up to 7-day and 14-day). Furthermore, significant differences of soil chemical profiles were detected between days of decomposition and soil regions. Soil moisture, NH 4 -N, PO 4 -P, and DOC were significantly higher when insects were excluded from the porcine carcass suggesting loss of tissue from larval feeding reduced the mass of nutrients entering the soil. This study provides useful information for forensic science in cases where insect colonization is delayed for a period of time postmortem and soil chemistry in the cadaver decomposition island is considered for estimating postmortem interval., (© 2020 American Academy of Forensic Sciences.)

The attraction and colonization of vertebrate remains by carrion-associated arthropods are processes largely governed by olfaction. As remains decompose, they emit a bouquet of volatile organic compounds (VOCs), which in part originate from endogenous and exogenous microbes surrounding the carcass or from the carcass itself. The composition and concentration of VOCs are influenced by the presence and abundance of microbial species and arthropods. Blowfly species, such as Cochliomyia macellaria, play a critical role in nutrient recycling and the decomposition process of carrion. Gas chromatography-mass spectroscopy analysis was used to identify and classify volatile emissions from insect-colonized (with C. macellaria) and uncolonized rat carcasses, as well as a standard Gainesville diet, over a 10-day period. There were significant differences in composition and abundance of compounds present in each treatment, with significant effects of time, and different compound composition between treatments. Notable indicator compounds included, but were not limited to, indole, dimethyl disulfide, and dimethyl trisulfide. A high compound richness, and a low compound diversity, was detected over the 10-day period. The indicator compounds detected across all treatments were found to be of microbial origin, highlighting the importance of microbes in decomposition processes and arthropod attraction to carrion. This study also discusses the significant impact of necrophagous arthropods to the VOC profile of carrion. The results of this study provide insight into the changes in decomposition VOCs over time, with an explanation of compounds in high concentration known to be attractive to carrion-colonizing arthropods.

Age determination of insects collected from vertebrate remains is an essential step in estimating time since colonization as related to the post-mortem interval. Long-established methods for making such estimates rely on determining age related to stage of development at the time of collection in relation to conditions experienced. However, such estimates are based on the completion of a stage of development. Methods allowing for more precise estimates of age (i.e., within a stage of development) are sorely needed. This study examined the potential of volatile organic compounds emitted by blow fly, Ch. rufifacies (Macquart), immatures to determine stage of development, which could potentially be used to estimate the age. Volatile organic compounds (VOCs) from the larval and pupal stages of Ch. rufifacies were collected by headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry (GC-MS). Analyses indicated 37 compounds shift quantitatively, as well as qualitatively, as the larvae and pupae age. Furthermore, compounds, such as 2-ethyl-1-hexanol, phenol, butanoic acid, hexadecanoic acid, octadecanoic acid, 2-methyl propanamide, and 2-methyl butanoic acid, serve as indicator compounds of specific stages within Ch. rufifacies development. This information could be important to determine if these compounds can be used in the field to predict the presence of certain developmental stages, in order to determine the potential of using volatile markers to estimate time of colonization.

Forensic practitioners analyzing entomological evidence are faced with numerous challenges when presenting their findings to law practitioners, particularly in terms of terminology used to describe insect age, what this means for colonization time of remains, and the limitations to estimates made. Due to varying legal requirements in different countries, there is no standard format for the entomological case report prepared, nor any guidelines as to the sections that are required, optional or unnecessary in a case report. The authors herein propose sections that should be considered when drafting an entomological case report. The criteria under which entomological evidence is analyzed are discussed, as well as the limitations for each criterion. The concept of a global, standardized entomological case report is impossible to achieve due to national legislative differences, but the authors here propose a basic template which can be adapted and changed according to the needs of the practitioner. Furthermore, while the discussion is fairly detailed, capturing all differences between nations could not be accomplished, and those initiating casework for the first time are encouraged to engage other practicing forensic entomologists or professional associations within their own nation or region, to ensure a complete report is generated that meets lab or national requirements, prior to generating a finalized report.