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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 m2 of surface area to 0.93 m3 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 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.

There are many benefits to producing insects for food and feed; they require fewer resources to produce, process, and distribute. The digested and undigested waste along with insect feces (i.e., frass) from the mass production of insects can be considerable. Black soldier fly larvae (BSFL) digestion of organic residue produces frass that is high in macronutrients that are desirable for plants, potentially serving as a partial replacement for fertilizer or growing media, such as peat. Arugula and lettuce were grown in greenhouse pot studies with treatments comprised of BSFL frass (BSF), vermicompost (VC), and peat, and compared to a 100% peat control (CP). Yield, productivity, greenness, and tissue nutrient concentrations were measured. Arugula and lettuce produced the highest fresh weight and dry weight in the BSFL treatments. Primary macronutrients (N, P, K) and Mg in both crops were also highest in the BSFL treatments. Secondary macronutrients (Ca, Mg, S) and micronutrients produced more variable results (B, Fe, Mn, Cu, Zn). Lettuce produced larger yields but had much lower concentrations of nutrients compared to arugula. In small amounts, 10–20%, BSFL frass can serve as a good replacement for peat in leafy green crops. It is an especially beneficial way to recycle organic side streams in different industries and reduce waste production overall.

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.

ABSTRACT Black soldier fly (BSF), Hermetia illucens (L.) (Diptera: Stratiomyidae), is a promising bio-agent to transform organic wastes into valuable insect biomass, and the intestinal microbes play an essential role in this process. Here, we report that Citrobacter amalonaticus, a Gram-negative gut commensal bacterium of BSF larvae, can colonize the whole intestines of the larva and significantly promote the larval growth and development. The bacterial association with the larva had a marked impact on the larval transcriptome and modulated the expression of genes involved in dozens of biological pathways including host macro-nutrient metabolism and immune response. We investigated the underlying mechanisms driving the interaction and found that C. amalonaticus association can directly promote the expression of two gene families related to intestinal protein metabolism: Hitryp serine protease trypsin family and Himtp metallopeptidase family. To determine the gene function, we developed a symbiont-mediated double-strand RNA interference approach and successfully achieved gene knockdown in larval midgut in situ. Knockdown of the two gene families and protease inhibition in larval intestines attenuated the promoting effects on larval growth significantly. The results indicate that the gut symbiont promotes BSF larval growth partly via modulating the expression of the genes involved in intestinal protein metabolism. Taken together, this study establishes a novel genetic tool to study the insect functional genomics and the host-symbiont interaction and sheds light on the important roles of intestinal protein metabolism on the interaction. 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.

Abstract 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.

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.

IntroductionIdentifying 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.MethodsBehavioral 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.ResultsUtilizing 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%.DiscussionOne 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.

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.

Abstract Human altered landscapes have caused declines in the diversity of wildlife where behaviorally plastic species (i.e., mesocarnivores and invasive species) tend to monopolize these areas and consume predictable and readily accessible food resources, such as human food waste and carrion. Increased consumption of carrion by vertebrates and invasive invertebrate species can alter population dynamics of native necrophagous insects relying on these resources. We tested the hypothesis that vertebrate scavengers and invasive species reduce blow fly (1) ability to use carrion and (2) reproduction in human‐impacted environments in central Texas, USA, with season, habitat (field and wooded landscapes), and carrion type (species of carrion and coat color) acting synergistically. Vertebrate scavengers in this habitat, of which 75% of the documented species were mesocarnivores and obligate scavengers, consumed 100% of carrion during the winter and 62% during summer despite having low species richness (2–5 species). Of the remaining carcasses available for arthropod activity during summer, the invasive red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae), monopolized 34%, and blow flies (e.g., Lucilia eximia and Chrysomya rufifacies [Diptera: Calliphoridae]) were only able to colonize 25%. Approximately 90% of carrion that was utilized by blow flies was co‐colonized by fire ants, and subsequent production of adult blow flies experienced up to a ninefold reduction in production compared with carcasses that were not scavenged by vertebrates or fire ants. Our results demonstrate oviposition resources used by blow flies in environments altered by human activity are reduced significantly by vertebrate scavengers and an invasive ant species. Future research should determine whether competitive interactions between vertebrate and invasive ant competitors for access to carrion resources have population‐level impacts to blow flies in human‐mediated ecosystems, or whether blow flies are able to shift to other resources to maintain sustainable populations and continue providing ecosystem services, such as pollination.

Abstract 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.

Abstract Background The black soldier fly (Diptera: Stratiomyidae, Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes (mitochondrial COI, nuclear ITS2 & 28S rDNA) and in order to gain insights into the phylogeography of the species. Results Our study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions (56 distinct haplotypes in total), but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian (N = 1), Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes. Conclusions Based on the results of this paper, we urge the black soldier fly community to apply molecular characterization (genotyping) of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species.

Reliability of forensic entomology analyses to produce relevant information to a given case requires an understanding of the underlying arthropod population(s) of interest and the factors contributing to variability. Common traits for analyses are affected by a variety of genetic and environmental factors. One trait of interest in forensic investigations has been species-specific temperature-dependent growth rates. Recent work indicates sexual dimorphism may be important in the analysis of such traits and related genetic markers of age. However, studying sexual dimorphic patterns of gene expression throughout immature development in wild-type insects can be difficult due to a lack of genetic tools, and the limits of most sex-determination mechanisms. Chrysomya rufifacies, however, is a particularly tractable system to address these issues as it has a monogenic sex determination system, meaning females have only a single-sex of offspring throughout their life. Using modified breeding procedures (to ensure single-female egg clutches) and transcriptomics, we investigated sexual dimorphism in development rate and gene expression. Females develop slower than males (9 h difference from egg to eclosion respectively) even at 30°C, with an average egg-to-eclosion time of 225 h for males and 234 h for females. Given that many key genes rely on sex-specific splicing for the development and maintenance of sexually dimorphic traits, we used a transcriptomic approach to identify different expression of gene splice variants. We find that 98.4% of assembled nodes exhibited sex-specific, stage-specific, to sex-by-stage specific patterns of expression. However, the greatest signal in the expression data is differentiation by developmental stage, indicating that sexual dimorphism in gene expression during development may not be investigatively important and that markers of age may be relatively independent of sex. Subtle differences in these gene expression patterns can be detected as early as 4 h post-oviposition, and 12 of these nodes demonstrate homology with key Drosophila sex determination genes, providing clues regarding the distinct sex determination mechanism of C. rufifacies. Finally, we validated the transcriptome analyses through qPCR and have identified five genes that are developmentally informative within and between sexes.

Abstract Effects of intraguild predation (IGP) on omnivores and detritivores are relatively understudied when compared to work on predator guilds. Functional genetic work in IGP is even more limited, but its application can help answer a range of questions related to ultimate and proximate causes of this behavior. Here, we integrate behavioral assays and transcriptomic analysis of facultative predation in a blow fly (Diptera: Calliphoridae) to evaluate the prevalence, effect, and correlated gene expression of facultative predation by the invasive species Chrysomya rufifacies. Field work observing donated human cadavers indicated facultative predation by C. rufifacies on the native blow fly Cochliomyia macellaria was rare under undisturbed conditions, owing in part to spatial segregation between species. Laboratory assays under conditions of starvation showed predation had a direct fitness benefit (i.e., survival) to the predator. As a genome is not available for C. rufifacies, a de novo transcriptome was developed and annotated using sequence similarity to Drosophila melanogaster. Under a variety of assembly parameters, several genes were identified as being differentially expressed between predators and nonpredators of this species, including genes involved in cell‐to‐cell signaling, osmotic regulation, starvation responses, and dopamine regulation. Results of this work were integrated to develop a model of the processes and genetic regulation controlling facultative predation.

Abstract Background Black soldier fly (BSF, Hermetia illucens L.) can efficiently degrade organic wastes and transform into a high fat containing insect biomass that could be used as feedstock for biodiesel production. Meanwhile, the molecular regulatory basis of fat accumulation by BSF is still unclear; it is necessary to identify vital genes and regulators that are involved in fat accumulation. Results This study analyzed the dynamic state of fat content and fatty-acid composition of BSF larvae in eight different stages. The late prepupa stage exhibited the highest crude fat, with lauric acid being the main component. Therefore, to provide insight into this unexplained phenomenon, the molecular regulation of rapid fat accumulation by BSF larvae was investigated. The twelve developmental stages of BSF were selected for transcriptome analysis, including the eight stages used for investigation of fat content and fatty-acid composition. By Illumina sequencing, 218,295,450,000 nt were generated. Through assembly by Trinity, 70,475 unigenes were obtained with an average length of 1064 nt and an N50 of 1749 nt. The differentially expressed unigenes were identified by DESeq, with 9159 of them being up-regulated and 10,101 of them were down-regulated. The several putative genes that are involved in the formation of pyruvate, acetyl-CoA biosynthesis, acetyl-CoA transcription, fatty-acid biosynthesis, and triacylglycerol biosynthesis were identified. The four vital metabolic genes that are associated with fat accumulation were validated by quantitative real-time PCR (qRT-PCR). The molecular mechanism of fat accumulation in BSF was clarified in this investigation through the construction of a detailed fat accumulation model from our results. Conclusion The study provides an unprecedented level of insight from transcriptome sequencing to reveal the crude fat accumulation mechanism in developing BSF. The finding holds considerable promise for insectival biodiesel production, and the fat content and fatty-acid composition can be altered by genetic engineering approaches in the future for the insect production industry.

Unlike for vertebrates, the impact of starvation on the gut microbiome of invertebrates is poorly studied. Deciphering shifts in metabolically active associated bacterial communities in vertebrates has led to determining the role of the associated microbiome in the sensation of hunger and discoveries of associated regulatory mechanisms. From an invertebrate perspective, such as the black soldier fly, such information could lead to enhanced processes for optimized biomass production and waste conversion. Bacteria associated with food substrates of black soldier fly are known to impact corresponding larval life-history traits (e.g., larval development); however, whether black soldier fly larval host state (i.e., starved) impacts the gut microbiome is not known. In this study, we measured microbial community structural and functional shifts due to black soldier fly larvae starvation. Data generated demonstrate such a physiological state (i.e., starvation) does in fact impact both aspects of the microbiome. At the phylum level, community diversity decreased significantly during black soldier fly larval starvation (p = 0.0025). Genus level DESeq2 analysis identified five genera with significantly different relative abundance (q < 0.05) across the 24 and 48 H post initiation of starvation: Actinomyces, Microbacterium, Enterococcus, Sphingobacterium, and Leucobacter. Finally, we inferred potential gene function and significantly predicted functional KEGG Orthology (KO) abundance. We demonstrated the metabolically active microbial community structure and function could be influenced by host-feeding status. Such perturbations, even when short in duration (e.g., 24 H) could stunt larval growth and waste conversion due to lacking a full complement of bacteria and associated functions.

Historically, research examining the use of microbes as a means to optimize black soldier fly (BSF) growth has explored few taxa. Furthermore, previous research has been done at the benchtop scale, and extrapolating these numbers to industrial scale is questionable. The objectives of this study were to explore the impact of microbes as supplements in larval diets on growth and production of the BSF. Three experiments were conducted to measure the impact of the following on BSF life-history traits on (1) Arthrobacter AK19 supplementation at benchtop scale, (2) Bifidobacterium breve supplementation at benchtop scale, and (3) Arthrobacter AK19 and Rhodococcus rhodochrous 21198 as separate supplements at an industrial scale. Maximum weight, time to maximum weight, growth rate, conversion level of diet to insect biomass, and associated microbial community structure and function were assessed for treatments in comparison to a control. Supplementation with Arthrobacter AK19 at benchtop scale enhanced growth rate by double at select time points and waste conversion by approximately 25–30% with no impact on the microbial community. Predicted gene expression in microbes from Arthrobacter AK19 treatment was enriched for functions involved in protein digestion and absorption. Bifidobacterium breve, on the other hand, had the inverse effect with larvae being 50% less in final weight, experiencing 20% less conversion, and experienced suppression of microbial community diversity. For those tested at the industrial scale, Arthrobacter AK19 and R. rhodochrous 21198 did not impact larval growth differently as both resulted in approximately 22% or more greater growth than those in the control. Waste conversion with the bacteria was similar to that recorded for the control. Diets treated with the supplemental bacteria showed increased percent difference in predicted genes compared to control samples for functions involved in nutritional assimilation (e.g., protein digestion and absorption, energy metabolism, lipid metabolism). Through these studies, it was demonstrated that benchtop and industrial scale results can differ. Furthermore, select microbes can be used at an industrial scale for optimizing BSF larval production and waste conversion, while others cannot. Thus, targeted microbes for such practices should be evaluated prior to implementation.

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.

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.

Abstract Background With increased availability of de novo assembly algorithms, it is feasible to study entire transcriptomes of non-model organisms. While algorithms are available that are specifically designed for performing transcriptome assembly from high-throughput sequencing data, they are very memory-intensive, limiting their applications to small data sets with few libraries. Results We develop a transcriptome assembly algorithm that recovers alternatively spliced isoforms and expression levels while utilizing as many RNA-Seq libraries as possible that contain hundreds of gigabases of data. New techniques are developed so that computations can be performed on a computing cluster with moderate amount of physical memory. Conclusions Our strategy minimizes memory consumption while simultaneously obtaining comparable or improved accuracy over existing algorithms. It provides support for incremental updates of assemblies when new libraries become available.

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.

Sensory cues predicting resource quality are drivers of key animal behaviors such as preference or aversion. Despite the abundance of behavioral choice studies across the animal kingdom, relatively few studies have tested whether these decisions are driven by preference for one choice or aversion to another. In the burying beetle Nicrophorus vespilloides, adult pairs exhibit parental care to raise their offspring on a small carrion resource. We tested whether carrion decomposition stage affected brood quantity and quality and found that mating pairs had significantly more offspring on fresher carcasses. To determine whether this observed reproductive benefit correlates with maternal preference behavior, we conducted a series of olfactory trials testing mated female preferences for mouse carcasses of differing decomposition stages. When given the option between fresh and older carcasses, females associated significantly more with fresher, 1-day old carcasses. However, this behavior may be driven by aversion, as females that were given a choice between the 7-day old carcass and a blank control spent significantly more time in the control chamber. We characterized volatile organic compound profiles of both carcass types, highlighting unique compounds that may serve as public information (sensu lato) conveying resource quality information to gravid beetles.

Meliponiculture, the keeping of domesticated stingless bees such as Geniotrigona thoracica (Smith, 1857) (Hymenoptera: Apidae), is an increasingly popular agricultural industry in Malaysia. This study reports the soldier fly (Diptera: Stratiomyidae) species of the genus Hermetia colonizing stingless bee colonies in Malaysia. The larvae were reared in the laboratory to the adult stage and identified through molecular and morphological approaches. Hermetia illucens (Linnaeus, 1758) and Hermetia fenestrata de Meijere, 1904 (Diptera: Stratiomyidae) were identified from the sample provided. Earlier records of stratiomyids in stingless bee nests were misidentified as H. illucens. This paper represents the first identified record of H. fenestrata colonizing a “spoiled” stingless bee colony. In addition, adult and larval morphological differences between both species and the roles of both species in bee nest decomposition are discussed.

Recent interest in the mass production of black soldier fly (BSF) larvae has resulted in many studies being generated. However, a majority of the studies are benchtop, or small-scale, experiments. Results generated from such studies may not translate to large-scale/industrial production. The current study was conducted at a conventional large-scale (10,000 larvae/treatment fed seven kg) to determine the impact on selected life-history traits when BSF were fed seven kg of manure (swine, dairy, or poultry) or a control diet (Gainesville diet: 50% wheat bran, 30% alfalfa meal, and 20% corn). Results showed larvae fed dairy manure took one to two days longer to develop to prepupation, with lower survivorship (45%) compared to those fed poultry or swine manure (>70%). Furthermore, the maximum larval weight was reached on day six for those fed swine manure, while other treatments achieved the maximum weight on day seven. However, larvae fed swine manure averaged 150 mg, while those fed the other diets ranged between 175 and 200 mg. Data from this study may be valuable for the industrialization of BSF. Companies using a scale varying from previously published work, including this study, should conduct pilot studies to optimize their system prior to implementation.

As vertebrate carrion decomposes, there is a release of nutrient-rich fluids into the underlying soil, which can impact associated biological community structure and function. How these changes alter soil biogeochemical cycles is relatively unknown and may prove useful in the identification of carrion decomposition islands that have long lasting, focal ecological effects. This study investigated the spatial (0, 1, and 5 m) and temporal (3–732 days) dynamics of human cadaver decomposition on soil bacterial and arthropod community structure and microbial function. We observed strong evidence of a predictable response to cadaver decomposition that varies over space for soil bacterial and arthropod community structure, carbon (C) mineralization and microbial substrate utilization patterns. In the presence of a cadaver (i.e., 0 m samples), the relative abundance of Bacteroidetes and Firmicutes was greater, while the relative abundance of Acidobacteria, Chloroflexi, Gemmatimonadetes, and Verrucomicrobia was lower when compared to samples at 1 and 5 m. Micro-arthropods were more abundant (15 to 17-fold) in soils collected at 0 m compared to either 1 or 5 m, but overall, micro-arthropod community composition was unrelated to either bacterial community composition or function. Bacterial community structure and microbial function also exhibited temporal relationships, whereas arthropod community structure did not. Cumulative precipitation was more effective in predicting temporal variations in bacterial abundance and microbial activity than accumulated degree days. In the presence of the cadaver (i.e., 0 m samples), the relative abundance of Actinobacteria increased significantly with cumulative precipitation. Furthermore, soil bacterial communities and C mineralization were sensitive to the introduction of human cadavers as they diverged from baseline levels and did not recover completely in approximately 2 years. These data are valuable for understanding ecosystem function surrounding carrion decomposition islands and can be applicable to environmental bio-monitoring and forensic sciences.

Distribution and occurrence of blow flies of forensic importance was performed during 2007 and 2008 in Chiang Mai and Lampang Provinces, northern Thailand. Surveys were conducted in forested areas for 30 minutes using a sweep net to collected flies attracted to a bait. A total of 2,115 blow flies belonging to six genera and 14 species were collected; Chrysomya megacephala (Fabricius) (44.7%), C. pinguis (Walker) (15.1%), C. chani Kurahashi (9.3%), C. thanomthini Kurahashi & Tumrasvin (0.3%); Achoetandrus rufifacies (Macquart) (10.5%), A. villeneuvi (Patton) (2.2%); Lucilia papuensis Macquart (2.2%), L. porphyrina (Walker) (12.4%), L. sinensis Aubertin (0.7%); Hemipyrellia ligurriens(Wiedemann) (1.3%), H. pulchra(Wiedemann) (0.1%); Hypopygiopsis infumata (Bigot) (0.6%), Hy. tumrasvini Kurahashi (0.2%) and Ceylonomyia nigripes Aubertin (0.4%). Among them, C. megacephala was the predominant species collected, particularly in the summer. The species likely to prevail in highland areas are C. pinguis, C. thanomthini, Hy. tumrasvini, L. papuensis and L. porphyrina.

Structural changes and growth of animal production systems have resulted in greater volumes of manure. Current manure storage methods pose a potential environmental threat. Lessening these issues is a key concern for the animal production industry. The primary aim of this research was to evaluate black soldier fly (BSF) performance when fed poultry, swine, or dairy manure at different rates (18 or 27 g/2 d until 40% prepupation). The results indicated that larvae fed with the control diet (Gainesville diet) were the heaviest (+31−70%); however, for other life-history traits, those fed the higher feed rate of poultry manure produced comparable results to the control. Larvae fed more resource, regardless of manure type, weighed more as larvae (+3−9%), pupae (+22−48%), and adults (+18−42%), developed faster (up to 3−4 d), had a higher percentage reach the prepupal stage (+2−16%), lived longer as adults (+1 d), and converted more resource to biomass (up to 1% more) than those fed at the lower rate. Yet, no difference was detected in dry matter (DM) reduction across feed rate for a given manure type. Based on these results, all three manure types can be digested by black soldier fly larvae, thus demonstrating their potential for waste management.

Blow flies of the subfamily Luciliinae (Diptera: Calliphoridae) are one of the main forensically important subfamilies globally. In addition to being used to estimate the minimum post-mortem interval (PMImin), assuming colonization occurred after death, blow fly specimens found infesting a human corpse are used to determine if the corpse was relocated or if the individual ingested narcotics prior to death. The presence of these blow flies in a given area is strongly influenced by abiotic and biotic factors, such as temperature, elevation, and habitat. Having this information, along with geographical distributions and the characteristics of preferred habitats, is necessary to better understand the biology of this group. This study aimed to characterize the spatial distribution of Luciliinae throughout 18 sampling sites within six ecozones (disturbed mixed deciduous forest, mixed deciduous forest, mixed orchard, paddy field, lowland village, and city/town) in central Chiang Mai Province, northern Thailand over one year (May 2009⁻May 2010). The purpose of the study was to elucidate the relationship of blow fly species composition with environmental abiotic factors (e.g., temperature, relative humidity, light intensity), and to predict the distribution of the common species within this subfamily using GIS. Adult collections were performed biweekly, baited with one-day-old beef offal. A total of 2331 Luciliinae flies trapped, comprising eight species, of which the four predominant species were Hemipyrellia ligurriens (Wiedemann) (n = 1428; 61.3%), Lucilia porphyrina (Walker) (n = 381; 16.3%), Hemipyrellia pulchra (Wiedemann) (n = 293; 12.6%), and Lucilia papuensis Macquart (n = 129; 5.5%). Population density across species varied seasonally, peaking in August 2009 coinciding with the rainy season. Predicting population composition was based on a model developed with ArcGIS 9.2, which utilized environmental variables (temperature, relative humidity, and light intensity) in conjunction with abundance data. Models indicated H. ligurriens had the most widespread geographic distribution, while H. pulchra was predicted to occur largely in mixed orchards and lowland villages. Lucilia porphyrina and L. papuensis were less widespread, restricted mainly to mixed deciduous forest. This model, along with knowledge of forensic information, may be useful under certain investigations where the corpse may have been relocated.

Blow flies (Diptera: Calliphoridae) are carrion-breeding flies that are commonly used as evidence in forensic investigation. An adequate knowledge of ecological and geographical data of blow fly has a direct application in forensic science, as far as estimating time of colonization or corpse relocation. The aim of this study was to evaluate the occurrence of four species of Chrysomyinae (Chrysomya pinguis, Chrysomya chani, Chrysomya villeneuvi, and Ceylonomyia nigripes) across six land use types in central Chiang Mai, northern Thailand. Eighteen study sites were selected for sampling across three districts of Chiang Mai province (Mueang Chiang Mai, Mae Rim, and Hang Dong). Adult flies were collected every two weeks using a funnel trap baited with 1-day tainted beef offal. The predicted geographic distributions of forensically important blow fly species were modeled using the computer program ArcGIS, based on selected climatic variables (temperature, relative humidity, and light intensity) recorded at study sites. During the study period, 1298 adult flies were collected, with peak fly occurrence during summer (April–May). Seasonal fluctuation patterns varied depending on fly species. Climatic factors displayed diverse impact on associated fly populations. Identified species were restricted mainly to mixed deciduous forests (MDF) especially in the mountainous area. None of these flies were trapped in an urban area.

This study examined the impact of diet protein and carbohydrate percentages as well as moisture on the immature development, survivorship, and resulting adult longevity and egg production of the black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae). Moisture impacted development and corresponding life-history traits more than protein:carbohydrate content; larvae were unable to develop on diets at 40% moisture. Larvae fed diets at 70% moisture developed faster, grew larger, and required less food than those reared on diets at 55% moisture. Larvae reared on the balanced diet (21% protein:21% carbohydrate) at 70% moisture developed the fastest on the least amount of food and had the greatest survivorship to the prepupal stage. Adult emergence and longevity were similar across treatments, indicating immature life-history traits were impacted the most. The control (Gainesville house fly) diet was superior to the artificial diets for all parameters tested. These differences could indicate that other constituents (e.g., associated microbes) serve a role in black soldier fly development. These data are valuable for industrialization of this insect as a “green” technology for recycling organic waste, which can be highly variable, to produce protein for use as feed in the livestock, poultry, and aquaculture industries, as well as for bioenergy production.

Black soldier fly (BSF) is a generalist species able to reduce large quantities of organic substrates and is thus considered as an interesting solution for waste management. Moreover, as BSF larvae accumulate high quantities of nutrients during their growth, they are valued because of their potential to produce products such as protein meal or fat for livestock feeds. Abiotic factors can influence larva growth, and a more detailed knowledge and control of these parameters can lead to the development of mass BSF breeding for the production of innovative products for animal feeds. As little information is available on the effects of the pH of substrates and feeding systems, the aim of this study was to evaluate the impact of these two factors on the activities of BSF larvae, prepupae, and adults. An experiment was performed with two fixed factors: i) pH (4.0; 6.1; 7.5; 9.5) and ii) feeding system (batch feeding system (TFS) or daily feeding system (DFS)). The pH treatments impacted larval weight on the first, third, and fifth day, but not at the end of the trial. Larval activity increased pH values from the fourth day onward, with final values of around 8.9-9.4 in all the treatments. The weight of the prepupae ranged from between 0.094 and 0.100 g. The final weight of the larvae and pupae, sex ratio, ingested food, larval mortality, percentage of emergence, and time to reach the pupa stadium were all affected by the feeding system. DFS showed the heaviest final larval weight (0.149 g), but required a longer time (11.3 d) than TFS to reach the prepupa stadium. The findings of this research could be useful for the mass production of BSF. Evaluation of an appropriate feeding system and initial pH value of the substrate are important parameters to reduce the time and to increase the weight in the production of larvae.