Guanjie Yan

Insect-mediated bioconversion is a promising strategy for livestock manure recycling, but its effects on antibiotic resistance genes (ARGs) in residual substrates remain insufficiently understood, especially under polystyrene particle exposure. In this study, swine manure was treated with Sarcophaga peregrina larvae under four concentrations of 100-nm polystyrene particles (PS particles: 0, 0.001, 0.01 and 0.1 mg/kg), and ARG profiles in air-dried residual manure were characterized. Larval treatment reduced manure mass by approximately 40% within 4 days, and reduction rates did not differ significantly among PS particle concentrations. However, larval treatment increased RPKM-normalized total ARG abundance relative to the corresponding controls, whereas the residue mass-adjusted relative ARG index remained below 1.0 in all larval treatment groups after incorporating residue mass. ARG richness and Shannon diversity were not significantly reduced. ARO-level turnover analysis showed both newly detected and no longer detected ARG types after larval treatment. PCoA and PERMANOVA indicated treatment-associated differences in resistome structure, with PERMDISP suggesting that dispersion also contributed. At class and mechanism levels, multidrug resistance and antibiotic efflux were dominant. These results indicate that S. peregrina larvae efficiently reduce manure mass, but their effects on ARGs are better described as residual resistome restructuring rather than ARG removal.

Intensive laboratory rearing often reduces key phenotypic traits in farmed insects. We tested royal jelly (RJ) as a nutritional intervention in Sarcophaga peregrina (Robineau-Desvoidy) (Diptera: Sarcophagidae), assessing life-history traits and gut microbiota. Larvae were supplemented with RJ at 0%, 1%, 5%, or 10% (w/w, of the total bovine liver paste diet); a subset of emerging adults continued to receive the same RJ dose, creating “larval-only” and “larval + adult” dietary regimens. Although RJ did not alter mean larval weight at dispersal, it significantly accelerated larval growth and increased pupation rate. Adult lifespan was extended by larval supplementation and further prolonged when RJ was provided to both larvae and adults. Fecundity followed the same pattern: the “larval + adult” regimen exceeded the corresponding “larval-only” dose across concentrations. Dose–response relationships were non-linear, with modest gains at 1% and a plateau between 5% and 10%, indicating a mid-range optimum. 16S rRNA gene sequencing showed that RJ reduced gut α-diversity and shifted community composition; control-enriched taxa were suppressed under RJ. Functional predictions indicated that RJ-treated larvae harbored a gut microbiota with reduced metabolic potential, suggesting less microbial nutrient use and a lower immune challenge to the host. Together, results suggest that RJ is a promising tool to improve mass-rearing efficiency. Under our experimental conditions, a 5% RJ diet applied at both larval and adult stages appears to be an effective starting formulation, with the observed gains likely reflecting both direct nutritional benefits and microbiome restructuring.

Coprophagous flies can convert livestock manure into protein-rich larval biomass for animal feed, but manure-based rearing raises biosafety concerns. This study characterized the internal bacterial community dynamics across development in Aldrichina grahami and Boettcherisca peregrina reared on swine manure, aiming to identify developmental stages with a lower microbial hazard profile. Using 16S rRNA gene amplicon sequencing of pooled internal samples, we analyzed communities from third-instar larvae, dispersing-stage larvae, pupae at multiple time points, and newly emerged adults. Developmental stage strongly structured bacterial composition and altered richness in both species. Communities were dominated by Bacillota and Pseudomonadota, reflecting substrate origin, with pronounced turnover during metamorphosis and stage-specific dominance patterns, indicating developmental filtering rather than uniform microbial clearance. Crucially, dispersing larvae did not show the marked dominance signatures seen in later pupal or adult stages, supporting this stage as a pragmatic harvest window with a comparatively lower microbial-hazard indicator profile. Since downstream processing such as drying or heating will further reduce viable hazards, stage selection serves as an effective upstream control to lower the initial hazard burden entering production.

To advance sustainable swine manure management through insect-mediated bioconversion, this study implemented a comparative evaluation of the flesh fly Boettcherisca peregrina (Robineau–Desvoidy) (Diptera: Sarcophagidae) and the blow fly Aldrichina grahami (Aldrich) (Diptera: Calliphoridae). We quantified: (i) larval viability and bioconversion efficiency under manure-fed conditions, (ii) nutritional profiles and heavy metal accumulation in larval biomass, and (iii) agronomic value and residual heavy metal speciation of processed residues. B. peregrina exhibited higher adaptability, achieving a 95% survival rate and maximum biomass within 72 h, achieving a 35.6% swine manure mass reduction, slightly outperforming A. grahami (34.3% over 84 h). Both species produced nutrient-dense larvae (56.2% to 56.4% crude protein, 24.5% to 25.2% lipid) and pupae (61.3% to 63.8% protein, 18.4% to 19.1% lipid), with balanced essential amino acid profiles suitable for animal feed. However, arsenic accumulation in larvae approached EU feed safety limits, highlighting the need for substrate optimization. Processed residues showed enhanced agronomic value, with available phosphorus increasing by 24% to 29%, while most heavy metals remained below phytotoxic thresholds; however, chromium approximately doubled after bioconversion. From an application perspective, the short development time (3 to 5 d) and relatively high conversion efficiency of both species suggest economic advantages over longer-cycle insects such as black soldier fly, although successful scale-up will require careful management of substrate variability, biosecurity, and trace-metal risks. Overall, these findings identify B. peregrina and A. grahami as promising candidates for circular agriculture systems.

In warmer environments, most ectotherms exhibit a plastic reduction in body size (the temperature–size rule, TSR). However, in such environments, growth is usually accelerated and would be expected to result in maturation at a larger body size, leading to increases in fecundity, survival, and mating success, compared to maturation at a smaller size (the ‘life-history puzzle’). To explore these mechanisms, we reared Aldrichina grahami at 20 °C, 25 °C, and 30 °C, and added a nutritional challenge by using dilutions of pork liver paste to provide diets that ranged in quality from high (undiluted) to moderate (1/8), low (1/16), and poor (1/24). Larvae were randomly sampled for weighing from hatching. Growth curves were fitted to the relationships between growth rate and weight for the third instar larvae. Our results showed that body size was affected by an interaction between temperature and diet, and that following or not following the TSR can vary depending on underfeeding. Moreover, when the TSR was followed as temperature increased, there was a cross-over point that divided the two growth curves into early and later stages, which could be used to help understand the life-history puzzle in warmer temperatures, with the instantaneous growth rate being faster in the early stages of development and then slower in later stages. This study reminds us that animals have evolved to cope with multiple simultaneous environmental changes, and it has thus offered a better understanding of life-history puzzles.

The blowfly, Aldrichina grahami (Diptera: Calliphoridae), is a well-known forensically important insect. Basic data related to A. grahami have been well documented; but despite the pivotal role of gut microbes in various facets of insect biology, little is known about its gut microbiome. To investigate the gut bacterial community of A. grahami and explore its stability, diet and developmental stage were selected as the two variables. Larvae were reared on bovine liver, swine manure, and chicken manure, and high-throughput sequencing of the 1st, 2nd, and 3rd instar larvae and the newly emerged adults was performed. According to our results, the alpha diversity of the gut bacterial community did not significantly vary among different diets and developmental stages. Principal coordinate analysis revealed that the gut microbiome of A. grahami clustered together among different diets and developmental stages. The main phyla in the gut microbial community of A. grahami were Firmicutes and Proteobacteria, and the dominant genera were Vagococcus, Providencia, Lactobacillus, and Morganella. These findings characterized the gut microbiome of A. grahami and demonstrated that the gut bacterial community is fairly stable. The dominant genera Vagococcus, Providencia, Lactobacillus, and Morganella have the potential to serve as core microbiomes within the gut microbial community of A. grahami.

Breech flystrike is a painful, debilitating and potentially lethal disease caused by the larvae of the blowfly, Lucilia cuprina, and, despite many years of research, it remains a serious financial and animal-welfare issue for the Merino sheep industry in Australia. The common methods of prevention, namely insecticides, crutching and ‘mulesing’, are problematical, so alternative approaches are needed. Breeding for resistance to breech strike is a fundamentally attractive proposition, but the trait itself is difficult and expensive to quantify in large numbers of sheep in extensive production systems. Several indirect traits are correlated with susceptibility to flystrike, but a large proportion of the variation in susceptibility remains unexplained. The common thread through those indirect traits is odour, so we turned to the biology of insect olfaction and its role in fly–sheep interactions. L. cuprina uses odours to detect and locate potential hosts over long distances, to guide orientation and landing behaviour, and to select egg-laying sites. Preliminary studies demonstrated the importance of confining our work to gravid female L. cuprina, and also validated the use of flies reared in the laboratory for experimentation. Using laboratory-reared flies and a combination of gas chromatography–mass spectrometry with electroantennographic detection, we identified odouriferous compounds from sheep that the antenna of L. cuprina can detect. To determine whether the identified compounds were attractive or repulsive, we needed to use a behaviour test. In preliminary studies, we compared four behaviour bioassays (Y-tube, landing time, visiting frequency, and trap) and found the trap to be the most effective. We observed that L. cuprina was attracted by several compounds in Merino wool, including octanal, nonanal and dimethyl trisulfide. We also found that the wool levels of octanal and nonanal are heritable in Merino sheep, suggesting that these compounds might be useful as traits in selection for flystrike resistance. Another possibility is that these olfactory-active compounds might guide efforts to modify the genome of sheep, or perhaps even L. cuprina. Success in these endeavours could save as much as A$200 m per year for the Australian Merino-based industries, while also improving the image of wool in world markets.

In warmer environments, most ectotherms reach maturity at a smaller body size (the temperature-size rule, TSR). However, in such environments, growth is usually accelerated and would be expected to result in maturation at a larger body size, leading to increases in fecundity, survival, and mating success, compared to maturation at a smaller size (the ‘life-history puzzle’). To explore mechanisms, we reared Aldrichina grahami at 20 ℃, 25 ℃ and 30 ℃, and added a nutritional challenge by using dilutions of pork liver paste to provide diets that ranged in quality from high (undiluted, control) to moderate (1/8), low (1/16) and poor (1/24). Larvae were randomly sampled for weighing from hatching. Growth curves were fitted to the relationships between growth rate and weight for the third instar larvae. Our results showed that body size was affected by an interaction between temperature and diet, and that TSR and TSR exceptions (E-TSR) could be interconverted by underfeeding. Moreover, when the TSR was followed as temperature increased, there was a cross-over point divided the two growth curves into early and later stages, which could be used to help understand the life-history puzzle in warmer temperatures, with the instantaneous growth rate being higher in the early stages of development and then lower in later stages. This study reminds us that animals evolved to cope with multiple simultaneous environmental changes, and offered a better understanding for life-history puzzles.

To identify flystrike‐related volatile compounds in wool from Merino sheep, the attractiveness of wool to Lucilia cuprina Wiedmann (Diptera: Calliphoridae) was examined. First, a selection of wool samples guided by previous knowledge of sheep lines, predicted to be more susceptible or more resistant to flystrike, was tested. The attractiveness of the 10 samples selected was not associated with field susceptibility: two samples from the more resistant line were identified as most attractive and two samples from the more susceptible line were identified as least attractive, based on the behavioural assays with gravid flies. Comparison of the headspace volatiles of these samples, using solid phase microextraction and gas chromatography‐mass spectrometry‐electroantennographic detection, revealed octanal and nonanal to be present in the attractive wool samples that elicited responses from the fly antenna. Furthermore, the two compounds were not present in wool that was least attractive to L. cuprina . In laboratory bioassays, octanal and nonanal evoked antennal and behavioural responses in gravid L. cuprina , thus confirming their potential role as semiochemicals responsible for attracting L. cuprina to Merino sheep.