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Abstract Background The gut microbiota of black soldier fly larvae (BSFL, Hermetia illucens) play a crucial role in recycling various organic waste streams. This capability is linked to the presence of a potential common core microbiota in BSFL. However, subjective thresholds for defining core taxa and the difficulty of separating genetic and environmental influences have prevented a clear consensus in the literature. We analysed the gut bacterial communities of two genetically distinct BSF lines (wild type (WT) and lab-adapted line (LD)) raised on ten different diets based on common agricultural by-products and food waste in Southeast Asia. Results High-throughput 16S rRNA gene sequencing revealed that gut bacterial communities were significantly influenced by genetics (p = 0.001), diet (plant/meat-dominated; p = 0.001), larval age (p = 0.001), and the interactions between all three (p = 0.002). This led us to investigate both common core taxa and lineage-specific core taxa. At a strict > 97% prevalence threshold, four core taxa were identified: Providencia_A_732258, an unclassified genus within the family Enterococcaceae, Morganella, and Enterococcus_H_360604. A relaxed threshold (> 80% prevalence) extended the core to include other potential common core taxa such as Klebsiella, Proteus, and Scrofimicrobium. Our data suggest that Proteus, Scrofimicrobium, Corynebacterium, Vagococcus_B, Lysinibacillus_304693 (all LD), and Paenibacillus_J_366884 (WT) are lineage-specific rather than members of a common core (> 90% prevalence in either LD or WT, with prevalence significantly different between lines (p ≤ 0.05)). Positive correlations were observed between several core genera and larval performance in LD, typical of a highly optimized lab-adapted line. Interestingly, only members of the genus Providencia appeared to play a crucial role in most aspects of larval performance in both genetic lineages. Conclusion Our study demonstrates that the gut microbiota of BSFL is influenced by genetic factors, diet composition, larval age, and their interactions. We identified a distinct lineage-specific core microbiota, emphasizing genetic background’s role. Future studies should apply a standardized high prevalence threshold of at least > 90% unless there is a valid reason for relaxation or sample exclusion. The consistent association of Providencia spp. with larval performance across both genetic lines highlights their crucial role in the BSFL gut ecosystem.

ABSTRACT Reproductive traits that mediate differential fitness associated with mate acquisition and fertilisation success are often strongly linked to the overall condition. We investigated the effects of resource quality and parental provisioning in the phenotypic expression of sexual and non‐sexual traits in a rainforest dung beetle, Onthophagus c.f. babirussa (Eschscholtz, 1822) from Singapore. F1 individuals were reared from wild‐caught beetles and paired up to produce offspring (F2), and F2 larvae from the same F1 parents were reared on two dung substrates (herbivore and omnivore) in a full‐sib design. Sexual traits displayed greater phenotypic variation in response to dung resource quality, with the precopulatory trait (horn length) responding more than the postcopulatory trait (testes weight). Notably, genotype‐by‐environment interactions between parental lines (genotype) and dung type (environment) affected male body size and horn length only, suggesting sex‐specific variance in plasticity associated with sexually selected precopulatory traits. Dung type had significant effects on all measured traits. Offspring that were provisioned higher quality resource (omnivore dung) had larger absolute and relative trait values. Parental lines only significantly affected female body size but none of the male traits, suggesting an important role of environment and resource partitioning in determining precopulatory success of male offspring. Parental provisioning of larval resource varied with resource quality and brood sequence. Parents provisioned more dung when herbivore dung was presented than when they were given omnivore dung and provisioned more dung for their earlier broods when using herbivore dung but not omnivore dung. This suggests a trade‐off between early offspring fitness and resource quality. We tested directly for genotype‐by‐environment (G × E) interactions in the expression of several morphological traits relevant to dung beetle fitness and documented that offspring with similar phenotypes may result from completely different parental resource allocation strategies. We discuss the importance of studying parental investment on trait variation and its implications on dung beetle ecology.

Juvenile environments can influence adult phenotypes in holometabolous insects. This study examines the effects of larval rearing substrates on the reproductive outcomes of Black Soldier Flies (BSFs) at a semi-industrial scale. Larvae were reared on five substrates. Larval growth, adult size, and reproductive performance were monitored, with a specific focus on egg production and hatching rates across 17 continuous egg collection days. Egg production was positively correlated with female body weight, while male body weight appeared to influence the egg production peak. Extended larval feeding stages and shorter non-feeding periods were associated with an earlier onset of egg-laying, suggesting life-history traits as predictors for reproductive timing. Diets high in carbohydrates but low in protein and fat positively influenced larval growth, egg production, and female body weight, while diets rich in both protein and carbohydrates negatively affected all measured parameters. Notably, higher fecundity did not necessarily correlate with fertility (i.e., more viable larvae for bioconversion), highlighting the need to optimize diets for both egg quantity and hatchability to maximize productivity. These findings provide valuable insights for optimizing larval nutrition and egg collection practices, enhancing reproductive efficiency and overall productivity in industrial BSF farming.

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

Summary: Aedes albopictus is a successful disease vector due to its ability to survive in a wide range of habitats. Despite its ubiquity and impact on public health, little is known about its differential gene flow capabilities across different city habitats. We obtained a comprehensive dataset of >27,000 genome-wide DNA markers across 105 wild-caught Ae. albopictus individuals from Singapore, a dengue-endemic tropical city with heterogeneous landscapes from densely populated urban areas to forests. Despite Singapore’s challenging small-scale heterogeneity, our landscape-genomic approach indicated that dense urban areas are characterized by higher Aedes gene flow rates than managed parks and forests. We documented the incidence of Wolbachia infections of Ae. albopictus involving two strains (wAlbA and wAlbB). Our results dispel the misconception that substantial dispersal of Ae. albopictus is limited to urban greenery, with wide implications for vector management and critical insights into urban planning strategies to combat dengue transmission.

Gene discovery has important implications for investigating phenotypic trait evolution, adaptation, and speciation. Male reproductive tissues, such as accessory glands (AGs), are hotspots for recruitment of novel genes that diverge rapidly even among closely related species/populations. These genes synthesize seminal fluid proteins that often affect post-copulatory sexual selection—they can mediate male-male sperm competition, ejaculate-female interactions that modify female remating and even influence reproductive incompatibilities among diverging species/populations. Although de novo transcriptomics has facilitated gene discovery in non-model organisms, reproductive gene discovery is still challenging without a reference database as they are often novel and bear no homology to known proteins. Here, we use reference-free GridION long-read transcriptomics, from Oxford Nanopore Technologies (ONT), to discover novel AG genes and characterize their expression in the widespread dung fly, Sepsis punctum. Despite stark population differences in male reproductive traits (e.g.: Body size, testes size, and sperm length) as well as female re-mating, the male AG genes and their secretions of S. punctum are still unknown. We implement a de novo ONT transcriptome pipeline incorporating quality-filtering and rigorous error-correction procedures, and we evaluate gene sequence and gene expression results against high-quality Illumina short-read data. We discover highly-expressed reproductive genes in AG transcriptomes of S. punctum consisting of 40 high-quality and high-confidence ONT genes that cross-verify against Illumina genes, among which 26 are novel and specific to S. punctum. Novel genes account for an average of 81% of total gene expression and may be functionally relevant in seminal fluid protein production. For instance, 80% of genes encoding secretory proteins account for 74% total gene expression. In addition, median sequence similarities of ONT nucleotide and protein sequences match within-Illumina sequence similarities. Read-count based expression quantification in ONT is congruent with Illumina’s Transcript per Million (TPM), both in overall pattern and within functional categories. Rapid genomic innovation followed by recruitment of de novo genes for high expression in S. punctum AG tissue, a pattern observed in other insects, could be a likely mechanism of evolution of these genes. The study also demonstrates the feasibility of adapting ONT transcriptomics for gene discovery in non-model systems.