Trehalase (Treh) is crucial for ovarian development as it directly regulates the energy supply by hydrolyzing trehalose into glucose. Juvenile hormone (JH) is also essential for ovarian development, but how it affects Treh2 activity remains unclear. This study, which employed Helicoverpa armigera as a model, showed that HaTreh2 transcription and enzymatic activity peaks coincided with the peak of JH titers (the 2 and 3 days after emergence). Compared to the dsGFP control, knockdown of HaTreh2 transcription severely impaired ovarian development. LC-MS/MS and site mutation experiments demonstrated that JH triggered the serine 345 phosphorylation of HaTreh2 via the GPCR-cAMP-PKA pathway, thereby activating its enzymatic activity. Additionally, HaTreh2 is directly bound with trehalose transporter (HaTreT) under JH induction, thus controlling intracellular trehalose and glucose contents as well as the transcription of HaTreT. TreT controls the amount of trehalose, which serves as a substrate for Treh1, entering the cell. Treh2, on the other hand, uses extracellular trehalose as substrate, and the hydrolysis product glucose is further transported into the cell. Here, HaTreh2 regulated the substrate that HaTreh1 can act upon in the cell by directly binding with HaTreT during ovarian development when JH is induced. Therefore, JH systematically regulated trehalose metabolism during ovarian development through regulating the activity of HaTreh2. This study sheds light on the coordinated interplay between JH pathway and sugar metabolism in ovarian development.

The Jun N-terminal kinase (JNK) signalling pathway has a key role in tissue remodelling during insect metamorphosis by regulating programmed cell death. However, multiple members of the JNK pathway in Lepidoptera remain uncharacterized. In this study, two key genes of the JNK pathway, BmJun and BmFos, were cloned from the silkworm Bombyx mori, a lepidopteran model insect, and their effects on reproductive development were investigated. BmJun and BmFos encode 239 and 380 amino acids, respectively. Both proteins have typical basic leucine zipper domains and form a BmJUN-BmFOS dimer activator protein to exert transcriptional regulation. During the wandering stage of silkworm development, interference in BmJun expression had no effect on pupation, whereas B. mori vitellogenin (BmVg) expression, which is essential for egg development, was suppressed in the fat body and egg laying was significantly reduced. Additionally, numerous eggs appeared shrivelled and deformed, suggesting that they were nutritionally stunted. Inhibition of the JNK pathway caused abnormal pupal metamorphosis, an increase in shrivelled, unfertilized eggs, a decrease in fat body synthesis, and accumulation of BmVg in the ovaries of female B. mori. The results indicated that BmJUN and BmFOS can form an AP-1 dimer. Interfering with BmJun or inhibiting the phosphorylation of BmJUN leads to a reduction in the synthesis of BmVg in the fat body and its accumulation in the ovaries, thereby affecting the quality and production of the progeny eggs. These findings suggest that regulating Jun in the JNK pathway could be a potential way to inhibit female reproduction in Lepidoptera.

Neuropeptide F (NPF) and short neuropeptide F (sNPF) are important neuropeptides and mainly affect feeding behaviour of insects. However, the regulation of insect feeding behaviour by NPF and sNPF appears to differ between species, and it is not clear how NPF and sNPF regulate the food intake of the brown planthopper (Nilaparvata lugens). Therefore, the functions of NPF and sNPF in regulating food intake and affecting the growth and antioxidant levels of N. lugens fed on host rice plants were investigated by knocking down NPF and sNPF respectively and simultaneously knocking down both of them by RNA interference. The results showed that NPF and sNPF were mainly expressed in the head of N. lugens, and N. lugens increased food intake after NPF and sNPF were knocked down, which was reflected in the prolonged duration of N4a and N4b waves in the electrical penetration graph (EPG) experiment after knocking down NPF and sNPF. In addition, knocking down NPF and sNPF led to the increase of body weight and mortality of N. lugens, and also led to the increase of antioxidant level of N. lugens. So it was concluded that NPF and sNPF could regulate food intake, maintain body weight stability and oxidative balance in N. lugens. Our study clarified the molecular mechanism of NPF and sNPF regulating feeding behaviour and affect the growth and antioxidant level of N. lugens.

Microorganisms are integral to ecosystem functioning and host adaptation, yet the understanding of microbiomes in diverse beetle taxa remains limited. We conducted a comprehensive study to investigate the microbial composition of two red flat bark beetle species, Cucujus haematodes and C. cinnaberinus, and assessed the influence of host taxonomic relatedness and host tree species on their microbiomes. We sampled 67 larvae of two Cucujus taxa taken from 11 host tree species. 16S rRNA V4 fragment sequencing revealed distinct microbial communities associated with each Cucujus species, with host tree species significantly influencing microbiome composition. Alpha and beta diversity metrics indicated significant differences between microbial communities in both beetle and host tree species. Principal component analysis indicated distinct clustering based on host tree species but not for beetle species. This overlap could be attributed to the similar ecology of both Cucujus species. The detection of various bacteria, among which some have already been reported in saproxylophagous beetles, suggests that the red flat bark beetles ingest the bacteria via foraging on other wood-dwelling invertebrates. Our findings show the complex interplay between host taxonomy, microhabitat and microbial composition in Cucujus, providing insights into their ecological roles and conservation implications. This research helps to fill the gap in understanding the microbial dynamics of saproxylic beetles, sheds light on factors shaping their microbiomes and highlights the importance of considering both host species and environmental conditions when studying insect-microbe interactions in forest ecosystems.

Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N-β-alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi-coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony), showing nucleotide deletion in the G and G generations. Remarkably, the resultant HaEbony mutants consistently displayed darker pigmentation than their wild-type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony individuals (G) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G and G generations of the HaEbony group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9-targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application.

The cAMP response element binding protein (CREB)-binding protein (CBP) is a histone acetyltransferase that plays an indispensable role in regulating the acetylation of histone and non-histone proteins. Recently, it has been discovered that chemical inhibitors A485 and C646 can bind to Bombyx mori's CBP (BmCBP) and inhibit its acetyltransferase activity. Notably, the binding ability of A485 with BmCBP showed a very low Kd value of 48 nM by surface plasmon resonance (SPR) test. Further identification showed that both A485 and C646 can decrease the acetylation level of known substrate H3K27 and only 1 μM of A485 can almost completely inhibit the acetylation of H3K27, suggesting that A485 is an effective inhibitor of BmCBP's acetyltransferase activity. Moreover, it was confirmed that A485 could downregulate the expression of acetylated Bm30K-24 protein at a post-translational level through acetylation modification by BmCBP. Additionally, it was found that A485 can downregulate the stability of Bm30K-24 and improve its ubiquitination level, suggesting that the acetylation modification by BmCBP could compete with ubiquitination modification at the same lysine site on Bm30K-24, thereby affecting its protein stability. Here, we predict that A485 may be a potent CBP acetyltransferase inhibitor which could be utilized to inhibit acetyltransferase activity in insects, including silkworms.

Sex pheromones emitted by female moths play important roles in mate attraction. The molecular mechanism underlying pheromone biosynthesis activating neuropeptide (PBAN)-regulated sex pheromone biosynthesis has been well elucidated in many moth species, although this mechanism is species-dependent. Spodoptera litura, an important pest, has caused serious economic losses to agricultural production, yet the mechanism for its sex pheromone biosynthesis has not been fully identified. The present study investigates in detail mechanism underlying PBAN-regulated sex pheromone biosynthesis in S. litura. The transcriptome sequencing of S. litura pheromone glands (PGs) was analysed to identify a serial of candidate genes potentially involved in sex pheromone biosynthesis. Further investigation revealed a bimodal pattern in both sex pheromone release and mating frequency. PBAN was found to regulate sex pheromone biosynthesis via its receptor by using Ca as a secondary messenger, as demonstrated by RNA interference and the application of pharmacological inhibitors. Furthermore, PBAN/Ca signalling activated calcineurin (CaN) and acetyl-CoA carboxylase (ACC), which mediated sex pheromone biosynthesis in response to PBAN stimulation. Mostly importantly, hexokinase 2 (HK2) was confirmed to be activated by PBAN/PBANR /Ca/PKC signalling via phosphorylation at two specific sites (ser and ser sites of HK2). Overall, our findings shed light on the intricate processes involved in sex pheromone production in S. litura, in which PBAN regulates sex pheromone biosynthesis through PBAN/PBANR/Ca/CaN/ACC and PBAN/PBANR/Ca/PKC/HK2 signalling pathways. These insights significantly contribute to our comprehension of the specific mechanisms underlying sex pheromone biosynthesis in this moth species.

Sugars play multiple critical roles in insects, serving as energy sources, carbon skeletons, osmolytes and signalling molecules. The transport of sugars from source to sink via membrane proteins is essential for the uptake, distribution and utilization of sugars across various tissues. Sugar supply and distribution are crucial for insect development, flight, diapause and reproduction. Insect sugar transporters (STs) share significant structural and functional similarities with those in mammals and other higher eukaryotes. However, they exhibit unique characteristics, including differential regulation, substrate selectivity and kinetics. Here, we have discussed structural diversity, evolutionary trends, expression dynamics, mechanisms of action and functional significance of insect STs. The sequence and structural diversity of insect STs, highlighted by the analysis of conserved domains and evolutionary patterns, underpins their functional differentiation and divergence. The review emphasizes the importance of STs in insect metabolism, physiology and stress tolerance. It also discusses how variations in transporter regulation, expression, selectivity and activity contribute to functional differences. Furthermore, we have underlined the potential and necessity of studying these mechanisms and roles to gain a deeper understanding of insect glycobiology. Understanding the regulation and function of sugar transporters is vital for comprehending insect metabolism and physiological potential. This review provides valuable insights into the diverse functionalities of insect STs and their significant roles in metabolism and physiology.

Protein disulphide isomerase (PDI) possesses disulphide isomerase, oxidoreductase and molecular chaperone activities, and is involved in regulating various physiological processes. However, there are few studies on the function in insect diapause. In this study, we cloned one novel member PDI family (TMX3, thioredoxin-related transmembrane protein 3) in Arma chinensis. The AcTMX3 encodes 426 amino acids that contains a predicted N-terminal signal sequence, a thioredoxin-like domain with the CXXC active site and a potential transmembrane region, which are typical sequence features of TMX3. RT-qPCR results showed that AcTMX3 was mainly expressed in the head under non-diapause conditions, while AcTMX3 was highly expressed in the fat body (central metabolic organ) under diapause conditions. Moreover, temporal expression profile showed that compared with non-diapause conditions, diapause conditions significantly induced AcTMX3 expression, and the expression of AcTMX3 was enhanced at 15°C. Silencing AcTMX3 in A. chinensis significantly inhibited the expression of antioxidant genes (AcTrx2 and AcTrx-like), increased the content of HO and ascorbate and reduced the survival rate of A. chinensis under diapause conditions. Our results suggested that AcTMX3 played an important role in the resistance of A. chinensis to oxidative stress under diapause conditions.

RNA interference (RNAi) has emerged as an eco-friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi-mediated pest control. We argue that the knowledge-based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome-wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large-scale RNAi screenings are typically challenging.

In insects, tyrosine hydroxylase (TH) plays essential roles in cuticle tanning and cuticle pigmentation. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a leaf-eating forest pest in salicaceous trees worldwide. However, the function of PverTH in P. versicolora is still unknown. In this study, we obtained a PverTH gene from transcriptome analysis. The expression analysis of PverTH showed that the highest expression was found in epidermis of larvae. In this study, we used RNA interference (RNAi) technology to knockdown the PverTH gene. The results showed that ingestion of dsTH led to cuticle coloration became lighter in larvae, pupae and adults. Knockdown of PverTH gene inhibited larval growth, and consequently caused higher mortality. In addition, RNAi of TH disrupted the cuticle tanning, caused lower pupation rate, lower eclosion rate and higher deformity rate. This study indicates that PverTH is vital for the cuticular pigments and cuticle tanning. Moreover, this research suggested that the development of PverTH gene as a potential target gene to control P. versicolora.

Deciding where to lay an egg is critical for the survival of insects' offspring. Compared with our understanding of the chemosensory assessment of egg-laying sites, the mechanisms of texture detection are largely unknown. Here, we show that Bactrocera dorsalis, a notoriously agricultural pest laying its eggs within ripening fruits, can discriminate substrate texture during the egg-laying process. Exposure to drugs targeting transient receptor potential vanilloid (TRPV) mechanosensory channels abolished their oviposition preference for hard textures. BdorNan and BdorIav are two members of the TRPV subfamily, and their transcripts were detected in the labellum, the foreleg tarsi and the ovipositor. Then, we successfully obtained knockout strains of each gene using the CRISPR/Cas9 technique. The results showed that BdorNan is required for the discrimination of stiffness difference. BdorIav knockout had no significant effect on the ability of B. dorsalis to choose harder substrates. Our study thus reveals that BdorNan plays a substantial role in the texture assessment of egg-laying behaviour in B. dorsalis.

Silkworm, Bombyx mori, an economically significant insect, plays a crucial role in silk production. However, silkworm breeding is highly susceptible to various pathogens, particularly the Bombyx mori nucleopolyhedrovirus (BmNPV), which poses a serious threat. Recent metabonomic studies have provided insights into the metabolic changes associated with BmNPV infection. BmNPV infection has obvious temporal characteristics. However, few studies have investigated the silkworms infected in different periods. This study employed gas chromatography-mass spectrometry (GC-MS) to perform a comprehensive analysis of haemolymph metabolites in silkworms at 48, 72, 96 and 120 h post-infection (h.p.i.). Through the integration of time-course analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, the study revealed distinct four-stage metabolic characteristics in the silkworm's response to BmNPV infection. At Stage 1 (48 h.p.i.), silkworms activate antioxidant defence mechanisms, with significant enrichment in metabolic pathways involving key antioxidants such as glutathione, to mitigate oxidative stress induced by viral invasion. By Stage 2 (72 h.p.i.), pathways related to amino acid metabolism and protein synthesis become active, indicating an increase in protein synthesis. In Stage 3 (96 h.p.i.), energy metabolism and substance transport pathways are significantly upregulated to support the rapid viral replication and the enhanced locomotor behaviour of silkworm. Finally, at Stage 4 (120 h.p.i.), there is a further enhancement of pathways related to energy metabolism, nucleic acid synthesis, and substance transport, which align with peak viral assembly and release. These findings contribute to an in-depth understanding of the biochemical basis of silkworm resistance to NPV.

Nearly all insects harbour bacterial communities that can have a profound effect on their life history, including regulating and shaping host metabolism, development, immunity and fitness. The bacteriomes of several coleopterans have been described; however, very little has been reported for wireworms. These long-lived larvae of click beetles (Coleoptera: Elateridae) are major agricultural pests of a variety of crops grown in the Canadian Prairies. Consequently, the goal of this study was to characterise the bacteriomes of five of the most significant pest species within the region: Limonius californicus, Hypnoidus abbreviatus, H. bicolor, Aeolus mellillus and Dalopius spp. To do this, we collected larvae from southern Manitoba fields (pre-seeding) and carried out 16S rRNA sequencing on individual specimens. Our results indicate wireworms have diverse and taxon-rich bacterial communities, with over 400 genera identified predominately from the phyla Proteobacteria, Actinobacteriota, Bacteroidota and Firmicutes. However, each species had nine or fewer genera comprising >80% of their bacteriome. Network analyses revealed some community structuring consistent among species, which may culminate in shaping/regulating host biology. Moreover, the microbial signatures were influenced by both ontogeny (early vs. late stage larvae) and reproductive strategy (sexual vs. parthenogenetic), with a myriad of other factors likely contributing to bacterial diversity that are impossible to resolve from our study. Overall, this metagenomics study represents the first to characterise the bacteriomes of wireworms in the Canadian Prairies and the findings could assist in the development of sustainable management strategies for these important agricultural pests.

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is a major migratory pest in rice agriculture. This pest is characterised by its larvae's ability to fold rice leaves using silk, a behaviour that culminates in the formation of a silken cocoon during the pupal stage. The fibroin light chain (CmFib-L) gene is crucial for silk production, yet its specific function in C. medinalis has reminded elusive. This study presents a comprehensive analysis of the CmFib-L gene, revealing its complete open reading frame (ORF) and expression patterns. Notably, the gene is highly expressed in the fifth-instar larvae and the silk gland, which are critical stages for silk production. Our experiments demonstrate that silencing the CmFib-L gene leads to a reduction in pupal weight, an extension of the pupal stage and a disorganised silk cocoon. Furthermore, the larval behaviour of leaf folding and spinning is significantly impaired when the expression of CmFib-L is downregulated. These findings not only show the importance of fibroin light chain in silk production but also reveal a new target gene to regulate and control the behaviour and development of C. medinalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F mutants. Specifically, F gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.

Social insects, particularly honey bees, have exceptionally high genomic frequencies of genetic recombination. This phenomenon and underlying mechanisms are poorly understood. To characterise the patterns of crossovers and gene conversion in the honey bee genome, a recombination map of 187 honey bee brothers was generated by whole-genome resequencing. Recombination events were heterogeneously distributed without many true hotspots. The tract lengths between phase shifts were bimodally distributed, indicating distinct crossover and gene conversion events. While crossovers predominantly occurred in G/C-rich regions and seemed to cause G/C enrichment, the gene conversions were found predominantly in A/T-rich regions. The nucleotide composition of sequences involved in gene conversions that were associated with or distant from crossovers corresponded to the differences between crossovers and gene conversions. These combined results suggest two types of DNA double-strand break repair during honey bee meiosis: non-canonical homologous recombination, leading to gene conversion and A/T enrichment of the genome, and the canonical homologous recombination based on completed double Holliday Junctions, which can result in gene conversion or crossover and is associated with G/C bias. This G/C bias may be selected for to balance the A/T-rich base composition of eusocial hymenopteran genomes. The lack of evidence for a preference of the canonical homologous recombination for double-strand break repair suggests that the high genomic recombination rate of honey bees is mainly the consequence of a high rate of double-strand breaks, which could in turn result from the life history of honey bees and their A/T-rich genome.

The olfactory system of above-ground insects is among the best described perceptual architectures. However, remarkably little is known about how below-ground insects navigate in the dark for foraging. Here, we investigated host plant preferences, olfactory sensilla and characterise olfactory proteins in below-ground larvae of the striped flea beetle (SFB) Phyllotreta striolata Fabricius (Coleoptera: Chrysomelidae). Both the adults and larvae of this coleopteran pest cause serious damage to Brassicaceous crops above and below ground, respectively. To elucidate the role of olfactory system in host location of below-ground larvae, we initially demonstrated that SFB larvae distinctly favoured Brassicaceae over other plant families by two-choice behavioural bioassay. Subsequently, scanning electron microscopy of sensilla in SFB larval head showed a significant reduction in the number of olfactory sensilla in larvae compared with adults. However, essential olfactory sensilla such as sensilla basiconica are underscoring the indispensability of the larval olfactory system. We selected four larval-specific odorant binding proteins for functional validation from our previous transcriptome data. Functional studies revealed that PstrOBP23 exhibits robust binding affinity to 24 volatiles of Brassicaceae plants, including seven isothiocyanate compounds. This suggests a pivotal role of PstrOBP23 in the foraging behaviour of the larvae below the ground. Moreover, two ligands displaying strong binding capacity exhibit apparent attractive or repellent activity towards SFB larvae. Our findings provide a crucial insight into the olfactory system of below-ground larvae in SFB, highlighting the highly selective tuning of larvae specific OBP to host plant volatiles. These results offer potential avenues for developing effective pest control strategies against SFB.

The Homeotic complex (Hox) genes play a crucial role in determining segment identity and appendage morphology in bilaterian animals along the antero-posterior axis. Recent studies have expanded to agricultural pests such as fall armyworm (FAW), scientifically known as Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae), which significantly threatens global agricultural productivity. However, the specific role of the hox gene Sfabd-B in FAW remains unexplored. This research investigates the spatial and temporal expression patterns of Sfabd-B in various tissues at different developmental stages using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, we explored the potential function of the Sfabd-B gene located in the FAW genome using CRISPR/Cas9 technology. The larval mutant phenotypes can be classified into three subgroups as compared with wild-type individuals, that is, an excess of pedis in the posterior abdomen, deficient pedis due to segmental fusion and deviations in the posterior abdominal segments. Importantly, significant differences in mutant phenotypes between male and female individuals were also evident during the pupal and adult phases. Notably, both the decapentaplegic (dpp) and cuticular protein 12 (cp 12) genes displayed a substantial marked decrease in expression levels in the copulatory organ of male mutants and the ovipositor of female mutants compared with the wild type. These findings highlight the importance of Sfabd-B in genital tract patterning, providing a potential target for improving genetic control.

Waprin, a WAP (Whey acidic protein) domain-containing extracellular secretory protein, is widely known for its antibacterial properties. In this study, a waprin homologue (Tc_wap) expressing in a female-specific manner was identified in Tribolium castaneum, through the analysis of sex-specific transcriptomes. Developmental- and tissue-specific profiling revealed the widespread expression of Tc_wap in adult female tissues, particularly in the ovary, gut and fatbody. This female-specific expression of Tc_wap is not regulated by the classical sex-determination cascade of T. castaneum, as we fail to get any attenuation in Tc_wap transcript levels in Tcdsx and Tctra (key players of sex determination cascade of T. castaneum) knockdown females. RNA interference-mediated knockdown of Tc_wap in females led to the non-hatching of eggs laid by these females, suggesting the crucial role of Tc_wap in the embryonic development in T. castaneum. This is the first report on the identification of a sex-specific waprin homologue in an insect and its involvement in embryonic development. Future investigations on the functional conservation of insect waprins and their mechanistic role in embryonic development can be exploited for improving pest management strategies.

Bumblebees are key pollinators with gut microbiotas that support host health. After bumblebee queens undergo winter diapause, which occurs before spring colony establishment, their gut microbiotas are disturbed, but little is known about community dynamics during diapause itself. Queen gut microbiotas also help seed worker microbiotas, so it is important that they recover post-diapause to a typical community structure, a process that may be impeded by pesticide exposure. We examined how bumblebee queen gut microbiota community structure and metabolic potential shift during and after winter diapause, and whether post-diapause recovery is affected by pesticide exposure. To do so, we placed commercial Bombus impatiens queens into diapause, euthanizing them at 0, 2 and 4 months of diapause. Additionally, we allowed some queens to recover from diapause for 1 week before euthanasia, exposing half to the common herbicide glyphosate. Using whole-community, shotgun metagenomic sequencing, we found that core bee gut phylotypes dominated queen gut microbiotas before, during and after diapause, but that two phylotypes, Schmidhempelia and Snodgrassella, ceased to be detected during late diapause and recovery. Despite fluctuations in taxonomic community structure, metabolic potential remained constant through diapause and recovery. Also, glyphosate exposure did not affect post-diapause microbiota recovery. However, metagenomic assembly quality and our ability to detect microbial taxa and metabolic pathways declined alongside microbial abundance, which was substantially reduced during diapause. Our study offers new insights into how bumblebee queen gut microbiotas change taxonomically and functionally during a key life stage and provides guidance for future microbiota studies in diapausing bumblebees.