Insect Molecular Biology最新文献

Cold shock proteins are relatively conserved in evolution and are involved in regulating various life activities, including cell proliferation, nutritional stress and cold adaptation. However, information about the function and regulation of cold shock proteins in ticks during cold response remains meagre. In the present study, six cold shock protein genes were identified from the important vector tick Haemaphysalis longicornis, which were named as HlY-box1, HlY-box2, HlY-box3, HlY-box4, HlY-box5 and HlY-box6. Spatiotemporal expression dynamics revealed dynamic expressions varied significantly after low-temperature treatment, with different expression patterns observed over prolonged exposure periods. Then the function and regulation of cold shock protein genes during the cold response of H. longicornis were explored. RNA interference (RNAi) efficiently knocked down these genes, significantly increasing tick mortality under cold stress. Transcriptomic analysis following HlY-box4 knockdown identified 336 differentially expressed genes (DEGs), which were mainly annotated in the MAPK signalling pathway and metabolism pathway. Proteomic analysis identified 632 differentially expressed proteins associated with ATP-dependent chromatin remodelling, metabolic pathway, spliceosome, ribosome and nucleoplasmic transport pathways. The results highlight the critical roles of cold shock proteins (CSPs) in tick cold responses, primarily through regulating metabolic pathways, and provide a foundation for further exploration of their molecular mechanisms.

The cuticle of insects serves as a crucial organ for preserving body composition, protecting against pathogen invasion, and retaining moisture in their bodies. Cuticular proteins (CPs) are the main constituents of insect cuticles and interact with chitin to form the cuticle's structural framework and mechanical properties. In this study, we investigated the role of a cuticular protein with R&R consensus (CPR), BmorCPR67, a member of the RR-2 subfamily, during the prepupal-to-pupal transition in Bombyx mori. The BmorCPR67 gene exhibited high expression levels during the prepupal stage, with the highest expression detected in the epidermis of the day-1 pupa in B. mori. The expression of the BmorCPR67 gene was induced by 20-hydroxyecdysone (20E). Chitin-binding assays indicated that the BmorCPR67 protein selectively binds to crystalline chitin and chitosan but not to amorphous chitin. Silencing the BmorCPR67 gene disrupted the moulting process from prepupa to pupa, resulting in silkworm mortality. Furthermore, the knockdown of BmorCPR67 altered the expression profiles of key genes involved in chitin metabolism. Notably, significant thinning of the endocuticle was observed 48–96 h after siRNA injection in BmorCPR67-silenced silkworms. These findings highlight the critical role of BmorCPR67 in cuticle development during the prepupal-to-pupal transition in B. mori, contributing to our understanding of the functions of CPs in insect metamorphosis.

Spinosad is a widely used insecticide effective in controlling Aedes aegypti populations, but the molecular mechanisms underlying resistance remain poorly understood. This study explores the role of a serine protease, AeaSP (AAEL002624), in the potential detoxification ability of spinosad. Our results showed the crude protein of Ae. aegypti degraded approximately 48% of spinosad in vitro within 1 h; based on our previous research, AeaSP was believed to be potentially involved in the degradation of spinosad. Subsequently, AeaSP was recombinantly expressed in vitro, and its enzymatic activity was tested using BAEE as a substrate, with a Michaelis constant (KM) of 0.88 mmol/L. Spatiotemporal expression profiles revealed that AeaSP expression peaked in third instar larvae and thoraxes. In vitro assays showed that AeaSP degraded approximately 63% of spinosad (500 ng/mL) within 6 h. RNAi knockdown of AeaSP significantly increased larval mortality under spinosad exposure and raised spinosad residue levels in larvae by 37% under 0.15 μg/mL spinosad. Our findings suggest AeaSP may play a critical role in detoxifying spinosad in Ae. aegypti and serve as a target for improving spinosad efficacy and mosquito control strategies.

Previous studies suggest that some insects require dietary arginine because they cannot synthesize this amino acid through the urea cycle. To determine whether this finding applies to all insects and what its metabolic implications are, we analysed the conservation of 20 genes involved in arginine biosynthesis and metabolism in the genomes of 150 species from 11 taxonomic orders. Our results showed that no insect can synthesize arginine via the urea cycle, as ornithine carbamoyltransferase is absent from all genomes analysed. While we found losses in other genes encoding urea cycle enzymes, nitric oxide synthase (NOS) was conserved across orders. However, the citrulline produced by NOS cannot be converted back to arginine in several insects due to the loss of argininosuccinate synthase and argininosuccinate lyase genes. Despite the inability to synthesize arginine, all insects (except some Hemiptera) can degrade it to ornithine and urea, as the arginase (ARG) gene is conserved across the orders analysed. For some Hemiptera that have lost ARG, we investigated how these insects produce or metabolize ornithine. Our results show that the genes for converting ornithine to glutamate, proline and putrescine are conserved across orders. However, while all insects have enzymes to synthesize putrescine and spermidine, some lack the ability to produce spermine due to the absence of the spermine synthase gene. Taken together, our results show that the loss of the urea cycle has led to significant changes in the pathways by which insects metabolize and recover arginine, which is particularly important for the diversification of hemipterans.

Sperm delivers genetic information from the male to the ovum, playing vital roles in sexual reproduction. Like other Lepidoptera, Bombyx mori exhibits dimorphic spermatogenesis, generating coexisting nucleated eupyrene sperm and anucleated apyrene sperm. The mechanism of dimorphic spermatogenesis is still to be clarified. In a previous study, we demonstrated that Gametocyte-specific factor1 (Gtsf1) is essential for female sex determination and PIWI-interacting RNA (piRNA) mediated transposon silencing in B. mori. Here, we performed functional analysis of the Gtsf1 paralog BmGtsf1L using a binary transgenic CRISPR/Cas9 system. BmGtsf1L is dispensable for sex determination but critical for fertility in both males and females. We separated different types of sperm and found that BmGtsf1L is highly expressed in both types of sperm. BmGtsf1L deficiency (△BmGtsf1L) impaired the formation and migration of eupyrene sperm, whereas the development and movement of apyrene sperm were normal. Furthermore, through a sperm culture experiment, we confirmed that eupyrene spermatogenesis defects appeared before the elongation stage. Double copulations of a female with △BmGtsf1L and Sex-lethal mutant males can rescue infertility phenotypes, revealing that the apyrene sperm of BmGtsf1L mutants is functional. We also found that the depletion of BmGtsf1L impacted proper oogenesis. This study provided the first functional analysis of Gtsf1 paralogs on physiology, demonstrating the critical role of BmGtsf1L in the development of eupyrene sperm and the ovary.

The Hox gene Sex combs reduced (Scr) is recognized as a key factor in the development of the head and thorax in insects. However, its function in the growth, development and morphogenesis of Gryllus bimaculatus remains poorly understood. This study aimed to explore the function of the Scr gene in G. bimaculatus by using CRISPR/Cas9 technology to generate an Scr gene knock-out strain. Intercrossing the G₀ generation knock-out individuals with wild-type individuals yielded the G₁ generation to screen the mutant strain. It was found that the knock-out of the Scr gene had a severe impact on the growth and development of G. bimaculatus, resulting in high mortality and making it difficult to obtain Scr^−/− mutants. Therefore, heterozygous individuals (Scr^+/−) with 1 bp deleted were obtained for investigation. The results showed that the Scr deletion led to ectopic segment formation in the G₀ generation. In the G₂ generation, it was observed that stable Scr^−/− strains displayed abnormal embryonic development, characterized by enlarged, blackened and lethal eggs during embryogenesis. During the post-embryonic stage, Scr^−/− mutants exhibited abnormalities in body segmentation, particularly in the head-thorax region, resulting in a dorsal ridge structure. Furthermore, some Scr^+/− individuals exhibited a dorsal ridge during the nymphal stage. Notably, this characteristic did not persist into the adult stage. Our findings highlight the distinct but crucial roles of the Scr gene in both embryonic and post-embryonic growth and development of G. bimaculatus.

RNA-based bioinsecticides that comprise a dsRNA active ingredient and function by RNA interference (RNAi) are being commercialised as insecticidal traits in transgenic crops and as sprayable biopesticides. These RNAi insecticidal technologies are valuable alternatives to conventional chemical insecticides due to their efficacy, high degree of specificity and favourable human and environmental safety profiles. As with all pesticides, appropriate insect resistance management (IRM) programmes are required to mitigate the selection for resistance in target insect populations and extend product durability in the field. IRM programmes for RNAi products follow the same guidelines that currently exist for insecticidal traits or conventional insecticidal sprays. These guidelines reflect the distinct exposure scenarios for traits versus sprays, that is, continuous exposure when dsRNA is expressed in the crop compared to intermittent exposure when sprayed on foliage. As such, IRM plans for dsRNA traits depend on pyramiding (stacking) non-cross-resistant traits along with a refuge of non-transgenic plants. On the other hand, IRM plans for dsRNA sprays rely on the timing of the application so that only a single generation of the pest is exposed, followed by the use of an insecticide from a different IRAC mode of action group.

Spodoptera frugiperda (fall armyworm) poses a substantial risk to crops worldwide, resulting in considerable economic damage. The gut microbiota of insects plays crucial roles in digestion, nutrition, immunity, growth and, sometimes, the degradation of insecticides. The current study examines the effect of synthetic insecticides on the gut microbiome of third instar S. frugiperda larvae using both culture-dependent techniques and 16S rRNA gene sequencing for bacterial community profiling and diversity analysis. In untreated larvae, the sequencing approach revealed a diverse microbiome dominated by the phyla Firmicutes, Proteobacteria and Bacteroidota, with key genera including Bacteroides, Faecalibacterium and Pelomonas. In parallel, 323 bacterial strains were isolated and assigned to the orders Bacillales, Burkholderiales, Enterobacterales, Flavobacteriales, Lactobacillales, Micrococcales, Neisseriaies, Pseudomonadales, Sphingobacteriales and Xanthomonadales. The prevailing culturable species included Serratia marcescens, Klebsiella variicola and Enterobacter quasiroggenkampii. Treatment with sublethal concentrations of three insecticides (broflanilide, spinosad and indoxacarb) caused significant changes in gut microbiome diversity and composition. Treated larvae showed a shift towards increased Proteobacteria abundance and decreased Firmicutes. Specifically, Acinetobacter and Rhodococcus were dominant in treated samples. Functional predictions highlighted significant metabolic versatility involving nutrient processing, immune response, detoxification, xenobiotic metabolism, and stress response, suggesting microbial adaptation to insecticide exposure. Network correlation analysis highlighted disrupted microbial interactions and altered community structures under insecticide treatment. These findings enhance our understanding of how insecticides impact the gut microbiota in S. frugiperda and may inform future strategies for managing pest resistance through microbiome-based approaches.

Insects are known to synthesise and secrete hundreds of unique defensive chemicals, including caustic acids, pungent phenolics and citrusy terpenes. Despite efforts to characterise the defensive chemistry of ground beetles (Coleoptera: Carabidae), our knowledge of semiochemical evolution within the family and how these compounds are biosynthesised remains limited. Few studies have demonstrated the likely biosynthetic precursors of select compounds in certain taxa and only one has demonstrated which genes may be involved in the biosynthesis of formic acid. Here, we characterise the defensive chemistry and generate defensive gland transcriptomes for ground beetle species representing two defensive chemical classes: the formic acid producer Platynus angustatus and the methacrylic acid producer Pterostichus moestus. Through comparative transcriptome analyses, we demonstrate that co-option of distinct primary metabolic pathways may be involved in formic acid and methacrylic acid biosynthesis in the defensive glands of these taxa. These results expand our knowledge of ground beetle defensive chemistry and provide additional evidence that co-option of existing primary metabolic pathways plays a major role in the evolution of ground beetle chemical defence.

Autophagy is a cellular mechanism that enhances cell survival in response to various stressors, including nutrient deprivation; however, it also plays a pivotal role in the regulation of programmed cell death. This study examined the effects of autophagy-related genes Atg3, Atg5 and Atg12 on apoptosis and autophagy during the degeneration of the posterior silk gland in Bombyx mori, employing RNA interference techniques. Apoptosis-specific markers and autophagic processes were evaluated in both control and treatment groups. The knockdown of all three genes resulted in a significant reduction in autophagy, modifications in the apoptosis process, aberrant expression of p53 and impaired lysosomal function. It was determined that Atg3 is involved in the regulation of intracellular mitochondrial homeostasis. Following the silencing of Atg5, evidence was obtained indicating the gene's role in regulating lysosomal pH. Notably, the loss of Atg3 and Atg5 was associated with an increase in apoptotic markers, whereas the silencing of Atg12 inhibited apoptosis. Elevated levels of the p53 transcription factor following gene silencing suggested a potential interaction between these genes and p53. Our findings further underscore the importance of autophagy-mediated cell death, involving Atg3, Atg5 and Atg12, in the proper progression of degeneration in the posterior silk gland. A comprehensive understanding of the molecular mechanisms that mediate the interaction between apoptosis and autophagy is essential for elucidating their roles in both physiological and pathological contexts.