Insect Molecular Biology最新文献

Dual oxidase (Duox) is well-known for its role in immunity and tyrosine cross-linking activity across various biological processes from mammals to holometabolous insects. Nevertheless, its function in hemimetabolous insects remains poorly understood. In this study, we explored the physiological roles of the Duox gene in a hemimetabolous insect, the brown planthopper, one of the most devastating rice pests. A comprehensive analysis of the spatiotemporal expression pattern of the Duox gene was conducted. RNA interference (RNAi)-mediated silencing of the Duox gene led to moulting defects in nymphs, wing abnormalities and impaired feeding in adults and reduced hatchability in eggs. Additionally, Duox knockdown significantly reduced hydrogen peroxide (H₂O₂) levels in premoulting nymphs and female ovaries. These findings highlight the indispensable role of Duox in moulting, hatching, wing expansion and feeding behaviours in the brown planthopper, shedding light on the relationship between H₂O₂ production and cuticle structural stability.

In insects, trehalose is critical for growth and development, as well as environmental stress response, which is mainly transported by trehalose transporters (TRETs). Over nearly two decades, the physiological functions of TRETs in insect growth, development, reproduction and environmental stress response have been well elucidated. However, the role of TRETs in chitin synthesis remains not fully understood. Here, we identified the HcTRET1 gene from Hyphantria cunea, a major Lepidoptera pest in agriculture and forestry. The role of HcTRET1 in growth and development, especially in chitin synthesis, was discussed by dsRNA-mediated HcTRET1 knockdown. Bioassay showed that HcTRET1 knockdown did not affect larval growth, development and survival in H. cunea, but it significantly reduced the pupa formation rate. Additionally, HcTRET1 silencing increased trehalose levels in the fat body but decreased them in the hemolymph, suggesting HcTRET1 plays a key role in trehalose homeostasis. Moreover, HcTRET1 knockdown significantly downregulated the genes for chitin synthesis (HcGFAT, HcUAP and HcCHSA), resulting in a remarkable reduction of chitin content in the epidermis. Moreover, HcTRET1 knockdown significantly reduced the survival of H. cunea larvae at 42°C. Taken together, these results demonstrated that HcTRET1 played a critical role in larva–pupa transition, in vivo trehalose homeostasis, especially in epidermal chitin biosynthesis in H. cunea larvae. In parallel, its important physiological function in response to high-temperature stress has been verified as well. The findings expand our understanding of the physiological functions of TRET1 in insects, providing a new perspective for trehalose transporters to regulate chitin biosynthesis.

The western flower thrips, Frankliniella occidentalis, is the principal thrips vector of Orthotospovirus tomatomaculae (order Bunyavirales, family Tospoviridae), a devastating plant-pathogenic virus commonly referred to as tomato spotted wilt virus (TSWV). The larval gut is the gateway for virus transmission by F. occidentalis adults to plants. In a previous report, gut expression at the transcriptome level was subtle but significant in response to TSWV in L1s. Since it has been well documented that the relationship between the expression of mRNA and associated protein products in eukaryotic cells is often discordant, we performed identical, replicated experiments to identify and quantify virus-responsive larval gut proteins to expand our understanding of insect host response to TSWV. While we documented statistically significant, positive correlations between the abundance of proteins (4189 identified) and their cognate mRNAs expressed in first and second instar guts, there was virtually no alignment of individual genes identified to be differentially modulated by virus infection at the transcriptome and proteome levels. Predicted protein–protein interaction networks associated with clusters of co-expressed proteins revealed wide variation in correlation strength between protein and cognate transcript abundance, which appeared to be associated with the type of cellular processes, cellular compartments and network connectivity represented by the proteins. In total, our findings indicate distinct and dynamic regulatory mechanisms of transcript and protein abundance (expression, modifications and/or turnover) in virus-infected gut tissues. This study provides molecular candidates for future functional analysis of thrips vector competence and underscores the necessity of examining complex virus-vector interactions at a systems level.

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.