Insect Molecular Biology最新文献

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.

Sex determination pathways regulate male and female-specific development and differentiation and offer potential targets for genetic pest management methods. Insect sex determination pathways are comprised of primary signals, relay genes and terminal genes. Primary signals of coleopteran, dipteran, hymenopteran and lepidopteran species are highly diverse and regulate the sex-specific splicing of relay genes based on the primary signal dosage, amino acid composition or the interaction with paternally inherited genes. In coleopterans, hymenopterans and some dipterans, relay genes are Transformer orthologs from the serine-arginine protein family that regulate sex-specific splicing of the terminal genes. Alternative genes regulate the splicing of the terminal genes in dipterans that lack Transformer orthologs and lepidopterans. Doublesex and Fruitless orthologs are the terminal genes. Doublesex and Fruitless orthologs are highly conserved zinc-finger proteins that regulate the expression of downstream proteins influencing physical traits and courtship behaviours in a sex-specific manner. Genetic pest management methods can use different mechanisms to exploit or disrupt female-specific regions of different sex determination genes. Female-specific regions of sex determination genes can be exploited to produce a lethal gene only in females or disrupted to impede female development or fertility. Reducing the number of fertile females in pest populations creates a male-biased sex ratio and eventually leads to the local elimination of the pest population. Knowledge on the genetic basis of sex determination is important to enable these sex determination pathways to be exploited for genetic pest management.

Starvation can induce autophagy and apoptosis in intestinal cells. To elucidate the underlying mechanisms, we investigated autophagy and apoptosis in the midgut of the model insect, silkworm (Bombyx mori), focusing on calcium homeostasis. The results indicated that the body weight of silkworms decreased, along with damage to the morphology of their digestive tracts and midguts after starvation treatment. Additionally, mitochondrial swelling, autophagy and apoptosis were observable. Further investigation revealed that starvation upregulated the transcription of Ca²⁺ release channel-associated genes (e.g., BmIP3R, BmRyR) but suppressed the expression of Ca²⁺ efflux genes (BmPMCA), resulting in Ca²⁺ overload in midgut cells and subsequent upregulation of BmCalpain transcription. In addition, starvation increased the transcription of key autophagy genes (BmATG5, BmATG7, BmATG8) and the expression of the LC3-II protein. Upon prolonged starvation, the NtATG5 protein levels increased, a process that facilitated the transition from autophagy to apoptosis. These results indicate that Ca²⁺ overload activates the calpain-mediated apoptosis pathway and promotes apoptosis of midgut cells. The present study reveals the significant role that Ca²⁺ plays in the occurrence and transformation of autophagy and apoptosis induced by starvation treatment, thus providing a new research strategy for investigating the damage caused by starvation in biological organisms.

Insect NF-κB-like factor, Relish, is activated by viral infection and induces the production of antiviral proteins. In this study, we performed a transcriptomic analysis of BmE cells expressing the active form of BmRelish (BmRelish_act) and identified BmVago-like as the most strongly-induced secreted-protein. Expression of BmVago-like was specifically triggered by Bombyx mori Nucleo Polyhedro Virus (BmNPV) infection and regulated by BmSTING-BmRelish pathway. Incubating the fresh culture of cells with supernatant medium of BmVago-like expressing cells or recombinant BmVago-like protein (rBmVago-like) significantly increased antiviral resistance. On the contrary, reducing the expression of Bmvago-like by RNA interference (RNAi) in BmE cells as well as in silkworm larvae impaired antiviral response. Furthermore, we constructed transgenic silkworm line over-expressing BmVago-like (BmVago-like^OV) and found they had markedly lower viral load and higher survival rate after BmNPV infection compared with the wild-type control. Co-immunoprecipitation assay showed Bmintegrin β1 interacts with BmVago-like and it was involved in BmVago-like mediated antiviral response. Finally, we found the expression level of signalling molecules in the JAK–STAT pathway increased in rBmVago-like-treated cells and BmVago-like^OV silkworm larvae but decreased in RNAi-treated cells. In summary, our research uncovered an inducible antiviral response in silkworm mediated by cytokine BmVago-like, which is the downstream effector of BmSTING-BmRelish pathway and functions as an antiviral cytokine.

Dermestes frischii Kugelann, 1792 is a storage pest worldwide, and is important for estimating the postmortem interval in forensic entomology. However, because of the lack of transcriptome and genome resources, population genetics and biological control studies on D. frischii have been hindered. Here, single-molecule real-time sequencing and next-generation sequencing were combined to generate the full-length transcriptome of the five developmental stages of D. frischii, namely egg, young larva, mature larva, pupa and adult. A total of 41,665 full-length non-chimeric sequences and 59,385 non-redundant transcripts were generated, of which 42,756 were annotated in public databases. Using the weighted gene co-expression network analysis, gene co-expression modules related to the five developmental stages were constructed and screened, and the genes in these modules were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression patterns of the differentially expressed genes (DEGs) related to olfaction and insect hormone biosynthesis were also explored. Transcription of most odorant binding proteins was up-regulated in the adult stage, suggesting they are important for foraging in adults. Many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage, may be mainly responsible for the tissue remodelling of metamorphosis. The results of the quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with the RNA-seq results. This is the first full-length transcriptome sequencing of dermestids, and the data obtained here are vital for understanding the stage-specific development and olfactory system of D. frischii, providing valuable resources for storage pest and forensic research.

Adipokinetic hormone (AKH), a crucial neuropeptide, participates in the important physiological processes by specially binding to its receptor to activate the AKH signalling pathway. AKH regulates energy metabolism. However, it remains unknown whether AKH affects larval development and adult reproduction by influencing energy metabolism. In the present study, the AKH was identified from Panonychus citri and contained the conserved functional domain ‘Q-[LIV]-[NT]-F-[ST]-X (2)-W’ that characterises the AKH family. The relative expression levels of PcAKH revealed different patterns of AKH expression at different developmental stages of P. citri. Feeding of double-standard RNA against PcAKH induced decreased fecundity and reduced survival, which was accompanied by the down-regulation of vitellogenin gene expression. In addition, after silencing the PcAKH, lipid metabolism and carbohydrate homeostasis were disrupted, manifested by increased body width and weight, and fasting phenomenon. Further investigation found that compared with the control, physiological changes in trehalose and triglyceride contents were accompanied by variations in the mRNA expression levels of genes related to lipid metabolism and carbohydrate metabolism. The disorder of lipid and carbohydrate metabolism may affect adult female reproduction, which may lead to insufficient vitellogenin deposition. Moreover, the silencing of PcAKH seriously affected the growth and development of larvae, which was manifested as delayed development period and difficulty in moulting. Conclusively, all these results in current study demonstrated that double-stranded RNA silencing system targeting PcAKH effectively inhibited larval development and female fecundity by disturbing lipid and carbohydrate metabolism, and PcAKH is a specific RNAi target for control of P. citri in the design and development of biopesticide in sustainable agriculture.

The GATA transcription factor gene, pannier (pnr), has been extensively studied in Drosophila, revealing its crucial role in dorsal closure, heart development and the regulation of cuticular bristle patterns in adults. However, studies on the functions of pnr in the development of coleopteran insects are still scarce. Herein, we identified the pnr gene in Henosepilachna vigintioctopunctata and discovered two splicing variants named Hvpnr-α and Hvpnr-β respectively. Temporal expression analysis revealed that Hvpnr, Hvpnr-α and Hvpnr-β were expressed at various stages including egg, larval, pupal and adult stages. To investigate the developmental role of Hvpnr in H. vigintioctopunctata, RNA interference (RNAi) assays were conducted on third-instar larvae. Injection of dsHvpnr, dsHvpnr-α and dsHvpnr-β and co-injection of dsHvpnr-α and dsHvpnr-β (dsRNAs mix) all resulted in significant downregulation of the target transcripts. In pupae developed from dsHvpnr-treated larvae, the symmetric black spots on both sides of the mesothorax, metathorax and tergites approached and connected. Pupal morphometric analysis revealed that dsHvpnr, dsHvpnr-α and dsRNAs mix injections significantly narrowed the spacing of dorsal symmetric spots, contracted spiracle distances on tergite sides, diminished pronotum width and markedly reduced inter-compound eye spacing compared to controls. In addition, injections of dsHvpnr and dsRNAs mix significantly reduced the oviposition in female adults. Silencing of Hvpnr led to the disappearance of the scutellum in adults, preventing the elytra from closing and properly attaching to the dorsal side of the abdomen. It is noteworthy that dsHvpnr-α or dsRNA mix induced scutellum formation defects in adults, while knockdown of Hvpnr-β had no impact. Furthermore, in stark contrast to previous studies on ladybird species such as Harmonia axyridis and Coccinella septempunctata, silencing Hvpnr did not affect melanin synthesis in pupae and adults in H. vigintioctopunctata. These findings demonstrate that among the splice variants of Hvpnr, Hvpnr-α plays a dominant regulatory role in the post-embryonic morphogenesis of H. vigintioctopunctata. This study also shows that Hvpnr is not involved in melanin synthesis, indicating significant functional differentiation of pnr during the evolution of ladybirds.

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 Ca²⁺ as a secondary messenger, as demonstrated by RNA interference and the application of pharmacological inhibitors. Furthermore, PBAN/Ca²⁺ 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 /Ca²⁺/PKC signalling via phosphorylation at two specific sites (ser⁴²³ and ser⁴³⁴ 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/Ca²⁺/CaN/ACC and PBAN/PBANR/Ca²⁺/PKC/HK2 signalling pathways. These insights significantly contribute to our comprehension of the specific mechanisms underlying sex pheromone biosynthesis in this moth species.

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.

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 G₀ and G₁ 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 (G₀) 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 G₀ and G₁ 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.