Insect Molecular Biology最新文献

Protaetia acuminata (Fabricius, 1775) (Coleoptera: Scarabaeidae) is widely distributed throughout Southeast Asia and plays a significant role in nutrient cycling by facilitating the decomposition of woody materials, a process that likely relies heavily on the contribution of symbiotic bacteria within their digestive system. However, their gut bacteria have not been thoroughly studied. By using V3-V4 amplicon sequencing, it was revealed that the midgut (MG) of Pr. acuminata larvae and fermented sawdust after rearing (FSD) share a similar microbial community, predominantly composed of Proteobacteria and Actinobacteriota, as well as functional genes associated with cellulolysis, nitrogen respiration, nitrate reduction and aerobic chemoheterotrophy. In contrast, the bacterial community in the hindgut (HG) was distinctly different, with anaerobic respiration being the dominant metabolic process. Agromyces, Altererythrobacter, Bacillus, Cellulomonas, Lysinibacillus, Pseudoxanthomonas and the family Promicromonosporaceae were the most common genera in MG, HG and FSD samples. The culture-based isolation method yielded 67 isolates from the larvae, with gram-positive bacteria predominating in HG and MG, whereas gram-negative bacteria were primarily found in the FSD. These microorganisms produce a range of lignocellulolytic enzymes including β-endoglucanase, laccase and xylanase that enable the beetles to digest their plant-based diet efficiently and also involve many biochemical pathways relating to biogeochemical cycling. Our results provide valuable insights into the gut-associated Pr. acuminata flower chafer larvae and could serve as a basis and reservoir for future studies on lignocellulolytic enzyme-producing bacteria.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in insect growth and development. However, the coordinated regulation of honeybee development by miRNAs and hormones remains poorly understood. In this study, the regulatory network of target genes for Apis mellifera miRNA-2161 (ame-miR-2161) was constructed, and its association with the survival and development of worker larvae was investigated. The results showed that ame-miR-2161 potentially targets 22 mRNAs, with particular emphasis on the target gene juvenile hormone acid methyltransferase (Jhamt), a key rate-limiting enzyme in the final step of the juvenile hormone (JH) biosynthesis. RT-qPCR analysis showed concordant expression patterns between ame-miR-2161 and Jhamt across larval developmental stages. Dual-luciferase assays confirmed that Jhamt is a direct target of ame-miR-2161. Functional studies revealed that overexpression of ame-miR-2161 upregulated the Jhamt expression, leading to a significant increase in JH titre in 4- to 6-day-old larvae, accompanied by a gradual upregulation of the JH downstream response gene Kr-h1. Conversely, inhibition of ame-miR-2161 downregulated the Jhamt expression, reducing JH titre and markedly suppressing Kr-h1 expression, indicating that ame-miR-2161 positively regulates the expression of Jhamt. Furthermore, ame-miR-2161 overexpression enhanced larval survival, whereas its inhibition decreased survival rates. Although pupation rates remained unaffected, ame-miR-2161 modulation influenced larval body weight changes. These results suggest that ame-miR-2161 regulates JH levels by targeting Jhamt, thereby modulating larval survival and development in honeybees. Our findings provide novel insights into the miRNA-mediated regulation of hormone signalling and metamorphic development in honeybees.

Juvenile hormone (JH) serves as a pivotal endocrine regulator of reproductive diapause (also known as adult diapause) in insects, though the molecular mechanisms underlying JH signalling remain poorly characterised. In this study, we investigate the roles of the histone acetyltransferase p300/CBP in regulating reproductive diapause through JH signalling in the cabbage beetle Colaphellus bowringi. RNA interference-mediated knockdown of p300/CBP in reproductive females induced diapause-like phenotypes, including arrested ovarian maturation, suppressed vitellogenesis, increased lipid accumulation and upregulation of diapause-associated genes. These effects were accompanied by downregulation of key JH biosynthetic enzymes and signalling components. Although exogenous JH application decreased lipid accumulation, it failed to rescue ovarian development. Similar results were observed in diapause females following p300/CBP knockdown: JH analog treatment reduced lipid accumulations but did not restore ovarian development. Collectively, these findings demonstrate that p300/CBP regulates photoperiodic reproductive diapause by modulating both JH biosynthesis and signal transduction pathways in C. bowringi. Our results establish p300/CBP as a critical epigenetic mediator of JH-mediated reproductive diapause and provide new insights into the molecular mechanisms underlying environmental adaptation in insects.

Heavy metals are prevalent environmental pollutants in habitats of phytophagous insects. This study investigates the tolerance of Lymantria dispar larvae to cadmium (Cd) and the associated mechanisms involving the ZIP family. Cd stress reduced larval body weight and extended the development duration without causing significant mortality. A significantly up-regulated expression of apoptosis genes Caspase-1, Caspase-3 and Caspase-7 was observed in Cd-exposed larvae. In Cd-exposed larvae, the expression of the apoptosis-inhibitory factor Bcl-2 in the mitochondrial pathway decreased, while apoptosis-inducing factors Bax and AIFM increased. Cd stress markedly elevated the expression of CHOP and Bip, key genes in the endoplasmic reticulum stress pathway. Among the ZIP family genes, LdZIP7 showed the highest up-regulation in response to Cd treatment. Silencing LdZIP7 intensified the negative impacts of Cd stress on L. dispar larvae and significantly reduced the tolerance of L. dispar larvae to Cd. The main manifestations were a further significant decrease in larval body weight, a further significant extension of developmental duration, and the further activation of the mitochondrial pathway and the endoplasmic reticulum stress pathway-triggered apoptosis in Cd-treated larvae. At the Sf9 cell level, LdZIP7 predominantly localises in the nuclear membrane and cell membrane. Overexpression of LdZIP7 mitigates Cd-induced cytotoxicity by inhibiting the Ca²⁺-MPTP opening degree-mitochondrial membrane potential-apoptosis pathway. Overall, LdZIP7 plays a pivotal role in alleviating the biotoxic effects of Cd and is a significant regulatory gene for Cd tolerance in L. dispar larvae.

Baculoviruses have been used to control different insect pests, but further research is needed to understand the mechanisms of their interactions with insect hosts. In this study, the importance of DNA methyltransferases (DNMTs) was investigated in Spodoptera frugiperda Sf9 cells following baculovirus infection. To do this, Sf9 cells were infected with Autographa californica nucleopolyhedrovirus (AcMNPV) and the transcript levels of DNMT1 and DNMT2 were quantified. The expression levels of both DNMTs were upregulated during AcMNPV infection, suggesting induction of the pathway during infection. To determine whether these genes function during virus infection, we treated Sf9 cells with the inhibitor of DNMTs, 5-azacytidine (5-AZA), and 24 h later, the cells were infected with AcMNPV. Quantitative PCR results showed that the viral DNA replication was significantly reduced in the treated cells compared with the control untreated cells. We also showed that 5-AZA modulates antiviral pathways in Sf9 cells. The expression levels of Toll and immune-deficiency (IMD) pathways genes (i.e., Relish and Dorsal) were increased at 24 h post AcMNPV infection, while the expression levels of Gloverin and Defensin antimicrobial peptides (AMPs) were downregulated. In the 5-AZA treated cells, however, the expression levels of both AMPs were elevated during AcMNPV infection. We also assessed the effect of 5-AZA on short interfering RNA and microRNA pathways and found that Dicer 2 expression level was increased in the 5-AZA treated cells, while the transcript levels of other key genes of the two pathways remained unchanged. Together, our results highlight the contribution of DNA methyltransferase activity in the immunity of Sf9 cells against AcMNPV infection through modulation of antiviral immune responses and shed more light on the molecular mechanisms underlying baculovirus-insect cell interactions.

Thermal stress significantly harms the survival, physiology and development of honeybees (Apis mellifera L.). miRNAs are emerging as key regulators of environmental stress responses. This study investigated honeybee's miRNA expression under heat stress and clarified the role of ame-miR-1-3p in thermotolerance. Global miRNA expression profiles were analysed in forager bees exposed to 45°C for 2 and 4 h using small RNA sequencing (sRNA-seq). To explore the effects of ame-miR-1-3p, young bees were fed miRNA mimics or antagonists to either overexpress or knock down its expression. The impact on target gene expression (Hsp90 and LOC413120) and the bees' heat susceptibility was then assessed. A dual luciferase reporter assay was used to confirm the direct targeting relationship between ame-miR-1-3p and LOC413120. The results showed that exposure to 45°C led to seven and five differentially expressed miRNAs (DEMs) at 2 and 4 h, respectively, all of which were suppressed. Bioinformatic analyses indicated that DEM target genes were enriched in stress-related signalling pathways including MAPK, phosphatidylinositol and Notch. ame-miR-1-3p, the most abundant DEM, was significantly down-regulated under heat exposure, as validated by qRT-PCR. Conversely, the expression of LOC413120 (encoding DnaJ homologue subfamily B member 12), a potential target of ame-miR-1-3p, was upregulated. Increasing ame-miR-1-3p expression significantly suppressed LOC413120 expression and reduced heat resistance, whereas decreasing ame-miR-1-3p expression significantly enhanced LOC413120 expression and increased heat resistance. Furthermore, dual luciferase analysis confirmed that LOC413120 is a direct target of ame-miR-1-3p. These findings reveal a novel function of miRNAs in honeybee heat resistance.

Sex determination is one of the fundamental questions in developmental and evolutionary biology. While the upstream regulatory mechanisms governing somatic sex determination in insects are highly divergent, the downstream transformer (tra) + transformer2 (tra2) → doublesex (dsx) pathway is relatively conserved. In the sweet potato whitefly, Bemisia tabaci, a global invasive pest, tra2 and dsx have been previously characterised, revealing their critical role in male genitalia formation. However, the role of tra in B. tabaci sex determination has remained unclear. Here, we characterised Bttra, the B. tabaci transformer, and identified multiple TRA/TRA-2 binding sites and several splice variants. A glutathione S-transferase (GST) pull-down assay confirmed direct interactions between BtTRA and BtTRA2 in vitro. Functional analysis through Bttra silencing demonstrated its regulatory impact on Btdsx expression and its essential role in female development. Our combined findings provide new insights into the function of tra in B. tabaci sex determination, highlighting variation in sex determination among haplodiploid insects. Moreover, this study identifies potential molecular targets for the future management of this devastating insect pest.

Many eye colour mutants have been identified in Drosophila melanogaster. Mutations in the sepia gene result in brown eyes due to a lack of PDA synthase, which is essential for production of the red drosopterin eye pigment. We previously used CRISPR/Cas9 to target the PDA synthase gene to establish sepia mutant strains for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an invasive global pest of soft skinned fruits. The fecundity and fertility of some of the sepia mutant strains were similar to wild-type. The goal of this study was to determine if the sepia gene could be used as a marker to identify transgenic D. suzukii. By using the sepia gene as a marker, we successfully developed lines expressing Streptomyces phage phiC31 integrase in the germline. For most of these lines, hemizygotes exhibited complete rescue of the sepia eye colour and relatively high levels of phiC31 RNA in ovaries. In contrast, lines with partial rescue showed low levels of sepia RNA in heads and phiC31 RNA in ovaries. These findings suggest that the sepia gene is an effective marker for D. suzukii transgenesis and its relatively small size (1.8 kb) makes it advantageous when assembling large gene constructs. The phiC31 integrase lines established in this study should serve as a valuable resource for future genetic research in D. suzukii, including the further development of strains for genetic biocontrol.

Genetic engineering is a formidable approach to studying biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to 10 times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques.

The development of adult body appendages is critical for insects. The homeobox gene Distal-less (Dll) is known for its role in proximodistal patterning, and its role in the development of the appendage has been well studied in the model organism Drosophila melanogaster. However, the biological function of Dll in other insects still remains to be further verified. In this study, we investigated the biological function of Dll in the lepidoptera model insect Bombyx mori. Similar to the expression patterns observed in D. melanogaster, BmDll was highly expressed in the tibia and tarsus of the legs. The BmDll mutants obtained through the binary transgenic CRISPR/Cas9 system appeared to thrive normally and no apparent deleterious phenotypes were observed throughout the embryonic and larval stages. Nevertheless, mutants exhibited severe defects in the development of the appendage, especially at the distal end of legs, from pupal stage to adult stage. RNA-seq analysis revealed that metabolism pathways, extracellular matrix (ECM)-receptor interaction and structural constituent of the cuticle were dysregulated in mutant silkworms. Our study provided genetic evidence that Dll plays an important role in the development of B. mori adult body appendages.