Precise regulation of Notch signalling is essential for proper eye development in Drosophila. Although the core components of Notch signalling are well characterised, the mechanisms modulating ligand activity remain less understood. Here, we identified microRNA miR-133 as a positive regulator of Notch signalling in a Drosophila eye model. In a Notch-sensitised background, miR-133 overexpression rescued the small-eye phenotype induced by fringe (fng), while miR-133 knockdown exacerbated this defect. miR-133 directly targets Synaptobrevin (Syb), a vesicle-associated SNARE protein, through a conserved site in its 3'UTR. Loss of Syb phenocopied miR-133 overexpression, whereas Syb overexpression enhanced Notch-related defects. miR-133 and Syb exhibited complementary expression patterns, and both miR-133 overexpression and Syb knockdown induced non-autonomous activation of Notch targets and accumulation of Delta and Serrate ligands at the plasma membrane. Furthermore, we show that the E3 ligase Godzilla (Gzl) and Rab11-dependent recycling are essential for Notch activation. Together, our findings revealed a miR-133/Syb/gzl/Rab11 regulatory axis that enhances Notch signalling by modulating ligand trafficking and distribution during development.

Dastarcus helophoroides is an important ectoparasitoid used in the biological control of longhorn beetles. Its first-instar larvae actively seek hosts and progress through four larval instars before pupation. Throughout these developmental stages, 20-hydroxyecdysone (20E) plays a pivotal role in initiating and regulating moulting and metamorphosis; however, the specific genes and underlying molecular mechanisms in D. helophoroides remain uncharacterized. In this study, comparative transcriptome analysis across larval development identified differentially expressed genes (DEGs) associated with cuticle remodelling, hormone metabolism and stage-specific metabolic and cellular reprogramming. RT-qPCR validation revealed stage-specific expression patterns of 20E biosynthesis and signalling pathway genes, with EcR, Br-C, E93 and HR39 peaking during the larval-prepupal transition, whereas CYP18A1, E74A, E75B, HR3 and FTZ-F1 were highly expressed in early instars. RNAi targeting the eight key genes (CYP315A1, CYP314A1, EcR, USP, Br-C, E93, HR39 and FTZ-F1) severely disrupted metamorphosis and reduced adult eclosion by 47.83-100%. The developmental arrests were accompanied by stage-specific morphological abnormalities, including abnormal pigmentation, wing deformities and malformed prepupae and pupae. This study identifies key ecdysteroid biosynthesis and signalling genes as critical regulators of D. helophoroides development, providing insights into growth regulation and artificial rearing optimization in this parasitoid biocontrol agent.

Peptidases play a key role in the metabolism of proteins and peptides. In this work, we studied the spectrum of midgut serine peptidases (SPs) and serine peptidase homologs (SPHs) in the IV instar larvae of the yellow mealworm Tenebrio molitor using biochemical and proteomic approaches. Seventeen SPs were isolated from the larval midgut of T. molitor and identified using MALDI-TOF MS analyses and genomic and transcriptomic data. The proteins were isolated separately from the anterior part (AM) and the posterior part of the midgut (PM) using size-exclusion chromatography, specific chromogenic and fluorogenic substrates and PAGE under native conditions. Of the 17 SPs, 3 were trypsins, 10 chymotrypsin-like, and 4 elastase-like. Three SPs were localized only in the AM, five SPs in the PM and nine SPs were identified both in AM and PM. In addition, six SPHs with 1-3 substitutions in the catalytic amino acid residues were identified. The majority of analysed proteins had a high level of mRNA expression specific to the feeding stages of development. The results obtained suggest that T. molitor larvae possess a wide range of digestive SPs and SPHs, which may provide them with high resistance to numerous dietary or insecticidal SP inhibitors, as well as indicate wide possibilities for their use for biotechnological production of T. molitor food/feed hydrolysates of this edible insect with increased digestibility, reduced allergenicity and possibly new biological activities.

Diapause is a crucial biological adaptation that enables many insects to endure unfavourable environmental conditions and maintain species survival. The silkworm, Bombyx mori, a classic model organism exhibiting embryonic diapause, provides an excellent system for investigating diapause mechanisms. In this study, we evaluated the effectiveness of electrical stimulation in terminating diapause and explored its underlying regulatory mechanisms during the termination process in silkworms. Specifically, electrical stimulation significantly improved the economic traits of silkworms compared to HCl treatment. Transcriptome sequencing was performed to analyse dynamic gene regulation during diapause termination following electrical stimulation in two silkworm strains: the Chinese lineage strain Qiufeng and the Japanese lineage strain Baiyu. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) revealed, for the first time, that the mechanism of diapause termination in silkworms is lineage-specific. Weighted gene co-expression network analysis (WGCNA) further indicated that this lineage-specific diapause release is primarily driven by the differential expression of genes involved in the carbohydrate biosynthesis pathway. These findings both validate the practical application of electrical stimulation in sericulture and reveal fundamental differences in diapause regulation between silkworm lineages, advancing our understanding of this crucial biological process.

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.

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.

Copper (Cu), a trace element with crucial roles in physiological processes, can exert detrimental effects when imbalanced. Despite growing research on Cu's impacts on biota, its effects on terrestrial organisms, particularly insects, at environmental concentrations remain poorly understood. This study aims to elucidate the mechanisms underlying Cu-induced damage in silkworms (Bombyx mori L., 1758 (Lepidoptera: Bombycidae)) and identify potential targets to mitigate such damage. Using an integrated approach of physiological, histopathological, biochemical and multi-omics analyses, we investigated the effects of Cu exposure throughout most of the silkworms' larval stage. Our findings reveal that Cu exposure significantly hampers the growth and development of silkworms, evidenced by reduced intestinal trehalose levels, compromised peritrophic membrane (PM) structure in the midgut (MG) and altered composition and diversity of the intestinal microbiota. Furthermore, Cu exposure leads to an increase in pathogenic bacteria and a decrease in probiotics and induces inflammation and apoptosis in the midgut and fatbody (FB) tissues. Biochemical and transcriptomic analyses indicate that Cu disrupts nutrient metabolism and energy homeostasis, resulting in decreased adenosine triphosphatase (ATP) levels. Chronic Cu exposure activates the PI3K/AKT/mTOR signalling pathway, triggering abnormal apoptosis and autophagy, altering detoxification processes, immune enzyme activities and gene expression. This study provides novel insights into the mechanisms of Cu toxicity in silkworms and establishes a foundation for identifying targets to reduce Cu's biotoxicity, offering valuable data for managing Cu pollution in insects.

Mealybugs are highly aggressive pests that infest various plants and cause substantial economic losses. Histone lysine methyltransferases (KMT) are evolutionarily conserved and proposed to be essential in early embryo development in animals. However, few KMTs have been reported in mealybugs. Here, we identified a novel KMT gene, PsASH2, in the cotton mealybug, Phenacoccus solenopsis Tinsley. This gene was highly expressed in the ovary of female adults. Through RNA interference (RNAi) of PsASH2 by dsRNA microinjection, we found a reduction in the number of male embryos and total embryos in the ovaries of pregnant females. Continuous downregulation of PsASH2 in mated females until their death resulted in few changes in sex ratio but significant decreases in the number of both male and female offspring. Therefore, we believe that PsASH2 plays essential roles in embryo survival for both sexes of the cotton mealybug which may provide a potential target gene for the management of cotton mealybug by disrupting embryo development.