Abstract Wolbachia bacteria are widespread maternally inherited symbionts of Nematoda and diverse Arthropoda hosts. Their evolutionary success is determined by the ability to affect the biology of the host in different ways, promoting the relative fitness of females harbouring Wolbachia , as well as sporadic cases of horizontal transmission of Wolbachia between different host species. Here, we revised Wolbachia infection in the Hymenoptera with respect to the symbiont occurrence in host taxa and Wolbachia genetics. The representatives of about half of the extant families and 1000 out of 140,000 non‐ant hymenopteran species have been tested for Wolbachia infection. We concluded that Wolbachia are found in all major hymenopteran families. More than 75% of Wolbachia diversity belongs to the A supergroup, whereas other variants belong to the B supergroup and only two isolates belong to the supergroup F. One of the main results of this study is the discovery of a specific Wolbachia genetic pattern (based on multilocus sequence typing [MLST]) in Apoidea hosts. Two haplotypes, ST‐479 and ST‐wH14, along with their alleles within other sequence types (STs), form the core of symbiont diversity, comprising 81% of unique host–Wolbachia ST associations. These haplotypes have not been reported beyond the Apoidea superfamily or Hymenoptera order. The reasons and mechanisms underlying this pattern in Apoidea remain unknown. Another important result of our study concerns the use of the MLST protocol, which has been previously criticised. We analysed 51 Wolbachia genomes for the average nucleotide identity (ANI) and MLST data, and found that genome and MLST variation are highly correlated. Therefore, the MLST protocol for Wolbachia remains reliable for many research tasks.
Parasitoidism is a trophic strategy that has evolved repeatedly in insects, but it has reached its greatest diversification and mechanistic sophistication in Hymenoptera. The ecological success of parasitoid wasps is strongly linked to their capacity to regulate host physiology through a temporally coordinated and compartmentalised arsenal of maternal and embryonic factors. Maternal components, including venom, calyx fluid and polydnaviruses, are delivered during oviposition, whereas embryonic factors, such as teratocytes and larval secretions, act throughout larval development within the host. Together, this diverse toolkit of molecular effectors manipulates host immunity, endocrine signalling, metabolism, development and behaviour, thereby ensuring survival of parasitoid larvae. This review first examines parasitoidism as an evolutionary and ecological strategy, highlighting diverse strategies of host exploitation. We then examine the principal molecular tools involved in host regulation and their mechanistic roles in host-parasitoid interactions. Finally, we critically evaluate the biotechnological potential of these molecules, with an emphasis on proteins and peptides that may serve as templates for peptide engineering, recombinant technologies and bioinspired molecular development, especially for insecticides and antimicrobials.
The decline in testicular function with age has raised significant concerns. Long non‐coding RNA (lncRNA) influences a wide array of physiological processes, including spermatogenesis. Nevertheless, the precise roles and regulatory mechanisms of lncRNA in testicular aging remain elusive. This investigation delves into the function of lncRNA:CR43306 in governing spermatogenesis during testicular aging. Depletion of lncRNA:CR43306 in 40‐day‐old Drosophila testes resulted in the hindrance of spermatogenesis, particularly affecting elongated spermatids, leading to functional senescence. Additionally, differentially expressed gene (DEG) expression analysis through bulk RNA‐seq unveiled genes linked to elongated spermatids and cell adhesion. Our findings underscore the pivotal role of lncRNA:CR43306 in testicular aging by influencing cell adhesion. These discoveries illuminate the regulatory pathways of lncRNA in testicular aging and offer valuable insights for potential studies and therapeutic interventions.
Chemosensory systems play key roles in the survival and reproductive success of insects. Two large and diverse chemosensory gene families, odorant receptors (ORs) and odorant-binding proteins (OBPs), play critical roles in insect chemosensation and mediate odour-guided behaviours. In the process of insect chemosensation, odorants from the environment pass through pores in the antennal sensilla and become soluble in the sensillar lymph, either directly on contact or by binding to an OBP. Solubilized odour molecules diffuse through the lymph until they reach and activate their cognate ORs, sending electrophysiological signals to the insect brain. To better understand the evolutionary roles of OR and OBP gene families among members of the Heliothinae, we systematically characterized these two gene families in Chloridea virescens (Lepidoptera: Noctuidae). A total of 81 ORs and 49 OBPs were identified genome-wide. Based on the number and positions of conserved cysteine residues, the OBPs were classified into three types: 34 Classic OBPs, 8 Minus-C OBPs and 7 Plus-C OBPs. Phylogenetic analyses identified potential gene duplications and losses within OR and OBP gene families among members of the Heliothinae, which may be associated with differences in their volatile sensation and olfactory behaviours. Further motif and structural analyses identified a conserved region that was unique among pheromone receptors and predicted as key residues of the binding pocket, implying its critical role in pheromone detection. Future work should focus on experimentally validating its function. Overall, our findings provide important insights into how chemosensory gene evolution contributes to ecological adaptation and reproductive isolation in the Heliothine moths.
Elcysma westwoodi is a major pest of cherry trees (Rosaceae), which contain high levels of cyanogenic glucosides that release toxic hydrogen cyanide upon tissue damage. To investigate the genomic basis of host-plant cyanogenic glucoside detoxification and the evolution of chemical defence genes, we generated a PacBio HiFi-based genome assembly of E. westwoodi. The final assembly spans 460.62 Mb with high contiguity (contig N50 > 15 Mb) and near-complete recovery of conserved genes (99.6% BUSCO completeness). We predicted 19,502 protein coding genes, with annotation completeness reaching 95.2% BUSCO. Comparative analyses of three Zygaenidae genomes revealed strong conservation of chromosomal synteny and showed that E. westwoodi shares a more similar detoxification gene repertoire with Achelura yunnanensis, another Rosaceae feeder, than with the more divergent, non-Rosaceae feeding Zygaena filipendulae. Genome-wide analysis identified 272 detoxification-related genes, indicating substantial capacity for xenobiotic metabolism. Phylogenetic analysis supported long-term conservation of a vertically inherited, single-copy β cyanoalanine synthase (bCAS) gene involved in cyanide detoxification. In addition, structural and phylogenetic analyses revealed parallel retention of CYP405A and CYP332A in the two Rosaceae feeding species, a pattern most parsimoniously explained by the inheritance of an ancestral cyanogenic toolkit rather than by independent convergent recruitment, whereas UGT33A retained the canonical GT-B fold characteristic of UDP glycosyltransferases, indicating the evolutionary stability of phase II conjugation enzymes. Together, these findings indicate that adaptation in E. westwoodi proceeds through targeted gene-level evolution within an otherwise conserved genomic framework.
Like other insects, coleopterans harbour dynamic bacteriomes that shape core aspects of their life history. The bacteriomes of several wireworm species (Coleoptera: Elateridae) have been described; however, little research has been undertaken to determine the factors that influence their structure and composition. These soil-dwelling larvae of click beetles are significant agricultural pests in the Canadian Prairies, with the most ubiquitous species, Hypnoidus bicolor, delineated into two genetically distinct clades and both sexual and parthenogenetic populations. In this study, we collected 69 H. bicolor adults and larvae from nine populations spanning three Prairie provinces and subjected them to Sanger and 16S rRNA gene sequencing to determine their clade and characterize their bacteriome, respectively. Combined with long-term surveillance, we provide compelling evidence that the parthenogenetic and sexual populations are associated with different clades. Development, sampling location and host genetics all contributed to the plasticity of H. bicolor bacteriomes. These differences are largely attributed to gut bacterial community composition of larvae, whereas, in adults, they appear driven by overall community structure as well as differences in the presence/absence of taxa and within-clade/population variance. Several notable genera emerged from our study, including Alphaproteobacteria and Rickettsiella endosymbionts that predominated in the parthenogenetic clade. Incorporation of this research into integrative pest management and reclassification of H. bicolor into a cryptic species complex is also discussed. Overall, this study advances our understanding of Elateridae bacteriomes, including factors that contribute to their richness and community composition.
Malaria-causing Plasmodium parasites are transmitted by some Anopheles mosquito species. The genetic and immunity basis for vector competence, the mosquitoes' intrinsic ability to transmit pathogens, has been extensively studied in major vectors such as Anopheles gambiae and Anopeles stephensi. In An. gambiae, Lola-type motifs were identified in immunity-related cis-regulatory elements; the Lola transcription factor is known to be involved in Drosophila development. Here, the potential role of lola in mosquito antimicrobial defence was investigated in Anopheles albimanus, a malaria vector in the Americas. The in vivo attenuation of lola was found to compromise mosquito survival to microbial infection. It was also found that the induction of antimicrobial defence genes requires lola upregulation. Genes for antimicrobial peptides, C-lectins, pattern recognition receptors, Clip-domain serine proteases and ML-domain proteins showed lola upregulation dependency. Enzymes involved in the generation and detoxification of reactive oxygen species, as well as components of the JNK immune signalling pathways, are also dependent on lola upregulation. These findings suggest that lola regulates the expression of immunity and detoxification genes and that there is a functional link between JNK signalling and lola expression in the mosquito midgut. Dysregulation of this pathway may contribute to increased pathogen invasion and malaria transmission.
The spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) is an important pest of forage grasses in South America, where its nymphs cause pasture damage by feeding on xylem sap and producing a characteristic foam that protects them against environmental stressors. To investigate the molecular basis of this adaptation, we integrated RNA-seq analysis of nymphs with LC-MS/MS proteomics of the Batelli gland, the primary source of foam secretion. De novo assembly of 100,666 unigenes revealed broad functional diversity, with strong representation of detoxification enzymes (CYP450s, GSTs, UGTs, carboxylesterases), transporters and ion pumps, cuticle proteins, and stress- and immunity-related genes. Nearly 16% of loci exhibited alternative splicing, particularly within detoxification, chemosensory and osmoregulatory gene families, highlighting evidence of transcriptomic variability. Signal peptide and secreted protein predictions identified 168 high-confidence candidate secreted proteins, including detoxification enzymes, proteases, structural proteins and immune-related factors, several of which are consistent with antimicrobial and surfactant-related functions. Proteomic profiling of the Batelli gland confirmed 500 proteins, enriched in chaperones, metabolic enzymes, detoxification pathways and osmoregulatory components, with the most abundant proteins corresponding to Hsp70 chaperones, ATP synthases, cuticle proteins and carbonic anhydrases. Together, these results provide an integrative transcriptomic and proteomic overview for M. spectabilis nymphs, highlighting genes and proteins associated with xylem feeding, foam production and responses potentially related to environmental stress tolerance. This comprehensive dataset not only advances the understanding of spittlebug biology but also identifies candidate molecular targets that may inform innovative strategies for controlling nymphal stages and mitigating spittlebug damage in forage systems.
DEET and citronella oil are two of the most widely used mosquito repellents; however, their impact on chemosensory tissues is not fully understood. In their vapour phase, the repellency effect involves the olfactory system, with mosquitoes detecting and responding to diverse odorants through their antennae and mouthparts (maxillary palps and proboscis). In this study, we first performed repellency behavioural assays that indicated both DEET and citronella effectively impaired female Aedes aegypti host-seeking capabilities (i.e. landing on a host and taking a blood meal). We next used RNA sequencing to assess global gene expression changes in Ae. aegypti antennae, mouthparts and headless bodies in response to volatile repellent exposure. The resultant transcriptional changes were considerably greater in the mosquito mouthparts than in the other tissues. The most notable responses in olfactory-related genes to both repellents were the suppressed expression of the three subunits comprising the mosquito CO2 receptor (Gr1, 2 and 3). Genes associated with mitochondrial function and metabolism were also suppressed. Exposure to DEET was further characterized by substantial inhibition of cytochrome P450 mRNA expression, the primary detoxification enzymes in insects. Overall, our study provides novel insights into the olfactory and physiological responses of Ae. aegypti chemosensory tissues in response to DEET and citronella exposure.
Wolbachia bacteria are widespread maternally inherited symbionts of Nematoda and diverse Arthropoda hosts. Their evolutionary success is determined by the ability to affect the biology of the host in different ways, promoting the relative fitness of females harbouring Wolbachia, as well as sporadic cases of horizontal transmission of Wolbachia between different host species. Here, we revised Wolbachia infection in the Hymenoptera with respect to the symbiont occurrence in host taxa and Wolbachia genetics. The representatives of about half of the extant families and 1000 out of 140,000 non-ant hymenopteran species have been tested for Wolbachia infection. We concluded that Wolbachia are found in all major hymenopteran families. More than 75% of Wolbachia diversity belongs to the A supergroup, whereas other variants belong to the B supergroup and only two isolates belong to the supergroup F. One of the main results of this study is the discovery of a specific Wolbachia genetic pattern (based on multilocus sequence typing [MLST]) in Apoidea hosts. Two haplotypes, ST-479 and ST-wH14, along with their alleles within other sequence types (STs), form the core of symbiont diversity, comprising 81% of unique host-Wolbachia ST associations. These haplotypes have not been reported beyond the Apoidea superfamily or Hymenoptera order. The reasons and mechanisms underlying this pattern in Apoidea remain unknown. Another important result of our study concerns the use of the MLST protocol, which has been previously criticised. We analysed 51 Wolbachia genomes for the average nucleotide identity (ANI) and MLST data, and found that genome and MLST variation are highly correlated. Therefore, the MLST protocol for Wolbachia remains reliable for many research tasks.
Nymphalid butterflies have unique leg morphology among Lepidoptera: they are the only family with greatly reduced forelegs (T1) in adults of both sexes, which are not used for walking. Previous studies have suggested that T1 legs may have chemosensory functions. To investigate which genes underpin this biology, we undertook a differential gene expression analysis in female Maniola jurtina. We find that nymphalid T1 legs have a distinct transcriptomic profile to T2 and T3 legs and also distinct from sensory palps. We find over 250 genes commonly expressed in nymphalid T1 legs and palps, but not in T2 legs. Despite this overlap, T1 legs are still more similar to T2 and T3 legs in their gene expression profiles than they are to palps. Genes expressed in common between palps and T1 legs include one encoding a trypsin-domain protein descendent from a nymphalid-specific duplication and several others involved in sensory functions, including genes with putative chemosensory roles. A blue-sensitive opsin gene is specifically expressed in T1 legs. Our findings indicate clear transcriptomic differences between T1 legs and walking legs in M. jurtina, pointing to the functional basis of these differences, including minor acquisition of palp-like gene expression.
Nuclear receptor genes play crucial roles in insect moulting. These genes, found in all insects, produce important proteins that control the expression of other genes, which in turn regulate the moulting process in larvae. This study investigated the roles of the nuclear receptor genes HcHR3 and HcβFTZ-F1 in the moulting development, pupation and eclosion of Hyphantria cunea. The mRNA expression of the two genes was detected at all developmental stages and in various adult tissues. A novel pET28a (+)-HT115 (DE3) RNase III-system was successfully constructed to efficiently express HcHR3-dsRNA and HcβFTZ-F1-dsRNA in bacteria. To explore the physiological functions of these genes, they were knocked down in larvae and pupae using the dsRNA extracted from bacteria, resulting in a decrease in the expression levels of HcHR3 and HcβFTZ-F1 by 13.70% and 99.01%, respectively. Moulting defects were observed in H. cunea larvae following HcHR3 and HcβFTZ-F1 silencing, with a phenotypic percentage ranging from 33.40% to 45.22%. Additionally, knockdown of these two genes in 5th instar larvae decreased weight, food intake and the survival rate, resulting in abnormal development during the larval-pupal transition. RNAi-mediated knockdown of HcHR3 and HcβFTZ-F1 prevented the eclosion of the pupae, with malformation rates ranging from 75.67% to 80.12%, and epidermal tanning defects were observed in pupae and adults. Our findings demonstrate that HcHR3 and HcβFTZ-F1 are indispensable for normal postembryonic development and provide validated molecular targets for RNAi-based pest management and insight into the outbreak mechanisms of H. cunea.
DNA methylation is an important epigenomic modification that significantly influences various cellular and organismal functions. In this study, we investigate the methylome of the small hive beetle, Aethina tumida. Our analysis reveals an average of 58,306 CpG methylation marks per beetle, representing approximately 0.99% of the genome's total CpGs. Notably, 85.4% of these methylation marks are located within genic regions on autosomes, with similar rates observed in both male and female beetles. However, male beetles exhibit a lower number of methylation marks and upregulated genes on Chromosome X when compared to female beetles. To evaluate the impact of epialleles on methylation, we identified 5828 associations between SNPs and methylation, with genotypes accounting for 39.2% of the variation observed at highly methylated sites. Interestingly, unfertilised eggs display slightly higher levels of DNA methylation compared to adult beetles, whereas embryos show methylation levels that are only about half of those in adults. This suggests that DNA methylation is dynamic during early development.
Insect guts host a diverse and abundant array of microorganisms. These microbes improve host fitness by extensively involving in a range of crucial physiological processes, which have mainly been revealed by high-throughput sequencing, particularly metagenomics. However, it is almost impossible to make an accurate and complete distinction between the genetic functions of microbial symbionts and insect hosts without host genome data. By comparing metagenomic data from gut germ-free and nonaxenic larvae, we accurately identified the data belonging to the gut microbiome of the onion maggot Delia antiqua (Diptera: Anthomyiidae). Besides, a correlation between bacteria of the genus Wohlfahrtiimonas (Gammaproteobacteria: Pseudomonadaceae) and vitamin B6 metabolism was detected through collinearity analysis. Furthermore, in vitro tests confirmed that the gut bacterium Wohlfahrtiimonas larvae contributed to the growth of D. antiqua larvae via the independent synthesis of vitamin B6. This study provides a comprehensive view of the gut bacterial diversity in D. antiqua and reveals a functional profile that is strictly specific to the gut microbiota of this species. It has preliminarily revealed the functional differentiation between insect hosts and their symbiotic microorganisms. This study also offers a technical reference for the study of microbial symbiotic functions in other insect-microbe symbioses without host genomic data.
The insect epidermis possesses the potential to generate remarkable diversity in exoskeletal cuticle traits, including colour, thickness and mechanical properties. Genetic manipulation is essential for investigating the molecular mechanisms; however, spatiotemporally controlled gene overexpression remains technically challenging despite the widespread use of loss-of-function approaches in many insects. In this study, we established an epidermal gene misexpression system in the model hemimetabolous insect Gryllus bimaculatus by inserting a gene expression cassette into the evolutionarily conserved yellow gene, which is involved in melanin synthesis, using genome editing. Insertion of the EGFP expression cassette into yellow gene resulted in fluorescence in the epidermis of pre-hatching embryos and post-moult individuals across developmental stages, corresponding to melanin pigmentation. Using this system, we induced the misexpression of Arylalkylamine N-acetyltransferase (aaNAT), an enzyme involved in the synthesis of N-acetyldopamine (NADA)-sclerotin, which serves as both a white pigment and a crosslinker in the cuticle. Compared to the background strain, the aaNAT misexpression strain exhibited a brighter body coloration, indicating that Gryllus aaNAT has the function of producing white NADA sclerotin and suppressing melanin pigment production. Wing thickness exhibited no substantial alteration, whereas a significant reduction in puncture resistance was observed. Collectively, these results suggest that an imbalance in the molecular components essential for cuticular crosslinking leads to alterations in the mechanical properties of the cuticle. This system serves as a gain-of-function analysis tool for cuticle research and has the potential to be applicable across a wide range of insect species, thereby helping to elucidate cuticle diversity.
The antioxidant defence system is crucial for herbivorous insects to adapt to various host plants. This study focused on catalase 2 gene (HcCAT2) to investigate the antioxidant mechanisms that underlie the ability of Hyphantria cunea to adapt to a wide range of hosts and to develop a disruptor for its polyphagous behaviour. Results indicated that, compared to larvae fed on the highly preferred host plant Morus alba, HcCAT2 expression increased 33.19- to 47.25-fold in larvae reared on Betula platyphylla and Tilia amurensis with moderate and low preference, respectively. Silencing HcCAT2 consistently reduced larval body weight and downregulated growth-related genes (e.g., Cyclin A2 and Decapentaplegic) across all host plant groups. Moreover, HcCAT2 silencing significantly downregulated key glycolytic genes (Hexokinase and Pyruvate kinase), the tricarboxylic acid cycle gene (Isocitrate Dehydrogenase 2) and lipid metabolism genes (e.g., Acetyl-CoA Carboxylase) in larvae fed on all three host plants. The redox response in larvae was complex, as HcCAT2 silencing led to a marked downregulation of negative regulators of oxidative damage and key ROS-producing genes. The nucleic acid pesticide CS-dsHcCAT2, designed to target HcCAT2 expression, demonstrated potent silencing efficacy. Treatment with CS-dsHcCAT2 suppressed larval body weight on all host plants. Collectively, the HcCAT2-mediated antioxidant defence system is critical to the host plant adaptation of H. cunea, and CS-dsHcCAT2, by inhibiting HcCAT2 expression, holds promise as an effective agent to disrupt the polyphagous behaviour of H. cunea.
Glycolipid metabolism is a core regulatory link in the energy homeostasis and physiological functions of insects, directly affecting their survival, development, reproduction and environmental adaptability. Catalpol, the main active component of Rehmannia glutinosa, possesses multiple pharmacological activities, such as antioxidation, anti-inflammation and regulation of glycolipid metabolism. However, the specific regulatory mechanism of its effect on insect metabolism remains unclear. In this study, Drosophila melanogaster was used as the model organism to systematically explore the effects of catalpol on glycolipid metabolism, growth and development, motor ability and lifespan of female adults, as well as its underlying molecular mechanism. At the molecular level, catalpol significantly enhances glucose catabolism, as demonstrated by increased activity of both soluble and membrane-bound trehalase and upregulation of the Tret1-1 gene. Concurrently, it dynamically regulated lipid metabolism, significantly increasing the content of free fatty acids and triglycerides. This finding, coupled with the upregulation of key lipolytic genes such as hormone-sensitive lipase (HSL), indicates accelerated fat turnover. These metabolic modulations translated into significant phenotypic improvements. Catalpol treatment promoted insect growth and development, as evidenced by increased body weight, abdominal size and wing area. Furthermore, it enhanced their motor ability, shown by improved climbing, and extended their average lifespan and survival rate, suggesting a delay in ageing. In conclusion, this study elucidates a novel regulatory model, wherein catalpol dynamically modulates both glucose and lipid metabolism, leading to improved growth, enhanced motor function and extended lifespan in D. melanogaster. Our findings provide initial insights into the intricate mechanism-phenotype connection and provide compelling experimental evidence for catalpol's potential as an insect physiological regulator. This research not only offers new perspectives on insect energy homeostasis but also lays a foundation for its application in agricultural pest control and beneficial insect breeding.
The suppression of Ku70 and Ku80 has been verified to increase homology-directed repair (HDR) efficiency in fruit fly, silkworm and mosquito, but not in other insects. In this study, PxKu70 and PxKu80 were identified from the Plutella xylostella genome. Domain analysis revealed that PxKu70 contained three conserved domains: Ku N, Ku and Ku C, while PxKu80 comprised the Ku N, Ku and Ku PK bind domains. Phylogenetic analysis and multiple-sequence alignment indicated strong conservation of both proteins among lepidopteran insects. RT-qPCR analysis showed that PxKu70 and PxKu80 were highly expressed in adult stages, particularly in reproductive tissues such as the ovary and testis, suggesting their role in maintaining genomic stability during gametogenesis. Two homozygous knockout lines (ΔPxKu70 and ΔPxKu80) were successfully generated through CRISPR/Cas9-mediated genome editing. These knockout lines remained viable and fertile without observable fitness effects. A donor construct carrying an EGFP cassette designed for insertion at the PxKmo locus was generated to assess HDR-mediated integration. The HDR insertion rate was significantly elevated in both knockout lines compared with the wild-type. These findings demonstrate that suppression of either PxKu70 or PxKu80 can enhance HDR in P. xylostella, offering an effective approach for precise genome editing in lepidopteran species.
RNA interference (RNAi) has emerged as a promising strategy for species-specific and environmentally friendly pest control, offering an alternative to conventional chemical insecticides that are increasingly constrained by resistance development and ecological concerns. RNAi-based approaches involve oral delivery of double-stranded RNA (dsRNA), which is processed into RNA-induced silencing complex (RISC)-bound small interfering RNA (siRNA) to silence essential genes of pests. This review synthesizes recent advances in experimental and bioinformatic methodologies that are facilitating and enhancing RNAi research in insect pest management. Particular emphasis is placed on molecular validation techniques that move beyond phenotype-based bioassays, including RISC-bound small RNA sequencing to resolve dsRNA processing and guide strand selection, RNA degradomics to map siRNA-mediated transcript cleavage events and transcriptomic and proteomic profiling to characterize genome-wide responses and compensatory effects. In parallel, dsRNA visualization methods provide mechanistic insight into uptake, intracellular trafficking and degradation dynamics, clarifying barriers that distinguish responsive from recalcitrant species. Complementing these experimental developments, emerging computational platforms enable insect-optimized target selection, dsRNA design and environmentally informed off-target prediction. Together, these innovations support a transition toward more predictive and mechanistically grounded RNAi-based pest control applications. The integration of high-resolution molecular tools with specialized bioinformatic pipelines is expected to enhance efficacy, safety and reproducibility, advancing RNAi-based pest control toward practical and scalable agricultural deployment.
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.
