Pheromones are crucial to intraspecific communication and reproduction in insects. However, the role of sex pheromones in the invasive pest, Callosobruchus maculatus, particularly regarding the conversion of chemical signals into electrical signals, remains unclear. In this study, we elucidate the sex pheromone system of C. maculatus by integrating chemosensory gene identification, ligand-receptor interaction modeling, and functional validation. First, we confirmed five sex pheromone components that evoked the electrophysiological and behavioral responses of males, suggesting roles in conspecific attraction: (Z)-3-methyl-2-heptenoic acid (Z32A), (Z)-3-methyl-3-heptenoic acid (Z33A), decanal, 2,4-dimethyldecane, and dodecane. Next, we annotated 42 odorant receptors (CmacORs) in the C. maculatus genome, including the obligate co-receptor, CmacOrco. To further detect the interactions between potential pheromones and CmacORs, we evaluated the predicted binding scores of pheromone-receptor interactions using multiple molecular docking algorithms. We heterologously co-expressed candidate CmacOR/Orco in adherent cell lines and found that CmacOR3 interacted with both Z32A and Z33A. Notably, RNA interference-mediated knockdown of CmacOR3 in male C. maculatus significantly attenuated antennal sensitivity to these pheromones, validating its role as a cognate receptor. Our results elucidate the molecular mechanisms underlying sex pheromone detection in C. maculatus, offering critical insights for the development of novel olfactory-mediated pest management strategies and advancing fundamental knowledge of insect chemosensory evolution.

BACKGROUND:Mosquitoes with aggressive biting behavior are important disease vectors threatening public health. Armigeres subalbatus, as an emerging arbovirus and filarial disease vector, exhibits aggressive host-seeking behavior and unique breeding preference for contaminated water. However, the molecular mechanisms underlying these biological characteristics remain poorly understood. This study aimed to generate a high-quality genome assembly and characterize the genetic basis of vector competence and environmental adaptation in Ar. subalbatus. METHODS:We sequenced and assembled the Ar. subalbatus genome using Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. Comparative genomic analysis was performed to identify gene families related to detoxification, diapause, innate immunity, and sex determination. Gene structure analysis focused on the male-determining factor and its evolutionary relationships with other mosquito vectors. RESULTS:The genome assembly consists of three chromosomes, with a total size of 1.33 Gbp and an N50 of 430.15 Mbp (GenBank assembly: GCA_024139115.2), displaying 99.4% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness. We identified the gene structure of the male-determining factor (AsuMf) and characterized its evolutionary relationship with other mosquito vectors. The analysis revealed expanded detoxification-related gene families including cytochrome P450s, which may facilitate adaptation to contaminated breeding sites. We characterized 566 putative diapause-related genes that could potentially contribute to geographical expansion, 334 innate immune genes, and 1673 endogenous viral elements, indicating complex virus-host interactions throughout evolution. CONCLUSIONS:Our study provides insights into the molecular basis of vector competence and adaptation in Ar. subalbatus. The expanded detoxification gene families may enable the species to survive in polluted environments, while the identified diapause-related genes could explain its geographical expansion capabilities. These findings establish a foundation for developing novel vector control strategies targeting this emerging disease vector.

The regulation of mosquito reproduction at the transcriptional level is well understood, but the translational mechanisms remain unclear. Here, we find that lysyl-tRNA synthetase (KARS) is essential for translational regulation and governs mosquito fecundity in a hormone-dependent manner. RNA interference silencing of KARS severely inhibits ovarian maturation and impairs mosquito fecundity. Polysome profiling shows enhanced translation initiation in KARS-deficient mosquitoes, as supported by increased phosphorylation of p70 ribosomal protein S6 kinase and decreased phosphorylation of the translation initiation factor eIF2α. By contrast, incorporated puromycin reveals a reduction in the synthesis of nascent polypeptides such as vitellogenin. Further analysis of ribosome profiling sequencing indicates that the dysregulation of mRNA translation in KARS-deficient mosquitoes was caused by ribosome stalling. Furthermore, the knockdown of KARS results in a significant increase in JH and a decline in 20E, thereby forcing the mosquitoes to maintain prereproductive status and preventing the 20E-activated vitellogenesis. This study demonstrates the function of KARS in regulating reproduction during the gonadotrophic cycle, hence providing insights into the potential control of disease-transmitting mosquitoes.

Callosobruchus maculatus is one of the most competitive stored grain pests, which causes a great loss to agricultural economy. However, due to an inadequacy of high-quality reference genome, the molecular mechanisms for olfactory and hypoxic adaptations to stored environments are unknown and require to be revealed urgently, which will contribute to the detection and prevention of the invasive pests C. maculatus. Here, we presented a high-quality chromosome-level genome of C. maculatus based on Illumina, Nanopore and Hi-C sequencing data. The total size was 1.2 Gb, and 65.17% (797.47 Mb) of it was identified to be repeat sequences. Among assembled chromosomes, chromosome 10 was considered the X chromosome according to the evidence of reads coverage and homologous genes among species. The current version of high-quality genome provides preferable data resources for the adaptive evolution research of C. maculatus.

Mosquitoes form a vital group of vector insects, which can transmit various diseases and filarial worms. The cuticle is a critical structure that protects mosquitoes from adverse environmental conditions and penetration resistance. Thus, cuticle proteins can be used as potential targets for controlling the mosquito population. In the present study, we found that AaCPR100A is a structural protein in the soft cuticle, which has flexibility and elasticity allowing insects to move or fly freely, of Aedes aegypti. RNA interference (RNAi) of AaCPR100A caused high mortality in Aedes aegypti larvae and adults and significantly decreased the egg hatching rate. Transmission electron microscopy (TEM) analysis revealed that the larval microstructure had no recognizable endocuticle in AaCPR100A-deficient mosquitoes. A yeast two-hybrid assay was performed to screen proteins interacting with AaCPR100A. We verified that the G12-like protein had the strongest interaction with AaCPR100A using yeast two-hybrid and GST pull-down assays. Knockdown of G12-like transcription resulted in high mortality in Ae. aegypti larvae, but not in adults. Interestingly, RNAi of G12-like rescued the high mortality of adults caused by decreased AaCPR100A expression. Additionally, adults treated with G12-like dsRNA were found to be sensitive to low temperature, and their eggshell formation and hatching were decreased. Overall, our results demonstrated that G12-like may interacts with AaCPR100A, and both G12-like and AaCPR100A are involved in Ae. aegypti cuticle development and eggshell formation. AaCPR100A and G12-like can thus be considered newly potential targets for controlling the Ae. aegypti mosquito.

AaCPR100A is a structural protein, found in the soft cuticle of Ae. Aegypti . RNAi of AaCPR100A resulted in high mortality in Ae. Aegypti and abnormal egg development in the surviving mosquitoes. Over thirty proteins that could interact with AaCPR100A were screened out by yeast two-hybrid assay, and subsequently, further verification by hybrid and GST pull-down assays identified that G12-like had the strongest interaction with AaCPR100A. RNAi of G12-like suggested it may be related to larva development. Interestingly, the adults in which the G12-like gene was knocked down were sensitive to low temperature, and their egg shell formation, production, and hatching were affected. G12-like has the opposite effect in the upstream expression of AaCPR100A , promoting AaCPR100A function in the larval stage and inhibiting AaCPR100A in the adult stage. In all, functional studies of AaCPR100A and its interaction protein G12-like provide insight into its involvement in cuticle development and formation and egg shell formation.

BackgroundThe cuticle is an indispensable structure that protects the mosquito against adverse environmental conditions and prevents pathogen entry. While most cuticles are hard and rigid, some parts of cuticle are soft and flexible to allow movement and blood-feeding. It has been reported that 3, 4-dihydroxyphenylacetaldehyde (DOPAL) synthase is associated with flexible cuticle formation in Aedes aegypti. However, the molecular function of DOPAL synthase in the ontogenesis of mosquito remains largely unknown. In this study, we characterized gene expression profiles of DOPAL synthase and investigated its functions in larvae and female adults of Aedes agypti by RNAi.ResultsOur results suggest that the expression of DOPAL synthase is different during development and the transcriptional level reached its peak at the female white pupal stage, and DOPAL synthase was more highly expressed in the cuticle and midgut than other tissues in the adult. The development process from larva to pupa was slowed down strikingly by feeding the first-instar larvae with chitosan/DOPAL synthase dsRNA nanoparticles. A qRT-PCR analysis confirmed that the dsRNA-mediated transcription of the DOPAL synthase was reduced >50% in fourth-instar larvae. Meanwhile, larval molt was abnormal during development. Transmission electron microscopy results indicated that the formation of endocuticle and exocuticle was blocked. In addition, we detected that the dsDOPAL synthase RNA caused significant mortality when injected into the female adult mosquitoes.ConclusionsOur findings demonstrate that DOPAL synthase plays a critical role in mosquito larval development and adult survival and suggest that DOPAL synthase could be a good candidate gene in RNAi intervention strategies in mosquito control.

AaCPR100A是本实验室从埃及伊蚊RNAseq数据库中获得的预测与表皮结构相关的基因.本文拟通过生物信息学和分子生物学技术,探究埃及伊蚊表皮AaCPR100A基因的序列特点,并分析其在埃及伊蚊的时空表达特征.埃及伊蚊表皮AaCPR100A蛋白序列AaCPR100A ORF长度为765 bp,编码254个氨基酸,蛋白分子量约为28.6 kDa,1～16位氨基酸残基为信号肽,其氨基酸序列中含有表皮蛋白CPR家族中的RR-1基序.埃及伊蚊与白纹伊蚊、 致倦库蚊、 冈比亚按蚊和黑腹果蝇的氨基酸同源性分别为95％、72％、61％、54％.qRT-PCR结果显示AaCPR00A基因在表皮和卵期表达量最高,并随发育阶段呈现显著递减变化.结果表明,埃及伊蚊AaCPR100A表达具有时空和组织特异性特征.