Abstract: 【Aim】Juvenile hormone epoxide hydrolase (JHEH) is a key enzyme in the juvenile hormone (JH) metabolic pathway, playing a crucial role in insect growth, development and reproductive regulation. Anopheles sinensis, a major vector of malaria, has not been well-studied regarding the function of its JHEH gene. This study aims to characterize the molecular properties and gene expression patterns of JHEH in An. sinensis, and to explore the role of JHEH in the reproduction of An. sinensis.【Methods】Based on the transcriptomic and genomic data of An. sinensis, the cDNA sequence of AsJHEH was amplified using PCR. Bioinformatics tools were employed to predict the physicochemical properties and structural features of AsJHEH, and a phylogenetic tree was constructed using MEGA11.0. The expression levels of AsJHEH in different developmental stages of An. sinensis (eggs, 1st-4th instar larvae, female pupae, male pupae, and female and male adults, 0, 10, 20 and 30 h after pupation, late female pupae, as well as female adults at 0, 6, 12, 24, 36, 48, 60 and 72 h after eclosion), different female and male adult tissues (head, thorax and abdomen), and different tissues of female adults (midgut, Malpighian tubules, fat body, ovaries and integument) were detected using RT-qPCR. The pET32a-AsJHEH prokaryotic expression vector was constructed to induce protein expression and purify the recombinant protein, followed by the preparation of AsJHEH polyclonal antibody. RNAi was performed by injecting dsAsJHEH into the late female pupae of An. sinensis. The gene silencing efficiency was verified by RT-qPCR and Western blotting, and the eclosion rate, ovarian development, number of eggs laid per female adult of An. sinensis and the expression levels of AsVg in eclosed adults at 24 and 48 h post blood-feeding were observed and determined.【Results】The cDNA sequence of AsJHEH (GenBank accession no.: PV466834) of An. sinensis was successfully cloned and obtained. It was 1 389 bp in length, encoding 462 amino acids. The predicted molecular weight and isoelectric point (pI) of the encoded protein were 53.02 kD and 6.20, respectively. AsJHEH contained conserved structural motifs, including a catalytic triad (Asp234-Glu411-His438), an oxyanion hole (Tyr306 and Tyr381), and the XWG and HGXP motifs. AsJHEH was most closely related to JHEHs from other Culicidae species. Spatio-temporal expression patterns revealed that AsJHEH was expressed in all developmental stages and adult tissues of An. sinensis, with the highest expression level in the 2nd instar larvae (888.98-fold as high as that in adult females) and male adult head (1 198.54-fold as high as that in the ovary of female adult). The recombinant AsJHEH was successfully prepared by prokaryotic expression system, yielding polyclonal antibody with a titer of 1∶128 000. After RNAi of AsJHEH, the expression levels of AsJHEH at the mRNA and protein levels in An. sinensis were significantly reduced as compared to those in the control group injected with dsEGFP. RNAi of AsJHEH led to a 26.67% decrease in eclosion rate, abnormal ovarian development, a 1.8-fold upregulation of AsVg expression level in eclosed adults at 48 h post blood-feeding, a 52.23% reduction in the number of eggs laid per female, and the production of elongated eggs (0.2-fold longer eggs) that failed to hatch compared to the control group injected with dsEGFP.【Conclusion】This study elucidates the critical role of AsJHEH in the growth, development and reproductive regulation in An. sinensis for the first time, not only laying a theoretical foundation for controlling An. sinensis through the JH degradation pathway but also supplying potential molecular targets for JH metabolism-based biocontrol strategies, which has important theoretical significance and practical value.

Key words: Anopheles sinensis, juvenile hormone epoxide hydrolase (JHEH), gene cloning, expression pattern, RNA interference, reproductive regulation