Aedes aegypti is an important vector for dengue virus and thus has been targeted with pyrethroid insecticides in many areas of the world. As such, resistance has been detected to several of these insecticides, including in China, but the mechanisms of the resistance are not well understood in this country. Using the World Health Organization larval mosquito bioassay, five field populations of Aedes aegypti from Southern China were characterized for their resistance to cypermethrin and cyhalothrin. RNA extraction with PCR amplification, cloning and sequencing of the sodium channel gene was followed by comparisons of susceptible and wild mosquito strains Additionally, genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the sodium channel genes to detect S989P, V1016G and F1534C mutations and allow for correlation analysis of resistance expression for the different mutations. All wild strains expressed resistance to cypermethrin and cyhalothrin and the resistance expression between the two insecticides was highly correlated suggesting cross-resistance between these two pyrethroids. The AS-PCR technique effectively distinguished individual genotypes for all three mutations. Among the five wild strains tested, two strains carried all three mutations. Although the S989P and V1016G mutations were positively correlated to resistance expression of both pyrethroids, the F1534C mutation was negatively correlated. Our methodology proved highly reliable and will aid future detection of kdr mutations. The three sodium channel mutations were common in the Ae. aegypti strains sampled from Southern China. The V1016G mutation appears to be the most important kdr mutation in Ae. aegypti strains in Southern China.

The midgut is the first barrier to dengue virus (DENV) infections of mosquitoes and therefore is a major bottleneck for the subsequent development of vector competence. However, the molecular mechanisms responsible for this barrier are unknown. We constructed three small RNA libraries from the midguts of adult Aedes albopictus females that had been fed on either sugar solution, an uninfected blood meal, or a blood meal infected with DENV-2, and112 conserved microRNAs represented by 173 miRNA sequences were identified, with 34 novel microRNAs predicted by Mireap, RNAfold and Sfold software. In addition, the expression of aal-miR-1174, aal-miR-2951 and aal-miR-956 was confirmed via stem-loop quantitative real-time PCR (qRT-PCR). Compared with microRNA expression profiles of mosquitoes that had ingested a regular blood meal, 43 microRNAs were upregulated and 4were downregulated in mosquitoes that had ingested a DENV-2-infected blood meal. Among the differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-4728-5p were verified via stem-loop qRT-PCR. Analyses indicated that the changing patterns in miRNA expression during DENV-2 infection were significant and varied at different time points post infection. Most miRNA were upregulated at 24 h but were downregulated at 48 h post DENV-2 intake. The aal-miR-4728-5p was chosen for an in vitro transient transfection assay, and the results show that this miRNA enhances DENV replication in C6/36 cells. This study provides the first information on microRNAs expressed in the midgut of Ae. albopictus and describes species-specific changes in their expression levels following infection by DENV-2.

Pesticide resistance due to sodium channel point mutations has been well documented in many mosquito species. We tested the resistance of six, wild, Chinese populations of the mosquito Culex pipiens quinquefasciatus to deltamethrin and cyhalothrin. The full length of the sodium channel gene was cloned and sequenced from a pooled sample of mosquitoes from each population. Seven amino acid alterations were found (V250M, R436K, M943V, I973T, L1035F, L1035S and E1901D). Correlation between the frequencies of these mutations and the level of pesticide resistance (LC50) associated with them indicates that those at position L1035 (corresponding to position L1014F in the house fly, Musca domestica; GenBank Accession No.: X96668) are associated with resistance to deltamethrin and cyhalothrin. The frequency of the L1035F mutation was significantly correlated with resistance to deltamethrin (R2 = 0.536, P = 0.049) and cyhalothrin (R2 = 0.626, P = 0.030), and the combined frequency of the L1035F and L1035S mutations was significantly correlated with resistance to both deltamethrin (R2 = 0.661, P = 0.025), and cyhalothrin (R2 = 0.803, P = 0.008). None of the other mutations were correlated with either deltamethrin or cyhalothrin resistance. Interestingly, an HWE test indicated significant linkage between the M943V and I973T mutations (P < 0.01), but further research is required to determine the biological significance of this linkage. Identifying these mutations may be of practical benefit to the development of pesticide resistance management programs.

Background: Mosquito metavirome research aims to comprehensively characterize the diversity of mosquito-associated viruses, particularly focusing on insect-specific viruses (ISVs) and their potential interactions with arboviruses of public health concern. Advances in next-generation sequencing (NGS) have significantly expanded our understanding of the viromic complexity within mosquito populations, revealing numerous novel viral species and genera. These studies not only contribute to viral taxonomy and evolutionary biology but also provide critical insights into the ecological dynamics between mosquitoes and their viromes. Methods: NGS was employed to characterize the metavirome of three epidemiologically significant mosquito vectors, Aedes albopictus , Culex tritaeniorhynchus , and Anopheles sinensis in Jiangxi Province, China. This study integrated bioinformatic workflows to conduct comparative analyses of viral composition and biological significance. Results: An analysis of the metavirome of three mosquito species in Jiangxi Province revealed 86 viruses. Of these, 49 belonged to 19 established families, while the remaining 37 were unclassified. The unclassified viruses had the highest relative abundance. The known virus families with relatively high abundances among the three mosquito species were: Solemoviridae , Xinmoviridae , Phasmaviridae , Flaviviridae , Rhabdoviridae , Peribunyaviridae and Orthomyxoviridae . Although the Shannon and Simpson diversity indices showed no significant differences between the three species ( p > 0.05), substantial compositional divergence was observed in the “top 30 viruses.” The most frequently detected viruses in the Ae. albopictus population include High Island virus, Usinis virus, Sichuan mosquito sobemo-like virus, Guangzhou sobemo-like virus, Barstukas virus , Piry virus (PIRYV), Aedes flavivirus (AEFV), and Aedes albopictus anphevirus (AealbAV). The most frequently detected viruses in the Cx. tritaeniorhynchus population include Hubei mosquito virus 2 , Quang Binh virus (QBV), Culex tritaeniorhynchus rhabdovirus (CTRV), Yongsan sobemo-like virus 1 (YSLV1), Bat sobemovirus (BSV), Wuhan Mosquito Virus 2 (WMV2), and Culex pseudovishnui bunya-like virus (CPBV). The most frequently detected viruses in the An. sinensis population include Hubei reo-like virus 12 , Xincheng mosquito virus (XCV), Wuhan mosquito virus 1 (WMV1), and Wuhan mosquito virus 5 (WMV5). Conclusions: The most frequently detected virus profiles of the three most important mosquito species for epidemiology in Jiangxi Province, Ae. albopictus , Cx. tritaeniorhynchus , and An. sinensis , exhibit evident differences. Further validation of the biological characteristics, pathogenicity, vector competence, and host relationships of the identified viruses (including ISVs) is required to gain a comprehensive understanding of their roles in host–virus interactions. This will provide theoretical support for vector control efforts in Jiangxi Province. Graphical