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.

There was no record of Aedes aegypti in Yunnan Province, China, until 2002, but this species is now continuously found in nine cities (or counties). Until now, little was known about the genetic diversity and population structure of this invasive species. Thus, a detailed understanding of the invasion strategies, colonisation and dispersal of this mosquito from a population genetics perspective is urgently needed for controlling and eliminating this disease vector. The genetic diversity and population structure of Ae. aegypti communities were analysed by screening nine microsatellite loci from 833 Ae. aegypti mosquitoes sampled from 28 locations in Yunnan Province. In total, 114 alleles were obtained, and the average polymorphic information content (PIC) value was 0.672. The value of the alleles per locus ranged from 2.90 to 5.18, with an average of 4.04. The value of He ranged from 0.353 to 0.681, and the value of Ho within populations ranged from 0.401 to 0.689. Of the 28 locations, two showed significant departures from the Hardy-Weinberg equilibrium (HWE) with P-values less than 0.05, and a bottleneck effect was detected among locations from Ruili and the border areas with the degree of 60% and 50%, respectively. Combined with the F-statistics (FIT = 0.222; FCT = 0.145), the analysis of molecular variance (AMOVA) revealed that there was substantial molecular variation among individuals, accounting for 77.76% of the sample, with a significant P-value (<0.0001). The results suggest that genetic differences in Ae. aegypti originated primarily among individuals rather than among populations. Furthermore, the STRUCTURE and UPGMA cluster analyses showed that Ae. aegypti from the border areas were genetically isolated compared to those from the cities Ruili and Jinghong, consistent with the results of the Mantel test (R 2 = 0.245, P < 0.0001). Continuous invasion contributes to the maintenance of Ae. aegypti populations’ genetic diversity and different invasion accidents result in the genetic difference among Ae. aegypti populations of Yunnan Province.

Background: Odorant-binding proteins (OBPs) play important roles in many physiological processes of mosquitoes. Previous high-throughput sequencing studies have revealed that some OBPs of Culex quinquefasciatus might be involved in the development of resistance to insecticides. Methods: Based on the results of sequencing analyses, the OBP28 gene was selected for evaluation in this study. Three laboratory strains of Cx. quinquefasciatus [susceptible strain (SS), deltamethrin-resistant strain 1 (HN) and deltamethrin-resistant strain 2 (RR)] were first examined by using the Centers for Disease Control and Prevention bottle bioassay, after which the expression level of the OBP28 gene in the susceptible and deltamethrin-resistant strains was determined by real-time quantitative polymerase chain reaction. The OBP28 gene in deltamethrin-resistant strain RR was silenced using RNA interference technology. The expression level of OBP28 and the resistance level were tested in the silenced strain and control strain after microinjection of double-stranded RNA for a 48-h interference period. Four field-collected strains (henceforth ‘field strains’) of Cx. quinquefasciatus were also examined for their resistance to deltamethrin and levels of OBP28 expression. Finally, a correlation analysis between deltamethrin resistance and gene expression was carried out for all seven strains, i.e. the four field strains and the three laboratory strains. Results: In the bioassay, the mortality of SS, HN and RR was 100%, 21.33% and 1.67%, respectively. The relative expression levels of OBP28 in strains HN and RR were 6.30- and 6.86-fold higher, respectively, than that of strain SS. After silencing of the OBP28 gene, the mortality of strain RR was 72.20% and that of the control strain 26.32%. The mortality of strain RR increased significantly after interference compared to that of the control strain. There was a negative correlation between OBP28 gene expression and mortality in adult mosquitoes after exposure to deltamethrin. Conclusions: To our knowledge, this study shows for the first time a correlation between the expression of a gene coding for OBP and insecticide resistance in mosquitoes. The potential resistance mechanism that was elucidated provides a new target gene for the surveillance of resistance in mosquitoes. Graphical

Background: Aedes aegypti and Ae. albopictus are important human arbovirus vectors that can spread arboviral diseases such as yellow fever, dengue, chikungunya and Zika. These two mosquito species coexist on Hainan Island and the Leizhou Peninsula in China. Over the past 40 years, the distribution of Ae. albopictus in these areas has gradually expanded, while Ae. aegypti has declined sharply. Monitoring their genetic diversity and diffusion could help to explain the genetic influence behind this phenomenon and became key to controlling the epidemic of arboviruses. Methods: To better understand the genetic diversity and differentiation of these two mosquitoes, the possible cohabiting areas on Hainan Island and the Leizhou Peninsula were searched between July and October 2021, and five populations were collected. Respectively nine and 11 microsatellite loci were used for population genetic analysis of Ae. aegypti and Ae. albopictus. In addition, the mitochondrial coxI gene was also selected for analysis of both mosquito species. Results: The results showed that the mean diversity index (PIC and SI values) of Ae. albopictus (mean PIC = 0.754 and SI = 1.698) was higher than that of Ae. aegypti (mean PIC = 0.624 and SI = 1.264). The same results were also observed for the coxI gene: the genetic diversity of all populations of Ae. albopictus was higher than that of Ae. aegypti (total H = 45 and Hd = 0.89958 vs. total H = 23 and Hd = 0.76495, respectively). UPGMA dendrogram, DAPC and STRUCTURE analyses showed that Ae. aegypti populations were divided into three clusters and Ae. albopictus populations into two. The Mantel test indicated a significant positive correlation between genetic distance and geographic distance for the Ae. aegypti populations (R² = 0.0611, P = 0.001), but the correlation was not significant for Ae. albopictus populations (R² = 0.0011, P = 0.250). Conclusions: The population genetic diversity of Ae. albopictus in Hainan Island and the Leizhou Peninsula was higher than that of Ae. aegypti. In terms of future vector control, the most important and effective measure was to control the spread of Ae. albopictus and monitor the population genetic dynamics of Ae. aegypti on Hainan Island and the Leizhou Peninsula, which could theoretically support the further elimination of Ae. aegypti in China. Graphical

Background: Aedes aegypti and Aedes albopictus are important vectors of human arboviruses, transmitting arboviral diseases such as yellow fever, dengue, chikungunya and Zika. These two mosquitoes coexist on Hainan Island and the Leizhou Peninsula in China. Over the past 40 years, the distribution of Ae. albopictus has gradually expanded in these areas, while the distribution of Ae. aegypti has declined dramatically mainly due to the ecological changes and some other factors such as heavy use of insecticide indoor based on endophagic bloodfeeding of the species.Methods: This study focused on the knockdown resistance (kdr) genes of both mosquitoes, investigated their mutations, and analyzed their haplotype and evolutionary diversity combined with population genetic features based on the ND4/ND5 genes to further elucidate the molecular mechanisms underlying the development of insecticide resistance in both mosquitoes.Results: Three mutations, S989P, V1016G and F1534C, were found to be present in Ae. aegypti populations, and the three mutations occurred synergistically. Multiple mutation types (F1534C/S/L/W) of the F1534 locus are found in Ae. albopictus populations, with the three common mutations F1534C, F1534S and F1534L all having multiple independent origins. The F1534W (TTC/TGG) mutation is thought to have evolved from the F1534L (TTC/TTG) mutation. The F1534S (TTC/TCG) mutation has evolved from the F1534S (TTC/TCC) mutation. The most common form of mutation at the F1534 locus found in this study was S1534C, accounting for 20.97%, which may have evolved from the F1534C mutation. In addition, a new non-synonymous mutation M1524I and 28 synonymous mutations were identified in Ae. albopictus populations. Correlation analysis showed that the genetic diversity of Ae. aegypti and Ae. albopictus populations did not correlate with their kdr haplotype diversity (P>0.05), but strong gene flow between populations may have contributed to the evolution of the kdr gene.Conclusion: The study of kdr gene evolution in the two mosquito species may help to identify the evolutionary trend of insecticide resistance at an early stage and provide a theoretical basis for improving the efficiency of biological vector control and subsequent research into new insecticides.