In total, 4,443 mosquito samples were captured from Hengyang (n = 14), Shaoyang (n = 499), Yueyang (n = 580), Changsha (n = 1,347), Zhuzhou (n = 353), Changde (n = 242), Xiangtan (n = 15), Yiyang (n = 949), Yongzhou (n = 95), Xiangxi (n = 82), Huaihua (n = 49) and Zhangjiajie (n = 218). They represented one family, four genera and nine species (Table 1). Culex pipiens pipiens (24%) was the most common mosquito species, followed by Armigeres subalbatus (23.4%) and Culex tritaeniorhynchus (20.6%) (Table 1). In addition to those shown in Table 1, a number of other mosquitos have been found in pig farms in Hunan province, namely Armigeres obturbans, Culex modestus and Anopheles lesteri, during previous pilot studies (Cao et al., 2019, Xu et al., 2022, Zhao et al., 2024). Taken together, a total of twelve different species have been recorded from pig farms in Hunan province. The number of mosquito species found in pig farms in Hunan province were not similar to those found in other Chinese provinces (Ren et al., 2017, Hameed et al., 2021, Dong et al., 2023), this is most likely due to difference in precipitation, temperature and rice cultivation.
Mosquito in Changsha, the capital of the province, was found to have the most abundant species (six species); however, three regions (Xiangxi, Xiangtan and Zhuzhou) of Hunan province only found one species (Table 2). The distribution of mosquitos in pig farms is undoubtedly closely related to the location of the natural environment and climatic conditions. The reason why there are more mosquito species in Changsha than other administrative regions in the province is likely because Changsha ranks among the top in annual precipitation in Hunan Province, reaching 1476 millimeters. It should also be noted that as the economic and trade center of Hunan province, Changsha city has the highest population density and the most frequent flow of goods, which facilitates mosquito blood feeding and population dispersal; this metropolis is also the most urbanized region in the province. The inability of urban precipitation to penetrate the impervious into the subsurface leads to stagnant water, and the resulting cesspools and tire ponds may provide a good breeding and developmental shelter for a variety of mosquitoes (including Ar subalbatus, Cx pipiens pallens and Cx quinquefasciatus). These profound urbanization features may have contributed to the expansion of mosquito populations in Changsha (Wilke et al., 2019).
Genomic DNA was extracted from 124 individual mosquito representing 12 geographical localities. No size variation was detected on agarose gel among any of the amplicons examined. The A+ T contents of the sequences were 62.6–63.5%, consistent with previous reports of the AT bias for other mosquitoes (Versteirt et al., 2015, Gao et al., 2017). While the intra-specific sequence variations within each of the mosquito species were 0-3.2%, the inter-specific sequence differences were significantly higher, being 2.4%-16.9%.
Phylogenetic analyses showed all mosquito species from present study grouping into distinct monophyletic groups corresponding to nine known mosquito species with strongly supported. In this tree, mosquitoes of the genus Culex were sister to the genus Anopheles, and the genera Culex and Anopheles was a sister group to the genus Aedes. The same species of mosquitoes with different geographical strains of the same species first clustered into a cluster, except for Cx. pipiens, which was mixed with Cx quinquefasciatus in the isolates of Cx pipiens pallens (Joyce et al., 2018, Aardema et al., 2020). It has been hypothesized that Cx pipiens pallens is the product of hybridization between Cx quinquefasciatus and Cx pipiens pipiens, that it is currently an independently evolved species, and that there is an extensive genetic exchange in the Culex species complex (Aardema et al., 2020). In addition, Cx tritaeniorhynchus, Ae vexans, Cx vishnui and An sinensis were divided into more than two branches, indicating that the four species may have been geographically isolated, with multiple geographical lineages (Fig. 3) (Kuwata et al., 2013).
The results of the present survey have indicated that mosquito species are abundant in intensive pig farms in Hunan province, China. A previous study has proven that the cox1 sequence provides genetic marker for the accurate identification of mosquitoes (Adeniran et al., 2021). Accurate identification of mosquito species can provide valuable clues for the prevention and control of mosquito-borne diseases (Wang et al., 2012, Schwab et al., 2018). In the present study, phylogenetic relationships based on partial cox1 sequences identified the mosquito species, reconfirming that cox1 is a suitable marker for molecular epidemiology.