关键词: 赫坎按蚊种团, 中华按蚊复合体, 分子鉴别, 区系分布

Abstract: Objective To improve the multiplex PCR molecular methods to identify species of Anopheles hyrcanus group, and investigate the faunal distribution of An. sinensis in China and the factors influencing its distribution. Methods The species-specific primers were designed based on rDNA-ITS2 sequences of Anopheles (An. sinensis, An. lesteri, An. yatsushiroensis, An. kleini and An. belenrae) to improve the specificity and sensitivity of the multiplex PCR method to identify the Anopheles mosquito species. Anopheles mosquitoes were collected from 18 locations in 8 provinces (municipalities or autonomous regions) in China using light trap and entomological aspirator from 2013 to 2018. Those mosquitoes identified as members of An. hyrcanus group based on morphological characteristics were used for further analysis. The genomic DNA of individual mosquito was extracted, and the multiplex PCR assay was used to determine the species of An. hyrcanus group. For those mosquitoes without multiplex PCR products, the rDNA-ITS2 was amplified and sequenced, and the sequences were subjected to BLAST of the NCBI GenBank database to determine the species. Based on the results of this study and the review of 17 publications that identified An. sinensis using molecular biological methods in China, South Korea and the Far East of Russian, as well as the collected geographical location and climate data, the influence q value was calculated using the geo-detector software. The effects of geographical longitude, latitude, annual average temperature and rainfall on the distribution of An. sinensis were analyzed. Results The improved multiplex PCR method was able to simultaneously identify five members of the An. hyrcanus group based on the size of the amplified fragments as An. sinensis (490 bp), An. lesteri (313 bp), An. yatsushiroensis (216 bp), An. kleini (386 bp) and An. belenrae (165 bp). In this study, the species of 365 Anopheles mosquitoes were identified by multiplex PCR, including An. sinensis (n = 114, collected from Shaanxi, Anhui and Shandong), An. yatsushiroensis (n = 34), An. lesteri (n = 9), An. kleini (n = 181), An. belenrae (n = 5). The 22 individuals without acquiring amplified products were identified as An. kweiyangensis (n = 2), An. sineroides (n = 1), An. koreicus (n = 8), An. lindesayi (n = 7) and An. pattoni (n = 4) by rDNA-ITS2 sequencing. The geographic and ecological data were collected from 101 sites, including 80 sites with An. sinensis and 21 sites without An. sinensis distribution. The calculated q values were 0.592 0 for annual average temperature, 0.507 2 for latitude, 0.351 2 for longitude and 0.214 4 for annual average rainfall. The results indicate that the most significant factor affecting the distribution of An. sinensis is temperature, followed by latitude. Comprehensive analysis of the impact of latitude and annual average temperature on the distribution of Anopheles revealed that the annual average temperature of 10 ℃ could be used as the reference to determine the northern boundary of An. sinensis distribution in China. An. sinensis was distributed in the entire provinces (municipalities or autonomous regions) of Yunnan, Guizhou, Chongqing, Henan, Shandong, Tianjin, Jiangsu, Anhui, Hubei, Zhejiang, Shanghai, Fujian, Jiangxi, Guangxi, Guangdong, Hainan, Taiwan, Hong Kong and Macao, and the southern parts of the Tibet, Sichuan, Gansu, Shaanxi, Shanxi, Hebei, Beijing and Liaoning. Conclusion The improved multiplex PCR assay used to identify An. hyrcanus group is fast, simple and reliable. The annual average temperature of 10 ℃ could be used as reference to determine the northern boundary for the geographical distribution of An. sinensis in China, and the distribution range is smaller than that in the previously recorded document.

Key words: Hyrcanus group, Anopheles sinensis complex, Molecular identification, Faunal distribution