Molecular identification of Anopheles hyrcanus group and faunal distribution of Anopheles sinensis (Diptera ∶ Culicidae)in China

doi:10.12140/j.issn.1000-7423.2020.01.009

Abstract

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

CLC Number:

R384.111

PENG Heng, CHEN Han-ming, CHEN Hui-ying, WANG Yan, LI Xiang-yu, LI Ji-xu, YANG Zhen-zhou, MA Ya-jun. Molecular identification of Anopheles hyrcanus group and faunal distribution of Anopheles sinensis (Diptera ∶ Culicidae)in China[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(1): 58-67.

References 46

[1]	Ma YJ, Xu JN.Progress of taxonomic study on the anopheline mosquitoes in China[J]. Chin J Vector Biol Control, 2015, 26(5): 433-438. (in Chinese)(马雅军, 徐建农. 中国按蚊的分类研究进展[J]. 中国媒介生物学及控制杂志, 2015, 26(5): 433-438.)
[2]	Lu BL.Zoology of China, insecta, volume Ⅷ Diptera, Culicidae (Book 2) [M]. Beijing: Science Press, 1997.(陆宝麟. 中国动物志昆虫纲第八卷双翅目蚊科(下卷)[M]. 北京: 科学出版社, 1997.)
[3]	Fang Y, Shi WQ, Zhang Yi.Research progress in classification of Anopheles hyrcanus group (Diptera ∶ Culicidae)[J]. Chin J Parasitol Parasit Dis, 2016, 34(6): 565-570. (in Chinese)(方圆, 施文琦, 张仪. 赫坎按蚊种团(双翅目 ∶ 蚊科)分类研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 565-570.)
[4]	Rueda LM.Two new species of Anopheles (Anopheles) hyrcanus group (Diptera ∶ Culicidae) from the Republic of South Korea[J]. Zootaxa, 2005, 941: 26.
[5]	Fang Y, Shi WQ, Zhang Y.Molecular phylogeny of Anopheles hyrcanus group (Diptera ∶ Culicidae) based on mtDNA COI[J]. Infect Dis Poverty, 2017, 6: 61.
[6]	Ma Y, Ma YJ, Lin L, et al.Phylogenetic relationship among some species of genus Anopheles subgenus Anopheles (Diptera ∶ Culicidae) in China: based on rDNA-ITS2 sequences[J]. Chin J Vector Biol Control, 2013, 24(5): 382-388. (in Chinese)(马颖, 马雅军, 林琳, 等. 基于rDNA-ITS2序列的中国按蚊属按蚊亚属部分种类的系统发育关系研究(双翅目 ∶ 蚊科)[J]. 中国媒介生物学及控制杂志, 2013, 24(5): 382-388.)
[7]	Ma Y, Ma YJ, Wang Y.Phylogenetic relationship of Anopheles species, subgenus Anopheles in China: based on mtDNA and rDNA sequences[J]. China Trop Med, 2015, 15(11): 1281-1288. (in Chinese)(马颖, 马雅军, 王琰. 基于mtDNA和rDNA基因序列的我国按蚊属按蚊亚属系统发育研究[J]. 中国热带医学, 2015, 15(11): 1281-1288.)
[8]	Wu T, Zhu DY, Wang YR, et al.A review of rDNA-ITS2 sequences as molecular identification features of Anopheles in China: ⅠSubgenus Anopheles[J]. Acta Parasitol Et Med Entomol Sin, 2013, 20(1): 16-24. (in Chinese)(伍桐, 诸德源, 王一然, 等. rDNA-ITS2序列作为中国按蚊分子鉴别特征的评述:Ⅰ按蚊亚属[J]. 寄生虫与医学昆虫学报, 2013, 20(1): 16-24.)
[9]	Ma YJ, Xu JN.The hyrcanus group of Anopheles (Anopheles) in China (Diptera ∶ Culicidae): species discrimination and phylogenetic relationships inferred by ribosomal DNA internal transcribed spacer 2 sequences[J]. J Med Entomol, 2005, 42(4): 610-619.
[10]	Gao Q, Beebe NW, Cooper RD.Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera ∶ Culicidae) in central China using polymerase chain reaction and appraisal of their position within the hyrcanus group[J]. J Med Entomol, 2004, 41(1): 5-11.
[11]	Hwang UW.Revisited ITS2 phylogeny of Anopheles (Anopheles) hyrcanus group mosquitoes: reexamination of unidentified and misidentified ITS2 sequences[J]. Parasitol Res, 2007, 101(4): 885-894.
[12]	Min GS, Choochote W, Jitpakdi A, et al. Intraspecific hybridization of Anopheles sinensis (Diptera ∶ Culicidae) strains from Thailand and Korea[J]. Mol Cells, 2002, 14(2): 198-204.
[13]	Ma YJ, Qu FY, Cao YC, et al. On molecular identification and taxonomic status of Anopheles lesteri and Anopheles anthropophagus in China (Diptera ∶ Culicidae)[J]. Chin J Parasitol Parasit Dis, 2000, 18(6): 325-328. (in Chinese)(马雅军, 瞿逢伊, 曹毓存, 等. 我国雷氏按蚊和嗜人按蚊的分子鉴别和分类地位的探讨[J]. 中国寄生虫学与寄生虫病杂志, 2000, 18(6): 325-328.)
[14]	Ma Y, Ma YJ, Meng XM, et al. Molecular identification of Anopheles hyrcanus group from Liaoning Province, China[J]. Acta Parasitol Et Med Entomol Sin, 2009, 16(4): 224-227. (in Chinese)(马颖, 马雅军, 孟祥梅, 等. 我国辽宁省赫坎按蚊种团的分子鉴别研究[J]. 寄生虫与医学昆虫学报, 2009, 16(4): 224-227.)
[15]	Li JX, Song ZH, Piao GM.Species identification of Anopheles mosquitoes from the Tumen river basin in Yanbian area of Jilin Province based on morphological and molecular characteristics[J]. Chin J Vector Biol Control, 2018, 29(1): 42-46. (in Chinese)(李基旭, 宋振海, 朴光明. 吉林省延边地区图们江流域按蚊形态学及基因特征分析[J]. 中国媒介生物学及控制杂志, 2018, 29(1): 42-46.)
[16]	Lin L, Yang MN, Nan CY, et al. Molecular taxonomy of Anopheles hyrcanus group in some regions of China[J]. Chin J Vector Biol Control, 2013, 24(2): 92-97. (in Chinese)(林琳, 杨曼尼, 南春燕, 等. 中国部分地区赫坎按蚊种团的分子鉴别研究[J]. 中国媒介生物学及控制杂志, 2013, 24(2): 92-97.)
[17]	Ma YJ, Chen Z, Zhang LT, et al. Anopheline biodiversity and dynamic state of mosquito population in Donggang, Liaoning[J]. Int J Med Parasit Dis, 2006, 33(2): 57-60. (in Chinese)(马雅军, 陈哲, 张立田, 等. 辽宁省东港市按蚊多样性和蚊虫种群动态变化研究[J]. 国际医学寄生虫病杂志, 2006, 33(2): 57-60.)
[18]	Chen XX, Li SZ, Deng XL, et al. Molecular identification of Anopheles hyrcanus complex in Shandong Province, China[J]. Chin J Parasit Dis Control, 2003, 16(4): 232-234. (in Chinese)(陈锡欣, 李石柱, 邓绪礼, 等. 山东省赫坎按蚊复合体成员种的分子鉴别研究[J]. 中国寄生虫病防治杂志, 2003, 16(4): 232-234.)
[19]	Shin EH, Lee WG, Chang KS, et al. Black light trap collections in Wangging County and Yanji City, Jilin Province, China, August 2006-2007[J]. Entomol Res, 2011, 41(1): 36-38.
[20]	Wang JF, Zhang TL, Fu BJ.A measure of spatial stratified heterogeneity[J]. Ecol Indic, 2016, 67: 250-256.
[21]	Wang Y, Yu WQ, Shi H, et al. Historical survey of the kdr mutations in the populations of Anopheles sinensis in China in 1996-2014[J]. Malar J, 2015, 14: 120.
[22]	Xu TL, Zhong DB, Tang LH, et al. Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements[J]. Parasit Vectors, 2014, 7: 54.
[23]	Chang XL, Zhong DB, Lo E, et al. Landscape genetic structure and evolutionary genetics of insecticide resistance gene mutations in Anopheles sinensis[J]. Parasit Vectors, 2016, 9: 228.
[24]	Ma YJ, Yang MN, Fan Y, et al. Population structure of the malaria vector Anopheles sinensis (Diptera ∶ Culicidae) in China: two gene pools inferred by microsatellites[J]. PLoS One, 2011, 6(7): e22219.
[25]	Makhawi AM, Liu XB, Yang SR, et al. Genetic variations of ND5 gene of mtDNA in populations of Anopheles sinensis (Diptera ∶ Culicidae) malaria vector in China[J]. Parasit Vectors, 2013, 6: 290.
[26]	Zhang HW, Liu Y, Hu T, et al. Knockdown resistance of Anopheles sinensis in Henan Province, China[J]. Malar J, 2015, 14: 137.
[27]	Zhu GD, Zhou HY, Li JL, et al. The colonization of pyrethroid resistant strain from wild Anopheles sinensis, the major Asian malaria vector[J]. Parasit Vectors, 2014, 7: 582.
[28]	Chang KS, Yoo DH, Ju YR, et al. Distribution of malaria vectors and incidence of vivax malaria at Korean army installations near the demilitarized zone, Republic of Korea[J]. Malar J, 2016, 15: 259.
[29]	Kang S, Jung J, Kim W.Population genetic structure of the malaria vector Anopheles sinensis (Diptera ∶ Culicidae) sensu stricto and evidence for possible introgression in the Republic of Korea[J]. J Med Entomol, 2015, 52(6): 1270-1281.
[30]	Foley DH, Klein TA, Lee IY, et al. Mosquito species composition and Plasmodium vivax infection rates on Baengnyeong-do (island), Republic of Korea[J]. Korean J Parasitol, 2011, 49(3): 313-316.
[31]	Kim HC, Rueda LM, Wilkerson RC, et al. Distribution and larval habitats of Anopheles species in northern Gyeonggi Province, Republic of Korea[J]. J Vector Ecol, 2011, 36(1): 124-134.
[32]	Khrabrova NV, Perevozkin VP, Andreeva YV, et al. Species composition of the mosquito Anopheles hyrcanus (Diptera ∶ Culicidae) group in the Russian far east[J]. J Vector Ecol, 2012, 37(2): 450-452.
[33]	Khrabrova NV, Andreeva YV, Sibataev AK, et al. Mosquitoes of Anopheles hyrcanus (Diptera ∶ Culicidae) group: species diagnostic and phylogenetic relationships[J]. Am J Trop Med Hyg, 2015, 93(3): 619-622.
[34]	Joshi D, Park MH, Saeung A, et al. Multiplex assay to identify Korean vectors of malaria[J]. Mol Ecol Resour, 2010, 10(4): 748-750.
[35]	Baimai V, Rattanarithikul R, Kijchalao U.Metaphase karyotypes of Anopheles of Thailand and Southeast Asia: Ⅰ. The hyrcanus group[J]. J Am Mosq Control Assoc, 1993, 9(1): 59-67.
[36]	Park MH, Choochote W, Kim SJ, et al. Nonreproductive isolation among four allopatric strains of Anopheles sinensis in Asia[J]. J Am Mosq Control Assoc, 2008, 24(4): 489-495.
[37]	Joshi D, Choochote W, Min GS.Short report: natural hybrid between Anopheles kleini and Anopheles sinensis[J]. Am J Trop Med Hyg, 2009, 81(6): 1020-1022.
[38]	Choochote W, Min GS, Intapan PM, et al. Evidence to support natural hybridization between Anopheles sinensis and Anopheles kleini (Diptera ∶ Culicidae): possibly a significant mechanism for gene introgression in sympatric populations[J]. Parasit Vectors, 2014, 7: 36.
[39]	Harrison BA, Scanlon JE.The subgenus Anopheles in Thailand (Diptera ∶ Culicidae)[J]. Contr Amer Ent Inst, 1975, 12(1): 45-50.
[40]	Tanaka K, Mizusawa K, Saugstad ES.A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawara Island) and Korea (Diptera ∶ Culicidae)[J]. Contr Am Entomol Ins, 1979, 16(1): 987.
[41]	Foley DH, Klein TA, Kim HC, et al. Geographic distribution and ecology of potential malaria vectors in the Republic of Korea[J]. J Med Entomol, 2009, 46(3): 680-692.
[42]	Joshi D, Kim JY, Choochote W, et al. Preliminary vivax malaria vector competence for three members of the Anopheles hyrcanus group in the Republic of Korea[J]. J Am Mosq Control Assoc, 2011, 27(3): 312-314.
[43]	Lee WJ, Klein TA, Kim HC, et al. Anopheles kleini, Anopheles pullus, and Anopheles sinensis: potential vectors of Plasmodium vivax in the Republic of Korea[J]. J Med Entomol, 2007, 44(6): 1086-1090.
[44]	Ubalee R, Kim HC, Schuster AL, et al. Vector competence of Anopheles kleini and Anopheles sinensis (Diptera∶ Culicidae) from the Republic of Korea to vivax malariainfected blood from patients from Thailand[J]. J Med Entomol, 2016, 53(6): 1425-1432.
[45]	Lu BL.Zoology of China, insecta, volume Ⅷ Diptera, Culicidae (Book 1) [M]. Beijing: Dcience Press, 1997. (in Chinese).(陆宝麟. 中国动物志昆虫纲第八卷双翅目蚊科(上卷)[M]. 北京: 科学出版社, 1997.)
[46]	Feng XY, Zhang SS, Huang F, et al. Biology, bionomics and molecular biology of Anopheles sinensis Wiedemann 1828 (Diptera : Culicidae), main malaria vector in China[J]. Front Microbiol, 2017, 8(1): 1473.