Landscape genetic structure and evolutionary genetics of insecticide resistance gene mutations in Anopheles sinensis.
BACKGROUND
Anopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis.


METHODS
Adults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns.


RESULTS
Three non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection.


CONCLUSIONS
Our results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.
Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, Anhui 233000, China.|Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.
Xuelian
X
Chang
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA. dzhong@uci.edu.
Daibin
D
Zhong
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.
Eugenia
E
Lo
Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, Anhui 233000, China. fq333@sohu.com.
Qiang
Q
Fang
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.
Mariangela
M
Bonizzoni
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.|Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Education Institutes, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
Xiaoming
X
Wang
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.
Ming-Chieh
MC
Lee
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA.
Guofa
G
Zhou
Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, Anhui 233000, China.
Guoding
G
Zhu
Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Education Institutes, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
Qian
Q
Qin
Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Education Institutes, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
Xiaoguang
X
Chen
Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA.
Liwang
L
Cui
Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA. guiyuny@uci.edu.|Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Education Institutes, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China. guiyuny@uci.edu.
Guiyun
G
Yan
D063147:Animal Distribution; D000818:Animals; D000852:Anopheles / Q000187:drug effects* / Q000235:genetics*; D005075:Biological Evolution; D002681:China; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007305:Insecticide Resistance / Q000235:genetics*; D009154:Mutation
D004272:DNA, Mitochondrial; D019476:Insect Proteins; D045304:Cytochromes c
Anopheles sinensis; Evolution; Knockdown resistance; Mitochondrial DNA; Mutation; Para-type sodium channel gene