Background
Anopheles sinensis is a major vector of malaria in China and countries of Southeast Asia. Chemical control of vector has played an important role in malaria control and elimination [
1]. In China, DDT has been widely used for indoor residual spray (IRS) since 1950s, and pyrethroids for insecticide-treated bed nets (ITNs) since 1980s [
2]. Unfortunately, long-term and large-scale use of pyrethroids has led to increasing insecticide resistance in Chinese
An. sinensis [
3,
4], which poses a major threat to malaria control.
The voltage-gated sodium channel protein is the major target for pyrethroids and DDT [
5]. Although there have been debates [
6‐
8], many studies have demonstrated that mutations at codon 1014 of
VGSC cause resistance to both pyrethroids and DDT in many arthropod species [
5,
6]. 1014F/S/H mutations can reduce sodium channel sensitivity to pyrethroids in
Xenopus oocytes [
9‐
15], and provide protection to pyrethroids and DDT [
9]. In
An. sinensis, significant positive correlations between
kdr allele frequency and bioassay-based resistance phenotype have been documented [
16‐
19]. Recent years,
kdr mutation has been used as a molecular mark for monitoring pyrethroid resistance in
An. sinensis in China [
19].
Guangxi Zhuang Autonomous Region was once a malaria-endemic region. Before 1949, there were more than 5 million malaria patients per year [
20]. Thanks to the “National Malaria Control Programme”, the “Basically Eliminating Malaria” strategy and the “Action Plan of Malaria Elimination (2010–2020)” implemented since 1955, 2000, and 2010 respectively in China [
21,
22], the malaria burden has been substantially reduced, with one indigenous and 2068 imported cases being reported from 2010 to 2015 in Guangxi [
22]. Although Guangxi has already achieved remarkable accomplishments in eliminating malaria, the risk of malaria re-emergence remains partly due to increasing cross-border population migration and the unique natural environment (e.g. rice fields) suitable for mosquito breeding in Guangxi [
22‐
25].
As the historical use in vector control and continuing use in agriculture of different insecticides, not only the geographical distribution and density of malaria vectors are likely to change, insecticide resistance is expected to be selected as well. Recent investigations have shown that
An. sinensis has replaced
Anopheles minimus to be the main malaria vector in Guangxi [
24]. However, the status of insecticide resistance in
An. sinensis is less understood in this region up to date. As an effort in this direction, the distribution and frequency of
VGSC mutations that possibly lead to resistance to DDT and pyrethroids were investigated in nine field populations of
An. sinensis collected extensively across Guangxi in this study.
Discussion
Sequence analysis reveals that the sodium channel gene of
An. sinensis has diverse genetic mutations. From 313 mosquitoes collected from Guangxi, 16 haplotypes were identified (Tables
1,
2). The number and frequency of
AS-
VGSC haplotypes are different among the nine populations from Guangxi. Overall, the 1014L1, 1014L2 and 1014C1 rank the top three haplotypes. Some haplotypes, for example 1014L2, 1014L3 and 1014L8, are widespread, and some haplotypes such as 1014L7 are restricted to a particular location (Table
2). Less number of haplotypes detected in Hezhou and Guigang may be related to the relative small sample size and/or strong selective pressure leading to 1014C1 in a high frequency. The high polymorphism of
AS-
VGSC may be partly explained by their large population size, wide distribution range of
An. sinensis [
14], diverse natural landscapes and different local insecticide selective pressure.
Three non-synonymous mutations (TTT for 1014F, TGT for 1014C and TCG for 1014S) were identified in this work. However, the TTC-encoding 1014F, previously found in Korea [
32] and in Anhui and Sichuan of China [
19], was not detected in Guangxi. The presence of L1014W substitution was reported based on the direct sequencing data of only one
An. sinensis heterozygote from Guiping of Guangxi [
17]. This result is questionable and needs reconfirmation, because 1014F/W is not the only possibility encoded by the individual genotype T(T/G)(T/G). In this and another study [
33], sequencing of cloned fragment with the T(T/G)(T/G) template resolved into two alleles, TTG and TGT, encoding 1014L/C rather than 1014F/W.
Differential
kdr distribution patterns were observed in the nine examined
An. sinensis populations from Guangxi. Overall, haplotypes carrying 1014C mutation are widespread and present in high frequencies in the northeast, while 1014S is rare and distributed in the west (Table
1; Fig.
2). Notably, four of the nine populations with
kdr allele frequencies higher than 50 % were located in the northeast of Guangxi, while only 1014S in a frequency less than 5 % was detected in the two western populations. Geographically, the frequency of
kdr mutations decreased towards south and west from northeast. This pattern is in keeping with previous observations that a high
kdr mutation frequency was detected in the central China which is located northeast to Guangxi [
19,
34,
40], and
kdr alleles were less prevalent in Yunnan and Hainan which are in the west and south to Guangxi respectively [
16,
18,
19,
34,
40].
Considering the fact that haplotype 1014C1 and 1014F1, detected in samples of this study, are also distributed in central China (Table
1), it is possible that migration of
An. sinensis population from central China through Hunan and Guizhou, may contribute to the occurrence of 1014C1 and 1014F1 in the northeast of Guangxi. The obvious distinction in the geographic distribution of each allele between populations in the west and in the northeast is likely a combined consequence of independent mutational events in different geographic locations and the geographical barriers limiting gene flow imposed by the mountainous landscapes of Guangxi. Theoretically, selective pressure may play a role in shaping the frequency of insecticide resistance-associated mutation in a population. However, it was not possible to assess the contribution of local selection of insecticides to the existing
kdr distribution pattern because application history of insecticides was not known for samples used in this study.
Previous studies have documented that the predominant
kdr allele is L1014 F [
4,
12,
13,
16,
19,
33‐
35,
40], the high frequency of 1014C1 in the five
An. sinensis populations in northeast of Guangxi (Fig.
3) is unexpected. 1014C was present in
An. sinensis samples collected in Anhui, Guizhou, Henan, Hubei, Hunan, Jiangsu and Shandong of China [
16,
19,
34,
40] and in Korea [
33], and was also found in
VGSC of other mosquito species, such as
Culex pipiens pipiens from China [
36] and
Anopheles albimanus from Mexico and Nicaragu [
37]. These observations indicate that 1014C is a conserved mutation in mosquitoes and widely distributed. Why 1014C1 is prevalent in the five populations remains to be investigated. One possibility is that 1014C1 may provide a better protection, or/and a lower fitness cost, than other
kdr alleles such as the classical 1014F.
The geographic distribution pattern and the genealogical analysis of
kdr haplotypes strongly suggests that
kdr mutations are not singly originated. For example, there are at least two independent mutation events giving rise to 1014F or 1014S haplotypes from a wild haplotype through a single mutational step (Table
1; Fig.
3). Multiple origins of resistance alleles via point mutations at the voltage-gated sodium channel gene have also been characterized in other insect species [
38,
39]. Interestingly, 1014C1 and 1014F1 is co-distributed in Guangxi and share the same intron (Tables
1,
2). The network analysis indicates that only one mutational step is able to change 1014F1 to 1014C1 (Fig.
3). Based on these observations, it is hypothesized that 1014C1 may represent a
kdr allele evolved via a sequential progression from 1014F1 and perhaps has gradually replaced 1014F1.
Authors’ contributions
XHQ, CY and XYF conceived the study. CY performed the experiment and XYF identified the species. CY, ZSH and XHQ analyzed the data. XHQ, CY, ZSH and XYF wrote the paper. XYF and ML contributed to sample collection. All authors read and approved the final manuscript.