Historical survey of the kdr mutations in the populations of Anopheles sinensis in China in 1996–2014

In China, DDT was largely used in 1950s-1970s, and pyrethroids have been utilized since 1980s when DDT was banned [27]. The applications of pyrethroids for IRS and ITNs have greatly contributed to the success of reducing malarial transmission. The occurrence of pyrethroid resistance in An. sinensis has been documented in many regions since 1980s [28,29]. For example, in Zhejiang [30], Hubei [31], Jiangsu [32], Shandong [33], Yunnan [34], Henan [34], Fujian [35], and Hainan [36]. Recently, Wang et al. conducted a survey of pyrethroid susceptibility in the An. sinensis populations from eight malaria endemic regions including Hubei, Henan, Hunan, Jiangsu, Jianxi, Sichuan, Shanghai and Yunnan [37]. They found that the An. sinensis populations in all examined regions were resistant to deltamethrin. The mortalities were in a range of 5.96-64.54% upon exposure to the diagnostic concentration of 0.25% deltamethrin [37]. The relationship of kdr genotypes and pyrethroid resistance has been investigated in recent years. At codon 1014 of the VGSC gene, five mutant alleles, TTT(F), TTG(F), TGT(C), TCG(S) and TGG(W), have been detected. The TTT(F) allele was the most prevalent mutant, followed by the alleles TGT(C) and TCG(S); the allele TGG(W) occurred rarely [7,16-19,21,34,38,39]. The kdr alleles were present with high frequency largely in the populations in central China, such as Jiangsu, Anhui, Shandong, Hubei, and Henan. For example, Qi and Cui detected the kdr alleles 1014F and 1014C in three populations from Henan, and the kdr allele frequency was associated with the resistant phenotype. However, no kdr alleles were detected in the two populations from Yunnan [34]. In the five populations collected from Jiangsu in 2009–2010, the kdr alleles 1014F and 1014C were detected and the frequency of allele 1014F was correlated with the resistance to beta-cypermethrin in these populations [16]. In the Guangxi collections, the kdr alleles 1014S, 1014F and 1014W were found [20]. In an investigation reported by Zhong et al. the kdr alleles 1014F and 1014C were present with high frequency (88.5-94.8%) in the resistant populations from Hunan, Hubei and Jiangsu in central China [17]. Both kdr alleles and the monooxygenase activity were significantly associated with the deltamethrin resistance, but the monooxygenase activity played a stronger role. On the other hand, no kdr alleles were detected in the two resistant populations in Yunnan, where the resistance was correlated with the monooxygenase activity [17]. A similar pattern was found in another study, in which the kdr mutation L1014F (70.0-88.9%) and L1014C (11.1-26.7%) were detected in the Anhui populations with higher frequency in the resistant mosquitoes [19]. Again, no kdr alleles were found in the mosquitoes from Yunnan. Based on a CART statistical analysis, metabolic detoxification enzymes (monooxygenases, glutathion S-transferase and carboxylesterases) played major roles in resistance to pyrethroids and DDT while kdr alleles weighed less in the context [19]. In Hainan Island, the L/F heterozygotes were present with low frequency (6.7-9.5%) in the DDT and pyrethroid-resistant individuals of the two An. sinensis populations, but no kdr alleles were detected in the sympatric An. vagus [18]. Overall, aforementioned studies demonstrated that the kdr mutations occurred largely in the An. sinensis populations in central China, where the resistance to DDT and pyrethroids was conferred primarily by the metabolic detoxification mechanisms as well as the kdr mutation. In the populations in other regions, the kdr alleles were less prevalent, and the pyrethroid resistance was conferred by the metabolic mechanisms.

In the current study, codon 1014 was examined in a large collection of the An. sinensis samples covering 17 provinces in a time span of 1996 to 2014. The kdr alleles 1014C and 1014F were found in the specimens sampled from Jiangsu and Shandong as early as 1997 (Additional file 1: Table S1). This clearly indicated that the kdr alleles already existed in the An. sinensis populations in 1990s. In line with the findings in the other reports mentioned above, the kdr alleles were more prevalent in central China. The occurrence of kdr alleles has been progressively increasing over time (Figures 1 and 2). For example, in the samples from Shandong that were collected in 1997, 2007, and 2012, the mutation frequency increased from 33.3% in 1997, 95.45% in 2007 to 100% in 2012 (Table 3). In the other regions, the kdr genotypes frequency was lower in Sichuan (0 in 1997 and 60.78% in 2014). Moreover, no kdr alleles were detected in the samples from Yunnan (2005–2013, except ZT06), Guangxi (2005), Fujian (1997), and Hainan (1997–2013) (Table 4). Apparently the pyrethroid resistance in these regions was conferred majorly by the mechanisms other than kdr mutations. Similar situations have been reported in An. gambiae in Africa. In south-western Nigeria, the kdr alleles were not found in the pyrethroid-resistant individuals of the molecular M form of An. gambiae [40]. In a survey conducted in south-western Chad, central Africa, the allele 1014F was frequently present in the resistant S form of An. gambiae, but was not found in the M form or An. arabiensis [41]. The frequent occurrence and accumulation of the kdr alleles in An. sinensis in central China may be explained by their large population size and wide distribution range. The mutant alleles may have had better chances to be selected and maintained in the An. sinensis populations in central China.

In this study, no kdr alleles were detected in all 232 specimens of the other eight member species of the An. hyrcanus group, including 167 specimens of An. lesteri. This resembles a finding reported by Kang et al. [42], in which the kdr alleles were only found in An. sinensis, not in the other five species (Anopheles pullus, Anopheles kleini, An. lesteri and An. belenrae) in the An. hyrcanus group from the Republic of Korea [42]. As a primary malaria vector in China, An. lesteri (previously known as An. anthropaphagus, which was rectified as An. lesteri based on the rDNA ITS2 sequences [23]), has been a major target of the vector control programmes. The extensive implementation of IRS and ITNs in 1990s-2010s has led to a remarkable reduction in An. lesteri abundance in China, which has resulted in a significant drop in malaria morbidity [43-45]. It is intriguing that the low kdr occurrence in some An. sinensis populations and absence in An. lesteri (in this study) given that both mosquitoes had been exposed to strong pyrethroid pressure. Further study is needed to investigate the resistant status and underlying mechanisms in these populations.