Plernsub Suriya, Saingamsook Jassada, Yanola Jintana, Lumjuan Nongkran, Tippawangkosol Pongsri, Sukontason Kom, Walton Catherine, Somboon Pradya
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Parasit Vectors. 2016 Jul 26;9(1):417. doi: 10.1186/s13071-016-1713-0.
Mutation in the voltage-gated sodium channel gene that results in knockdown resistance (kdr), is a major mechanism of pyrethroid resistance in several mosquito species. In Aedes aegypti, V1016G (occurring with and without S989P) and F1534C mutations are common and widely distributed throughout Asia. The G1016 allele is known to be associated with resistance to type I and II pyrethroids. The C1534 allele is primarily associated with resistance to type I pyrethroids and is known to be a recessive allele in conferring kdr.
We performed crossing experiments using a P989 + G1016 homozygous mutant strain (UPK-R), a C1534 homozygous mutant strain (PMD-R) and a pyrethroid susceptible strain (PMD) to determine the insecticide susceptibility of different genotypic hybrids. Allele-specific PCR methods were used to confirm the genotypes. Metabolic resistance caused by oxidative enzymes and esterase enzymes was ruled out by the addition of piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate, BNPP), respectively.
The median lethal concentration (LC50) of deltamethrin susceptibility of a S/P989 + V/G1016 + F/F1534 double heterozygous hybrid from the UPK-R × PMD cross was 0.57 (95 % CI: 0.51-0.63) μgl(-1), which was about 12-fold lower than for UPK-R, 6.98 (6.10-8.04) μgl(-1), and only about 4-fold greater than the susceptible PMD, 0.13 (0.12-0.15) μgl(-1). This resistance returned to 0.08 (0.07-0.09) μgl(-1) on the addition of PBO suggesting that the P989 + G1016 kdr alleles are recessive. The LC50 of the S/P989 + V/G1016 + F/C1534 triple heterozygous hybrid was 3.58 (3.21-3.95) μgl(-1), which was intermediate between that of the homozygous mutant genotypes, being 2-fold higher than the C1534 homozygote and 2-fold lower than the P989 + G1016 homozygote. These minor differences and the high LC50 values of the triple mutated heterozygote indicate there is some degree of functional equivalence of the P989 + G1016 and C1534 alleles in the heterozygote. Addition of PBO decreased the LC50 values by 2-fold, from 3.58 (3.21-3.95) to 1.52 (1.35-1.73) μgl(-1), suggesting that oxidase enzymes play a partial role in resistance. The results are consistent with the median lethal time (LT50) of the triple mutated heterozygote against 0.05 % deltamethrin paper. An adult susceptibility test also revealed that the triple mutated heterozygote was resistant to deltamethrin and permethrin.
The combination of the three kdr alleles in the triple mutated heterozygote, S/P989 + V/G1016 + F/C1534, confers high resistance to pyrethroids. This heterozygous form is common in Ae. aegypti populations throughout Thailand and may have an adverse effect on the efficacy of a mosquito control program using insecticide-based approaches.
电压门控钠通道基因突变导致击倒抗性（kdr），是几种蚊虫对拟除虫菊酯产生抗性的主要机制。在埃及伊蚊中，V1016G（伴有或不伴有S989P）和F1534C突变很常见，且在亚洲广泛分布。已知G1016等位基因与对I型和II型拟除虫菊酯的抗性有关。C1534等位基因主要与对I型拟除虫菊酯的抗性有关，且已知在赋予kdr抗性方面是隐性等位基因。
我们使用P989 + G1016纯合突变株（UPK - R）、C1534纯合突变株（PMD - R）和拟除虫菊酯敏感株（PMD）进行杂交实验，以确定不同基因型杂种的杀虫剂敏感性。采用等位基因特异性PCR方法确认基因型。分别添加胡椒基丁醚（PBO）和双（4 - 硝基苯基）磷酸酯（BNPP）排除了氧化酶和酯酶引起的代谢抗性。
UPK - R×PMD杂交产生的S/P989 + V/G1016 + F/F1534双杂合杂种对溴氰菊酯的半数致死浓度（LC50）为0.57（95%置信区间：0.51 - 0.63）μg l-1，比UPK - R的6.98（6.10 - 8.04）μg l-1低约12倍，仅比对敏感的PMD的0.13（0.12 - 0.15）μg l-1高约4倍。添加PBO后，这种抗性恢复到0.08（0.07 - 0. )9）μg l-1，表明P989 + G1016 kdr等位基因是隐性的。S/P989 + V/G1016 + F/C1534三杂合杂种的LC50为3.58（3.21 - 3.95）μg l-1，介于纯合突变基因型之间，比C1534纯合子高2倍，比P989 + G1016纯合子低2倍。这些微小差异和三突变杂合子的高LC50值表明，P989 + G1016和C1534等位基因在杂合子中有一定程度的功能等效性。添加PBO使LC50值降低了2倍，从3.58（3.21 - 3.95）降至1.52（1.35 - 1.73）μg l-1，表明氧化酶在抗性中起部分作用。结果与三突变杂合子对0.05%溴氰菊酯纸片的半数致死时间（LT50）一致。成虫敏感性试验还表明，三突变杂合子对溴氰菊酯和氯菊酯具有抗性。
三突变杂合子S/P989 + V/G1016 + F/C1534中三种kdr等位基因的组合赋予了对拟除虫菊酯的高抗性。这种杂合形式在泰国各地的埃及伊蚊种群中很常见，可能会对使用基于杀虫剂的方法进行的蚊虫控制计划的效果产生不利影响。
