中华按蚊对拟除虫菊酯类杀虫剂击倒抗性（knockdown resistance, kdr）的机理研究
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摘要
过去几十年来,DDT和拟除虫菊酯类杀虫剂被大量用来浸泡蚊帐、滞留喷洒灭蚊防疟,随着拟除虫菊酯类杀虫剂的广泛、长时间的使用,媒介昆虫的抗药性日趋明显。对媒介昆虫抗性监测显示,昆虫对杀虫剂的抗药性问题日益突出,部分地区已到了严重制约杀虫剂有效使用的程度。为了加强抗性种群的治理,指导合理使用杀虫剂,延缓抗性的发生发展,迫切需要对媒介昆虫的抗药性及其机制进行更深入的研究。中华按蚊作为我国和东南亚地区重要的疟疾媒介,其抗性研究过去主要集中在生物抗性的发生发展,对其分子机制的研究较少。本研究以野外采集的16个中华按蚊种群为研究对象,在现场测定了其生物抗性,在实验室运用分子生物学方法,研究了中华按蚊自然种群对拟除虫菊酯类杀虫剂击倒抗性的机理,以中华按蚊七个微卫星DNA位点为遗传标记,分析遗传学参数与kdr频率的相关性,推测中华按蚊种群kdr抗性基因频率的现状及发展趋势。获得了如下的结果:
1.中华按蚊幼虫和成蚊抗药性的生物测定
1.1幼虫致死中浓度(LC50)测定
对5个幼虫种群的生物测定结果显示,从徐州、淮阴、南京、常熟和苏州采集的5个中华按蚊幼虫种群均为高抗种群,其对高效氯氰菊酯的致死中浓度(LC50)分别为0.894 mg/L、1.264 mg/L、1.670mg/L、2.067 mg/L和0.677 mg/L,实验室敏感种群致死中浓度测试结果为0.001 mg/L,抗性种群的抗药性指数分别为894、1264、1670、2067和677倍。
1.2成虫击倒中时(KT50)测定
11个种群的中华按蚊成蚊对三种杀虫剂的平均击倒中时从8.95min到47.62min不等;其中,对高效氯氰菊酸的击倒中时为8.95-26.87min;对溴氰菊酯的击倒中时为10.03-37.03min;对氯菊酯是击倒中时为16.13-47.62min。对高效氯氰菊酯抗药性指数从2.439至9.191倍,对溴氰菊酯抗药性指数从3.310至12.221倍,对二氯苯醚菊酯抗药性指数从3.226至9.524倍;从抗性种群的地理分布来看,镇江当涂种群(抗药性指数8.64-12.221)、潜山王河种群(抗药性指数7.322-9.524)的抗药性较高,丰县肖埝种群(抗药性指数3.226-3.703)、徐州云龙种群(抗药性指数2.439-4.444)的抗药性较低。
2.中华按蚊击倒抗性的研究
2.1敏感中华按蚊钠通道基因序列分析
根据已知近缘种群钠通道基因序列设计简并引物,采用RT-PCR,巢式PCR方法,蓝白斑筛选阳性克隆,提取阳性克隆的质粒送测序,获得中华按蚊钠通道部分cDNA序列,该序列包含L1014(kdr)和M918(super-kdr)经典突变位点。将cDNA序列翻译成氨基酸序列,在GeneBank中对序列同源性进行比较,发现其与GenBank中公布的淡色库蚊(Culex pipiens pallens)、埃及伊蚊(Aedes egypti)、冈比亚按蚊(Anopheles gambiae )、黑腹果蝇(Drosphila melanogaster)、家蝇(Musca domestica)及德国小蠊(Blattella germanica)的氨基酸序列具有较高的同源性,其同源性分别为98.3%, 98.3%, 98.3%, 96.7%, 94.2%和95.8%。确认了实验室繁殖的中华按蚊敏感株ⅡS4-ⅡS6区段的基因序列结构与特征,获得了358bp的特异序列,为编码中华按蚊钠通道的119个氨基酸残基,并将序列登录Genbank,登录号为:JN002364。
2.2抗性中华按蚊钠通道基因cDNA序列分析
根据已知的敏感株中华按蚊钠通道基因序列设计特异引物,采用RT-PCR方法,提取了三个野外按蚊种群ⅡS4-ⅡS 6区段的cDNA序列,部分直接测序,部分突变株用于单克隆测序,发现抗性中华按蚊的钠通道cDNA序列的1014位点氨基酸存在两种类型的突变,对应的cDNA序列存在两种类型的碱基改变,翻译成氨基酸后的突变类型为L1014F和L1014C。
2.3 AS-PCR检测方法的建立
根据中华按蚊钠通道基因组ⅡS4-ⅡS6序列(敏感及抗性),设计一对非特异性外引物CD1和CD2(扩增对照条带)。以1014位点第3个碱基为3’末端,分别设计三条特异性内引物Cgd3、Cgd4和Cgd5(扩增特异条带)。其中,Cgd3用于扩增非突变等位基因,Cgd4用于扩增突变F等位基因,Cgd5用于扩增突变C等位基因。AS-PCR设计为平行三管法。检测了50只中华按蚊的kdr分型,与直接测序相比,灵敏度和特异度都在94%以上。
2.4 PCR-RFLP检测方法的建立
根据中华按蚊突变位点特征(T[T/G][T/G]),通过在PCR引物引入突变碱基的办法,建立针对这两个突变碱基的限制性长度酶切法(PCR-RFLP),分别读出这两个碱基的突变类型。针对第一个突变位点的G/G型、T/T型、G/T型酶切后分别呈现196bp单一带,154bp和42bp两条带,196bp、154bp和42bp三条带,针对第二个突变位点的T/T型、G/G型、G/T型酶切后分别呈现155bp单一带,123bp和32bp两条带,155bp、123bp和32bp三条带,结合两个碱基的突变类型即可得出该氨基酸位点(L1014)的突变类型。本研究建立的PCR-RFLP方法,检测了50只中华按蚊,与直接测序相比,对kdr基因突变的检出灵敏度和特异度都在96%以上。
2.5 Taqman-MGB检测方法的建立
针对突变位点的三种突变类型,设计三条TaqMan-MGB探针,分别与含有TTG/TTT/TGT三种突变类型的等位基因序列相结合,在突变位点的上下游分别设计两条非特异引物,组成TaqMan-MGB探针法检测体系。结果显示,对50只中华按蚊的kdr突变的检测灵敏度和特异度都在98%以上。
2.6中华按蚊kdr基因频率的测定
2.6.1中华按蚊幼虫组kdr基因频率的测定
利用AS-PCR法,对5个中华按蚊自然种群和一个中华按蚊敏感种群进行突变类型的测定。实验室敏感株在L1014位点没有发现突变。徐州、淮阴、常熟、南京和苏州5个中华按蚊种群L1014F的频率分别为75%、75%、77.27%、75.51%和84.00%,L1014C的频率分别为19.64%、23.68%、21.59%、23.47%和16.00%。在检测的5个种群中,敏感纯合子只在徐州按蚊中发现1只,频率为1.19%,L1014F和L1014C的敏感杂合子都很少;基因型频率以RR-F/F和RR/FC最高,分别为51.02%-69.33%,28.57%-46.94%;其他各种基因型的频率都在5%以下。
2.6.2中华按蚊成蚊组kdr基因频率的测定
中华按蚊成蚊试验所用的689只中华按蚊,运用AS-PCR法,进行了kdr基因频率的测定,在1014位点,L、F、C三种等位基因均被检出;主要的基因型是RR-F/F和RR-F/C,没有检出敏感的纯合子,RS-F/L和RR-F/C两种杂合子仅在少数种群检出,且基因型频率都很低(5%以下),只有沙庄和潘镇两个种群有RR-C/C基因型检出;沙庄kdr-F的突变频率最高,潘镇的kdr-C的突变频率最高;沙庄的RR-F/F基因型频率最高,云龙的RR-F/C基因型频率最高。
kdr-F的突变频率最高,为75.00%-88.54%,kdr-C的突变频率为9.36%-23.47%;L1014的频率范围为0-4.35%,在肖埝、丹阳和奔牛三个种群没有检出;RR-F/F的基因型频率为48.48%-79.17%,RR-F/C的基因型频率为14.58%-46.15%。
2.7哈迪-温伯格平衡检验
2.7.1幼虫抗性组的哈迪-温伯格平衡检验
根据各种群的kdr基因检测频率及孟德尔遗传规律,计算出该种群各种基因型的理论频率,与该种群kdr基因型频率的实际测得值,进行配对T检验,根据P值的大小,得出测得值与观察值之间差异的显著与否,通过对5个中华按蚊种群的检验,种群的P值为0.900-0.999,T值的范围为-0.001-0.014,双侧检验差异不显著,说明各种群测定的kdr基因型频率与预测值之间没有显著性差异,种群处于哈迪-温伯格平衡状态。
2.7.2成虫抗性组的哈迪-温伯格平衡检验
对成虫抗性相关的11个中华按蚊种群的哈迪-温伯格平衡检验结果显示,11个种群的P值分别为0.335(YL),0.23(XY),0.853(SZ),0.535(DT),0.998(DY),0.706(CS),0.997(BN),0.403(PZ),0.989(FC),1(CZ)和0.995(WH),T值的范围为-0.912-0.666,p值均大于0.05,双侧检验显示各种群等位基因频率的观察值和预测值之间差异不显著,提示种群现kdr基因型频率与预测值之间没有显著性差异,种群处于遗传平衡状态。
3.中华按蚊抗性水平与突变频率的相关性分析
3.1致死中浓度(LC50)与突变频率的关系
等级相关分析5个中华按蚊幼虫种群的抗性水平与L1014F、L1014C和RR-F突变频率的关系,均显示相关显著,P≤0.01;5个种群抗性水平与各突变频率和基因型频率之间的相关分析表明,kdr(F+C)、kdr-F、kdr-C、RR-F/F、RR-F/C与LC50之间的对数关系明显,R2分别为0.981、0.951、0.955、0.862和0.840。因此提示该等位基因突变(L1014F,L1014C)可以作为中华按蚊高效氯氰菊酯抗性的分子标记。
3.2击倒中时(KT50)与突变频率的关系
中华按蚊KT50与kdr等位基因突变频率和基因型频率的相关性分析结果显示,高效氯氰菊酯的击倒中时与kdr-F突变频率和RR-F/F基因型频率的相关性明显,相关系数分别为0.6195和0.6166,与kdr-F和kdr-C总的突变频率达到了0.6202;溴氰菊酯和二氯苯醚菊脂与kdr-F的相关性结果显示,不及前者高,其相关系数均为0.4662,二者与F和C总的突变频率也达到了0.4721。
4.中华按蚊微卫星序列分析
4.1荧光标记引物扩增及测序结果
根据文献针对中华按蚊15个微卫星位点的侧翼序列,设计引物,并在上游引物5’端用FAM, HEX和TAMARA荧光进行标记,PCR产物用毛细管法测定微卫星序列的多态性。结果表明AN1, AN2,AN5,AN11,AN12,AN13和AN15七个位点的条带清晰,测序结果可靠,可以用于微卫星数据分析。
4.2各微卫星位点的多态性
本研究10个地理种群7个微卫星座位平均多态信息含量为0.55(0.21-0.75),提示这7个基因座所能提供的遗传信息比较丰富。多态信息含量及杂合度较高的5个位点是AN1, AN2,AN5,AN11和AN15。
4.3哈迪-温伯格平衡检验
从对种群遗传结构的分析来看,7个微卫星位点的哈迪温伯格平衡检验表明,全部位点的杂合子过剩的分析均符合哈迪-温伯格平衡,而部分位点的杂合子缺失的哈迪-温伯格平衡分析不符合哈迪-温伯格平衡。
4.4各位点和种群的连锁不平衡分析
对不同种群的7个位点之间以及相同种群的不同位点之间的连锁不平衡分析表明,在每个位点上各种群之间为非连锁关系,各种群独立遗传;各位点之间也是非连锁关系,一个位点的基因型与另一个位点的基因型彼此独立(P > 0.05),配子之间自由组合,未显示对某种基因型有选择作用。
4.5基因流及其与kdr抗性关系
本研究所涉及的各自然种群的迁移率(Nm=1.12)较低,基因流与kdr基因突变频率相关性不明显(R2=0.2251,0.3086,0.3059),提示kdr基因突变可能主要来自杀虫剂的选择压力和种群内部突变个体之间的交流。用7个位点估计的固定系数F值结果显示:个体相对于全部种群的遗传差异是34.29%,其中来自种群间的是11.56%,来自种内的是25.75%。成对比较的种群间的差异表明,差异最大的DT种群和BN种群,为28.63%,差异最小的是XN和SZ种群,为1.6%。将成对种群间的基因流与kdr等位基因频率的差做散点图,江苏7个种群间的kdr基因频率的差异有随着基因流增大而减小的趋势,但这种趋势不明显,而安徽4个种群没有随着基因流增大而减小的趋势,全部种群的kdr等位基因频率的差异有随着基因流的增大而减小的趋势,但这种趋势更小,进一步说明了kdr基因主要来自种群内部突变个体之间的基因交流,选择压力是影响抗药性种群结构的主要因素。
Insectcides play a main role in control of major vectors of diseases. However, development of resistance in many insect species represents a significant and increasing thread to their continued effective use in the recent years. It is important and urgent to elucidate the resistance mechanism in order to slow down the spread and development of resistance of vector populations. Most studies on the resistance-related mechanism of Anopneles sinensis were mainly focused on the occurrence, development and biochemistry of the resistance. There are no research based on molecular biology of An.sinensis as it being very important vector species associated with malaria. In the past thirty years, the mainly use of DDT and pyrethroids insecticides for the impregnation of bet nets and indoor residual spraying in China caused the resistance increased rapidly. But there is no kdr resistance reported, as we all know that was associated with pyrethroids and DDT resistance. In this research, 16 resistance strains which had been collected in Jiangsu and Anhui provinces were studied, a biochemistry and three molecular methods have been adopted to find out the resistance-related mechanism and relationship between the para-knockdown frequencies and bioassay outcome. STR on seven locuses were analyzed. Here are the results.
1 Bioassay analyses on larval mosquitoes and adult mosquitoes.
1.1 Bioassay analyses on larval mosquitoes of An.sinensis.
Larval bioassay results on the five strains from XZ, HY, CS, NJ and SZ all showed resistance to beta-cypermethrin. LC50 of the five strains were 0.894 mg/L, 1.264 mg/L, 1.670mg/L, 2.067 mg/L and 0.677 mg/L,respectively. R/S were 894, 1264, 1670, 2067 and 677-folds,respectively.
1.2 Bioassay analyses on adult mosquitoes of An.sinensis.
Adult mosquitoes bioassay on the 11 strains from Jiangsu and Anhui provinces with beta-cypermethrin, deltapermethrin and permethrin showed the average KT50 were 8.95-47.62 min. KT50 to beta-cypermethrin was 8.95-26.87min. KT50 to deltamethrin was 10.03-37.03 min. KT50 to permethrin was 16.13-47.62 min. R/S of KT50 of the three kinds of pyrethroids to 11 populations ranged from 2.439- to 12.221-folds. The populations of DT and WH showed the higher R/S, they were 8.64-12.221-folds in DT population and 7.322-9.524-folds in WH population. The lower R/S occurred in XN and YL populations.
2. The action of knockdown resistance (kdr) in An.sinensis.
2.1 Sequence of the voltage-dependent sodium channel in An.sinensis from S strain.
A total 358bp of sodium channel gene had been amplified by RT-PCR, nest-PCR and sequenced from Ss strain. It has been demonstrated that 119 amino acid are encoded including four homologous domains and the hydrophilic linking peptides of varying length without 3` UTR. Although it is incomplete in this para-like cDNA sequence in An. sinensis, but it is long enough to find out the kdr mutation in An.sinensis because it contains all of the mutations loci of L1014 and M989 that have been reported to be related with kdr. It was found that the closest sequence homology to the Culex pipes pallens sodium channel with 98.3%, and with 96.7% sequence identity with Drosophila para, and with 94.2% sequence identity with M domestica para-like (Vssc1),and with 98.3% sequence identity with An.gambiae(para), and with 95.8% sequence identity with B germanica para-lik sodium channel.
2.2 cDNA sequence analysis of the sodium channel of the beta-pyrethroid-resistance strain of An.sinensis.
The cDNA sequence of IIS4-IIS6 in which kdr mutation located and the partial cDNA sequence have been analyzed from the resistance specimen. Resistance-related mutation of L1014F and L1014C had been found.
2.3 Method of AS-PCR was adapted to detect the para-knockdown resistance mutations.
Method of AS-PCR was adapted to detect the L1014F ,L1014C mutation in sodium channel . 263bp longer fragment amplified by two outer non allele-specific primers CD1 and CD2. The special primers of Cgd3, Cgd4, Cgd5 were used to detect the three alleles of TTG, TTT, TGT in three parallel tubes with the anti-sense primer CD2 to amplify a 169bp fragment. The sensitivity and specificity of this genotype method was showed to exceed 94%.
2.4 Methods of PCR-RFLP were adapted to detect the para-knockdown resistance mutations.
According to the mutation sites, after the first PCR based on the primers of C→T submission and digestion with HindⅢ, the presence of sequence with a (G/G) was inferred by the appearance of a single 196-bp fragment, a (T/T) was inferred by the appearance of a 154-bp and a 42-bp fragment and a (G/T) was inferred by the three fragments of 196-bp, 154-bp and 42-bp. In the same way, the second (T/G) involving (T/T),(G/G),(G/T) were inferred by the three fragments of 155-bp, 123-bp and 32-bp(Figure 3). Direct sequencing was used for validating the results of cPASA and PCR-FRLP. The sensitivity and specificity of this genotype method was showed to exceed 96%.
2.5 Methods of TaqMan-MGB were adapted to detect the para-knockdown resistance mutations.
According to the mutation sites of the para-knockdown resistance gene, three Probees were desired with a Report group of FAM and VIC in the 5’,a Quencher group and MGB group in the 3’, to detect the three sequences with the 1014 loci of TTG, TTT, TGT. Two non-specific primers were desired overstriding the para-kdr mutations sites. The high sensitivity and specificity of this genotype method leaded to the 99% in sensitivity and 100% in specificity.
2.6 Assay of the kdr mutation frequencies and genotyping frequencies.
3.6.1 Assay of the kdr mutation frequencies and genotyping frequencies on the larval mosquitoes.
In the larval group, a total of five resistance populations from XZ, SZ, CS, NJ and HY cities rural and an Ss population, 290 specimens were tested with cPASA. Kdr frequencies ranged from 16.01% to 84.00%. The kdr alleles existed mainly in kdr-F/F and kdr-F/C genotype, the resistant homozygous form, and only a small portion (1.33%-3.57%) of the mosquitoes possessed kdr-C homozygous (RR-C) kdr alleles, 2.63%-4.76% of the kdr-C and kdr-F heterozygous (RS-C and RS-F) kdr genotype on the five populations. Kdr allelic frequency ranged from 73.68% to 84.00% on the kdr-F, and 16.01% to 23.68% on kdr-C. The genotype frequency of RR-F and RR-F/C ranged from 51.02% to 69.33%, 28.57% to 46.94 respectively.
2.6.2 Assay of the kdr mutation frequencies and genotyping frequencies on the adult mosquitoes.
A total of 689 An.sinensis specimens were genotyped in AS-PCR method. There did also co-exist the co-occurring knockdown resistance mutations in the eleven strains. There were mainly two genotype exist RR-F/F and RR-F/C. No Ss genotype was found. The para-knockdown resistance mutation frequencies of KDR-F and KDR-C ranged from 75.00%-88.54% and 9.36%-23.47%, respectively. No s-allele on 1014 loci was found in XN, DY and BN strains. Genotyping frequency ranged from 14.58% to 79.17%, which including 48.48%-79.17% in RR-F/F and 14.58%-46.15% in RR-F/C. The highest KDR-F and KDR-C frequencies were found in SZ and PZ population. The highest genotype frequency of RR-F/F and RR-F/C were found in SZ and YL populations.
2.7 Hardy-Weinberg equilibrium test
2.7.1 Hardy-Weinberg equilibrium test of larval mosquito group.
Hardy-Weinberg equilibrium test was accepted for kdr alleles in each population using the T-paired test, the unbiased estimate of P-value of the five populations were 0.989(XZ), 0.930(HY), 0.991(NJ), 0.999(CS), 0.900(SZ), respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The five populations were all present genetic equilibrium.
2.7.2 Hardy-Weinberg equilibrium test of adult mosquito group.
Hardy-Weinberg equilibrium test was also accepted for kdr alleles in the adult mosquito population using the T-paired test, the unbiased estimate of P-value of the eleven populations were 0.335(YL),0.23(XN),0.853(SZ),0.535(DT),0.998(DY),0.706(CS),0.997(BN),0.403(PZ),0.989(FC),1(CZ),0.995(WH),respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The eleven populations were all present genetic equilibrium. The P-value of YL and XN populations showed the two populations were in rim of balance.
3. Frequency of L1014F and L1014C mutations in response to pyrethroid .
3.1 Frequency of L1014F and L1014C mutations in response to beta-cypermethrin.
Spearman's rank correlation analysis based on the correlate analysis showed significant correlations between the LC50 and the kdr allele frequencies of kdr-F, kdr-C (p≤0.01). Regression analysis revealed a significant correlation between LC50 estimates and the frequency of kdr-(F+C) ( R2=0.981), kdr-C (R2=0.9548), kdr-F (R2=0.9513) and F/C genotype (R2=0.8399) . So we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin in An.sinensis.
3.2 Frequency of L1014F and L1014C mutations in response to KT50 of beta-cypermethrin, deltamethrin and permethrin .
Regression analysis revealed a significant correlation between KT50 estimates to beta-cypermethrin and the frequency of kdr-F ( R2=0.6195), RR-F/F (R2=0.0.6166), kdr (F+C) (R2=0.6202). Regression analysis revealed a correlation between KT50 estimates to deltamethrin and permethrin and the frequency of kdr-F ( R2=0.4662), kdr (F+C) (R2=0.4712), so we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin, deltamethrin and permethrin in An.sinensis in adult mosquitoes.
4. Microsatellite loci of An.sinensis analyses
4.1 Results of amplification and sequenced.
The 15 paired-primers marked with FAM, HEX and TAMARA fluor groups based on the flanking sequence of microsatellite were used to amplify the microsatellite sequence. Only 7 paired primers amplified and sequenced with capillary tube method successfully. These results from sequencing would be used for analysis.
4.2 Polymorphism on the microsatellite location.
Polymorphism on the seven microsatellite locations arranged from 0.21 to 0.75 and average was 0.55. The results showed the seven microsatellite locations being in high genetic information. The five higher polymorphism information content locations exist in AN1,AN2,AN5,AN11,AN15.
4.3 Hardy-weinberg equilibrium tests .
Hardy-weinberg equilibrium tests of the seven microsatellite locations showed that heterozygote excess on different locations were in equilibrium and parts of the locations out of equilibrium in heterozygote deficit test.
4.4 Linkage disequilibrium analysis
Linkage disequilibrium analysis was done between the different populations and microsatellite locations. The results showed non linkage relationship between different populations and different microsatellite locations based on the P-values. We further suggested that there was independent assortment ship in the gametes based on the results, No special selection ships were found from one genotype to another.
4.5 Gene flow and kdr mutations
The result showed a low transport ratio in the 10 populations. No direct correlation relationship exist between the frequencies of geneflow and kdr frequencies (R2=0.2251,0.3086,0.3059). The F statistic values also showed Fit, Fst and Fis were 34.29%,11.56% and 25.75%. The paired-comparing results showed the signifcant difference of gene loci existing in the populations form DT and BN(28.63%),the minimal difference existing in the populations from XN and SZ(1.6%)。The difference of kdr frequencies between the different populations weren’t accordant to the change of increasing or decreasing of gene flow and Nm estimations. According to the pre-results we suggested that the kdr allele mostly coming from the transporting of inner exchanging between An.sinensis specimens in the same populations. It was concluded that selection played an important role on the population structure.
1.中华按蚊幼虫和成蚊抗药性的生物测定
1.1幼虫致死中浓度(LC50)测定
对5个幼虫种群的生物测定结果显示,从徐州、淮阴、南京、常熟和苏州采集的5个中华按蚊幼虫种群均为高抗种群,其对高效氯氰菊酯的致死中浓度(LC50)分别为0.894 mg/L、1.264 mg/L、1.670mg/L、2.067 mg/L和0.677 mg/L,实验室敏感种群致死中浓度测试结果为0.001 mg/L,抗性种群的抗药性指数分别为894、1264、1670、2067和677倍。
1.2成虫击倒中时(KT50)测定
11个种群的中华按蚊成蚊对三种杀虫剂的平均击倒中时从8.95min到47.62min不等;其中,对高效氯氰菊酸的击倒中时为8.95-26.87min;对溴氰菊酯的击倒中时为10.03-37.03min;对氯菊酯是击倒中时为16.13-47.62min。对高效氯氰菊酯抗药性指数从2.439至9.191倍,对溴氰菊酯抗药性指数从3.310至12.221倍,对二氯苯醚菊酯抗药性指数从3.226至9.524倍;从抗性种群的地理分布来看,镇江当涂种群(抗药性指数8.64-12.221)、潜山王河种群(抗药性指数7.322-9.524)的抗药性较高,丰县肖埝种群(抗药性指数3.226-3.703)、徐州云龙种群(抗药性指数2.439-4.444)的抗药性较低。
2.中华按蚊击倒抗性的研究
2.1敏感中华按蚊钠通道基因序列分析
根据已知近缘种群钠通道基因序列设计简并引物,采用RT-PCR,巢式PCR方法,蓝白斑筛选阳性克隆,提取阳性克隆的质粒送测序,获得中华按蚊钠通道部分cDNA序列,该序列包含L1014(kdr)和M918(super-kdr)经典突变位点。将cDNA序列翻译成氨基酸序列,在GeneBank中对序列同源性进行比较,发现其与GenBank中公布的淡色库蚊(Culex pipiens pallens)、埃及伊蚊(Aedes egypti)、冈比亚按蚊(Anopheles gambiae )、黑腹果蝇(Drosphila melanogaster)、家蝇(Musca domestica)及德国小蠊(Blattella germanica)的氨基酸序列具有较高的同源性,其同源性分别为98.3%, 98.3%, 98.3%, 96.7%, 94.2%和95.8%。确认了实验室繁殖的中华按蚊敏感株ⅡS4-ⅡS6区段的基因序列结构与特征,获得了358bp的特异序列,为编码中华按蚊钠通道的119个氨基酸残基,并将序列登录Genbank,登录号为:JN002364。
2.2抗性中华按蚊钠通道基因cDNA序列分析
根据已知的敏感株中华按蚊钠通道基因序列设计特异引物,采用RT-PCR方法,提取了三个野外按蚊种群ⅡS4-ⅡS 6区段的cDNA序列,部分直接测序,部分突变株用于单克隆测序,发现抗性中华按蚊的钠通道cDNA序列的1014位点氨基酸存在两种类型的突变,对应的cDNA序列存在两种类型的碱基改变,翻译成氨基酸后的突变类型为L1014F和L1014C。
2.3 AS-PCR检测方法的建立
根据中华按蚊钠通道基因组ⅡS4-ⅡS6序列(敏感及抗性),设计一对非特异性外引物CD1和CD2(扩增对照条带)。以1014位点第3个碱基为3’末端,分别设计三条特异性内引物Cgd3、Cgd4和Cgd5(扩增特异条带)。其中,Cgd3用于扩增非突变等位基因,Cgd4用于扩增突变F等位基因,Cgd5用于扩增突变C等位基因。AS-PCR设计为平行三管法。检测了50只中华按蚊的kdr分型,与直接测序相比,灵敏度和特异度都在94%以上。
2.4 PCR-RFLP检测方法的建立
根据中华按蚊突变位点特征(T[T/G][T/G]),通过在PCR引物引入突变碱基的办法,建立针对这两个突变碱基的限制性长度酶切法(PCR-RFLP),分别读出这两个碱基的突变类型。针对第一个突变位点的G/G型、T/T型、G/T型酶切后分别呈现196bp单一带,154bp和42bp两条带,196bp、154bp和42bp三条带,针对第二个突变位点的T/T型、G/G型、G/T型酶切后分别呈现155bp单一带,123bp和32bp两条带,155bp、123bp和32bp三条带,结合两个碱基的突变类型即可得出该氨基酸位点(L1014)的突变类型。本研究建立的PCR-RFLP方法,检测了50只中华按蚊,与直接测序相比,对kdr基因突变的检出灵敏度和特异度都在96%以上。
2.5 Taqman-MGB检测方法的建立
针对突变位点的三种突变类型,设计三条TaqMan-MGB探针,分别与含有TTG/TTT/TGT三种突变类型的等位基因序列相结合,在突变位点的上下游分别设计两条非特异引物,组成TaqMan-MGB探针法检测体系。结果显示,对50只中华按蚊的kdr突变的检测灵敏度和特异度都在98%以上。
2.6中华按蚊kdr基因频率的测定
2.6.1中华按蚊幼虫组kdr基因频率的测定
利用AS-PCR法,对5个中华按蚊自然种群和一个中华按蚊敏感种群进行突变类型的测定。实验室敏感株在L1014位点没有发现突变。徐州、淮阴、常熟、南京和苏州5个中华按蚊种群L1014F的频率分别为75%、75%、77.27%、75.51%和84.00%,L1014C的频率分别为19.64%、23.68%、21.59%、23.47%和16.00%。在检测的5个种群中,敏感纯合子只在徐州按蚊中发现1只,频率为1.19%,L1014F和L1014C的敏感杂合子都很少;基因型频率以RR-F/F和RR/FC最高,分别为51.02%-69.33%,28.57%-46.94%;其他各种基因型的频率都在5%以下。
2.6.2中华按蚊成蚊组kdr基因频率的测定
中华按蚊成蚊试验所用的689只中华按蚊,运用AS-PCR法,进行了kdr基因频率的测定,在1014位点,L、F、C三种等位基因均被检出;主要的基因型是RR-F/F和RR-F/C,没有检出敏感的纯合子,RS-F/L和RR-F/C两种杂合子仅在少数种群检出,且基因型频率都很低(5%以下),只有沙庄和潘镇两个种群有RR-C/C基因型检出;沙庄kdr-F的突变频率最高,潘镇的kdr-C的突变频率最高;沙庄的RR-F/F基因型频率最高,云龙的RR-F/C基因型频率最高。
kdr-F的突变频率最高,为75.00%-88.54%,kdr-C的突变频率为9.36%-23.47%;L1014的频率范围为0-4.35%,在肖埝、丹阳和奔牛三个种群没有检出;RR-F/F的基因型频率为48.48%-79.17%,RR-F/C的基因型频率为14.58%-46.15%。
2.7哈迪-温伯格平衡检验
2.7.1幼虫抗性组的哈迪-温伯格平衡检验
根据各种群的kdr基因检测频率及孟德尔遗传规律,计算出该种群各种基因型的理论频率,与该种群kdr基因型频率的实际测得值,进行配对T检验,根据P值的大小,得出测得值与观察值之间差异的显著与否,通过对5个中华按蚊种群的检验,种群的P值为0.900-0.999,T值的范围为-0.001-0.014,双侧检验差异不显著,说明各种群测定的kdr基因型频率与预测值之间没有显著性差异,种群处于哈迪-温伯格平衡状态。
2.7.2成虫抗性组的哈迪-温伯格平衡检验
对成虫抗性相关的11个中华按蚊种群的哈迪-温伯格平衡检验结果显示,11个种群的P值分别为0.335(YL),0.23(XY),0.853(SZ),0.535(DT),0.998(DY),0.706(CS),0.997(BN),0.403(PZ),0.989(FC),1(CZ)和0.995(WH),T值的范围为-0.912-0.666,p值均大于0.05,双侧检验显示各种群等位基因频率的观察值和预测值之间差异不显著,提示种群现kdr基因型频率与预测值之间没有显著性差异,种群处于遗传平衡状态。
3.中华按蚊抗性水平与突变频率的相关性分析
3.1致死中浓度(LC50)与突变频率的关系
等级相关分析5个中华按蚊幼虫种群的抗性水平与L1014F、L1014C和RR-F突变频率的关系,均显示相关显著,P≤0.01;5个种群抗性水平与各突变频率和基因型频率之间的相关分析表明,kdr(F+C)、kdr-F、kdr-C、RR-F/F、RR-F/C与LC50之间的对数关系明显,R2分别为0.981、0.951、0.955、0.862和0.840。因此提示该等位基因突变(L1014F,L1014C)可以作为中华按蚊高效氯氰菊酯抗性的分子标记。
3.2击倒中时(KT50)与突变频率的关系
中华按蚊KT50与kdr等位基因突变频率和基因型频率的相关性分析结果显示,高效氯氰菊酯的击倒中时与kdr-F突变频率和RR-F/F基因型频率的相关性明显,相关系数分别为0.6195和0.6166,与kdr-F和kdr-C总的突变频率达到了0.6202;溴氰菊酯和二氯苯醚菊脂与kdr-F的相关性结果显示,不及前者高,其相关系数均为0.4662,二者与F和C总的突变频率也达到了0.4721。
4.中华按蚊微卫星序列分析
4.1荧光标记引物扩增及测序结果
根据文献针对中华按蚊15个微卫星位点的侧翼序列,设计引物,并在上游引物5’端用FAM, HEX和TAMARA荧光进行标记,PCR产物用毛细管法测定微卫星序列的多态性。结果表明AN1, AN2,AN5,AN11,AN12,AN13和AN15七个位点的条带清晰,测序结果可靠,可以用于微卫星数据分析。
4.2各微卫星位点的多态性
本研究10个地理种群7个微卫星座位平均多态信息含量为0.55(0.21-0.75),提示这7个基因座所能提供的遗传信息比较丰富。多态信息含量及杂合度较高的5个位点是AN1, AN2,AN5,AN11和AN15。
4.3哈迪-温伯格平衡检验
从对种群遗传结构的分析来看,7个微卫星位点的哈迪温伯格平衡检验表明,全部位点的杂合子过剩的分析均符合哈迪-温伯格平衡,而部分位点的杂合子缺失的哈迪-温伯格平衡分析不符合哈迪-温伯格平衡。
4.4各位点和种群的连锁不平衡分析
对不同种群的7个位点之间以及相同种群的不同位点之间的连锁不平衡分析表明,在每个位点上各种群之间为非连锁关系,各种群独立遗传;各位点之间也是非连锁关系,一个位点的基因型与另一个位点的基因型彼此独立(P > 0.05),配子之间自由组合,未显示对某种基因型有选择作用。
4.5基因流及其与kdr抗性关系
本研究所涉及的各自然种群的迁移率(Nm=1.12)较低,基因流与kdr基因突变频率相关性不明显(R2=0.2251,0.3086,0.3059),提示kdr基因突变可能主要来自杀虫剂的选择压力和种群内部突变个体之间的交流。用7个位点估计的固定系数F值结果显示:个体相对于全部种群的遗传差异是34.29%,其中来自种群间的是11.56%,来自种内的是25.75%。成对比较的种群间的差异表明,差异最大的DT种群和BN种群,为28.63%,差异最小的是XN和SZ种群,为1.6%。将成对种群间的基因流与kdr等位基因频率的差做散点图,江苏7个种群间的kdr基因频率的差异有随着基因流增大而减小的趋势,但这种趋势不明显,而安徽4个种群没有随着基因流增大而减小的趋势,全部种群的kdr等位基因频率的差异有随着基因流的增大而减小的趋势,但这种趋势更小,进一步说明了kdr基因主要来自种群内部突变个体之间的基因交流,选择压力是影响抗药性种群结构的主要因素。
Insectcides play a main role in control of major vectors of diseases. However, development of resistance in many insect species represents a significant and increasing thread to their continued effective use in the recent years. It is important and urgent to elucidate the resistance mechanism in order to slow down the spread and development of resistance of vector populations. Most studies on the resistance-related mechanism of Anopneles sinensis were mainly focused on the occurrence, development and biochemistry of the resistance. There are no research based on molecular biology of An.sinensis as it being very important vector species associated with malaria. In the past thirty years, the mainly use of DDT and pyrethroids insecticides for the impregnation of bet nets and indoor residual spraying in China caused the resistance increased rapidly. But there is no kdr resistance reported, as we all know that was associated with pyrethroids and DDT resistance. In this research, 16 resistance strains which had been collected in Jiangsu and Anhui provinces were studied, a biochemistry and three molecular methods have been adopted to find out the resistance-related mechanism and relationship between the para-knockdown frequencies and bioassay outcome. STR on seven locuses were analyzed. Here are the results.
1 Bioassay analyses on larval mosquitoes and adult mosquitoes.
1.1 Bioassay analyses on larval mosquitoes of An.sinensis.
Larval bioassay results on the five strains from XZ, HY, CS, NJ and SZ all showed resistance to beta-cypermethrin. LC50 of the five strains were 0.894 mg/L, 1.264 mg/L, 1.670mg/L, 2.067 mg/L and 0.677 mg/L,respectively. R/S were 894, 1264, 1670, 2067 and 677-folds,respectively.
1.2 Bioassay analyses on adult mosquitoes of An.sinensis.
Adult mosquitoes bioassay on the 11 strains from Jiangsu and Anhui provinces with beta-cypermethrin, deltapermethrin and permethrin showed the average KT50 were 8.95-47.62 min. KT50 to beta-cypermethrin was 8.95-26.87min. KT50 to deltamethrin was 10.03-37.03 min. KT50 to permethrin was 16.13-47.62 min. R/S of KT50 of the three kinds of pyrethroids to 11 populations ranged from 2.439- to 12.221-folds. The populations of DT and WH showed the higher R/S, they were 8.64-12.221-folds in DT population and 7.322-9.524-folds in WH population. The lower R/S occurred in XN and YL populations.
2. The action of knockdown resistance (kdr) in An.sinensis.
2.1 Sequence of the voltage-dependent sodium channel in An.sinensis from S strain.
A total 358bp of sodium channel gene had been amplified by RT-PCR, nest-PCR and sequenced from Ss strain. It has been demonstrated that 119 amino acid are encoded including four homologous domains and the hydrophilic linking peptides of varying length without 3` UTR. Although it is incomplete in this para-like cDNA sequence in An. sinensis, but it is long enough to find out the kdr mutation in An.sinensis because it contains all of the mutations loci of L1014 and M989 that have been reported to be related with kdr. It was found that the closest sequence homology to the Culex pipes pallens sodium channel with 98.3%, and with 96.7% sequence identity with Drosophila para, and with 94.2% sequence identity with M domestica para-like (Vssc1),and with 98.3% sequence identity with An.gambiae(para), and with 95.8% sequence identity with B germanica para-lik sodium channel.
2.2 cDNA sequence analysis of the sodium channel of the beta-pyrethroid-resistance strain of An.sinensis.
The cDNA sequence of IIS4-IIS6 in which kdr mutation located and the partial cDNA sequence have been analyzed from the resistance specimen. Resistance-related mutation of L1014F and L1014C had been found.
2.3 Method of AS-PCR was adapted to detect the para-knockdown resistance mutations.
Method of AS-PCR was adapted to detect the L1014F ,L1014C mutation in sodium channel . 263bp longer fragment amplified by two outer non allele-specific primers CD1 and CD2. The special primers of Cgd3, Cgd4, Cgd5 were used to detect the three alleles of TTG, TTT, TGT in three parallel tubes with the anti-sense primer CD2 to amplify a 169bp fragment. The sensitivity and specificity of this genotype method was showed to exceed 94%.
2.4 Methods of PCR-RFLP were adapted to detect the para-knockdown resistance mutations.
According to the mutation sites, after the first PCR based on the primers of C→T submission and digestion with HindⅢ, the presence of sequence with a (G/G) was inferred by the appearance of a single 196-bp fragment, a (T/T) was inferred by the appearance of a 154-bp and a 42-bp fragment and a (G/T) was inferred by the three fragments of 196-bp, 154-bp and 42-bp. In the same way, the second (T/G) involving (T/T),(G/G),(G/T) were inferred by the three fragments of 155-bp, 123-bp and 32-bp(Figure 3). Direct sequencing was used for validating the results of cPASA and PCR-FRLP. The sensitivity and specificity of this genotype method was showed to exceed 96%.
2.5 Methods of TaqMan-MGB were adapted to detect the para-knockdown resistance mutations.
According to the mutation sites of the para-knockdown resistance gene, three Probees were desired with a Report group of FAM and VIC in the 5’,a Quencher group and MGB group in the 3’, to detect the three sequences with the 1014 loci of TTG, TTT, TGT. Two non-specific primers were desired overstriding the para-kdr mutations sites. The high sensitivity and specificity of this genotype method leaded to the 99% in sensitivity and 100% in specificity.
2.6 Assay of the kdr mutation frequencies and genotyping frequencies.
3.6.1 Assay of the kdr mutation frequencies and genotyping frequencies on the larval mosquitoes.
In the larval group, a total of five resistance populations from XZ, SZ, CS, NJ and HY cities rural and an Ss population, 290 specimens were tested with cPASA. Kdr frequencies ranged from 16.01% to 84.00%. The kdr alleles existed mainly in kdr-F/F and kdr-F/C genotype, the resistant homozygous form, and only a small portion (1.33%-3.57%) of the mosquitoes possessed kdr-C homozygous (RR-C) kdr alleles, 2.63%-4.76% of the kdr-C and kdr-F heterozygous (RS-C and RS-F) kdr genotype on the five populations. Kdr allelic frequency ranged from 73.68% to 84.00% on the kdr-F, and 16.01% to 23.68% on kdr-C. The genotype frequency of RR-F and RR-F/C ranged from 51.02% to 69.33%, 28.57% to 46.94 respectively.
2.6.2 Assay of the kdr mutation frequencies and genotyping frequencies on the adult mosquitoes.
A total of 689 An.sinensis specimens were genotyped in AS-PCR method. There did also co-exist the co-occurring knockdown resistance mutations in the eleven strains. There were mainly two genotype exist RR-F/F and RR-F/C. No Ss genotype was found. The para-knockdown resistance mutation frequencies of KDR-F and KDR-C ranged from 75.00%-88.54% and 9.36%-23.47%, respectively. No s-allele on 1014 loci was found in XN, DY and BN strains. Genotyping frequency ranged from 14.58% to 79.17%, which including 48.48%-79.17% in RR-F/F and 14.58%-46.15% in RR-F/C. The highest KDR-F and KDR-C frequencies were found in SZ and PZ population. The highest genotype frequency of RR-F/F and RR-F/C were found in SZ and YL populations.
2.7 Hardy-Weinberg equilibrium test
2.7.1 Hardy-Weinberg equilibrium test of larval mosquito group.
Hardy-Weinberg equilibrium test was accepted for kdr alleles in each population using the T-paired test, the unbiased estimate of P-value of the five populations were 0.989(XZ), 0.930(HY), 0.991(NJ), 0.999(CS), 0.900(SZ), respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The five populations were all present genetic equilibrium.
2.7.2 Hardy-Weinberg equilibrium test of adult mosquito group.
Hardy-Weinberg equilibrium test was also accepted for kdr alleles in the adult mosquito population using the T-paired test, the unbiased estimate of P-value of the eleven populations were 0.335(YL),0.23(XN),0.853(SZ),0.535(DT),0.998(DY),0.706(CS),0.997(BN),0.403(PZ),0.989(FC),1(CZ),0.995(WH),respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The eleven populations were all present genetic equilibrium. The P-value of YL and XN populations showed the two populations were in rim of balance.
3. Frequency of L1014F and L1014C mutations in response to pyrethroid .
3.1 Frequency of L1014F and L1014C mutations in response to beta-cypermethrin.
Spearman's rank correlation analysis based on the correlate analysis showed significant correlations between the LC50 and the kdr allele frequencies of kdr-F, kdr-C (p≤0.01). Regression analysis revealed a significant correlation between LC50 estimates and the frequency of kdr-(F+C) ( R2=0.981), kdr-C (R2=0.9548), kdr-F (R2=0.9513) and F/C genotype (R2=0.8399) . So we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin in An.sinensis.
3.2 Frequency of L1014F and L1014C mutations in response to KT50 of beta-cypermethrin, deltamethrin and permethrin .
Regression analysis revealed a significant correlation between KT50 estimates to beta-cypermethrin and the frequency of kdr-F ( R2=0.6195), RR-F/F (R2=0.0.6166), kdr (F+C) (R2=0.6202). Regression analysis revealed a correlation between KT50 estimates to deltamethrin and permethrin and the frequency of kdr-F ( R2=0.4662), kdr (F+C) (R2=0.4712), so we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin, deltamethrin and permethrin in An.sinensis in adult mosquitoes.
4. Microsatellite loci of An.sinensis analyses
4.1 Results of amplification and sequenced.
The 15 paired-primers marked with FAM, HEX and TAMARA fluor groups based on the flanking sequence of microsatellite were used to amplify the microsatellite sequence. Only 7 paired primers amplified and sequenced with capillary tube method successfully. These results from sequencing would be used for analysis.
4.2 Polymorphism on the microsatellite location.
Polymorphism on the seven microsatellite locations arranged from 0.21 to 0.75 and average was 0.55. The results showed the seven microsatellite locations being in high genetic information. The five higher polymorphism information content locations exist in AN1,AN2,AN5,AN11,AN15.
4.3 Hardy-weinberg equilibrium tests .
Hardy-weinberg equilibrium tests of the seven microsatellite locations showed that heterozygote excess on different locations were in equilibrium and parts of the locations out of equilibrium in heterozygote deficit test.
4.4 Linkage disequilibrium analysis
Linkage disequilibrium analysis was done between the different populations and microsatellite locations. The results showed non linkage relationship between different populations and different microsatellite locations based on the P-values. We further suggested that there was independent assortment ship in the gametes based on the results, No special selection ships were found from one genotype to another.
4.5 Gene flow and kdr mutations
The result showed a low transport ratio in the 10 populations. No direct correlation relationship exist between the frequencies of geneflow and kdr frequencies (R2=0.2251,0.3086,0.3059). The F statistic values also showed Fit, Fst and Fis were 34.29%,11.56% and 25.75%. The paired-comparing results showed the signifcant difference of gene loci existing in the populations form DT and BN(28.63%),the minimal difference existing in the populations from XN and SZ(1.6%)。The difference of kdr frequencies between the different populations weren’t accordant to the change of increasing or decreasing of gene flow and Nm estimations. According to the pre-results we suggested that the kdr allele mostly coming from the transporting of inner exchanging between An.sinensis specimens in the same populations. It was concluded that selection played an important role on the population structure.
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