海南岛传疟蚊媒种群密度、杀虫剂抗性及发生机制研究
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摘要
研究背景：疟疾(Malaria)是危害公众健康和阻碍社会经济发展一个重要问题,传播媒介是按蚊(Anopheles)到目前为止,疟疾不再是不可防治的疾病,人类在疟疾控制方面取得重大成就之一是传疟媒介得到了有效控制。使用浸泡过除虫菊酯类药物的蚊帐和室内喷洒杀虫剂是控制蚊媒的主要措施。然而,长期大量使用杀虫剂在大大削减传疟蚊虫种群密度的同时也改变了其种群结构,并引起抗药性。为了彻底消除疟疾,研究抗药机制势在必行。
众所周知,蚊媒对杀虫剂的抗性机制主要包含两大类：代谢解毒和靶标敏感性下降。代谢解毒产生的抗药性(代谢抗药性)主要是蚊虫通过体内解毒代谢酶基因扩增或过量表达,增强解毒代谢酶活性,从而降低杀虫剂毒性,阻断杀虫剂作用到抗性昆虫的靶标。其中,细胞色素单加氧酶P450氧化酶家族(单加氧酶P450s)、非专一性酯酶系(ESTs)和谷胱甘肽转移酶-S-转移酶系(GSTs)为主要的代谢性解毒酶。靶标敏感性降低产生的抗药性(靶标抗药性)主要是指由于抗性蚊体内的杀虫剂作用靶标基因发生突变,降低了其与杀虫剂的结合,从而使蚊虫对杀虫剂的敏感性降低而产生的抗性。杀虫剂对蚊媒作用的靶标主要有3种：有机磷和氨基甲酸酯类杀虫剂的作用靶标乙酰胆碱酯酶(AChE); DDT和拟除虫菊酯类杀虫剂的作用靶标神经轴突钠离子通道(SC)；环戊二烯类和吡唑类、二环磷酯类、二环苯甲酸酯类杀虫剂的作用靶标Y-氨基丁酸(GABA)受体-氯离子通道复合体。
中国的恶性疟疾发病主要集中在海南岛和云南省。海南岛是被30公里宽的琼州海峡与中国大陆隔离的一个孤岛,南部主要为山脉,也是曾经全岛疟疾流行的高发区域。海南岛主要以种植水稻为主,一年三季,水稻田有利于传疟按蚊的孳生。十年前,微小按蚊(Anopheles minimums)作为海南岛主要传播疟疾的蚊媒,经过大量杀虫剂的使用已经得到了很好的控制,其种群几乎灭绝。而中华按蚊(Anopheles sinensis)、棋斑按蚊(Anopheles tessellatus)、迷走按蚊(Anopheles vagus)等其他传播疟疾的蚊媒种群却在十年后的今天大量被报道。为了彻底控制海南岛传疟媒介,本课题对目前海南岛主要传疟媒介的种群密度、对杀虫剂的抗药性及其发生机制进行了如下研究：
一、研究方法：
1.海南岛主要传疟蚊媒种群密度及杀虫剂使用情况调查
2012年7月到8月,根据海南岛不同的地势和海拔,对海南岛12个地点传疟蚊媒种群密度进行了调查,最后在临高、陵水、文昌、澄迈、定安、屯昌、保亭和三亚8个调查点收集到了研究所需的按蚊幼虫、蛹和成虫,我们按照采集到的幼虫数量研究种群密度。采集到的主要传疟按蚊有三种：中华按蚊、棋斑按蚊和迷走按蚊。幼虫采集采用幼虫勺定点捞法,在采集点随机选取5个按蚊孳生地,采用5个孳生地的350m1勺子舀水中所含幼虫个数的平均数计为幼虫的密度。所有幼虫均采自水稻田。成虫采集自农户家中牛或猪圈内,用专用吸蚊器吸取,取单位时间内捕捉到的成虫个数,除以单位时间的平均数计为当地成蚊的密度。蚊种的鉴定方面,我们主要根据幼虫室内孵化为成蚊肉眼形态观察和实验室PCR扩增蚊ITS2和28S D3区域确定。在采集样本的同时,我们询问农户及周边出售农药的供销社调查海南目前各地使用的农药。
2.海南岛主要传疟蚊媒杀虫剂抗性的生物学测定
我们采用WHO推荐的标准成蚊筒内药膜接触法进行生物学活性测定。药膜采用WHO规定的浓度：溴氰菊酯0.05%,DDT4.0%和马拉硫磷5%,同时制备对照组药膜(均由中国CDC提供)。测试按蚊为我们从海南8个地点采集的幼虫和蛹进行室内孵化培养,选取羽化后三天的未吸血雌蚊,20只为一批,每个地点每种药膜均测定5批,一个地点的一种药物选用一张对照药膜测试20只未吸血的雌蚊作为对照。操作方法参照由中国标准出版社出版的《蚊虫抗药性检测方法生物测定法(GB/T26347-2010)》：分别取20只未吸血的雌蚊吸入装有实验药膜和对照药膜的接触筒中,观察第10、15、20、30、40、50和60分钟被击倒的蚊虫个数,并记录首次击倒时间。1蚊虫小时后仍未被击倒的蚊虫转入恢复筒,用8%的葡萄糖溶液喂养,观察24小时后继续存活的蚊虫为杀虫剂抗性蚊,24小时内被击倒的蚊虫则为杀虫剂敏感蚊。根据击倒顺序标记每只蚊虫,击倒和存活的每只蚊虫单管保存在1.5ml EP管内,-20℃保存备用。
3.海南岛主要传疟蚊媒代谢解毒酶活性测定
3.1KP04蚊酶液制备
在冰上取下每只蚊虫的头和足,放入装有2000L无水乙醇的0.5mL EP管内,按照原来的标记进行标记,-20℃保存。将每只蚊虫的剩余部分在试管内加入3000LKPO4(0.25M)缓冲液在冰上充分研磨,1,3000rpm离心5分钟后取上清液转入2.0mL EP管内,并用KP04缓冲液稀释至1.5mL,即为蚊虫酶液。残渣加入200p L无水乙醇,也按照原来的标记对应标记-20℃保存。蚊虫酶液用于测定谷胱甘肽转移酶转移酶(GST)、单加氧酶P450、羧酸酯酶(COE,又名CES)代谢解毒酶活性测定及蛋白含量。
3.2蛋白含量的测定
取单只蚊虫的40μ L蚊酶液移入一次性比色杯(d=1),再取0.25M KPO4缓冲液40pL移入另一只一次性比色杯作为空白对照。分别加入9000L考马斯亮蓝染色液,5分钟后分别在波长为595nm的分光光度计上读取OD值两次。两次读数的平均值(即平均OD值)分别代入根据牛血清白蛋白制定的标准曲线方程Y=28.977X2-22.201X+5.1657计算蛋白含量,其中,X为OD,Y为蛋白含量。实验样品与空白对照之差即为所测40uL蚊酶液的蛋白含量。然后根据公式M=Y×1500/4000计算单只蚊虫的总蛋白含量M(单位：mg)。该数值用于三种酶活性最终计算。
3.3谷胱甘肽转移酶(GST)酶活性的测定
将蚊虫酶液、GST缓冲液、1-氯-2,4-二硝基笨(cDNB)缓冲液各2000L混合加入一次性比色杯内,同时将0.25M KP04缓冲液、GST缓冲液、1-氯-2,4-二硝基笨(cDNB)缓冲液各2000L混合加入另一只一次性比色杯内作为空白对照,分别在波长为340nm的分光光度计上读取OD值4次,每次间隔2-4分钟。记录每次读取的时间和每次读取的OD值。两次OD读数之差除以间隔时间获得每分钟0D变化值。四次读数标记为1,2,3,4,按照读数4-1,4-2,4-3,3-1,3-2,2-1的顺序各自除以间隔时间得到6个数值。用实验样品的平均值减去空白对照的平均值,即获得校正后的实验样品每分钟OD变化值。然后根据每只蚊虫总蛋白含量计算出其GST酶活性值(单位：μmol cDNB/min/mg protein)。共测得956个样本的GST酶活性。
3.4单加氧酶P450酶活性的测定
在一次性比色杯中加入350pL蚊虫酶液和8μL7-4-乙氧基香豆素(7EC)同时取0.25M KP04缓冲液350pL代替蚊虫酶液与8μL7EC加入另一只比色杯中作为空白对照,然后在30℃水浴培养4小时。分别加入560pL氨基乙酸缓冲液,在波长调至450nm的分光光度计上读取OD值两次。然后将实验样品将两次OD读数的平均值减去空白对照的平均值得到校正后的蚊虫酶液的OD值,再除以水浴培养的时间获得每分钟蚊虫酶液OD的变化值。所有操作重复一次。然后,取两遍操作获得的OD变化值的平均值作为该蚊虫最后的OD变化值；再根据7-3-羟基香豆素(7-HC)制定的标准曲线方程Y=88.470D+4.88计算出最后的单加氧酶P450酶活性值(单位：7-HC/min/mg protein)。共测得938个样本的P450酶活性。
3.5羧酸酯酶(COE)酶活性的测定
取900pL对硝基苯基醋酸盐缓冲液放入1.5ml EP管内,30℃水浴培养5分钟,加入100pL蚊虫酶液13,000转离心5秒,转入比色杯中在分光光度计405nm波长处读取OD值,测定读取两遍,所有操作重复一次。同时将0.25M KP04缓冲液100pL代替蚊虫酶液重复上述操作获得空白对照的OD值。将然后将实验样品将两次OD读数的平均值减去空白对照的平均值得到校正后的蚊虫酶液的OD值,再除以水浴培养的时间获得每分钟蚊虫酶液OD值。所有操作重复一次。然后,取两遍操作获得的OD值的平均值作为该蚊虫最后的OD变化值。根据公式0D/(16.4X0.05×protein(mg/mL))×1000计算出最终COE酶活性值(单位：nmoL/min/mg protein)。共测得903个样本的COE酶活性。
4.海南岛主要传疟蚊媒靶标抗性测定
溴氰菊酯和DDT作用的靶标为神经轴突钠离子通道,而马拉硫磷作用靶标为乙酰胆碱酯酶(AChE),因此我们对前者进行击倒抗性(Knockdown resistance, Kdr)靶标L1014位点PCR扩增验证实验,对后者进行PCR-RFLP检测Ace-1靶标G119位点基因实验。共用150只迷走按蚊、137只中华按蚊进行实验。蚊虫DNA采用专用试剂盒提取,按说明操作。
4.1击倒抗性(Knockdown resistance, Kdr)靶标L1014位点验证实验
Para钠离子通道具有快速击倒效应,昆虫对此产生的抗药性称为击倒抗药性(Knock down resisters, Kdr)。按蚊最常见的是位于通道第二功能区的S6疏水区1014位点的亮氨酸突变为苯丙氨酸(LeuyPhe)、丝氨酸(serine)、半胱氨酸(cysteine)等,即L1014F、L1014S.L1014C三个位点的突变,我们重点检测L1014位点的突变。采用PCR扩增中华按蚊,其引物5’→3’为Kdr-F1023(TGCCACTCCGTGTGTTTAGA)和Kdr-R1347(GAGCGATGATGATCCGAAAT)的S6区域,目的片段大小为325bp, PCR反应条件为：反应条件为94℃C初始变性3分钟,(94℃变性1分钟,50℃复性30秒,72℃延伸30秒)×45个循环,72℃最后延伸10分钟。产物通过凝胶电泳检测并测序。迷走按蚊采用引物Agdlmi-F和Agd2h-R进行PCR扩增,反应条件仅改变复性温度50℃为47℃。
4.2聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)检测Ace-1靶标G119区域实验
PCR扩增中华按蚊和迷走按蚊G119区域,观察是否发生突变。所用引物5’→3’为Ace-1-F22F(TAGGTCACGGTGAGTCCGYA)和Ace-1-R660(ACCACGATCACGTTCTCCTC),反应条件为94℃初始变性3分钟,(94℃变性45秒,55℃复性30秒,72℃延伸30秒)×35个循环,72℃C最后延伸5分钟。2.5%凝胶电泳测得中华按蚊产物为602bp,迷走按蚊产物为571bp。所得产物继续加入AIM限制性核酸酶进行反应(按AIU1限制性核酸酶试剂盒说明书操作),若为纯合型(AGC/AGC),将得到118bp和75bp两条产物,若为野生型(GGC/GGC),只有193bp一条产物,若为杂合型(GGC/AGC),将得到193bp,118bp和75bp三条产物。根据突变个数计算突变频率。
5.统计学分析
所有数据采用SPSS13.0软件进行统计分析。蚊虫的死亡率根据每个蚊虫种群接触每种杀虫剂24小时后的死亡数量进行计算。以蚊虫接触无杀虫剂但有对应溶剂的空白膜为对照进行校正。将每个重复校正后的死亡率用于计算每个蚊虫种群对每种杀虫剂处理后蚊虫的平均死亡率和标准差。将每个种群每种杀虫剂处理后24小时内死亡的蚊虫分为敏感性蚊虫(敏感组S),24小时后活着的蚊虫分为抗药性蚊虫(抗性组R)。用卡方检验(Chi-square)或Fisher确切概率检验分析蚊虫抗药性与基因突变(kdr靶标L1014位点和ace-1靶标G119位点基因)的关联。用两独立样本t检验分析抗性组与敏感组蚊虫酶活性的差异。当P<0.05时有统计学意义。同时计算每个蚊虫种群对每种杀虫剂处理后获得的抗性组与敏感组蚊虫酶活性之比值(R/S ratio),抗性组与敏感组比值的标准差用下列公式计算：SD=SQRT[(SDR/MR)2+(SDs/Ms)2].
二、实验结果
1.海南岛主要传疟媒介种群密度及使用杀虫剂调查结果
在海南岛8个点中,中华按蚊主要分布在三亚(0.923±0.280条/勺)和保亭(1.056±0.665条/勺)；棋斑按蚊主要分布在三亚(1.979±1.470条/勺)、保亭(4.572±1.855条/勺)和陵水(1.143±0.595条/勺)；迷走按蚊主要分布在海南岛北部,其中澄迈(1.363±0.980条/勺)、临高(1.012±0.595条/勺)、定安(1.275±0.175条/勺)、屯昌(1.099±0.420条/勺)和文昌(4.792±1.085条/勺)均有分布。
海南岛各地杀虫剂的使用情况：除虫菊酯类如溴氰菊酯、甲醚菊酯、高效氯氰菊酯、联苯菊酯、氯氰菊酯等在采样的8个点均有使用。有机磷酸类如三唑磷等在保亭、临高、屯昌均有使用。中国政府一直禁用的有机氯类如DDT,却在保亭、定安、文昌发现使用。其他的杀虫剂如烟碱类药物吡虫啉发现在三亚、保亭、澄迈、文昌使用。
2.海南岛主要传疟媒介生物学活性的测定结果
因采集的按蚊幼虫孵化为成蚊数量不足,本研究根据各地样本成蚊孵化数量及各地杀虫剂使用情况,选择性的在不同的地点用不同的药膜进行了生物测定。不同地点不同药膜对照组的致死率均为0。生物学活性共测定1725个样本,各点各药膜测定结果如下：
2.10.05%溴氰菊酯的测试结果
中华按蚊具有初步抗性,尤其在三亚(85.78%±17.54%)、保亭(90.98%±9.39%)。在澄迈(97.87%±4.31%)、定安(100%),迷走按蚊为敏感群。棋斑按蚊也为敏感群,其中在三亚和陵水,蚊虫在0.05%溴氰菊酯测试中死亡率均为100%。
2.24%DDT的测试结果
中华按蚊具有抗性：三亚(78.38%±15.83%)、保亭(72.71%±21.20%)。迷走按蚊在屯昌(67.06%±7.54%)具有抗性,在澄迈(84.00%±11.68%)、定安(88.82%±10.07%)具有初步抗性。
2.35%马拉硫磷测试结果
迷走按蚊具有初步抗性：定安(77.29%±10.57%)、澄迈(88.87%±4.26%)、屯昌(78.86%±9.49%)。
3.海南岛主要传疟媒介代谢解毒酶活性测定结果
3.1谷胱甘肽转移酶(GST)酶活性测定结果
GST酶对定安采集的迷走按蚊抗5%马拉硫磷的影响有统计学意义(R/S=1.1361±0.3145,t=2.806,P=0.0060),对屯昌采集的迷走按蚊抗5%马拉硫磷影响亦有统计学意义(R/S=1.1141±0.1853,t=2.501,P=0.0150)。
3.2单加氧酶(P450)酶活性测定结果
单加氧酶P450酶对三亚采集的中华按蚊抗0.05%溴氰菊酯影响有统计学意义(R/S=1.5391±0.8130,t=2.738,P=0.0076),单加氧酶P450酶活性对保亭采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.5278±0.6072,t=2.575,P=0.0115),保亭(R/S=1.2662±0.7366,t=2.145,P=0.0346)和三亚(R/S=1.2732±0.5853,t=2.317,P=0.0226)采集的中华按蚊抗4%DDT,定安采集的迷走按蚊抗5%马拉硫磷(R/S=1.3143±0.7058,t=2.417,P=0.0175)影响具有统计学意义。
3.3羧酸酯酶(COE)酶活性测定结果
COE酶活性对三亚采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.2580±0.3507,t=3.478,P=0.0010)影响有统计学意义。对保亭采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.2738±0.4677,t=2.009,P=0.0470)影响有统计学意义。
4.海南岛主要传疟媒介靶标抗性测定结果
4.1击倒抗性(Knockdown resistance, KDR)靶标验证结果
中华按蚊抗溴氰菊酯(三亚采集,突变频率7.5%,P=0.025<0.05),抗DDT(三亚采集,突变频率为9.5%,P=0.015<0.05)、(保亭采集,突变频率为7.8%,P=0.011<0.05)Para钠离子第二功能区的S6疏水区1014位点的亮氨酸发生突变为苯丙氨酸(L1014),中华按蚊抗DDT可能与其抗性有关。迷走按蚊未发现L1014位点突变。
4.2PCR-RFLP检测Ace-1靶标G119位点基因实验
我们发现迷走按蚊G119位点未发生突变,中华按蚊在各地采集的抗马拉硫磷与敏感蚊虫G119位点均有突变,突变频率在53%到72%之间,有抗性的蚊虫突变频率略高于敏感蚊虫。迷走按蚊均未发生G119位点突变。
三、结论
1.海南岛在7、8月间主要传疟按蚊为中华按蚊、迷走按蚊和棋斑按蚊,在海南岛南部主要传疟媒介以中华按蚊和棋斑按蚊种群为主,北部主要以迷走按蚊分布为主。除虫菊酯类杀虫剂在海南应用最为广泛。
2.生物学活性测定结果表明海南岛中华按蚊(保亭和三亚)对溴氰菊酯具有初步抗性,中华按蚊、迷走按蚊对DDT具有初步抗性,迷走按蚊(澄迈、定安、屯昌)对马拉硫磷具有初步抗性。PCR-RFLP检测Ace-1靶标G119位点基因实验结果表明,中华按蚊(保亭和三亚)对马拉硫磷有初步抗性。
3.海南中华按蚊和迷走按蚊代谢性解毒酶活性和分子生物学击倒抗性实验(Kdr)测试结果表明,代谢性解毒酶产生抗药性机制的在海南较为普遍。由于击倒抗性实验我们只在海南的中华按蚊中发现有L1014位点的突变,突变类型为L1014F,说明迷走按蚊的抗药性机制只与代谢性解毒酶的活性有关。而中华按蚊抗药性可能是酶生物学活性和靶标基因发生突变共同作用的结果。
Background:Malaria is a significant public health problem and impediment to socioeconomic development in world. So far, malaria is no longer a non-preventable disease. Vector control is a fundamental element of the existing global strategy to fight malaria. Use pyrethroid treated bed nets and indoor residue sprays insecticides are the main components of malaria control strategies. However, these practices have resulted in pyrethroid resistance, and greatly reshaped the mosquito community and population structures. In order to completely eliminate malaria, study the mechanism of malaria vector insecticide resistance is imperative.
As we all know, study on the mechanism of Pyrethroids resistance of mosquitoes include two categories:metabolic detoxification and reduced sensitivity of the target. Metabolic resistance mainly through in metabolic enzyme gene of malaria vector insecticide-resistance mosquito vivo amplification or over-expression, enhance the activity of metabolic enzyme, thereby degrading pesticide toxicity and blocking the insecticides arrived to resistant insects target. Such as cytochrome P450oxidase (P450s), non-specific esterase system (ESTs) and glutathione S-transferase (GSTs) are the main metabolic detoxification enzymes. Target resistance mainly refers to mutations in target gene of malaria vector insecticide-resistance mosquito, reduce its combination with insecticides and result reduce the malaria vector sensitivity to insecticide, then produces insecticide resistance. There are3main target insecticide actions:the target of organophosphate and carbamate insecticides is acetylcholinesterase (AChE); DDT and pyrethroid insecticides target is the para-type sodium channel (SC); target of cyclopentadienyl and pyrazole kind, Bicyclic phosphate ester kind, Bicyclic benzoate kind insecticides are γ-aminobutyric acid (GABA) receptor chloride ion channel complex.
In China, Plasmodium falciparum malaria is mainly found in two tropical provinces, Hainan and Yunnan. Hainan Island, separated by the30km-wide Qiongzhou Strait from the mainland of China, The southern part of the island is mountainous, and has been the most malarias region of the island. Hainan Island is mainly rice farming, three quarters of a year, It's favorable of Anopheles vectors breeding. As the main malaria vector in Hainan Island, Anopheles minimums have been controlled well by insecticide used more than a decade, the population of Anopheles minimums almost facing extinction. However, the other important malaria vectors populations have been reported more and more, just as Anopheles sinensis, Anopheles tessellatus and Anopheles vagus, et al. To completely eliminate Hainan Island malaria vector, our study the population surveillance, current status and mechanisms of insecticide resistance of the malaria vector Anopheles mosquito in Hainan Island as follow:
Methods
1. Investigation the main malaria vector population surveillance and insecticides were used in Hainan Island.
In July and August2012, based on the different topography and elevation, we investigated12sites in Hainan Island. Finally, sampled enough malaria vector Anopheles number to study from Lingao, Linshui, Wenchang, Chengmai, Dinan, Tunchang, Baoting and Sanya8sites, Only larval own enough number to study larval population density. There are main three Anopheles type:Anopheles sinensis, Anopheles tessellatus and Anopheles vagus. We sampled Anopheles larva by350ml spoon to gain. Usually, we choose5points in one site and take average larvae of total5points to calculate larva population density. Each point use sized-spoon gain larva once a time. ALL larvae sampled from rice field. Adult mosquitoes sampled from cattle and pigsty by definite mosquito-sucker, and use the sampled adult numbers in unite time divided unite time, then take the average as adult population density For the reason cannot sampled enough adult number, we decided use larva population density as local mosquito population density. We accord the Anopheles different appearance to object and using PCR amply mosquito ITS2and D3region to assay Anopheles type. Always larval cannot be decided type, so we identification their type after they were incubated adult. At the same time, we also investigated each country used insecticide current in Hainan Island.
2. Bioassay tests the main malaria vector in Hainan Island.
Bioassay tests play an important role in study mechanisms of insecticide resistance of the malaria vector mosquito Anopheles, which can tell from the mosquito resistance or sensitivity to insecticide. According to WHO, we used the standard tube with sensitive layer contact adult method to test mosquito bioassay. Bioassay with0.05%Deltamethrin,4.0%DDT and5%Malathion impregnated papers, and we also made control papers (they all made in China CDC). Test mosquitoes were acquired form we collected larvae and pupae from Hainan Island8sites rearing in door and three days after emergence without fed blood.20mosquitoes once a time and total5times a site to satisfied statistical significance. At the same time, we put another20mosquitoes into control insecticide paper tested tube in each site, the mortality data of the control groups only to test the effection of the test paper. Procedure reference the "mosquito resistance bioassay method (GB/T26347-2010)" published by the China Standard Press. we put the test paper on the inner wall of the cylinder liner, as the contact tube, each time suck20female mosquitoes without fed blood in close contact cylinder clapboard, and we also suck20female mosquitoes without fed blood in close contact cylinder clapboard in the control tuber with each insecticide control paper. The numbers of knockdown and dead mosquitoes were recorded after10,15,20,30,40,50and60minutes of exposure. After1h exposure to insecticide impregnated papers, the surviving mosquitoes were blown recovery cylinder, with8%glucose water on the inside to rear,24h after recording tested mosquitoes mortality, all mosquitoes were immediately collected in EP tube single and preserved at-20℃. We sign the mosquito number based on the mosquito death time, marked one by one.
3. Analysis metabolic detoxification enzyme activity of the main malaria vector in Hainan Island.
3.1Mosquito enzyme with KPO4make
Take legs and head from each mosquito, signed and preserved at filled with200μL alcoholic0.5ml EP tuber, marked as before,-20℃. The other individuals were homogenized in a1.5-ml tube with300μL of phosphate KPO4buffer (0.25M, pH7.2) and using a connect the grinder (Argos, UK) grinding rod to grind the residue of the mosquito enough at4℃, the tube was mixed and centrifuged at1,3000rpm for5min, and the supernatant was removed to2.0EP tube and diluted by adding1,200ml of phosphate buffer, witch was used to test the activity of GST, P450and COE metabolic enzyme activity assays and total protein assay. Residues add200μL alcoholic were marked as before,-20℃preserved.
3.2Total protein assay
Two replicates of40μL of each mosquito homogenate were placed in a disposable colorimetric cup (d=1), using0.25M KPO4buffer40μL as negative control, add900μL Coomassie protein assay reagent was mixed enough. The colorimetric cup was incubated for5min and read at595nm using biophotometer plus. Take two times average and CK, According bovine serum albumin was used to prepare a typical standard curve Y=28.977X2-22.201X+5.1657to calculate protein,(X:OD, Y:Bovine serum albumin protein), which subdue CK then times1500, divide40, and divide1000, to get the total protein (Unite:mg). We can use the total protein to calculate three assay activities.
3.3Glutathione-S-transferase (GST) activity
A total of200μL reduced glutathione solution and200μL cDNB solution, added200μL of mosquito homogenate (Glutathione solution:cDNB solution:mosquito homogenate=1:1:1) were mixed enough in a disposable colorimetric cup (d=1). Using0.25M KPO4buffer as the negative control, then every minute for4minutes at340nm, repeated again. The four times reading OD marked1,2,3,4, as4-1,4-2,4-3,3-1,3-2,2-1divide each reading inter time to get6number, take average subduce the average of CK divide time which result is one time operation, take two times operation result to make average, then times20as the final GST activity (Unite:μmol cDNB/min/mg protein).Total test956samples GST assay activities.
3.4Cytochrome P450(Cyt P450) monooxygenases activity
A total of8μL of the7-ethoxycoumarine (7-EC)(Sigma, E1379-25MG) solution was added to400μL of mosquito supernatant and samples were incubated at30℃for4h in a disposable colorimetric cup (d=1). Using0.25M KPO4buffer as the negative control, the reaction was stopped by the addition of560μL glycine buffer (pH10.4,0.1mM) and sample absorbance was read at450nm two times, repeated again. Take average of two times reading OD divided interval time once a time, then get two times operation average subdue CK, according to a standard regression based on a serial dilution of 7-hydroxycoumarin and its relevant OD values Y=88.47OD+4.88calculate final P450activity (Unite:7-HC/min/mg protein). Total test938samples P450assay activities.
3.5Carboxylesterase (COE) activity assay
A total of900μL p-nitrophenyl acetate solution to1.5ml EP tube and place the tube for5min in a30℃water bath, add100μL mosquito homogenates and then centrifuged at1,3000rpm for5min. Transfer the reaction mixture to a disposable colorimetric cup (d=1), read at405nm two times, using0.25M KPO4buffer as negative control, repeat again. Take the two times reading OD subdue the first times reading OD once a time, get the average two times subdue CK as final OD, according to the formula OD/(16.4X0.05Xprotein(mg/ml))X1000to calculate COE activity (Unites:nmol/min/mg protein). Total test903samples COE assay activities.
4. Analysis target resistance of the main malaria vector in Hainan Island.
For the reason DDT and pyrethroid insecticides target is the para-type sodium channel (SC), the target of organophosphate and carbamate insecticides is acetylcholinesterase (AChE). We did Knockdown resistance test before, and did ace-1test behind. One leg of each mosquito was used for DNA extraction with the E.Z.N. A. TM Micro Elute Genomic DNA Kit (Operate as Product).
4.1PCR amply the target of the Knockdown resistance L1014site test
Para sodium channels with rapid knockdown effect of insecticide resistance, this is called knockdown resistance (Knock down resisters, Kdr). Anopheles sodium channel second functional areas in the hydrophobic region of S6, leucine to phenylalanine mutation at codon1014. Usually leucine was replaced by phenylalanine (LeuyPhe) or serine or cysteine mutation at codon1014, and in turn was named L1014F, L1014S and L1014C. So we test the codon1014. PCR primers5'→3'of An. sinensis are Kdr-F1023(TGCCACTCCGTGTGTTTAGA)and Kdr-R1347(GAGCGATGATGATCCGAAAT), the cycling conditions of An. sinensis was as follows:initial denaturation at94℃for3min,45cycles of lmin denaturation at94℃,30s annealing at50℃and30s extension at72℃followed by a final extension of10min at72℃. We amplified a325bp fragment product and sequenced. PCR primers5'→3'of An. vagus are Agd1mi-F (TAGGTCACGGTGAGTCCGYA) and Agd2h-R (ACCACGATCACGTTCTCCTC), The cycling conditions of An. vagus was only change the temperate of annealing from50℃to47℃, others are same as An. sinensis.
4.2PCR-RFLP amplify the target of Ace-1gene at positions119test
Use PCR-RFLP amplify test to determine point mutations of Anopheles sinensis and Anopheles Vagus the ace-1gene at positions119. The PCR primes are Ace-1-F22F and Ace-1-R660. The cycling conditions were as follows:initial denaturation at94℃for3min,35cycles of45s denaturation at94℃,30s annealing at55℃and30s extension at72℃followed by a final extension of5min at72℃.2.5%agarose gel electrophoresis test the product, we get602bp product of Anopheles sinensis, and571bp product of Anopheles Vagus. Then we use the restriction end nuclease AIU1of PCR products were digested by enzyme (Operate as Product). Homozygous mutations type was amplified two bands (118bp and75bp), Wild type was only amplified one band (193bp), Heterozygous mutations type was amplified three bands (193bp,118bp and75bp), and the gene type simply:wild type is GGC/GGC, heterozygous mutations type is GGC/AGC and homozygous mutations type is AGC/AGC. We accorded mutation number to calculate frequency of G119mutation.
5. Statistical analysis
All data were statistical analyzed by SPSS13.0software. Mosquito mortality rates after the24hr recovery period were calculated for each insecticide and each population. Mosquito contact no pesticides but blank film corresponding solvent as control correction. Each repeated corrected mortality to calculate the average mortality and standand devation of each mosquito population after each insecticide treatment. Each population handling pesticides each within24hours after the death of the mosquito is divided into sensitive mosquitoes (sensitive group S),24hours after the live mosquitoes into resistant mosquitoes (resistance group R).Chi-square tests or Fisher exact probability tests were used to examine the association between target site mutations and the resistance phenotype. Two independent samples t test was used to compare the enzyme activities between resistant and susceptible mosquitoes for each population. When P<0.05had statistical significance. At the same time is calculated for each of the mosquito population resistance to insecticide treatment group for each site and sensitive group of mosquito enzyme activity ratio (R/S ratio), calculated using the following formula between resistant and susceptible Ratio Standards:SD=SQRT [(SDR/MR)2+(SDs/Ms)2].
Results
1. Result of Investigation the main malaria vector population surveillance and insecticides were used in Hainan Island.
8sites of Hainan Island we found that Anopheles sinensis are main distribute at Sanya (0.923±0.280number/spoon) and Baoting (1.056±0.665number/spoon), Anopheles tessellatus are main distribute at Sanya (1.979±1.470number/spoon), Baoting (4.572±1.855number/spoon) and linshui (1.143±0.595number/spoon), Anopheles vagus are main distribute at the Northeast in Hainan Island, including Chengmai (1.363±0.980number/spoon), Lingao (1.012±0.595number/spoon), Dingan (1.275±0.175number/spoon), Tunchang (1.099±0.420number/spoon) and Wenchang (4.792±1.085number/spoon). Pyrethroids insecticide were used in8sites we sampled, just as Deltamethrin, Methothrin, Beta cypermethrin, Bifenthrin, Lambda-cyhalothrin, et al. Organophosphates insecticide just as Imidacloprid were used in Baoting, Lingao, Tunchang. Organochlorines insecticide just as DDT was forbidden used in China, but we surprised found they were used in Baoting, Dinan, Wenchang. Imidacloprid were found used in Sanya, Baoting, Chengmai, Wenchang.
2. Result of bioassay tests the main malaria vector in Hainan Island.
For the reason we cannot get enough adult number and each site use different insecticide in Hainan Island, we did bioassay use different insecticide paper to different sites. ALL control groups mortality is zero. Total tested1725samples and result of each site each insecticide as follow:
2.1Result of0.05%Deltamethrin bioassay
Anopheles sinensis have initial0.05%Deltamethrin resistance in Sanya (85.78%±17.54%) and Baoting (90.98%±9.39%). Anopheles vagus has0.05%Deltamethrin resistant in Chenmai (97.87%±4.31%) and Dinan (100%). Anopheles tessellates have0.05%Deltamethrin resistant, the mosquito mortality is100%in Sanya and Lingshui.
2.2Result of4%DDT bioassay
Anopheles sinensis have4%DDT resistant in Sanya (78.38%±15.83%) and Baoting (72.71%±21.20%). Anopheles vagus have4%DDT resistant in Tunchang (67.057%±7.54%) and have initial4%DDT resistance in Chengmai (84.00%±11.68%) and Dinan (88.82%±10.07%).
2.3Result of5%Malathion bioassay
Anopheles vagus has5%Malathion initial resistance in Dinan (77.29%±10.57%), Chenmai (88.87%±4.26%) and Tunchang (78.86%±9.49%).
3. Result of analysis metabolic detoxification enzyme activity of the main malaria vector in Hainan Island.
3.1Result of Glutathione-S-transferase (GST) activity
We found the GST metabolic enzyme activities R/S ratio of Anopheles vagus was significantly higher in the two sites from Hainan Island (Dinan, Tunchang), where mosquitoes were tested bioassay by5%Malathion. The ratio of Dinan and Tunchang respectively was1.1361±0.3145(t=2.806, P=0.0060) and1.1141±0.1853(t=2.501, P=0.0150).(Unite:μmol cDNB/min/mg protein).
3.2Result of Cytochrome P450(Cyt P450) monooxygenases activity
We found the P450metabolic enzyme activities R/S ratio of Anopheles sinensis was significantly higher in the two sites from Hainan Island, the ratio of Baoting and Sanya (used0.05%Deltamethrin to test bioassay) respectively was1.5278±0.6072(t=2.575,F=6.6300, P=0.0115) and1.5391±0.8130(t=2.738, P=0.0076), and the ratio of Baoting and Sanya (used4%DDT to test bioassay) respectively was1.2662±0.7366(t=2.145, P=0.0346) and1.2732±0.5853(t=2.317,P=0.0226). The P450metabolic enzyme activities R/S ratio of Anopheles vagus was significantly higher in the one site from Hainan Island (Dingan), where mosquitoes were tested bioassay by5%Malathion. The ratio was1.3143±0.7058(t=2.417, P=0.0175).(Unite:7-HC/min/mg protein).
3.3Result of Carboxylesterase (COE) activity assay
We found the COE metabolic enzyme activities R/S ratio of Anopheles sinensis was significantly higher in the two sites from Hainan Island (Baoting, Sanya), where mosquitoes were tested bioassay by0.05%Deltamethrin. The ratio of Baoting and Sanya respectively was1.2738±0.4677(t=2.009, P=0.0470) and1.2580±0.3507(t=3.478, P=0.0010).(Unite:nmol/min/mg protein).
4. Result of Analysis target resistance of the main malaria vector in Hainan Island.
4.1Result of PCR amply the target of Knockdown resistance L1014site test
The kdr mutation of An. Sinensis only detected one heterozygous mutations type (TTG/TTT):Leucine to Phenylalanine substitution. The kdr mutation frequency of Deltamethrin-resistant mosquitoes from Sanya and DDT-resistant mosquitoes from Sanya and Baoting individuate is7.5%(P=0.025),9.5%(P=0.015) and7.8%(P=0.011),they are all L1014F mutation, which maybe related with An. sinensis insecticide resistance. No kdr mutations were detected in An. vagus populations from Hainan Island, China.
4.2Result of PCR-RFLP amplified the target of Ace-1gene at positions119test
Modest to high (53%-72%) ace-1mutation frequency was found in all sites we sampled An. sinensis populations, the resistance group mutation frequency higher than the sensitive group. But no ace-1mutation was detected in An. vagus populations.
Conclusions:
1. During7-8months, the main malaria vectors were An. sinensis, An. vagus and An. tessellates in Hainan Island. An. sinensis and An. tessellates populations were main sampled in Southern of Hainan Island, An. vagus population were main found in Northest of Hainan Island. Pyrethroid mosquito coils insecticides were the most propular insecticide to spray in rice frield in Hainan.
2. Result of Bioassay tests shows that Deltamethrin resistance was widespread over the Anopheles sinensis populations of Sanya and Baoting, DDT resistance was widespread over the Anopheles sinensis and Anopheles vagus populations, Malathion resistance was widespread over the Anopheles vagus populations of Chengmai, Dinan and Tunchang. Result of PCR-RFLP amplified the target of Ace-1gene at positions119test shows that Malathion resistance was widespread over the Anopheles sinensis populations of Sanya and Baoting.
3. The Kdr and enzyme assay results show that metabolic detoxification mechanisms are wide-spread in An. Sinensis and An. vagus populations from Hainan Island China, the target site mutations(L1014F) was only detected in An. Sinensis suggest the mechanisms of An. vagus insecticide resistant from Hainan Island is not relations to Kdr, but consider maybe relation with the metabolic detoxification enzyme activity. Then the mechanisms of An. Sinensis insecticide resistant maybe the common function with Kdr and metabolic detoxification enzyme activity. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes.
众所周知,蚊媒对杀虫剂的抗性机制主要包含两大类：代谢解毒和靶标敏感性下降。代谢解毒产生的抗药性(代谢抗药性)主要是蚊虫通过体内解毒代谢酶基因扩增或过量表达,增强解毒代谢酶活性,从而降低杀虫剂毒性,阻断杀虫剂作用到抗性昆虫的靶标。其中,细胞色素单加氧酶P450氧化酶家族(单加氧酶P450s)、非专一性酯酶系(ESTs)和谷胱甘肽转移酶-S-转移酶系(GSTs)为主要的代谢性解毒酶。靶标敏感性降低产生的抗药性(靶标抗药性)主要是指由于抗性蚊体内的杀虫剂作用靶标基因发生突变,降低了其与杀虫剂的结合,从而使蚊虫对杀虫剂的敏感性降低而产生的抗性。杀虫剂对蚊媒作用的靶标主要有3种：有机磷和氨基甲酸酯类杀虫剂的作用靶标乙酰胆碱酯酶(AChE); DDT和拟除虫菊酯类杀虫剂的作用靶标神经轴突钠离子通道(SC)；环戊二烯类和吡唑类、二环磷酯类、二环苯甲酸酯类杀虫剂的作用靶标Y-氨基丁酸(GABA)受体-氯离子通道复合体。
中国的恶性疟疾发病主要集中在海南岛和云南省。海南岛是被30公里宽的琼州海峡与中国大陆隔离的一个孤岛,南部主要为山脉,也是曾经全岛疟疾流行的高发区域。海南岛主要以种植水稻为主,一年三季,水稻田有利于传疟按蚊的孳生。十年前,微小按蚊(Anopheles minimums)作为海南岛主要传播疟疾的蚊媒,经过大量杀虫剂的使用已经得到了很好的控制,其种群几乎灭绝。而中华按蚊(Anopheles sinensis)、棋斑按蚊(Anopheles tessellatus)、迷走按蚊(Anopheles vagus)等其他传播疟疾的蚊媒种群却在十年后的今天大量被报道。为了彻底控制海南岛传疟媒介,本课题对目前海南岛主要传疟媒介的种群密度、对杀虫剂的抗药性及其发生机制进行了如下研究：
一、研究方法：
1.海南岛主要传疟蚊媒种群密度及杀虫剂使用情况调查
2012年7月到8月,根据海南岛不同的地势和海拔,对海南岛12个地点传疟蚊媒种群密度进行了调查,最后在临高、陵水、文昌、澄迈、定安、屯昌、保亭和三亚8个调查点收集到了研究所需的按蚊幼虫、蛹和成虫,我们按照采集到的幼虫数量研究种群密度。采集到的主要传疟按蚊有三种：中华按蚊、棋斑按蚊和迷走按蚊。幼虫采集采用幼虫勺定点捞法,在采集点随机选取5个按蚊孳生地,采用5个孳生地的350m1勺子舀水中所含幼虫个数的平均数计为幼虫的密度。所有幼虫均采自水稻田。成虫采集自农户家中牛或猪圈内,用专用吸蚊器吸取,取单位时间内捕捉到的成虫个数,除以单位时间的平均数计为当地成蚊的密度。蚊种的鉴定方面,我们主要根据幼虫室内孵化为成蚊肉眼形态观察和实验室PCR扩增蚊ITS2和28S D3区域确定。在采集样本的同时,我们询问农户及周边出售农药的供销社调查海南目前各地使用的农药。
2.海南岛主要传疟蚊媒杀虫剂抗性的生物学测定
我们采用WHO推荐的标准成蚊筒内药膜接触法进行生物学活性测定。药膜采用WHO规定的浓度：溴氰菊酯0.05%,DDT4.0%和马拉硫磷5%,同时制备对照组药膜(均由中国CDC提供)。测试按蚊为我们从海南8个地点采集的幼虫和蛹进行室内孵化培养,选取羽化后三天的未吸血雌蚊,20只为一批,每个地点每种药膜均测定5批,一个地点的一种药物选用一张对照药膜测试20只未吸血的雌蚊作为对照。操作方法参照由中国标准出版社出版的《蚊虫抗药性检测方法生物测定法(GB/T26347-2010)》：分别取20只未吸血的雌蚊吸入装有实验药膜和对照药膜的接触筒中,观察第10、15、20、30、40、50和60分钟被击倒的蚊虫个数,并记录首次击倒时间。1蚊虫小时后仍未被击倒的蚊虫转入恢复筒,用8%的葡萄糖溶液喂养,观察24小时后继续存活的蚊虫为杀虫剂抗性蚊,24小时内被击倒的蚊虫则为杀虫剂敏感蚊。根据击倒顺序标记每只蚊虫,击倒和存活的每只蚊虫单管保存在1.5ml EP管内,-20℃保存备用。
3.海南岛主要传疟蚊媒代谢解毒酶活性测定
3.1KP04蚊酶液制备
在冰上取下每只蚊虫的头和足,放入装有2000L无水乙醇的0.5mL EP管内,按照原来的标记进行标记,-20℃保存。将每只蚊虫的剩余部分在试管内加入3000LKPO4(0.25M)缓冲液在冰上充分研磨,1,3000rpm离心5分钟后取上清液转入2.0mL EP管内,并用KP04缓冲液稀释至1.5mL,即为蚊虫酶液。残渣加入200p L无水乙醇,也按照原来的标记对应标记-20℃保存。蚊虫酶液用于测定谷胱甘肽转移酶转移酶(GST)、单加氧酶P450、羧酸酯酶(COE,又名CES)代谢解毒酶活性测定及蛋白含量。
3.2蛋白含量的测定
取单只蚊虫的40μ L蚊酶液移入一次性比色杯(d=1),再取0.25M KPO4缓冲液40pL移入另一只一次性比色杯作为空白对照。分别加入9000L考马斯亮蓝染色液,5分钟后分别在波长为595nm的分光光度计上读取OD值两次。两次读数的平均值(即平均OD值)分别代入根据牛血清白蛋白制定的标准曲线方程Y=28.977X2-22.201X+5.1657计算蛋白含量,其中,X为OD,Y为蛋白含量。实验样品与空白对照之差即为所测40uL蚊酶液的蛋白含量。然后根据公式M=Y×1500/4000计算单只蚊虫的总蛋白含量M(单位：mg)。该数值用于三种酶活性最终计算。
3.3谷胱甘肽转移酶(GST)酶活性的测定
将蚊虫酶液、GST缓冲液、1-氯-2,4-二硝基笨(cDNB)缓冲液各2000L混合加入一次性比色杯内,同时将0.25M KP04缓冲液、GST缓冲液、1-氯-2,4-二硝基笨(cDNB)缓冲液各2000L混合加入另一只一次性比色杯内作为空白对照,分别在波长为340nm的分光光度计上读取OD值4次,每次间隔2-4分钟。记录每次读取的时间和每次读取的OD值。两次OD读数之差除以间隔时间获得每分钟0D变化值。四次读数标记为1,2,3,4,按照读数4-1,4-2,4-3,3-1,3-2,2-1的顺序各自除以间隔时间得到6个数值。用实验样品的平均值减去空白对照的平均值,即获得校正后的实验样品每分钟OD变化值。然后根据每只蚊虫总蛋白含量计算出其GST酶活性值(单位：μmol cDNB/min/mg protein)。共测得956个样本的GST酶活性。
3.4单加氧酶P450酶活性的测定
在一次性比色杯中加入350pL蚊虫酶液和8μL7-4-乙氧基香豆素(7EC)同时取0.25M KP04缓冲液350pL代替蚊虫酶液与8μL7EC加入另一只比色杯中作为空白对照,然后在30℃水浴培养4小时。分别加入560pL氨基乙酸缓冲液,在波长调至450nm的分光光度计上读取OD值两次。然后将实验样品将两次OD读数的平均值减去空白对照的平均值得到校正后的蚊虫酶液的OD值,再除以水浴培养的时间获得每分钟蚊虫酶液OD的变化值。所有操作重复一次。然后,取两遍操作获得的OD变化值的平均值作为该蚊虫最后的OD变化值；再根据7-3-羟基香豆素(7-HC)制定的标准曲线方程Y=88.470D+4.88计算出最后的单加氧酶P450酶活性值(单位：7-HC/min/mg protein)。共测得938个样本的P450酶活性。
3.5羧酸酯酶(COE)酶活性的测定
取900pL对硝基苯基醋酸盐缓冲液放入1.5ml EP管内,30℃水浴培养5分钟,加入100pL蚊虫酶液13,000转离心5秒,转入比色杯中在分光光度计405nm波长处读取OD值,测定读取两遍,所有操作重复一次。同时将0.25M KP04缓冲液100pL代替蚊虫酶液重复上述操作获得空白对照的OD值。将然后将实验样品将两次OD读数的平均值减去空白对照的平均值得到校正后的蚊虫酶液的OD值,再除以水浴培养的时间获得每分钟蚊虫酶液OD值。所有操作重复一次。然后,取两遍操作获得的OD值的平均值作为该蚊虫最后的OD变化值。根据公式0D/(16.4X0.05×protein(mg/mL))×1000计算出最终COE酶活性值(单位：nmoL/min/mg protein)。共测得903个样本的COE酶活性。
4.海南岛主要传疟蚊媒靶标抗性测定
溴氰菊酯和DDT作用的靶标为神经轴突钠离子通道,而马拉硫磷作用靶标为乙酰胆碱酯酶(AChE),因此我们对前者进行击倒抗性(Knockdown resistance, Kdr)靶标L1014位点PCR扩增验证实验,对后者进行PCR-RFLP检测Ace-1靶标G119位点基因实验。共用150只迷走按蚊、137只中华按蚊进行实验。蚊虫DNA采用专用试剂盒提取,按说明操作。
4.1击倒抗性(Knockdown resistance, Kdr)靶标L1014位点验证实验
Para钠离子通道具有快速击倒效应,昆虫对此产生的抗药性称为击倒抗药性(Knock down resisters, Kdr)。按蚊最常见的是位于通道第二功能区的S6疏水区1014位点的亮氨酸突变为苯丙氨酸(LeuyPhe)、丝氨酸(serine)、半胱氨酸(cysteine)等,即L1014F、L1014S.L1014C三个位点的突变,我们重点检测L1014位点的突变。采用PCR扩增中华按蚊,其引物5’→3’为Kdr-F1023(TGCCACTCCGTGTGTTTAGA)和Kdr-R1347(GAGCGATGATGATCCGAAAT)的S6区域,目的片段大小为325bp, PCR反应条件为：反应条件为94℃C初始变性3分钟,(94℃变性1分钟,50℃复性30秒,72℃延伸30秒)×45个循环,72℃最后延伸10分钟。产物通过凝胶电泳检测并测序。迷走按蚊采用引物Agdlmi-F和Agd2h-R进行PCR扩增,反应条件仅改变复性温度50℃为47℃。
4.2聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)检测Ace-1靶标G119区域实验
PCR扩增中华按蚊和迷走按蚊G119区域,观察是否发生突变。所用引物5’→3’为Ace-1-F22F(TAGGTCACGGTGAGTCCGYA)和Ace-1-R660(ACCACGATCACGTTCTCCTC),反应条件为94℃初始变性3分钟,(94℃变性45秒,55℃复性30秒,72℃延伸30秒)×35个循环,72℃C最后延伸5分钟。2.5%凝胶电泳测得中华按蚊产物为602bp,迷走按蚊产物为571bp。所得产物继续加入AIM限制性核酸酶进行反应(按AIU1限制性核酸酶试剂盒说明书操作),若为纯合型(AGC/AGC),将得到118bp和75bp两条产物,若为野生型(GGC/GGC),只有193bp一条产物,若为杂合型(GGC/AGC),将得到193bp,118bp和75bp三条产物。根据突变个数计算突变频率。
5.统计学分析
所有数据采用SPSS13.0软件进行统计分析。蚊虫的死亡率根据每个蚊虫种群接触每种杀虫剂24小时后的死亡数量进行计算。以蚊虫接触无杀虫剂但有对应溶剂的空白膜为对照进行校正。将每个重复校正后的死亡率用于计算每个蚊虫种群对每种杀虫剂处理后蚊虫的平均死亡率和标准差。将每个种群每种杀虫剂处理后24小时内死亡的蚊虫分为敏感性蚊虫(敏感组S),24小时后活着的蚊虫分为抗药性蚊虫(抗性组R)。用卡方检验(Chi-square)或Fisher确切概率检验分析蚊虫抗药性与基因突变(kdr靶标L1014位点和ace-1靶标G119位点基因)的关联。用两独立样本t检验分析抗性组与敏感组蚊虫酶活性的差异。当P<0.05时有统计学意义。同时计算每个蚊虫种群对每种杀虫剂处理后获得的抗性组与敏感组蚊虫酶活性之比值(R/S ratio),抗性组与敏感组比值的标准差用下列公式计算：SD=SQRT[(SDR/MR)2+(SDs/Ms)2].
二、实验结果
1.海南岛主要传疟媒介种群密度及使用杀虫剂调查结果
在海南岛8个点中,中华按蚊主要分布在三亚(0.923±0.280条/勺)和保亭(1.056±0.665条/勺)；棋斑按蚊主要分布在三亚(1.979±1.470条/勺)、保亭(4.572±1.855条/勺)和陵水(1.143±0.595条/勺)；迷走按蚊主要分布在海南岛北部,其中澄迈(1.363±0.980条/勺)、临高(1.012±0.595条/勺)、定安(1.275±0.175条/勺)、屯昌(1.099±0.420条/勺)和文昌(4.792±1.085条/勺)均有分布。
海南岛各地杀虫剂的使用情况：除虫菊酯类如溴氰菊酯、甲醚菊酯、高效氯氰菊酯、联苯菊酯、氯氰菊酯等在采样的8个点均有使用。有机磷酸类如三唑磷等在保亭、临高、屯昌均有使用。中国政府一直禁用的有机氯类如DDT,却在保亭、定安、文昌发现使用。其他的杀虫剂如烟碱类药物吡虫啉发现在三亚、保亭、澄迈、文昌使用。
2.海南岛主要传疟媒介生物学活性的测定结果
因采集的按蚊幼虫孵化为成蚊数量不足,本研究根据各地样本成蚊孵化数量及各地杀虫剂使用情况,选择性的在不同的地点用不同的药膜进行了生物测定。不同地点不同药膜对照组的致死率均为0。生物学活性共测定1725个样本,各点各药膜测定结果如下：
2.10.05%溴氰菊酯的测试结果
中华按蚊具有初步抗性,尤其在三亚(85.78%±17.54%)、保亭(90.98%±9.39%)。在澄迈(97.87%±4.31%)、定安(100%),迷走按蚊为敏感群。棋斑按蚊也为敏感群,其中在三亚和陵水,蚊虫在0.05%溴氰菊酯测试中死亡率均为100%。
2.24%DDT的测试结果
中华按蚊具有抗性：三亚(78.38%±15.83%)、保亭(72.71%±21.20%)。迷走按蚊在屯昌(67.06%±7.54%)具有抗性,在澄迈(84.00%±11.68%)、定安(88.82%±10.07%)具有初步抗性。
2.35%马拉硫磷测试结果
迷走按蚊具有初步抗性：定安(77.29%±10.57%)、澄迈(88.87%±4.26%)、屯昌(78.86%±9.49%)。
3.海南岛主要传疟媒介代谢解毒酶活性测定结果
3.1谷胱甘肽转移酶(GST)酶活性测定结果
GST酶对定安采集的迷走按蚊抗5%马拉硫磷的影响有统计学意义(R/S=1.1361±0.3145,t=2.806,P=0.0060),对屯昌采集的迷走按蚊抗5%马拉硫磷影响亦有统计学意义(R/S=1.1141±0.1853,t=2.501,P=0.0150)。
3.2单加氧酶(P450)酶活性测定结果
单加氧酶P450酶对三亚采集的中华按蚊抗0.05%溴氰菊酯影响有统计学意义(R/S=1.5391±0.8130,t=2.738,P=0.0076),单加氧酶P450酶活性对保亭采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.5278±0.6072,t=2.575,P=0.0115),保亭(R/S=1.2662±0.7366,t=2.145,P=0.0346)和三亚(R/S=1.2732±0.5853,t=2.317,P=0.0226)采集的中华按蚊抗4%DDT,定安采集的迷走按蚊抗5%马拉硫磷(R/S=1.3143±0.7058,t=2.417,P=0.0175)影响具有统计学意义。
3.3羧酸酯酶(COE)酶活性测定结果
COE酶活性对三亚采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.2580±0.3507,t=3.478,P=0.0010)影响有统计学意义。对保亭采集的中华按蚊抗0.05%溴氰菊酯(R/S=1.2738±0.4677,t=2.009,P=0.0470)影响有统计学意义。
4.海南岛主要传疟媒介靶标抗性测定结果
4.1击倒抗性(Knockdown resistance, KDR)靶标验证结果
中华按蚊抗溴氰菊酯(三亚采集,突变频率7.5%,P=0.025<0.05),抗DDT(三亚采集,突变频率为9.5%,P=0.015<0.05)、(保亭采集,突变频率为7.8%,P=0.011<0.05)Para钠离子第二功能区的S6疏水区1014位点的亮氨酸发生突变为苯丙氨酸(L1014),中华按蚊抗DDT可能与其抗性有关。迷走按蚊未发现L1014位点突变。
4.2PCR-RFLP检测Ace-1靶标G119位点基因实验
我们发现迷走按蚊G119位点未发生突变,中华按蚊在各地采集的抗马拉硫磷与敏感蚊虫G119位点均有突变,突变频率在53%到72%之间,有抗性的蚊虫突变频率略高于敏感蚊虫。迷走按蚊均未发生G119位点突变。
三、结论
1.海南岛在7、8月间主要传疟按蚊为中华按蚊、迷走按蚊和棋斑按蚊,在海南岛南部主要传疟媒介以中华按蚊和棋斑按蚊种群为主,北部主要以迷走按蚊分布为主。除虫菊酯类杀虫剂在海南应用最为广泛。
2.生物学活性测定结果表明海南岛中华按蚊(保亭和三亚)对溴氰菊酯具有初步抗性,中华按蚊、迷走按蚊对DDT具有初步抗性,迷走按蚊(澄迈、定安、屯昌)对马拉硫磷具有初步抗性。PCR-RFLP检测Ace-1靶标G119位点基因实验结果表明,中华按蚊(保亭和三亚)对马拉硫磷有初步抗性。
3.海南中华按蚊和迷走按蚊代谢性解毒酶活性和分子生物学击倒抗性实验(Kdr)测试结果表明,代谢性解毒酶产生抗药性机制的在海南较为普遍。由于击倒抗性实验我们只在海南的中华按蚊中发现有L1014位点的突变,突变类型为L1014F,说明迷走按蚊的抗药性机制只与代谢性解毒酶的活性有关。而中华按蚊抗药性可能是酶生物学活性和靶标基因发生突变共同作用的结果。
Background:Malaria is a significant public health problem and impediment to socioeconomic development in world. So far, malaria is no longer a non-preventable disease. Vector control is a fundamental element of the existing global strategy to fight malaria. Use pyrethroid treated bed nets and indoor residue sprays insecticides are the main components of malaria control strategies. However, these practices have resulted in pyrethroid resistance, and greatly reshaped the mosquito community and population structures. In order to completely eliminate malaria, study the mechanism of malaria vector insecticide resistance is imperative.
As we all know, study on the mechanism of Pyrethroids resistance of mosquitoes include two categories:metabolic detoxification and reduced sensitivity of the target. Metabolic resistance mainly through in metabolic enzyme gene of malaria vector insecticide-resistance mosquito vivo amplification or over-expression, enhance the activity of metabolic enzyme, thereby degrading pesticide toxicity and blocking the insecticides arrived to resistant insects target. Such as cytochrome P450oxidase (P450s), non-specific esterase system (ESTs) and glutathione S-transferase (GSTs) are the main metabolic detoxification enzymes. Target resistance mainly refers to mutations in target gene of malaria vector insecticide-resistance mosquito, reduce its combination with insecticides and result reduce the malaria vector sensitivity to insecticide, then produces insecticide resistance. There are3main target insecticide actions:the target of organophosphate and carbamate insecticides is acetylcholinesterase (AChE); DDT and pyrethroid insecticides target is the para-type sodium channel (SC); target of cyclopentadienyl and pyrazole kind, Bicyclic phosphate ester kind, Bicyclic benzoate kind insecticides are γ-aminobutyric acid (GABA) receptor chloride ion channel complex.
In China, Plasmodium falciparum malaria is mainly found in two tropical provinces, Hainan and Yunnan. Hainan Island, separated by the30km-wide Qiongzhou Strait from the mainland of China, The southern part of the island is mountainous, and has been the most malarias region of the island. Hainan Island is mainly rice farming, three quarters of a year, It's favorable of Anopheles vectors breeding. As the main malaria vector in Hainan Island, Anopheles minimums have been controlled well by insecticide used more than a decade, the population of Anopheles minimums almost facing extinction. However, the other important malaria vectors populations have been reported more and more, just as Anopheles sinensis, Anopheles tessellatus and Anopheles vagus, et al. To completely eliminate Hainan Island malaria vector, our study the population surveillance, current status and mechanisms of insecticide resistance of the malaria vector Anopheles mosquito in Hainan Island as follow:
Methods
1. Investigation the main malaria vector population surveillance and insecticides were used in Hainan Island.
In July and August2012, based on the different topography and elevation, we investigated12sites in Hainan Island. Finally, sampled enough malaria vector Anopheles number to study from Lingao, Linshui, Wenchang, Chengmai, Dinan, Tunchang, Baoting and Sanya8sites, Only larval own enough number to study larval population density. There are main three Anopheles type:Anopheles sinensis, Anopheles tessellatus and Anopheles vagus. We sampled Anopheles larva by350ml spoon to gain. Usually, we choose5points in one site and take average larvae of total5points to calculate larva population density. Each point use sized-spoon gain larva once a time. ALL larvae sampled from rice field. Adult mosquitoes sampled from cattle and pigsty by definite mosquito-sucker, and use the sampled adult numbers in unite time divided unite time, then take the average as adult population density For the reason cannot sampled enough adult number, we decided use larva population density as local mosquito population density. We accord the Anopheles different appearance to object and using PCR amply mosquito ITS2and D3region to assay Anopheles type. Always larval cannot be decided type, so we identification their type after they were incubated adult. At the same time, we also investigated each country used insecticide current in Hainan Island.
2. Bioassay tests the main malaria vector in Hainan Island.
Bioassay tests play an important role in study mechanisms of insecticide resistance of the malaria vector mosquito Anopheles, which can tell from the mosquito resistance or sensitivity to insecticide. According to WHO, we used the standard tube with sensitive layer contact adult method to test mosquito bioassay. Bioassay with0.05%Deltamethrin,4.0%DDT and5%Malathion impregnated papers, and we also made control papers (they all made in China CDC). Test mosquitoes were acquired form we collected larvae and pupae from Hainan Island8sites rearing in door and three days after emergence without fed blood.20mosquitoes once a time and total5times a site to satisfied statistical significance. At the same time, we put another20mosquitoes into control insecticide paper tested tube in each site, the mortality data of the control groups only to test the effection of the test paper. Procedure reference the "mosquito resistance bioassay method (GB/T26347-2010)" published by the China Standard Press. we put the test paper on the inner wall of the cylinder liner, as the contact tube, each time suck20female mosquitoes without fed blood in close contact cylinder clapboard, and we also suck20female mosquitoes without fed blood in close contact cylinder clapboard in the control tuber with each insecticide control paper. The numbers of knockdown and dead mosquitoes were recorded after10,15,20,30,40,50and60minutes of exposure. After1h exposure to insecticide impregnated papers, the surviving mosquitoes were blown recovery cylinder, with8%glucose water on the inside to rear,24h after recording tested mosquitoes mortality, all mosquitoes were immediately collected in EP tube single and preserved at-20℃. We sign the mosquito number based on the mosquito death time, marked one by one.
3. Analysis metabolic detoxification enzyme activity of the main malaria vector in Hainan Island.
3.1Mosquito enzyme with KPO4make
Take legs and head from each mosquito, signed and preserved at filled with200μL alcoholic0.5ml EP tuber, marked as before,-20℃. The other individuals were homogenized in a1.5-ml tube with300μL of phosphate KPO4buffer (0.25M, pH7.2) and using a connect the grinder (Argos, UK) grinding rod to grind the residue of the mosquito enough at4℃, the tube was mixed and centrifuged at1,3000rpm for5min, and the supernatant was removed to2.0EP tube and diluted by adding1,200ml of phosphate buffer, witch was used to test the activity of GST, P450and COE metabolic enzyme activity assays and total protein assay. Residues add200μL alcoholic were marked as before,-20℃preserved.
3.2Total protein assay
Two replicates of40μL of each mosquito homogenate were placed in a disposable colorimetric cup (d=1), using0.25M KPO4buffer40μL as negative control, add900μL Coomassie protein assay reagent was mixed enough. The colorimetric cup was incubated for5min and read at595nm using biophotometer plus. Take two times average and CK, According bovine serum albumin was used to prepare a typical standard curve Y=28.977X2-22.201X+5.1657to calculate protein,(X:OD, Y:Bovine serum albumin protein), which subdue CK then times1500, divide40, and divide1000, to get the total protein (Unite:mg). We can use the total protein to calculate three assay activities.
3.3Glutathione-S-transferase (GST) activity
A total of200μL reduced glutathione solution and200μL cDNB solution, added200μL of mosquito homogenate (Glutathione solution:cDNB solution:mosquito homogenate=1:1:1) were mixed enough in a disposable colorimetric cup (d=1). Using0.25M KPO4buffer as the negative control, then every minute for4minutes at340nm, repeated again. The four times reading OD marked1,2,3,4, as4-1,4-2,4-3,3-1,3-2,2-1divide each reading inter time to get6number, take average subduce the average of CK divide time which result is one time operation, take two times operation result to make average, then times20as the final GST activity (Unite:μmol cDNB/min/mg protein).Total test956samples GST assay activities.
3.4Cytochrome P450(Cyt P450) monooxygenases activity
A total of8μL of the7-ethoxycoumarine (7-EC)(Sigma, E1379-25MG) solution was added to400μL of mosquito supernatant and samples were incubated at30℃for4h in a disposable colorimetric cup (d=1). Using0.25M KPO4buffer as the negative control, the reaction was stopped by the addition of560μL glycine buffer (pH10.4,0.1mM) and sample absorbance was read at450nm two times, repeated again. Take average of two times reading OD divided interval time once a time, then get two times operation average subdue CK, according to a standard regression based on a serial dilution of 7-hydroxycoumarin and its relevant OD values Y=88.47OD+4.88calculate final P450activity (Unite:7-HC/min/mg protein). Total test938samples P450assay activities.
3.5Carboxylesterase (COE) activity assay
A total of900μL p-nitrophenyl acetate solution to1.5ml EP tube and place the tube for5min in a30℃water bath, add100μL mosquito homogenates and then centrifuged at1,3000rpm for5min. Transfer the reaction mixture to a disposable colorimetric cup (d=1), read at405nm two times, using0.25M KPO4buffer as negative control, repeat again. Take the two times reading OD subdue the first times reading OD once a time, get the average two times subdue CK as final OD, according to the formula OD/(16.4X0.05Xprotein(mg/ml))X1000to calculate COE activity (Unites:nmol/min/mg protein). Total test903samples COE assay activities.
4. Analysis target resistance of the main malaria vector in Hainan Island.
For the reason DDT and pyrethroid insecticides target is the para-type sodium channel (SC), the target of organophosphate and carbamate insecticides is acetylcholinesterase (AChE). We did Knockdown resistance test before, and did ace-1test behind. One leg of each mosquito was used for DNA extraction with the E.Z.N. A. TM Micro Elute Genomic DNA Kit (Operate as Product).
4.1PCR amply the target of the Knockdown resistance L1014site test
Para sodium channels with rapid knockdown effect of insecticide resistance, this is called knockdown resistance (Knock down resisters, Kdr). Anopheles sodium channel second functional areas in the hydrophobic region of S6, leucine to phenylalanine mutation at codon1014. Usually leucine was replaced by phenylalanine (LeuyPhe) or serine or cysteine mutation at codon1014, and in turn was named L1014F, L1014S and L1014C. So we test the codon1014. PCR primers5'→3'of An. sinensis are Kdr-F1023(TGCCACTCCGTGTGTTTAGA)and Kdr-R1347(GAGCGATGATGATCCGAAAT), the cycling conditions of An. sinensis was as follows:initial denaturation at94℃for3min,45cycles of lmin denaturation at94℃,30s annealing at50℃and30s extension at72℃followed by a final extension of10min at72℃. We amplified a325bp fragment product and sequenced. PCR primers5'→3'of An. vagus are Agd1mi-F (TAGGTCACGGTGAGTCCGYA) and Agd2h-R (ACCACGATCACGTTCTCCTC), The cycling conditions of An. vagus was only change the temperate of annealing from50℃to47℃, others are same as An. sinensis.
4.2PCR-RFLP amplify the target of Ace-1gene at positions119test
Use PCR-RFLP amplify test to determine point mutations of Anopheles sinensis and Anopheles Vagus the ace-1gene at positions119. The PCR primes are Ace-1-F22F and Ace-1-R660. The cycling conditions were as follows:initial denaturation at94℃for3min,35cycles of45s denaturation at94℃,30s annealing at55℃and30s extension at72℃followed by a final extension of5min at72℃.2.5%agarose gel electrophoresis test the product, we get602bp product of Anopheles sinensis, and571bp product of Anopheles Vagus. Then we use the restriction end nuclease AIU1of PCR products were digested by enzyme (Operate as Product). Homozygous mutations type was amplified two bands (118bp and75bp), Wild type was only amplified one band (193bp), Heterozygous mutations type was amplified three bands (193bp,118bp and75bp), and the gene type simply:wild type is GGC/GGC, heterozygous mutations type is GGC/AGC and homozygous mutations type is AGC/AGC. We accorded mutation number to calculate frequency of G119mutation.
5. Statistical analysis
All data were statistical analyzed by SPSS13.0software. Mosquito mortality rates after the24hr recovery period were calculated for each insecticide and each population. Mosquito contact no pesticides but blank film corresponding solvent as control correction. Each repeated corrected mortality to calculate the average mortality and standand devation of each mosquito population after each insecticide treatment. Each population handling pesticides each within24hours after the death of the mosquito is divided into sensitive mosquitoes (sensitive group S),24hours after the live mosquitoes into resistant mosquitoes (resistance group R).Chi-square tests or Fisher exact probability tests were used to examine the association between target site mutations and the resistance phenotype. Two independent samples t test was used to compare the enzyme activities between resistant and susceptible mosquitoes for each population. When P<0.05had statistical significance. At the same time is calculated for each of the mosquito population resistance to insecticide treatment group for each site and sensitive group of mosquito enzyme activity ratio (R/S ratio), calculated using the following formula between resistant and susceptible Ratio Standards:SD=SQRT [(SDR/MR)2+(SDs/Ms)2].
Results
1. Result of Investigation the main malaria vector population surveillance and insecticides were used in Hainan Island.
8sites of Hainan Island we found that Anopheles sinensis are main distribute at Sanya (0.923±0.280number/spoon) and Baoting (1.056±0.665number/spoon), Anopheles tessellatus are main distribute at Sanya (1.979±1.470number/spoon), Baoting (4.572±1.855number/spoon) and linshui (1.143±0.595number/spoon), Anopheles vagus are main distribute at the Northeast in Hainan Island, including Chengmai (1.363±0.980number/spoon), Lingao (1.012±0.595number/spoon), Dingan (1.275±0.175number/spoon), Tunchang (1.099±0.420number/spoon) and Wenchang (4.792±1.085number/spoon). Pyrethroids insecticide were used in8sites we sampled, just as Deltamethrin, Methothrin, Beta cypermethrin, Bifenthrin, Lambda-cyhalothrin, et al. Organophosphates insecticide just as Imidacloprid were used in Baoting, Lingao, Tunchang. Organochlorines insecticide just as DDT was forbidden used in China, but we surprised found they were used in Baoting, Dinan, Wenchang. Imidacloprid were found used in Sanya, Baoting, Chengmai, Wenchang.
2. Result of bioassay tests the main malaria vector in Hainan Island.
For the reason we cannot get enough adult number and each site use different insecticide in Hainan Island, we did bioassay use different insecticide paper to different sites. ALL control groups mortality is zero. Total tested1725samples and result of each site each insecticide as follow:
2.1Result of0.05%Deltamethrin bioassay
Anopheles sinensis have initial0.05%Deltamethrin resistance in Sanya (85.78%±17.54%) and Baoting (90.98%±9.39%). Anopheles vagus has0.05%Deltamethrin resistant in Chenmai (97.87%±4.31%) and Dinan (100%). Anopheles tessellates have0.05%Deltamethrin resistant, the mosquito mortality is100%in Sanya and Lingshui.
2.2Result of4%DDT bioassay
Anopheles sinensis have4%DDT resistant in Sanya (78.38%±15.83%) and Baoting (72.71%±21.20%). Anopheles vagus have4%DDT resistant in Tunchang (67.057%±7.54%) and have initial4%DDT resistance in Chengmai (84.00%±11.68%) and Dinan (88.82%±10.07%).
2.3Result of5%Malathion bioassay
Anopheles vagus has5%Malathion initial resistance in Dinan (77.29%±10.57%), Chenmai (88.87%±4.26%) and Tunchang (78.86%±9.49%).
3. Result of analysis metabolic detoxification enzyme activity of the main malaria vector in Hainan Island.
3.1Result of Glutathione-S-transferase (GST) activity
We found the GST metabolic enzyme activities R/S ratio of Anopheles vagus was significantly higher in the two sites from Hainan Island (Dinan, Tunchang), where mosquitoes were tested bioassay by5%Malathion. The ratio of Dinan and Tunchang respectively was1.1361±0.3145(t=2.806, P=0.0060) and1.1141±0.1853(t=2.501, P=0.0150).(Unite:μmol cDNB/min/mg protein).
3.2Result of Cytochrome P450(Cyt P450) monooxygenases activity
We found the P450metabolic enzyme activities R/S ratio of Anopheles sinensis was significantly higher in the two sites from Hainan Island, the ratio of Baoting and Sanya (used0.05%Deltamethrin to test bioassay) respectively was1.5278±0.6072(t=2.575,F=6.6300, P=0.0115) and1.5391±0.8130(t=2.738, P=0.0076), and the ratio of Baoting and Sanya (used4%DDT to test bioassay) respectively was1.2662±0.7366(t=2.145, P=0.0346) and1.2732±0.5853(t=2.317,P=0.0226). The P450metabolic enzyme activities R/S ratio of Anopheles vagus was significantly higher in the one site from Hainan Island (Dingan), where mosquitoes were tested bioassay by5%Malathion. The ratio was1.3143±0.7058(t=2.417, P=0.0175).(Unite:7-HC/min/mg protein).
3.3Result of Carboxylesterase (COE) activity assay
We found the COE metabolic enzyme activities R/S ratio of Anopheles sinensis was significantly higher in the two sites from Hainan Island (Baoting, Sanya), where mosquitoes were tested bioassay by0.05%Deltamethrin. The ratio of Baoting and Sanya respectively was1.2738±0.4677(t=2.009, P=0.0470) and1.2580±0.3507(t=3.478, P=0.0010).(Unite:nmol/min/mg protein).
4. Result of Analysis target resistance of the main malaria vector in Hainan Island.
4.1Result of PCR amply the target of Knockdown resistance L1014site test
The kdr mutation of An. Sinensis only detected one heterozygous mutations type (TTG/TTT):Leucine to Phenylalanine substitution. The kdr mutation frequency of Deltamethrin-resistant mosquitoes from Sanya and DDT-resistant mosquitoes from Sanya and Baoting individuate is7.5%(P=0.025),9.5%(P=0.015) and7.8%(P=0.011),they are all L1014F mutation, which maybe related with An. sinensis insecticide resistance. No kdr mutations were detected in An. vagus populations from Hainan Island, China.
4.2Result of PCR-RFLP amplified the target of Ace-1gene at positions119test
Modest to high (53%-72%) ace-1mutation frequency was found in all sites we sampled An. sinensis populations, the resistance group mutation frequency higher than the sensitive group. But no ace-1mutation was detected in An. vagus populations.
Conclusions:
1. During7-8months, the main malaria vectors were An. sinensis, An. vagus and An. tessellates in Hainan Island. An. sinensis and An. tessellates populations were main sampled in Southern of Hainan Island, An. vagus population were main found in Northest of Hainan Island. Pyrethroid mosquito coils insecticides were the most propular insecticide to spray in rice frield in Hainan.
2. Result of Bioassay tests shows that Deltamethrin resistance was widespread over the Anopheles sinensis populations of Sanya and Baoting, DDT resistance was widespread over the Anopheles sinensis and Anopheles vagus populations, Malathion resistance was widespread over the Anopheles vagus populations of Chengmai, Dinan and Tunchang. Result of PCR-RFLP amplified the target of Ace-1gene at positions119test shows that Malathion resistance was widespread over the Anopheles sinensis populations of Sanya and Baoting.
3. The Kdr and enzyme assay results show that metabolic detoxification mechanisms are wide-spread in An. Sinensis and An. vagus populations from Hainan Island China, the target site mutations(L1014F) was only detected in An. Sinensis suggest the mechanisms of An. vagus insecticide resistant from Hainan Island is not relations to Kdr, but consider maybe relation with the metabolic detoxification enzyme activity. Then the mechanisms of An. Sinensis insecticide resistant maybe the common function with Kdr and metabolic detoxification enzyme activity. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes.
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