淡色库蚊对残杀威抗性机制的研究--酯酶与抗性的关系

为探讨酯酶与抗性的关系，采用生物测定法和微滴定板法，检测淡色库蚊抗残杀威品系各世代的抗性指数及蚊体现人酯酶的活性。淡色库蚊经过43代的汰选，抗性指数达到11.26倍；蚊体内的乙酰胆碱酯酶（AchE)活性的平均抑制率随世代的增加而变低，低于30%的个体频率随世代的增加而增加，呈正相关关系；非特异性酯酶（NSE）的活力随世代的增加而增强，而高于或等于0.9的个体频率逐渐增高，与生物测试法基本一致。抗残杀威品系AchE的不敏感性与抗性呈正相关关系，NSE的活力与抗性亦呈相关关系。     

不同品系淡色库蚊各发育阶段的非特异性酯酶和乙酰胆碱酯酶活力变化

研究并探讨非特异性酯酶（NSE）和乙酰胆碱酯酶（AChE）活力在不同品系淡色库蚊（Cules pipiens pallens）各发育阶段的变化，为应用生物化学方法检测蚊虫抗药性及科学合理地进行蚊虫化学防治提供依据。用微量滴定板法测定单个蚊虫NSE和AChE活力，结果显示，敏感品系、DDVP抗性品系和残杀威抗性品系淡色库蚊NSE活力水平在不同发育阶段有变化，I－Ⅲ龄幼虫较低，Ⅳ龄幼虫、蛹和3日龄雌成蚊较高，在各发育阶段均是DDVP抗性品系NSE活力最高，残杀威抗性品系次之，敏感品系最低，其中Ⅳ龄幼虫、蛹和3日龄雌成蚊阶段三个品系间NSE活力差别更明显；三个品系淡色库蚊AChE活力在I－Ⅳ龄幼虫和蛹期较低，3日龄雌成蚊较高，三个品系间AChE活力稍有差异，从高到低依次为残杀威抗性品系、DDVP抗性品系和敏感品系。测定蚊虫NSE活力判断抗药性可选择Ⅳ龄幼虫、蛹和3日龄未吸血雌成蚊；测定蚊虫AChE不敏感性判断抗药性可选择3日龄未吸血雌成蚊；蚊虫化学防治中应选择低龄幼虫阶段施药。     

河南省淮滨县中华按蚊的抗药性研究

用成蚊接触筒法和幼虫浸渍法测定河南省淮滨县中华按蚊子1代Ⅳ龄幼虫和3~5日龄成蚊对敌敌畏、溴氰菊酯和残杀威的抗药性,用色度法测定幼虫和成蚊的乙酰胆碱酯酶(AChE)在加入上述3种杀虫剂前后的活性,计算抑制率。结果显示,0.05%溴氰菊酯(0.017 8 g/m2)药纸测定中华按蚊成蚊的死亡率为51.2%(<80%),判断为抗性群体(R);中华按蚊幼虫对敌敌畏、溴氰菊酯和残杀威等3种杀虫剂的半数致死浓度(LC50)分别为2.76、320.85和754.32 mg/L。0.005 mmol/L敌敌畏、溴氰菊酯和残杀威对幼虫AChE的抑制率分别为77.8%、62.8%和58.9%,对成蚊的抑制率分别为76.1%、62.2%和58.2%,对幼虫和成虫的AChE活性均有显著的抑制作用(均P<0.01)。     

酯酶滤纸法检测淡色库蚊抗药性的研究

为探讨简捷灵敏的蚊虫抗药性早期测报技术,以α-乙酸萘酯为底物,坚硬固蓝B盐为显色剂,用滤纸法测定实验室和现场淡色库蚊（Cx.pipien pallens)酯酶活力,结果显示三个抗性品系淡色库蚊以抗DDVP品系抗性个体频率最高,其次为抗残杀威品系,抗氯氰菊酯品系较低,与敏感品系相近;三个现场淡色库蚊均对DDVP和残杀威产生不同程度抗性,其抗性个体频率均较高,表明酯酶滤纸法可用于检测蚊虫对有机磷和氨基甲酸酯类杀虫剂的抗性水平,该方法操作简便快速,观察结果直观灵敏,具有实用价值。     

非特异性酯酶在淡色库蚊对不同杀虫剂抗性中的作用研究

目的：探讨非特异性酯酶（Non-specific esterase,NSE)在谈色库蚊对不同杀虫剂抗性中的作用。方法：以β－乙酸萘酯为底物,坚固蓝B盐为显色剂,测定室内5个品系淡色库蚊的NSE活力。结果：5个品系淡色库蚊中以抗DDVP品系NSE活力水平最高,其次为抗DDVP降解品系和抗残杀威品系,抗氯氰菊酯品系较低,与敏感品系相近。结论：NSE在淡色库蚊对有机磷类杀虫剂的抗性中起重要作用,也是对氨基甲酸酯类杀虫剂产生的机制之一,与对拟除虫菊类杀虫剂的抗性关系不大。     

非特异性酯酶在淡色库蚊对不同杀虫剂抗性中的作用研究

目的　探讨非特异性酯酶（Non-specific esterase,NSE）在淡色库蚊对不同杀虫剂抗性中的作用。方法　以β-乙酸萘酯为底物,坚固蓝B盐为显色剂,测定室内5个品系淡色库蚊的NSE活力。结果　5个品系淡色库蚊中以抗DDVP品系NSE活力水平最高,其次为抗DDVP降解品系和抗残杀威品系。抗氯氰菊酯品系较低,与敏感品系相近。结论　NSE在淡色库蚊对有机磷类杀虫剂的抗性中起重要作用,也是对氨基甲酸酯类杀虫剂产生抗性的机制之一,与对拟除虫菊酯类杀虫剂的抗性关系不大。     

微板法测定淡色库蚊体内非特异性酯酶检测其对杀虫剂抗性的研究

目的　探讨简捷灵敏的蚊虫抗药性早期测报技术。方法　以 β -乙酸萘酯为底物 ,坚固蓝B盐为显色剂 ,用微板法测定实验室和现场单个淡色库蚊体内非特异性酯酶活力。结果　实验室五个品系淡色库蚊以抗DDVP品系非特异性酯酶活力水平最高 ,其次为抗残杀威品系和抗DDVP降解品系 ,抗氯氰菊酯品系较低 ,与敏感品系相近 ;三个现场淡色库蚊种群对DDVP抗性为 4 15～ 9 36倍 ,对残杀威抗性为 1 0 2～ 3 81倍 ,其非特异性酯酶活力水平均较高。结论　微板法测定非特异性酯酶能够检测蚊虫对有机磷和氨基甲酸酯类杀虫剂的抗性水平。方法简便快速 ,结果客观准确 ,可重复性强 ;可测出抗性频率 ,早期发现抗性的存在 ;可测知抗性机制 ,便于制订抗药性克服对策     

微板法测定抗药性醋酶检测淡色库蚊抗药性的研究

目的 研究简捷灵敏的蚊虫抗药性早期测报技术。方法 以β-乙酸萘酯为底物,坚固蓝B盐为显色剂,用酯酶微板法测定实验室和现场单个淡色库蚊体内非特异性酯酶活力。结果 实验室5个品系淡色库蚊以抗DDVP品系非特异性酯酶活力水平最高,其次为抗残杀威品系和抗DDVP降解品系,抗氯氰菊酯品系较低,与敏感品系相近;3个现场淡色库蚊种群对DDVP抗性为4.15～9.36倍,对残杀威抗性为1.02～3.81倍,其非特异性酯酶活力水平均较高。结论 酯酶微板法测定非特异性酯酶能够检测蚊虫对有机磷和氨基甲酸酯类杀虫剂的抗性水平,能够早期预测抗性发展趋势,便于制订抗药性克服对策。     

微板法测定抗药性酯酶检测淡色库蚊抗药性的研究

目的研究简捷灵敏的蚊虫抗药性早期测报技术. 方法以β-乙酸萘酯为底物,坚固蓝B盐为显色剂,用酯酶微板法测定实验室和现场单个淡色库蚊体内非特异性酯酶活力. 结果实验室5个品系淡色库蚊以抗DDVP品系非特异性酯酶活力水平最高,其次为抗残杀威品系和抗DDVP降解品系,抗氯氰菊酯品系较低,与敏感品系相近;3个现场淡色库蚊种群对DDVP抗性为4.15～9.36倍,对残杀威抗性为1.02～3.81倍,其非特异性酯酶活力水平均较高. 结论酯酶微板法测定非特异性酯酶能够检测蚊虫对有机磷和氨基甲酸酯类杀虫剂的抗性水平,能够早期预测抗性发展趋势,便于制订抗药性克服对策.     

温度对酯酶测定法检测淡色库蚊对DDVP的抗药性的影响

目的：用室内多年选育的抗DDVP淡色库蚊抗性株在室内和现场进行试验，以比较WHO标准法和微板测定法在检测蚊虫对DDVP抗药性的差别。方法：微板测定法、生理测定法。结果：用两种方法在温度为23－32℃范围内分别对淡色库蚊进行DDVP抗药性检测，WHO法结果与温度无关，而NSE法结果与温度有关。为消除温度对检测的影响，在缩短孵育时间后，在27－31℃对野外淡色库蚊对DDVP抗性测定，NSE法测定为WHO法结果相同。结论：NSE法检测蚊虫的DDVP抗药性可随外界温度升高而增加，实际应用时应缩短孵育时间加以纠正。     

Insecticide resistance status of Aedes aegypti in 10 localities in Colombia

Insecticide resistance is one of the major threats to the effectiveness of vector control programs. In order to establish a baseline susceptibility profile of Aedes aegypti in the southwest of Colombia, 10 localities in four Departments (States) were evaluated. Standardized WHO bioassay, CDC bottle bioassay and microplate biochemical assays of non-specific β-esterase (NSE), mixed function oxidases (MFO) and acetylcholinesterase were used. Cross resistance was evaluated with field collected mosquitoes that underwent selection pressure in the laboratory from DDT, propoxur and lambdacyhalothrin during three alternate generations. Mosquitoes with mortality rates below 80% in bioassays were considered resistant. Insecticide resistance varied geographically. Insecticide resistance was observed in 100% of localities in which mosquitoes were exposed to DDT, bendiocarb and temephos using both assays. WHO bioassays showed susceptibility to pyrethroids in all the localities evaluated, however CDC bottle bioassays showed decreases in susceptibility especially with lambdacyhalothrin. All localities showed susceptibility to the organophosphate malathion. Mosquitoes from eight regions with evidence of resistance to any of the insecticide evaluated were also evaluated biochemically. Mosquitoes from five of these regions had increased levels of NSE and two regions had increased levels of MFO. Increase levels of NSE explain partially the low susceptibility to temephos found in all the localities. However, the biochemical mechanisms evaluated do not explain all the resistance observed. Cross resistance was observed between the DDT-selected strain and lambdacyhalothrin, and between the lambdacyhalothrin-selected strain and propoxur and vice versa. The selected strains do not show changes in the biochemical assays evaluated, therefore the observed cross-resistance suggests different biochemical mechanisms. This study shows that Ae. aegypti from Colombia can develop resistance to most of the insecticide classes in the market. Periodic surveillance of insecticide resistance is necessary in order to maintain effective interventions. This study helped to establish the National Network for the surveillance of the insecticide resistance in Colombia.     

Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa

Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1^R mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.     

Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (approximately 800 times) are due to the esterase B1 gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase B1 protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase B1 gene. It is suggested that these repetitive sequences may have a role in the amplification process.     

Pyrethroid Resistance in Aedes aegypti from Grand Cayman

The Grand Cayman population of Aedes aegypti is highly resistant to DDT and pyrethroid insecticides. Glutathione transferase, cytochrome P450, and esterase levels were increased in the Grand Cayman population relative to a susceptible laboratory strain, but synergist studies did not implicate elevated insecticide detoxification as a major cause of resistance. The role of target site resistance was therefore investigated. Two substitutions in the voltage-gated sodium channel were identified, V1016I in domain II, segment 6 (IIS6) (allele frequency = 0.79) and F1534C in IIIS6 (allele frequency = 0.68). The role of the F1534C mutation in conferring resistance to insecticides has not been previously established and so a tetraplex polymerase chain reaction assay was designed and used to genotype mosquitoes that had been exposed to insecticides. The F1534C mutation was strongly correlated with resistance to DDT and permethrin.     

Laboratory selection of carbofuran tolerant line of Culex quinquefasciatus Say, the filarial vector at Mysore

Carbofuran is a systemic insecticide/nematicide extensively employed in modern agriculture and public health to combat various insect pests and vectors. The development of insecticide resistance due to natural selection pressure by Anopheline and Culicine mosquitoes has created serious repercussions in mosquito control and disease eradication campaigns. In this regard Culex quinquefasciatus, a widely dispersed domestic mosquito and the only vector of lymphatic filariasis in India, was selected for the present experiment. In order to assess the resistance development in this species, it was subjected to carbofuran bioassay by employing WHO method for 10 generations. The results of larval selection test up to F10 revealed a significant increase in tolerance development in every generation compared to the susceptible F1. The results revealed up to 16.225 times tolerance after 10 generations. The LC50 observed in F10 is 1.298 ppm compared to 0.08 ppm in the F1. The implications of the observations will be discussed.     

Characterization of insecticide resistance in Trinidadian strains of Aedes aegypti mosquitoes

Bioassays and biochemical assays were conducted on eight Trinidadian strains of Aedes aegypti larvae to determine the involvement of biochemical mechanisms in resistance to insecticides. Larval strains were assayed to dichlorodiphenyltrichloroethane (DDT), bendiocarb, temephos and permethrin, using the Centers for Disease Control and Prevention (CDC) time-mortality bioassay method. A Resistance Threshold (RT) was calculated for each insecticide in relation to the CAREC reference susceptible Ae. aegypti strain and larval strains with <80% mortality were considered to be resistant. Biochemical assays were performed to determine the activities of nonspecific esterases (α- and β-), PNPA-esterases, mixed function oxidases (MFO), glutathione-S-transferases (GST) and acetylcholinesterase (AChE) enzymes which are involved in insecticide resistance in mosquitoes. Enzyme profiles of each strain were compared with those of the CAREC reference susceptible strain by Kruskal–Wallis and Dunn's multiple comparison tests (p < 0.05). The CAREC 99th percentile was calculated for each enzyme and the percentage of individuals with enzyme activities above that of the CAREC 99th percentile was calculated. Activities were classified as unaltered (<50%), incipiently altered (15–50%) or altered (>50%) for each strain.The established RTs for permethrin and bendiocarb were 30 and 75 min, respectively; and 120 min for DDT and temephos. All strains were resistant to DDT (1.00–40.25% mortality) and temephos (11.50–74.50% mortality) while six strains were resistant to bendiocarb (51.50–78.50% mortality) and five to permethrin (6.50–42.50% mortality). Biochemical assays revealed that the median activity levels for all enzymes varied significantly (p < 0.05). The Curepe strain had incipiently altered levels of α-esterase while the other seven strains had altered activity with five of them registering 100%. The St Clair strain showed altered activity levels of β-esterase while three strains had incipiently altered levels. The majority of strains had altered activity of MFO enzymes but only the St Clair strain showed altered activity of GST. PNPA-esterases activity was unaltered in all strains and only the Haleland Park strain showed altered remaining AChE activity in the presence of propoxur.Elevated levels of enzymes (incipiently altered or altered), except in the case of PNPA-esterases, show that biochemical resistance may play an important role in the manifestation of insecticide resistance in Trinidadian populations of Ae. aegypti. It is therefore important for insecticide resistance surveillance to be ongoing as the detection of resistance before it spreads throughout an entire population makes it possible for early intervention.     

Determination of insecticide susceptibility in Culex quinquefasciatus Say adults by rapid enzyme microassays.

Rapid enzyme microassays for the detection of resistance due to organophosphate and carbamate in individual field-collected strains of Culex quinquefasciatus adults were conducted. These tests allowed accurate differentiation by eye, on the basis of color changes of susceptible and resistant individuals. Two separate tests were conducted for the biochemical assays. In the insensitive acetylcholinesterase (AChE) test, acetylthiocholine iodide (ACTH) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were used as substrate and coupling agent respectively. The resulting yellow chromophore indicated AChE activity. Test results showed that the color intensity decreased as increasing concentrations of propoxur were added, thereby confirming the susceptibility of the enzyme to inhibitor. Assay of non-specific esterase however, indicated elevated levels which were correlated with degree of malathion resistance. Electrophoretic data revealed the presence of 2 esterase bands in all strains. It was concluded that such a pattern was not contributory to malathion resistance in adults.     

Bottle and biochemical assays on temephos resistance in Aedes aegypti in Thailand

The bottle bioassay measuring the time-mortality rate is a simplified procedure for detecting insecticide resistance. It can be used with a biochemical microplate assay to identify the mechanism involved. This integrated approach was used to detect temephos resistance in Aedes aegypti from Nonthaburi (lowest use) and Roi Et (highest use). Ae. aegypti BKK1 laboratory strain was used as the susceptible reference strain. The appropriate concentration of insecticide for bottle bioassay was determined empirically for Ae. aegypti BKK1 strain and found to be in the range of 800-1,050 microg/bottle. The time-mortality rate at 800 microg/bottle was 170 +/- 8.66 minutes, significantly different from the time-mortality rates in the 850, 900, 950, and 1,050 microg/bottle (p = 0.008) concentrations, which were 135 +/- 15.00, 140 +/- 8.66, 135 +/- 15.00, and 125 +/- 8.66 minutes, respectively. The cut-off concentration selected for resistance detection was 850 microg/bottle. The time-mortality rate for the Roi Et strain was 382 +/- 26.41 minutes, significantly higher than the Nonthaburi (150 +/- 25.10 minutes) and BKK1 strains (145 +/- 20.49 minutes) (p < 0.001). The temephos resistance ratio (RR100) for the Ae. aegypti Roi Et strain was 2.64-fold higher at lethal time (LT100) than for the reference Ae. aegypti BKK1 strain. The mean optical density (OD) value from the biochemical microplate assay for the non-specific esterase of the Roi Et strain was higher than the mean OD for the non-specific esterase of both the Nonthaburi and BKK1 strains. Insensitive acetylcholinesterase was not found to be responsible for the resistance in the field-collected mosquitos. This study suggests that esterase detoxification is the primary cause of resistance in the Ae. aegypti population from Roi Et. Both the bottle bioassay and the biochemical microplate assay were proven to be promising tools for initial detection and field surveillance for temephos resistance.