代谢解毒酶活性变化和击倒抗性基因突变在白纹伊蚊菊酯类杀虫剂抗性产生中的作用
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摘要
目的探讨代谢解毒酶活性变化和击倒抗性(kdr)基因突变在白纹伊蚊菊酯类抗性机制中的作用。方法 2017年8月至9月分别在山东省济南市千佛山公园(JN)、浙江省杭州市上茅家埠(HZ)、上海市宝山区宝山六村(BS)、上海市杨浦区共青森林公园(YP)和海南省海口市美兰区居民区(HK)采集现场白纹伊蚊(生物测定均为抗性种群),检测其代谢解毒酶谷胱甘肽S-转移酶(GST)和多功能氧化酶(MFO)的活性并与敏感品系的白纹伊蚊比较,采用分类回归树方法(CART)分析GST和MFO活性变化及kdr突变(I1532和F1534)在抗性产生中的贡献率。结果白纹伊蚊敏感品系的GST和MFO的活性基线水平均高于现场抗性种群BS和HK(P均<0.01)。BS种群接触溴氰菊酯后与未接触杀虫剂的基线相比,GST和MFO变化不明显(P>0.05),接触氯菊酯后GST活性升高(P<0.05),MFO活性也升高(P<0.01);HK种群接触溴氰菊酯后GST活性差异不明显(P>0.05)、MFO活性升高(P<0.01),接触氯菊酯后GST和MFO活性变化均不明显(P均>0.05)。5个现场抗性种群接触溴氰菊酯和氯菊酯后的GST和MFO活性与敏感品系的基线相比,变化无规律。CART分析结果显示,白纹伊蚊对溴氰菊酯的抗性产生中GST活性和kdr F1534突变的贡献率较大,其次是MFO活性,kdr I1532突变贡献率最小;对氯菊酯的抗性产生中,kdr F1534突变的贡献率最大,其次是GST和MFO活性,kdr I1532突变无贡献。结论代谢解毒酶GST和MFO活性水平不适合作为判断白纹伊蚊种群对菊酯类杀虫剂抗性的单因素指标;代谢解毒酶活性变化和kdr突变可能是白纹伊蚊对菊酯类杀虫剂抗性产生中相互协同的2种机制。
Objective To explore the role of metabolic detoxification enzyme activity and knockdown resistance(kdr) gene mutations in the pyrethroid resistance of Aedes(Ae.) albopictus. Methods From Aug. to Sep. in 2017, the Ae.albopictus samples were collected in Qianfoshan Park, Jinan City, Shandong Province(JN), Shangmaojiabu, Hangzhou City,Zhejiang Province(HZ), Baoshan Sixth Village, Baoshan District, Shanghai(BS), Gongqing Forest Park, Yangpu District,Shanghai(YP), and Meilan District Residential Area, Haikou City, Hainan Province(HK). The above five field populations were all resistant to insecticide. The activities of metabolic detoxification enzymes(glutathione-S transferase [GST] and mixed function oxidase [MFO]) were detected and compared with the Ae. albopictus susceptible strain(JS). The contribution rates of activity changes of GST and MFO and kdr mutations(I1532 and F1534) in the resistance formation were analyzed by the classification and regression trees(CART). Results The baseline enzyme activities of GST and MFO in Ae. albopictus JS were both significantly higher than those in the BS and HK resistant populations(both P<0.01). There were no significant difference in the activities of GST and MFO between the BS population unexposed and exposed to deltamethrin(P>0.05).After exposure to permethrin of BS population, the activities of GST and MFO were significantly increased(P<0.05,P<0.01). After exposure to deltamethrin, the GST activity was not significantly changed in the HK population(P>0.05),while the MFO activity was significantly increased(P<0.01). However, after exposure to permethrin in the HK population,there were no significant changes in the GST and MFO activities(both P>0.05). In the 5 field resistant populations exposed to deltamethrin and permethrin, the changes of GST and MFO activities were irregular compared with baseline of Ae. albopictus JS strain. CART analysis showed that in the resistance formation of Ae. albopictus against deltamethrin, the contribution rates of GST activity and kdr F1534 mutation were the greatest, followed by MFO activity, and the kdr I1532 mutation was the smallest. In the resistance formation of Ae. albopictus against permethrin, the kdr F1534 mutation had the highest contribution rate, followed by the GST and MFO activities, and the kdr I1532 mutation had no contribution. Conclusion The activity levels of metabolic detoxification enzymes(GST and MFO) are not suitable as single markers for detecting the resistance of Ae. albopictus to pyrethroids. The activity changes of metabolic detoxification enzymes and kdr mutations may be two synergistic mechanisms in the resistance formation of Ae. albopictus to pyrethroid insecticides.
Objective To explore the role of metabolic detoxification enzyme activity and knockdown resistance(kdr) gene mutations in the pyrethroid resistance of Aedes(Ae.) albopictus. Methods From Aug. to Sep. in 2017, the Ae.albopictus samples were collected in Qianfoshan Park, Jinan City, Shandong Province(JN), Shangmaojiabu, Hangzhou City,Zhejiang Province(HZ), Baoshan Sixth Village, Baoshan District, Shanghai(BS), Gongqing Forest Park, Yangpu District,Shanghai(YP), and Meilan District Residential Area, Haikou City, Hainan Province(HK). The above five field populations were all resistant to insecticide. The activities of metabolic detoxification enzymes(glutathione-S transferase [GST] and mixed function oxidase [MFO]) were detected and compared with the Ae. albopictus susceptible strain(JS). The contribution rates of activity changes of GST and MFO and kdr mutations(I1532 and F1534) in the resistance formation were analyzed by the classification and regression trees(CART). Results The baseline enzyme activities of GST and MFO in Ae. albopictus JS were both significantly higher than those in the BS and HK resistant populations(both P<0.01). There were no significant difference in the activities of GST and MFO between the BS population unexposed and exposed to deltamethrin(P>0.05).After exposure to permethrin of BS population, the activities of GST and MFO were significantly increased(P<0.05,P<0.01). After exposure to deltamethrin, the GST activity was not significantly changed in the HK population(P>0.05),while the MFO activity was significantly increased(P<0.01). However, after exposure to permethrin in the HK population,there were no significant changes in the GST and MFO activities(both P>0.05). In the 5 field resistant populations exposed to deltamethrin and permethrin, the changes of GST and MFO activities were irregular compared with baseline of Ae. albopictus JS strain. CART analysis showed that in the resistance formation of Ae. albopictus against deltamethrin, the contribution rates of GST activity and kdr F1534 mutation were the greatest, followed by MFO activity, and the kdr I1532 mutation was the smallest. In the resistance formation of Ae. albopictus against permethrin, the kdr F1534 mutation had the highest contribution rate, followed by the GST and MFO activities, and the kdr I1532 mutation had no contribution. Conclusion The activity levels of metabolic detoxification enzymes(GST and MFO) are not suitable as single markers for detecting the resistance of Ae. albopictus to pyrethroids. The activity changes of metabolic detoxification enzymes and kdr mutations may be two synergistic mechanisms in the resistance formation of Ae. albopictus to pyrethroid insecticides.
引文
[1]BONIZZONI M,GASPERI G,CHEN X,JAMES A A.The invasive mosquito species Aedes albopictus:current knowledge and future perspectives[J].Trends Parasitol,2013,29:460-468.
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[8]孟凤霞,王义冠,冯磊,刘起勇.我国登革热疫情防控与媒介伊蚊的综合治理[J].中国媒介生物学及控制杂志,2015,26:4-10.
[9]王晓花,陈辉莹,杨新艳,林怡,蔡芳,钟汶兵,等.海口市白纹伊蚊对菊酯类杀虫剂的抗药性及击倒抗性基因突变分析[J].第二军医大学学报,2015,36:832-838.WANG X H,CHEN H Y,YANG X Y,LIN Y,CAI F,ZHONG W B,et al.Resistance to pyrethroid insecticides and analysis of knockdown resistance(kdr)gene mutations in Aedes albopictus from Haikou City[J].Acad J Sec Mil Med Univ,2015,36:832-838.
[10]曾林海,孙定炜,赵伟,李善干,杨霞.海南省埃及伊蚊和白纹伊蚊对常用杀虫剂的敏感性测定[J].中国媒介生物学及控制杂志,2010,21:148-149.
[11]SMITH L B,KASAI S,SCOTT J G.Pyrethroid resistance in Aedes aegypti and Aedes albopictus:important mosquito vectors of human diseases[J].Pestic Biochem Physiol,2016,133:1-12.
[12]HEMINGWAY J,HAWKES N J,MCCARROLLL,RANSON H.The molecular basis of insecticide resistance in mosquitoes[J].Insect Biochem Mol Biol,2004,34:653-665.
[13]PETHUAN S,JIRAKANJANAKIT N,SAENGTHARATIP S,CHAREONVIRIYAPHAP T,KAEWPA D,RONGNOPARUT P.Biochemical studies of insecticide resistance in Aedes(Stegomyia)aegypti and Aedes(Stegomyia)albopictus(Diptera:Culicidae)in Thailand[J].Trop Biomed,2007,24:7-15.
[14]ISHAK I H,RIVERON J M,IBRAHIM S S,STOTT R,LONGBOTTOM J,IRVING H,et al.The cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdrfree Malaysian populations of the dengue vector Aedes albopictus[J/OL].Sci Rep,2016,6:24707.doi:10.1038/srep24707.
[15]史琦琪,程鹏,公茂庆.蚊虫抗药性分子机制研究进展[J].中国媒介生物学及控制杂志,2016,27:515-519.
[16]WILLIAMSON M S,MARTINEZ-TORRES D,HICK CA,DEVONSHIRE A L.Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance(kdr)to pyrethroid insecticides[J].Mol Gen Genet,1996,252(1/2):51-60.
[17]SAAVEDRA-RODRIGUEZ K,URDANETA-MARQUEZ L,RAJATILEKA S,MOULTON M,FLORES A E,FERNANDEZ-SALAS I,et al.A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti[J].Insect Mol Biol,2007,16:785-798.
[18]GAO J P,CHEN H M,SHI H,PENG H,MA Y J.Correlation between adult pyrethroid resistance and knockdown resistance(kdr)mutations in Aedes albopictus(Diptera:Culicidae)field populations in China[J/OL].Infect Dis Poverty,2018,7:86.doi:10.1186/s40249-018-0471-y.
[19]BROGDON W G,BARBER A M.Microplate assay of glutathione S-transferase activity for resistance detection in single-mosquito triturates[J].Comp Biochem Physiol B,1990,96:339-342.
[20]POLSON K A,RAWLINS S C,BROGDON W G,CHADEE D D.Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypt[J].Bull Entomol Res,2011,101:435-441.
[21]A?KPON R,S?ZONLIN M,OSS?R,AKOGB?TO M.Evidence of multiple mechanisms providing carbamate and organophosphate resistance in field An.gambiae population from Atacora in Benin[J/OL].Parasit Vectors,2014,7:568.doi:10.1186/s13071-014-0568-5.
[22]CHANG X,ZHONG D,FANG Q,HARTSEL J,ZHOUG,SHI L,et al.Multiple resistances and complex mechanisms of Anopheles sinensis mosquito:a major obstacle to mosquito-borne diseases control and elimination in China[J/OL].PLoS Negl Trop Dis,2014,8:e2889.doi:10.1371/journal.pntd.0002889.
[23]SANGBA M L,DEKETRAMETE T,WANGO SP,KAZANJI M,AKOGBETO M,NDIATH M O.Insecticide resistance status of the Anopheles funestus population in Central African Republic:a challenge in the war[J/OL].Parasit Vectors,2016,9:230.doi:10.1186/s13071-016-1510-9.
[24]李袁飒,孟凤霞,蔡松武,刘起勇.广东省湛江市抗溴氰菊酯白纹伊蚊代谢酶活性及其特征研究[J].中国媒介生物学及控制杂志,2013,24:103-107.
[25]吴家红,赵彤言,董言德.五株白纹伊蚊对溴氰菊酯抗性生物测定和生化检测的研究[J].中国人兽共患病杂志,2005,21:851-854.
[26]NGOAGOUNI C,KAMGANG B,BRENGUES C,YAHOUEDO G,PAUPY C,NAKOUN?E,et al.Susceptibility profile and metabolic mechanisms involved in Aedes aegypti and Aedes albopictus resistant to DDTand deltamethrin in the Central African Republic[J/OL].Parasit Vectors,2016,9:599.doi:10.1186/s13071-016-1887-5.
[27]VERSCHOYLE R D,ALDRIDGE W N.Structureactivity relationships of some pyrethroids in rats[J].Arch Toxicol,1980,45:325-329.
[28]NARAHASHI T.Neuronal ion channels as the target sites of insecticides[J].Pharmacol Toxicol,1996,79:1-14.
[29]LIU N.Insecticide resistance in mosquitoes:impact,mechanisms,and research directions[J].Annu Rev Entomol,2015,60:537-559.
[30]ISHAK I H,JAAL Z,RANSON H,WONDJI C S.Contrasting patterns of insecticide resistance and knockdown resistance(kdr)in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia[J/OL].Parasit Vectors,2015,8:181.doi:10.1186/s13071-015-0797-2.
[31]KUSHWAH R B,MALLICK P K,RAVIKUMAR H,DEV V,KAPOOR N,ADAK T P,et al.Status of DDTand pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance(kdr)mutation[J].JVector Borne Dis,2015,52:95-98.
[2]杨舒然,刘起勇.白纹伊蚊的全球分布及扩散趋势[J].中国媒介生物学及控制杂志,2013,24:1-4.
[3]GUO C,ZHOU Z,WEN Z,LIU Y,ZENG C,XIAO D,et al.Global epidemiology of dengue outbreaks in 1990-2015:a systematic review and meta-analysis[J/OL].Front Cell Infect Microbiol,2017,7:317.doi:10.3389/fcimb.2017.00317.
[4]LOUNIBOS L P,KRAMER L D.Invasiveness of Aedes aegypti and Aedes albopictus and vectorial capacity for chikungunya virus[J].J Infect Dis,2016,214(Suppl 5):S453-S458.
[5]EPELBOIN Y,TALAGA S,EPELBOIN L,DUSFOUR I.Zika virus:an updated review of competent or naturally infected mosquitoes[J/OL].PLoS Negl Trop Dis,2017,11:e0005933.doi:10.1371/journal.pntd.0005933.
[6]KULARATNE S A.Dengue fever[J].BMJ,2015,351:h4661.
[7]LI M T,SUN G Q,YAKOB L,ZHU H P,JIN Z,ZHANGW Y.The driving force for 2014 dengue outbreak in Guangdong,China[J/OL].PLoS One,2016,11:e0166211.doi:10.1371/journal.pone.0166211.
[8]孟凤霞,王义冠,冯磊,刘起勇.我国登革热疫情防控与媒介伊蚊的综合治理[J].中国媒介生物学及控制杂志,2015,26:4-10.
[9]王晓花,陈辉莹,杨新艳,林怡,蔡芳,钟汶兵,等.海口市白纹伊蚊对菊酯类杀虫剂的抗药性及击倒抗性基因突变分析[J].第二军医大学学报,2015,36:832-838.WANG X H,CHEN H Y,YANG X Y,LIN Y,CAI F,ZHONG W B,et al.Resistance to pyrethroid insecticides and analysis of knockdown resistance(kdr)gene mutations in Aedes albopictus from Haikou City[J].Acad J Sec Mil Med Univ,2015,36:832-838.
[10]曾林海,孙定炜,赵伟,李善干,杨霞.海南省埃及伊蚊和白纹伊蚊对常用杀虫剂的敏感性测定[J].中国媒介生物学及控制杂志,2010,21:148-149.
[11]SMITH L B,KASAI S,SCOTT J G.Pyrethroid resistance in Aedes aegypti and Aedes albopictus:important mosquito vectors of human diseases[J].Pestic Biochem Physiol,2016,133:1-12.
[12]HEMINGWAY J,HAWKES N J,MCCARROLLL,RANSON H.The molecular basis of insecticide resistance in mosquitoes[J].Insect Biochem Mol Biol,2004,34:653-665.
[13]PETHUAN S,JIRAKANJANAKIT N,SAENGTHARATIP S,CHAREONVIRIYAPHAP T,KAEWPA D,RONGNOPARUT P.Biochemical studies of insecticide resistance in Aedes(Stegomyia)aegypti and Aedes(Stegomyia)albopictus(Diptera:Culicidae)in Thailand[J].Trop Biomed,2007,24:7-15.
[14]ISHAK I H,RIVERON J M,IBRAHIM S S,STOTT R,LONGBOTTOM J,IRVING H,et al.The cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdrfree Malaysian populations of the dengue vector Aedes albopictus[J/OL].Sci Rep,2016,6:24707.doi:10.1038/srep24707.
[15]史琦琪,程鹏,公茂庆.蚊虫抗药性分子机制研究进展[J].中国媒介生物学及控制杂志,2016,27:515-519.
[16]WILLIAMSON M S,MARTINEZ-TORRES D,HICK CA,DEVONSHIRE A L.Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance(kdr)to pyrethroid insecticides[J].Mol Gen Genet,1996,252(1/2):51-60.
[17]SAAVEDRA-RODRIGUEZ K,URDANETA-MARQUEZ L,RAJATILEKA S,MOULTON M,FLORES A E,FERNANDEZ-SALAS I,et al.A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti[J].Insect Mol Biol,2007,16:785-798.
[18]GAO J P,CHEN H M,SHI H,PENG H,MA Y J.Correlation between adult pyrethroid resistance and knockdown resistance(kdr)mutations in Aedes albopictus(Diptera:Culicidae)field populations in China[J/OL].Infect Dis Poverty,2018,7:86.doi:10.1186/s40249-018-0471-y.
[19]BROGDON W G,BARBER A M.Microplate assay of glutathione S-transferase activity for resistance detection in single-mosquito triturates[J].Comp Biochem Physiol B,1990,96:339-342.
[20]POLSON K A,RAWLINS S C,BROGDON W G,CHADEE D D.Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypt[J].Bull Entomol Res,2011,101:435-441.
[21]A?KPON R,S?ZONLIN M,OSS?R,AKOGB?TO M.Evidence of multiple mechanisms providing carbamate and organophosphate resistance in field An.gambiae population from Atacora in Benin[J/OL].Parasit Vectors,2014,7:568.doi:10.1186/s13071-014-0568-5.
[22]CHANG X,ZHONG D,FANG Q,HARTSEL J,ZHOUG,SHI L,et al.Multiple resistances and complex mechanisms of Anopheles sinensis mosquito:a major obstacle to mosquito-borne diseases control and elimination in China[J/OL].PLoS Negl Trop Dis,2014,8:e2889.doi:10.1371/journal.pntd.0002889.
[23]SANGBA M L,DEKETRAMETE T,WANGO SP,KAZANJI M,AKOGBETO M,NDIATH M O.Insecticide resistance status of the Anopheles funestus population in Central African Republic:a challenge in the war[J/OL].Parasit Vectors,2016,9:230.doi:10.1186/s13071-016-1510-9.
[24]李袁飒,孟凤霞,蔡松武,刘起勇.广东省湛江市抗溴氰菊酯白纹伊蚊代谢酶活性及其特征研究[J].中国媒介生物学及控制杂志,2013,24:103-107.
[25]吴家红,赵彤言,董言德.五株白纹伊蚊对溴氰菊酯抗性生物测定和生化检测的研究[J].中国人兽共患病杂志,2005,21:851-854.
[26]NGOAGOUNI C,KAMGANG B,BRENGUES C,YAHOUEDO G,PAUPY C,NAKOUN?E,et al.Susceptibility profile and metabolic mechanisms involved in Aedes aegypti and Aedes albopictus resistant to DDTand deltamethrin in the Central African Republic[J/OL].Parasit Vectors,2016,9:599.doi:10.1186/s13071-016-1887-5.
[27]VERSCHOYLE R D,ALDRIDGE W N.Structureactivity relationships of some pyrethroids in rats[J].Arch Toxicol,1980,45:325-329.
[28]NARAHASHI T.Neuronal ion channels as the target sites of insecticides[J].Pharmacol Toxicol,1996,79:1-14.
[29]LIU N.Insecticide resistance in mosquitoes:impact,mechanisms,and research directions[J].Annu Rev Entomol,2015,60:537-559.
[30]ISHAK I H,JAAL Z,RANSON H,WONDJI C S.Contrasting patterns of insecticide resistance and knockdown resistance(kdr)in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia[J/OL].Parasit Vectors,2015,8:181.doi:10.1186/s13071-015-0797-2.
[31]KUSHWAH R B,MALLICK P K,RAVIKUMAR H,DEV V,KAPOOR N,ADAK T P,et al.Status of DDTand pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance(kdr)mutation[J].JVector Borne Dis,2015,52:95-98.
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