外来入侵物种细足捷蚁的SS-PCR快速检测技术研究
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摘要
细足捷蚁Anoplolepis gracilipes为我国南部地区新发现的外来入侵物种,对入侵地区生物多样性已造成严重威胁。本研究针对其个体小、表型特征较难鉴定等问题,建立基于SS-PCR技术的细足捷蚁快速分子检测鉴定技术体系。以细足捷蚁为阳性对照,以其他7种形态相似种和常见种为阴性对照,采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ(mt DNA COI)基因序列的种特异性PCR方法 (species-specific PCR,SS-PCR),研究其快速分子检测鉴定技术的关键指标和反应条件。利用昆虫mt DNA COI基因通用型引物LCO-1490/HCO-2198获得细足捷蚁及其他7种形态相似种和常见蚂蚁种的COI序列,根据测序比对及筛选得到1对高灵敏度引物APGPF/APGPR,其扩增片段大小为374 bp。种特异性检验结果表明,该引物只对细足捷蚁的COI基因具有扩增能力,对其他7种形态相似种和常见种包括拟光腹弓背蚁Camponotus irritans(Smith,1857)、费氏弓背蚁Camponotus fedtschenkoi(Mayr,1877)、尼科巴弓背蚁Camponotus nicobarensis(Mayr,1865)、黄猄蚁Oecophylla smaragdina(Fabricius,1775)、大阪举腹蚁Crematogaster osakensis(Forel,1900)、宽结大头蚁Pheidole noda(Smith,1874)、大头蚁Pheidole sp.不具有特异性扩增效果。灵敏性检验的结果表示,该对引物不仅对成年工蚁具有良好的扩增能力,对不同品级与龄期的蚂蚁卵、蛹、幼虫也具有同样的扩增能力,其检测阈值为22.00 pg/μL(相当于1/51 200头成虫)。建立的细足捷蚁对其他7种形态相似种和常见种的物种特异性检测鉴定技术,可用于细足捷蚁的快速识别、监测预警和控制清除等相关研究与应用。
Yellow crazy ant,Anoplolepis gracilipes,is a newly recorded invasive species in southern China,which poses a serious threat to the native biodiversity.Morphological identification of A.gracilipes is limited by small size,high degree of similarity to related species.This study aims to develop a technique based on mitochondrial DNA cytochrome c oxidase subunit I(mt DNA COI) gene sequence for the rapid identification of A.gracilipes.The fragments of mt DNA COI gene of A.gracilipes and sevenother ant species were amplified and sequenced using COI gene universal primers LCO-1490/HCO-2198.One pair of species specific COI(SS-COI) primers,APGPF and APGPR,was designed.The target fragment amplified by the SS-COI primers(APGPF/APGPR) was 374 bp in length.The species specificity and sensitivity of the primer pair were tested.The species specificity of the primer pair was validated using seven other ant species,i.e.,Camponotus irritans(Smith.1857),Camponotus fedtschenkoi(Mayr.1877),Camponotus nicobarensis(Mayr.1865),Oecophylla smaragdina(Fabricius.1775),Crematogaster osakensis(Forel.1900),Pheidole noda(Smith,1874),Pheidole sp.All A.gracilipes specimens were successfully detected,and no cross reaction with other ant species was observed.The method was tested using individual worker or queen,individual 1 stinstar,2 ndinstar larva,and even one grain of egg,and proved to be applicable for all of these life stages and grades.Sensitivity test results demonstrated that successful amplification could be obtained with the concentration of template DNA as low as 22.00 pg/μL,equal to 1/51 200 of a whole worker of A.gracilipes.The SS-COI method developed here provides a quick,simple and reliable molecular technique for the identification and monitoring of A.gracilipes,which would be useful in detecting,monitoring and control of this new invasive ant species.
Yellow crazy ant,Anoplolepis gracilipes,is a newly recorded invasive species in southern China,which poses a serious threat to the native biodiversity.Morphological identification of A.gracilipes is limited by small size,high degree of similarity to related species.This study aims to develop a technique based on mitochondrial DNA cytochrome c oxidase subunit I(mt DNA COI) gene sequence for the rapid identification of A.gracilipes.The fragments of mt DNA COI gene of A.gracilipes and sevenother ant species were amplified and sequenced using COI gene universal primers LCO-1490/HCO-2198.One pair of species specific COI(SS-COI) primers,APGPF and APGPR,was designed.The target fragment amplified by the SS-COI primers(APGPF/APGPR) was 374 bp in length.The species specificity and sensitivity of the primer pair were tested.The species specificity of the primer pair was validated using seven other ant species,i.e.,Camponotus irritans(Smith.1857),Camponotus fedtschenkoi(Mayr.1877),Camponotus nicobarensis(Mayr.1865),Oecophylla smaragdina(Fabricius.1775),Crematogaster osakensis(Forel.1900),Pheidole noda(Smith,1874),Pheidole sp.All A.gracilipes specimens were successfully detected,and no cross reaction with other ant species was observed.The method was tested using individual worker or queen,individual 1 stinstar,2 ndinstar larva,and even one grain of egg,and proved to be applicable for all of these life stages and grades.Sensitivity test results demonstrated that successful amplification could be obtained with the concentration of template DNA as low as 22.00 pg/μL,equal to 1/51 200 of a whole worker of A.gracilipes.The SS-COI method developed here provides a quick,simple and reliable molecular technique for the identification and monitoring of A.gracilipes,which would be useful in detecting,monitoring and control of this new invasive ant species.
引文
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Blancafort X,Gómez C.Consequences of the Argentine ant,Linepithema humile(Mayr),invasion on pollination of Euphorbia characias(Euphorbiaceae)[J].Acta Oecologica,2005,28(1):49-55.
Brühl CA,Eltz T.Fuelling the biodiversity crisis:Species loss of ground-dwelling forest ants in oil palm plantations in Sabah,Malaysia(Borneo)[J].Biodiversity and Conservation,2010,19(2):519-529.
Buczkowski G,Vargo EL,Silverman J.The diminutive supercolony:The Argentine ants of the southeastern United States[J].Molecular Ecology,2004,13(8):2235-2242.
Chen CR.A dangerous pest-Anoplolepis gracilipes was intercepted in Zhuhai port[J].Pesticide Market News,2009,6:41.[陈楚荣.珠海口岸首次截获有害生物细足捷蚁[J].农药市场信息,2009,6:41]
Chen MM,Guo R,Zhang JL,et al.Rapid identification of Aleyrodes proletella(Hemiptera:Aleyrodidae),a new invasive whitefly species in mainland China,based on SS-COI marker[J].Acta Entomologica Sinica,2015,58(5):579-586.[陈苗苗,郭荣,张金良.基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术[J].昆虫学报,2015,58(5):579-586]
Cheng XT,Shataer A,Zhang W,et al.Species-specific PCR primers for identification of Carpomyia vesuviana[J].Scientia Silvae Sinicae,2013,49(11):98-102.[程晓甜,阿地力·沙塔尔,张伟.枣实蝇特异引物PCR鉴定技术[J].林业科学,2013,49(11):98-102]
Christian CE.Consequences of a biological invasion reveal the importance of mutualism for plant communities[J].Nature,2001,413(6856):635-639.
Darling JA,Blum MJ.DNA-based methods for monitoring invasive species:A review and prospectus[J].Biological Invasions,2007,9(7):751-765.
Davidson DW.Resource discovery versus resource domination in ants:Afunctional mechanism for breaking the trade-off[J].Ecological Entomology,1998,23(4):484-490.
Davis NE,O'dowd DJ,Green PT,et al.Effects of an alien ant invasion on abundance,behavior,and reproductive success of endemic island birds[J].Conservation Biology,2008,22(5):1165-1176.
Davis NE,O'Dowd DJ,Mac Nally R,et al.Invasive ants disrupt frugivory by endemic island birds[J].Biology Letters,2010,6(1):85-88.
Espadaler X,Rey S,Bernal V.Queen number in a supercolony of the invasive garden ant,Lasius neglectus[J].Insectes Sociaux,2004,51(3):232-238.
Folmer RA,Nilges M,Folkers PM,et al.A model of the complex between single-stranded DNA and the single-stranded DNAbinding protein encoded by gene V of filamentous bacteriophage M13[J].Journal of Molecular Biology,1994,240(4):341-357.
Hebert PN,Cywinska A,Ball SL,et al.Biological identifications through DNA barcodes[J].Proceedings of the Royal Society of London B:Biological Sciences,2003,270(1512):313-321.
Holldobler,B,Wilson,EO.Host tree selection by the neotropical ant Paraponera clavata(Hymenoptera:Formicidae)[J].Biotropica,1990,22:213-214.
Holway DA.Competitive mechanisms underlying the displacement of native ants by the invasive Argentine ant[J].Ecology,1999,80(1):238-251.
Huang KS,Lee SE,Yeh Y,et al.Taqman real-time quantitative PCRfor identification of western flower thrips(Frankliniella occidentalis)for plant quarantine[J].Biol.Lett.,2010,6,555-557.
Human KG,Gordon DM.Behavioral interactions of the invasive Argentine ant with native ant species[J].Insectes Sociaux,1999,46(2):159-163.
Le Breton J,Delabie JC,Chazeau J,et al.Experimental evidence of large-scale unicoloniality in the tramp ant Wasmannia auropunctata(Roger)[J].Journal of insect Behavior,2004,17(2):263-271.
Lowe S,Browne M,Boudjelas S,et al.100 of the World's Worst Invasive Alien Species:A Selection from the Global Invasive Species Database[M].Auckland:Invasive Species Specialist Group,2000:S1-S12.
Ma LB,Xu SQ.A method of extracting ant genomic DNA[J].Journal of Shaanxi Normal University(Natural Science Edition),2006,34(1):85-87.[马丽滨,许升全.蚂蚁DNA提取方法的研究[J].陕西师范大学学报(自科版),2006,34(1):85-87]
Mack RN,Simberloff D,Mark LW,et al.Biotic invasions:Causes,epidemiology,global consequences,and control[J].Ecological Applications,2000,10(3):689-710.
Mc Natty A,Abbott KL,Lester PJ.Invasive ants compete with and modify the trophic ecology of hermit crabs on tropical islands[J].Oecologia,2009,160(1):187-194.
O'Dowd DJ,Green PT,Lake PS.Status,Impact and Recommendations for Research and Management of Exotic Invasive Ants in Christmas Island National Park.Report to Environment Australia[M].Melbourne:Monash University,1999.
O'Dowd DJ,Green PT,Lake PS.Invasional‘meltdown’on an oceanic island[J].Ecology Letters,2003,6(9):812-817.
Paran I,Michelmore RW.Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce[J].Theoretical and Applied Genetics,1993,85(8):985-993.
Room PM.Relative distributions of ant species in cocoa plantations in Papua New Guinea[J].The Journal of Applied Ecology,1975,12:47-61.
Rowles AD,O’Dowd DJ.Interference competition by Argentine ants displaces native ants:Implications for biotic resistance to invasion[J].Biological Invasions,2007,9(1):73-85.
Suarez AV,Yeh P,Case TJ.Impacts of Argentine ants on avian nesting success[J].Insectes Sociaux,2005,52(4):378-382.
Sun BJ,Li ZL,Li ZX,et al.Reasons for SCAR conversion failure and its remedies[J].Molecular Plant Breeding,2010,8(3):589-594.[孙保娟,李植良,黎振兴.SCAR标记转化失败的原因和对策[J].分子植物育种,2010,8(3):589-594]
Tian H,Li XF,Wan FH,et al.Identification of Phenacoccus solenopsis Tinsley(Hemiptera:Pseudococcidae)with species-specific COI(SS-COI)primers[J].Acta Entomologica Sinica,2013,6(6):689-696.[田虎,李小凤,万方浩.利用种特异性COI引物(SS-COI)鉴别扶桑绵粉蚧[J].昆虫学报,2013,6(6):689-696]
Walters AC,Mackay DA.Importance of large colony size for successful invasion by Argentine ants(Hymenoptera:Formicidae):Evidence for biotic resistance by native ants[J].Austral Ecology,2005,30(4):395-406.
Wetterer JK,Moore JA.Red imported fire ants(Hymenoptera:Formicidae)at gopher tortoise(Testudines:Testudinidae)burrows[J].Florida Entomologist,2005,88(4):349-354.
Wetterer JK.Ants on Cecropia in Hawaii[J].Biotropica,1997,29(1):128-132.
Wetterer JK.Worldwide distribution and potential spread of the longlegged ant,Anoplolepis gracilipes(Hymenoptera:Formicidae)[J].Sociobiology,2005,45(45):77-97.
Xu M,Zhu HB,Yang XJ,et al.A dangerous pest-Anoplolepis gracilipes was intercepted in Jiangsu port[J].Plant Quarantine,2007,21(3):177-180.[徐梅,朱宏斌,杨晓军.江苏局首次截获危险性有害生物---细足捷蚁[J].植物检疫,2007,21(3):177-180]
Yu DJ,Deng ZP,Chen ZL,et al.Method of the polymerase chain reaction for quarantine and identification of Bactrocera correcta[J].Plant Quarantine,2004,18(2):73-76.[余道坚,邓中平,陈志粦.PCR法检疫鉴定番石榴实蝇[J].植物检疫2004,18(2):73-76]
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