(7.4)--ISPM第27号附件29_Bactroceradorsalis.pdf

This diagnostic protocol was adopted by the Standards Committee on behalf of the Commission on Phytosanitary Measures in February 2019.The annex is a prescriptive part of ISPM 27.International Plant Protection Convention DP 29-1 ISPM 27 Diagnostic protocols for regulated pests DP 29:Bactrocera dorsalis Adopted 2019;published 2019 CONTENTS 1.Pest Information.2 2.Taxonomic Information.3 3.Detection.4 3.1 Trapping.4 3.2 Inspection of fruits.5 3.3 Rearing larvae to obtain adults.5 4.Identification.5 4.1 Preparation of adults for identification.6 4.1.1 Preparation of adults for microscopic examination of genitalia.6 4.2 Morphological identification of adults.7 4.2.1 Characters to identify the subgenus Bactrocera(Bactrocera).7 4.2.2 Characters to identify the Bactrocera dorsalis complex.7 4.2.3 Morphological identification of six economically important species belonging to the Bactrocera dorsalis complex.8 4.2.4 Diagnostic key to six economically important species belonging to the Bactrocera dorsalis complex(adult).12 4.3 Molecular identification of Bactrocera carambolae.12 4.3.1 DNA extraction for molecular tests.13 4.3.2 ITS1 PCR and DNA sequencing to distinguish B.carambolae from B.dorsalis s.l.13 4.3.3 Controls for molecular tests.14 4.3.4 Interpretation of molecular test results.14 4.4 Other molecular methods of identification.15 5.Records.15 6.Contact Points for Further Information.15 7.Acknowledgements.16 8.References.16 9.Figures.19 DP 29 Diagnostic protocols for regulated pests DP 29-2 International Plant Protection Convention 1.Pest Information Fruit flies of the family Tephritidae represent an economically important insect group with a worldwide distribution.The biology of these fruit flies is dependent on host plants that can serve as mating locations,oviposition sites for eggs,and nutrient resources for developing larvae.The genus Bactrocera Macquart consists of over 460 described species that are distributed mostly in regions of Asia and Australasia and subtropical islands of the southern Pacific Ocean(Drew and Romig,2013;Doorenweerd etal.,2018).A few Bactrocera species are native to Africa and several pest species were introduced to that continent.Within the genus is a group of flies named the Bactrocera dorsalis complex(Drew and Hancock,1994;Drew,2004;Clark etal.,2005).This complex comprises 85 described species(Vargas etal.,2015)that share a very similar appearance,but the complex as a whole does not represent a monophyletic lineage and is merely a group of convenience(Leblanc etal.,2015).The complex is named after one of its member species,Bactrocera dorsalis(Figure 1),which is a polyphagous pest of commercial fruits.Several other species in the complex are also recognized as pests,based on plant host use and pest records(White and Elson-Harris,1992;Clarke etal.,2005;Vargas etal.,2015;Plant Health Australia,2016).The scope of the current protocol is to diagnose adult B.dorsalis fruit flies.Five economically important species of the Bactrocera dorsalis complex that are found in commercial fruits and vegetables associated with international trade and that can be confused with B.dorsalis during an identification are also included in the protocol.These five species are:B.carambolae,B.caryeae,B.kandiensis,B.occipitalis and B.pyrifoliae.Distributions of these species are mapped with their pest status and invasion history by Vargas etal.(2015).A lack of characters that can be used reliably to distinguish B.dorsalis from two other species(i.e.B.papayae Drew and Hancock,1994 and B.invadens Drew etal.,2005)has resulted in debate regarding the valid taxonomy of the species(Clarke etal.,2005;Chen and Hui,2007;Schutze etal.,2015a,b;Drew&Romig,2016;Schutze etal.,2017).These three species have been treated as members of a sibling species complex,not to be confused with the Bactrocera dorsalis complex(Clarke and Schutze,2014).It is not possible to reliably distinguish among these three species because an accurate identification requires both evaluation of species distribution information and analysis of morphological characters that are not discrete for the species.Species distribution information may not be reliable when examining specimens collected outside the species known range.Published molecular data cannot distinguish these species(Schutze etal.,2015a).In a review of available evidence,Schutze etal.(2015a)concluded that these three species are in fact a single biological species called Bactrocera dorsalis.Drew and Romig(2016)disagree with that revision and reversed the synonymy;however,Schutze etal.(2017)published a rebuttal to Drew and Romig(2016)that supports the synonymy by Schutze etal.(2015a).In this protocol,the three species are collectively treated as B.dorsalis sensu lato.Evidence has been reported of hybridization among some of these six Bactrocera species under laboratory conditions(McInnis etal.,1999;Ebina and Ohto,2006;Schutze etal.,2013)and of morphological intermediates in the wild(Delomen etal.,2013;Jalani etal.2014).The frequency of hybrids between these species in nature has not been estimated.Although methods for detecting hybrids between B.dorsalis and B.carambolae have been reported(Ebina and Ohto,2006)it is currently not possible to measure impacts of hybridization events over time,such as genome introgression or detection of progeny of backcrossed populations.B.carambolae attacks a wide range of fruits from 20 plant families,particularly Averrhoa carambola(carambola)(CABI,n.d.).It is found in the southern peninsular area of southeast Asia through Indonesia and several islands in the Bay of Bengal(Drew and Romig,2013).It is also present in some South American countries(CABI,n.d.).B.caryeae is known to attack Mangifera spp.(mango),Malpighia emarginata(acerola),Psidium spp.(guava),Citrus spp.and Pouteria spp.(mamey sapote),and is endemic to southern India(CABI,n.d.).Diagnostic protocols for regulated pests DP 29 International Plant Protection Convention DP 29-3 B.dorsalis s.l.attacks over 270 plant species(Vargas etal.,2015)in over 50 families of commercial fruits and wild fruits(CABI,n.d.).It has the largest species range of the six pests included in this protocol,and is found on some islands in the Pacific Ocean and most of continental Africa(sub-Saharan countries)in addition to its original Asian range(Drew and Hancock,1994;Drew etal.,2005;White,2006;Drew and Romig,2013;Schutze etal.,2015a,b).B.kandiensis attacks a wide range of fruits including Mangifera indica(mango),Garcinia spp.,Carica papaya(papaya),Persea americana(avocado)and Psidium spp.(guava)(CABI,n.d.).It has a limited distributional range,being endemic to Sri Lanka.B.occipitalis attacks Mangifera spp.(mango),Psidium spp.(guava),Spondias purpurea(red mombin),Averrhoa carambola(carambola),Citrus spp.and Manilkara zapota(sapodilla)(CABI,n.d.).It has a relatively narrow range in southeast Asia(Drew and Romig,2013).B.pyrifoliae attacks Psidium spp.(guava)and Prunus persica(peach)(Allwood etal.,1999).It is known from parts of southeast Asia(Drew and Romig,2013).2.Taxonomic Information Name:Bactrocera dorsalis complex Synonyms:None Taxonomic position:Insecta,Diptera,Tephritidae,Dacinae,Bactrocera The species included in the Bactrocera dorsalis complex are in the subgenus Bactrocera(Bactrocera).According to ICZN(1999),three species are treated as synonyms under B.dorsalis s.l.:B.papayae,B.invadens and B.philippinensis.Drew and Romig(2013)placed B.philippinensis as a synonym of B.papayae.Revision by Schutze etal.(2015a)placed B.invadens and B.papayae as junior synonyms of B.dorsalis.Drew and Romig(2016)provide an argument for treating these as separate species,but Schutze etal.(2017)published a rebuttal of this argument.Note that B.invadens was placed in the Bactrocera dorsalis complex by Drew etal.(2008)but then removed from the complex by Drew and Romig(2013).Based on Schutze etal.(2015a),B.invadens is considered a sibling species,or synonym,of Bactrocera dorsalis.The current protocol treats these names(B.papayae,B.invadens and B.philippinensis)as part of B.dorsalis s.l.DP 29 Diagnostic protocols for regulated pests DP 29-4 International Plant Protection Convention Table 1.Common names and synonyms of six species in the Bactrocera dorsalis complex included in the protocol Bactrocera species Common name Synonyms Bactrocera(Bactrocera)carambolae Drew and Hancock,1994 Carambola fruit fly None Bactrocera(Bactrocera)caryeae(Kapoor,1971)Chaetodacus ferrugineus incises Bezzi,1916 Dacus(Strumeta)caryeae Kapoor,1971 Bactrocera(Bactrocera)dorsalis s.l.(Hendel,1912)Oriental fruit fly Bactrocera conformis Doleschall,1858 Dacus dorsalis Hendel,1912 Chaetodacus ferrugineus var.okinawanus Shiraki,1933 Dacus(Bactrocera)semifemoralis Tseng,Chen&Chu,1992 Dacus(Bactrocera)yilanensis Tseng,Chen&Chu,1992 Bactrocera papayae Drew and Hancock,1994 Bactrocera philippinensis Drew and Hancock,1994 Bactrocera invadens Drew etal.,2005 Bactrocera(Bactrocera)kandiensis Drew and Hancock,1994 None Bactrocera(Bactrocera)occipitalis(Bezzi,1919)Chaetodacus ferrugineus var.occipitalis Bezzi,1919;Drew and Hancock,1994 Dacus(Strumeta)dorsalis var.occipitalis(Bezzi,1919);Hardy and Adachi,1954;Hardy,1969 Dacus(Strumeta)occipitalis(Bezzi,1919);Hardy,1974 Bactrocera(Bactrocera)pyrifoliae Drew and Hancock,1994 None 3.Detection Fruit flies of the genus Bactrocera are detected mainly by male lure trap or in fruits.Only male adult fruit flies are captured by male lure trapping,while all immature stages such as eggs(Figure 2(a),early to final instar larvae(Figures 2(b)to(d),puparia and pupae(Figures 2(e)and(f)can be found during inspection of fruits.3.1 Trapping Guidance on trapping Bactrocera fruit flies is given in Appendix 1 of ISPM 26(Establishment of pest free areas for fruit flies(Tephritidae).Additional information on trapping methods is provided by Drew(1982),Drew and Romig(2010),and FAO and IAEA(2018).The Bactrocera dorsalis complex includes species that respond to different male lures.When the lure responsiveness information is available,it can be used as supporting information for species identification.Five of the target species in this diagnostic protocol are methyl eugenol responding species.The only exception is B.pyrifoliae,which has been reported to respond to an alternative lure:cue lure(Drew and Romig,2013).Diagnostic protocols for regulated pests DP 29 International Plant Protection Convention DP 29-5 Additional information on attractants for trapping,such as synthetic food attractants and hydrolysed protein substances,are available in Appendix 1 of ISPM 26.3.2 Inspection of fruits Fruits with soft areas,dark stains,dark pin spots,rot,orifices or injuries that might have originated from female oviposition or larval feeding activities should be targeted for inspection.In order to detect punctures made by female flies during oviposition,fruits should be examined under a microscope by an expert.If larval exit holes are observed,the fruit containers should be inspected for pupae.Second and third instar larvae and pupae are not likely to occur when unripe fruits are collected and packed;however,these fruits might host eggs and first instar larvae,which are more difficult to detect.Potentially infested fruits that show typical punctures made by ovipositioning female flies(Figure 3)should be cut open to search for eggs or larvae inside.The success of detection depends on careful sampling and examination of fruits.Once detected,immature larvae can be reared to adults for identification(section 3.3).Rearing of adults is required to accurately identify a fly to species level or as part of the Bactrocera dorsalis complex.The incubation of infested fruits is a common practice to obtain adult flies,which is necessary to identify species in this protocol.Even if there are no signs of fruit fly infestation,an incubation could be conducted as an oviposition mark is often difficult to recognize.3.3 Rearing larvae to obtain adults Larvae can be reared to adults by placing infested fruits in cages containing a pupation medium(e.g.damp vermiculite,sand or sawdust)at the bottom.The cages are covered with cloth or fine mesh.Once the larvae emerge from the fruit,they will move to the pupation medium.Each sample should be observed and pupae gathered daily.The pupae are placed in containers with the pupation medium,and the containers are covered with a tight lid that enables proper ventilation.Once the adults emerge,they must be kept alive for several days to ensure that the tegument and wings acquire the rigidity and characteristic coloration of the species.Flies can be fed with honey(sugar)and water.The adults are then killed by freezing,or by exposure to ethyl acetate or other killing agents appropriate for morphological examination,and then mounted on pins.Prior to mounting(before they harden),it is useful to gently squeeze the apical part of the preabdomen with forceps,then squeeze the base and apex of the oviscape to expose the aculeus tip for females,and to pull out the aedeagus for males.Alternatively,this will need to be dissected later in flies.4.Identification Identification at the level of the species or the Bactrocera dorsalis complex requires morphological examination of adult flies.It is generally difficult and not reliable to morphologically identify eggs,larvae or pupae to the species level.It is not possible to identify a fly to the Bactrocera dorsalis complex using immature life stages.Molecular methods of Bactrocera species identification have been reported and provide additional information to support morphological identifications of specimens.DNA sequencing of the cytochrome oxidase I DNA barcode does not provide adequate resolution to identify many species in the B.dorsalis complex(details in section 4.4).Other molecular methods lack the specificity data needed to demonstrate that a test is accurate for species identification.For example,the molecular profiles of all six pest species targeted in this protocol are not known using ribosomal DNA analysis(section 4.4).DNA can be used to distinguish B.carambolae from B.dorsalis s.l.and a method for doing this is provided in this protocol(section 4.3.2).The use of a fly leg for DNA extraction is recommended when molecular data are to be collected.For guidance on preparing a specimen for molecular study,see section 4.3.1.DP 29 Diagnostic protocols for regulated pests DP 29-6 International Plant Protection Convention 4.1 Preparation of adults for identification Proper preparation of specimens is essential for accurate morphological identification.General instructions on preparation of adult fruit fly specimens are given by Drew(1991)and White and Elson-Harris(1992).Every attempt should be made to preserve all characters on at least one side of the centre line,regardless of the mounting method(Foote etal.,1993).Characters on the head,wing,leg,thorax and abdomen of a fly can be examined from pinned specimens under magn