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An introduction to diagnostic method for Bactrocera species

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1 An introduction to diagnostic method for Bactrocera species
International Plant Protection Convention Secretariat _ECBD_2014_Dec Agenda item 3.2 Expert Consultation on Phytosanitary Treatments in Bactrocera dorsalis complex (ECBD) An introduction to diagnostic method for Bactrocera species 1-5 December, 2014 Okinawa, Japan Deuk-Soo Choi Dept. of Plant Quarantine, Animal and Plant Quarantine Agency/ MAFRA Republic of Korea

2 Phylogeny of Diptera (>100 Families)
Crane fly Suborder NEMATOCERA Horse fly Infraorders Tabanomorpha ? Asilomorpha Suborder BRACHYCERA Muscomorpha (>70 families) House fly Tephritidae belongs here

3 Outgroup Relationships of Family Tephritidae
Superfamily Tephritoidea Outgroup Relationships of Family Tephritidae Hennig, Griffiths, McAlpine, 1989 Lonchaeidae PALLOPTEROIDEA ? Conopidae ? Conopidae Conopidae ? ? Lonchaeidae Pallopteridae Lonchaeidae (in part) Pallopteridae TEPHRITIDAE FAMILY GROUP Richardiidae Richardiidae Piophilidae ? Pallopteridae Piophilidae Richardiidae Piophilidae Pallopteridae (in part) TEPHRITOIDEA Otitidae Otitidae OTITOIDEA Otitidae Platystomatidae Platystomatidae Platystomatidae Tephritidae Tephritidae Tephritidae Pyrgotidae Pyrgotidae Pyrgotidae Tachiniscidae Tachiniscidae Tachiniscidae

4 Superfamily Tephritoidea
Include eight acalyptrate families Lonchaeidae, Richardiidae, Pallopteridae, Piophilidae Ulidiidae, Platystomatidae, Pyrgotidae, Tephritidae (incl. Tachiniscidae) Diverse biology and behavior Phytophagous: Tephritidae Parasitic: Pyrgotidae, (Tachiniscidae) Saprophagous: Most others Defined as monophyletic group based on morphology (e.g., Korneyev 1999) Male tergum 6 strongly reduced or absent Inner lobe of sustylus with prensisetae Female tergosternum 7 consisting of oviscape and taeniae baring eversible membrane…

5 Nemeurinus leucocelis
Magnimyiolia sp. 3 of 40 Family Tephritidae Itosigo bellus Trypeta binotata

6 Family Pyrgotidae

7 Family Platystomatidae

8 Family Ulidiidae

9 Family Pallopteridae Temnosira reducta

10 Family Tephritoidea Synapomorphy: subcostal vein apically
About 5,000 known species but actual number may be 2-3 times higher Almost exclusively phytophagous - includes many serious pest species Well defined monophyletic group Synapomorphy: subcostal vein apically sharply angled

11 Tephritidae Higher Classification
Dacini Higher Taxa Defined but Relationships not Known DACINAE Ceratitini Trypetini, s.str. Carpomyini Toxotrypanini Euphrantini Adramini Dithrycini Myopitini Terelliini Acrotaeniini Eutretini Schistopterini Tephrellini Platensinini Tephritini Many unplaced taxa TRYPETINAE Phytalmiini Acanthonevrini Rivelliomimini Zaceratini Many unplaced taxa TEPHRITINAE Inter-tribal relationships not known

12 Tephritid Classification
Tephritidae Higher Classification Poor Resolution in Tephritid Classification Too many taxa, too few systematists. Majority of higher taxa have not been phylogenetically analyzed. Many morphological characters are highly homoplastic.

13 Genus Bactrocera more than 500 species currently described.
cell bm size at least two times as wide as cell cup cell bm cell cup

14 Korean Bactrocera species
B. depressa

15 Korean Bactrocera species
B. scutellata

16 DNA barcoding of fruit flies
DNA barcoding is based on a relatively simple concept - Most eukaryote cells contain mitochondria (maternal inheritance), and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variation in mtDNA sequences between species. In insects, a comparatively small variance within species. A 658-bp region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was proposed as a potential 'barcode'.

17 DNA barcoding of fruit flies
collecting samples pinning dried (2weeks) STEP 1 Label Species information Host plant information Date, location, collector dissection making voucher specimens observation dried species wings Identification STEP 2 abdomen & genitalia Species information

18 DNA barcoding of fruit flies
DNA extraction PCR amplification sequencing STEP 3 STEP 4 Combined data Molecular analyses alignment homogenate Usually a leg

19 DNA barcoding of fruit flies

20 Case: dried persimmon intercepted date: Nov. 20, 2008
origin and quantity: China, 5kg

21 Case: dried persimmon Abdominal tergites with broadly
contiguous dark areas on lateral margins of T3-T5. ratio of width of apical band in cell r4+5 to length of r-m 0.5.

22 Case: dried persimmon Bactrocera verbascifoliae philippinensis
dorsalis papayae zonata carambolae invadens cacuminata

23 Case: dried persimmon

24 Primers TY-J-1466 JCO1A 5'-GCCTAAACTTCAGCCATTTAA-3' TY-J-1468 JCO1F
Gene Primer Abbr. Sequence COI TY-J-1466 JCO1A 5'-GCCTAAACTTCAGCCATTTAA-3' TY-J-1468 JCO1F 5'-GCCTAAACTTCAGCCATTTAATC-3' C1-J-1514 JCO1M 5'-TTTTCAACAAATCATAAAGATATTGG-3' C1-N-2221 NCO1B 5'-GTCTAATAATATGTGAAATTATTCC-3' C1-N-2286 NCO1E 5'-AATTTATGCTATATTAGCTATTGG-3' COII TL2-J-3045 JCO2A 5'-GATTAGTGCAATGGATTTAAGC-3' TL2-J-3032 JCO2C 5'-CTAATATGGCAGATTAGTGC-3' TK-N-3768 NCO2B 5'-ACTTGCTTTCAGTCATCTAATG-3' TK-N-3814 NCO2D 5'-TTAGAAGTAAGTGCTAATTTAC-3'

25 COI 738 bp + COII 669 bp COII Remark Korean species Quarantine pest
Indvid. Individ. Korean species 41 65 Quarantine pest 32 43 44 Total 73 108 109

26 COI 738bp 73 species 109 individuals Acidiella issikii
Acidiella pachypogon COI 738bp 73 species 109 individuals Acidiostigma polyfasciatum Stemonocera mica Acidiostigma s-nigrum Aischrocrania multipilosa Oreurinus cuspidatus Pseudhemilea longistigma Calosphenisca aliquantula Itosigo bellus Chetostoma continuans Anastrepha serpentina Anastrepha ludens Anastrepha suspensa Rhagoletis pomonella Ceratitis rosa Acanthonevra pteropleuralis Acanthonevra trigona Bactrocera cucurbitae Bactrocera depressa Bactrocera scutellata Bactrocera dosalis Terellia caerulea Chaetostomella vibrissata Xyphosia miliaria Campiglossa hirayamae Campiglossa messalina Campiglossa deserta Orotava hamula Urophora mandschurica Urophora misakiana Sphaeniscus atilius Tephritis majuscula Acanthiophilus helianthi Trupanea pseudovicina

27 COI 738bp 73 species 109 individuals Acidiella Anastrepha Rhagoletis
Ceratitis Euphranta Acanthonevra Bactrocera Chaetostomella Campiglossa Urophora Trupanea

28 COII 669bp 73 species 108 individuals Bactrocera dosalis
Bactrocera cucurbitae Bactrocera depressa Bactrocera scutellata Acanthonevra pteropleuralis Acanthonevra trigona Anastrepha striata Anastrepha ludens Anastrepha suspensa Ceratitis rosa Rhagoletis pomonella Chetostoma continuans Pseudhemilea longistigma Acidiella issikii Oreurinus cuspidatus Acidiostigma s-nigrum Aischrocrania multipilosa Acidiella pachypogon Acidiostigma polyfasciatum Stemonocera mica Calosphenisca aliquantula Itosigo bellus Chaetostomella vibrissata Terellia caerulea Xyphosia miliaria Urophora mandschurica Urophora misakiana Tephritis majuscula Trupanea pseudovicina Sphaeniscus atilius Campiglossa deserta Orotava hamula Campiglossa hirayamae Campiglossa messalina

29 COII 669bp 73 species 108 individuals Bactrocera Acanthonevra
Anastrepha Euphranta Ceratitis Rhagoletis Acidiella Chaetostomella Urophora Trupanea Campiglossa

30 COI + COII 1407bp 72 species 108 individuals
Bactrocera COI + COII bp 72 species 108 individuals Acanthonevra Euphranta Anastrepha Acidiella Ceratitis Rhagoletis Chaetostomella Urophora Trupanea Campiglossa

31 [ISPM 28: Phytosanitary treatments for regulated pests]
I. Introduction Official definition under the IPPC Treatment(s) are… for the control of regulated pests on regulated articles, primarily those moving in international trade. [ISPM 28: Phytosanitary treatments for regulated pests] Need of international standards - To suggest further experiments needed in future for harmonized scheme of cold treatment for pest species - To develop ISPM on minimum effective requirement of temperature and duration for each pest species with available data

32 I. Introduction All plants and plant products could be imported
except for the prohibited articles - Prohibited articles are… • Host plants and plant products from the origin of the prohibited pest (prohibited area) including those in transit through the prohibited area * Prohibited pests: Fruit fly, Codling moth, PCN, Fire blight, etc. Lift a ban after PRA/IRA on the prohibited articles - Prohibited articles from the relevant countries cannot be imported without PRA/IRA

33 Ⅱ. Approved cold treatment condition
PRA/IRA including Pest Risk Management on pests of concern - Pest Risk Managements are… • Hot water treatment: Mango, Papaya… • Fumigation (Methyl bromide): Cherry, Mangosteen • KD treatment: Pinus spp. • Cold treatment: Orange, Lemon, Grapefruit, Grape… Lift a ban after PRA/IRA - Total : 24 countries, 27 items, 46 cases - Cold treatment : 11 countries, 9 items, 19 cases

34 Ⅱ. Approved cold treatment condition
Introduction to cold treatment in Korea - What kind of pests can be applied? * Prohibited pests: Fruit fly Examples for Fruit importation - Items: Citrus spp. (orange, lemon, grapefruit) - Countries: Argentina, USA(Florida), Spain, South Africa, Taiwan, Israel, Australia, Egypt Why harmonization? - Items (Countries): Orange (8 countries) - Cold treatments: 15 conditions

35 Ⅱ. Approved cold treatment condition
Market access in Korea Market access with cold treatment Argentina: Orange Peru: Grape Israel: Sweetie, Orange, Grapefruit Taiwan: Pongkang orange, Litchi Australia: Orange, Lemon South Africa: Orange, Lemon, Grapefruit Egypt: Orange Spain: Orange Italy: Kiwi France: Kiwi USA(Florida): Orange, Grapefruit, Tangerine NORTH AMERICA EUROPE 11 countries 9 items ASIA AFRICA SOUTH AMERICA OCEANIA

36 Ⅱ. Approved cold treatment condition
Condition on Citrus spp. Country Since Item Core temp. Duration Target pests USA 2000 Orange Grapefruit Tangerine 0.6℃ 0.8℃ 1.1℃ 1.4℃ 1.7℃ 14 days 16 days 17 days 19 days 20 days Anastrepha suspensa Australia 1±0.5℃ 2±0.5℃ 3±0.5℃ 18 days Bactrocera aquilonis B. halfordiae B. jarvisi B. neohumevalis B. trivitis B. tryoni Ceratitis capitata Lemon Taiwan 1995 Pongkang orange 1.0℃ Bactrocera dorsalis B. cucurbitae B. tsuneonsis B. caudatus B. latifrons B. Tau

37 Ⅱ. Approved cold treatment condition
Condition on Citrus spp. (continued) Country Since Item Temperature Duration Target pests Spain 2004 Orange 2℃ 16 days Ceratitis capitata Egypt 2007 1.7℃ Bactrocera zonata Argentina 2010 2±0.5℃ 21 days Anastrepha fraterculus A. obliqua A. serpentina Israel 1999 2011 Sweetie Grapefruit 1.5℃ 2.2℃ 18 days Cryptophlebia leucotreta(FCM) South Africa 2012 Lemon -0.6±0.6℃ 24 days C. quinaria C. rosa

38 Ⅱ. Approved cold treatment condition
Condition on other fresh fruits Country Since Item Core temp. Duration Target pests Taiwan 1997 Litchi 2℃ 42 hrs Bactrocera dorsalis Peru 2011 Grape 1.1℃ 1.6℃ 15 days 17 days Anastrepha fraterculus A. obliqua Ceratitis capitata Italy 2012 Kiwi 2.2℃ 14 days 16 days 18 days France 2013

39 Ⅱ. Approved cold treatment condition
Conditions on fresh orange from 6 countries Country Since Item Core temp. Duration Target pests Australia 2000 Orange 1±0.5℃ 2±0.5℃ 3±0.5℃ 16 days 18 days 20 days Ceratitis capitata Bactrocera aquilonis B. halfordiae B. jarvisi B. neohumevalis B. trivitis B. tryoni Spain 2004 2℃ Egypt 2007 1.7℃ Bactrocera zonata Argentina 2010 21 days Anastrepha fraterculus A. obliqua A. serpentina Israel 1999 1.5℃ 2.2℃ Cryptophlebia leucotreta(FCM) South Africa -0.6±0.6℃ 24 days C. qinaria C. rosa

40 Ⅲ. Need of harmonization
Cold treatment scheme for fruit fly in fresh fruits Present Too many conditions - The required temperatures are various from -0.6℃ to 3℃ - The required durations are various from 16days to 24days Future ? Subject to consideration - Target pests - Items - Production area - Others

41 Thank you! ありがとうございます

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