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Streito: a Barcode Database to Identify the Vectors of Xylella Fastidiosa in Europe

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Streito: a Barcode Database to Identify the Vectors of Xylella Fastidiosa in Europe A barcode database to identify the vectors of Xylella fastidiosa in Europe. By Streito Jean-Claude, Pierre Eric, Genson Guénaëlle, Bellifa Maxime, Chartois Marguerite, †Germain Jean-François, Astrid Cruaud & Jean-Yves Rasplus. Ajaccio 29/10/2019 _01 Introduction Why DNA barcoding of vectors of Xf is important? .02 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcoding? 1) Potential vectors of Xf are diversified 4 Hemiptera famillies, 120 spp. for Europe Aphrophoridae (29 spp.) Cercopidae (7 spp.) Cicadellidae (9 spp.) Cicadidae (79 spp.) For an overview of Xf vectors I invite you to go and see the poster P58 by Wilson et al. .03 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcoding? 2) Main vectors must be dissected to be identified It is possible and reliable but time-consuming and not suitable for mass screening of Xf in insects .04 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcoding? .05 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcoding? 1) Potential vectors of Xf are diversified 2) Main vectors must be dissected to be identified 3) The taxonomy of Xf’s vectors is not 100% satisfactory Barcoding is known to be adequate to check for taxonomical problems: cryptic species and synonymies This is why we started to barcode the vectors of Xf when the bacterium was detected in Corsica in summer 2015 .06 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 _02 Materials & Methods Our main concern is to avoid misidentification and to provide reliable sequences .07 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: multiple quality controls Species to be Sampling conditioning 1) IdentificationIdentification by Databases included storage a taxonomist Web portal Philaenus italosignus Philaenus lukasi Philaenus maghresignus Philaenus signatus Philaenus spumarius Philaenus tarifa Philaenus tesselatus 2)DNA Non extraction destructive and Collections extractionstorage ADN 3) Vouchers PCR amplification COI Sequencing Phylogenetic analysis 1st quality control: sequences 2nd quality control .08 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: Standard Barcode: COI 658 bp (Herbert et al., 2003) First: Classical PCR and Sanger sequencing Now: two-step PCR and Miseq sequencing .09 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: multiple quality controls Species to be Sampling conditioning 1) IdentificationIdentification by Databases included storage a taxonomist Web portal Philaenus italosignus Philaenus lukasi Philaenus maghresignus Philaenus signatus Philaenus spumarius Philaenus tarifa Philaenus tesselatus 5) Consistency 2)DNA Non extraction destructive and Collections extractionstorage ADN morpho/molecular 3) Vouchers identification PCR amplification COI Sequencing 4) Pipeline to check Phylogenetic analysis 1st quality control: sequences sequences (non-coding, 2nd quality control contaminants, numts…) .010 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Photography of all species available Help for morphological identification On the CBGP collection platform: with a Keyence VHX5000 microscope .011 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Database : validated sequences and photos Data are stored in the Database Arthemis http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home Freely available in Arthemis : - Pictures - Sequences validated Arthemis DB@se focuses on pests and their natural enemies. To date it contains 21 169 COI sequences of Arthropods .012 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 _03 Results .013 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Sampling North American vectors European vectors Total putative vectors : 22 849 - Aphrophoridae : 11 199 - Cercopidae : 165 Xylella samples - Cicadellidae : 11 364 QBOL samples - Cicadidae : 121 .014 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Number of barcodes and pictures Number of Xf vectors included: http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home European vectors: 21(+16) spp. 234 (+151) COI Non European vectors: 23 spp. 173 COI Total number of spp. 44(+32) 407(+151) COI Number of pictures : 271 (24 European spp. and 5 non European spp.) .015 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: Philaenus 7 spp. in Europe Number of Bold Espèces / sous-espèces Pictures Séquences 5 spp. sequenced and GenBank (in process) imaged Philaenus italosignus 7 4 1 Philaenus loukasi 0 0 1 + Philaenus maghresignus 9 1 2 few sequences available Philaenus signatus (7) (7) 1 Philaenus spumarius 32 39 (11) > 100 in international Philaenus tarifa 0 0 2 databases Philaenus tesselatus 11 29 (8) 2 Philaenus arslani (Liban) 0 0 1 Philaenus elbursianus (Iran) 0 0 0 All European spp. Philaenus iranicus (Iran) 0 0 0 sequenced .016 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns Philaenus COI separates all spp. studied except spumarius/tesselatus .017 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns spumarius/tesselatus Philaenus Philaenus spp. : - same variable habitus - aedeagus diagnostic but difficult between spumarius/tesselatus but Is P. tesselatus a good species ? For more details I invite you to go and see the poster P60 by Seabra et al. !! .018 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Neophilaenus Number of Bold 11 spp. in Europe Espèces / sous-espèces Pictures Séquences GenBank (in process) 2 spp. sequenced Neophilaenus albipennis 2 (1) 0 5 imaged Neophilaenus angustipennis 0 0 0 Neophilaenus campestris 9 31 (8) 3 + Neophilaenus exclamationis 9 (1) 0 Nearly no Neophilaenus infumatusA difficult genus0 , more0 0 barcodes Neophilaenus limpidusbarcodes are 0needed0 0 Neophilaenus lineatus 9 8 (6) >100 in international db Neophilaenus longiceps 0 0 0 Neophilaenus minor 2 (2) 0 Neophilaenus modestus 0 0 0 <50% European Neophilaenus pallidus 0 0 0 spp. sequenced .019 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Aphrophora 7 spp. in Europe Number of Bold Espèces / sous-espèces Pictures Séquences 4 spp. sequenced GenBank (in process) and imaged Aphrophora alni 7 11 (6) >100 Aphrophora corticea 9 5 (5) 3 AphrophoraA lot ofmajor taxonomic problems0 0 remains0 In international db Aphrophorabeforepectoralisreliable barcode13 -3based 1 confusion between Aphrophora salicina 13 3 (13) 7 Aphrophoraidentificationsimilis can0 be performed0 0 alni/paralella and Aphrophora willemsi 0 0 0 salicina/pectoralis .020 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Lepyronia and other genus Number of Bold Espèces / sous-espèces Pictures Séquences GenBank (in process) Lepyronia coleoptrata 15 7 (9) 14 Lepyroniella petrovi (Pologne, Russie…) 0 0 0 Paraphilaenus notatus [F, Ukraine, Russie…] 0 0 0 Peuceptyelus coriaceus 0 0 0 Lepyronia coleoptrata : COI and morpho ok Other genus are missing .021 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Cercopidae Number of Bold Espèces / sous-espèces Pictures Séquences 7 spp. in Europe GenBank (in process) 5 spp. sequenced Cercopis arcuata (15) (7) 0 Cercopis intermedia 7 15 0 and 6 imaged Cercopis sabaudiana 0 (1?) 0 + Cercopis sanguinolenta 7 2(12) 5 Cercopis vulnerata 9 19 8 misidentifications in Haematoloma dorsata 5 15 (3?) 2 international db Triecphorella geniculata (10) 0 0 .022 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns Cercopidae C. intermedia C. sanguinolenta COI discriminates all spp. studied Sequences of C. vulnerata C. sabaudiana and Triecphorella Only one taxonomic problem remains : are missing is C. sabaudiana a good speciesH. dorsata ? C. arcuata .023 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Cicadellidae putative vectors of Xf Number of Bold 9 spp. listed for Espèces / sous-espèces Pictures Séquences GenBank (in process) Europe Ledra aurita 8 11 0 5 spp. sequenced Cicadella viridis 13 14 81 Cicadella lasiocarpae 0 0 0 and 6 imaged Graphocephala fennahi 1 6 21 + Evacanthus acuminatus 2 2 (1) 10 Evacanthus interruptus 7 5 (4) 11 G. fennahi/G. Evacanthus rostagnoi 0 0 0 coccinea confused Anoterostemma ivanoffi 0 0 0 in international db Errhomenus brachypterus (4) 0 0 .024 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Cicadidae (1/79 spp. Europe ; 1/20 ssp. France) Aestuansella aestuans (Fabricius 1794) Cicada barbara ssp. barbara (Stål, 1866) Cicada barbara ssp. lusitanica Boulard, 1982 Cicada cretensis Quartau & Simões, 2005 Cicada mordoganensis Boulard, 1979 Results Cicada orni Linné, 1758 [F, C] Number of Cicadatra alhageos (Kolenati, 1857) Cicadatra atra (Olivier, 1790) [F] Bold Cicadatra glycyrrhizae (Kolenati 1857) Espèces / sous-espèces Pictures Sequences Cicadatra hyalina (F., 1798) Cicadatra icari Simões, Sanborn & Quartau, 2013 GenBank Cicadatra karpathoensis Simões, Sanborn & Quartau, 2013 (in process) Cicadatra persica Kirkaldy, 1909 Cicadatra platyptera Fieber, 1876 Cicadetta albipennis Fieber, 1876 Cicadetta anapaistica ssp. anapaistica Hertach 2011 Cicadidae
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