Culicoides species from the subgenus Culicoides in Catalonia (NE Spain) Pagès, N., Muñoz-Muñoz, F., Talavera, S., Sarto, V., Lorca, C. and Nuñez, J.I.
Identification Background Identification of Culicoides based on morphology Metric traits Size dependent: individual metric characters Non-size dependent: indexes Non metric traits Presence/absence: sensilla coeloconica, other Number of: sensilla coeloconica, teeth, other Shape: sensory pit, aedeagus, other Colour Wing pattern Spots distribution and intensity Colour Macrotrichia Identification of Culicoides based on molecular methods Sequencing PCR based diagnostics
Identification troubleshooting Morphological troubles Intraspecific variability? Metric traits Non-metric traits Wing pattern Results: Discrepancy in literature Useful differential characters? Synonym species? Molecular concerns Selection and standardization of the target diagnostic region (ITS, COI) Lack of morpho-molecular tandem analyses. Few Culicoides sequences available. Few works focussed on design of new PCR based diagnostics for Culicoides Limitations and errors of PCR based diagnostics advantages of sequencing.
Identification troubleshooting Limitations and errors of PCR based diagnostics
Aims Perform: Analyses species diversity within the subgenus Culicoides Many individuals Regional scale To: 1. Determine the presence of sibling species within the subgenus. 2. Determine useful characters to distinguish them. 3. Develop species-specific diagnostic assays.
Analyses in Catalonia (NE Spain) Culicoides capture: Surveillance area of 31.114Km 2 Individuals of the subgenus Culicoides trapped in 10 sheep/goat farms. CDC blacklight traps Culicoides identification: Midge identification according to wing morphology C. punctatus C. pulicaris, C. lupicaris C. fagineus C. newsteadi C. flavipulicaris Storage (-20ºC) Dissection Sequencing (COI gene) Morphological analyses n=118
Neighbour-Joining tree COI sequencing of Culicoides 91 81 87 dq792577 91 63 64 A53 C.pulicarisP1 100 A80 C.punctatusP1 A352 C.pulicarisP1 A432 C.pulicarisP1 A431 C.pulicarisP3 A97 C.pulicarisP3 100 A349 C.pulicarisP3 52 A350 C.pulicarisP3 A96 C.pulicarisP3 A347 C.pulicarisP3 A52 C.lupicaris A423 C.lupicaris A426 C.lupicaris A428 C.lupicaris A430 C.lupicaris A427 C.lupicaris A729 C.lupicaris A422 C.lupicaris 70 A81 C.lupicaris 100 A425 C.lupicaris A448 C.lupicaris A101 C.lupicaris A366 C.newsteadiN3 A92 C.newsteadiN3 A363 C.newsteadiN3 100 A91 C.newsteadiN3 A90 C.newsteadiN3 92 A361 C.newsteadiN3 A362 C.newsteadiN3 A360 C.newsteadiN3 A95 C.newsteadiN3 A367 C.newsteadiN2 A86 C.newsteadiN2 100 A58 C.newsteadiN2 84 A89 C.newsteadiN2 70 A57 C.newsteadiN2 65 A88 C.newsteadiN2 79 A46 C.newsteadiN1 100 A364 C.newsteadiN1 A87 C.newsteadiN1 A365 C.newsteadiN1 A340 C.punctatus 91 58 A341 C.punctatus A115 C.punctatus 100 A337 C.punctatus A343 C.punctatus A116 C.punctatus 87 A48 C.punctatus A50 C.punctatus A111 C.punctatus A113 C.punctatus A342 C.punctatus A336 C.punctatus A338 C.punctatus A339 C.punctatus A344 C.punctatus 59 A345 C.punctatus A112 C.punctatus A47 C.punctatus A261 C.punctatus A51 C.punctatus A119 C.flavipulicaris A370 C.flavipulicaris 65 A371 C.flavipulicaris A369 C.flavipulicaris A121 C.flavipulicaris A122 C.flavipulicaris A120 C.flavipulicaris A375 C.flavipulicaris A118 C.flavipulicaris A374 C.flavipulicaris A44 C.flavipulicaris A368 C.flavipulicaris 100 A372 C.flavipulicaris A373 C.flavipulicaris A376 C.flavipulicaris A449 C.subfagineus A451 C.subfagineus 61 A382 C.subfagineus A438 C.subfagineus 100 A439 C.subfagineus A450 C.subfagineus A440 C.subfagineus 71 A444 C.fagineus A441 C.fagineus A442 C.fagineus 63 A183 C.fagineus 97 A180 C.fagineus A182 C.fagineus 100 A380 C.fagineus A381 C.fagineus A377 C.fagineus 58 99 A379 C.fagineusF1 A445 C.fagineusF1 A446 C.fagineusF1 100 A181 C.fagineusF1 A378 C.fagineusF1 A31 C.imicola 65 1 3 2 11 3 2 C. pulicaris P1 C. pulicaris P3 C. lupicaris 0.02
Neighbour-Joining tree COI sequencing of Culicoides 91 81 87 dq792577 91 63 64 A53 C.pulicarisP1 100 A80 C.punctatusP1 A352 C.pulicarisP1 A432 C.pulicarisP1 A431 C.pulicarisP3 A97 C.pulicarisP3 100 A349 C.pulicarisP3 52 A350 C.pulicarisP3 A96 C.pulicarisP3 A347 C.pulicarisP3 A52 C.lupicaris A423 C.lupicaris A426 C.lupicaris A428 C.lupicaris A430 C.lupicaris A427 C.lupicaris A729 C.lupicaris A422 C.lupicaris 70 A81 C.lupicaris 100 A425 C.lupicaris A448 C.lupicaris A101 C.lupicaris A366 C.newsteadiN3 A92 C.newsteadiN3 A363 C.newsteadiN3 100 A91 C.newsteadiN3 A90 C.newsteadiN3 92 A361 C.newsteadiN3 A362 C.newsteadiN3 A360 C.newsteadiN3 A95 C.newsteadiN3 A367 C.newsteadiN2 A86 C.newsteadiN2 100 A58 C.newsteadiN2 84 A89 C.newsteadiN2 70 A57 C.newsteadiN2 65 A88 C.newsteadiN2 79 A46 C.newsteadiN1 100 A364 C.newsteadiN1 A87 C.newsteadiN1 A365 C.newsteadiN1 A340 C.punctatus 91 58 A341 C.punctatus A115 C.punctatus 100 A337 C.punctatus A343 C.punctatus A116 C.punctatus 87 A48 C.punctatus A50 C.punctatus A111 C.punctatus A113 C.punctatus A342 C.punctatus A336 C.punctatus A338 C.punctatus A339 C.punctatus A344 C.punctatus 59 A345 C.punctatus A112 C.punctatus A47 C.punctatus A261 C.punctatus A51 C.punctatus A119 C.flavipulicaris A370 C.flavipulicaris 65 A371 C.flavipulicaris A369 C.flavipulicaris A121 C.flavipulicaris A122 C.flavipulicaris A120 C.flavipulicaris A375 C.flavipulicaris A118 C.flavipulicaris A374 C.flavipulicaris A44 C.flavipulicaris A368 C.flavipulicaris 100 A372 C.flavipulicaris A373 C.flavipulicaris A376 C.flavipulicaris A449 C.subfagineus A451 C.subfagineus 61 A382 C.subfagineus A438 C.subfagineus 100 A439 C.subfagineus A450 C.subfagineus A440 C.subfagineus 71 A444 C.fagineus A441 C.fagineus A442 C.fagineus 63 A183 C.fagineus 97 A180 C.fagineus A182 C.fagineus 100 A380 C.fagineus A381 C.fagineus A377 C.fagineus 58 99 A379 C.fagineusF1 A445 C.fagineusF1 A446 C.fagineusF1 100 A181 C.fagineusF1 A378 C.fagineusF1 A31 C.imicola 65 4 5 6 4 5 6 C. newsteadi N3 C. newsteadi N2 C. newsteadi N1 0.02
Neighbour-Joining tree COI sequencing of Culicoides 91 81 87 dq792577 91 63 64 A53 C.pulicarisP1 100 A80 C.punctatusP1 A352 C.pulicarisP1 A432 C.pulicarisP1 A431 C.pulicarisP3 A97 C.pulicarisP3 100 A349 C.pulicarisP3 52 A350 C.pulicarisP3 A96 C.pulicarisP3 A347 C.pulicarisP3 A52 C.lupicaris A423 C.lupicaris A426 C.lupicaris A428 C.lupicaris A430 C.lupicaris A427 C.lupicaris A729 C.lupicaris A422 C.lupicaris 70 A81 C.lupicaris 100 A425 C.lupicaris A448 C.lupicaris A101 C.lupicaris A366 C.newsteadiN3 A92 C.newsteadiN3 A363 C.newsteadiN3 100 A91 C.newsteadiN3 A90 C.newsteadiN3 92 A361 C.newsteadiN3 A362 C.newsteadiN3 A360 C.newsteadiN3 A95 C.newsteadiN3 A367 C.newsteadiN2 A86 C.newsteadiN2 100 A58 C.newsteadiN2 84 A89 C.newsteadiN2 70 A57 C.newsteadiN2 65 A88 C.newsteadiN2 79 A46 C.newsteadiN1 100 A364 C.newsteadiN1 A87 C.newsteadiN1 A365 C.newsteadiN1 A340 C.punctatus 91 58 A341 C.punctatus A115 C.punctatus 100 A337 C.punctatus A343 C.punctatus A116 C.punctatus 87 A48 C.punctatus A50 C.punctatus A111 C.punctatus A113 C.punctatus A342 C.punctatus A336 C.punctatus A338 C.punctatus A339 C.punctatus A344 C.punctatus 59 A345 C.punctatus A112 C.punctatus A47 C.punctatus A261 C.punctatus A51 C.punctatus A119 C.flavipulicaris A370 C.flavipulicaris 65 A371 C.flavipulicaris A369 C.flavipulicaris A121 C.flavipulicaris A122 C.flavipulicaris A120 C.flavipulicaris A375 C.flavipulicaris A118 C.flavipulicaris A374 C.flavipulicaris A44 C.flavipulicaris A368 C.flavipulicaris 100 A372 C.flavipulicaris A373 C.flavipulicaris A376 C.flavipulicaris A449 C.subfagineus A451 C.subfagineus 61 A382 C.subfagineus A438 C.subfagineus 100 A439 C.subfagineus A450 C.subfagineus A440 C.subfagineus 71 A444 C.fagineus A441 C.fagineus A442 C.fagineus 63 A183 C.fagineus 97 A180 C.fagineus A182 C.fagineus 100 A380 C.fagineus A381 C.fagineus A377 C.fagineus 58 99 A379 C.fagineusF1 A445 C.fagineusF1 A446 C.fagineusF1 100 A181 C.fagineusF1 A378 C.fagineusF1 A31 C.imicola 65 7 8 9 7 8 9 C. subfagineus C. fagineus C. fagineus F1 0.02
Neighbour-Joining tree COI sequencing of Culicoides 91 81 87 dq792577 91 63 64 A53 C.pulicarisP1 100 A80 C.punctatusP1 A352 C.pulicarisP1 A432 C.pulicarisP1 A431 C.pulicarisP3 A97 C.pulicarisP3 100 A349 C.pulicarisP3 52 A350 C.pulicarisP3 A96 C.pulicarisP3 A347 C.pulicarisP3 A52 C.lupicaris A423 C.lupicaris A426 C.lupicaris A428 C.lupicaris A430 C.lupicaris A427 C.lupicaris A729 C.lupicaris A422 C.lupicaris 70 A81 C.lupicaris 100 A425 C.lupicaris A448 C.lupicaris A101 C.lupicaris A366 C.newsteadiN3 A92 C.newsteadiN3 A363 C.newsteadiN3 100 A91 C.newsteadiN3 A90 C.newsteadiN3 92 A361 C.newsteadiN3 A362 C.newsteadiN3 A360 C.newsteadiN3 A95 C.newsteadiN3 A367 C.newsteadiN2 A86 C.newsteadiN2 100 A58 C.newsteadiN2 84 A89 C.newsteadiN2 70 A57 C.newsteadiN2 65 A88 C.newsteadiN2 79 A46 C.newsteadiN1 100 A364 C.newsteadiN1 A87 C.newsteadiN1 A365 C.newsteadiN1 A340 C.punctatus 91 58 A341 C.punctatus A115 C.punctatus 100 A337 C.punctatus A343 C.punctatus A116 C.punctatus 87 A48 C.punctatus A50 C.punctatus A111 C.punctatus A113 C.punctatus A342 C.punctatus A336 C.punctatus A338 C.punctatus A339 C.punctatus A344 C.punctatus 59 A345 C.punctatus A112 C.punctatus A47 C.punctatus A261 C.punctatus A51 C.punctatus A119 C.flavipulicaris A370 C.flavipulicaris 65 A371 C.flavipulicaris A369 C.flavipulicaris A121 C.flavipulicaris A122 C.flavipulicaris A120 C.flavipulicaris A375 C.flavipulicaris A118 C.flavipulicaris A374 C.flavipulicaris A44 C.flavipulicaris A368 C.flavipulicaris 100 A372 C.flavipulicaris A373 C.flavipulicaris A376 C.flavipulicaris A449 C.subfagineus A451 C.subfagineus 61 A382 C.subfagineus A438 C.subfagineus 100 A439 C.subfagineus A450 C.subfagineus A440 C.subfagineus 71 A444 C.fagineus A441 C.fagineus A442 C.fagineus 63 A183 C.fagineus 97 A180 C.fagineus A182 C.fagineus 100 A380 C.fagineus A381 C.fagineus A377 C.fagineus 58 99 A379 C.fagineusF1 A445 C.fagineusF1 A446 C.fagineusF1 100 A181 C.fagineusF1 A378 C.fagineusF1 A31 C.imicola 65 Culicoides pulicaris group (n=3 spp.) Culicoides newsteadi group (n=3 spp.) Culicoides punctatus (n=1 sp.) Culicoides flavipulicaris (n=1 sp.) Culicoides fagineus group (n=3 spp.) 0.02
Morphological analyses Morphological data recorded Eye data: 3 records Mouth parts: 11 records Wing metric data: 5 records Legs: 7 records Spermathecae: 2-6 records Antenna: 29 records Wing pattern analysis Few differences found within groups of species No differences found between C. pulicaris P1 and C. pulicaris P3 Statistical analyses grouping individuals according COI sequence data PCA: principal component analyses Useful morphological diagnostic characters found for: - C. pulicaris group - C. newsteadi group - C. fagineus group
Design of PCR based diagnostic assays. Single PCRs Species found: C. pulicaris P1, C. pulicaris P3, C. lupicaris, C. newsteadi N1, C. newsteadi N2, C. newsteadi N3, C. fagineus, C. fagineus F1, C. subfagineus, C. flavipulicaris and C. punctatus (n=11) Design of 15 species-specific primers and validation of 18 single diagnostic PCRs
Design of PCR based diagnostic assays. Multiplex PCR for pulicaris group. C. pulicaris P1: 197bp 410bp C. pulicaris P3: 310bp C. lupicaris: 405bp Diagnostic bands Primers
Design of PCR based diagnostic assays. Multiplex PCR for fagineus group. C. fagineus F1: 242bp 522bp C. fagineus: 415bp 522bp Control band (522bp) Diagnostic bands Primer C. subfagineus: 379bp 522bp
Design of PCR based diagnostic assays. Multiplex PCR for newsteadi group. C. newsteadi N1: 415bp 522bp C. newsteadi N2: 153bp 522bp Control band (522bp) Diagnostic bands C. newsteadi N3 : 231bp 522bp
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