RESUMO
Until the relatively recent application of molecular identification tools, identification of Culex pipiens f. pipiens and Cx. pipiens f. molestus relied on expressed ecological characteristics, including autogeny, host preference and stenogamy. Herein we test two DNA assays, one based on the microsatellite locus CQ11 and the other on species-diagnostic nucleotide bases in the mtDNA cytochrome oxidase I, on 322 wild-caught Cx. pipiens s.l. collected in above ground habitats from 6 counties across southern England and Wales. Of the 322 Culex pipiens s.l. screened using the CQ11 assay, 205 were identified as Cx. pipiens f. pipiens, 95 as Cx. pipiens f. molestus and 22 were determined as hybrids. Neither above ground Cx. pipiens f. molestus, nor hybrids have previously been reported in UK. However, comparison of COI barcodes (658bp) from 30 individuals from the above defined grouping indicated that inadvertent inclusion of specimens of Cx. torrentium resulted in the expected product sizes purportedly diagnostic for Cx. pipiens f. molestus, Cx. pipiens f. pipiens and hybrids in the CQ11 assay. COI sequences showed Cx. torrentium was misidentified as Cx. pipiens s.l. in more than 50% of cases and that all above ground Cx. pipiens s.l. collected in this study were in fact Cx. pipiens f. pipiens. Thus in regions of the Palearctic where Cx. torrentium and Cx. pipiens s.l. are sympatric, we showed that the CQ11 assay produces misleading results and should not be used.
Assuntos
Distribuição Animal , Culex/genética , Código de Barras de DNA Taxonômico/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Culex/anatomia & histologia , Culex/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Proteínas de Insetos/genética , Masculino , Repetições de Microssatélites/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Reino UnidoRESUMO
The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.
Assuntos
Anopheles/genética , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Animais , Anopheles/classificação , Anopheles/enzimologia , Colômbia , DNA Intergênico/genética , DNA Mitocondrial/genética , DNA Ribossômico/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
BACKGROUND: Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. METHODS: DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. RESULTS: Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. CONCLUSIONS: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.
Assuntos
Anopheles/classificação , Animais , Anopheles/genética , Análise por Conglomerados , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , América do SulRESUMO
The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.