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Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae).

ABSTRACT: Culex bidens and C. interfor, implicated in arbovirus transmission in Argentina, are sister species, only distinguishable by feature of the male genitalia; however, intermediate specimens of the species in sympatry have been found. Fourth-instar larvae and females of both species share apomorphic features, and this lack of clear distinction creates problems for specific identification. Geometric morphometric traits of these life stages also do not distinguish the species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using two mitochondrial genes and to determine the degree of their reproductive isolation using microsatellite loci. Sequences of the ND4 and COI genes were concatenated in a matrix of 993 nucleotides and used for phylogenetic and distance analyses. Bayesian and maximum parsimony inferences showed a well resolved and supported topology, enclosing sequences of individuals of C. bidens (0.83 BPP, 73 BSV) and C. interfor (0.98 BPP, 97 BSV) in a strong sister relationship. The mean K2P distance within C. bidens and C. interfor was 0.3% and 0.2%, respectively, and the interspecific variation was 2.3%. Bayesian clustering also showed two distinct mitochondrial lineages. All sequenced mosquitoes were successfully identified in accordance with the best close match algorithm. The low genetic distance values obtained indicate that the species diverged quite recently. Most morphologically intermediate specimens of C. bidens from Córdoba were heterozygous for the microsatellite locus GT51; the significant heterozygote excess observed suggests incomplete reproductive isolation. However, C. bidens and C. interfor should be considered good species: the ventral arm of the phallosome of the male genitalia and the ND4 and COI sequences are diagnostic characters.

SUBMITTER: Laurito M

PROVIDER: S-EPMC5325596 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae).

Laurito Magdalena M Ayala Ana M AM Almirón Walter R WR Gardenal Cristina N CN

PloS one 20170224 2

Culex bidens and C. interfor, implicated in arbovirus transmission in Argentina, are sister species, only distinguishable by feature of the male genitalia; however, intermediate specimens of the species in sympatry have been found. Fourth-instar larvae and females of both species share apomorphic features, and this lack of clear distinction creates problems for specific identification. Geometric morphometric traits of these life stages also do not distinguish the species. The aim of the present ...[more]

PMID: 28235083

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Project description:BackgroundCulex tritaeniorhynchus and Culex pipiens pallens are the major vectors of the Japanese encephalitis virus and Wuchereria bancrofti, the causative agent of filariasis. The knowledge of mitochondrial genomes has been widely useful for the studies on molecular evolution, phylogenetics and population genetics.MethodsIn this study, we sequenced and annotated the mitochondrial (mt) genomes of Cx. tritaeniorhynchus and Cx. p. pallens, and performed a comparative analysis including four known mt genomes of species of the subgenus Culex (Culex). The phylogenetic relationships of Cx. tritaeniorhynchus, Cx. p. pallens and four known Culex mt genome sequences were reconstructed by maximum likelihood based on concatenated protein-coding gene sequences.ResultsCulex tritaeniorhynchus and Cx. p. pallens mt genomes are 14,844 bp and 15,617 bp long, both consists of 13 PCGs, 22 tRNAs, 2 rRNAs and 1 CR (not sequenced for Cx. tritaeniorhynchus). The initiation and termination codons of PCGs are ATN and TAA, respectively, except for COI starting with TCG, and COI and COII terminated with T. tRNAs have the typical clover-leaf secondary structures except for trnS ((AGN)) that is lacking the DHU stem. 16S rRNA and 12S rRNA secondary structures were drawn for the first time for mosquito mt genomes. The control region of Cx. p. pallens mt genome is 747 bp long and with four tandem repeat structures. Phylogenetic analyses demonstrated that the mt genome of Cx. tritaeniorhynchus was significantly separated from the remaining five mt genomes of Culex spp. Culex p. pipiens, Cx. p. pallens and Cx. p. quinquefasciatus formed a monophyletic clade with Cx. p. quinquefasciatus linked in the middle of the clade, and Cx. p. pallens should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus.ConclusionsThe mt genomes of Cx. tritaeniorhynchus and Cx. p. pallens share the same gene composition and order with those of two other Culex species. Culex p. pallens of the Pipiens complex should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus investigated. We enriched the Culex mt genome data and provided a reference basis for further Culex mt genome sequencing and analyses.

Project description:BackgroundFlaviviruses are important pathogens for humans and animals (Dengue viruses, Yellow fever virus, Zika virus and West Nile virus). Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus, detected in a wide variety of mosquito species.ObjectiveTo detect Flavivirus in mosquitoes of a tropical region of the Colombian Caribbean.MethodsIn 2014, an entomological surveillance of arboviruses was conducted in the department of Cordoba area of the Caribbean, Colombia. A total of 8270 mosquitoes were captured as follow: Mansonia (n = 3271/39.5%), Culex (n = 2668/32.26%), Anopheles (n = 840/10.15%), Aedeomyia (n = 411/4.9%), Psorophora (n = 397/4.8%), Coquilletidia (n = 369/4.46%), Uranotaenia (n = 261/3.15%) and Aedes (n = 53/0.6%). All mosquito species were collected in dry tropical forest of the Caribbean area. Universal primers for NS5 gene (958 pb), RT-PCR for flavivirus and sequencing were used for molecular identification of viruses detected.ResultsTwo pools belonging to Culex coronator were positive for flavivirus RNA sequence by RT-PCR. The sequences of the PCR amplicons, matched that of the Culex flaviviruses, CxFv COL PM_149 (GenBank: KR014201) and CxFv COL PM_212 (GenBank: KT307717). Phylogenetic analysis of the NS5 protein sequences of the Culex flaviviruses sequences with those of reference sequences available in GenBank indicated viruses of Genotype II, closely related to the Brazilian strain, BR_SJRP_01_ (GenBank: KT726939), from Culex sp. The alignment of Culex flavivirus sequences CxFv COL_ PM 212 and CxFv COL_ PM 149 with sequences of strains detected in different geographical regions grouped the strains in a Latin American clade reported in Brazil, Argentina and Mexico.ConclusionsThe present work illustrated that CxFV was circulating among vectors of human pathogenic arboviruses in Colombia, but the impact of CxFV on other flaviviruses which are endemic in the study area still remains to be explored.

Dataset Information

Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae).

Publications

Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae).

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