Population genetics of Aedes albopictus (Diptera: Culicidae) in its native range in Lao People's Democratic Republic.

BACKGROUND: The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse) is an important worldwide invasive species and can be a locally important vector of chikungunya, dengue and, potentially, Zika. This species is native to Southeast Asia where populations thrive in both temperate and tropical climates. A better understanding of the population structure of Ae. albopictus in Lao PDR is very important in order to support the implementation of strategies for diseases prevention and vector control. In the present study, we investigated the genetic variability of Ae. albopictus across a north-south transect in Lao PDR. METHODS: We used variability in a 1337-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), to assess the population structure of Ae. albopictus in Lao PDR. For context, we also examined variability at the same genetic locus in samples of Ae. albopictus from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA. RESULTS: We observed very high levels of genetic polymorphism with 46 novel haplotypes in Ae. albopictus from 9 localities in Lao PDR and Thailand populations. Significant differences were observed between the Luangnamtha population and other locations in Lao PDR. However, we found no evidence of isolation by distance. There was overall little genetic structure indicating ongoing and frequent gene flow among populations or a recent population expansion. Indeed, the neutrality test supported population expansion in Laotian Ae. albopictus and mismatch distribution analyses showed a lack of low frequency alleles, a pattern often seen in bottlenecked populations. When samples from Lao PDR were analyzed together with samples from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA, phylogenetic network and Bayesian cluster analysis showed that most populations from tropical/subtropical regions are more genetically related to each other, than populations from temperate regions. Similarly, most populations from temperate regions are more genetically related to each other, than those from tropical/subtropical regions. CONCLUSIONS: Aedes albopictus in Lao PDR are genetically related to populations from tropical/subtropical regions (i.e. Thailand, Singapore, and California and Texas in the USA). The extensive gene flow among locations in Lao PDR indicates that local control is undermined by repeated introductions from untreated sites.

The complete mt genomes of Lutzia halifaxia, Lt. fuscanus and Culex pallidothorax (Diptera: Culicidae) and comparative analysis of 16 Culex and Lutzia mt genome sequences.

BACKGROUND: Despite the medical importance of the genus Culex, the mitochondrial genome (mt genome) characteristics of Culex spp. are not well understood. The phylogeny of the genus and particularly the generic status of the genus Lutzia and the subgenus Culiciomyia remain unclear. METHODS: The present study sequenced and analyzed the complete mt genomes of Lutzia halifaxia, Lutzia fuscanus and Cx. (Culiciomyia) pallidothorax and assessed the general characteristics and phylogenetics of all known 16 mt genome sequences for species in the genera Culex and Lutzia. RESULTS: The complete mt genomes of Lt. halifaxia, Lt. fuscanus and Cx. pallidothorax are 15,744, 15,803 and 15,578 bp long, respectively, including 13 PCGs, 22 tRNAs, two tRNAs and a control region (CR). Length variations in the Culex and Lutzia mt genomes involved mainly the CR, and gene arrangements are the same as in other mosquitoes. We identified four types of repeat units in the CR sequences, and the poly-T stretch exists in all of these mt genomes. The repeat units of CR are conserved to different extent and provide information on their evolution. Phylogenetic analyses demonstrated that the Coronator and Sitiens groups are each monophyletic, whereas the monophyletic status of the Pipiens Group was not supported; Cx. pallidothorax is more closely related to the Sitiens and Pipiens groups; and both phylogenetics analysis and repeat unit features in CR show that Lutzia is a characteristic monophyletic entity, which should be an independent genus. CONCLUSIONS: To our knowledge, this is the first comprehensive review of the mt genome sequences and taxonomic discussion based on the mt genomes of Culex spp. and Lutzia spp. The research provides general information on the mt genome of these two genera, and the phylogenetic and taxonomic status of Lutzia and Culiciomyia.

A Multi-Gene Analysis and Potential Spatial Distribution of Species of the Strodei Subgroup of the Genus Nyssorhynchus (Diptera: Culicidae).

Nyssorhynchus strodei (Root) is an understudied species of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum Welch, Plasmodium vivax Grassi & Feletti, and Plasmodium malariae Feletti & Grassi. It belongs to the strodei subgroup that includes Nyssorhynchus albertoi (Unti), Nyssorhynchus arthuri (Unti), Nyssorhynchus rondoni (Neiva & Pinto), Nyssorhynchus striatus (Sant'Ana & Sallum), and three unnamed species, Nyssorhynchus arthuri B, Nyssorhynchus arthuri C, and Nyssorhynchus arthuri D. As the accurate identification of vector species is of fundamental importance for public health entomology, the aim of the study was to provide additional information for the presence of seven species that had been previously misidentified as Ny. strodei. Bayesian phylogenetic analyses using DNA sequences of the genes COI mtDNA, white, CAD and CAT nuclear genes confirmed Ny. albertoi, Ny. arthuri, Ny. strodei, and Ny. striatus as distinct groups within the strodei subgroup and corroborated the presence of three undescribed species under the name Ny. arthuri. Results of the GMYC model analysis corroborated Ny. arthuri B, Ny. arthuri C, and Ny. arthuri D; however, they did not distinguish between Ny. strodei and Ny. albertoi. Predicted distribution of seven species based on maximum entropy in MaxEnt showed that each species has its specific ecological niche suitability.

A multi-gene analysis and potential spatial distribution of species of the Strodei subgroup of the genus Nyssorhynchus (Diptera: Culicidae)

Nyssorhynchus strodei (Root) is an understudied species of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum Welch, Plasmodium vivax Grassi & Feletti, and Plasmodium malariae Feletti & Grassi. It belongs to the strodei subgroup that includes Nyssorhynchus albertoi (Unti), Nyssorhynchus arthuri (Unti), Nyssorhynchus rondoni (Neiva & Pinto), Nyssorhynchus striatus (Sant’Ana & Sallum), and three unnamed species, Nyssorhynchus arthuri B, Nyssorhynchus arthuri C, and Nyssorhynchus arthuri D. As the accurate identification of vector species is of fundamental importance for public health entomology, the aim of the study was to provide additional information for the presence of seven species that had been previously misidentified as Ny. strodei. Bayesian phylogenetic analyses using DNA sequences of the genes COI mtDNA, white, CAD and CAT nuclear genes confirmed Ny. albertoi, Ny. arthuri, Ny. strodei, and Ny. striatus as distinct groups within the strodei subgroup and corroborated the presence of three undescribed species under the name Ny. arthuri. Results of the GMYC model analysis corroborated Ny. arthuri B, Ny. arthuri C, and Ny. arthuri D; however, they did not distinguish between Ny. strodei and Ny. albertoi. Predicted distribution of seven species based on maximum entropy in MaxEnt showed that each species has its specific ecological niche suitability.

A multi-gene analysis and potential spatial distribution of species of the Strodei subgroup of the genus Nyssorhynchus (Diptera: Culicidae)

Nyssorhynchus strodei (Root) is an understudied species of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum Welch, Plasmodium vivax Grassi & Feletti, and Plasmodium malariae Feletti & Grassi. It belongs to the strodei subgroup that includes Nyssorhynchus albertoi (Unti), Nyssorhynchus arthuri (Unti), Nyssorhynchus rondoni (Neiva & Pinto), Nyssorhynchus striatus (Sant’Ana & Sallum), and three unnamed species, Nyssorhynchus arthuri B, Nyssorhynchus arthuri C, and Nyssorhynchus arthuri D. As the accurate identification of vector species is of fundamental importance for public health entomology, the aim of the study was to provide additional information for the presence of seven species that had been previously misidentified as Ny. strodei. Bayesian phylogenetic analyses using DNA sequences of the genes COI mtDNA, white, CAD and CAT nuclear genes confirmed Ny. albertoi, Ny. arthuri, Ny. strodei, and Ny. striatus as distinct groups within the strodei subgroup and corroborated the presence of three undescribed species under the name Ny. arthuri. Results of the GMYC model analysis corroborated Ny. arthuri B, Ny. arthuri C, and Ny. arthuri D; however, they did not distinguish between Ny. strodei and Ny. albertoi. Predicted distribution of seven species based on maximum entropy in MaxEnt showed that each species has its specific ecological niche suitability.

Wing Morphometry and Genetic Variability Between Culex coronator and Culex usquatus (Diptera: Culicidae), Two Sibling Species of the Coronator Group.

Culex coronator Dyar and Knab and Culex usquatus Dyar belong to the Coronator Group of the subgenus Culex. Culex coronator and Cx. usquatus are widespread and sympatric throughout their distribution range, which includes Brazil. Morphological identification of these species is based primarily on the characteristics of the male genitalia; females are indistinguishable using the qualitative characteristics employed in identification keys. The primary objective of this study was to distinguish females of Cx. coronator from those of Cx. usquatus employing both wing geometric morphometrics, and DNA sequences (NADH5, COI, Hunchback, and CAD). Additionally, we employed the isolation with migration model (IMa) to evaluate: 1) the migration rates and 2) the divergence time, between Cx. coronator and Cx. usquatus. Specimens were captured in Pariquera-Açu and Cananéia south-eastern São Paulo, Ribeira Valley, Brazil. Canonical variate analysis (CVA) demonstrated two groups in the morphospace. The accuracy of species recognition was moderate (82.6%) for Cx. coronator and low (60.8%) for Cx. usquatus. Bayesian analyses of concatenated gene sequences recovered from specimens of Cx. coronator separated the species into three lineages (herein referred to as Culex coronator A, B, and C), whereas Cx. usquatus specimens clustered into a single lineage. Lineages A and B of Cx. coronator intermixed with specimens of Cx. usquatus, and one specimen of Cx. coronator clustered into the Cx. usquatus lineage. The IMa analysis indicated that the divergence of Cx. coronator and Cx. usquatus is a slow process, with some degree of gene flow between the two species.

Mitochondrial genomes and comparative analyses of Culex camposi, Culex coronator, Culex usquatus and Culex usquatissimus (Diptera:Culicidae), members of the coronator group.

BACKGROUND: The Coronator Group currently encompasses six morphologically similar species (Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex usquatus Dyar, Culex usquatissimus Dyar, and Culex ousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to provide genetic information about these species. RESULTS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi varied from 15,573 base pairs in Cx. usquatus to 15,576 in Cx. coronator. They contained 37 genes (13 protein-encoding genes, 2 rRNA genes, and 22 tRNA genes) and the AT-rich control region. Comparative analyses of the 37 genes demonstrated the mitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plus NADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control region contained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and the Culex pipens complex are monophyletic taxa. CONCLUSIONS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi share the same gene composition and arrangement features that match to those reported for most Culicidae species. They are composed of the same 37 genes and the AT-rich control region, which contains poly-T stretches that may be involved in the functional role of the mitochondrial genome. Taken together, results of the dN/dS ratios, the sliding window analyses and the Bayesian phylogenetic analyses suggest that ATP6, ATP8 and NADH5 are promising genes to be employed in phylogenetic studies involving species of the Coronator Group, and probably other species groups of the subgenus Culex. Bayesian topology corroborated the morphological hypothesis of the Coronator Group as monophyletic lineage within the subgenus Culex.

Wing geometry of Culex coronator (Diptera: Culicidae) from South and Southeast Brazil.

BACKGROUND: The Coronator Group encompasses Culex coronator Dyar & Knab, Culex camposi Dyar, Culex covagarciai Forattini, Culex ousqua Dyar, Culex usquatissimus Dyar, Culex usquatus Dyar and Culex yojoae Strickman. Culex coronator has the largest geographic distribution, occurring in North, Central and South America. Moreover, it is a potential vector-borne mosquito species because females have been found naturally infected with several arboviruses, i.e., Saint Louis Encephalitis Virus, Venezuelan Equine Encephalitis Virus and West Nile Virus. Considering the epidemiological importance of Cx. coronator, we investigated the wing shape diversity of Cx. coronator from South and Southeast Brazil, a method to preliminarily estimate population diversity. METHODS: Field-collected immature stages of seven populations from a large geographical area in Brazil were maintained in the laboratory to obtain both females and males linked with pupal and/or larval exuviae. For each individual female, 18 landmarks of left wings were marked and digitalized. After Procrustes superimposition, discriminant analysis of shape was employed to quantify wing shape variation among populations. The isometric estimator centroid size was calculated to assess the overall wing size and allometry. RESULTS: Wing shape was polymorphic among populations of Cx. coronator. However, dissimilarities among populations were higher than those observed within each population, suggesting populational differentiation in Cx. coronator. Morphological distances between populations were not correlated to geographical distances, indicating that other factors may act on wing shape and thus, determining microevolutionary patterns in Cx. coronator. Despite the population differentiation, intrapopulational wing shape variability was equivalent among all seven populations. CONCLUSION: The wing variability found in Cx. coronator populations brings to light a new biological problem to be investigated: the population genetics of Cx. coronator. Because of differences in the male genitalia, we also transferred Cx. yojoae to the Apicinus Subgroup.

Taxonomic and phylogenetic relationships between species of the genus Culex (Diptera: culicidae) from Brazil inferred from the cytochrome c oxidase I mitochondrial gene.

Species of the genus Culex Linnaeus have been incriminated as the main vectors of lymphatic filariases and are important vectors of arboviruses, including West Nile virus. Sequences corresponding to a fragment of 478 bp of the cytochrome c oxidase subunit I gene, which includes part of the barcode region, of 37 individuals of 17 species of genus Culex were generated to establish relationships among five subgenera, Culex, Phenacomyia, Melanoconion, Microculex, and Carrollia, and one species of the genus Lutzia that occurs in Brazil. Bayesian methods were employed for the phylogenetic analyses. Results of sequence comparisons showed that individuals identified as Culex dolosus, Culex mollis, and Culex imitator possess high intraspecific divergence (3.1, 2.3, and 3.5%, respectively) when using the Kimura two parameters model. These differences were associated either with distinct morphological characteristics of the male genitalia or larval and pupal stages, suggesting that these may represent species complexes. The Bayesian topology suggested that the genus and subgenus Culex are paraphyletic relative to Lutzia and Phenacomyia, respectively. The cytochrome c oxidase subunit I sequences may be a useful tool to both estimate phylogenetic relationships and identify morphologically similar species of the genus Culex.

Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae).

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.

Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae)

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.