Phylogeny of Anophelinae using mitochondrial proteincoding genes

Malaria is a vector-borne disease that is a great burdenon the poorest and most marginalized communities of thetropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the aminoacid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culexspecies as out group taxa, supplemented with 23 mitogenomes from GenBank...

Mitochondrial COI gene as a tool in the taxonomy of mosquitoes Culex subgenus Melanoconion

The subgenus Melanoconion is the second largest subgenus within the genus Culex, with 160 described species. Several of the species are proven vectors of arboviruses, including West Nile virus, Venezuelan equine encephalitis virus complex and Eastern equine encephalomyelitis virus. Species of Melanoconion are well distributed from southern North America to most countries of South America and display the highest species diversity in tropical regions. Taxonomical identification within this group has been primarily based on morphological characters, with the male genitalia as the source of the most solid diagnostic features. The difficulty in reaching accurate species determinations when studying specimens of Culex (Melanoconion) has been extensively documented as a real limitation to expand knowledge of these insects. We tested the utility of the mitochondrial gene COI as a complementary tool in the taxonomy of Melanoconion. Using a data set of 120 COI sequences from Culex specimen captured in several localities in Brazil, the utility of COI barcodes for species delimitation is discussed through the evaluation of genetic divergences among specimens and the clustering patterns of species in three topologies obtained with Neighbor Joining, Maximum Likelihood and Bayesian phylogenetic inference. For all specimens included in this study a previous morphological examination was performed, and most of the taxonomical determinations were corroborated using the COI barcode. We generated COI sequences that belong to 48 species of Melanoconion, with a mean intraspecific K2P genetic divergence of 3%; and all interspecific divergence values higher than the intraspecific divergence values. This is the first comprehensive study of subgenus Melanoconion, with evidence of COI as a useful and accessible DNA barcode...

Mitochondrial Genomes of Anopheles (Kerteszia) (Diptera: Culicidae) From the Atlantic Forest, Brazil

Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, such as the Anopheles albitarsis complex. Except for a few studies that employed a limited number of nuclear or mitochondrial loci to address the genetic structure and species status of Anopheles cruzii, Anopheles bellator, and Anopheles homunculus, little is known about genetic markers that can be employed instudies focusing on Kerteszia species. The complete mitochondrial genomes of seven specimens of An. bellator,An. cruzii, An. homunculus, and Anopheles laneanus were sequenced using long-range polymerase chain reaction and Illumina sequencing. The mitochondrial genomes varied from 15,446 to 15,738 bp in length and contained 37 genes (13 protein-encoding genes, 2 rRNA genes [12S rRNA and 16S rRNA] and 22 tRNA genes), and the AT-rich control region, as all do other Anopheles mitochondrial genomes sequenced to date. Specimens from four populations of An. cruzii showed differences in codon composition...

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

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 Culexousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint LouisEncephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome ofCx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to providegenetic information about these species.Results: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi varied from 15,573base pairs in Cx. usquatus to 15,576 in Cx. coronator. They contained 37 genes (13 protein-encoding genes, 2 rRNAgenes, and 22 tRNA genes) and the AT-rich control region. Comparative analyses of the 37 genes demonstrated themitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plusNADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control regioncontained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and theCulex pipens complex are monophyletic taxa...

Malaria vectors in South America: current and future scenarios

Malaria remains a significant public health issue in South America. Future climate change mayinfluence the distribution of the disease, which is dependent on the distribution of those Anopheles mosquitoes competent to transmit Plasmodium falciparum. Here in, predictive niche models of the habitat suitability for P.falciparum, the current primary vector Anopheles darlingi and nine other known and/or potential vector species of the Neotropical Albitarsis Complex, were used to document the current situation and project future scenarios underclimate changes in South America in 2070. Methods: To build each ecological niche model, we employed topography, climate and biome, and the currently defined distribution of P. falciparum, An. darlingi and nine species comprising the Albitarsis Complex in South America. Current and future (i.e., 2070) distributions were forecast by projecting the fitted ecological niche model on to the current environmental situation and two scenarios of simulated climate change. Statistical analyses were performed between the parasite and each vector in both the present and future scenarios to address potential vector roles in the dynamics of malaria transmission...

Brazilian Anopheles darlingi Root (Diptera: Culicidae) Clusters by Major Biogeographical Region

The major drivers of the extensive biodiversity of the Neotropics are proposed to be geologicaland tectonic events together with Pliocene and Pleistocene environmental and climatic change. Geographical barriers represented by the rivers Amazonas/Solimões, the Andesand the coastal mountain ranges in eastern Brazil have been hypothesized to lead to diversification within the primary malaria vector, Anopheles (Nyssorhynchus) darlingi Root, which primarily inhabits rainforest. To test this biogeographical hypothesis, we analyzed 786 single nucleotide polymorphisms (SNPs) in 12 populations of An. darlingi from across the complex Brazilian landscape. Both model-based (STRUCTURE) and non-model-based (Principal Components and Discriminant Analysis) analysis of population structure detected three major genetic clusters that correspond with newly described Neotropical biogeographical regions: 1) Atlantic Forest province (= southeast population); 2) Parana Forest province (= West Atlantic forest population, with one Chacoan population - SP); and 3) Brazilian dominion population (= Amazonian population with one Chacoan population - TO)...

Anopheles goeldii Rozeboom & Gabaldón (Diptera, Culicidae): a species of the Nuneztovari Complex of Anopheles Meigen

Anopheles (Nyssorhynchus) goeldii Rozeboom & Gabaldón, 1941, a species of the Nuneztovari Complex, was described based on morphological characteristics of the male, female, larva, pupa, and eggs. The typelocality is Boa Vista (= Fordlândia), a district in the vicinity of Rio Tapajós, in the municipality of Aveiro, in the state of Pará, Brazil. Anopheles goeldii is redescribed based on morphological traits of the fourth instar larva, pupa, egg, and male and female. DNA sequences from the cytochrome oxidase subunit I (COI barcoderegion) of the mitochondrial genome were utilized for species characterization. Specimens of An. goeldiifrom the Pará, Amapá, and Amazonas states were employed to redescribe the species and to compare with morphologically similar taxa...

Morphometric comparisons of the scanning electron micrographs of the eggs of Anopheles (Nyssorhynchus) darlingi Root(Diptera: Culicidae)

Anopheles darlingi Root is the principal vector of Plasmodium in Brazil, but its biological variability is notwell known. Morphometric analyses of scanning electron microscopy images of the eggs of An. darlingiwere conducted using individuals collected in nine states of Brazil (Acre, Amapá, Espírito Santo, Pará,Paraná, Rio de Janeiro, Rondônia, São Paulo, and Tocantins). Ten attributes of the eggs (seven continuous variables and three discrete variables) were respectively measured or counted and analyzed to determine if populations from different geographical regions or biomes could be distinguished. Univariate analysisshowed that the eggs from Espírito Santo were the narrowest where as representatives from Tocantins populations had the smallest floats. Results of multivariate analyses of continuous variables showed that the first principal component (PC1), mainly represented by all four float attributes, helped to differentiate populations. The second principal component (PC2) comprised roughly the length and width of the egg. PC1 of discrete variables corresponded to the number of ribs on the float where as PC2 was approximately equivalent to the number of discs on the micropyle. Based on those variables (continuous and discrete separately), multivariate discriminant analysis indicated that eggs from individuals collected in Tocantins were distinct from the other populations. Among sampled localities, the one from the state of Tocantins was situated within the Cerrado biome whereas the locality from São Paulo state was at the border of Cerrado, within a transition zone of the Atlantic Forest biome...

A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes.Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup. Methods: Larvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches...

Phylogenetic Analysis and DNA-based Species Confirmation in Anopheles (Nyssorhynchus)

Specimens of neotropical Anopheles (Nyssorhynchus) were collected and identified morphologically. We amplified three genes for phylogenetic analysis–the single copy nuclear white and CAD genes, and the COI barcode region. Since we had multiple specimens for most species we were able to test how well the single or combined genes were able to corroborate morphologically defined species by placing the species into exclusive groups. We found that single genes, including the COI barcode region, were poor at confirming species, but that the three genes combined were able to do so much better. This has implications for species identification, species delimitation, and species discovery, and we caution that single genes are not enough. Higher level groupings were partially resolved with some well-supported groupings, where as others were found to be either polyphyletic or paraphyletic. There were examples of known groups, such as the Myzorhynchella Section, which were poorly supported with single genes but were well supported with combined genes. From this we can infer that more sequence data will be needed in order to show more higher-level groupings with good support. We got unambiguously good support (0.94–1.0 Bayesian posterior probability) from all DNA-based analyses for a grouping of An.dunhami with An. nuneztovari and An. goeldii, and because of this and because of morphological similarities we propose that An. dunhami be included in the Nuneztovari Complex. We obtained phylogenetic corroboration for new species which had been recognised by morphological differences; these will need to be formally described and named...

Wing geometry of Anopheles darlingi Root (Diptera: Culicidae) in five major Brazilian ecoregions

We undertook geometric morphometric analysis of wing venation to assess this character’s ability to distinguish Anopheles darlingi Root populations and to test the hypothesis that populations from coastalareas of the Brazilian Atlantic Forest differ from those of the interior Atlantic Forest, Cerrado, and the regions South and North of the Amazon River. Results suggest that populations from the coastal and interior Atlantic Forest are more similar to each other than to any of the other regional populations. Notably,the Cerrado population was more similar to that from north of the Amazon River than to that collected of south of the River, thus showing no correlation with geographical distances. We hypothesize that environmentaland ecological factors may affect wing evolution in An. darlingi. Although it is premature to associate environmental and ecological determinants with wing features and evolution of the species,investigations on this field are promising...

New records of anopheles homunculus in central and serra do mar biodiversity corridors of the Atlantic forest, Brazil

Two new records of Anopheles homunculus in the eastern part of the Atlantic Forest are reported. This species was found for the first time in Barra do Ouro district, Maquiné municipality, Rio Grande do Sul state, located in the southern limit of the Atlantic Forest. The 2nd new record was in the Serra Bonita Reserve, Camacan municipality, southeast Bahia state. These records extend the geographical distribution of An. homunculus, suggesting that the species may be widely distributed in coastal areas of the Atlantic Forest. It is hypothesized that the disjunct distribution of the species may be caused by inadequate sampling, and also difficulties in species identification based only on female external characteristics. Species identification was based on morphological characters of the male, larva, and pupa, and corro borated by DNA sequence analyses, employing data from both 2nd internal transcribed spacer of nuclear ribosomal DNA and of mitochondrial cytochrome c oxidase subunit I...

Systematic notes of anopheles konderi and its first record in paraná state, brazil

We analyzed nuclear (second internal transcribed spacer and white gene) and mitochondrial(cytochrome c oxidase subunit I) data from Anopheles konderi collected in the Amazonian states of Acre,Amapa´, and Rondoˆnia and the southern Brazilian state of Parana´ . This was the first record of An. konderi inthe state of Parana´ . We found a high degree of genetic divergence within the Amazonian region and supportfor An. konderi as a species complex, possibly consisting of 3 species...

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferredfrom the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayesian phylogeneticmethods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis,Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyleticat the combined genes. Within the Albimanus Section the monophyly of the Strodei Subgroup wasstrongly supported and within the Myzorhynchella Section Anopheles antunesi and An. lutzii formed astrongly supported monophyletic group. The epidemiologically significant Albitarsis Complex showedevidence of paraphyly (relative to An. lanei-Myzorhynchella) and discordance across gene trees, and thepreviously synonomized species of An. dunhami and An. goeldii were recovered as sister species. Finally,there was evidence of complexes in several species, including An. antunesi, An. deaneorum, and An. strodei...