Plasmodium infection in Kerteszia cruzii (Diptera: Culicidae) in the Atlantic tropical rain forest, southeastern Brazil.

In Southeastern Brazil, Kerteszia cruzii (former Anopheles cruzii), a bromeliad mosquito species, is considered an efficient human Plasmodium spp. vector. In this region, recent studies showed asymptomatic or sub-patent Plasmodium falciparum infection. In areas of the Atlantic coast in Rio de Janeiro, Plasmodium simium infection was recently reported in both human and howler monkey. Considering that (1) few malaria cases are reported each year in areas across the tropical Atlantic rain forest in southeastern Brazil; (2) malaria elimination in Atlantic forest is challenged by circulation of P. falciparum and P. simium in humans; (3) the complexity of malaria epidemiology in this region; and (4) the public health importance of Kerteszia cruzii as a sylvatic vector; the major goal of this study is to evaluate Plasmodium infection in Ke. cruzii. Mosquito sampling collections were conducted in Esteiro do Morro and Sítio Itapuan, in Cananeia municipality, and Tapiraí municipality in Ribeira Valley, southeastern São Paulo state, Brazil. Influence of climate and landscape factors in Plasmodium infection in Ke. cruzii was addressed. Among the 1719 mosquitoes tested, 3 females collected in Sítio Itapuan and three from Tapiraí were found infected with either P. vivax or P. simium. Results of statistical analyses did not demonstrate association between Plasmodium infection in mosquito and the landscape. Mosquito infection was found in two landscape clusters, with Plasmodium detected in forest fringe mosquitoes. This finding shows that Ke. cruzii can facilitate transmission among human and non-human primates. Plasmodium falciparum was not identified in the samples analyzed. Spatiotemporal variation in local malaria incidence, low prevalence of Plasmodium, variations in humidity and temperature can explain the absence of mosquitoes infected with P. falciparum in the study.

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...

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...

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...