Tatiane M. P. de Oliveira

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, whereas 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.

COI barcode versus morphological identification of Culex (Culex) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil

Sequences of the cytochrome c oxidase subunit I (COI) mitochondrial gene from adults of 22 Culex ( Culex ) species from Argentina and Brazil were employed to assess species identification and to test the usefulness of COI for barcoding using the best close match (BCM) algorithm. A pairwise Kimura two-parameter distance matrix including the mean intra and interspecific distances for 71 COI barcode sequences was constructed. Of the 12 COI lineages recovered in the Neighbour-joining topology, five confirmed recognised morphological species ( Cx. acharistus , Cx. chidesteri , Cx. dolosus , Cx. lygrus and Cx. saltanensis ) with intraspecific divergences lower than 1.75%. Cx. bilineatus is formally resurrected from the synonymy of Cx. dolosus . Cx. maxi , Cx. surinamensis and the Coronator group species included were clustered into an unresolved lineage. The intraspecific distance of Cx. pipiens (3%) was almost twice the interspecific between it and Cx. quinquefasciatus (1.6%). Regarding the BCM criteria, the COI barcode successfully identified 69% of all species. The rest of the sequences, approximately 10%, 18% and 3%, remained as ambiguously, mis and unidentified, respectively. The COI barcode does not contain enough information to distinguish Culex ( Cux. ) species.

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

BackgroundThe 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.MethodsLarvae, 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.ResultsBayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach.ConclusionsOur study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.

Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm?

BackgroundRecently an unexpectedly high prevalence of Plasmodium falciparum was found in asymptomatic blood donors living in the southeastern Brazilian Atlantic forest. The bromeliad-malaria paradigm assumes that transmission of Plasmodium vivax and Plasmodium malariae involves species of the subgenus Kerteszia of Anopheles and only a few cases of P. vivax malaria are reported annually in this region. The expectations of this paradigm are a low prevalence of P. vivax and a null prevalence of P. falciparum. Therefore, the aim of this study was to verify if P. falciparum is actively circulating in the southeastern Brazilian Atlantic forest remains.MethodsIn this study, anophelines were collected with Shannon and CDC-light traps in seven distinct Atlantic forest landscapes over a 4-month period. Field-collected Anopheles mosquitoes were tested by real-time PCR assay in pools of ten, and then each mosquito from every positive pool, separately for P. falciparum and P. vivax. Genomic DNA of P. falciparum or P. vivax from positive anophelines was then amplified by traditional PCR for sequencing of the 18S ribosomal DNA to confirm Plasmodium species. Binomial probabilities were calculated to identify non-random results of the P. falciparum-infected anopheline findings.ResultsThe overall proportion of anophelines naturally infected with P. falciparum was 4.4% (21/480) and only 0.8% (4/480) with P. vivax. All of the infected mosquitoes were found in intermixed natural and human-modified environments and most were Anopheles cruzii (22/25 = 88%, 18 P. falciparum plus 4 P. vivax). Plasmodium falciparum was confirmed by sequencing in 76% (16/21) of positive mosquitoes, whereas P. vivax was confirmed in only 25% (1/4). Binomial probabilities suggest that P. falciparum actively circulates throughout the region and that there may be a threshold of the forested over human-modified environment ratio upon which the proportion of P. falciparum-infected anophelines increases significantly.ConclusionsThese results show that P. falciparum actively circulates, in higher proportion than P. vivax, among Anopheles mosquitoes of fragments of the southeastern Brazilian Atlantic forest. This finding challenges the classical bromeliad-malaria paradigm, which considers P. vivax circulation as the driver for the dynamics of residual malaria transmission in this region.

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

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.

Geographic distribution, evolution, and disease importance of species within the Neotropical Anopheles albitarsis Group (Diptera, Culicidae)

ABSTRACT: The Anopheles albitarsis group of mosquitoes comprises eight recognized species and one mitochondrial lineage. Our knowledge of malaria vectorial importance and the distribution and evolution of these taxa is incomplete. We constructed ecological niche models (ENMs) for these taxa and used hypothesized phylogenetic relationships and ENMs to investigate environmental and ecological divergence associated with speciation events. Two major clades were identified, one north (Clade 1) and one south (Clade 2) of the Amazon River that likely is or was a barrier to mosquito movement. Clade 1 species occur more often in higher average temperature locations than Clade 2 species, and taxon splits within Clade 1 corresponded with a greater divergence of variables related to precipitation than was the case within Clade 2. Comparison of the ecological profiles of sympatric species and sister species support the idea that phylogenetic proximity is related to ecological similarity. Anopheles albitarsis I, An. janconnae, and An. marajoara ENMs had the highest percentage of their predicted suitable habitat overlapping distribution models of Plasmodium falciparum and P. vivax, and warrant additional studies of the transmission potential of these species. Phylogenetic proximity may be related to malaria vectorial importance within the Albitarsis Group.

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.

Phylogeny of Anophelinae using mitochondrial protein coding genes

Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical 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 amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.

First Record of Anopheles oryzalimnetes, Anopheles argyritarsis, and Anopheles sawyeri (Diptera: Culicidae) in the Caatinga Biome, Semiarid Scrubland of Sergipe State, Brazil

ABSTRACT Caatinga is one of the least known biomes of Brazil in relation to biodiversity. The dry condition of semiarid areas has been associated in the past with low richness of fauna and flora, not encouraging studies in this region. There is a lack of mosquito records including anophelines. Thus, to investigate the biodiversity of Anopheles mosquitoes in the Caatinga biome, we collected immature mosquitoes in aquatic habitats in a conservation reserve located in the northwestern portion of Sergipe state. The captured specimens were initially identified as Anopheles albitarsis l.s. and Anopheles argyritarsis l.s. To confirm the morphological identification, sequences were generated by cytochrome oxidase subunit I mitocondrial gene. The results showed that the specimens belong to the species Anopheles oryzalimnetes, An. argyritarsis, and Anopheles sawyeri. These are the first records of these species in this region. The presence of Anopheles in the Caatinga biome, which is characterized by arid and semiarid climatic conditions, encourages the interest in the study of biological, physiological, and behavioral adaptations, selected over time, which allow these mosquito populations to survive through the long periods of drought that is characteristic of this region.

Mosquitoes of the Caatinga: 1. Adults stage survey and the emerge of seven news species endemic of a dry tropical forest in Brazil

The Caatinga is the least known Brazilian biome in terms of the diversity of Culicidae. No systematic study of the diversity or ecology of the mosquitoes of this biome is available, despite the importance of vector diseases in Brazil. The present study addressed the mosquito biodiversity in the Caatinga biome by sampling adult populations. Specimens were sampled monthly from March 2013 to September 2014 in a Caatinga conservation unit located in the Brazilian semiarid zone. Mosquito collections were carried out in Shannon traps from late afternoon to early evening, and manual aspiration was used to capture diurnal species as well. A total of 4,692 mosquitoes were collected. The most dominant and constant species were all undescribed species belonging to the genera Wyeomyia and Runchomyia, which together represented 80% of the specimens. The most abundant species of epidemiological importance was Haemagogus (Con.) leucocelaenus. The abundance of mosquitoes was positively associated with the relative humidity and temperature recorded during the month preceding the collection date. In the Caatinga, the diversity of adult mosquitoes was associated with the availability (quantity and diversity) of natural larval habitats found in the different phytophysiognomies of the biome, which vary according to temperature and humidity. The number of species unknown to science reflects the levels of endemism that exist in the study area, and reinforces the need to further taxonomic investigation in the biome.