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Population subdivision among several neotropical malaria vectors has been widely evaluated; however, few studies have analyzed population variation at a microgeographic scale, wherein local environmental variables may lead to population differentiation. The aim of the present study was to evaluate the genetic and geometric morphometric structure of Anopheles nuneztovari and Anopheles albimanus in endemic localities of northwestern Colombia. Genetic and phenetic structures were evaluated using microsatellites markers and wing geometric morphometrics, respectively. In addition, entomological indices of importance in transmission were calculated. Results showed that the main biting peaks of Anopheles nuneztovari were between 20:00 and 22:00, whereas Anopheles albimanus exhibited more variation in biting times among localities. Infection in An. nuneztovari by Plasmodium spp. (IR: 4.35%) and the annual entomological inoculation rate (30.31), indicated high vector exposure and local transmission risk. We did not detect Plasmodium-infected An. albimanus in this study. In general, low genetic and phenetic subdivision among the populations of both vectors was detected using a combination of phenotypic, genetic and environmental data. The results indicated high regional gene flow, although local environmental characteristics may be influencing the wing conformation differentiation and behavioral variation observed in An. albimanus. Furthermore, the population subdivision detected by microsatellite markers for both species by Bayesian genetic analysis provides a more accurate picture of the current genetic structure in comparison to previous studies. Finally, the biting behavior variation observed for both vectors among localities suggests the need for continuous malaria vector surveys covering the endemic region to implement the most effective integrated local control interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Vale do Rio Juruá in western Acre, Brazil, is a persistent malaria transmission hotspot partly due to fish farming development that was encouraged to improve local standards of living. Fish ponds can be productive breeding sites for Amazonian malaria vector species, including Nyssorhynchus darlingi, which, combined with high human density and mobility, add to the local malaria burden.This study reports entomological profile of immature and adult Ny. darlingi at three sites in Mâncio Lima, Acre, during the rainy and dry season (February to September, 2017). From 63 fishponds, 10,859 larvae were collected, including 5,512 first-instar Anophelinae larvae and 4,927 second, third and fourth-instars, of which 8.5% (n = 420) were Ny. darlingi. This species was most abundant in not-abandoned fishponds and in the presence of emerging aquatic vegetation. Seasonal analysis of immatures in urban landscapes found no significant difference in the numbers of Ny. darlingi, corresponding to equivalent population density during the rainy to dry transition period. However, in the rural landscape, significantly higher numbers of Ny. darlingi larvae were collected in August (IRR = 5.80, p = 0.037) and September (IRR = 6.62, p = 0.023) (dry season), compared to February (rainy season), suggesting important role of fishponds for vector population maintenance during the seasonal transition in this landscape type. Adult sampling detected mainly Ny. darlingi (~93%), with similar outdoor feeding behavior, but different abundance according to landscape profile: urban site 1 showed higher peaks of human biting rate in May (46 bites/person/hour), than February (4) and September (15), while rural site 3 shows similar HBR during the same sampling period (22, 24 and 21, respectively). This study contributes to a better understanding of the larvae biology of the main malaria vector in the Vale do Rio Juruá region and, ultimately will support vector control efforts., Competing Interests: The authors have declared that no competing interests exist.

Inter-relationships among mosquito vectors, Plasmodium parasites, human ecology, and biotic and abiotic factors, drive malaria risk. Specifically, rural landscapes shaped by human activities have a great potential to increase the abundance of malaria vectors, putting many vulnerable people at risk. Understanding at which point the abundance of vectors increases in the landscape can help to design policies and interventions for effective and sustainable control. Using a dataset of adult female mosquitoes collected at 79 sites in malaria endemic areas in the Brazilian Amazon, this study aimed to (1) verify the association among forest cover percentage (PLAND), forest edge density (ED), and variation in mosquito diversity; and to (2) test the hypothesis of an association between landscape structure (i.e., PLAND and ED) and Nyssorhynchus darlingi (Root) dominance. Mosquito collections were performed employing human landing catch (HLC) (peridomestic habitat) and Shannon trap combined with HLC (forest fringe habitat). Nyssorhynchus darlingi abundance was used as the response variable in a generalized linear mixed model, and the Shannon diversity index (H') of the Culicidae community, PLAND, and the distance house-water drainage were used as predictors. Three ED categories were also used as random effects. A path analysis was used to understand comparative strengths of direct and indirect relationships among Amazon vegetation classes, Culicidae community, and Ny. darlingi abundance. Our results demonstrate that Ny. darlingi is negatively affected by H´ and PLAND of peridomestic habitat, and that increasing these variables (one-unit value at β0 = 768) leads to a decrease of 226 (P < 0.001) and 533 (P = 0.003) individuals, respectively. At the forest fringe, a similar result was found for H' (β1 = -218; P < 0.001) and PLAND (β1 = -337; P = 0.04). Anthropogenic changes in the Amazon vegetation classes decreased mosquito biodiversity, leading to increased Ny. darlingi abundance. Changes in landscape structure, specifically decreases in PLAND and increases in ED, led to Ny. darlingi becoming the dominant species, increasing malaria risk. Ecological mechanisms involving changes in landscape and mosquito species composition can help to understand changes in the epidemiology of malaria., Competing Interests: The authors have declared that no competing interests exist.

The relationship between deforestation and malaria in Amazonian Brazil is complex, and a deeper understanding of this relationship is required to inform effective control measures in this region. Here, we are particularly interested in characterizing the impact of land use and land cover change on the genetics of the major regional vector of malaria, Nyssorhynchus darlingi (Root). We used nextera-tagmented, Reductively Amplified DNA (nextRAD) genotyping-by-sequencing to genotype 164 Ny. darlingi collected from 16 collection sites with divergent forest cover levels in seven municipalities in four municipality groups that span the state of Amazonas in northwestern Amazonian Brazil: São Gabriel da Cachoeira, Presidente Figueiredo, four municipalities in the area around Cruzeiro do Sul, and Lábrea. Using a dataset of 5,561 Single Nucleotide Polymorphisms (SNPs), we investigated the genetic structure of these Ny. darlingi populations with a combination of model- and non-model-based analyses. We identified weak to moderate genetic differentiation among the four municipality groups. There was no evidence for microgeographic genetic structure of Ny. darlingi among forest cover levels within the municipality groups, indicating that there may be gene flow across areas of these municipalities with different degrees of deforestation. Additionally, we conducted an environmental association analysis using two outlier detection methods to determine whether individual SNPs were associated with forest cover level without affecting overall population genetic structure. We identified 14 outlier SNPs, and investigated functions associated with their proximal genes, which could be further characterized in future studies., Competing Interests: The authors have declared that no competing interests exist.

West Nile virus (WNV; Flaviviridae: Flavivirus) is a widely distributed arthropod-borne virus that has negatively affected human health and animal populations. WNV infection rates of mosquitoes and human cases have been shown to be correlated with climate. However, previous studies have been conducted at a variety of spatial and temporal scales, and the scale-dependence of these relationships has been understudied. We tested the hypothesis that climate variables are important to understand these relationships at all spatial scales. We analyzed the influence of climate on WNV infection rate of mosquitoes and number of human cases in New York and Connecticut using Random Forests, a machine learning technique. During model development, 66 climate-related variables based on temperature, precipitation and soil moisture were tested for predictive skill. We also included 20-21 non-climatic variables to account for known environmental effects (e.g., land cover and human population), surveillance related information (e.g., relative mosquito abundance), and to assess the potential explanatory power of other relevant factors (e.g., presence of wastewater treatment plants). Random forest models were used to identify the most important climate variables for explaining spatial-temporal variation in mosquito infection rates (abbreviated as MLE). The results of the cross-validation support our hypothesis that climate variables improve the predictive skill for MLE at county- and trap-scales and for human cases at the county-scale. Of the climate-related variables selected, mean minimum temperature from July-September was selected in all analyses, and soil moisture was selected for the mosquito county-scale analysis. Models demonstrated predictive skill, but still over- and under-estimated WNV MLE and numbers of human cases. Models at fine spatial scales had lower absolute errors but had greater errors relative to the mean infection rates., Competing Interests: The authors have declared that no competing interests exist.

The major drivers of the extensive biodiversity of the Neotropics are proposed to be geological and tectonic events together with Pliocene and Pleistocene environmental and climatic change. Geographical barriers represented by the rivers Amazonas/Solimões, the Andes and 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). Significant levels of pairwise genetic divergences were found among the three clusters, allele sharing among clusters was negligible, and geographical distance did not contribute to differentiation. We infer that the Atlantic forest coastal mountain range limited dispersal between the Atlantic Forest province and the Parana Forest province populations, and that the large, diagonal open vegetation region of the Chacoan dominion dramatically reduced dispersal between the Parana and Brazilian dominion populations. We hypothesize that the three genetic clusters may represent three putative species.

Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) sequences were used to evaluate initial identification and to investigate phylogenetic relationships of seven Anopheles morphospecies of the Arribalzagia Series from Colombia. Phylogenetic trees recovered highly supported clades for An. punctimaculas.s., An. calderoni, An. malefactor s.l., An. neomaculipalpus, An. apicimacula s.l., An. mattogrossensis and An. peryassui. This study provides the first molecular confirmation of An. malefactorfrom Colombia and discovered conflicting patterns of divergence for the molecular markers among specimens from northeast and northern Colombia suggesting the presence of two previously unrecognized Molecular Operational Taxonomic Units (MOTUs). Furthermore, two highly differentiated An. apicimacula MOTUs previously found in Panama were detected. Overall, the combined molecular dataset facilitated the detection of known and new Colombian evolutionary lineages, and constitutes the baseline for future research on their bionomics, ecology and potential role as malaria vectors.

The Colombian Pacific region is second nationally in number of malaria cases reported. This zone presents great ecological heterogeneity and Anopheles species diversity. However, little is known about the current spatial and temporal distribution of vector species. This study, conducted in three ecologically different localities of the Pacific region, aimed to evaluate the composition and distribution of Anopheles species and characterize transmission intensity. A total of 4,016 Anopheles mosquitoes were collected representing seven species. The composition and dominant species differed in each locality. Three species were infected with malaria parasites: Anopheles darlingi and An. calderoni were infected with Plasmodium falciparum and An. nuneztovari with Plasmodium vivax VK210 and VK247. Annual EIRs varied from 3.5-7.2 infective bites per year. These results confirm the importance of the primary vector An. nuneztovari in areas disturbed by human interventions, of An. darlingi in deforested margins of humid tropical rainforest and An. albimanus and the suspected vector An. calderoni in areas impacted by urbanization and large-scale palm oil agriculture close to the coast. This constitutes the first report in the Colombia Pacific region of naturally infected An. darlingi, and in Colombia of naturally infected An. calderoni. Further studies should evaluate the epidemiological importance of An. calderoni in the Pacific region.