The Influence of the pH and Salinity of Water in Breeding Sites on the Occurrence and Community Composition of Immature Mosquitoes in the Green Belt of the City of São Paulo, Brazil.

The physicochemical parameters of water, such as pH, salinity, conductivity, and total dissolved solids, can influence mosquito larval development, survival, and abundance. Therefore, it is important to elucidate how these factors influence mosquito occurrence. We hypothesized that the occurrence and community composition of immature mosquito species are driven not only by the availability of suitable aquatic habitats, but also by the physicochemical factors of these habitats. The primary objective of this study was therefore to investigate the influence of the physicochemical parameters of water in different types of aquatic habitats on the occurrence of mosquito species in two remnants of Atlantic Forest in the city of São Paulo, Brazil. Collections of immature mosquitoes and assessment of the physicochemical characteristics of the water in the collection sites were carried out for twelve months. The variation in species composition and occurrence with the measured physicochemical parameters and the type of breeding site was assessed using constrained ordination methods. The results indicate that there was a statistically significant difference in species composition as a function of the different types of aquatic habitats, and that pH had an influence on species occurrence even when the variance explained by the type of aquatic habitat was removed from the analysis. There was a statistically significant association between mosquito species occurrence and pH and salinity, and the former had a significant influence on the mosquito species collected regardless of the type of aquatic habitat, showing that the pH of the breeding site water is an important factor in driving mosquito population dynamics and species distribution.

The influence of anthropogenic habitat fragmentation on the genetic structure and diversity of the malaria vector Anopheles cruzii (Diptera: Culicidae).

Fragmentation of natural environments as a result of human interference has been associated with a decrease in species richness and increase in abundance of a few species that have adapted to these environments. The Brazilian Atlantic Forest, which has been undergoing an intense process of fragmentation and deforestation caused by human-made changes to the environment, is an important hotspot for malaria transmission. The main vector of simian and human malaria in this biome is the mosquito Anopheles cruzii. Anthropogenic processes reduce the availability of natural resources at the tree canopies, An. cruzii primary habitat. As a consequence, An. cruzii moves to the border of the Atlantic Forest nearing urban areas seeking resources, increasing their contact with humans in the process. We hypothesized that different levels of anthropogenic changes to the environment can be an important factor in driving the genetic structure and diversity in An. cruzii populations. Five different hypotheses using a cross-sectional and a longitudinal design were tested to assess genetic structure in sympatric An. cruzii populations and microevolutionary processes driving these populations. Single nucleotide polymorphisms were used to assess microgeographic genetic structure in An. cruzii populations in a low-endemicity area in the city of São Paulo, Brazil. Our results show an overall weak genetic structure among the populations, indicating a high gene flow system. However, our results also pointed to the presence of significant genetic structure between sympatric An. cruzii populations collected at ground and tree-canopy habitats in the urban environment and higher genetic variation in the ground-level population. This indicates that anthropogenic modifications leading to habitat fragmentation and a higher genetic diversity and structure in ground-level populations could be driving the behavior of An. cruzii, ultimately increasing its contact with humans. Understanding how anthropogenic changes in natural areas affect An. cruzii is essential for the development of more effective mosquito control strategies and, on a broader scale, for malaria-elimination efforts in the Brazilian Atlantic Forest.

Wing Geometric Morphometrics as a Tool for the Identification of Culex Subgenus Mosquitoes of Culex (Diptera: Culicidae).

Culex is the largest subgenus within the genus Culex that includes important vectors of diseases. The correct identification of mosquitoes is critical for effective control strategies. Wing geometric morphometrics (WGM) has been used to identify mosquito species alongside traditional identification methods. Here, WGM was used for eleven Culex species from São Paulo, Brazil, and one from Esquel, Argentina. Adult mosquitoes were collected using CDC (Centers for Disease Control) traps, morphologically identified and analyzed by WGM. The canonical variate analysis (CVA) was performed and a Neighbor-joining (NJ) tree was constructed to illustrate the patterns of species segregation. A cross-validated reclassification test was also carried out. From 110 comparisons in the cross-validated reclassification test, 87 yielded values higher than 70%, with 13 comparisons yielding 100% reclassification scores. Culexquinquefasciatus yielded the highest reclassification scores among the analyzed species, corroborating with the results obtained by the CVA, in which Cx. quinquefasciatus was the most distinct species. The high values obtained at the cross-validated reclassification test and in the NJ analysis as well as the segregation observed at the CVA made it possible to distinguish among Culex species with high degrees of confidence, suggesting that WGM is a reliable tool to identify Culex species of the subgenus Culex.

Wing geometric morphometrics for identification of mosquito species (Diptera: Culicidae) of neglected epidemiological importance.

Culicidae comprises more than 3500 species, some of which are responsible for the spread of various human diseases, causing millions of deaths worldwide. Correct identification of these species is essential for the development of surveillance and control strategies. The most common method of mosquito identification is based on specific traits of the external morphology of species. However, identification of mosquitoes by morphological characters can be inaccurate or even unfeasible if the specimen is damaged or there is a lack of distinguishing features, as in the case of cryptic species complexes. Wing geometric morphometrics is a reliable, affordable tool for the identification of mosquito species, including sibling species. More importantly, it can be used in addition to both traditional morphologic identification methods as well as genetic approaches. Here, wing geometric morphometrics was used to identify sixteen mosquito species from eight genera: Aedes, Coquillettidia, Culex, Limatus, Mansonia, Psorophora, Runchomyia, and Wyeomyia. The 390 specimens used here were collected in São Paulo, Brazil using CDC traps, aspiration, and Shannon traps. Allometry was assessed by multivariate regression of the Procrustes coordinates on centroid size followed by canonical variate analysis and a pairwise cross-validated reclassification test. A Neighbor-Joining tree based on Mahalanobis distances was constructed with 1,000 bootstrap replicates using MorphoJ 1.02 and Past 2.17c. The canonical variate analysis of genera resulted in distinct clusters for Culex, Limatus, and Psorophora and partial overlapping between Aedes, Coquilettidia, and Mansonia, and between Runchomyia and Wyeomyia. Pairwise cross-validated reclassification tests indicated that genera were identified with an accuracy of at least 99% and subgenera with a mean accuracy of 96% and that in 160 of the 240 possible comparisons species were identified with an accuracy of 100%. Our results show that the eight genera in the study were correctly distinguished by wing shape, as were subgenera and most species, demonstrating that wing geometric morphometrics can be used for the identification of the mosquito species studied here.

Neotropical Anopheles (Kerteszia) mosquitoes associated with bromeliad-malaria transmission in a changing world.

Kerteszia is a neotropical subgenus of Anopheles composed of 12 species. The species in this subgenus are strongly associated with humid forests rich in epiphytic bromeliads. Forest fragmentation and anthropogenic changes can therefore have a negative impact on the abundance and survival of these mosquito species. Within this subgenus, four species are considered primary vectors of malaria: An. cruzii, An. bellator, An. homunculus and An. neivai. Malaria cases associated with Kerteszia species are often referred to as bromeliad malaria, a type of malaria reported to be endemic in the coastal rainforest of the neotropical region since the end of the nineteenth century. Although the incidence of bromeliad-malaria cases has decreased since the middle of the last century, autochthonous malaria cases continue to be registered every year. The complexity of the epidemiology of bromeliad malaria appears to be increasing as asymptomatic plasmodial infections and transmission of simian Plasmodium to humans have recently been reported. Kerteszia vector species have a great affinity for human beings and can be found in human-modified areas close to forest fragments such as in the Extra-Amazonian region of Brazil, Colombian pacific coast, and the Caribbean coast. Deforestation and forest fragmentation have been occurring continuously in the biomes of the neotropical region, and findings of Kerteszia species in dwellings are frequent in this region. Controlling the species in the Kerteszia subgenus is particularly difficult because they move frequently from natural to rural and peri-urban areas in search of blood sources, posing a challenge for the development of control strategies based on integrated vector management. Furthermore, as it has been shown that some Kerteszia species share similar morphological and genetic characteristics, the existence of a species complex formed by cryptic, sibling species within the Kerteszia group in different areas in the South and Southeast of Brazil cannot be ruled out. The existence of such a complex could represent an obstacle to the control of Kerteszia species and consequently to the elimination of bromeliad-malaria transmission in these regions. Here, we review publications that focus on the biology and ecology of Kerteszia malaria vectors and their association with human-modified areas and bromeliad-malaria transmission.

Wing geometric morphometrics as a tool for the Identification of culex subgenus mosquitoes of culex (Diptera: Culicidae)

Culex is the largest subgenus within the genus Culex that includes important vectors of diseases. The correct identification of mosquitoes is critical for effective control strategies. Wing geometric morphometrics (WGM) has been used to identify mosquito species alongside traditional identification methods. Here, WGM was used for eleven Culex species from São Paulo, Brazil, and one from Esquel, Argentina. Adult mosquitoes were collected using CDC (Centers for Disease Control) traps, morphologically identified and analyzed by WGM. The canonical variate analysis (CVA) was performed and a Neighbor-joining (NJ) tree was constructed to illustrate the patterns of species segregation. A cross-validated reclassification test was also carried out. From 110 comparisons in the cross-validated reclassification test, 87 yielded values higher than 70%, with 13 comparisons yielding 100% reclassification scores. Culexquinquefasciatus yielded the highest reclassification scores among the analyzed species, corroborating with the results obtained by the CVA, in which Cx. quinquefasciatus was the most distinct species. The high values obtained at the cross-validated reclassification test and in the NJ analysis as well as the segregation observed at the CVA made it possible to distinguish among Culex species with high degrees of confidence, suggesting that WGM is a reliable tool to identify Culex species of the subgenus Culex.

Diversidade genética e fenotípica do vetor de malária Anopheles cruzii (Diptera: Culicidae) em São Paulo, Brasil / Genetic and phenotypic diversity in the malaria vector Anopheles cruzii (Diptera: Culicidae) in São Paulo, Brazil

Mudanças antropogênicas no ambiente podem ser um fator poderoso na evolução das espécies. A fragmentação de áreas naturais devido às interferências humanas é associada à diminuição na riqueza e aumento na abundância de espécies de mosquitos adaptadas a ambientes urbanos. A Mata Atlântica brasileira tem passado por um processo intenso de fragmentação e desmatamento devido às alterações antropogênicas no ambiente. Esse bioma é um importante local de transmissão de malária, no qual o principal vetor de patógenos que causam a malária humana e simiana é o Anopheles cruzii. Desse modo, é possível que alterações antropogênicas no meio ambiente possam modular o fenótipo e genótipo de An. cruzii. Neste estudo, foram usados a geometria alar e marcadores moleculares SNPs para acessar a estrutura micro-geográfica de populações de An. cruzii coletadas em uma área hipoendêmica de malária na cidade de São Paulo, Brasil. Nesta região, as modificações antropogênicas suprimem a Mata Atlântica a fragmentos, fazendo com que An. cruzii, Plasmodium e seres humanos vivam em simpatria. Diferentes hipóteses para ambos os marcadores foram usadas em um desenho de estudo transversal e longitudinal para acessar se estruturação simpátrica e processos microevolutivos estão modulando as populações de An. cruzii. No geral, as hipóteses testadas foram divididas em: há estrutura entre populações de An. cruzii de copa e solo, há estrutura entre populações de An. cruzii em áreas com diferentes níveis de modificações antropogênicas e há estrutura populacional em relação ao tempo em An. cruzii. Portanto, foram investigadas a variação alar e genética em populações de An. cruzii coletadas em áreas silvestre, periurbana e urbana durante um período de três anos. Os principais resultados da análise de geometria da asa mostraram que as populações de copa e solo do ambiente urbano são distintas de acordo com a análise de variáveis canônicas. Os resultados também mostraram uma sutil, mas significativa variação fenotípica nas populações das três áreas durante os três anos de estudo. As variações temporal e simpátrica do formato alar foram maiores no ambiente urbano, sugerindo que mudanças antropogênicas no ambiente podem modular a variação no formato alar de An. cruzii. Os resultados da análise de 1.235 SNPs considerados neutros e independentes mostraram baixa estruturação genética entre as populações de An. cruzii no geral. Porém, as análises sugeriram que as populações de copa e solo da área urbana são significativamente estruturadas. A população do solo da área urbana apresentou maior diversidade genética que a de copa, corroborando os resultados da morfometria alar. Esses resultados indicam que modificações antropogênicas no ambiente levando à fragmentação de áreas naturais podem estar modulando a acrodendrofilia de An. cruzii e mantendo estrutura e diversidade genética na população de solo da área urbana.

Anthropogenic changes in the environment can be a powerfull aspect in the evolution of the species. Fragmentation of natural areas due to human interference has been associated with the decrease of species richness and increase in the abundance of species that are adapted to these environments. The Brazilian Atlantic Forest has been undergoing an intense process of fragmentation and deforestation due to anthropogenic alterations in the environment. This biome is an important hotspot of malaria transmission, in which the main vector of simian and human malaria is the mosquito Anopheles cruzii. We hypothesized that anthropogenic alterations in the environment are an important driver for the phenotypic and genetic structure and diversity of An. cruzii. We tested five different hypotheses using both a cross-sectional and longitudinal design to assess the sympatric structure and microevolution process driving An. cruzii populations. We used both wing geometry and SNPs to assess the microgeographical genetic structure of An. cruzii populations in a malaria hypo-endemic area in the city of São Paulo, Brazil. In this region, anthropogenic modifications in the environment suppress the Atlantic Forest into fragments, resulting in mosquitoes An. cruzii, Plasmodium and humans living in simpatry. Our results show a slight but significant phenotypic variation in all three populations over the study period. Time was a more powerful driver for wing variation than geographic distance. Temporal wing-shape variation appears to be positively associated with urbanization, suggesting that anthropogenic changes in the environment may be a strong driver for wing-shape variation in An. cruzii. After filtering and removal of outlier loci, 1,235 SNPs were considered independent and neutral, and therefore, suitable to perform the analyses. The results showed an overall weak genetic structure among populations with a significant sympatric structure between ground and tree canopies populations from the urban area, with the population from ground level showing higher genetic diversity. Our findings indicate that anthropogenic modifications leading to habitat fragmentation could be driving the acrodendrophily of An. cruzii and maintaining genetic diversity and structure in populations from ground level.

Population structuring of the invasive mosquito Aedes albopictus (Diptera: Culicidae) on a microgeographic scale.

Aedes albopictus is an invasive mosquito species that has spread globally and can transmit several arboviruses, including dengue, chikungunya and yellow fever. The species was first reported in Brazil in 1986 and since then has been found in 24 of the 27 Brazilian states, often in peri-urban environments close to highly urbanized areas. To date, population genetics of this important mosquito in areas in the city of São Paulo has not been investigated. In this study, we used 12 microsatellite loci to investigate the microgeographic population genetics of Ae. albopictus, which is present throughout the city of São Paulo. All the analyses revealed structuring of the populations studied, divided into two groups with restricted gene flow between them and without evidence of isolation by distance. We propose two hypotheses to explain the results: (i) low dispersal capability-limited gene flow between populations is due to the low dispersal capability inherent to Ae. albopictus; and (ii) multiple introductions-the structure identified here results from multiple introductions, which led to different dispersal patterns within the city and more genetic heterogeneity. The ability of Ae. albopictus to invade new areas and expand may explain why these mosquito populations appear to be well established and thriving in the city of São Paulo.

Urbanization as a driver for temporal wing-shape variation in Anopheles cruzii (Diptera: Culicidae).

Anopheles cruzii is the main vector of human and simian malaria in the Brazilian Atlantic Forest. This biome, which is an important hotspot of malaria transmission, has suffered fragmentation and deforestation as a result of urban expansion. Fragmentation and deforestation occur continually in the south of the city of São Paulo, Brazil, and findings of An. cruzii in the peridomicile have consequently become more frequent in this part of the city. Although An. cruzii is of considerable epidemiological importance, the impact of urbanization on the microevolution of this species in this malaria-endemic region has not been investigated to date. In this study, we investigated temporal variation in wing shape and size in An. cruzii populations collected in sylvatic, peri-urban and urban areas over a three-year period. Our results show a slight but significant phenotypic variation in all three populations over the study period. Time was a more powerful driver for wing variation than geographic distance. Temporal wing-shape variation appears to be positively associated with urbanization, suggesting that anthropogenic changes in the environment may be a strong driver for wing-shape variation in An. cruzii. Further studies using genetic markers are needed to assess genetic differentiation in these populations.

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.

Mosquito Population Diversity and Abundance Patterns In Two Parks In São Paulo, Brazil.

Temperature and rainfall are important drivers of mosquito abundance and have been used in previous studies as the basis for predictive models. To elucidate patterns of mosquito population dynamics in urban environments, the variation in mosquito abundance over a year and its association with climatic variables were analyzed. The Akaike information criterion was used to analyze the correlations between abundance and climate variables in mosquito populations collected in 2 urban parks: Alfredo Volpi Park and Burle Marx Park. Our findings suggest that both climatic and density-dependent variations may have an important impact on fluctuations in mosquito abundance, modulating population dynamics in urban parks.

Composition and diversity of mosquitoes (Diptera: Culicidae) in urban parks in the South region of the city of São Paulo, Brazil / Composição e diversidade de mosquitos (Diptera: Culicidae) em parques urbanos localizados na região Sul do município de São Paulo, Brasil

Abstract Many parks in the city of São Paulo contain remnants of Atlantic Forest. Of the 30 municipal parks in the South of the city, we investigated two in this study (Santo Dias Park and Shangrilá Park) in order to survey their mosquito fauna and investigate the presence of potential bioindicators of environmental conditions and vectors of human pathogens. Mosquitoes were collected monthly between March 2011 and February 2012 using aspirators, Shannon and CDC traps for adult mosquitoes and larval dippers and suction samplers for immature forms. Sampling effort was evaluated by plotting a species accumulation curve, and total richness was estimated using the first-order jackknife. To compare the diversity between the two parks Shannon and Simpson diversity indexes were calculated. Species similarity was compared by the Sorensen similarity index. In all, 8,850 specimens were sampled in both parks. Collections in Santo Dias Park yielded 1,577 adult mosquitoes and 658 immature individuals distributed in seven genera (Aedes, Anopheles, Culex, Limatus, Mansonia, Toxorhynchites and Wyeomyia) and 27 taxonomic units. Among the adult mosquitoes collected, Culex nigripalpus .and Aedes fluviatilis were the most abundant, while the most abundant immature forms were Cx. imitator, Wy. davisi, Wy. galvaoi and Ae. albopictus. Collections in Shangrilá Park yielded 4,952 adult specimens and 1,663 immature forms distributed in eight genera (Aedes, Anopheles, Culex, Limatus, Mansonia, Toxorhynchites, Uranotaenia and Wyeomyia) and 36 taxonomic units. Species accumulation curves in both parks were close to the asymptote, and the total richness estimate was close to the observed richness. Although the observed species richness was higher in the Shangrilá Park, there was no statistically significant difference between the diversity indexes measured. Regarding species composition, the two sites shared 16 species, including those of epidemiological importance such as Culex nigripalpus, Cx. quinquefasciatus, Aedes albopictus and Ae. aegypti. As some of the mosquito taxa found are bioindicators of environmental conditions and have epidemiological potential to carry pathogens, we recommend that urban parks should be included in official mosquito surveillance programs, and regular surveys carried out to detect circulating arboviruses.

Resumo Parques urbanos do município de São Paulo contêm remanescentes de Mata Atlântica. No sul da cidade há 30 parques municipais, sendo os parques Santo Dias e Shangrila alvos deste estudo. Este estudo teve a proposta de levantamento da fauna de culicídeos desses dois parques no sul da cidade de São Paulo e avaliar a presença de potenciais bioindicadores e espécies vetoras de patógenos aos seres humanos. Os mosquitos foram coletados mensalmente entre março de 2011 e fevereiro de 2012, com aspiradores, armadilhas de Shannon e CDCs para mosquitos adultos e conchas entomológicas e bombas manuais de sucção para os imaturos. O esforço amostral foi avaliado por traçar uma curva de acumulação de espécies, e a riqueza total foi estimada pelo método jackknife de primeira ordem. Para comparar a diversidade entre os dois parques, foram calculados os índices de diversidade de Shannon e de Simpson. A similaridade na composição de espécies foi comparada pelo índice de similaridade de Sorensen. Foram coletados um total de 8.850 espécimes de culicídeos em ambos os parques. Coletas no parque Santo Dias renderam 1.577 mosquitos adultos e 658 imaturos, distribuídos em sete gêneros (Aedes, Anopheles, Culex, Limatus, Mansonia, Toxorhynchites e Wyeomyia) e 27 unidades taxonômicas: Culex nigripalpus, e Aedes fluviatilis foram as mais abundantes unidades taxonômicas coletadas como adultos, enquanto em formas imaturas, as espécies mais abundantes coletadas foram Cx. imitator, Wy. davisi, Wy. galvaoi e Ae. albopictus. Coletas no parque Shangrilá renderam 4.952 espécimes como adultos e 1.663 formas imaturas, distribuídas em oito gêneros (Aedes, Anopheles, Culex, Limatus, Mansonia, Toxorhynchites, Uranotaenia e Wyeomyia) e 36 unidades taxonômicas.. As curvas de acúmulo de espécies em ambos os parques ficaram perto da assíntota, e as estimativas de riqueza total foram próximas às riquezas observadas. Apesar da riqueza observada ter sido maior no parque Shangrilá, não houve diferença estatisticamente significante entre os índices de diversidade mensurados. Em relação à composição de espécies os dois locais compartilharam 16 espécies, incluindo as de maior importância epidemiológica como Culex nigripalpus, Cx. quinquefasciatus, Aedes albopictus e Ae. aegypti. Alguns táxons de culicídeos são bioindicadores de condições ambientais nas áreas ou possuem potencial para veicular patógenos. Atenção deve ser dada a parques urbanos, com inclusão destes locais nos programas oficiais de vigilância entomológica e investigações periódicas na circulação de arbovírus.

Population Genetic Structure of Aedes fluviatilis (Diptera: Culicidae).

Although Aedes fluviatilis is an anthropophilic mosquito found abundantly in urban environments, its biology, epidemiological potential and genetic characteristics are poorly understood. Climate change and urbanization processes that result in environmental modifications benefit certain anthropophilic mosquito species such as Ae. fluviatilis, greatly increasing their abundance in urban areas. To gain a better understanding of whether urbanization processes modulate the genetic structure of this species in the city of São Paulo, we used eight microsatellite loci to genetically characterize Ae. fluviatilis populations collected in nine urban parks in the city of São Paulo. Our results show that there is high gene flow among the populations of this species, heterozygosity deficiency and low genetic structure and that the species may have undergone a recent population expansion. There are two main hypotheses to explain these findings: (i) Ae. fluviatilis populations have undergone a population expansion as a result of urbanization; and (ii) as urbanization of the city of São Paulo occurred recently and was quite intense, the structuring of these populations cannot be observed yet, apart from in the populations of Ibirapuera and Piqueri parks, where the first signs of structuring have appeared. We believe that the expansion found in Ae. fluviatilis populations is probably correlated with the unplanned urbanization of the city of São Paulo, which transformed green areas into urbanized areas, as well as the increasing population density in the city.

Morphometric Wing Characters as a Tool for Mosquito Identification.

Mosquitoes are responsible for the transmission of important infectious diseases, causing millions of deaths every year and endangering approximately 3 billion people around the world. As such, precise identification of mosquito species is crucial for an understanding of epidemiological patterns of disease transmission. Currently, the most common method of mosquito identification relies on morphological taxonomic keys, which do not always distinguish cryptic species. However, wing geometric morphometrics is a promising tool for the identification of vector mosquitoes, sibling and cryptic species included. This study therefore sought to accurately identify mosquito species from the three most epidemiologically important mosquito genera using wing morphometrics. Twelve mosquito species from three epidemiologically important genera (Aedes, Anopheles and Culex) were collected and identified by taxonomic keys. Next, the right wing of each adult female mosquito was removed and photographed, and the coordinates of eighteen digitized landmarks at the intersections of wing veins were collected. The allometric influence was assessed, and canonical variate analysis and thin-plate splines were used for species identification. Cross-validated reclassification tests were performed for each individual, and a Neighbor Joining tree was constructed to illustrate species segregation patterns. The analyses were carried out and the graphs plotted with TpsUtil 1.29, TpsRelw 1.39, MorphoJ 1.02 and Past 2.17c. Canonical variate analysis for Aedes, Anopheles and Culex genera showed three clear clusters in morphospace, correctly distinguishing the three mosquito genera, and pairwise cross-validated reclassification resulted in at least 99% accuracy; subgenera were also identified correctly with a mean accuracy of 96%, and in 88 of the 132 possible comparisons, species were identified with 100% accuracy after the data was subjected to reclassification. Our results showed that Aedes, Culex and Anopheles were correctly distinguished by wing shape. For the lower hierarchical levels (subgenera and species), wing geometric morphometrics was also efficient, resulting in high reclassification scores.

Microsatellite loci cross-species transferability in Aedes fluviatilis (Diptera:Culicidae): a cost-effective approach for population genetics studies.

BACKGROUND: Aedes fluviatilis is a neotropical mosquito species thought to be a potential vector of Yellow Fever viruses and can be infected with Plasmodium gallinaceum in laboratory. A better understanding of its genetic structure is very important to understand its epidemiologic potential and how it is responding to urbanization. The objective of this study was to survey the transferability of microsatellites loci developed for other Aedes to Ae. fluviatilis. FINDINGS: We tested in Ae. fluviatilis 40 pairs of primers known to flank microsatellite regions in Aedes aegypti, Aedes albopictus and Aedes caspius, and found eight loci that amplified consistently. The number of alleles per locus ranged from 2 to 15, and the expected heterozygosity ranged from 0.09 to 0.85. CONCLUSIONS: We found that several microsatellite primers successfully transferred to Ae. fluviatilis. This finding opens avenues for cost-effective optimization of high-resolution population genetic tools.