Altitudinal population structure and microevolution of the malaria vector Anopheles cruzii (Diptera: Culicidae).

BACKGROUND: In Brazil, the autochthonous transmission of extra-Amazonian malaria occurs mainly in areas of the southeastern coastal Atlantic Forest, where Anopheles cruzii is the primary vector. In these locations, the population density of the mosquito varies with altitude (5-263 m above sea level), prompting us to hypothesise that gene flow is also unevenly distributed. Describing the micro-geographical and temporal biological variability of this species may be a key to understanding the dispersion of malaria in the region. We explored the homogeneity of the An. cruzii population across its altitudinal range of distribution using wing shape and mtDNA gene analysis. We also assessed the stability of wing geometry over time. METHODS: Larvae were sampled from lowland (5-20 m) and hilltop (81-263 m) areas in a primary Atlantic Forest region, in the municipality of Cananéia (State of São Paulo, Brazil). The right wings of males and females were analysed by standard geometric morphometrics. Eighteen landmarks were digitised for each individual and a discriminant analysis was used to compare samples from the hilltop and lowland. A 400-bp DNA fragment of the mitochondrial cytochrome oxidase gene subunit I (CO-I) was PCR-amplified and sequenced. RESULTS: Wing shapes were distinct between lowland and hilltop population samples. Results of cross-validated tests based on Mahalanobis distances showed that the individuals from both micro-environments were correctly reclassified in a range of 54-96%. The wings of hilltop individuals were larger. The CO-I gene was highly polymorphic (haplotypic diversity = 0.98) and altitudinally structured (Фst = 0.085 and Jaccard = 0.033). We found 60 different haplotypes but only two were shared by the lowland and hilltop populations. Wing shape changed over the brief study period (2009-2013). CONCLUSIONS: Wing geometry and CO-I gene analysis indicated that An. cruzii is vertically structured. Wing shape varied rapidly, but altitude structure was maintained. Future investigations should identify the biotic/abiotic causes of these patterns and their implications in the local epidemiology of malaria.

Morphometrical diagnosis of the malaria vectors Anopheles cruzii, An. homunculus and An. bellator.

BACKGROUND: Anopheles (Kerteszia) cruzii is a primary vector of Plasmodium parasites in Brazil's Atlantic Forest. Adult females of An. cruzii and An. homunculus, which is a secondary malaria vector, are morphologically similar and difficult to distinguish when using external morphological characteristics only. These two species may occur syntopically with An. bellator, which is also a potential vector of Plasmodium species and is morphologically similar to An. cruzii and An. homunculus. Identification of these species based on female specimens is often jeopardised by polymorphisms, overlapping morphological characteristics and damage caused to specimens during collection. Wing geometric morphometrics has been used to distinguish several insect species; however, this economical and powerful tool has not been applied to Kerteszia species. Our objective was to assess wing geometry to distinguish An. cruzii, An. homunculus and An. bellator. METHODS: Specimens were collected in an area in the Serra do Mar hotspot biodiversity corridor of the Atlantic Forest biome (Cananeia municipality, State of Sao Paulo, Brazil). The right wings of females of An. cruzii (n= 40), An. homunculus (n= 50) and An. bellator (n= 27) were photographed. For each individual, 18 wing landmarks were subjected to standard geometric morphometrics. Discriminant analysis of Procrustean coordinates was performed to quantify wing shape variation. RESULTS: Individuals clustered into three distinct groups according to species with a slight overlap between representatives of An. cruzii and An. homunculus. The Mahalanobis distance between An. cruzii and An. homunculus was consistently lower (3.50) than that between An. cruzii and An. bellator (4.58) or An. homunculus and An. bellator (4.32). Pairwise cross-validated reclassification showed that geometric morphometrics is an effective analytical method to distinguish between An. bellator, An. cruzii and An. homunculus with a reliability rate varying between 78-88%. Shape analysis revealed that the wings of An. homunculus are narrower than those of An. cruzii and that An. bellator is different from both of the congeneric species. CONCLUSION: It is possible to distinguish among the vectors An. cruzii, An. homunculus and An. bellator based on female wing characteristics.

Culicídeos associados às bromélias na Mata Atlântica do município de Cananéia, São Paulo, Brasil / Bromeliad associated mosquitoes from Atlantic Forest, in Cananéia, São Paulo, Brasil

A Mata Atlântica não possui fisionomia uniforme, pois seu relevo determina diferentes condições ambientais que definem a composição de sua fauna e flora. Em uma mesma região, a diversidade de espécies pode variar de acordo com a heterogeneidade dos habitats. Objetivo: Caracterizar a fauna de Culicidae em fitotelmatas de bromélias (Bromeliaceae), no município de Cananéia, São Paulo, Brasil. Métodos: Larvas e pupas de culicídeos foram obtidas, mensalmente, do conteúdo aquático de bromélias de três ambientes: planície, encosta e morro. As coletas ocorreram entre julho de 2008 e junho de 2009. Para estimar a variedade de espécies foram utilizados os índices de Margalef e de Menhinick. O índice de Jaccard e o de Sorensen indicaram a semelhança entre bromélias de dois ambientes. Para avaliar a dominância, empregou-se o índice de Simpson e de Berger-Parker. A equidade na distribuição das espécies foi avaliada pelo índice de Pielou e a constância em cada ambiente pelo índice c. Os testes estatísticos empregados foram: análise de variância (ANOVA) com o teste de Fisher; teste Kruskal-Wallis e valor p; teste de associação qui-quadrado e regressão linear simples por meio do coeficiente de correlação de Pearson. Resultados: O teste Kruskal-Wallis mostrou diferenças significantes para o volume hídrico das bromélias de cada ambiente (p=0,002, IC= 95 por cento) mas não para o valor do pH (p= 0,775). Foram identificados 2024 mosquitos, 786 (38,83 por cento) na planície, 606 (29,94 por cento) na encosta e 632 (31,23 por cento) no morro, dentre eles: Culex (Microculex) (62,25 por cento), Culex ocellatus (21,20 por cento), Anopheles (Kerteszia) (15,17 por cento), Wyemoyia (Phoniomyia) (1,33 por cento) e Runchomyia (Runchomyia) (0,05 por cento). O índice de riqueza de Margalef foi maior na planície (d1=2,55) e menor no morro (d1=1,86), e o de Sorensen indicou encosta e morro como os ambientes mais similares (QS=0,79) e planície e morro como os menos similares (QS=0,65)...

Atlantic Forest is not uniform and its relief determines different environmental conditions that define the composition of its fauna and flora. Thus, in the same region, species diversity may vary according to habitat heterogeneity. Objective: To assess mosquito (Culicidae) fauna in bromeliads phytotelmate (Bromeliaceae), in Cananéia, São Paulo, Brazil. Methods: The larvae and pupae of mosquitoes were collected monthly in the water content of bromeliads from three regions: the lowland, hillside and hilltop. Sampling occurred between July 2008 and June 2009. To estimate richness, Margalef and Menhinick indices were used. Jaccard and Sorensen indices indicated the similarity between bromeliads from two regions. To assess dominance, we employed Simpson and Berger-Parker índices. Equity in the distribution of species was assessed by Pielou index and c index evaluated species constancy in each region. Statistical analyses applied were: ANOVA with Fisher's exact test, Kruskal-Wallis test and p value; Chi-square association test and linear regression using Pearson correlation coefficient. Results: Kruskal-Wallis test showed significant differences in water volume for bromeliads from each region (p=0,002, CI=95 per cent) but not for the pH value (p=0,775). In all, 2024 mosquitoes were identified, 786 (38,83 per cent) in the lowland, 606 (29,94 per cent) on the hillside and 632 (31,23 per cent) on the hilltop, among them: Culex (Microculex) (62,25 per cent), Culex ocellatus (21,20 per cent), Anopheles (Kerteszia) (15,17 per cent), Wyemoyia (Phoniomyia) (1,33 per cent) and Runchomyia (Runchomyia) (0,05 per cent). Margalef richness index was higher in the lowland (d1=2,55) and lower on the hilltop (d1=1,86). According to the Sorensen similarity index, species from the hillside are more likely to be found on the hilltop (QS=0,79) than in the lowland (QS=0,73). The greatest differences occurred between the hilltop and the lowland (QS=0,65). The 2 test showed...