Assessment of wing geometric morphometrics of urban Culex quinquefasciatus (Diptera: Culicidae) populations.

Culex quinquefasciatus is a cosmopolitan species distributed throughout tropical and subtropical areas of the world. The species is of great epidemiological importance as it is responsible for vectoring the causative agent of lymphatic filariasis and several arboviruses, including West Nile virus. Wing geometric morphometrics has been widely used to assess phenotypic variations in mosquito species. Here, we hypothesize that Cx. quinquefasciatus populations in urban parks in the city of São Paulo, Brazil, have been subjected to anthropogenic selective pressures that are responsible for driving their ecology and behavior. Mosquitoes were collected by CDC traps in five municipal parks in the city of São Paulo. Eighteen anatomical landmark coordinates on each female right wing were digitized. Canonical variate analysis, wireframe graphs, cross-validated reclassification tests and the neighbor-joining method were used to assess phenotypical dissimilarity in wing shape between populations. Centroid size was calculated to assess differences in wing size between populations, which can result from different environmental conditions during immature mosquito development. Moderately heterogeneous wing shape and wing size patterns were found in the populations analyzed, indicating that selective pressures in the urban environment are affecting the wing patterns of Cx. quinquefasciatus populations in the city of São Paulo, Brazil.

Relationship between vertical stratification and feeding habits of mosquito (Diptera: Culicidae) assemblages collected in conservation units in the green belt of the city of São Paulo, Brazil.

São Paulo is one of the largest cities in the world and has several characteristics that favor a diversity of urban and wild mosquitoes. Little is known about how variations in mosquito diversity and feeding preferences for different hosts in different vegetation strata can influence the risk of pathogen transmission to humans. We investigated vertical stratification of mosquitoes and its relationship with vertebrate hosts in environments with different degrees of conservation in two conservation units in the city of São Paulo. Adult mosquitoes were collected using CDC traps, aspiration and Shannon traps. After morphological identification, host blood in engorged females was analyzed by PCR with a vertebrate-specific primer set based on mitochondrial cytochrome b DNA of vertebrates commonly found in the two conservation units. Although a higher abundance of the species Anopheles cruzii and Culex nigripalpus was found in the canopy, blood not only from birds but also from humans and rodents was identified in these mosquitoes. In one of the units, Wyeomyia confusa and Limatus durhamii were found occupying mainly niches at ground level while Culex vaxus was frequently found in the canopy. Haemagogus leucocelaenus, the main vector of yellow fever, was found in low abundance at all collection points, particularly in the canopy. Species richness and composition tended to vary little between canopy and ground level in the same environment, but the abundance between canopy and ground level varied more depending on the species analyzed, the most abundant and frequent species exhibiting a predilection for the canopy. Even those mosquito species observed more frequently in the canopy did not show an association with hosts found in this stratum as most of the blood identified in these species was from humans, suggesting opportunist feeding behavior, i.e., feeding on the most readily available host in the environment. The two most common species in the study, An. cruzii and Cx. nigripalpus, may be able to act as bridge vectors for pathogens to circulate between the forest canopy and ground level.

Retention of ancestral polymorphism in Culex nigripalpus (Diptera: Culicidae) from São Paulo, Brazil.

Culex nigripalpus Theobald (Diptera: Culicidae) is a native species of Brazil that is well adapted to urban environments and found extensively in the city of São Paulo, Brazil. As a native species, it has been present in this region since long before the foundation of the city, but over time Cx. nigripalpus populations have been affected by man-made changes to the environment. We hypothesize that the populations analyzed in this study constituted a large Cx. nigripalpus population that separated into smaller populations as a result of increased levels of urbanization in the city, and that such high levels of urbanization would result in a genetic homogenization effect. We therefore investigated the microgeographic genetic structure and microevolutionary processes in Cx. nigripalpus populations from seven different locations in the city of São Paulo using a set of six microsatellite primers originally developed for Culex quinquefasciatus and Culex pipiens. Our results indicate that Cx. nigripalpus did not benefit from urbanization and is currently under selective pressures caused by anthropogenic changes and that populations from areas with higher levels of urbanization exhibited similar genetic patterns and low levels of polymorphism, contrasting with the more sylvatic SHA population. These findings are likely to contribute to a better understanding of how anthropogenic selective pressures are driving population genetics and, to some extent, the dynamics of Cx. nigripalpus populations. They should also help elucidate the effects that urbanization processes have on the ecology and behavior of these mosquito populations.

Microsatellite Markers Transferability in Culex nigripalpus (Diptera: Culicidae).

Microsatellite loci specifically designed and optimized for secondary vectors and neglected species of mosquitoes are lacking. Cross-species transferability of microsatellite loci between close taxa has been used as a reliable option, being successfully done for Aedes taeniorhynchus Wiedemann (Diptera: Culicidae), Triatoma sordida Stål (Hemiptera: Reduviidae), and Aedes fluviatilis Lutz (Diptera: Culicidae). Nonetheless, prior to the utilization of transferred microsatellites, they must be assessed for their performance and reliability on the target species. Therefore, the goal of this study was to transfer and validate SSR primers previously developed for the Culex pipiens complex to Culex (Culex) nigripalpus Theobald (Diptera: Culicidae). Culex nigripalpus were collected in seven different locations throughout the city of São Paulo using CDC traps baited with dry ice between 2011 and 2013, yielding a total of 223 specimens. Six SSR primers were successfully transferred to Cx. nigripalpus yielding satisfactory results for allele richness, ranging from 5 to 12 alleles per locus. The expected heterozygosity values were higher than the observed heterozygosity (P < 0.05) for the loci CxqCTG10, CxqGT51, and Cxpq59. Linkage disequilibrium results were statistically significant only for the comparison between the loci CxqCA115 and CxqCTG10, and the estimation for the probability of null alleles was also low, ranging from 0 to 0.052. These results substantially validate the SSRs transference to Cx. nigripalpus as well as their subsequent use in microevolution studies comprising this species.

Microgeographic Population Structuring of Culex quinquefasciatus (Diptera: Culicidae) From São Paulo, Brazil.

The continuing worldwide increase in urbanization can potentially have a major impact on the epidemiology of vector-borne diseases, as anthropogenic changes to the environment are known to favor a few species of mosquitoes that can thrive in urban environments. Culex quinquefasciatus Say (Diptera: Culicidae) is found extensively in urban habitats, where it lives in degraded, polluted areas and is often the only species of mosquito capable to surviving under such conditions. Traditional mosquito control strategies no longer have the desired effect due to the several factors such as insecticide resistance, abundance of breeding sites, lack of proper sewage and sanitation, and absence of natural predator, leading Cx. quinquefasciatus populations to increase its numbers in cities. In this study, five Cx. quinquefasciatus populations were analyzed using 12 microsatellite markers to investigate whether the dynamics of these populations are being modulated by urbanization and how they are structured in the city of São Paulo, Brazil. Our results indicate that only one of the study populations (the population from Anhanguera Park) exhibited evidence of expansion. The populations from Ibirapuera Park and Piqueri Park, the most urbanized regions of the areas studied, did not show signs of expansion. A better understanding of the mechanisms involved in the dispersal of Cx. quinquefasciatus and its colonization of new areas, as well as the species' demographic patterns and how these are associated with urbanization, particularly in areas undergoing a rural-to-urban transformation, such as Anhanguera Park, is of great importance for mosquito control.

Wing variation in Culex nigripalpus (Diptera: Culicidae) in urban parks.

BACKGROUND: Culex nigripalpus has a wide geographical distribution and is found in North and South America. Females are considered primary vectors for several arboviruses, including Saint Louis encephalitis virus, Venezuelan equine encephalitis virus and Eastern equine encephalitis virus, as well as a potential vector of West Nile virus. In view of the epidemiological importance of this mosquito and its high abundance, this study sought to investigate wing variation in Cx. nigripalpus populations from urban parks in the city of São Paulo, Brazil. METHODS: Female mosquitoes were collected in seven urban parks in the city of São Paulo between 2011 and 2013. Eighteen landmark coordinates from the right wing of each female mosquito were digitized, and the dissimilarities between populations were assessed by canonical variate analysis and cross-validated reclassification and by constructing a Neighbor-Joining (NJ) tree based on Mahalanobis distances. The centroid size was calculated to determine mean wing size in each population. RESULTS: Canonical variate analysis based on fixed landmarks of the wing revealed a pattern of segregation between urban and sylvatic Cx. nigripalpus, a similar result to that revealed by the NJ tree topology, in which the population from Shangrilá Park segregated into a distinct branch separate from the other more urban populations. CONCLUSION: Environmental heterogeneity may be affecting the wing shape variation of Cx. nigripalpus populations.

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.

Species Composition and Ecological Aspects of Immature Mosquitoes (Diptera: Culicidae) in Bromeliads in Urban Parks in the City of São Paulo, Brazil.

BACKGROUND: Bromeliads can be epiphytic, terrestrial or saxicolous and use strategies to allow water to be retained in their leaf axils, where various arthropods can be found. These include mosquitoes, whose larvae are the most abundant and commonly found organisms in the leaf axils. The objective of this study was to look for immature forms of mosquitoes (the larval and pupal stages) in bromeliads in municipal parks in São Paulo and to discuss the ecological and epidemiological importance of these insects. METHODS: From October 2010 to July 2013, immature mosquitoes were collected from bromeliads in 65 municipal parks in the city of São Paulo, Brazil, using suction samplers. The immature forms were maintained until adult forms emerged, and these were then identified morphologically. RESULTS: Two thousand forty-two immature-stage specimens belonging to the genera Aedes, Culex, Trichoprosopon, Toxorhynchites, Limatus and Wyeomyia were found in bromeliads in 15 of the 65 parks visited. Aedes albopictus was the most abundant species (660 specimens collected), followed by Culex quinquefasciatus (548 specimens) and Cx. (Microculex) imitator (444). The taxa with the most widespread distribution were Ae. aegypti and Toxorhynchites spp, followed by Ae. albopictus and Cx. quinquefasciatus. CONCLUSION: Bromeliads in urban parks are refuges for populations of native species of Culicidae and breeding sites for exotic species that are generally of epidemiological interest. Hence, administrators and surveillance and mosquito-control agencies must constantly monitor these microenvironments as the presence of these species endangers the health of park users and employees as well as people living near the parks.

Blood meal sources of mosquitoes captured in municipal parks in São Paulo, Brazil.

The aim of this study was to investigate blood meal sources of mosquitoes captured in municipal parks in the city of São Paulo, Brazil, and to identify possible associations between mosquito species and their food preferences. Fourteen species of blood hosts of 510 engorged adult female mosquitoes were identified using PCR assays with a vertebrate-specific primer set based on cytochrome b mitochondrial DNA of the following vertebrates: birds, dogs, cats, rodents, humans, and other primates. Mosquitoes were captured using a manual aspirator, CDC traps in the canopy, CDC traps at ground level, and Shannon traps. With the exception of cats, all other vertebrates were used as hosts by mosquitoes in the parks. Statistical analysis failed to show any trend toward association between most culicid species captured and the sources of blood meals. Instead, they revealed random patterns, indicating that the mosquitoes fed on the most abundant or convenient blood meal sources. Although feeding preferences were observed in two species (birds in the case of Cx. nigripalpus and dogs in the case of Cx. quinquefasciatus), our results highlight the opportunistic feeding habits of the female mosquitoes in this study.

Mosquito fauna in municipal parks of São Paulo City, Brazil: a preliminary survey.

A mosquito faunal survey was conducted from October 2010 to February 2011 in the municipal parks of São Paulo City, Brazil. A total of 7,015 specimens of 53 taxonomic categories grouped into 12 genera (Aedes, Anopheles, Coquilletidia, Culex, Limatus, Lutzia, Mansonia, Psorophora, Toxorhynchites, Trichoprosopon, Uranotaenia, and Wyeomyia) were collected. The largest and most peripheral parks showed greater species richness compared to smaller and more centralized parks.