Genetic Structuring of One of the Main Vectors of Sylvatic Yellow Fever: Haemagogus (Conopostegus) leucocelaenus (Diptera: Culicidae).

Genetic diversity and population structuring for the species Haemogogus leucocelaenus, a sylvatic vector of yellow fever virus, were found to vary with the degree of agricultural land use and isolation of fragments of Atlantic Forest in municipalities in the state of São Paulo where specimens were collected. Genotyping of 115 mitochondrial SNPs showed that the populations with the highest indices of genetic diversity (polymorphic loci and mean pairwise differences between the sequences) are found in areas with high levels of agricultural land use (northeast of the State). Most populations exhibited statistically significant negative values for the Tajima D and Fu FS neutrality tests, suggesting recent expansion. The results show an association between genetic diversity in this species and the degree of agricultural land use in the sampled sites, as well as signs of population expansion of this species in most areas, particularly those with the highest forest edge densities. A clear association between population structuring and the distance between the sampled fragments (isolation by distance) was observed: samples from a large fragment of Atlantic Forest extending along the coast of the state of São Paulo exhibited greater similarity with each other than with populations in the northwest of the state.

The role of forest fragmentation in yellow fever virus dispersal.

The intense process of deforestation in tropical forests poses serious challenges for the survival of biodiversity, as well as for the human species itself. This scenario is supported by the increase in the incidence of epidemics of zoonotic origin observed over the last few decades. In the specific case of sylvatic yellow fever (YF), it has already been shown that an increase in the transmission risk of the causative agent (yellow fever virus - YFV) is associated with areas with a high degree of forest fragmentation, which can facilitate the spread of the virus. In this study we tested the hypothesis that areas with more fragmented landscapes and a higher edge density (ED) but a high degree of connectivity between forest patches favor YFV spread. To this end, we used YF epizootics in non-human primates (NHPs) in the state of São Paulo to build direct networks, and used a multi-selection approach to analyze which landscape features could facilitate YFV spread. Our results showed that municipalities with the potential to spread the virus exhibited a higher amount of forest edge. Additionally, the models with greater empirical support showed a strong association between forest edge density and the risk of occurrence of epizootic diseases, as well as the need for a minimum threshold of native vegetation cover to restrict their transmission. These findings corroborate our hypothesis that more fragmented landscapes with a higher degree of connectivity favor the spread of YFV, while landscapes with fewer connections tend to act as dead zones for the circulation of the virus.

The influence of landscape structure on the dispersal pattern of yellow fever virus in the state of São Paulo.

Man-made changes to the landscape play a crucial role in altering the epidemiologic patterns of infectious diseases, mainly as a result of pathogen spillover. Sylvatic yellow fever is ideally suited to modeling of this phenomenon as the risk of transmission of the disease as well as its circulation and dispersal are associated with forest fragmentation. In this study we investigated the temporal dispersal pattern of yellow fever virus (YFV) by means of confirmed cases of epizootics in non-human primates in municipalities in the state of São Paulo where there was no recommendation for vaccination in 2017. We analyzed the resistance to dispersal associated with different classes of land use and the geographic distances between the different locations where epizootics were recorded. The model that best explained the temporal dispersal pattern of YFV in the study area indicated that this was influenced by the geographic distance between collection locations and by the permeability of the forest edges (150 m) at the interface with the following core areas: Water, Agricultural, Non-Forest Formation and Forestry. Water, Agricultural, Urban and Forest core areas and the interfaces between the latter two formed important barriers to circulation of the virus. These findings indicate that fragmentation of vegetation tends to decrease the time taken for pathogens to spread, while conservation of forest areas has the opposite effect.

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.

Diversity analysis and an updated list of mosquitoes (Diptera: Culicidae) found in Cantareira State Park, São Paulo, Brazil.

Cantareira State Park (CSP) is located in the Metropolitan Region of São Paulo, one of the most densely populated areas on the planet. Recently, a yellow-fever epidemic practically annihilated the howler monkey population in this park, and human infections were reported in the vicinity. As simian and human plasmodia also circulate in CSP, the present study sought to provide an update on the mosquito fauna in this park, including an analysis of the diversity in areas with different degrees of conservation and a comparison of the yields achieved with different collection techniques. From October 2015 to April 2017, adult mosquitoes were collected with CDC traps, hand-held battery-powered aspirators and Shannon traps, and larvae and pupae were collected with larval dippers and suction samplers in natural and artificial breeding sites. In total, 11,038 specimens distributed in 103 taxa represented by 16 genera were collected. Both the observed species richness and diversity were greater in the environments with the highest degree of preservation. The 'wild' (most preserved) area in CSP had the greatest species richness, followed by the transition area and human-impacted area. The estimated richness indicated that the three environments may have a greater number of species than observed in this study, and Sorensen's index showed that the average degree of similarity varies little between areas. In the inventory of local species, the Shannon trap was the most efficient collection technique for adult mosquitoes, and the suction sampler the most efficient for immatures. The results highlight the increase in the number of different taxa collected as different mosquito capture techniques were included, confirming the importance of using several strategies to sample the local mosquito fauna satisfactorily when exploring a greater number of ecotopes. CSP is a refuge and shelter for native and introduced mosquito species where new biocenoses including pathogens, vertebrate hosts and vectors can form, allowing zoonotic outbreaks in the local human population to occur.

Influence of landscape composition and configuration on the richness and abundance of potential sylvatic yellow fever vectors in a remnant of Atlantic Forest in the city of São Paulo, Brazil.

The landscape's structure can play a relevant role in epidemic patterns of arboviruses, influencing factors such as abundance, movement, and dispersal ability in arthropod vectors and vertebrate hosts, besides promoting alterations in the rate of potential infectious contacts between these organisms. In the Americas, yellow fever (YF) exhibits only the sylvatic cycle, in which the virus circulates in sylvatic areas among non-human primates, being transmitted by mosquitoes of the Haemagogus and Sabethes genera. In this study, we investigate some aspects of the landscape in relation to diversity and abundance of culicid species associated with YF transmission. Studies were performed in the Cantareira State Park, a remnant of the Atlantic Forest located in Greater Metropolitan São Paulo, Brazil, where the YF virus circulated recently with dozens of deaths in howler monkeys (Alouatta guariba), in addition to reported human cases. Mosquito collections were carried out monthly from February 2015 to April 2017. Mosquitoes were collected from three sites using battery-powered aspirator (12-volt battery), CDC, and Shannon traps for adults, and suction samplers and entomological spoons in breeding sites to collect immature forms. 703 mosquitoes belonging to 12 species of the Aedini and Sabethini tribes were collected. Aedes scapularis and Psorophora ferox exhibited higher abundance, while Haemagogus leucocelaenus, the main vector of YF in São Paulo state, showed lower abundance in all sampled areas. The site with longer edge between forest area and anthropic area presented more richness and abundance of YF vector species, while the site with larger forest cover area and shorter edges between forest and anthropic areas exhibited an inverse pattern. Statistically significant differences were observed between the composition of potential YF vector species among the investigated sites. Although Hg. leucocelaenus occurred in all sampled sites, the different patterns of distribution and abundance of other mosquitoes such as Aedes scapularis and Psorophora ferox suggest that these species may be involved in the transmission of sylvatic YF in the study area.

Wing morphometric variability in Aedes aegypti (Diptera: Culicidae) from different urban built environments.

BACKGROUND: Aedes aegypti is the main vector of the dengue, Zika and several other arboviruses. It is highly adapted to urbanized environments and can be found worldwide. Mosquito population control is considered the best strategy for fighting mosquito-borne diseases, making an understanding of their population dynamics vital for the development of more effective vector control programs. This study therefore sought to investigate how different levels of urbanization affect Aedes aegypti populations and modulate population structure in this species with the aid of wing geometric morphometrics. METHODS: Specimens were collected from eleven locations in three areas with distinct levels of urbanization in the city of São Paulo, Brazil: conserved, intermediate and urbanized. The right wings of female mosquitoes collected were removed, and photographed and digitized. Canonical variate analysis and Mahalanobis distance were used to investigate the degree of wing-shape dissimilarity among populations. Thin-plate splines were calculated by regression analysis of Canonical Variation Analysis scores against wing-shape variation, and a cross-validated reclassification was performed for each individual; a neighbor-joining tree was then constructed. RESULTS: Metapopulation and individual population analysis showed a clear segregation pattern in the Canonical Variation Analysis. Pairwise cross-validated reclassification yielded relatively high scores considering the microgeographical scale of the study and the fact that the study populations belong to the same species. The neighbor-joining tree showed that mosquitoes in the intermediate urban area segregated in the metapopulation and individual population analyses. Our findings show significant population structuring in Aedes aegypti mosquitoes in the areas studied. This is related to the different degrees of urbanization in the areas where the specimens were collected along with their geographical location. CONCLUSIONS: Urbanization processes in the study areas appear to play an important role in microevolutionary processes triggered by man-made modifications in the environment, resulting in a previously unknown population structuring pattern of major epidemiological importance.

Microgeographic population structuring of Aedes aegypti (Diptera: Culicidae).

Aedes aegypti is one of the species most favored by changes in the environment caused by urbanization. Its abundance increases rapidly in the face of such changes, increasing the risk of disease transmission. Previous studies have shown that mosquito species that have adapted to anthropogenic environmental changes benefit from urbanization and undergo population expansion. In light of this, we used microsatellite markers to explore how urbanization processes may be modulating Ae. aegypti populations collected from three areas with different levels of urbanization in the city of São Paulo, Brazil. Specimens were collected at eleven sites in three areas with different degrees of urbanization in the city of São Paulo: conserved, intermediate and urbanized. Ten microsatellite loci were used to characterize the populations from these areas genetically. Our findings suggest that as urbanized areas grow and the human population density in these areas increases, Ae. aegypti populations undergo a major population expansion, which can probably be attributed to the species' adaptability to anthropogenic environmental changes. Our findings reveal a robust association between, on the one hand, urbanization processes and densification of the human population and, on the other, Ae. aegypti population structure patterns and population expansion. This indicates that this species benefits from anthropogenic effects, which are intensified by migration of the human population from rural to urban areas, increasing the risk of epidemics and disease transmission to an ever-increasing number of people.

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.

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.