Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil.

BACKGROUND: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. METHODS: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. RESULTS: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). CONCLUSIONS: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil

ABSTRACT Background: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. Methods: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. Results: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). Conclusions: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Locomotor activity in Aedes aegypti with different insecticide resistance profiles.

OBJECTIVE: To evaluate locomotor activity in four field populations of Ae. aegypti with different insecticide resistance profiles from the state of São Paulo for two years. METHODS: This study comprised the susceptible Rockefeller strain and four populations from São Paulo, Brazil: two considered populations with "reduced susceptibility" to pyrethroids (Campinas and Marília), and two "resistant populations" (Santos and Ribeirão Preto). First, 2016 and 2017 eggs from these five populations were hatched in laboratory. Virgin females underwent experiments under laboratory conditions at 25°C, with 12:12h light/dark (LD) photoperiod; 24-hour individual activity was recorded using a locomotor activity monitor (LAM). RESULTS: In females from 2016 field populations, both resistant populations showed significant more locomotor activity than the two reduced susceptibility populations and the Rockefeller strain (p < 0.05). As for females from 2017 field populations, reduced susceptibility populations showed a significant increased locomotor activity than the Rockefeller strain, but no significant difference when compared to Santos resistant population (p > 0.05). CONCLUSIONS: Our results indicate that insecticide-resistant Ae. aegypti populations show increased locomotor activity, which may affect the transmission dynamics of their arboviruses.

Silent circulation of dengue virus in Aedes albopictus (Diptera: Culicidae) resulting from natural vertical transmission.

Vertical transmission in Aedes aegypti and Aedes albopictus is considered a maintenance mechanism for dengue virus (DENV) during unfavorable conditions and may be implicated in dengue outbreaks. Since DENV infection dynamics vary among wild-type viruses and vector populations, vertical transmission rates can also vary between regions. However, even though São Paulo is the most populous city in the Americas and has experienced major dengue epidemics, natural vertical transmission had never been detected in this area before. Here we confirm and describe for the first time natural vertical transmission of DENV-3 in two pools of male Ae. albopictus from the city of São Paulo. The detection of DENV-3 in years when no human autochthonous cases of this serotype were recorded suggests that silent circulation of DENV-3 is occurring and indicates that green areas may be maintaining serotypes that are not circulating in the human population, possibly by a vertical transmission mechanism.

Influence of water's physical and chemical parameters on mosquito (Diptera: Culicidae) assemblages in larval habitats in urban parks of São Paulo, Brazil.

Water's physical and chemical characteristics are important constraints in aquatic ecosystems, acting on the development, survival, and adaptation of different organisms. Immature forms of mosquitoes develop in widely diverse aquatic environments and are mainly found in permanent or temporary freshwater bodies with little or no movement. The current study aimed to investigate whether variations in larval habitats' pH, salinity, dissolved oxygen, and water temperature influence the composition of Culicidae assemblages and the presence and abundance of Aedes albopictus and Ae. aegypti. From August 2012 to July 2013, captures of immature forms and measurement of water's physical and chemical profiles were performed monthly in natural and artificial breeding sites in four urban parks in the city of São Paulo, Brazil. Changes in species composition related to the parameters' variation were assessed by multivariate analysis. Regression trees were performed to evaluate the effect of breeding sites' physical and chemical variations on the presence and abundance of Ae. albopictus and Ae. aegypti. The observations suggest ranges of conditions for the measured variables in which most species tend to be found more frequently, and pH and salinity are the variables most closely associated with variations in mosquito composition. Ae. aegypti and Ae. albopictus were present in both natural and artificial breeding sites and were observed under significantly varying conditions of pH, salinity, dissolved oxygen, and temperature. For Ae. albopictus, larval habitat type and pH were the best predictors of incidence and abundance. For Ae. aegypti, pH and salinity were the best predictors of abundance, while dissolved oxygen and larval habitat type were better predictors of presence. This information broadens our understanding of the ecology and interaction of the investigated species with abiotic factors in the aquatic environments, providing useful data for studies that seek to elucidate the underlying mechanisms of selection and colonization of breeding sites by these mosquitoes. This study also reinforces previous observations indicating that Ae. albopictus and Ae. aegypti can colonize diverse types of larval habitats with widely varying physical and chemical conditions.

Gonotrophic discordance in Culex quinquefasciatus (Diptera: Culicidae) in the city of São Paulo, Brazil

Abstract INTRODUCTION: This study aimed to assess the occurrence of gonotrophic discordance in females of Culex quinquefasciatus in São Paulo, Brazil. METHODS: Resting females were collected monthly for 8 months. Females of Cx. quinquefasciatus were identified, and their midgut and ovaries were dissected. RESULTS: Two hundred females were dissected, out of which, 27.5% were nulliparous and 57% were parous. Most females had no blood in the midgut, but gonotrophic discordance was found in 21% females. CONCLUSIONS: Females of Cx. quinquefasciatus showed a high parity rate and gonotrophic discordance, which could favor the vector capacity of this species.

Gonotrophic discordance in Culex quinquefasciatus (Diptera: Culicidae) in the city of São Paulo, Brazil.

INTRODUCTION: This study aimed to assess the occurrence of gonotrophic discordance in females of Culex quinquefasciatus in São Paulo, Brazil. METHODS: Resting females were collected monthly for 8 months. Females of Cx. quinquefasciatus were identified, and their midgut and ovaries were dissected. RESULTS: Two hundred females were dissected, out of which, 27.5% were nulliparous and 57% were parous. Most females had no blood in the midgut, but gonotrophic discordance was found in 21% females. CONCLUSIONS: Females of Cx. quinquefasciatus showed a high parity rate and gonotrophic discordance, which could favor the vector capacity of this species.

Parity and gonotrophic discordance of females of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the city of São Paulo, SP, Brazil.

The objective of this study was to assess the parity, presence of blood in the stomach, and the gonotrophic discordance of females of Aedes aegypti and Aedes albopictus captured in two areas of the city of São Paulo. The captures were undertaken monthly, by aspiration, in the period from January, 2015 to August, 2017. All the females of the two species had their midguts and ovaries dissected to determine the presence of blood and the parity/stage of maturation. With regard to parity, 27% and 34% of the females of Ae. aegypti and Ae. albopictus, respectively, were parous or were in advanced stages of the development of their ovaries (33% and 27%, respectively). The larger part of the females of Ae. aegypti and Ae. albopictus contained blood in their stomachs (77% and 60%, respectively), beyond which 36% and 27% of the females of Ae. aegypti and Ae. albopictus, respectively, were in gonotrophic discordance. Our results indicate favorable conditions in the study areas because of the presence of parous females. Moreover, this frequent and multiple contact of Ae. aegypti and Ae. albopictus females with vertebrate hosts, such as humans, increases the possibility of transmitting the viruses they may be carrying.

Seasonal and spatial distribution of Aedes aegypti and Aedes albopictus in a municipal urban park in São Paulo, SP, Brazil.

Dengue, yellow fever, chikungunya and Zika are important arboviruses present in various countries of the world, the etiological agents of which are transmitted to human-beings by the bite of infected females of Aedes aegypti and Aedes albopictus. Biological aspects of these vectors, such as their distribution and abundance, are influenced by climatic variables such as rainfall and temperature. We assess the spatial and seasonal distribution of Ae. aegypti and Ae. albopictus, during spring 2014 and spring 2015 and autumn 2015 and autumn 2016, in an urban Municipal Park, São Paulo (SP, Brazil), using 36 ovitraps. The Park was divided into three areas: internal, intermediate and peripheral, and 12 geo-referenced ovitraps were randomly installed in each area. We evaluated the association between the environmental variables maximum and minimum temperatures and rainfall with oviposition rates in the park using negative binomial regression models. Further, to estimate the distribution of the species in the three areas during the seasons, we employed the geostatic interpolation method with the use of kriging. Our results show the presence of the two species in the area in both the seasons but with a greater predominance of Ae. albopictus. Both species were significantly more abundant in spring than autumn. However, our results suggested that this seasonal variation was mediated by the maximum and minimum temperatures, which were significantly associated with the oviposition rate of both species, in all regression models. Cumulative rainfall of the week of collection was not associated with the abundance of the vectors in the multiple models. Moreover, regardless of climatic variables, the oviposition of Ae. aegypti was positively associated with the peripheral area of the park compared with the internal area (oviposition rate ratio [ORR]: 4.92; 95% CI: 2.46-9.83). On the other hand, the oviposition of Ae. albopictus was negatively associated with the peripheral area as compared with the internal one (ORR: 0.59; 95% CI: 0.38-0.91). The spatial distribution revealed a pattern of spatial segregation, confirming the ecological preferences of each species. Green areas in urban centers can serve as important habitats for various mosquito species, including especially Ae. albopictus. Thus it is that our study highlights the importance of maintaining surveillance for the targeting of control strategies in green areas as well, since most control strategies are focused on Ae. aegypti and urban residential centers.

Aedes aegypti breeding site in an underground rainwater reservoir: a warning.

We describe the discovery of Aedes aegypti underground breeding site in the Pinheiros neighborhood of São Paulo, SP, during an entomological survey program performed in 2016. Even with intense surveillance and vector control, large numbers of mosquitoes were present in this area. A detailed investigation allowed for the detection of Ae. aegypti in an underground reservoir used for rainwater storage. After the implementation of protection screens in the accesses, the presence of the vector was no longer detected. In this study, we discuss the frequent use of this type of reservoir structure and its risk for mosquito production.

Aedes aegypti breeding site in an underground rainwater reservoir: a warning / Criadouro de Aedes aegypti em reservatório subterrâneo de água da chuva: um alerta

ABSTRACT We describe the discovery of Aedes aegypti underground breeding site in the Pinheiros neighborhood of São Paulo, SP, during an entomological survey program performed in 2016. Even with intense surveillance and vector control, large numbers of mosquitoes were present in this area. A detailed investigation allowed for the detection of Ae. aegypti in an underground reservoir used for rainwater storage. After the implementation of protection screens in the accesses, the presence of the vector was no longer detected. In this study, we discuss the frequent use of this type of reservoir structure and its risk for mosquito production.

RESUMO Descreve-se o encontro de criadouro subterrâneo de Aedes aegypti no bairro de Pinheiros, São Paulo, SP, ocorrido durante um programa de levantamento entomológico, realizado em 2016. Mesmo com intensa vigilância e controle vetorial, grande quantidade de mosquitos estava presente nessa área. Investigação minuciosa permitiu a detecção de Ae. aegypti em reservatório subterrâneo para armazenamento de água pluvial. Após a implantação de telas de proteção nos acessos, não foi mais detectada a presença do vetor. O uso frequente desse tipo de estrutura e o seu risco para a produção de mosquitos é discutido.

Finding Aedes aegypti in a natural breeding site in an urban zone, Sao Paulo, Southeastern Brazil.

This is the description of how nine Aedes aegypti larvae were found in a natural breeding site in the Pinheiros neighborhood, city of Sao Paulo, SP, Southeastern Brazil. The record was conducted in December 2014, during an entomological surveillance program of dengue virus vectors, with an active search of potential breeding sites, either artificial or natural. FindingAe. aegypti larvae in a tree hole shows this species' ability to use both artificial and natural environments as breeding sites and habitats, which points towards the importance of maintaining continuous surveillance on this mosquito in all kinds of water-holding containers.

Twenty-eight years of Aedes albopictus in Brazil: a rationale to maintain active entomological and epidemiological surveillance.

INTRODUCTION: Aedes albopictus was first detected in Brazil in 1986. This mosquito species presents a major threat to public health because Brazilian populations have shown substantial vector competence for arboviruses such as dengue and chikungunya. METHODS: We updated the records of Ae. albopictus in several States of Brazil, focusing on areas in which its presence had been reported after 2002. RESULTS: Twenty-eight years after its arrival in Brazil, Ae. albopictus has been detected in 24 of 27 States. CONCLUSIONS: The rapid spread of this species and its high vector competence demonstrate the danger of Ae. albopictus in Brazil.

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.

Biodiversidade de mosquitos (Diptera: Culicidae) nos parques da cidade de São Paulo I / Mosquito (Diptera: Culicidae) survey in parks of São Paulo City I

Diante da escassez de informações sobre mosquitos (Diptera: Culicidae) na cidade de São Paulo, foi proposto um projeto para estudar esse grupo de insetos nas áreas verdes representadas pelos parques municipais da cidade. Foram investigados 35 desses parques distribuídos nas regiões sul, norte e centro-oeste da cidade, entre outubro de 2010 e fevereiro de 2011 em período diurno. Os imaturos foram coletados dos criadouros por meio de conchas entomológicas e bomba de sucção e os adultos foram capturados em seus abrigos por aspirador elétrico (bateria de 12V). A identificação e catalogação de espécimes foram feitas no Laboratório de Entomologia da Faculdade de Saúde Pública, Universidade de São Paulo. Nesta primeira fase do projeto, coletou-se um total de 5.129 espécimes distribuídos em 11 gêneros e 41 categorias taxonômicas. Culex (Cux.) quinquefasciatus foi a espécie mais abundante. O gênero Aedes foi representado principalmente por Ae. (Och.) fluviatilis e Ae. (Ste.) albopictus. Ae. (Ste.) aegypti e Ae. (Och.)scapularis também foram frequentes em alguns parques. Os demais gêneros apresentaram-se pouco abundantes. Dos parques, 25,7% apresentaram mais de dez grupos, com destaque para o Anhanguera com 26; em contrapartida, 57,1% apresentaram cinco ou menos grupos. Apesar da pressão antrópica sobre esses ambientes, diversas espécies de culicídeos se utilizam destes habitats para a manutenção e refúgio de suas populações. É recomendado que estes ambientes estejam sob constante vigilância epidemiológica, visto que algumas das espécies coletadas possuem importância em saúde pública como vetoras de patógenos à população humana.

Given the scarcity of information on mosquitoes (Diptera: Culicidae) in the city of São Paulo, led the authors to propose a project to investigate this group of insects in some of the green areas, represented by municipal parks. The captures were undertaken in 35 municipal parks in the south, north and central-west of the city, between October 2010 and February 2011, during daylight. Immature forms were collected from breeding places with entomological ladles and suction pumps and adults from resting places with electric aspirators (12V battery). The identification of the specimens was undertaken in the Culicid laboratory of the Faculdade de Saúde Pública, Universidade de São Paulo. In this first phase of the project, a total of 5,129 specimens distributed in 11 genera and 41 taxonomic categories were captured. Culex (Cux) quinquefasciatus was the most abundant species. The genus Aedes was represented mainly by Ae. (Och) fluviatilis and Ae. (Ste) albopictus. Ae. (Ste) aegypti and Ae. (Och) scapularis were frequent in some parks. Other genera were less abundant. Of the parks investigated, 25.7% presented more than ten groups of species, notably the Anhanguera with 26; on the other hand, 57.1% had five or fewer groups. Despite the anthropic pressure on these environments, several culicid species make use of these habitats as refuges. It is recommended that these environments be kept under constant epidemiological surveillance as some of the species collected constitute public health threats as pathogen vectors to the human population.

Malaria outside the Amazon region: natural Plasmodium infection in anophelines collected near an indigenous village in the Vale do Rio Branco, Itanhaém, SP, Brazil.

A few cases of Plasmodium vivax malaria in which anophelines of subgenus Kerteszia were incriminated as vectors have been reported outside the Amazon region, in the Atlantic Forest. This study was carried out near an indigenous Guarani village in the Curucutu reserve, an environmental protection area in the municipality of Itanhaém in the state of São Paulo, Brazil, on November 30, 2009, February 18, 2010, April 29, 2010 and May 26, 2010. Mosquitoes were collected along the route to the Guarani village where the edge of the Branco river floodplain meets the forests on the mountain slopes. Adult forms were collected with CO(2)-baited CDC traps and Shannon traps from twilight to 10:00 P.M. Anopheles cruzii predominated in both traps. The other species collected in the CDC traps were An. pseudomaculipes/maculipes, An. fluminensis and An. mediopunctatus/forattinii/costai. In addition to the latter three species, An. apicimacula/intermedius and An. strodei were also found in the Shannon traps. All but An. cruzii and An. strodei belong to subgenus Anopheles. A total of 506 mosquitoes were assayed by PCR to detect natural infection by Plasmodium species. In the CDC traps, An. fluminensis and An. pseudomaculipes/maculipes were positive for Plasmodium malariae, while in the Shannon traps An. pseudomaculipes/maculipes was positive for Plasmodium vivax and Plasmodium malariae and An. cruzii was positive for P. malariae, resulting in a minimum infection rate of 0.24%. Our findings suggest that An. cruzii may be incriminated in the transmission of malaria between monkeys and humans, as this species was found to be infected by P. malariae. They also highlight the need for an understanding of the role of anophelines from outside subgenus Kerteszia in the transmission of malaria in the Atlantic Forest, as these were also found to be naturally infected by P. vivax and P. malariae.

Brazilian mosquito (Diptera: Culicidae) fauna: I. Anopheles species from Porto Velho, Rondônia state, western Amazon, Brazil / Fauna brasileira de mosquitos (Diptera: Culicidae): I. Espécies de Anopheles de Porto Velho, estado de Rondônia, oeste da Amazônia, Brasil

This study contributes to knowledge of Anopheles species, including vectors of Plasmodium from the western Brazilian Amazon in Porto Velho, Rondônia State. The sampling area has undergone substantial environmental changes as a consequence of agricultural and hydroelectric projects, which have caused intensive deforestation and favored habitats for some mosquito species. The purpose of this study was to diagnose the occurrence of anopheline species from collections in three locations along an electric-power transmission line. Each locality was sampled three times from 2010 to 2011. The principal adult mosquitoes captured in Shannon trap were Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An.gilesi and An. costai. In addition, larvae were collected in ground breeding sites for Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. and An. oswaldoi-konderi. Anopheles darlingi was the most common mosquito in the region. We discuss Culicidae systematics, fauna distribution, and aspects of malaria in altered habitats of the western Amazon.

Este estudo contribui para o conhecimento de espécies de Anopheles, incluindo vetores de Plasmodium do oeste da Amazônia brasileira, em Porto Velho, no estado de Rondônia. Esta região vem passando por mudanças ambientais, como consequência de agricultura extensiva e projetos hidroelétricos que causam desmatamento, favorecendo o desenvolvimento de algumas espécies de mosquitos. Assim, a proposta deste estudo é registrar a presença de espécies de anofelinos na área, sendo conduzidas coletas de mosquitos em três locais, ao longo de uma linha de transmissão de energia elétrica. Cada uma das localidades foi amostrada três vezes, no período de 2010 a 2011. Os principais mosquitos adultos capturados em armadilhas de Shannon foram Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An.gilesi e An. costai. Assim como as formas larvárias Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. e An. oswaldoi-konderi, coletadas em criadouros. Anopheles darlingi foi a espécie mais coletada na região. Em adição, discutiu-se sistemática de Culicidae, distribuição de fauna e aspectos da malária em ambientes modificados do oeste da Amazônia brasileira.

Mosquitoes in degraded and preserved areas of the Atlantic Forest and potential for vector-borne disease risk in the municipality of São Paulo, Brazil.

In order to assess the epidemiological potential of the Culicidae species in remaining areas of the Brazilian Atlantic Forest, specimens of this family were collected in wild and anthropic environments. A total of 9,403 adult mosquitoes was collected from May, 2009 to June, 2010. The most prevalent among species collected in the wild environment were Anopheles (Kerteszia) cruzii, the Melanoconion section of Culex (Melanoconion), and Aedes serratus, while the most common in the anthropic site were Coquillettidia chrysonotum/albifera, Culex (Culex) Coronator group, and An. (Ker.) cruzii. Mosquito richness was similar between environments, although the abundance of individuals from different species varied. When comparing diversity patterns between environments, anthropic sites exhibited higher richness and evenness, suggesting that environmental stress increased the number of favorable niches for culicids, promoting diversity. Increased abundance of opportunistic species in the anthropic environment enhances contact with culicids that transmit vector-borne diseases.

Brazilian mosquito (Diptera: Culicidae) fauna: I. Anopheles species from Porto Velho, Rondônia state, western Amazon, Brazil.

This study contributes to knowledge of Anopheles species, including vectors of Plasmodium from the western Brazilian Amazon in Porto Velho, Rondônia State. The sampling area has undergone substantial environmental changes as a consequence of agricultural and hydroelectric projects, which have caused intensive deforestation and favored habitats for some mosquito species. The purpose of this study was to diagnose the occurrence of anopheline species from collections in three locations along an electric-power transmission line. Each locality was sampled three times from 2010 to 2011. The principal adult mosquitoes captured in Shannon trap were Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An.gilesi and An. costai. In addition, larvae were collected in ground breeding sites for Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. and An. oswaldoi-konderi. Anopheles darlingi was the most common mosquito in the region. We discuss Culicidae systematics, fauna distribution, and aspects of malaria in altered habitats of the western Amazon.