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.

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.

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.

Body size does not affect locomotor activity of Aedes aegypti and Aedes albopictus females (Diptera:Culicidae).

Intraspecific competition between mosquito larvae can affect several adult traits, particularly size. This study tested the hypothesis that intraspecific competition during the larval stage affects wing length in Ae. aegypti and Ae. albopictus adults, in turn influencing locomotor activity. L1 larvae of both species were reared in trays under conditions of low and high competition. After adults had emerged, the locomotor activity of virgin females of Ae. aegypti and Ae. albopictus was evaluated under light-dark cycles of 12:12 h at 25 °C and 70% relative humidity. At the end of the locomotor activity experiment, the left wings of the mosquitoes were removed to be measured, and wing length was used as an indicator of adult female size. Although the results showed that the wing lengths of Ae. aegypti and Ae. albopictus females reared under low larval competition were significantly greater than those of females reared under high larval competition, this difference did not affect locomotor activity in females of either species, demonstrating that locomotor activity in small Ae. aegypti and Ae. albopictus females is not lower than in larger females. Our findings reinforce the idea that intraspecific competition alters the wing length of Ae. aegypti and Ae. albopictus females and provide new evidence about this effect on the locomotor activity of these species.

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.

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.

Biodiversity and public health interface / Biodiversidade e a interface com a saúde pública

Abstract Alongside modernity, the human activity has been a key factor in global environmental risks, with worldwide anthropic modification being the cause of the emergence of diseases for wild and livestock animals, and even humans. In special, the increase in the spatial distribution and in the incidence of some emerging infectious diseases (EID) are directly associated to deforestation and global climate changes. Moreover, the arise of new EID agents, such as the SARS-COV-2 have been reported for the last 30 years. On the other hand, biodiversity has been shown to be a key indicator for ecosystem health, and to pose a role to increase the promotion of human public health. In neotropical regions, and in special, in Brazil, several infectious diseases have been demonstrated to be directly affected for the biodiversity loss, such as malaria, hantavirus pulmonary syndrome, yellow fever, urban arboviruses, spotted fever, amongst other. To better understand the ecosystem capacity of regulation of infectious diseases, FAPESP BIOTA program have supported researchers and research projects to increase knowledge about Brazilian biodiversity and the ecosystems, such as diversity of bird bioagents, venomous animals biodiversity, diversity of mosquitos species in forest patches inside urban areas, propagation of the yellow fever virus over fragmented forest territories, loss of ecological corridors and occurrence of spotted fever and malaria, amongst others. It is noteworthy that FAPESP BIOTA is a successful program and must be expanded as an important tool for present and future public health promotion.

Resumo Junto à modernidade, a atividade humana tem sido um fator chave ligada aos riscos ambientais globais, as modificações antrópicas em âmbito mundial têm sido causa do surgimento de doenças para os animais silvestres e domésticos, bem como para o ser humano. Em especial, o incremento na distribuição espacial e incidência de doenças infecciosas emergentes (DIE) estão diretamente associados ao desmatamento e às mudanças climáticas globais, além disso, o surgimento de novos agentes de DIE, como o SARS-COV-2, tem sido relatado nos últimos 30 anos. Por outro lado, a biodiversidade tem se mostrado um indicador chave para a saúde dos ecossistemas, além de representar um papel importante na promoção da saúde pública humana. Nas regiões neotropicais, e em especial, no Brasil, várias doenças infecciosas têm demonstrado ser diretamente afetadas pela perda de biodiversidade, como a malária, a síndrome pulmonar por hantavírus, a febre amarela, as arboviroses urbanas, a febre maculosa, entre outras. Para entender melhor a capacidade ecossistêmica de regulação de doenças infecciosas, o programa BIOTA FAPESP tem apoiado pesquisadores e projetos de pesquisa para aumentar o conhecimento sobre a biodiversidade e os ecossistemas brasileiros, como a diversidade de bioagentes de aves, a biodiversidade de animais peçonhentos, a diversidade de espécies de mosquitos em fragmentos florestais dentro de áreas urbanas, a propagação do vírus da febre amarela em território florestal fragmentado, perda e isolamento de remanescentes florestais e a ocorrência de febre maculosa e malária, entre outros. Ressalta-se que o BIOTA FAPESP é um programa de sucesso e deve ser ampliado como importante ferramenta de promoção da saúde pública presente e futura.

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.

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.

The toxic effects of chloroquine and hydroxychloroquine on skeletal muscle: a systematic review and meta-analysis.

The aim of this systematic review was to perform qualitative and quantitative analysis on the toxic effects of chloroquine (CQ) and hydroxychloroquine (HCQ) on skeletal muscles. We designed the study according to PRISMA guidelines. Studies for qualitative and quantitative analyses were selected according to the following inclusion criteria: English language; size of sample (> 5 patients), adult (> age of 18) patients, treated with CQ/HCQ for inflammatory diseases, and presenting and not presenting with toxic effects on skeletal muscles. We collected data published from 1990 to April 2020 using PubMed, Cochrane Library, EMBASE, and SciELO. Risk of bias for observational studies was assessed regarding the ROBIN-I scale. Studies with less than five patients (case reports) were selected for an additional qualitative analysis. We used the software Comprehensive Meta-Analysis at the confidence level of 0.05. We identified 23 studies for qualitative analysis (17 case-reports), and five studies were eligible for quantitative analysis. From case reports, 21 patients presented muscle weakness and confirmatory biopsy for CQ/HCQ induced myopathy. From observational studies, 37 patients out of 1,367 patients from five studies presented muscle weakness related to the use of CQ/HCQ, and 252 patients presented elevated levels of muscle enzymes (aldolase, creatine phosphokinase, and lactate dehydrogenase). Four studies presented data on 34 patients with confirmatory biopsy for drug-induced myopathy. No study presented randomized samples. The chronic use of CQ/HCQ may be a risk for drug-induced myopathy. There is substantiated need for proper randomized trials and controlled prospective studies needed to assess the clinical and subclinical stages of CQ/HCQ -induced muscle myopathy.

Confirmation of the first report of Psammolestes tertius Lent and Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) in Paraná State, Brazil.

INTRODUCTION: This study confirms the occurrence of Psammolestes tertius Lent & Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) in the state of Paraná, Brazil. METHODS: In 2002, a male specimen of P. tertius was collected in the municipality of Porto Rico, Paraná, Brazil. RESULTS: This finding adds to the data on the geographical distribution of P. tertius from 14 to 15 known occurrences in Brazilian states and, therefore, reports the increase in the diversity of triatomines in Paraná. CONCLUSIONS: The presence of P. tertius in the state of Paraná demonstrates that the biodiversity of these insects may have been underestimated.

A mathematical model for zoonotic transmission of malaria in the Atlantic Forest: Exploring the effects of variations in vector abundance and acrodendrophily.

Transmission foci of autochthonous malaria caused by Plasmodium vivax-like parasites have frequently been reported in the Atlantic Forest in Southeastern and Southern Brazil. Evidence suggests that malaria is a zoonosis in these areas as human infections by simian Plasmodium species have been detected, and the main vector of malaria in the Atlantic Forest, Anopheles (Kerteszia) cruzii, can blood feed on human and simian hosts. In view of the lack of models that seek to predict the dynamics of zoonotic transmission in this part of the Atlantic Forest, the present study proposes a new deterministic mathematical model that includes a transmission compartment for non-human primates and parameters that take into account vector displacement between the upper and lower forest strata. The effects of variations in the abundance and acrodendrophily of An. cruzii on the prevalence of infected humans in the study area and the basic reproduction number (R0) for malaria were analyzed. The model parameters are based on the literature and fitting of the empirical data. Simulations performed with the model indicate that (1) an increase in the abundance of the vector in relation to the total number of blood-seeking mosquitoes leads to an asymptotic increase in both the proportion of infected individuals at steady state and R0; (2) the proportion of infected humans at steady state is higher when displacement of the vector mosquito between the forest strata increases; and (3) in most scenarios, Plasmodium transmission cannot be sustained only between mosquitoes and humans, which implies that non-human primates play an important role in maintaining the transmission cycle. The proposed model contributes to a better understanding of the dynamics of malaria transmission in the Atlantic Forest.

Complexity of malaria transmission dynamics in the Brazilian Atlantic Forest.

Plasmodium malariae and Plasmodium vivax are protozoan parasites that can cause malaria in humans. They are genetically indistinguishable from, respectively, Plasmodium brasilianum and Plasmodium simium, i.e. parasites infecting New World non-human primates in South America. In the tropical rainforests of the Brazilian Atlantic coast, it has long been hypothesized that P. brasilianum and P. simium in platyrrhine primates originated from P. malariae and P. vivax in humans. A recent hypothesis proposed the inclusion of Plasmodium falciparum into the transmission dynamics between humans and non-human primates in the Brazilian Atlantic tropical rainforest. Herein, we assess the occurrence of human malaria in simians and sylvatic anophelines using field-collected samples in the Capivari-Monos Environmental Protection Area from 2015 to 2017. We first tested simian blood and anopheline samples. Two simian (Aloutta) blood samples (18%, n = 11) showed Plasmodium cytb DNA sequences, one for P. vivax and another for P. malariae. From a total of 9,416 anopheline females, we found 17 pools positive for Plasmodium species with a 18S qPCR assay. Only three showed P. cytb DNA sequence, one for P. vivax and the others for rodent malaria species (similar to Plasmodium chabaudi and Plasmodium berghei). Based on these results, we tested 25 rodent liver samples for the presence of Plasmodium and obtained P. falciparum cytb DNA sequence in a rodent (Oligoryzomys sp.) liver. The findings of this study indicate complex malaria transmission dynamics composed by parallel spillover-spillback of human malaria parasites, i.e. P. malariae, P. vivax, and P. falciparum, in the Brazilian Atlantic forest.

Phenotypic and genetic variation of Triatoma costalimai (Hemiptera: Reduviidae)

Abstract INTRODUCTION: We aimed to study intraspecific variation in Triatoma costalimai, a potential vector of Chagas disease present in Brazil and Bolivia. METHODS: We analyzed phenotypic (connexivum color patterns, wing morphometrics) and genetic variation (16S mtDNA) of three Brazilian T. costalimai populations. We compared 16S sequences with those of putative Bolivian T. costalimai and its sister species, T. jatai. RESULTS: Brazilian populations had different connexivum color patterns and forewing shapes. A 16S mtDNA haplotype network showed a clear separation of Brazilian T. costalimai from both T. jatai and Bolivian T. costalimai. CONCLUSIONS: We report considerable variability in T. costalimai populations.

Confirmation of the first report of Psammolestes tertius Lent and Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) in Paraná State, Brazil

Abstract INTRODUCTION This study confirms the occurrence of Psammolestes tertius Lent & Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) in the state of Paraná, Brazil. METHODS: In 2002, a male specimen of P. tertius was collected in the municipality of Porto Rico, Paraná, Brazil. RESULTS: This finding adds to the data on the geographical distribution of P. tertius from 14 to 15 known occurrences in Brazilian states and, therefore, reports the increase in the diversity of triatomines in Paraná. CONCLUSIONS: The presence of P. tertius in the state of Paraná demonstrates that the biodiversity of these insects may have been underestimated.

Phenotypic and genetic variation of Triatoma costalimai (Hemiptera: Reduviidae).

INTRODUCTION: We aimed to study intraspecific variation in Triatoma costalimai, a potential vector of Chagas disease present in Brazil and Bolivia. METHODS: We analyzed phenotypic (connexivum color patterns, wing morphometrics) and genetic variation (16S mtDNA) of three Brazilian T. costalimai populations. We compared 16S sequences with those of putative Bolivian T. costalimai and its sister species, T. jatai. RESULTS: Brazilian populations had different connexivum color patterns and forewing shapes. A 16S mtDNA haplotype network showed a clear separation of Brazilian T. costalimai from both T. jatai and Bolivian T. costalimai. CONCLUSIONS: We report considerable variability in T. costalimai populations.

Microgeographic Wing-Shape Variation in Aedes albopictus and Aedes scapularis (Diptera: Culicidae) Populations.

Aedes albopictus and Aedes scapularis are vectors of several arboviruses, including the dengue, chikungunya, and Rocio virus infection. While Ae. albopictus is a highly invasive species native to Asia and has been dispersed by humans to most parts of the world, Ae. scapularis is native to Brazil and is widely distributed in the southeast of the country. Both species are highly anthropophilic and are often abundant in places with high human population densities. Because of the great epidemiological importance of these two mosquitoes and the paucity of knowledge on how they have adapted to different urban built environments, we investigated the microgeographic population structure of these vector species in the city of São Paulo, Brazil, using wing geometric morphometrics. Females of Ae. albopictus and Ae. scapularis were collected in seven urban parks in the city. The right wings of the specimens were removed and digitized, and eighteen landmarks based on vein intersections in the wing venation patterns were used to assess cross-sectional variation in wing shape and size. The analyses revealed distinct results for Ae. albopictus and Ae. scapularis populations. While the former had less wing shape variation, the latter had more heterogeneity, indicating a higher degree of intraspecific variation. Our results indicate that microgeographic selective pressures exerted by different urban built environments have a distinct effect on wing shape patterns in the populations of these two mosquito species studied here.