Arbovirus surveillance in mosquitoes: Historical methods, emerging technologies, and challenges ahead.

Arboviruses cause millions of infections each year; however, only limited options are available for treatment and pharmacological prevention. Mosquitoes are among the most important vectors for the transmission of several pathogens to humans. Despite advances, the sampling, viral detection, and control methods for these insects remain ineffective. Challenges arise with the increase in mosquito populations due to climate change, insecticide resistance, and human interference affecting natural habitats, which contribute to the increasing difficulty in controlling the spread of arboviruses. Therefore, prioritizing arbovirus surveillance is essential for effective epidemic preparedness. In this review, we offer a concise historical account of the discovery and monitoring of arboviruses in mosquitoes, from mosquito capture to viral detection. We then analyzed the advantages and limitations of these traditional methods. Furthermore, we investigated the potential of emerging technologies to address these limitations, including the implementation of next-generation sequencing, paper-based devices, spectroscopic detectors, and synthetic biosensors. We also provide perspectives on recurring issues and areas of interest such as insect-specific viruses.

Back to Where It Was First Described: Vectors of Sylvatic Yellow Fever Transmission in the 2017 Outbreak in Espírito Santo, Brazil.

Evidence of sylvatic yellow fever was first reported in Atlantic Forest areas in Espírito Santo, Brazil, during a yellow fever virus (YFV) outbreak in 1931. An entomological survey was conducted in six forest sites during and after an outbreak reported ~80 years after the last case in the area. Among 10,658 mosquitoes of 78 species, Haemagogus leucocelaenus, and Hg. janthinomys/capricornii were considered the main vectors as they had a relatively high abundance, co-occurred in essentially all areas, and showed high YFV infection rates. Sabethes chloropterus, Sa. soperi, Sa. identicus, Aedes aureolineatus, and Shannoniana fluviatilis may have a secondary role in transmission. This is the first report of Sa. identicus, Ae. aureolineatus, and Sh. fluviatilis infected with YFV. Our study emphasizes the importance of entomological monitoring and maintenance of high vaccination coverage in receptive areas to YFV transmission.

Fast surveillance response reveals the introduction of a new yellow fever virus sub-lineage in 2021, in Minas Gerais, Brazil.

BACKGROUND: In Brazil, the yellow fever virus (YFV) is maintained in a sylvatic cycle involving wild mosquitoes and non-human primates (NHPs). The virus is endemic to the Amazon region; however, waves of epidemic expansion reaching other Brazilian states sporadically occur, eventually causing spillovers to humans. OBJECTIVES: To report a surveillance effort that led to the first confirmation of YFV in NHPs in the state of Minas Gerais (MG), Southeast region, in 2021. METHODS: A surveillance network was created, encompassing the technology of smartphone applications and coordinated actions of several research institutions and health services to monitor and investigate NHP epizootics. FINDINGS: When alerts were spread through the network, samples from NHPs were collected and YFV infection confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and genome sequencing at an interval of only 10 days. Near-complete genomes were generated using the Nanopore MinION sequencer. Phylogenetic analysis indicated that viral genomes were related to the South American genotype I, clustering with a genome detected in the Amazon region (state of Pará) in 2017, named YFVPA/MG sub-lineage. Fast YFV confirmation potentialised vaccination campaigns. MAIN CONCLUSIONS: A new YFV introduction was detected in MG 6 years after the beginning of the major outbreak reported in the state (2015-2018). The YFV strain was not related to the sub-lineages previously reported in MG. No human cases have been reported, suggesting the importance of coordinated surveillance of NHPs using available technologies and supporting laboratories to ensure a quick response and implementation of contingency measures to avoid YFV spillover to humans.

Serological Evidence of Orthopoxvirus Infection in Neotropical Primates in Brazil.

The genus Orthopoxvirus (OPXV) of the family Poxviridae comprises several viruses that are capable of infecting a wide range of hosts. One of the most widespread OPXVs is the Vaccinia virus (VACV), which circulates in zoonotic cycles in South America, especially in Brazil, infecting domestic and wild animals and humans and causing economic losses as well as impacting public health. Despite this, little is known about the presence and/or exposure of neotropical primates to orthopoxviruses in the country. In this study, we report the results of a search for evidence of OPVX infections in neotropical free-living primates in the state of Minas Gerais, southeast Brazil. The sera or liver tissues of 63 neotropical primates were examined through plaque reduction neutralization tests (PRNT) and real-time PCR. OPXV-specific neutralizing antibodies were detected in two sera (4.5%) from Callithrix penicillata, showing 55% and 85% reduction in plaque counts, evidencing their previous exposure to the virus. Both individuals were collected in urban areas. All real-time PCR assays were negative. This is the first time that evidence of OPXV exposure has been detected in C. penicillata, a species that usually lives at the interface between cities and forests, increasing risks of zoonotic transmissions through spillover/spillback events. In this way, studies on the circulation of OPXV in neotropical free-living primates are necessary, especially now, with the monkeypox virus being detected in new regions of the planet.

Ecological and environmental factors affecting transmission of sylvatic yellow fever in the 2017-2019 outbreak in the Atlantic Forest, Brazil.

BACKGROUND: Yellow fever virus (YFV) is an arbovirus that, despite the existence of a safe and effective vaccine, continues to cause outbreaks of varying dimensions in the Americas and Africa. Between 2017 and 2019, Brazil registered un unprecedented sylvatic YFV outbreak whose severity was the result of its spread into zones of the Atlantic Forest with no signals of viral circulation for nearly 80 years. METHODS: To investigate the influence of climatic, environmental, and ecological factors governing the dispersion and force of infection of YFV in a naïve area such as the landscape mosaic of Rio de Janeiro (RJ), we combined the analyses of a large set of data including entomological sampling performed before and during the 2017-2019 outbreak, with the geolocation of human and nonhuman primates (NHP) and mosquito infections. RESULTS: A greater abundance of Haemagogus mosquitoes combined with lower richness and diversity of mosquito fauna increased the probability of finding a YFV-infected mosquito. Furthermore, the analysis of functional traits showed that certain functional groups, composed mainly of Aedini mosquitoes which includes Aedes and Haemagogus mosquitoes, are also more representative in areas where infected mosquitoes were found. Human and NHP infections were more common in two types of landscapes: large and continuous forest, capable of harboring many YFV hosts, and patches of small forest fragments, where environmental imbalance can lead to a greater density of the primary vectors and high human exposure. In both, we show that most human infections (~ 62%) occurred within an 11-km radius of the finding of an infected NHP, which is in line with the flight range of the primary vectors. CONCLUSIONS: Together, our data suggest that entomological data and landscape composition analyses may help to predict areas permissive to yellow fever outbreaks, allowing protective measures to be taken to avoid human cases.

Fast surveillance response reveals the introduction of a new yellow fever virus sub-lineage in 2021, in Minas Gerais, Brazil

BACKGROUND In Brazil, the yellow fever virus (YFV) is maintained in a sylvatic cycle involving wild mosquitoes and non-human primates (NHPs). The virus is endemic to the Amazon region; however, waves of epidemic expansion reaching other Brazilian states sporadically occur, eventually causing spillovers to humans. OBJECTIVES To report a surveillance effort that led to the first confirmation of YFV in NHPs in the state of Minas Gerais (MG), Southeast region, in 2021. METHODS A surveillance network was created, encompassing the technology of smartphone applications and coordinated actions of several research institutions and health services to monitor and investigate NHP epizootics. FINDINGS When alerts were spread through the network, samples from NHPs were collected and YFV infection confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and genome sequencing at an interval of only 10 days. Near-complete genomes were generated using the Nanopore MinION sequencer. Phylogenetic analysis indicated that viral genomes were related to the South American genotype I, clustering with a genome detected in the Amazon region (state of Pará) in 2017, named YFVPA/MG sub-lineage. Fast YFV confirmation potentialised vaccination campaigns. MAIN CONCLUSIONS A new YFV introduction was detected in MG 6 years after the beginning of the major outbreak reported in the state (2015-2018). The YFV strain was not related to the sub-lineages previously reported in MG. No human cases have been reported, suggesting the importance of coordinated surveillance of NHPs using available technologies and supporting laboratories to ensure a quick response and implementation of contingency measures to avoid YFV spillover to humans.

Survey on Non-Human Primates and Mosquitoes Does not Provide Evidences of Spillover/Spillback between the Urban and Sylvatic Cycles of Yellow Fever and Zika Viruses Following Severe Outbreaks in Southeast Brazil.

In the last decade, Flaviviruses such as yellow fever (YFV) and Zika (ZIKV) have expanded their transmission areas. These viruses originated in Africa, where they exhibit both sylvatic and interhuman transmission cycles. In Brazil, the risk of YFV urbanization has grown, with the sylvatic transmission approaching the most densely populated metropolis, while concern about ZIKV spillback to a sylvatic cycle has risen. To investigate these health threats, we carried out extensive collections and arbovirus screening of 144 free-living, non-human primates (NHPs) and 5219 mosquitoes before, during, and after ZIKV and YFV outbreaks (2015-2018) in southeast Brazil. ZIKV infection was not detected in any NHP collected at any time. In contrast, current and previous YFV infections were detected in NHPs sampled between 2017 and 2018, but not before the onset of the YFV outbreak. Mosquito pools screened by high-throughput PCR were positive for YFV when captured in the wild and during the YFV outbreak, but were negative for 94 other arboviruses, including ZIKV, regardless of the time of collection. In conclusion, there was no evidence of YFV transmission in coastal southeast Brazil before the current outbreak, nor the spread or establishment of an independent sylvatic cycle of ZIKV or urban Aedes aegypti transmission of YFV in the region. In view of the region's receptivity and vulnerability to arbovirus transmission, surveillance of NHPs and mosquitoes should be strengthened and continuous.

Howler monkeys are the reservoir of malarial parasites causing zoonotic infections in the Atlantic forest of Rio de Janeiro.

BACKGROUND: Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015-2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts. METHODOLOGY: A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques. PRINCIPAL FINDINGS: In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms-the only available genetic markers that differentiate P. simium from P. vivax-were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum. CONCLUSIONS/SIGNIFICANCE: Our data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons.

BACKGROUND: In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES: To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS: We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS: Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS: Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Haemagogus leucocelaenus and Haemagogus janthinomys are the primary vectors in the major yellow fever outbreak in Brazil, 2016-2018.

The yellow fever virus (YFV) caused a severe outbreak in Brazil in 2016-2018 that rapidly spread across the Atlantic Forest in its most populated region without viral circulation for almost 80 years. A comprehensive entomological survey combining analysis of distribution, abundance and YFV natural infection in mosquitoes captured before and during the outbreak was conducted in 44 municipalities of five Brazilian states. In total, 17,662 mosquitoes of 89 species were collected. Before evidence of virus circulation, mosquitoes were tested negative but traditional vectors were alarmingly detected in 82% of municipalities, revealing high receptivity to sylvatic transmission. During the outbreak, five species were found positive in 42% of municipalities. Haemagogus janthinomys and Hg. leucocelaenus are considered the primary vectors due to their large distribution combined with high abundance and natural infection rates, concurring together for the rapid spread and severity of this outbreak. Aedes taeniorhynchus was found infected for the first time, but like Sabethes chloropterus and Aedes scapularis, it appears to have a potential local or secondary role because of their low abundance, distribution and infection rates. There was no evidence of YFV transmission by Aedes albopictus and Aedes aegypti, although the former was the most widespread species across affected municipalities, presenting an important overlap between the niches of the sylvatic vectors and the anthropic ones. The definition of receptive areas, expansion of vaccination in the most affected age group and exposed populations and the adoption of universal vaccination to the entire Brazilian population need to be urgently implemented.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons

BACKGROUND In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Genomic and structural features of the yellow fever virus from the 2016-2017 Brazilian outbreak.

Southeastern Brazil has been suffering a rapid expansion of a severe sylvatic yellow fever virus (YFV) outbreak since late 2016, which has reached one of the most populated zones in Brazil and South America, heretofore a yellow fever-free zone for more than 70 years. In the current study, we describe the complete genome of 12 YFV samples from mosquitoes, humans and non-human primates from the Brazilian 2017 epidemic. All of the YFV sequences belong to the modern lineage (sub-lineage 1E) of South American genotype I, having been circulating for several months prior to the December 2016 detection. Our data confirm that viral strains associated with the most severe YF epidemic in South America in the last 70 years display unique amino acid substitutions that are mainly located in highly conserved positions in non-structural proteins. Our data also corroborate that YFV has spread southward into Rio de Janeiro state following two main sylvatic dispersion routes that converged at the border of the great metropolitan area comprising nearly 12 million unvaccinated inhabitants. Our original results can help public health authorities to guide the surveillance, prophylaxis and control measures required to face such a severe epidemiological problem. Finally, it will also inspire other workers to further investigate the epidemiological and biological significance of the amino acid polymorphisms detected in the Brazilian 2017 YFV strains.

Oviposition behavior of Haemagogus leucocelaenus (Diptera: culicidae), a vector of wild yellow fever in Brazil.

Haemagogus leucocelaenus, which is considered a major vector of wild yellow fever, exhibits acrodendrophilic habits and mainly deposits its eggs in treeholes and bamboo internodes. The selection of nursery sites is essential in the life history and reproductive success of mosquitoes. The present work investigated the preferred oviposition height and period of Hg. leucocelaenus in an Atlantic forest area in Rio de Janeiro. Sampling was performed using oviposition traps that were placed on plant material at 0, 2, 4, 6, and 8 m above the ground, from August 2015 to July 2016. Eggs were more abundant during October and May, and the height of traps placement had no significant effect on the eggs number indicating that Hg. leucocelaenus explores different levels of forest habitats, a behavior that may favor the transmission of pathogens among arboreal animals including primates and humans. The findings of the present study are discussed from an ecological and epidemiological point of view.

Genome analysis of yellow fever virus of the ongoing outbreak in Brazil reveals polymorphisms.

The current yellow fever outbreak in Brazil is the most severe one in the country in recent times. It has rapidly spread to areas where YF virus (YFV) activity has not been observed for more than 70 years and vaccine coverage is almost null. Here, we sequenced the whole YFV genome of two naturally infected howler-monkeys (Alouatta clamitans) obtained from the Municipality of Domingos Martins, state of Espírito Santo, Brazil. These two ongoing-outbreak genome sequences are identical. They clustered in the 1E sub-clade (South America genotype I) along with the Brazilian and Venezuelan strains recently characterised from infections in humans and non-human primates that have been described in the last 20 years. However, we detected eight unique amino acid changes in the viral proteins, including the structural capsid protein (one change), and the components of the viral replicase complex, the NS3 (two changes) and NS5 (five changes) proteins, that could impact the capacity of viral infection in vertebrate and/or invertebrate hosts and spreading of the ongoing outbreak.

Influence of breeding site availability on the oviposition behaviour of Aedes aegypti

Despite the importance of the mosquito Aedes aegypti in the transmission of arboviruses, such as yellow fever, Chikungunya fever and dengue fever, some aspects of their behaviour remain unknown. In the present study, the oviposition behaviour of Ae. aegypti females that were exposed to different densities of breeding sites (2, 4, 8 and 16) was evaluated in laboratory and semi-field conditions. The number of breeding sites that were used was proportional to the number available, but tended towards stabilisation. Females used four-six breeding sites on average, with a maximum of 11. A high percentage of eggs was observed in the water, along with the presence of a breeding site termed “favourite”, which received at least 40% of the eggs. The results are discussed in ecological, evolutionary and epidemiological approaches.

Influence of breeding site availability on the oviposition behaviour of Aedes aegypti.

Despite the importance of the mosquito Aedes aegypti in the transmission of arboviruses, such as yellow fever, Chikungunya fever and dengue fever, some aspects of their behaviour remain unknown. In the present study, the oviposition behaviour of Ae. aegypti females that were exposed to different densities of breeding sites (2, 4, 8 and 16) was evaluated in laboratory and semi-field conditions. The number of breeding sites that were used was proportional to the number available, but tended towards stabilisation. Females used four-six breeding sites on average, with a maximum of 11. A high percentage of eggs was observed in the water, along with the presence of a breeding site termed "favourite", which received at least 40% of the eggs. The results are discussed in ecological, evolutionary and epidemiological approaches.

Study of oviposition behavior of aedes aegypti in two neighborhoods under the influence of semi-arid climate in the municipality of salinas, state of Minas Gerais, Brazil

The mosquito Aedes aegypti (Diptera: Culicidae) is the main vector of human arborvirus in Brazil.Oviposition behavior is important for the survival and dispersal of A. aegypti. The objective of this studywas to evaluate the oviposition behavior and its relationship to meteorological factors. Twenty ovitrapswere installed in two neighborhoods in the urban area of the city of Salinas, MG, southeastern Brazil.The traps were inspected weekly for the presence of eggs in the water and on the paddle. Simultaneously,regional weather data were analyzed. The results showed the Ovitrap Positivity Index (OPI) variedgreatly, from 0 to 90 percent, between the weeks and boroughs sampled, demonstrating the vector’s greatpopulation fluctuation. Among the analyzed climatic factors, rainfall strongly influenced oviposition,followed by relative air humidity. The number of eggs found on the paddle was significantly higher thanin the water (p<0.05). However, a significant increase of eggs in the water was noticed when the relativehumidity dropped below 40 percent. This seems to be an important behavior to maintain the population inperiods of drought. Through the low number of eggs (<30) found in 56 percent of positive traps, we concludedthat skip-oviposition behavior occurred, contributing to the prevalence of mosquitoes during thesampled period. It is concluded that the occurrence of water oviposition may be higher than described inthe literature, and should be studied due to its evolutionary and epidemiological importance...

O mosquito Aedes aegypti é o principal vetor de arborvírus humanos no Brasil. O comportamentode oviposição é importante para a sobrevivência e dispersão do A. aegypti. O objetivo deste trabalho foi avaliar estes comportamentos e sua relação com fatores climáticos. Instalaram-se vintearmadilhas ovitrampa em dois bairros de Salinas-MG. Todas foram inspecionadas semanalmentepara contagem e identificação dos ovos presentes na água e na palheta. Paralelamente, analisaram-sedados meteorológicos da região. Os resultados mostraram que o Índice de Positividade deOvitrampa (IPO) variou muito (0 por cento a 90 por cento) entre as semanas/bairros amostrados, o que demonstraa grande flutuabilidade populacional do vetor. Dentre os fatores climáticos analisados, o que maisintensamente influenciou a oviposição foi a precipitação, seguida da umidade relativa. O númerode ovos encontrados na palheta foi significativamente maior que na água (p<0,05). Entretanto,observou-se aumento significativo de ovos na água quando a umidade relativa esteve abaixo de 40 por cento.Este parece ser um dado importante para a manutenção das populações em períodos de seca. O baixonúmero de ovos (<30) encontrados em 56 por cento das armadilhas positivas demonstrou a ocorrência docomportamento de skip-oviposition que pareceu contribuir para a prevalência do mosquito em todoo período amostrado. Concluiu-se que a ocorrência de oviposição na água pode ser maior do que odescrito na literatura e deve ser investigada em virtude de sua importância evolutiva e epidemiológica...