Change in the faunal composition of mosquitoes (Diptera: Culicidae) along a heterogeneous landscape gradient in the Brazilian Amazon.

This study aimed to evaluate the influence of different anthropic landscape profiles on the diversity and distribution of mosquito species in a rural settlement of the Brazilian Amazon. Eight field collections were conducted at 18 sampling points interspersed throughout 2020-2021. Plastic containers, bamboo internodes, and tires were used as traps to capture immature mosquitoes in three distinct habitats: forest, forest edge, and peridomicile. A total of 15,547 individuals, distributed in 26 species of culicids, were collected. The most abundant species were Culex urichii (8,376 specimens), Culex (Melanoconion) (2,473 specimens), and Aedes albopictus (1,252 specimens). Forest habitat showed the highest abundance, and forest edge showed the highest species richness. Different types of environments influenced both the abundance and richness of mosquitoes. The species composition was also significantly different between the analyzed sites, mainly between forest and peridomicile environments. The change in species dominance could largely explain this change in mosquito community composition. Haemagogus janthinomys, an important sylvatic arbovirus vector, was found in peridomicile habitats and Ae. albopictus, a vector associated with human environments, was found in forest habitats, thus providing evidence of species spillover. Our results indicated that landscape changes affect mosquito communities, influencing their richness and abundance. These changes may have implications for future arboviral outbreaks in this rural settlement due to the possible establishment of sylvatic vector species in anthropic environments.

Blood-stage antiplasmodial activity and oocyst formation-blockage of metallo copper-cinchonine complex.

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes' midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite.

Amazonian Anopheles with low numbers of oocysts transmit Plasmodium vivax sporozoites during a blood meal.

Anopheles darlingi is the main malarial vector in the Brazilian Amazon region. An. nuneztovari s.l., An. triannulatus s.l., An. evansae, and An. benarrochi s.l. do not have a defined role as malarial vectors, although they have been found to be naturally infected with Plasmodium vivax, and some develop oocysts. In this study, we evaluated the importance of low numbers of oocysts in sporozoite salivary gland invasion and transmission. Field-collected mosquitoes were experimentally infected with P. vivax. The infection rates and oocyst and sporozoite infection intensities were evaluated and compared with those of An. aquasalis. We found the highest number of oocysts in An. darlingi (mean = 39.47) and the lowest in An. nuneztovari s.l. (mean = 2). The highest number of sporozoites was observed in An. darlingi (mean = 610) and lowest in An. benarrochi s.l. (mean = 30). Plasmodium vivax DNA was detected in the saliva of all mosquito species after a blood meal. Regardless of the number of oocysts, all species transmitted sporozoites during blood meals. Considering the abundance of these mosquitoes and transmission of sporozoites, it is logical to assume that An. nuneztovari s.l. and An. triannulatus s.l. are involved in the transmission of P. vivax.

Presence of trypanosomatids, with emphasis on Leishmania, in Rodentia and Didelphimorphia mammals of a rural settlement in the central Amazon region.

BACKGROUND: Trypanosomatids are widespread and cause diseases - such as trypanosomiasis, sleeping sickness, Chagas disease, and cutaneous and visceral leishmaniasis - in animals and humans. These diseases occur in both rural and urban regions due to unplanned growth and deforestation. Thus, wild and synanthropic reservoir hosts living in residential areas are risk factors. OBJECTIVE: We aimed to evaluate the diversity of small mammals (rodents and marsupials), and the occurrence of trypanosomatids, especially Leishmania, in the rural settlement of Presidente Figueiredo, Amazonas. METHODS: Animals were collected using Sherman, Tomahawk, and Pitfall traps along 16 trails in four landscapes: continuous forest, forest with planting, planting, and peridomiciliar. Leishmania sp. was detected in liver samples by polymerase chain reaction targeting kDNA. FINDINGS: Diversity was higher in forests with planting and lower around residences. In total, 135 mammals (81 rodents and 54 marsupials covering 14 genera) were captured. Rodents presented infection rates (IR) of 74% and marsupials of 48%. Rodents in domicile landscapes presented a higher IR (92.9%), while marsupials showed a higher IR in forests (53.3%). MAIN CONCLUSIONS: The results suggest high prevalence of trypanosomatids across 12 mammalian genera possibly involved as reservoir hosts in the enzootic transmission of leishmaniasis in the Amazon's rural, peridomiciliar landscape.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Distribution and diversity of mosquitoes and Oropouche-like virus infection rates in an Amazonian rural settlement.

Mosquito diversity and disease transmission are influenced by landscape modifications, i.e., vectors and pathogens previously found only in forests are now found close to human environments due to anthropic changes. This study determined the diversity and distribution of mosquitoes in forest environments in order to analyze the potential vectors of Amazonian forest arboviruses. Mosquitoes were collected by 1) vertical stratification from forest canopy and ground areas using Hooper Pugedo (HP) light traps and human attraction and 2) horizontal stratification using HP light traps in peridomicile, forest edge, and forest environments near the Rio Pardo rural settlement, Amazonas, Brazil. A total of 3,750 mosquitoes were collected, representing 46 species. 3,139 individuals representing 46 species were sampled by vertical stratification. Both the Shannon-Weaver diversity index (H') and equitability (J') were higher in the canopy than on the ground. 611 individuals representing 13 species were sampled by horizontal stratification. H' decreased in the following order: forest edge > forest > peridomicile, and J' was greater at the forest edge and smaller in the peridomicile environment. Moreover, H' was higher for the human attraction collection method than the HP traps. A total of 671 pools were analyzed by RT-qPCR; three species were positive for Oropouche-like viruses (Ochlerotatus serratus, Psorophora cingulata, and Haemagogus tropicalis) and the minimum infection rate was 0.8%. The composition of mosquito species did not differ significantly between anthropic and forest environments in Rio Pardo. Some mosquito species, due to their abundance, dispersion in the three environments, and record of natural infection, were hypothesized to participate in the arbovirus transmission cycle in this Amazonian rural settlement.

Long-lasting infectivity of Plasmodium vivax present in malarial patient blood to Anopheles aquasalis.

Experimental studies for understanding the relationship between Plasmodium vivax and its vector hosts are difficult, because of to the lack of a long-term, in vitro continuous culture system unavailability of infected blood samples, seasonality of the disease, and the concentration of most cases in remote areas. This study evaluates the duration of the infectivity of P. vivax to Anopheles aquasalis after collecting blood from malaria-infected patients. Blood was collected from patients and stored at 4 °C and 37 °C. Every day, for 4 days, the blood was fed to An. aquasalis adult females, and a Giemsa-stained thick blood smear was mounted to account for sexual (gametocytes) and asexual (trophozoites and schizonts) stages and calculate parasitemia. Oocysts in the midgut of the mosquitoes were counted on the seventh day after feeding. Kruskal-Wallis test was used to compare the mean number of oocysts (MO) and the parasite density (PD) in each storage condition and post-infection time-points. The Mann-Whitney test was used to compare the number of oocysts for each day between temperatures. The results show that P. vivax stored at 4 °C and at 37 °C has its infectivity to An. aquasalis preserved for 2 days and 3 days, respectively. Infection rate (IR), PD and MO were higher on the day of blood collection and decreased gradually over time. The parasite density (number of parasites/µL) diminished faster at 4 °C than at 37 °C. In this study, a preservation protocol is shown for long-lasting infectivity of P. vivax in a blood sample taken from malaria-infected patients. These results show that infectivity of P. vivax stored at 4 °C and at 37 °C to An. aquasalis persist until 3 days after blood collection, but parasite density, infection rate, and mean of oocysts decreased 24h after blood collection. Since the malaria cases are increasingly far from the urban areas these results indicate that is possible, losing some infectivity, to realize experimental infections several dozen hours after the blood collection. However, it is necessary to improve the procedures for preserving P. vivax gametocytes for mosquito infection in the laboratory.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Diversity of mosquitoes (Diptera: Culicidae) collected in different types of larvitraps in an Amazon rural settlement.

Anthropogenic environments provide favorable conditions for some species, which is especially true of mosquitoes that present eclecticism at the moment of choice for the site of oviposition. In the present study, the diversity of mosquitoes was assessed by providing plastic containers, bamboo internodes, and tires in a forest, the forest edge, and peridomicile environments in a rural settlement area. Eighteen sampling points were chosen, delimited by a buffer of 200 m, placed in three environments: forest, forest edge, and peridomicile. In each environment, larvitraps were installed, separated by a minimum distance of 7 m and 1 m from the ground. A total of 10,131 immature mosquitoes of 20 species were collected. The most abundant species was Culex urichii (29.5%), followed by Trichoprosopon digitatum (27.1%), and Cx. (Melanoconion) spp. (10.4%). There was a difference in the composition of immature mosquito populations between larvitraps (p < 0.0005), and the plastic container hosted a greater diversity of species, whereas tires presented a greater abundance of individuals. The forest, forest edge, and peridomicile environments were also different with regard to diversity of immature mosquito populations (p < 0.0010). The forest edge was the environment with the greatest diversity of species, followed by the peridomicile and forest environments. In the forest and peridomicile, plastic container larvitraps had the greatest diversity, whereas the forest edge tire presented the largest number of individuals. Further, tire larvitraps collected the largest number of individuals in all environments. Ten species associated with the bamboo internode and tire were identified. The preference of species for artificial larvitraps, such as the plastic container and tire, even in wild environments was noted. These artificial objects may represent a risk factor for the population living in this region, as all vector species found in the study were present in plastic containers and tires.

Diversity of biting midges Culicoides (Diptera: Ceratopogonidae), potential vectors of disease, in different environments in an Amazonian rural settlement, Brazil.

INTRODUCTION: The Culicoides transmit a variety of pathogens. Our aim was to survey the Culicoides species occurring in an Amazonian rural settlement, comparing abundance, richness, and diversity in different environments. METHODS: Culicoides were captured using CDC light traps. The Shannon-Wiener (H') and Rényi indices were used to compare species diversity and evenness between environments, the equitability (J') index was used to calculate the uniformity of distribution among species, and similarity was estimated using the Jaccard similarity index. A permutational multivariate analysis of variance was applied to assess the influence of environment on species composition. A non-metric dimensional scale was used to represent the diversity profiles of each environment in a multidimensional space. RESULTS: 6.078 Culicoides were captured, representing 84 species (45 valid species/39 morphotypes). H' values showed the following gradient: forest > capoeira > peridomicile > forest edge. The equitability J' was greater in capoeira and forests compared to peridomiciles and the forest edge. The population compositions of each environment differed statistically, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness. Species of medical and veterinary importance were found primarily in peridomiciles: C. paraensis, vector of Oropouche virus; C. insignis and C. pusillus, vectors of Bluetongue virus; C. filariferus, C. flavivenula, C. foxi, and C. ignacioi, found carrying Leishmania DNA. CONCLUSIONS: This study indicates that diversity was higher in natural environments than in anthropized environments, while abundance and richness were highest in the most anthropized environment. These findings suggest that strictly wild Culicoides can adapt to anthropized environments.

Diversity of biting midges Culicoides (Diptera: Ceratopogonidae), potential vectors of disease, in different environments in an Amazonian rural settlement, Brazil

Abstract INTRODUCTION: The Culicoides transmit a variety of pathogens. Our aim was to survey the Culicoides species occurring in an Amazonian rural settlement, comparing abundance, richness, and diversity in different environments. METHODS: Culicoides were captured using CDC light traps. The Shannon-Wiener (H') and Rényi indices were used to compare species diversity and evenness between environments, the equitability (J') index was used to calculate the uniformity of distribution among species, and similarity was estimated using the Jaccard similarity index. A permutational multivariate analysis of variance was applied to assess the influence of environment on species composition. A non-metric dimensional scale was used to represent the diversity profiles of each environment in a multidimensional space. RESULTS: 6.078 Culicoides were captured, representing 84 species (45 valid species/39 morphotypes). H' values showed the following gradient: forest > capoeira > peridomicile > forest edge. The equitability J' was greater in capoeira and forests compared to peridomiciles and the forest edge. The population compositions of each environment differed statistically, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness. Species of medical and veterinary importance were found primarily in peridomiciles: C. paraensis, vector of Oropouche virus; C. insignis and C. pusillus, vectors of Bluetongue virus; C. filariferus, C. flavivenula, C. foxi, and C. ignacioi, found carrying Leishmania DNA. CONCLUSIONS: This study indicates that diversity was higher in natural environments than in anthropized environments, while abundance and richness were highest in the most anthropized environment. These findings suggest that strictly wild Culicoides can adapt to anthropized environments.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence.

INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence

Abstract INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon.

BACKGROUND: Asymptomatic individuals are one of the major challenges for malaria elimination programs in endemic areas. In the absence of clinical symptoms and with a lower parasite density they constitute silent reservoirs considered important for maintaining transmission of human malaria. Studies from Brazil have shown that infected individuals may carry these parasites for long periods. RESULTS: Patients were selected from three periurban endemic areas of the city of Manaus, in the western Brazilian Amazon. Symptomatic and asymptomatic patients with positive thick blood smear and quantitative real-time PCR (qPCR) positive for Plasmodium vivax were invited to participate in the study. A standardised pvs25 gene amplification by qPCR was used for P. vivax gametocytes detection. Anopheles aquasalis were fed using membrane feeding assays (MFA) containing blood from malaria patients. Parasitemia of 42 symptomatic and 25 asymptomatic individuals was determined by microscopic examination of blood smears and qPCR. Parasitemia density and gametocyte density were assessed as determinants of infection rates and oocysts densities. A strong correlation between gametocyte densities (microscopy and molecular techniques) and mosquito infectivity (P < 0.001) and oocysts median numbers (P < 0.05) was found in both groups. The ability to infect mosquitoes was higher in the symptomatic group (41%), but infectivity in the asymptomatic group was also seen (1.42%). CONCLUSIONS: Although their infectivity to mosquitoes is relatively low, given the high prevalence of P. vivax asymptomatic carriers they are likely to play and important role in malaria transmission in the city of Manaus. The role of asymptomatic infections therefore needs to be considered in future malaria elimination programs in Brazil.

Lutzomyia umbratilis from an area south of the Negro River is refractory to in vitro interaction with Leishmania guyanensis

BACKGROUND Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.

Lutzomyia umbratilis from an area south of the Negro River is refractory to in vitro interaction with Leishmania guyanensis.

BACKGROUND: Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES: This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS: Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS: Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS: The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes.

BACKGROUND: Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES: In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS: Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS: The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION: Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

Analysis of the genetic structure of allopatric populations of Lutzomyia umbratilis using the period clock gene.

In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and an isolated population has been identified in the state of Pernambuco, Northeastern region. This study assessed the phylogeographic structure of three allopatric Brazilian populations of L. umbratilis. Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the period gene. Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and one clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations revealed high indices of interpopulational divergence. Phylogenetic analysis indicated that geographical distance and environmental differences have not modified the ancestral relationship shared by the Recife and Rio Preto da Eva populations. Genetic similarities suggest that, in evolutionary terms, these populations are more closely related to each other than to the Manacapuru population. These results confirm the existence of an L. umbratilis species complex composed of at least two incipient species.

Phenotypic and genotypic variations among three allopatric populations of Lutzomyia umbratilis, main vector of Leishmania guyanensis.

BACKGROUND: In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and in the state of Pernambuco, Northeastern region, where an isolated population has been identified. This study assessed the phylogeographic structure and size and shape differences of the wing of three Brazilian populations. METHODS: Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the Cytochrome Oxidase I mitochondrial gene. Geometrics morphometry analysis of the right wing shape of the three populations was made using discriminate canonical analysis. RESULTS: Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and the other clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations generated high indices of interpopulational divergence. Geometric morphometry analysis indicated two distinct groups between the studied populations. Canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and both populations were genetically distant from Manacapuru. CONCLUSION: The polymorphic sites and geometric morphometry analysis indicate that the distance, lack of continuity and environmental differences have not modified the ancestral relationship between Recife and Rio Preto da Eva populations. The genetic and morphological similarities shared by the Recife and Rio Preto da Eva populations suggest that these populations are more closely related evolutionarily. These results confirm the existence of an L. umbratilis species complex in the North and Northeast regions.