Where boundaries become bridges: Mosquito community composition, key vectors, and environmental associations at forest edges in the central Brazilian Amazon.

Risk of spillover and spillback of mosquito-borne viruses in the neotropics, including yellow fever, dengue, Zika (Flaviviridae: Flavivirus), chikungunya, and Mayaro (Togaviridae: Alphavirus) viruses, is highest at ecotones where humans, monkeys, and mosquitoes coexist. With a view to identifying potential bridge vectors, we investigated changes in mosquito community composition and environmental variables at ground level at distances of 0, 500, 1000, and 2000 m from the edge of a rainforest reserve bordering the city of Manaus in the central Brazilian Amazon. During two rainy seasons in 2019 and 2020, we sampled 9,467 mosquitoes at 244 unique sites using BG-Sentinel traps, hand-nets, and Prokopack aspirators. Species richness and diversity were generally higher at 0 m and 500 m than at 1000 m and 2000 m, while mosquito community composition changed considerably between the forest edge and 500 m before stabilizing by 1000 m. Shifts in environmental variables mainly occurred between the edge and 500 m, and the occurrence of key taxa (Aedes albopictus, Ae. scapularis, Limatus durhamii, Psorophora amazonica, Haemagogus, and Sabethes) was associated with one or more of these variables. Sites where Ae. aegypti and Ae. albopictus were detected had significantly higher surrounding mean NDBI (Normalized Difference Built-up Index) values than sites where they were not detected, while the opposite was true for Sabethes mosquitoes. Our findings suggest that major changes in mosquito communities and environmental variables occur within 500 m of the forest edge, where there is high risk for contact with both urban and sylvatic vectors. By 1000 m, conditions stabilize, species diversity decreases, and forest mosquitoes predominate. Environmental variables associated with the occurrence of key taxa may be leveraged to characterize suitable habitat and refine risk models for pathogen spillover and spillback.

Towards the Laboratory Maintenance of Haemagogus janthinomys (Dyar, 1921), the Major Neotropical Vector of Sylvatic Yellow Fever.

Haemagogus (Haemagogus) janthinomys (Dyar, 1921), the major neotropical vector of sylvatic yellow fever virus, is notoriously difficult to maintain in captivity. It has never been reared beyond an F1 generation, and almost no experimental transmission studies have been performed with this species since the 1940s. Herein we describe installment hatching, artificial blood feeding, and forced-mating techniques that enabled us to produce small numbers of F3 generation Hg. janthinomys eggs for the first time. A total of 62.8% (1562/2486) F1 generation eggs hatched during ≤10 four-day cycles of immersion in a bamboo leaf infusion followed by partial drying. Hatching decreased to 20.1% (190/944) in the F2 generation for eggs laid by mosquitoes copulated by forced mating. More than 85% (79/92) female F2 mosquitoes fed on an artificial blood feeding system. While we were unable to maintain a laboratory colony of Hg. janthinomys past the F3 generation, our methods provide a foundation for experimental transmission studies with this species in a laboratory setting, a critical capacity in a region with hyper-endemic transmission of dengue, Zika, and chikungunya viruses, all posing a risk of spillback into a sylvatic cycle.

Microclimate and the vertical stratification of potential bridge vectors of mosquito­borne viruses captured by nets and ovitraps in a central Amazonian forest bordering Manaus, Brazil.

In the Americas, some mosquito-borne viruses such as Zika, chikungunya, and dengue circulate among humans in urban transmission cycles, while others, including yellow fever and Mayaro, circulate among monkeys in sylvatic cycles. The intersection of humans and wildlife at forest edges creates risk for zoonotic virus exchange. We built a scaffold tower at the edge of a treefall gap in rainforest bordering Manaus, Brazil, to identify vectors that may bridge transmission between humans and monkeys. We vertically sampled diurnally active, anthropophilic mosquitoes using handheld nets at 0, 5, and 9 m and container-breeding mosquitoes in ovitraps at 0, 5, 10, and 15 m. Haemagogus janthinomys and Psorophora amazonica were present in high relative abundance in nets at each height sampled, while anthropophilic species were uncommon in ovitraps. Hg. janthinomys was more abundant at elevated heights than at ground level, while Ps. amazonica abundance was not significantly stratified across heights. The presence of each species increased with increasing 7-day rainfall lagged at 1 week, and at 1 and 4 weeks prior to collection, respectively. In addition, Hg. janthinomys was most frequently collected at 29.9 °C, irrespective of height. These data provide insight into the potential role of each species as bridge vectors.

The vertical stratification of potential bridge vectors of mosquito-borne viruses in a central Amazonian forest bordering Manaus, Brazil.

The emergence of Zika virus (ZIKV) in Latin America brought to the fore longstanding concerns that forests bordering urban areas may provide a gateway for arbovirus spillback from humans to wildlife. To bridge urban and sylvatic transmission cycles, mosquitoes must co-occur with both humans and potential wildlife hosts, such as monkeys, in space and time. We deployed BG-Sentinel traps at heights of 0, 5, 10, and 15 m in trees in a rainforest reserve bordering Manaus, Brazil, to characterize the vertical stratification of mosquitoes and their associations with microclimate and to identify potential bridge vectors. Haemagogus janthinomys and Sabethes chloropterus, two known flavivirus vectors, showed significant stratification, occurring most frequently above the ground. Psorophora amazonica, a poorly studied anthropophilic species of unknown vector status, showed no stratification and was the most abundant species at all heights sampled. High temperatures and low humidity are common features of forest edges and microclimate analyses revealed negative associations between minimum relative humidity, which was inversely correlated with maximum temperature, and the occurrence of Haemagogus and Sabethes mosquitoes. In this reserve, human habitations border the forest while tamarin and capuchin monkeys are also common to edge habitats, creating opportunities for the spillback of mosquito-borne viruses.

Into the woods: Changes in mosquito community composition and presence of key vectors at increasing distances from the urban edge in urban forest parks in Manaus, Brazil.

Mosquito-borne Zika virus (ZIKV) was recently introduced into the Americas and now has the potential to spill back into a sylvatic cycle in the region, likely involving non-human primates and Aedes, Haemagogus, and Sabethes species mosquitoes. We investigated potential routes of mosquito-borne virus exchange between urban and sylvatic transmission cycles by characterizing mosquito communities in three urban forest parks that receive heavy traffic from both humans and monkeys in Manaus, Brazil. Parks were stratified by both distance from the urban-forest edge (0, 50, 100, and 500 m) and relative Normalized Difference Vegetation Index (NDVI) (low, medium, or high), and mosquitoes were sampled at randomly selected sites within each stratum using BG-Sentinel traps. Additionally, temperature, relative humidity, and other environmental data were collected at each site. A total of 1,172 mosquitoes were collected from 184 sites sampled in 2018, of which 98 sites were resampled in 2019. Using park as the unit of replication (i.e. 3 replicates per sampling stratum), a two-way ANOVA showed no effect of distance or NDVI on the mean number of identified species (P > 0.05 for both comparisons) or on species diversity as measured by the Shannon-Wiener diversity index (P > 0.10 for both comparisons). However, the Morisita overlap index revealed that mosquito communities changed substantially with increasing distance from edge, with communities at 0 m and 500 m being quite distinct. Aedes albopictus and Ae. aegypti penetrated at least 100 m into the forest, while forest specialists including Haemagogus janthinomys, Sabethes glaucodaemon, and Sa. tridentatus were detected in low numbers within 100 m from the forest edge. Trichoprosopon digitatum and Psorophora amazonica were among the most abundant species collected, and both showed distributions extending from the forest edge to its interior. Our results show overlapping distributions of urban and forest mosquitoes at park edges, which highlights the risk of arbovirus exchange via multiple bridge vectors in Brazilian urban forest parks. These parks may also provide refugia for both Ae. albopictus and Ae. aegypti from mosquito control programs.