Since the appearance of the outbreak of yellow fever virus (Flaviviridae) (YFV) by Haemagogus spp. and forest related mosquitos in the Midwest region of Brazil in 2016 (Abreu et al., 2019; de Olivera et al., 2020), considered the most important sylvatic outbreak in the last eight decades, the risk of expansion from the sylvatic cycle by sylvan species of mosquitos to the urban cycle by Aedes aegypti (L‎)‎ and Aedes albopictus (Sukse) (Diptera: Culicidae) was predicted many times as the epidemic wave spread south out of the Amazon basin and approached densely populated cities (Marques et al., 1998; Massad et al., 2018; Rezende et al., 2018; Childs et al., 2019; de Olivera et al., 2020). However, no infected females of Ae. aegypti were near recent YFV foci (Abreu et al., 2020). On the other hand, the risk of YFV transmission to humans by the sylvan mosquitoes Haemagogus leucocelaenus (Dyar & Shannon) (Culicidae) and Aedes scapularis (Rondani) was associated with forest-rural-urban interfaces and green patches within the cities that harbor nonhuman primates (NHP) (Cunha et al., 2020; Sacchetto, 2020). The spatial scales used to characterize this interface-related risk range from national trends to 2 km buffers, which are useful scales for strategic programmatic planning; however, the landscape's structural patterns and mosquito behavior research in sylvan-urban ecotones at the microscale could be used to design better preventive and operational interventions at the foci level (de Thoisy et al., 2020; Wilk-da-Silva et al., 2020). Cardoso (2010) isolated YFV from Hg. leucocelaenus and Aedes serratus (Theobald) in southeastern Brazil, almost at the border with Misiones, Argentina. Abreu et al. (2019) confirmed that Hg. leucocelaenus was the main vector in central Brazil in the 2016-2018 outbreak along with Haemagogus janthinomys (Dyar). The last YF sylvatic outbreak in Argentina occurred between 2007 and 2008, with epizootics in monkeys and confirmed human cases. One of these epizootics occurred in a private reserve in Puerto Iguazú in 2007, which borders Iguazú National Park. Subsequently, five human cases and one death were confirmed in the departments of Guaraní, San Pedro and Eldorado, Misiones province (Ministerio de Salud, 2008). Although YFV was isolated in Sabethes alviprivus (Theobald) (Culicidae) in 2009 in southern Misiones (Goenaga et al., 2012), the ecoepidemiology of this disease in Argentina is unknown as well as the role of Hg. leucocelaenus and the other species considered transmitters. However, its role as a vector of YFV was reported in southern Brazil, including states bordering the province of Misiones (Cardoso et al., 2010; Abreu et al., 2019). The present study aims to report the space-time occurrence of Hg. leucocelaenus at the microscale in the wild-periurban fringe of Puerto Iguazú in Argentina, using environmentally stratified sampling.

Entomological monitoring was carried out in Puerto Iguazú (25°36′39″S; 54°34′49″W), Misiones Province (Fig. 1), in the three-country border area of Argentina, Brazil and Paraguay. This area is included in the ecoregion of the Paranaense Forest where NHP troops can move between countries and where YFV epizootics approach from the Brazilian border (SESP, 2020). The climate is subtropical with an annual average temperature of 21°C (15°C-25°C) and rainfall between 1,000 and 2,200 mm / year (Oyarzabal et al., 2018).

Monthly samples were taken from April 2019 to February 2020. The study area was classified into three types of environments as follows, based on visual analysis of satellite images and socio-demographic data (INDEC, 2010): urban densely populated areas, periurban areas with subsistence agricultural family farms, and wild environments in the public use and trail areas of Iguazú National Park (INP) (Fig. 1). Based on data collected by Silver (2008) we modified the methodology using type oviposition traps which consist of black plastic containers (1L) with the interior lined with corrugated cardboard as egg supports. The corrugated cardboard was tested in a preliminary experiment to ensure that withstands rainwater and collects eggs successfully. These traps were filled with clean water covering 50% of their capacity. The ovitraps were distributed randomly in each of the defined environmental strata, placed between 0.5 and 1.5 meters from the ground (Alencar et al., 2010). The areas from selected environments were not homogeneous in shape and size, so they were divided randomly into quadrants of 400 m² and the quadrants amount to cover the 10% necessary of the surface of each environment was selected. Then, three or four ovitraps were randomly distributed within each of the selected quadrants. A total of 35 ovitraps were placed in the urban environment with a distance to the edge from 0 m to 1,174.62 m, 30 ovitraps in the periurban environment with a distance to the edge from 45 m to 1,346 m and 16 in the wild environment with a distance from 13.55 m to 248.29 m from the edge, each remaining active for seven days per sampling. The egg supports were transferred to the laboratory dried and preserved in an incubator (19 ± 2 °C) until immersion. Larvae were fed with a standard laboratory diet (Puggioli et al., 2013) in 250 ml plastic containers until the pupal stage. Pupae were placed in smaller containers (20 ml) in emergence cages. Fourth-instar larvae that did not reach the adult stage were placed in 80% ethanol for later identification. The emerged adults were sacrificed with acetone after 48 hs and mounted as described by Rossi et al. (2006) Species identification was performed using keys of Forattini (2002) and Zavortink (1972). Preserved specimens were incorporated into the entomological collection of the National Institute of Tropical Medicine (INMeT).

Of the 81 total ovitraps, eight were positive for Hg. leucocelaenus: six in the wild environment and two in the periurban environment (Fig. 1, Table I). Regarding the time distribution, 98.5% of the occurrence was concentrated in November and December towards the beginning of the rainy season, with accumulated rainfall for these months of 110.4 mm and 220.5 mm, respectively, and average temperatures of 30.5 °C in November and 26.8 °C in December. Also, 1.5% of the captures were in May at the end of the rainy season, with accumulated rainfall of 174 mm and an average temperature of 21.4°C. Haemagogus leucocelaenus was not found in the urban environment.

Haemagogus leucocelaenus has been recorded in Puerto Iguazú in the area characterized here as wild environments since the 1940s (Duret, 1950), even in the last report, which included one specimen (25°35'28" S; 54°33'38" W) (D’Oria et al., 2010).

Our study of the occurrence of Hg. leucocelaenus confirms the habitat plasticity of this species, and also shows the potential of the females in their wild source populations to deposit fertile eggs in periurban artificial containers at least up to 300 m from the wild-periurban fringe. Abreu et al. (2019) proposed intensified human vaccination against YFV in a buffer of 12 km from the forest-urban ecotone due to the colonization capability of secondary forest patches by Haemagogus species and its flight range and endophagic behavior. However, from a metapopulation dynamics perspective, our results at the microscale suggest that intensive monitoring of wild vectors of YFV should be performed at least in a 500 m buffer from the sylvan edge. The presence of ovitraps in the periurban-wild or wild-urban edge or in its proximity without oviposition of Hg. leucocelaenus could be due to a patch colonization of the species corresponding to source and sink population dynamics (Hanski et al., 1997; Hanski, 1999). According to Alencar et al. (2013), Hg. leucocelaenus has an eclectic behavior in its feeding habits. This could be observed directly related to its mobility between the canopy and the ground in search of food or breeding sites to deposit its eggs. In the study area, the presence of Ae­. albopictus (Lizuain et al., 2019) has been proposed as a bridge between the sylvatic and domestic YFV cycles (Pereira Dos Santos et al., 2018). On the other hand, vertical transmission of YFV has been observed in Hg. janthinomys by natural infection and in Haemagogus equinus (Theobald) by experimental infection (Mondet et al., 2002), but this transmission has not been observed in field-collected Haemagogus spp. in recent outbreaks of the region (Abreu et al., 2019). Oviposition by Hg. leucocelaenus in periurban environments in our study mainly occurred at the beginning of the rainy season. The best predictor for the positivity of ovitraps and the number of eggs of Hg. leucocelaenus in Rio de Janeiro State, Brazil, was the mean temperature above 27 ºC and accumulated precipitation above 100 mm, with a time lag of four weeks between the climatic record and the ovitrap collection (Couto-Lima et al., 2020). In conclusion, our study indicates the appropriateness of the surveillance of sylvan synanthropic mosquitoes, such as Hg. leucocelaenus, with artificial containers in wild-periurban fringes and around urban green patches large enough to harbor NHP. Also, this work provides insight into the temporal and spatial risk scenario of YFV transmission in the region, based on distribution patterns as a function of the urban-periurban-wild ecotone associated with the forest border and climatic variables such as rainfall, which would affect the presence of Hg. leucocelaenus. Nonetheless, to provide better operational protocols for monitoring, additional studies at the microscale are still required, taking into account the limitations of ovitraps (Alencar et al., 2016).

ACKNOWLEDGMENTS

We thank P. Adler for their corrections and suggestions, M. Casafús and E. Burgos for their support and accompaniment, and the INMeT technicians D. Soto and J. Molina for their work at both laboratory and field.

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Notas de autor

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