Characterization and Host-Feeding Patterns of s.l. Taxa in a West Nile Virus-Endemic Area in Southeastern Romania.

sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of namely and , are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for , and human settings and animal farmlands for . In the latter habitats, and were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by biotype, while was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of . These findings highlight the role of biotype as enzootic/epizootic vector, and specifically show as the bridge vector for WNV. The and biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.