Comparative Phylogeography and Integrative Taxonomy of Ochlerotatus caspius (Dipera: Culicidae) and Ochlerotatus dorsalis.

Given that accurately identifying pathogen vectors is vital for designing efficient mosquito control programs based on the proper surveillance of the epidemiologically important species, it has been suggested the complementary use of independently evolving genes and morphometric traits as a reliable approach for the characterization and delimitation of related species. Hence, we examined the spatial distribution of COI mtDNA and ITS2 rDNA variation from the historical perspective of Ochlerotatus caspius (Pallas, 1771) and O. dorsalis (Meigen, 1830), while simultaneously testing the utility of the two markers in integrative species delimitation when combined with phenotypic character analyses of larvae and adults. Despite the striking difference in haplotype diversity (high in COI mtDNA, low in ITS2 rDNA), no evident phylogeographic structure was apparent in the Palearctic O. caspius. The Holarctic O. dorsalis species was subdivided into two highly distinctive COI mtDNA phylogroups which corresponded to the Nearctic and Palearctic regions. Strong support for the independence of the two allopatric evolutionary lineages suggested that geographical barrier and climatic changes during Pleistocene caused vicariance of the ancestral range. COI mtDNA reliably distinguished O. caspius and O. dorsalis, while ITS2 rDNA yet again lacked the proper resolution for solving this problem. An integrative approach based on the larval and adult morphological traits have varying taxonomic applications due to their differential diagnostic values. Thus, by the implementation of an integrative taxonomic approach, we successfully detected species borders between the two epidemiologically relevant species and uncovered the presence of cryptic diversity within O. dorsalis.

The First Molecular and Phenotypic Characterization of the Invasive Population of Aedes albopictus (Diptera: Culicidae) from the Central Balkans.

Aedes (Stegomya) albopictus (Skuse 1984), the Asian tiger mosquito, represents the most invasive and one of the medically most important mosquito vectors. Although native to South East Asia, the species has recently spread globally, and was registered in the city of Novi Sad (Serbia, Central Balkans) in August 2018. We characterized the invasive population using phenotypic (wing size and shape) and molecular (nuclear, internal transcribed spacer 2- ITS2, and mitochondrial, cytochrome c oxidase subunit I- COI) markers. The results of phenotypic analyses indicated that the Serbian population could be differentiated from the native (Thailand) and invasive (Hawaii and Florida) populations due to restricted gene flow, founder effect, and supposed different strain origin. The Serbian population showed genetic homogeneity, indicative of a small founder number (bottleneck invasion model). Despite the incorporation of ITS2 GenBank sequences into the data set, neither spatial (Geneland) nor nonspatial (BAPS) genetic structuring analyses helped infer the Serbian population origin. However, the comparison of the retrieved COI haplotype with previously characterized mitogenomes indicated a temperate strain origin, capable of overwintering. Such findings suggest that the newly registered Ae. albopictus population could be able to establish itself since previous studies outlined Novi Sad as a suitable area.

First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014.

Since its first appearance in Europe, Usutu virus (USUV) diverged to several different genetic lineages. The virus was reported to date from multiple countries across Europe (Hungary, Italy, Switzerland, Spain, Germany, Czech Republic and Belgium). Considering the more frequently published impact of the virus on humans it is crucial to investigate locally circulating genetic variants and trace its evolution. We retrospectively analyzed mosquito samples from Serbia Vojvodina region, collected during 2014. In this study we report the results of the screening of 23,753 female mosquitoes (753 pools) for USUV-specific nucleic-acid. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August. Based on their partial NS5 sequence, all strains were identical, therefore we adjusted one representative strain for complete genome sequencing. Based on phylogenetic analysis the Serbian USUV sequences were most closely related to the virus that emerged in Austria in 2001, in Hungary in 2005 and was circulating until 2015 in Hungary. This data presents a wider geographic distribution of this genetic variant and provides the first genetic data from this region.

Genetic and phenotypic variation in central and northern European populations of Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera, Culicidae).

The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape genetics approach (FST , STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations, while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation-by-Distance patterns. Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low-to-moderate FST values on a broader geographical scale indicate a continuous between-population exchange of individuals, including considerable gene flow on the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed data considering population structure in the light of vector control strategies of the mosquito from public health importance.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

Genetic diversity and differentiation between Palearctic and Nearctic populations of Aedimorphus (=Aedes) vexans (Meigen, 1830) (Diptera, Culicidae).

Genetic diversity was studied at allozyme loci in two Palearctic and one Nearctic population of Aedimorphus (=Aedes) vexans, a species of public health and veterinary importance. The population from Serbia was the most polymorphic (P= 35%) with the highest observed heterozygosity (H(o) = 0.027). The lowest observed heterozygosity (H(o) = 0.010) was obtained for the Nearctic population. All analyses based on individual (STRUCTURE analysis) and population level (pairwise F(ST), Nm values, AMOVA, Nei's D value) revealed significant structuring between Nearctic and Palearctic populations, indicating a lack of gene flow and thus, the presence of independent gene pools. Taxon-specific alleles at the diagnostic Ao, Hk-2, Hk-3, Hk-4, Idh-1, and Idh-2 loci were used for identification and separation of Nearctic and Palearctic populations. Population genetics study provided valuable information on the correct distinction of Am. vexans populations and their adaptive potential that could find a future use in the studies of vector competence and development of vector-control strategies.