Xenomonitoring of Mosquitoes (Diptera: Culicidae) for the Presence of Filarioid Helminths in Eastern Austria

Information on mosquito-borne filarioid helminths in Austria is scarce, but recent discoveries of Dirofilaria repens indicate autochthonous distribution of this parasite in Eastern Austria. In the current xenomonitoring study, more than 48,000 mosquitoes were collected in Eastern Austria between 2013 and 2015, using different sampling techniques and storage conditions, and were analysed in pools with molecular tools for the presence of filarioid helminth DNA. Overall, DNA of D. repens, Setaria tundra, and two unknown filarioid helminths were documented in twenty mosquito pools within the mitochondrial cox1 gene (barcode region). These results indicate that S. tundra, with roe deer as definite hosts, is common in Eastern Austria, with most occurrences in floodplain mosquitoes (e.g., Aedes vexans). Moreover, DNA of D. repens was found in an Anopheles plumbeus mosquito close to the Slovakian border, indicating that D. repens is endemic in low prevalence in Eastern Austria. This study shows that xenomonitoring is an adequate tool to analyse the presence of filarioid helminths, but results are influenced by mosquito sampling techniques, storage conditions, and molecular protocols.


Introduction
In Europe, filarioid helminths of veterinary and/or medical relevance have mainly been documented in Mediterranean regions, but increasingly these pathogens are being reported in temperate climate zones in Central and Northern Europe as well [1][2][3].
Mosquito-borne filarioid helminths of the genus Setaria mainly parasitize in the abdominal cavities of artiodactyls, hyracoids, and equines. Mosquitoes of the genus Aedes are thought to be the main vectors of these parasites (e.g., Ae. vexans for S. labiatopapillosa) [12,13]. Setaria tundra is a parasite of roe deer documented in several European countries such as Austria, Switzerland, Germany, France, Italy, Hungary, Poland, Spain, and Denmark (summarized in Enemark et al. [14]). In Northern Europe, S. tundra can also be found in domestic reindeer, wild forest reindeer, and moose [15]. is species is associated with climate changes and causes severe outbreaks of periodontitis in semidomestic reindeer in Finland [16]. e aim of this study was to xenomonitor Eastern Austrian mosquitoes for the presence of DNA of filarioid helminths-with the main focus on Dirofilaria repens and D. immitis but also on Setaria tundra and other mosquitoborne filarioid helminths.

Materials and Methods
e present study combines the analysis for filarioid helminth DNA in mosquitoes sampled in two independent mosquito monitoring programs using two different storage conditions (dry and −80°C) conducted between 2013 and 2015. was carried out on a daily basis for 24 hours from March to October. Mosquitoes were killed using the insecticide dichlorvos as soon as they entered the trap. Once a week, the traps were emptied, and Culicidae were dried and stored at room temperature until further processing [17].

Mosquito Sampling Method 2.
Mosquitoes were monitored across three provinces of Eastern Austria (Burgenland, Lower Austria, and Vienna) at 35 permanent and 23 nonpermanent trapping sites. At permanent sampling sites, mosquitoes were monitored on a regular basis every second week for a 24-hour time period from April to October 2014-2015 using Biogents Sentinel Traps (Regensburg, Germany) equipped with carbon dioxide as attractant. Nonpermanent sampling sites were investigated at least once and up to six times during the summer months using Biogents Sentinel Traps (Regensburg, Germany) or exhausters. All mosquitoes were stored at −80°C until further processing [18].
Mosquitoes were identified morphologically using the identification key of Becker et al. [19] and pooled by species, collection site, and date, with a maximum number of 50 individuals per pool. To each pool, 400 µl of DNA/RNA lysis buffer (Zymo Research Corp., USA) and two ceramic beads (Precellys Ceramic Beads, Peqlab Biotechnologie GmbH) were added, and the samples were homogenized in a TissueLyser II (Qiagen, Germany). Approximately 350 µl of  [20]. e primers used target a 724 bp fragment of the mitochondrial cytochrome oxidase subunit I gene and are specific for various filarioid helminths (e.g., Dirofilaria, Wuchereria, Brugia, Onchocerca, Setaria, and Acanthocheilonema). PCR products were separated by electrophoresis in 2% agarose gels stained with Midori Green Advance DNA stain (Nippon Genetics Europe, Germany). Finally, purified PCR products were sequenced by a commercial company (LGC Genomics GmbH, Germany). Sequences thus obtained were compared for similarity to sequences available in GenBank ® database (http://www.ncbi.nlm.nih.gov/BLAST).

Results and Discussion
45,848 mosquitoes representing 25 mosquito species were analysed for the presence of filarioid DNA in this xenomonitoring survey (Table 1), resulting in the identification of DNA from D. repens, S. tundra, and two unknown filarioid helminths in 20 of the mosquito pools (Table 2; Figure 1). DNA of D. repens was only found in 2015 in a single Anopheles plumbeus mosquito in Marchegg (Lower Austria) close to the Slovakian border. Although several mosquito Hook Company CO 2 baited mosquito traps; b analysis of maximum identity to GenBank Entries was performed on August 4, 2017; c sequences were not uploaded to GenBank (e.g., short sequences or poor sequence quality).
species of different genera are proven as potential vectors of D. repens [21], DNA of this parasite has so far only been detected in other Anopheles species (An. algeriensis and An. maculipennis complex) in Austria [2]. To date, all D. repens positive mosquitoes have been collected in close proximity to the Slovakian (this study) and the Hungarian borders [2]. In both Slovakia and Hungary, D. repens is known to be endemic with a prevalence above 10% in dogs in the Bratislava area, close to the Austrian border [22,23]. Previous metadata analysis has shown that most reported but also potential autochthonous findings in dogs were in Eastern Austria [10]. Furthermore, Duscher et al. [24] described the examination of D. repens positive dogs in the same districts where positive mosquitoes were documented (Gänserndorf and Neusiedl am See). is indicates that D. repens might be endemic with low prevalence in this area. Simon et al. [4] postulated that two preconditions are required for a successful establishment of D. repens and D. immitis in a novel area: (i) the presence of competent mosquito vectors, which is the case in Austria, and (ii) a certain number of positive dogs shedding microfilaria. e second precondition seems to limit the distribution of D. repens (but also D. immitis) because there are almost no stray dogs, and kennel holding is not common in Austria.
Dirofilaria immitis was not identified in the present large-scale survey, confirming previous results that this parasite has not yet established itself in Eastern Austria [10].
is pathogen has however been confirmed in dogs [22,23,25] and in mosquitoes [26,27] in Slovakia and Hungary in the vicinity of our study area. e most commonly found filarioid helminth within the present study area in Eastern Austria was S. tundra, with most occurrences of S. tundra DNA in mosquitoes of the genus Aedes, especially Ae. vexans. Similarly, prevalences of up to 12.3% have been reported in roe deer in Central Europe (e.g., northern Bavaria [28]). is parasite has also been recorded in Ae. vexans in studies in Germany and Hungary [1,27,29,30], suggesting that S. tundra is a common parasite of roe deer in Eastern Austria. e discovery of DNA of unknown filarioid helminths in ornithophilic Culex mosquitoes (Cx. modestus and Cx. pipiens complex) is not surprising because several avian filarioid helminths (with low pathogenicity for bird hosts) are present in Central Europe [29].

Conclusions
is xenomonitoring survey confirms the presence of DNA of certain filarioid helminths in mosquitoes in Eastern Austria and indicates possible vector competence of select mosquito species. However, comparison of the two sampling techniques and storage schemes used here suggests that storage of dried mosquitoes at room temperature increases the number of false negative pools because of a decrease of DNA quality, a circumstance that has also been noticed during analyses of mosquito DNA itself [31]. Moreover, different techniques for mosquito sampling (certain mosquito species are attracted by certain traps) and the use of different PCR protocols also influence the outcome of xenomonitoring studies [32]. Nevertheless, xenomonitoring is an effective tool to examine if certain pathogens are present in an area (e.g., [27]). It can be concluded that D. repens, S. tundra, and unknown filarioid helminths (most probably avian parasites) are present in Eastern Austria. Further studies are needed to monitor in more detail the situation of D. repens and D. immitis in Austria and neighbouring countries.

Conflicts of Interest
e authors declare that they have no conflicts of interest.