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Trypanosomatid flagellates have not been studied in Austria in any detail. In this study, specific nested PCR, targeted on the ribosomal small subunit, was used to determine the occurrence and diversity of trypanosomatids in wild-caught mosquitoes sampled across Eastern Austria in the years 2014-2015. We collected a total of 29,975 mosquitoes of 19 species divided in 1680 pools. Of these, 298 (17.7%), representing 12 different mosquito species, were positive for trypanosomatid DNA. In total, seven trypanosomatid spp. were identified (three Trypanosoma, three Crithidia and one Herpetomonas species), with the highest parasite species diversity found in the mosquito host Coquillettidia richiardii. The most frequent parasite species belonged to the mammalian Trypanosoma theileri/cervi species complex (found in 105 pools; 6.3%). The avian species T. culicavium (found in 69 pools; 4.1%) was only detected in mosquitoes of the genus Culex, which corresponds to their preference for avian hosts. Monoxenous trypanosomatids of the genus Crithidia and Herpetomonas were found in 20 (1.3%) mosquito pools. One third (n = 98) of the trypanosomatid positive mosquito pools carried more than one parasite species. This is the first large scale study of trypanosomatid parasites in Austrian mosquitoes and our results are valuable in providing an overview of the diversity of these parasites in Austria.

Aedes pulcritarsis is a tree-hole breeding species with its main distribution in the Mediterranean area. Within the scope of two independent monitoring programmes, this mosquito species was detected for the first time in Austria, in the province of Lower Austria (2018, districts Mistelbach and Gaenserndorf; 2020, district Bruck an der Leitha). As the climatic and habitat situation in Central Europe seems to be generally suitable for this species, the most likely explanation for the species not being recorded previously is that it might have been overlooked in the past due to its specialized breeding habitat. However, further research on the distribution of Ae. pulcritarsis in Austria would be needed to support this hypothesis. The results from this study will contribute to the investigation of the northern distribution limit of Ae. pulcritarsis in Europe and possible changes thereof.

Mosquitoes of the genus Culex are important vectors of a variety of arthropod-borne viral infections. In most of the northern parts of the USA, Cx. pipiens/restuans is the predominant representative of this genus. As vectors, they play a key role in the spreading of arboviruses and thus, knowledge of the population dynamic of mosquitoes is important to understand the disease ecology of these viruses. As poikilotherm animals, the vital rates of mosquitoes are highly dependent on ambient temperature, and also on precipitation. We present a compartmental model for the population dynamics of Cx. pipiens/restuans. The model is driven by temperature, precipitation, and daytime length (which can be calculated from the geographic latitude). For model evaluation, we used long-term mosquito capture data, which were averaged from multiple sites in Cook County, Illinois. The model fitted the observation data and was able to reproduce between-year differences in the abundance of the Cx. pipiens/restuans mosquitoes, as well as the different seasonal trends. Using this model, we evaluated the effectiveness of targeting different vital rates for mosquito control strategies. The final model is able to reproduce the weekly mean Cx. pipiens/restuans abundance for Cook County with a high accuracy, and over a long time period of 20 years.

In the past few decades, the relevance of Dirofilaria immitis and Dirofilaria repens, causing cardiopulmonary and subcutaneous dirofilariosis in dogs and cats, and of Angiostrongylus vasorum, causing canine angiostrongylosis, has steadily increased in Central and Northern Europe. In this review, a summary of published articles and additional reports dealing with imported or autochthonous cases of these parasites is provided for Central (Austria, Czechia, Germany, Hungary, Luxemburg, Poland, Slovakia, Slovenia, and Switzerland) and Northern (Denmark, Finland, Iceland, Norway, and Sweden) Europe. Research efforts focusing on Dirofilaria spp. and A. vasorum have varied by country, and cross-border studies are few. The housing conditions of dogs, pet movements, the spread of competent vectors, and climate change are important factors in the spread of these nematodes. Dogs kept outside overnight are a major factor for the establishment of Dirofilaria spp. However, the establishment of invasive, diurnal, synanthropic, competent mosquito vectors such as Aedes albopictus may also influence the establishment of Dirofilaria spp. The drivers of the spread of A. vasorum remain not fully understood, but it seems to be influenced by habitats shared with wild canids, dog relocation, and possibly climatic changes; its pattern of spreading appears to be similar in different countries. Both Dirofilaria spp. and A. vasorum merit further monitoring and research focus in Europe.

Native to East Asia, the Asian bush mosquito ( Aedes japonicus ) has recently expanded its global range, with established invasive populations in Europe and North America. Given its potential role as a vector of various arboviruses, understanding its invasion process and ecological dynamics is crucial for managing its spread and mitigating public health risks. In the Iberian Peninsula, the species was first detected in Asturias in 2018 and has since expanded to neighbouring regions. Here, we elucidate the invasion pathways and possible origins of Ae. japonicus populations in Spain using sequence data and microsatellite markers, and by screening for the presence of maternally transmitted bacteria of the genus Wolbachia . We analysed 635 Ae. japonicus from 14 countries, including Japan (native range), the United States, and 12 European countries. No clear association between haplotypes and geographical location was detected in any of the three genes analysed (nuclear ITS2, mitochondrial COI and ND4). Wolbachia was not detected in any of the screened samples. In contrast, microsatellite-based population structure analyses showed that most Spanish samples clustered closely with those from College Park, Maryland (USA), located near the Port of Baltimore, one of the largest ports in the United States and a recognised gateway for invasive species introductions. Northern Spain hosts major seaports such as Bilbao and Gijón, whereas the nearest established Ae. japonicus population in Europe lies over 1,000 km away in northeastern France. Taken together, these findings suggest that the most plausible invasion route of Ae. japonicus into Spain involves maritime transport from the eastern coast of the United States to northern Spanish ports, likely accompanied by additional minor introductions of European origin. The inclusion of additional microsatellite loci originally developed for Ae. albopictus yielded results consistent with those obtained using Ae. japonicus -specific loci, reinforcing the robustness of the observed patterns. This work provides new insights into the invasion process of Ae. japonicus in Europe and highlights the need for continuous monitoring and tailored interventions at key ports of entry.

Background Insect vectors, namely mosquitoes (Diptera: Culicidae), are compulsory for malaria parasites ( Plasmodium spp.) to complete their life cycle. Despite this, little is known about vector competence of different mosquito species for the transmission of avian malaria parasites. Methods In this study, nested PCR was used to determine Plasmodium spp. occurrence in pools of whole individuals, as well as the diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across Eastern Austria in 2013–2015. Results A total of 45,749 mosquitoes in 2628 pools were collected, of which 169 pools (6.43%) comprising 9 mosquito species were positive for avian Plasmodium , with the majority of positives in mosquitoes of Culex pipiens s.l./ Culex torrentium . Six different avian Plasmodium lineages were found, the most common were Plasmodium vaughani SYAT05, Plasmodium sp. Linn1 and Plasmodium relictum SGS1. In 2014, mosquitoes of the Culex pipiens complex were genetically identified and Culex pipiens f. pipiens presented with the highest number of avian Plasmodium positives (n = 37; 16.74%). Despite this, the minimum infection rate (MIR) was highest in Culex torrentium (5.36%) and Culex pipiens f. pipiens/ f. molestus hybrids (5.26%). During 2014 and 2015, seasonal and annual changes in Plasmodium lineage distribution were also observed. In both years P. vaughani SYAT05 dominated at the beginning of the sampling period to be replaced later in the year by P. relictum SGS1 (2014) and Plasmodium sp. Linn1 (2015). Conclusions This is the first large-scale study of avian Plasmodium parasites in Austrian mosquitoes. These results are of special interest, because molecular identification of the taxa of the Cx. pipiens complex and Cx. torrentium enabled the determination of Plasmodium prevalence in the different mosquito taxa and hybrids of this complex. Since pools of whole insects were used, it is not possible to assert any vector competence in any of the examined mosquitoes, but the results are nonetheless valuable in providing an overview of avian Plasmodium species and lineages present in Austria.

Background Sand flies are vectors of the protozoan Leishmania spp. and phleboviruses. In Europe, several species are widely distributed in the Mediterranean region and a northward spread can be observed. They can be found regularly also in some regions of Central Europe, with Phlebotomus mascittii being the most cold-tolerant and northerly distributed species, but the knowledge on their distribution in countries such as Germany, Austria, Czechia, Slovakia, and Hungary remains fragmentary because of a lack of comprehensive field studies and a poor understanding of the ecological requirements and phylogeographic history. Methods A comprehensive literature review of sand fly occurrence in five Central European countries was complemented by entomological surveys, including sand fly and rodent screening for sand fly-borne pathogens. Nucleic acid extraction, COI barcoding, blood meal analysis, and phylogenetic and environmental analyses incorporating unsupervised machine learning techniques were conducted. Results This study significantly advances the understanding of the current distribution of six sand fly species in Central Europe. Among them, only Ph. mascittii was present in all analyzed countries, except Czechia, with its seasonal activity peaking in July. Phlebotomus papatasi , Ph. perfiliewi and Ph. neglectus were recorded in Hungary, while Ph. perniciosus and Phlebotomus simici were found in Germany and Austria, respectively. Although Leishmania DNA was absent in sand flies and rodents, DNA from two distinct Trypanosoma lineages was detected in several specimens, suggesting Ph. mascittii feeds on both birds and ruminants. Trypanosomatid lineages identified in local rodents differed, indicating distinct lineages between sand flies and rodents. Environmental analysis identified 15 Corine land cover classes associated with sand fly presence, with urban locations being the most frequently occupied. Linear regression models comparing presence versus absence revealed significantly higher sand fly presence in forested and urban landscapes. Furthermore, Ph. mascittii populations formed four distinct ecological clusters, which broadly grouped into two main geographic groups: one in the Upper Rhine Valley of southwestern Germany and the other spanning the Carpathian Basin. Conclusions This study provides new insights into the current distribution, ecological preferences, seasonal activity, and potential vector capacity of sand fly species in Central Europe. Graphical Abstract

Background Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. Methods For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus , Aedes japonicus, Aedes koreicus, and Aedes geniculatus . For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. Results Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. Conclusions In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps. Graphical Abstract

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.

In Austria, only fragmented information on the occurrence of alien and potentially invasive mosquito species exists. The aim of this study is a nationwide overview on the situation of those mosquitoes in Austria. Using a nationwide uniform protocol for the first time, mosquito eggs were sampled with ovitraps at 45 locations in Austria at weekly intervals from May to October 2020. The sampled eggs were counted and the species were identified by genetic analysis. The Asian tiger mosquito Aedes albopictus was found at two sites, once in Tyrol, where this species has been reported before, and for the first time in the province of Lower Austria, at a motorway rest stop. The Asian bush mosquito Aedes japonicus was widespread in Austria. It was found in all provinces and was the most abundant species in the ovitraps by far. Aedes japonicus was more abundant in the South than in the North and more eggs were found in habitats with artificial surfaces than in (semi-) natural areas. Further, the number of Ae. japonicus eggs increased with higher ambient temperature and decreased with higher wind speed. The results of this study will contribute to a better estimation of the risk of mosquito-borne disease in Austria and will be a useful baseline for a future documentation of changes in the distribution of those species.