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.

Department for Vector Borne Diseases Austrian Agency for Health and Food Safety Vienna Austria

Department of Parasitology Faculty of Science Charles University Prague Prague Czechia

Division of Science Research and Development Federal Ministry of Defence Vienna Austria

Independent Researcher Karlsdorf Neuthard Germany

Institute of Specific Prophylaxis and Tropical Medicine Center for Pathophysiology Infectiology and Immunology Medical University Vienna Vienna Austria

Sustainability Solutions Research Lab University of Pannonia Veszprém Hungary

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