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Background Phlebotomus mascittii is one of the most widespread but least studied sand fly species in Europe, occurring from Mediterranean to Central European regions. Despite its broad distribution, its potential role in Leishmania transmission remains unknown, mainly due to the lack of laboratory colonies. This study provides the first experimental assessment of the vector competence of P. mascittii for Leishmania infantum and Leishmania martiniquensis . Methods Wild-caught P. mascittii females from Styria, Austria, were experimentally infected using membrane feeding with blood containing L. infantum and L. martiniquensis isolates of different geographical origins. Infections were evaluated 7 days post-blood meal (PBM) by microscopy and polymerase chain reaction (PCR). Morphological forms of L. infantum were quantified and compared with infections in Phlebotomus perniciosus , a known competent vector. Results Fifteen (94% of dissected) P. mascittii females developed L. infantum infections, all showing colonization of the stomodeal valve, whereas L. martiniquensis failed to establish infection. Infection patterns and parasite localization closely resembled those in P. perniciosus . Morphometric analysis revealed a significantly higher proportion of metacyclic and leptomonad forms and fewer nectomonads in P. mascittii than in P. perniciosus . Conclusions Our findings demonstrate that P. mascittii supports full development of L. infantum to the transmissible metacyclic stage and colonization of the stomodeal valve, confirming its potential vector competence. This study provides the first experimental evidence on P. mascittii vectorial competence and highlights its epidemiological relevance in Europe. Graphical Abstract

We report 4 cases of equine cutaneous leishmaniasis caused by Leishmania martiniquensis in Czech Republic and Austria, outside the known endemic range of leishmaniases. The parasite should be considered as a potential cause of cutaneous lesions in horses; the risk for zoonotic transmission to immunocompromised humans is anticipated throughout central Europe.

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae) worldwide. The subgenus Adlerius is taxonomically challenging and currently comprises about 20 species with a wide geographic distribution from eastern Asia to southeastern Europe. Some species are confirmed or suspected vectors of Leishmania donovani / infantum , L. major , and L. tropica , and are thus of high medical and veterinary relevance. A single record of Phlebotomus ( Adlerius ) simici in Austria from 2018 marks its sporadic northernmost and westernmost occurrence, with the origin of its appearance remaining unclear. To better understand Adlerius diversification and particularly post-glacial spread of Ph. simici to northern parts of Europe, we combined phylogenetic analyses with climatic suitability modelling. Divergence time estimates well supported the currently observed geographic distribution of the studied species and revealed several taxonomic challenges in the subgenus. We clearly delineated three distinct genetic and geographic Ph. simici lineages and phylogeographically assessed diversification that were well supported by climatic models. This study provides a comprehensive phylogenetic analysis of the subgenus Adlerius , enhancing our understanding of the diversification in relation to changing climate of this understudied group, and we present new insights into the post-glacial spread of Ph. simici , a suspected vector of L. infantum .

Background Phlebotomine sand flies are the key vectors for phleboviruses (order Hareavirales and family Phenuiviridae), of which some are associated with febrile diseases and nervous system infections. In the Mediterranean Basin, Toscana virus (TOSV; Phlebovirus toscanaense ) and sandfly fever Sicilian viruses (SFSV; Phlebovirus siciliaense ) are important human pathogens, and their endemicity has been known for decades, particularly in the Balkan countries. While the circulation of both viruses is highly evident among humans and livestock in the Central Balkan country Kosovo, data from companion animals are scarce; however, it might help to further assess the distribution of both viruses in the country. Methods Sera of dogs from all seven districts of Kosovo were screened for TOSV and SFSV antibodies by seroneutralization assays. Results Altogether, 45 of 288 (15.6%) samples showed anti- Phlebovirus antibodies, of which 36 (12.5%) were against TOSV, 11 (3.8%) were against SFSV, and 2 (0.7%) were positive for antibodies against both viruses. Conclusions Phlebovirus seroprevalence was observed in all seven districts of the country, generally being higher for TOSV compared with SFSV. Our study presents the first assessment of neutralization-based seroprevalence of two medically important phleboviruses among dogs in the Republic of Kosovo. Although healthy dogs are unsusceptible to Phlebovirus infection, dogs with leishmaniasis can be potential amplifying hosts. Given the high number of stray dogs, frequent uncontrolled spreading of phleboviruses in dogs, and potential spillover in populated regions of the country, these findings should be taken into consideration. Graphical Abstract

Background Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis. Since 2008, the Ministry of Health and the Ministry of Environmental Protection of Israel have conducted nationwide, periodic sand fly surveys. Initially, these surveys focused on localities with known endemic transmission of cutaneous leishmaniasis, but in recent years, entomological trapping has expanded to areas where prior information on sand flies was unavailable. Here we report the first confirmed occurrence of Phlebotomus (Larroussius) orientalis (Parrot, 1936) in Israel and place the Israeli material in a comparative phylogeographic context. Methods Entomological surveys by CO 2 trapping were conducted in the Negev Desert, southern Israel, between 2020 and 2024. Morphological sand fly identification was confirmed by sequencing fragments of the mitochondrial COI and Cytb / NADH1 genes. For a newly reported species, we inferred intraspecific phylogenetic relationships and divergence times between major clades. A subset of females was additionally screened for Leishmania DNA and vertebrate blood-meal sources by real-time polymerase chain reaction (PCR) coupled with high-resolution melt analysis. Results Targeted surveys and routine surveillance in the Negev region between 2020 and 2024 yielded 269 Phlebotomus orientalis (96 males, 173 females) among other species of local sand fly fauna from multiple wadi systems in the central Negev. These detections constitute the first confirmed records of Ph. orientalis in Israel. Species identification was confirmed through both morphological examination and molecular analyses of partial COI and Cytb / NADH1 genes. Phylogenetic analysis indicated that the Israeli Ph. orientalis specimens constitute a distinct lineage that diverged from East African conspecifics during the Early to Middle Pleistocene. Blood-meal analysis of engorged Ph. orientalis females identified the European hare as a vertebrate host, and none of the tested Ph. orientalis specimens were positive for Leishmania DNA. Conclusions Phlebotomus orientalis is a confirmed vector of L. donovani , the main agent of visceral leishmaniasis in East Africa. Its detection as a distinct and apparently long-established lineage in the Negev, in a region where parasites of the L. donovani complex are already involved in cutaneous leishmaniasis transmission, highlights the need to clarify the distribution, ecology, and host preferences of Ph. orientalis in Israel. Further studies are required to characterize its spatial and seasonal occurrence, evaluate its vector competence for L. donovani and L. infantum , and assess its potential contribution to current and future leishmaniasis transmission risks.

Background Tahyna orthobunyavirus (TAHV) is a mosquito-borne virus that may cause mild flu-like symptoms or neurological symptoms in humans. It is historically associated with floodplain habitats in Central Europe, and the mammalophilic floodwater mosquito, Aedes vexans , is thought to be the principal vector. There are few contemporary reports of TAHV transmission ecology within mosquitoes or their vertebrate hosts, and virus infections are rarely reported (and probably seldom diagnosed). The objectives of this study were to survey the mosquito population for TAHV in three floodwater habitats and describe host usage by the predominant floodwater mosquito species to potentially define TAHV transmission at these foci. Methods We performed longitudinal mosquito sampling along three major rivers in eastern Austria to characterize the mosquito community in floodplain habitats, and tested for the presence of TAHV in pools of mosquitoes. We characterized TAHV rescued from mosquito pool homogenate by sequencing. We surveyed mosquito host selection by analyzing mosquito blood meals. Results We identified TAHV in two pools of Ae. vexans captured along the Leitha River. This mosquito, and other floodwater mosquitoes, used large mammals (red deer, roe deer, wild boar) as their hosts. The sequence of the rescued virus was remarkably similar to other TAHV isolates from the region, dating back to the first isolate of TAHV in 1958. Conclusions In general, we confirmed that TAHV is most likely being transmitted by Ae. vexans , although the precise contribution of vertebrate-amplifying hosts to the ecological maintenance of the virus is unclear. The pattern of host selection matches the estimated exposure of the same large mammal species in the region to TAHV based on a recent serosurvey, but hares were also hosts at the site where TAHV was detected. We also confirm humans as hosts of two floodwater mosquito species, providing a potential mechanism for spillover of TAHV or other mosquito-borne viruses. Graphical Abstract

Background Canine vector-borne pathogens (CVBP) are transmitted by arthropod vectors such as ticks, fleas, mosquitoes, and phlebotomine sand flies and are of global veterinary and medical importance. Dogs are important reservoir hosts, which may develop potentially life-threatening clinical signs. The Balkan area harbors diverse vector fauna and associated CVBPs, and data, particularly from the Republic of Kosovo, are scarce. Considering the high number of stray and privately owned dogs primarily kept outside, living in close contact with dogs might promote spillover of zoonotic pathogens to human populations. To combat these diseases, a One Health approach is required. Therefore, our study molecularly analyzed samples of dogs for CVBP. Methods Blood samples of 276 dogs originating from all seven districts of Kosovo collected from 2021 to 2022 were screened using polymerase chain reaction (PCR) and sequencing for a substantial set of pathogens, including Anaplasma spp., Babesia spp., Bartonella spp., Ehrlichia spp., Filarioidea, Hepatozoon spp., Mycoplasma spp., Rickettsia spp., and Trypanosoma spp. Prevalence rates were statistically assessed on the basis of various factors such as sex, breed, age, and district. Results In total, 150 (54.3%) dogs tested positive for at least one pathogen, comprising eight species of five genera. The most prevalent pathogens detected were Candidatus Mycoplasma haematoparvum (55; 19.9%), Hepatozoon canis (52; 18.8%), and Mycoplasma haemocanis (49; 17.8%). We also detected double (32; 11.6%) and triple (5; 1.8%) infections, with the latter involving combinations of Mycoplasma spp., Dirofilaria repens , Dirofilaria immitis , H. canis , or Babesia vulpes. In addition, prevalence rates were calculated and mapped by district. Of all included factors, significant prevalence differences were found for purebred/mixed breed dogs as well as between age groups. Conclusions This study provides the first comprehensive polymerase chain reaction (PCR)-based screening and detection of vector-borne pathogens in dogs from Kosovo and highlights the circulation of pathogens with high veterinary importance and zoonotic potential. Graphical Abstract

The mutualism between the thioautotrophic bacterial ectosymbiont Candidatus Thiobius zoothamnicola and the giant ciliate Zoothamnium niveum thrives in a variety of shallow-water marine environments with highly fluctuating sulfide emissions. To persist over time, both partners must reproduce and ensure the transmission of symbionts before the sulfide stops, which enables carbon fixation of the symbiont and nourishment of the host. We experimentally investigated the response of this mutualism to depletion of sulfide. We found that colonies released some initially present but also newly produced macrozooids until death, but in fewer numbers than when exposed to sulfide. The symbionts on the colonies proliferated less without sulfide, and became larger and more rod-shaped than symbionts from freshly collected colonies that were exposed to sulfide and oxygen. The symbiotic monolayer was severely disturbed by growth of other microbes and loss of symbionts. We conclude that the response of both partners to the termination of sulfide emission was remarkably quick. The development and the release of swarmers continued until host died and thus this behavior contributed to the continuation of the association.

Background Sand flies are principal vectors of the protozoan parasites Leishmania spp. and are widely distributed in all warmer regions of the world, including the Mediterranean parts of Europe. In Central European countries, the sand fly fauna is still under investigation. Phlebotomus mascittii , a suspected but unproven vector of Leishmania infantum , is regarded as the most widely distributed species in Europe. However, many aspects of its biology and ecology remain poorly known. The aim of this study was to provide new data on the biology and ecology of Ph. mascittii in Austria to better understand its current distribution and potential dispersal. Methods Sand flies were collected by CDC light traps at four localities in Austria for 11 (2018) and 15 weeks (2019) during the active sand fly season. Climatic parameters (temperature, relative humidity, barometric pressure and wind speed) were retrospectively obtained for the trapping periods. Sand flies were identified by a combined approach (morphology, DNA barcoding, MALDI-TOF protein profiling), and blood meals of engorged females were analysed by DNA sequencing and MALDI-TOF mass spectrometry. Results In total, 450 individuals of Ph. mascittii were caught. Activity was observed to start at the beginning of June and end at the end of August with peaks in mid-July at three locations and early August at one location. Increased activity was associated with relatively high temperatures and humidity. Also, more individuals were caught on nights with low barometric pressure. Analysis of five identified blood meals revealed chicken ( Gallus gallus ) and equine ( Equus spp.) hosts. Sand fly abundance was generally associated with availability of hosts. Conclusion This study reports unexpectedly high numbers of Ph. mascittii at selected Austrian localities and provides the first detailed analysis of its ecology to date. Temperature and humidity were shown to be good predictors for sand fly activity. Blood meal analyses support the assumption that Ph. mascittii feeds on mammals as well as birds. The study significantly contributes to understanding the ecology of this sand fly species in Central Europe and facilitates prospective entomological surveys. Graphical Abstract

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