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While previous studies have examined the elevational limits of Ixodes ricinus in Central Europe, this study is the first to investigate the influence of slope orientation on tick distribution in Slovenia. Our results provide new insights into how two important microclimatic factors, temperature and humidity, vary between the northern and southern slopes, and how these differences affect the abundance of I. ricinus , a factor that has not been studied in this region before. We found that nymph density was highest on the southern slope at intermediate elevations (720 m) and increased with temperature. In contrast, fewer adult ticks were found on the southern slope compared to the northern slope, most of them at 720 m elevations. The significantly higher abundance of adult ticks on the northern slope is probably related to the cooler temperatures, higher relative humidity, denser ground vegetation and greater availability of hosts such as roe deer. Although these results are regionally specific, they contribute to a more comprehensive understanding of the ecological factors influencing the distribution of I. ricinus in Central Europe. Des études antérieures ont examiné les limites d’altitude d’ Ixodes ricinus en Europe centrale, mais cette étude est la première à examiner l’influence de l’orientation des pentes sur la répartition des tiques en Slovénie. Nos résultats apportent de nouvelles informations sur la façon dont deux facteurs microclimatiques importants, la température et l’humidité, varient entre les versants nord et sud, et sur l’impact de ces différences sur l’abondance d’ I. ricinus , un facteur qui n’avait jamais été étudié dans cette région auparavant. Nous avons constaté que la densité de nymphes était maximale sur le versant sud, à des altitudes intermédiaires (720 m), et qu’elle augmentait avec la température. En revanche, moins de tiques adultes ont été trouvées sur le versant sud que sur le versant nord, la plupart à 720 m d’altitude. L’abondance significativement plus élevée de tiques adultes sur le versant nord est probablement liée aux températures plus fraîches, à une humidité relative plus élevée, à une végétation au sol plus dense et à une plus grande disponibilité d’hôtes tels que les chevreuils. Bien que ces résultats soient spécifiques à chaque région, ils contribuent à une compréhension plus complète des facteurs écologiques influençant la distribution d’ I. ricinus en Europe centrale.

Background Sand flies (Diptera: Phlebotominae) are vectors of Leishmania spp., protozoan parasites that cause leishmaniasis, a zoonosis endemic in the Mediterranean region. Although Slovenia is not considered endemic, its proximity to affected areas and the presence of competent vectors underscore the importance of entomological surveillance. As part of the CLIMOS project, we investigated sand fly species composition, seasonal abundance, and the presence of Leishmania parasites at two sites in southwestern Slovenia: Cetore and Velike Žablje. Methods From May to October 2023, adult sand flies were collected using Centers for Disease Control and Prevention (CDC) miniature light traps. Specimens were morphologically identified and screened for Leishmania spp. DNA using quantitative polymerase chain reaction (PCR) targeted kinetoplast DNA, followed by species confirmation with ITS-1 nested PCR and restriction fragment length polymorphism (RFLP) analysis. Results A total of 274 sand flies were collected, predominantly Phlebotomus neglectus , P. perniciosus , and P. mascittii . Abundance peaked in July in Cetore and August in Velike Žablje, likely reflecting local climatic differences. Among 25 pools of female sand flies analyzed, Leishmania DNA was detected in three pools (12.0%), specifically in P. neglectus and P. mascittii . The cycle threshold values (Ct 35–37) indicated low parasite DNA loads. ITS-1 amplification was unsuccessful, likely due to low DNA concentration. Conclusions This study provides the first molecular evidence of Leishmania DNA in sand flies from Slovenia. The detection of Leishmania in P.  mascittii , a species with uncertain vector competence, is particularly noteworthy and warrants further investigation. Although species identification of the parasite was not possible, the findings suggest potential for autochthonous transmission and highlight the need for continued surveillance and research in the region. Graphical Abstract

Mosquitoes are of public health interest as many of them are vectors of pathogens that can cause diseases in humans. If they are not perfectly preserved, their identification can be difficult. Therefore, we tested the use of an alternative, advanced method of geometric morphometrics for the analysis of wing morphological variability in five ( Anopheles , Aedes , Coquillettidia , Culex , Culiseta ) genera and ten Slovenian mosquito species alone, seven autochthonous and three invasive. We analysed the intragenic and intraspecific variability in wing size and shape. The results showed that the analysed mosquito species differ in wing size and have different levels of variability, which also affects the correct identification of each taxon. Different classification approaches reach up to 80% ( Culex and Coquillettidia ), 98% ( Aedes ) and 100% ( Anopheles and Culiseta ) accuracy, respectively. Furthermore, additional information on wing size can help in identification when the data and wing venation are less reliable. The distances between taxa centroids in morphological space reflect genetic distances and taxonomic hierarchy: on average, they are greatest between the two subfamilies (Anophelinae, Culicinae), followed by the distances between genera within the Culicinae family and finally by the distances between species within the genera Aedes and Culex . Nevertheless, the topology of the similarity tree based on morphological data reflects the topology of the genetic phylogeny only at the subfamily level. We assessed that geometric morphometrics on wings can be used as an alternative method when other structures otherwise traditionally used for species identification are broken.

The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

The first record of Aedes koreicus was made in the village of Lovrenc na Dravskem Polju, north-eastern part of the country . The discovery of Ae. koreicus in various continental European countries motivated us to revise samples of the collected Aedes japonicus japonicus . We found Ae. koreicus in samples from 2013, where the larvae were misidentified as Ae. j. japonicus . The species was identified morphologically and molecularly. The first discovery of Ae. koreicus advocates an urgent need for a nationwide mosquito surveillance programme.

The Transphlebotomus subgenus has been drawing attention in last decade due to the uncertain vector capacity and cryptic life history of the group. During a 2015 entomological survey, Phlebotomine flies were collected in western locations of Slovenia, with a total of 22 specimens of the Transphlebotomus subgenus collected. In addition to morphological identifications, we confirmed the presence of Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) by nucleotide sequences of the mitochondrial cytochrome b gene. During the study, we found that P. mascittii is most abundant in the area of coastal–karst region of Slovenia, while in rare occasions can also be collected in Northeastern part of the country, near the Austrian border. Vector competence of P. mascitii, like all other member species of the Transphlebotomus subgenus, is unclear, but potential to transmit Leishmania (Kinetoplastida: Trypanosomatidae) pathogens or phleboviruses is strongly suspected.

Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling has emerged over the last decade as a method of choice for species identification of many medically important arthropods. However, the influence of intraspecific variability on the performance of this popular technique has seldom been tested. This study provides the first standardized comparison of different geographical populations of Phlebotomus perniciosus , a vector of Leishmania infantum and Toscana virus in the western Mediterranean, by MALDI-TOF MS protein profiling. Methods Phlebotomus perniciosus males were collected in five countries (Portugal, Spain, France, Italy, Croatia) that represent most of its distribution in Europe. All samples were trapped, stored and processed according to a highly standardized protocol to avoid effects other than geographical origin on their protein spectra acquired by MALDI-TOF MS protein profiling. The obtained protein spectra were compared with laboratory-reared specimens of Ph. perniciosus . Results Twenty-two analysed specimens from five geographical populations provided protein spectra that were highly similar, species-specific and clustering according to their quality. No grouping according to geographical origin was observed, and the protein spectra of field-collected specimens showed similar composition and complexity to spectra from Ph. perniciosus laboratory colony-reared in captivity for several decades. Conclusions Our findings demonstrate that in samples of a same sex, with the same collection method and storage time, MALDI-TOF MS protein profiling does not reflect the geographical origin of analysed specimens, confirming the value of this technique for high-fidelity and reproducible species identification of sand flies regardless of their geographical origin. Graphical Abstract

Apart from being against the law, illegal waste dumping also poses a threat to human health and to the environment. Solid and decomposing waste is an ideal breeding ground for a number of rodents, insects, and other vermin that pose a health risk through the spread of infectious diseases. The main objective of this study was to survey disease vectors and rodents for the presence of sp. from waste sites along the Istrian Peninsula in Slovenia and Croatia. During the survey five sandfly ( , ) and five rodent species were collected ( , , , and ). Sandflies and rodents were screened using a molecular probe to amplify an approximately 120 bp fragment of the kinetoplast DNA (kDNA) minicircle for the detection of sp. parasites. DNA was detected in the spleen of one juvenile black rat ( ). Despite few published records on sp. infection in black rats, the addition of our record highlights the importance of further investigation into the frequency and distribution of such occurrences so that we may better classify the role of rodents as potential reservoirs of leishmaniasis in the Mediterranean basin.

To reduce the population of the Asian tiger mosquito Aedes albopictus (Skuse, 1894) (Diptera: Culicidae), a mosquito control campaign was organized by five Slovenian municipalities in 2020. The campaign focused on the application of Aquatain AMFTM in public water containers. The main objective of our study was to test the efficacy of the agent by directly testing the water drains for mosquito larvae and monitoring the density of the mosquito population. The drains were inspected before and after the application of the product from May to September. A water sample was taken from the drains using a dipper. For mosquito monitoring, the number of mosquito eggs and adult mosquitoes was counted from June to October. Up to two weeks after the application of the product, we observed the lethal effect of Aquatain on mosquito larvae, pupae and adult mosquitoes. After rainfall, the product was washed away and the live mosquitoes were sampled in the inspected water drains. The average number of eggs collected per site was twice as high in the area without mosquito control as in the threated locations. Nevertheless, we assessed the mosquito control campaign as partially successful, as mosquito abundance was still high in the locations with mosquito control. However, we believe that this is a good starting point and that mosquito control should be continued in the future together with the citizen education campaign.

Toxoplasma gondii is one of the most successful parasites on Earth, infecting a wide array of mammals including one third of the global human population. The obligate intracellular protozoon is not capable of synthesizing cholesterol (Chl), and thus depends on uptake of host Chl for its own development. To explore the genetic regulation of previously observed lipid metabolism alterations during acute murine T. gondii infection, we here assessed total Chl and its fractions in serum and selected tissues at the pathophysiological and molecular level, and integrated the observed gene expression of selected molecules relevant for Chl metabolism, including its biosynthetic and export KEGG pathways, with the results of published transcriptomes obtained in similar murine models of T. gondii infection. The serum lipid status as well as the transcript levels of relevant genes in the brain and the liver were assessed in experimental models of acute and chronic toxoplasmosis in wild-type mice. The results showed that acute infection was associated with a decrease in Chl content in both the liver and periphery (brain, peripheral lymphocytes), and a decrease in Chl reverse transport. In contrast, in chronic infection, a return to normal levels of Chl metabolism has been noted. These changes corresponded to the brain and liver gene expression results as well as to data obtained via mining. We propose that the observed changes in Chl metabolism are part of the host defense response. Further insight into the lipid metabolism in T. gondii infection may provide novel targets for therapeutic agents.