﻿Molecular characterisation of three Ixodes (Pholeoixodes) species (Ixodida, Ixodidae) and the first record of Ixodes (Pholeoixodes) kaiseri from Slovakia

﻿Abstract A study of ticks on wildlife was carried out in the area of Levice, Bratislava, Stupava, and Vrbovce (south-western Slovakia) during 2021 and 2022. Overall, 512 ticks were collected from 51 individuals of six wild mammalian species. Eight tick species were identified, namely Dermacentorreticulatus, D.marginatus, Haemaphysalisinermis, H.concinna, Ixodesricinus, I.hexagonus, and two Ixodes spp. Ixodeshexagonus were collected from northern white-breasted hedgehogs (Erinaceusroumanicus), females belonging to Ixodes spp. were collected from red fox (Vulpesvulpes) and nymphs from European badger (Melesmeles). Ixodeshexagonus and the Ixodes spp. were identified morphologically and molecularly based on sequences of fragments of two mitochondrial genes, COI and 16S rRNA. Molecular analysis of Ixodes spp. confirmed the identity of Ixodeskaiseri Arthur, 1957 and I.canisuga (Johnston, 1849). Sequence analyses show that the I.kaiseri isolate from Slovakia is identical to I.kaiseri isolates from Romania, Poland, Germany, Turkey, and Croatia. We demonstrate for the first time the presence of I.kaiseri in Slovakia using both morphological and molecular methods.


Introduction
Ticks (Ixodida) belong to the most important ectoparasites of terrestrial vertebrate species. Hard ticks of the subgenus Pholeoixodes Schulze, 1942 (Ixodes, Ixodidae) are usually associated with burrow-dwelling mammals and terrestrial birds that nest in cavities (tree holes or burrows). All representatives of the subgenus Pholeoixodes are endophilic three-host ticks with a rather uniform circle of hosts for all active ontogenetic stages. For a long time, the taxonomy of the subgenus Pholeoixodes was rather confused (Estrada-Peña et al. 2017a;Tsapko 2018). This can be explained by the large intraspecific morphological variability of the species, when morphologically differing specimens of the same species were described under different names or were misdiagnosed. At the same time, as mentioned by Filippova (1977) and Emelyanova (1979), new findings from previously unknown hosts were described as new species. In the Western Palaearctic the Ixodes (Pholeoixodes) group currently includes I. (P.) kaiseri Arthur, 1957, collected from the Indian crested porcupine Hystrix indica and from the northern (Erinaceus roumanicus) and southern (E. concolor) white-breasted hedgehogs. Hornok et al. (2017) analysed the phylogenetic relationships by using two mitochondrial genes and morphological differences in females of three Ixodes species of the subgenus Pholeoixodes including I. kaiseri. Furthermore, Hornok et al. (2021) published pictorial keys for identification of I. kaiseri, I. canisuga, and I. hexagonus males, nymphs, and larvae. Ixodes kaiseri was found to harbour several tick-borne pathogens of veterinary and medical importance (Hornok et al. 2020b;Wodecka et al. 2022); however, it is not clear if and to what extent this species contributes to pathogen circulation in nature.
Ixodes canisuga, considered by some authors as a synonym of I. crenulatus (e.g. Filippova and Uspenskaya 1973;Filippova 1977;Siuda 1993), is associated with mammals which inhabit burrows. The most infested species are medium-sized mustelids and canids such as the red fox and European badger, among others (Cornely and Schultz 1992;Santos-Silva et al. 2011). This tick species is also a common parasite of domestic dogs and has also been found on cats Földvári and Farkas 2005;Hornok et al. 2017). Overall, I. canisuga is distributed from the Spanish Pyrenees through Russia, Iran, Afghanistan, India (Kashmir), to eastern China (Estrada-Peña et al. 2017a). In Europe, this species has been recorded in almost all countries, from United Kingdom, Ireland, France, Austria, and Germany in the west Cornely and Schultz 1992;Ogden et al. 2000), Portugal in the south (Santos-Silva et al. 2011), and in countries of central and south-eastern Europe, i.e., Hungary, Poland, Romania, Croatia, Serbia, and Bosnia and Herzegovina (Hornok et al. 2017;Krčmar et al. 2022). It was possibly recorded also in the former Czechoslovakia, but under different names (Černý 1972).
Ixodes hexagonus is a common species in the Western Palaearctic. However, the species was sometimes mistakenly identified as I. canisuga (Hornok et al. 2017). Ixodes hexagonus is associated with hedgehogs, Erinaceus europaeus and E. roumanicus, which are the main hosts among the broad spectrum of medium-sized burrow-inhabiting mammals. The species was also found on wild carnivores such as red foxes, mustelids, and the European badger (Estrada-Peña et al. 2017a;Karbowiak et al. 2020). In addition, I. hexagonus can frequently be encountered on domestic pets (cats and dogs) and harbours various tick-borne pathogens (Walker 2018). The distribution of I. hexagonus covers almost the whole of Europe, ranging from the British Islands in the west, across all the countries in central and eastern Europe, including Slovakia (Černý 1972;Estrada-Peña et al. 2017a;Karbowiak et al. 2020). The species was also found outside this range, e.g. in north-western Iran (Tavassoli and Mohamadi 2015).

Ethics statement
The study complies with current laws of the Slovak Republic and with species conservation guidelines. The animals were killed for hunting reasons during the legal hunting season and not specifically for this study. Collections of ticks from hedgehogs were in accordance with Decision No. 8711/2022-6.3 -Exemption from Act No. 543/2022 on Nature and Landscape Protection.

Tick collection and identification
We collected ticks individually with tweezers directly from the skin of hunted mammals. The searches for hedgehogs were performed at night (10 p.m. to 3 a.m.) by two persons. The equipment consisted of headlamps and thick welding gloves for hedgehog handling. After removing the ticks, all the hedgehogs were released back in their original capture location without significant manipulation.
Ticks were stored in 80% ethanol at 4 °C. They were examined morphologically; adult ixodid ticks are usually easier to identify to species than immature stages, and therefore morphological comparisons followed Siuda (1993), Slovák (2010Slovák ( , 2014, and Bristol University Tick ID (http://www.bristoluniversitytickid.uk). Species of the subgenus Pholeoixodes are the most problematic group in the genus Ixodes. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Moreover, identification of engorged ticks is even more difficult. Nymphs of Ixodes spp. were morphologically identified according to Hornok et al. (2017Hornok et al. ( , 2021 using a stereomicroscope (Olympus SZ61). Photographs were taken using a Leica M205C stereo microscope and a Leica Flexacam C1 camera, including LAS X software with a Z-stack projection tool. The photographed specimens were immersed in 80% ethanol, because they were stored for further molecular detections of microorganisms.

Molecular analysis
Genomic DNA was isolated individually from legs of females and nymphs of I. hexagonus and Ixodes spp. by the method of alkaline hydrolysis with modifications (Guy and Stanek 1991) and from whole engorged ticks with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. DNA samples were stored at −20 °C until further analyses. Fragments of the mitochondrial 16S rRNA and the COI genes were chosen for molecular analyses and tick species identification (Black and Piesman 1994; Hornok et al. 2017). PCR amplified approximately 710 bp of the COI gene using the primers LCO1490 and HCO2198 (Hornok et al. 2017) and approximately 460 bp of the 16S rRNA gene of Ixodidae, using the primers 16S+1 and 16S-1 (Black and Piesman 1994). PCR products were analysed by electrophoresis in 1.5% agarose gel stained with GoodView Nucleic Acid Stain (SBS Genetech, Beijing, China) and visualized under UV light. Amplicons were purified using a QIAquick Spin PCR Purification Kit (Qiagen, Hilden, Germany) as described by the manufacturer. The sequencings were performed by Eurofins Genomics Europe (https://www.eurofinsgenomics.eu). DNA sequences were compared with available databases in GenBank using the Basic Local Alignment Search Tool (BLAST) NCBI. The MEGA model selection method was applied to choose the appropriate model for phylogenetic analyses. Phylogenetic analyses were conducted using the maximum likelihood method based on the Tamura-Nei model Nei 1993) using MEGA v. 7.0 (Kumar et al. 2016

Data availability statement
The data presented in this study are available upon request from the corresponding author. Nucleotide sequences of COI genes derived from the study are available in BOLD: The Barcode of Life Data System (http://www.barcodinglife.org).

Results
During 2021 and 2022, we obtained ticks from hunted wild mammals belonging to five species in the Levice region of south-western Slovakia. In terms of species (Table 1), I. ricinus was the most frequent and abundant species, accounting for 71.1% of all specimens collected, and it was found on five hosts (wild boar, red deer, roe deer, red fox, and hedgehog). Dermacentor reticulatus accounted for 7% of all specimens and D. marginatus for 5.1%; both were confirmed on one host, wild boar. Fewer H. concinna (4.3%) and H. inermis (5.9%) were found on two hosts (wild boar and red deer), and I. hexagonus (4.5%) was found on hedgehogs only. The remaining specimens (2.2%) belonged to Ixodes spp. and were collected from red fox and European badger.

Morphological diagnosis
In total, three female ticks and one male were collected from red fox and nine nymphs from European badger. Initially, based on the existence of an anal groove and the typical structure of the mouthparts, they were morphologically identified as Ixodes spp. One of the females and the male from red fox were further identified as I. ricinus and the other two females from the red fox and all nymphs from the European badger as Ixodes spp. Because mouthparts of the females and part of the nymphs were damaged during their removal from the host skin, morphological identification was only possible in six undamaged nymphs. Two of them were identified as I. canisuga and four as I. kaiseri (Figs 1, 2) according to detailed morphological examinations showing that the nymphs have the characters described by Hornok et al. (2021).

Molecular identification
By amplification of the COI gene, two Ixodes spp. females from the red fox and four nymphs from the European badger were identified as I. kaiseri and four nymphs as I. canisuga. Amplification was not successful for one nymph morphologically identified as I. canisuga. By amplification of fragments of the 16S rRNA gene in DNA samples from one female and two nymphs of I. kaiseri and three nymphs of I. canisuga the identification of the species was confirmed. Molecular analyses of amplified fragments of the COI and 16S rRNA genes in DNA samples derived from two females and one nymph of I. hexagonus from hedgehogs confirmed the correct identification of the species based on morphology.

Host/tick species I. ricinus I. canisuga I. kaiseri I. hexagonus D. reticulatus D. marginatus H. inermis H. concinna
Molecular identification with markers COI and 16S rRNA confirmed the morphological identification of I. kaiseri, I. canisuga, and I. hexagonus from Slovakia (Figs 3, 4). We demonstrated the presence of I. kaiseri for the first time in Slovakia using both morphological and molecular methods. Consequently, Slovakia was added to the list of countries where I. kaiseri was reported from red fox and European badger.

Discussion
We recorded the co-occurrence of two endophilic Ixodes spp. of the subgenus Pholeoixodes, I. kaiseri and I. canisuga on European badger, and of I. kaiseri and I. ricinus on red fox. For the first time, the occurrence of I. kaiseri is confirmed in Slovakia. The presence of I. kaiseri in this country was predictable (Karbowiak et al. 2020), as it was found to parasitize wild carnivores and dogs in neighbouring countries of Hungary, Poland, and Ukraine, but also in Germany and south-eastern Europe (Romania, Croatia, Serbia) (Akimov and Nebogatkin 2002;Hornok et al. 2017Hornok et al. , 2021Dwużnik et al. 2020;Krčmar et al. 2022). Meyer-Kayser et al. (2012) found on foxes in Germany predominance of tick larvae (48%), followed by adults (34%), and nymphs (18%). Ixodes ricinus was the most frequent tick species, followed by I. canisuga and I. hexagonus. In previous studies from Slovakia, D. reticulatus, H. concinna, I. ricinus, and two Pholeoixodes species, I. hexagonus and I. crenulatus identified based on morphology, had been found to parasitize red foxes (Kočišová et al. 2006;Karbowiak et al. 2020). Dwużnik et al. (2020) investigated ectoparasites of red foxes in three regions of Poland. Ixodes ricinus and D. reticulatus were the dominant tick species on adult foxes, but I. kaiseri was also recorded. We found I. canisuga on European badger, but not on red fox, and I. kaiseri on red fox and European badger. However, we examined only one individual of each host species for ticks, which is not enough to draw conclusions on their parasitofauna in Slovakia.
Ixodes ricinus and I. hexagonus are common ectoparasites of hedgehogs in urban and suburban areas of Europe. For example, by examining hedgehogs in a city park in Budapest (Hungary), the high prevalence (93.7%) of I. ricinus and presence of I. hexagonus were recorded. Nymphs prevailed in both species (Földvári et al. 2011). In four urban habitats in Cluj-Napoca city, Romania, in addition to birds and small mammals, ticks were collected from northern white-breasted hedgehogs. Ixodes ricinus prevailed (89.7%), followed by I. hexagonus (7.7%) and Haemaphysalis punctata (2.6%). With regards to life stages, larvae dominated in I. ricinus (67%) and H. punctata (71.4%) and females (75.9%) in I. hexagonus (Borşan et al. 2020). We identified I. ricinus and I. hexagonus on hedgehogs from three urbanized areas of Slovakia. Immature stages (nymphs and larvae) prevailed in both species. Thus, the ratios of adult ticks and immature stages differ between sites and are probably affected by microclimate and the presence of other hosts for ticks.
In general, changes in land usage patterns and climate, i.e., milder winters and earlier onset of spring in the northern hemisphere, can significantly affect the geographic distribution, phenology, and population density of some tick species and the occurrence of tick-borne zoonoses (Gray et al. 2009;Gilbert 2021). In addition, ticks can easily spread and colonize new regions via international pet trade, domestic animal transport, or bird migration (Földvári et al. 2016). Given the vector role of ticks, accurate species identification is very important, but it requires time and considerable experience. Among hard ticks (Ixodidae), the genus Ixodes contains the most species, which are grouped into subgenera. Species of the subgenus Phloeixodes belong to a problematic group, as they share morphologic and ecologic characters (Estrada-Peña et al. 2017a, b). In a recent review of available taxonomic literature, Guglielmone et al. (2020) pointed to the problems and importance of correct tick identification, mainly for species of medical, veterinary, and evolutionary importance. For example, a comparative test of tick species identification conducted by a network of European researchers in 14 laboratories specialising in ticks provided an overall misidentification rate of almost 29.6% (Estrada-Peña et al. 2017c), which highlighted the need for molecular methods to identify ticks. DNA barcoding methods serve for accurate and rapid identification of tick species and provide the basis for a molecular data platform for the family Ixodidae (Krčmar et al. 2022). Recently, molecular methods have increasingly been used for accurate tick identification, especially in groups of morphologically very similar species (Hornok et al. 2017(Hornok et al. , 2021Krčmar et al. 2022). Thanks to the DNA barcoding method, the occurrence of I. kaiseri was recorded in Croatia, Germany, Serbia, Hungary, and Romania (Hornok et al. 2017(Hornok et al. , 2021Krčmar et al. 2022), Turkey (Orkun and Karaer 2018), and now Slovakia (this study). Subsequently, Slovakia was added to the list of countries where I. kaiseri has been recorded. Moreover, by using DNA barcoding, the identity of I. canisuga and I. hexagonus was also confirmed.
We hypothesize that I. kaiseri has been present in Slovakia but was misidentified because of the variability of morphological characters in species of the subgenus Pholeoixodes. Moreover, due to the endophilic mode of life, only engorged individuals can be collected from hosts and their identification is generally more difficult than unfed ticks. Therefore, further studies of species of the subgenus Pholeoixodes are needed, and morphological identifications in previous studies should be confirmed by molecular methods.