Research Article |
Corresponding author: Mária Kazimírová ( maria.kazimirova@savba.sk ) Academic editor: Dmitry Apanaskevich
© 2023 Zuzana Krumpálová, Barbara Mangová, Slávka Purgatová, Yuliya M. Didyk, Mária Kazimírová.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Krumpálová Z, Mangová B, Purgatová S, Didyk YuM, Kazimírová M (2023) Molecular characterisation of three Ixodes (Pholeoixodes) species (Ixodida, Ixodidae) and the first record of Ixodes (Pholeoixodes) kaiseri from Slovakia. ZooKeys 1158: 147-162. https://doi.org/10.3897/zookeys.1158.101936
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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 Dermacentor reticulatus, D. marginatus, Haemaphysalis inermis, H. concinna, Ixodes ricinus, I. hexagonus, and two Ixodes spp. Ixodes hexagonus were collected from northern white-breasted hedgehogs (Erinaceus roumanicus), females belonging to Ixodes spp. were collected from red fox (Vulpes vulpes) and nymphs from European badger (Meles meles). Ixodes hexagonus 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 Ixodes kaiseri 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.
European badger, hedgehog, Ixodes canisuga, Ixodes kaiseri, red fox, ticks
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 (
Ixodes kaiseri was for the first time described from Common Egyptian fox from Burg EJ Arab, Mariut, Western Desert Governorate, Egypt (
Ixodes canisuga, considered by some authors as a synonym of I. crenulatus (e.g.
Ixodes hexagonus is a common species in the Western Palaearctic. However, the species was sometimes mistakenly identified as I. canisuga (
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.
Hunted animals originated from the area of Žemberovce (south-western Slovakia). The village of Žemberovce (48°15'30"N, 18°44'30"E) borders with the town of Levice. There are extensive thermophilous forest communities, mainly oak–hornbeam Carpathian forests with Carpinus betulus and Quercus petraea. Mixed oak forests (Quercus cerris, Acer campestre, Cerasus avium, and Tilia cordata) are also found in suitable habitats. Hedgehogs were captured in parks within residential zones of Bratislava (48°14'85"N, 17°10'77"E), Stupava (48°27'89"N, 16°99'55"E), and in the village of Vrbovce (48°79'98"N, 17°46'89"E) (western Slovakia).
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
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 (
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).
During 2021 and 2022, we obtained ticks from hunted wild mammals belonging to five species in the Levice region of south-western Slovakia. The wildlife consisted of red fox Vulpes vulpes, European badger Meles meles, wild boar Sus scrofa, red deer Cervus elaphus, and European roe deer Capreolus capreolus. At the same time, we collected ticks from hedgehogs (Erinaceus roumanicus) in green areas of Bratislava, Stupava, and Vrbovce.
In total, 335 ticks (54 nymphs, 191 females, and 90 males) were collected from 35 hunted wild mammalian individuals and 178 ticks (48 larvae, 77 nymphs, 44 females, and 9 males) from hedgehogs. We identified the presence of eight tick species, namely Dermacentor reticulatus, D. marginatus, Haemaphysalis inermis, H. concinna, Ixodes ricinus, I. canisuga, I. hexagonus, and I. kaiseri.
In terms of species (Table
Total number of ticks collected from wildlife (all life stages combined, identification comprises also results of molecular analyses).
Host/tick species | I. ricinus | I. canisuga | I. kaiseri | I. hexagonus | D. reticulatus | D. marginatus | H. inermis | H. concinna |
---|---|---|---|---|---|---|---|---|
Vulpes vulpes | 2 | 2 | ||||||
Meles meles | 5 | 4 | ||||||
Capreolus capreolus | 19 | |||||||
Cervus elaphus | 146 | 3 | 7 | |||||
Sus scrofa | 43 | 36 | 26 | 27 | 15 | |||
Erinaceus roumanicus | 155 | 23 | ||||||
Total | 365 | 5 | 6 | 23 | 36 | 26 | 30 | 22 |
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
Partially engorged Ixodes canisuga and I. kaiseri nymphs (dorsal views – upper panels, ventral views – lower panels) collected from Meles meles in Slovakia. Differences are seen in the shape of scutum (a) which is shorter in I. canisuga than in I. kaiseri. There is a broad internal spur on coxa I (b) in both species. Photo: Ľ. Vidlička
Gnathosoma of Ixodes canisuga and I. kaiseri nymphs (dorsal views – upper panels, ventral views – lower panels) collected from Meles meles in Slovakia. Ixodes canisuga – anteriorly flattened basis capituli (a); dorsally absence of cornua (b); long, moderately thick auricular ridge (c). Ixodes kaiseri: cornua well developed (b); short, thin auricular ridge (c); anteriorly rounded, sclerotized protuberance on palpal segment I (d).
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.
The isolates UZINS204 23 SK, UZINS205 23 SK, and UZINS206 23 SK were uniform based on the COI sequences. BLAST search showed 100% identity of the Slovak isolates with COI gene sequences of I. kaiseri isolates from Turkey (ON527576), Croatia (MZ305531), and Romania (KY962020), 99.84% identity with isolate from Hungary (KY962015), and 99.52% with isolate from Serbia (KY962033). Isolates UZINS193 23 SK, UZINS194 23 SK, and UZINS195 were uniform and showed 100% identity with the COI gene sequences in isolates of I. hexagonus from Hungary (OM200350), Croatia (MZ305530), and Germany (KY962046), and 99.54% identity with isolates from Italy (MG432679) and Portugal (LC508366). Isolate UZINS197 23 SK showed 100% identity with the COI gene sequence of I. canisuga isolate from Romania (KY962023), and 99.84% identity with isolates from France (KY962049), United Kingdom (KY962048), Germany (KY962045), and 99.53% with isolate from Hungary (KX218106). Samples UZINS198 23 SK and UZINS199 23 SK showed 100% identity with the COI gene sequences of I. canisuga isolate from Hungary (KX218106) and 99.68% with isolates from France (KY962049), United Kingdom (KY962048), and Germany (KY962045) (Fig.
Sequence of the 16S rRNA gene in our samples UZINS205 23 SK and UZINS206 23 SK showed 100% identity with the 16S rRNA gene sequences in I. kaiseri isolates from Poland (MK613135), Romania (MT658766), and Germany (MT658770), 99.76% identity with isolate from Turkey (ON540356), and 99.51% identity with isolate from China (MG763864). Sequence in our sample UZINS204 23 SK showed 100% identity with the 16S rRNA gene sequences in I. kaiseri isolate from Turkey (ON540356), 99.76% identity with isolates from Poland (MK613135) and China (MG763864), and 99.75% identity with isolates from Germany (MT658770) and Romania (MT658766). Sequences in samples UZINS193 23 SK, UZINS194 23 SK and UZINS195 23 SK were uniform and showed 100% identity with the 16S rRNA gene sequences in I. hexagonus isolates from Croatia (KY962077), Austria (KY962058), Germany (JF928502), and Poland (AF001400) and 99.76% identity with isolate from Italy (KY319189). Sequence of the 16S rRNA gene in our sample UZINS197 23 SK was 100% identical with the 16S rRNA gene sequences of I. canisuga isolate from Poland (MK613137), France (KY962074), and United Kingdom (KY962071), and 99.75% identical with isolates from Germany (KY962068) and Croatia (KY962072). Sequences in samples UZINS198 23 SK and UZINS199 23SK showed 100% identity with the 16S rRNA gene sequences in I. canisuga isolates from Croatia (KY962072) and Germany (KY962068), 99.76% identity with isolate from Poland (MK613137), and 99.75% with isolates from France (KY962074) and United Kingdom (KY962071) (Fig.
Molecular identification with markers COI and 16S rRNA confirmed the morphological identification of I. kaiseri, I. canisuga, and I. hexagonus from Slovakia (Figs
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 (
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 (
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 (
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.
This study was carried out with the financial support of various institutions. As representative for all those who contributed in one way or another, we would like to express our gratitude to the Slovak Grant Agencies VEGA No. 1/0604/20 (Environmental assessment of specific habitats of the Danubian Lowland), KEGA No. 019UKF-4/2021 (Creation and innovation of education – Zoology for Ecologists, part – Invertebrates) and VEGA No. 2/0004/22, as well as the European Fund for Regional Development (EFRD) ITMS2014 + 313021W683-DNA barcoding of Slovakia (SK-BOL), as a part of international initiative International Barcode of Life (iBOL). The study was funded by the statutory activities of Faculty of Natural Sciences, Constantine the Philosopher University, Nitra (Slovakia) and the Slovak Academy of Sciences Institute of Zoology (Slovakia). We thank Assoc. prof. Ľubomír Vidlička for taking photographs.
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.