2.1.2. Tick Collection

A total of 1115 questing ticks were collected for a previous study by flagging 100 m² simultaneously with small mammal trapping at 17 of the 21 sites, once per season (spring, summer, and autumn) in 2018 and 2019 [32] (Figure A1). The study sites for ticks were composed of one plot in the forest and one in the bordering grassland–forest ecotone, as described before [32]. Collected ticks were specified for sex, life stage, and species under a light microscope (Motic^® SMZ–171, Moticeurope, S.L.U., Barcelona, Spain) according to taxonomic keys [33,34]. DNA was extracted using a QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions for DNA isolation. Extracted DNA of 1094 ticks was available from the study of Król et al., where further details of tick handling, taxonomic identification, and DNA extraction procedure are described [32]. In addition to the given dataset, four ticks (three nymphs and one female of I. ricinus) that were all collected in spring 2019 were processed after the same protocol. Seventeen individuals (four I. ricinus nymphs and thirteen D. reticulatus adults) had to be excluded from further molecular biological analyses due to insufficient material conservation. Data on I. ricinus and I. inopinatus (presumably 16 individuals) were merged under the terminus “I. ricinus complex” [35].

In total, 1115 ticks belonging to 2 species were collected (Table A1). The most prevalent species was the I. ricinus complex (91.7%), followed by D. reticulatus (8.3%). The most frequently found life stages were the nymphs of the I. ricinus complex (74.9%). Most ticks were flagged in spring (74.1%) in comparison to summer (18.3%) and autumn (7.6%). In ecotones less ticks (35.2%) were flagged than in forests (64.8%). After excluding the above-mentioned ticks, 1018 ticks of the I. ricinus complex and 80 individuals of D. reticulatus were further processed (Table 1).

Rickettsia spp. prevalence in ticks tested per habitat.

No.: number. ¹ Ixodes ricinus and Ixodes inopinatus.