DNA of I. hookeri was detected in ticks collected from locations broadly distributed across the Netherlands. Although this is the first notice of this species in the Netherlands the broad distribution and the cosmopolitan distribution of this insect indicates that the wasp is a native species. The prevalence varies strongly with sampling location as had been reported from other studies as well [26, 27]. The prevalence might influenced by biological or climatic factors. Tick density, which in turn is affected by tick-host abundance, has been suspected to be a major determinant for the prevalence of I. hookeri. This corresponds with the finding that the highest I. hookeri prevalence in this study was found in 'Duin & Kruidberg' (Figure 4), an area with exceptional high tick density (data not shown). However, another area with comparable tick density, 'Kop van Schouwe', had a much lower I. hookeri prevalence. Other factors like (micro)climate, vegetation and prevalence of different vertebrate species in the habitats might play a role, but such data were not collected during this study.
The molecular data suggest that I. hookeri, as many other insects, may harbour at least one Wolbachia strain. The specimen studied here was infected with a Wolbachia strain similar to that of other Hymenoptera but also closely matched Wolbachia endosymbionts of insects from different orders. Although the specimen hatched in our laboratory was apparently infected with a single strain, several I. hookeri positive ticks harboured multiple Wolbachia strains. Multiple infections with Wolbachia are known to occur in insects [28, 29] and the combination of these might have different biological impacts on their hosts. Wolbachia have been linked to parasitoid specialization  and different Wolbachia strains in I. hookeri might be one explanation for apparent differences in its biology in different countries [18, 19, 31].
Detection of Wolbachia DNA in ticks in this study is strongly correlated with I. hookeri infestation. The small deviation between PCR results might be explained by different sensitivities of PCRs. On the one hand, some I. hookeri might have no or only a low Wolbachia load while others might have a high bacterial load. On the other hand, the copy number of the 28S rRNA gene, which was used to detect I. hookeri in this study, is not known for this species and will influence the PCR sensitivity for this target. Furthermore PCR products from the prevalence study were not sequenced. Therefore, some of the wsp and 28S rRNA positive PCR results might have been due to environmental contamination of samples with Wolbachia or insect species not related to infestation of ticks.
Data on spread and prevalence of I. hookeri around the world is scarce. The specificity of the I. hookeri PCR could be increased for future studies by the use of primers with high specificity, which can now be designed based on the 16S rRNA and 28S rRNA sequence data from this study. In future, molecular methods can be used to examine the spread and prevalence of I. hookeri in different tick species and regions.