Intra-individual polymorphisms in the mitochondrial COI gene of tick-killing Ixodiphagus wasps parasitizing Haemaphysalis flava ticks.

Ixodid ticks are significant vectors of pathogens affecting both humans and animals. Biological control with natural enemies represents a sustainable tool for managing ticks. However, there is a substantial lack of knowledge about the natural enemies of ticks. Wasps of the genus Ixodiphagus (Encyrtidae) are currently the only known tick-specific parasitoids. While these wasps have been sporadically recorded worldwide, their presence in Eastern Asia is poorly documented. In this study, we investigated the occurrence of Ixodiphagus wasps in field-collected ticks reared on rabbits under laboratory conditions. Ticks were collected from the Hokkaido, Hokuriku, Chugoku, Shikoku, and Kyushu regions in Japan. Out of 1,933 Haemaphysalis ticks infested on rabbits, adult wasps emerged from 15 engorged ticks. All the ticks from which wasps emerged were morphologically and molecularly identified as Haemaphysalis flava. Additionally, wasp DNA was detected in unfed H. flava nymphs using a newly designed Ixodiphagus-specific PCR assay. Among nine experimental sites in the Chugoku region, Ixodiphagus wasps were detected at three sites, with parasitism rates ranging from 1.8 % to 8.1 %. Finally, the mitochondrial COI gene sequences of four wasp and two tick samples were characterized using shotgun sequencing, direct sequencing, and in-fusion cloning approaches. Multiple intra-individual polymorphisms were observed in all the tested samples. Further studies are needed to investigate the relationship between Ixodiphagus wasps and Ixodid ticks. An increased understanding of these parasitoid wasps could contribute to future biological control measures against ticks.