Molecular characterisation of Australasian Ixodiphagus (Hymenoptera; Encyrtidae; Encyrtinae) reveals unexpected diversity and a potential novel host switch

Ticks are important medical and veterinary parasites that represent a substantial health threat to humans, companion animals, and livestock. Ixodiphagus wasps (Hymenoptera; Encyrtidae) are known endoparasitoids of ixodid (hard) and argasid (soft) ticks, with potential utility as natural biocontrol agents. Two species, Ixodiphagus brunneus and Ixodiphagus mysorensis, are previously recorded from Australia, however, the genus lacks formal revisionary work in Australia, and the validity and host ranges of these species remain uncertain. This work aimed to investigate the diversity of Ixodiphagus in Australasia and provide a molecular data resource for future work on these understudied endoparasitoids. We extracted DNA from archival Ixodiphagus specimens from Australian and New Zealand insect collections and performed high-throughput sequencing which resulted in complete or mostly complete mitochondrial genome sequences from 11 specimens, including I. brunneus, Ixodiphagus taiaroaensis, and a novel Ixodiphagus sp. reared from Rhipicephalus linnaei from Townsville, Australia. In addition, approximately 70% of the genome of the Wolbachia endosymbiont of I. brunneus was recovered. Finally, we screened 178 recently collected pooled tick samples from southern New South Wales, Australia, for Ixodiphagus spp. using 28S rRNA and cytochrome c oxidase subunit 1(COI) gene PCR, and recovered 14 positive samples. Phylogenetic analysis of Australasian Ixodiphagus spp. based on 28S rRNA and complete mitochondrial genome sequences determined that members of the Australasian fauna are distinct from Ixodiphagus hookeri (the only other Ixodiphagus species for which genetic data exists), and that at least two distinct species are present in Australia; I. brunneus identified from Ixodes holocyclus and Haemaphysalis bancrofti ticks, and an uncharacterised Ixodiphagus sp. found in Rhipicephalus linnaei ticks from northern Queensland. Furthermore, there was substantial genetic diversity at the 28S rRNA loci among I. brunneus samples, which may represent normal genetic variability or a secondary cryptic species. The molecular data generated here represents the first known for the genus Ixodiphagus in Australasia, doubling that of the world fauna, and provides the first known complete mitochondrial genomes for these important tick parasitoids.