Unbiased Characterization of the Microbiome and Virome of Questing Ticks

Abstract

Due to their vector capacity, ticks are ectoparasites of medical and veterinary significance. Modern sequencing tools have facilitated tick-associated microbiota studies, but these have largely focused on bacterial pathogens and symbionts. By combining 16S rRNA gene sequencing with total RNA-sequencing methods, we aimed to determine the complete microbiome and virome of questing, female <i>Ixodes holocyclus</i> recovered from coastal, north-eastern New South Wales (NSW), Australia. We present, for the first time, a robust and unbiased method for the identification of novel microbes in ticks that enabled us to identify bacteria, viruses, fungi and eukaryotic pathogens. The dominant bacterial endosymbionts were <i>Candidatus</i> Midichloria sp. Ixholo1 and <i>Candidatus</i> Midichloria sp. Ixholo2. <i>Candidatus</i> Neoehrlichia australis and <i>Candidatus</i> Neoehrlichia arcana were also recovered, confirming that these bacteria encompass <i>I. holocyclus</i>' core microbiota. In addition, seven virus species were detected-four previously identified in <i>I. holocyclus</i> and three novel species. Notably, one of the four previously identified virus species has pathogenic potential based on its phylogenetic relationship to other tick-associated pathogens. No known pathogenic eukaryotes or fungi were identified. This study has revealed the microbiome and virome of female <i>I. holocyclus</i> from the environment in north-eastern NSW. We propose that future tick microbiome and virome studies utilize equivalent methods to provide an improved representation of the microbial diversity in ticks globally.