Molecular barcoding of Australian ticks

Globally, ticks (Acari: Ixodida) are one of the most important vectors of disease due to their ability to transmit a wide variety of pathogenic bacteria, protozoa, and viruses during blood feeding. The microbes transmitted by ticks varies by species, and so it is essential that ticks are able to be identified correctly. However, the identification and discrimination of tick species still relies on traditional morphological techniques, which can at times be ambiguous, particularly in the case of subadult ticks. This study tested previously developed molecular barcoding assays to facilitate the identification of Australian ticks using DNA sequences in the case that morphology is inconclusive. Using reference ticks from eight native species of medical and veterinary importance (Amblyomma triguttatum, Bothriocroton auruginans, Haemaphysalis bancrofti, Haemaphysalis humerosa, Ixodes cornuatus, Ixodes hirsti, Ixodes holocyclus and Ixodes tasmani), four potential barcoding genes were trialled (Cytochrome c oxidase (COI), Internal transcribed spacer 2 (ITS2), 16S rRNA and 12S rRNA). Amplification was successful in 98.3% of samples (n=58) for COI, 89.6% (n=48) of samples for ITS2, and 100% of samples for both 16S rRNA and 12S rRNA (n=58 and n=48 respectively). Following Sanger sequencing, all four genes were found to be suitable for specimen identification using BLAST when genetic data was available. However, analysis of the sequences generated using Automatic Barcode Gap Discovery (ABGD) indicated that COI was the most efficient gene for species delimitation. This analysis revealed three new species group hypotheses and pairwise distances confirmed high levels of genetic divergence within these species. Further research is required to investigate the validity of these species groups, and to ensure that the assays tested in this study are suitable for all Australian ticks. Lastly, this study provided new genetic information for nine species based on 17 sequences, and these data will become publicly available on the genetic database, GenBank