Nanopore-based 18S rRNA metabarcoding reveals Hepatozoon lineages from ectoparasites

Inverse surveillance seeks to identify potential pathogens before diseases emerge in populations. In this study, we conducted a molecular survey of eukaryotic vector-borne pathogens (VBPs) within ectoparasite species, to anticipate disease outbreaks in animals and humans. We analysed fleas, lice, mites, ticks and triatomines collected from rodents, birds, carnivores and from the environment across Chile. After extracting genomic DNA, we employed an 18S rRNA gene amplicon-based sequencing strategy using Oxford Nanopore Technologies to characterise eukaryotic VBPs. Our findings revealed a narrow taxonomic range of microorganisms, with Hepatozoon as a well-supported taxon. A single sequence matching the genus Babesia was additionally confirmed via BLAST. The bioinformatic pipeline allowed the recovery of high-quality Hepatozoon consensus sequences, enabling robust phylogenetic and population genetic analyses and providing the first molecular record of Hepatozoon in triatomines and the first genetic detection of this parasite genus in trombiculid and macronyssid mites in the Americas. Three different Hepatozoon lineages were detected, which clustered with others found in rodents and reptiles. Analyses of molecular variance (AMOVA) indicate that host identity may play a more influential role than geographic region in shaping the genetic differentiation of Hepatozoon lineages in Chile. Our study provides new data on ectoparasite-host-parasite associations, demonstrating the utility of Nanopore metabarcoding data for exploring VBPs diversity, thereby expanding the known arthropod associations of the Hepatozoon genus and suggesting the role of these ectoparasites as its potential vectors. As some Hepatozoon species can cause animal disease, these findings constitute an early warning for veterinarians.