Viromics in Flat Mites from Hawaii Shows Abundant Arrays of Viruses, Expands the Evolutionary Origin of Plant Viruses, and Provides a Surveillance Tool for Brevipalpus -Transmitted Viruses

ABSTRACT Flat mites (Tenuipalpidae) are diverse phytophagous arthropods, among which Brevipalpus species are economically important pests capable of transmitting plant viruses. Brevipalpus-transmitted viruses (BTVs) cause localized infections in plants and are classified into two major groups based on cytopathology and genome organization: BTV-C (genera Cilevirus and Higrevirus , family Kitaviridae ) and BTV-N (genus Dichorhavirus , family Rhabdoviridae ). Despite their significance, the virome of tenuipalpid mite vectors remains poorly characterized. Using high-throughput sequencing (HTS), we analyzed virus populations associated with Brevipalpus and Dolichotetranychus mites collected from multiple plant hosts across two Hawaiian Islands. We identified a diverse assemblage of viral sequences affiliated with Kitaviridae , negeviruses, Picornavirales , Narnaviridae , Tombusviridae , Solemoviridae , Ourmiaviridae , Reoviridae , and Potyviridae . Near-complete genomes of citrus leprosis virus C2H and hibiscus green spot virus 2 (both BTV-C) were recovered, highlighting the utility of HTS-based viromics for surveillance of BTVs in mite vectors. In addition, multiple divergent virus-like contigs were identified based on viral hallmark genes and sequence divergence, including Brevipalpus-associated negevirus, Brevipalpus-associated bluner-like virus, and Dolichotetranychus-associated cile-like virus, all showing evolutionary affinities to BTV-C-related viruses. Phylogenetic analyses support evolutionary links between negeviruses and kitavirids, consistent with the hypothesis that Kitaviridae evolved from arthropod-associated ancestors. While some detected plant viruses may reflect ingestion rather than active replication in mites, this study establishes a robust framework for virome-based surveillance of tenuipalpid mites, advancing our understanding of plant virus evolution and supporting agricultural biosecurity and pest management efforts.