Reef-building coral species harbour Chromera velia and apicomplexan “type-N” (cf. Gemmocystis cylindrus) on the Great Barrier Reef, Australia based on combined small-subunit rRNA gene amplicon pyrosequencing assays Targeted Locus (Loci)
Background: The coral holobiont is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with a yet undefined large number of symbiotic eukaryotes, bacteria and viruses. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring.  Methods & Principal Findings: We investigated the diversity of eukaryotic community associated with a common reef-building coral species from the southern Great Barrier Reef, Australia. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene assays to demonstrate the presence of the “type-N” (cf. Gemmocystis cylindrus) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) in all examined coral and a photosynthetic sister species to Apicomplexa - Chromera velia - in Montipora digitata. Eukaryotic small-subunit rRNA gene clone library confirmed presence of Symbiodinium sp. Clade C corroborating findings using amplicon pyrosequencing and diagnostic PCR confirmed presence of apicomplexan type-N. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus) in M. digitata DNA. We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively.  Significance: This study proved that eukaryote tag encoded amplicon pyrosequencing is capable of demonstrating presence of C. velia and apicomplexan type-N in total coral DNA. This is the first time apicomplexan type-N has been observed in the Great Barrier Reef. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease.