Isolation and genotyping of novel T4 cyanophages associated with diverse coral reef invertebrates

Abstract

In an effort to facilitate virus isolation-based studies on coral reefs, we describe here a simple holobiont virus extraction protocol that is effective at separating and concentrating virus particles from coral reef invertebrates. We demonstrate the application of this protocol by isolating and barcoding cyanophages from invertebrate holobionts associated with coral reefs in southwest Puerto Rico. Cyanophages were also isolated and barcoded from adjacent coral reef seawater. Barcoding of cyanophage isolates was carried out with the cyanomyovirus DNA polymerase (g43) or major capsid protein (g23) marker genes. We then utilized cyanophage sequences from Puerto Rico, along with those published previously associated with Rhode Island and Bermuda seawater, to assess the presence of cyanophage-like sequences in reef invertebrate virome, metagenome, and transcriptome sequencing libraries. The detailed holobiont virus extraction protocol successfully separated and concentrated virus particles from the tissue of 20 different species of coral reef invertebrate. Cyanophages were isolated and barcoded from 15 of these species: three scleractinian corals, a gorgonian, a corallimorpharian, a zoantharian, two hydrozoans, four species of sponges, two tunicates, and a nudibranch. In total, there were 146 cyanophages isolated and barcoded from seawater (n = 46) or invertebrate tissue (n = 100). The majority (69%) of cyanomyovirus sequences reported in this study were novel, sharing rather low similarity (< 98% nucleotide similarity) with publicly available sequences in the NCBI nucleotide database. Sequence library mining efforts revealed evidence of cyanophage-like sequences in 23 next-generation sequencing datasets, representing 17 species of coral reef invertebrates which included seven species of stony corals, one scyphozoan, seven species of sponges, and two species of copepods. This is the first investigation into cyanophage diversity on Puerto Rico reefs and is a relevant step in coral reef virology, ideally stimulating holobiont-associated isolation efforts to further explore virus genetic and functional diversity within invertebrate tissue on the reef.