Development of a standardized holobiont virus extraction protocol and its first application to coral reef environments

Abstract

Coral reef invertebrates are not, nor have ever been, completely autonomous entities; rather they exist as a network of inter-dependent species, living and evolving as a “holobiont”. A major, yet relatively understudied, resident of coral reef holobionts and environment, are the viruses. Viruses have just recently been recognized for their roles in animal health and understanding their importance within coral reef holobionts remains unknown until we are able to isolate, identify and know what viruses do. This study therefore, had two objectives: 1. To develop a simple virus extraction protocol for marine invertebrates, that is effective across a wide range of coral reef invertebrate taxa and 2. To utilize this new pipeline to test the feasibility of isolating invertebrate-associated viruses on non-invertebrate associated host strains. The novel virus separation pipeline dubbed Isolation of Holobiont Viruses (ISO-HOV) developed in this study contains five major stages: (1). Rinse and rest, (2). Tissue separation, (3). Physical cell disruption and homogenization, (4). Homogenate cleaning and (5). Extraction validation. Viruses were successfully extracted from tissue of 19 different species of coral reef invertebrates using the ISO-HOV pipeline. For testing objective (2), Synechococcus-infecting cyanophages were targeted for isolation, culturing and genotyping using the two marine strains WH7803 and WH8101. A total of 136 cyanomyoviruses were genotyped using either DNA polymerase or major capsid protein marker genes. The cyanomyoviruses were associated with coral reef invertebrate tissue as well as, seawater collected 1 m above coral reefs substrata. The majority of sequences (~65%) in this study represent previously unreported or novel cyanophage genotypes. The ISO-HOV protocol is a highly effective procedure across several invertebrate taxa facilitating future coral reef-based virus isolation studies. Continued isolation and culturing of viruses will enable full investigations into virus genomes and roles in coral reef invertebrate biology, ecology and resilience.