Abstract
Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.
Highlights
Marine viruses are the most abundant biological agents in the world’s oceans, with quantities exceeding bacterial abundances by at least one order of magnitude (Wommack and Colwell, 2000; Suttle, 2005, 2007), and can infect members of all domains of life (Rohwer et al, 2009)
Over the past decade a range of methods have been employed to study viruses associated with coral reefs and the coral holobiont, including transmission electron microscopy (TEM) (Wilson et al, 2001, 2005a; Davy et al, 2006; Davy and Patten, 2007; Lohr et al, 2007; Patten et al, 2008a), flow cytometry (Seymour et al, 2005; Patten et al, 2006, 2008b; Lohr et al, 2007), and metagenomics (Angly et al, 2006; Dinsdale et al, 2008; Marhaver et al, 2008; Thurber et al, 2008; Hewson et al, 2012)
While these studies have revealed a diverse array of viruses, coral virology is in its infancy and inherent methodological challenges still exist in this research field
Summary
Marine viruses are the most abundant biological agents in the world’s oceans, with quantities exceeding bacterial abundances by at least one order of magnitude (Wommack and Colwell, 2000; Suttle, 2005, 2007), and can infect members of all domains of life (Rohwer et al, 2009). Over the past decade a range of methods have been employed to study viruses associated with coral reefs and the coral holobiont, including transmission electron microscopy (TEM) (Wilson et al, 2001, 2005a; Davy et al, 2006; Davy and Patten, 2007; Lohr et al, 2007; Patten et al, 2008a), flow cytometry (Seymour et al, 2005; Patten et al, 2006, 2008b; Lohr et al, 2007), and metagenomics (Angly et al, 2006; Dinsdale et al, 2008; Marhaver et al, 2008; Thurber et al, 2008; Hewson et al, 2012). There are no established coral cell lines and only two bacteriophages have been isolated and characterized; they infect the coral-associated bacterial pathogens, Vibrio coralliilyticus (Efrony et al, 2007) and Thallasomonas loyana (Efrony et al, 2009)
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