Marine Diatom Virus Investigations: Methodology for the isolation and characterization of environmentally relevant laboratory systems

As the most abundant biological entities in aquatic environments, virus impact through their infection dynamics has been estimated to have stimulatory effects on microbial food webs. Major primary producers in coastal ecosystems are diatoms, eukaryotic phytoplankton that are capable of rapid nutrient uptake and increased abundances. Diatom viruses are unique compared to many other phytoplankton in that, to date, all isolates contain ssDNA or +ssRNA genomes. Additionally, they are highly specific for their host, many at the level of strain-specificity and have largely been underrepresented in laboratory studies with only ~20 isolated and characterized. To increase our understanding of the fundamental role viruses have on diatom community structure and physiology, strains of diatoms have been isolated from coastal Southern California waters. Also, methods for the robust and routine isolation, characterization and evaluation of diatom viruses have been developed. Isolation of viruses is generally specific for the ‘life’ cycle used, ranging from lytic viruses that are released into the extracellular matrix to cell-associated viruses that remain persistent as intracellular units. Preliminary data indicated that both viral ‘life’ strategies were present for marine diatoms, therefore three different methods were evaluated, including 1) physical disruption, 2) filtration and concentration, and 3) hypotonic or water lysis. To assess presence of +ssRNA viruses, RT-PCR and qRT-PCR approaches were implemented using an inhouse sequence database from a global marine RNA virus study and existing laboratory strains. Established RNA isolation and sequencing methods were modified to gain insights into free and intracellular viruses as well as assess host perturbations during infection. Upon isolation strain-specificity was determined using host-range assays. Together, these methods represent a robust mechanism to obtain laboratory systems of relevant marine diatoms and their viruses.