UBC Theses and Dissertations
The molecular analysis of marine algal virus communities Short, Steven Michael
Viruses are abundant members of marine microbial communities and important components of marine food webs and geochemical cycles. Previously, PCR was used to amplify DNA polymerase gene fragments from algal viruses belonging to the family Phycodnaviridae. The Phyconaviridae are described by the International Committee on Taxonomy of Viruses as large (genomes > 300 kbp) dsDNA viruses lacking envelopes that infect algae. In order to examine algal virus communities, I developed a denaturing gradient gel electrophoresis (DGGE) protocol to rapidly fingerprint gene fragments amplified from marine algal viruses. This thesis describes the development of this fingerprinting method and its application to the study of marine algal viruses in nature. Initially, PCR and DGGE were used to resolve similar sized products amplified from related but relatively dissimilar virus templates. PCR with degenerate algal-virus-specific primers was used to amplify pol gene fragments from three cultured viruses that infect microalgae and a naturally occurring virus community. Although amplification from all samples resulted in PCR products approximately 700 bp in length, the fragments from cultured viruses focused at different locations in a denaturing gradient gel and several bands were resolved in the natural sample. This study demonstrated that PCR and DGGE could be used to resolve genetically distinct viruses from artificial and natural communities. To determine if pol fragments of similar sequence could be amplified from geographically distant areas, natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic Peninsula. Genetic fingerprints of algal virus communities were generated using DGGE. DNA polymerase gene fragments were recovered and sequenced from 25 bands extracted from the gradient gel. All 25 sequences fell outside the clusters of known algal viruses, but were within the Phycodnaviridae. In addition, similar virus sequences (>98 % sequence identity) were recovered from British Columbia and Antarctica indicating that closely related viruses occur both in the Southern Ocean and the NE Pacific. The temporal variability of natural algal virus communities and the co-occurring eukaryotic plankton were studied at a single location on a weekly basis over fourteen months. The changes in the community were related to physical and biological characteristics of the environment. Comparison of algal virus fingerprints with environmental conditions revealed that, at certain times, changes in algal virus community composition were coincident with changes in tide height, salinity, or chlorophyll a concentration. Overall, algal virus community fingerprints were temporally less variable than eukaryotic community fingerprints. While the algal virus fingerprint patterns were stable throughout most of the study, stable eukaryote fingerprint patterns were observed only during the winter months. It appeared that specific taxa of algal viruses could persist in fluctuating physical and biological environments suggesting that the production of, and mortality from, some algal viruses was constantly occurring. My research has demonstrated that fingerprinting techniques can be used to investigate the geographic and temporal variability of marine algal virus communities. The results show that some marine algal viruses are geographically widespread and that some persist through several seasons. These findings corroborate previous studies showing that viruses are important to the mortality of marine phytoplankton and are therefore ecologically important members of marine food webs.
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