| Summary: | 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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