Marine microbes are tremendously abundant and are major players and driving forces in global biogeochemical cycles of carbon, nitrogen, phosphorus, and iron. We learned over the past two decades that viruses are pervasive elements in marine systems, with significant ecological, biogeochemical, genetic, and evolutionary effects on cellular marine organisms, but we have remarkably little information about the dynamics of marine viral community structure and how it relates to the community structure of their hosts (largely bacteria and phytoplankton). Such information is critical for developing proper conceptual and practical models of the roles of viruses and how these change over time and space. The goals of this project are (1) primarily, to characterize a significant subset of the natural virus community and its dynamics, along with bacterial host communities, as they change over daily to monthly time scales at the USC well-studied marine Microbial Observatory site (midway between Los Angeles and Santa Catalina Island), testing hypotheses regarding repeating patterns, host range effects, and taxa-time relationships, and (2) secondarily, to incorporate these viruses into microbial association networks by statistically connecting particular types of viruses to specific potential hosts. Approaches for this study include: (a) nested daily, weekly, and monthly collection of bacteria and viruses for nucleic acid samples, (b) amplification of conserved genes, as proxy phylogenetic markers, from a few moderately-well-characterized broad viral groups previously readily found in seawater (i.e. the T4-like myoviruses, T7-like podoviruses), as well as bacterial rRNA genes (c) extensive sequencing, after screening by community fingerprinting, from the mixed amplified products, (d) binning of the sequences or fingerprint fragments into operational taxonomic units (OTUs) at different levels of resolution, (e) evaluation of the results with statistical approaches to examine temporal patterns, relationships (including time-lagged ones) with other viral OTUs, bacteria, protists (monthly only), and environmental parameters, (f) incorporating the viral OTUs mathematically into microbial association networks. Data on environmental parameters, bacteria, and protists are already being collected monthly for an existing Microbial Observatory, so the viral work is complementary to this project, providing a major value-added component. Similarly, this project will add selected daily and weekly microbial data to the Microbial Observatory. Data from the literature and from the PI's preliminary results show they have the technology and capability to meet the first goal, and to our knowledge this would be the first such data set of its scope and kind. The investigators have already published in 2006 that the bacterial communities at the 5m depth of this site show a predictable repeating annual cycle in bacterial community composition, so the expectation of a predictable repeating viral community is not unreasonable. They also have some preliminary data showing some repeated viral occurrences. The second goal requires that there are indeed significant statistical relationships between the viruses and other measured parameters, which the PI anticipates to be the case, but of course cannot predict; if they cannot be demonstrated, this result itself would be informative and would constrain the possible modes of microbial/viral interactions. BROADER IMPACTS: The project will have a significant training component, with a graduate student doing a considerable amount of the work (field, lab, bioinformatic, and writing components) as part of her dissertation research. The investigators also will link the project to a local secondary school science teacher training program, incorporating results into a training module, and also to community outreach at our marine lab, often visited by secondary school students. Outreach to the broader global community will include a project website with protocols, nontechnical and technical descriptions of the work, results, and conclusions.
Marine microorganisms are enormously abundant (Fig 1), and they generate and process the large majority of ocean productivity, essential for ecosystem function. The least understood part of marine microbial systems are the viruses, whose many roles include infection and mortality of bacteria, archaea, and protists, but also the augmentation of functions of the system and transfer of genes. This project aimed to study the viruses as an integrated part of marine microbial systems. Studies centered around a well-established Microbial Observatory, the San Pedro Ocean Time Series (SPOT), and included monthly as well as daily studies of the changes in naturally-occurring bacteria, archaea, protists, and viruses. We used molecular genetic characterization of these microbes to track them, and microbial association network analysis to demonstrate interactions among these organisms. Thousands of statistically significant correlations (positive and negative) were found, indicative of many interactions. This project was the first to study these interactions this way. Among our findings, we found that the Association Networks of bacteria and viruses were much more interconnected than the networks of bacteria and protists. We interpreted this as indicative that viruses and bacteria have many specific interactions, where viruses can infect only one or a few bacteria; this contrasts to the less-specific interactions between bacteria and protists, whereby protists include grazing organisms that can consume many species of bacteria, or alternatively some protists are photosynthetic and can essentially feed multiple species of bacteria. The daily time series showed that changes from day to day were generally gradual, but many associations were found in the changes of the various microbial types. Most correlations were among bacteria or among viruses, but interestingly the correlations between bacteria and viruses tended to have time lags of 2-5 days, suggesting these correlations result from infection processes that take a few days to develop. Overall, this project considerably expanded our knowledge of marine viruses and their potential interacitons with the rest of the marine ecosystem. Broader impacts of the project included training of students from a local Los Angeles high school, USC undergraduates and graduate students, as well as technicians. Results have been integrated into websites for broad dissemination.
