Viruses are an integral component of the marine food web contributing to the disease and mortality of essentially every type of marine life, yet the diversity of marine viral assemblages remains very poorly characterized. This is especially true of the RNA containing viruses. There are several reports of isolations of RNA containing viruses that infect marine protists, but cultivation-independent surveys of RNA viral diversity are still very limited. Previous studies in coastal British Columbia and in coastal Oahu have shown RNA viruses are diverse and persistent in both temperate and subtropical waters. These molecular surveys have made it clear that the few available isolates of marine RNA viruses are just the tip of the iceberg; novel RNA viruses are still being discovered with each new sample analyzed. Many of these novel gene sequences appear to derive from viruses of marine protists and their high diversity suggests that viral infections are a persistent force shaping protistan community composition in the sea. Quantifying the abundance, diversity, and dynamics of these viruses is an important first step toward incorporating them into the ecology of the sea. The immediate goals with this proposal, therefore, are focused on answering some important first order ecological questions, namely, 1) Do RNA viruses make a significant numerical contribution to the total virus pool?, 2) On what spatial and temporal scales does the diversity and specific composition of the marine RNA virus community in this ecosystem vary?, 3) What are the decay and production rates of RNA viruses and how do they compare to those of DNA containing viruses?
The investigators will estimate the absolute and relative abundance of RNA viruses using epifluorescence and electron microscopy in parallel with RNA and DNA assays of purified and fractionated viral assemblages harvested from seawater and we will document the genomic diversity of RNA viruses in a subtropical environment using a metagenomics approach. New bioinformatic tools and approaches specifically designed to handle metagenomic data will also be tested and implemented in this project. The investigators will employ DGGE fingerprinting and sequencing of the RdRP gene to determine on what scales (meters to kilometers, hours to years) the community composition varies and how this variability relates to measured environmental gradients. They will also quantify the spatial distribution and temporal dynamics of individual phylotypes of RNA viruses by qRT PCR. The sampling program will resolve the relevant scales of variability in community composition. This is a fundamental issue that is frequently ignored in investigations of microbial diversity. Finally, the investigators will attempt to make the first estimates of decay and production rates of RNA viruses relative to DNA containing viruses in different plankton size fractions. These estimates will provide a basis for estimating the contribution of RNA viruses to mortality among the plankton.
It is expected that the output from this project will be of interest and value to researchers in a range of disciplines including microbial oceanographers, evolutionary biologists, virologists, and protistan ecologists. This study will provide the first information on the dynamics of a poorly studied group of pathogens, which are likely to have significant influence on structure and function of the planktonic food web. The project is almost certain to dramatically expand the known range of genomic diversity among RNA viruses, will provide new insights into their evolution, ecology, and biology, and will contribute to the development of new bioinformatics tools of broad applicability for metagenomics.
The PIs lab participates routinely in the bi-annual open house held by the School of Ocean and Science and Technology at the University of Hawaii, which draws thousands of visitors over two days including K12 school groups from over 50 Oahu schools. This opportunity is used to educate students and teachers about the presence and importance of microbes in the sea and to motivate student interest in scientific research. The PI and post-doc on this project are also affiliated with a recently established Science and Technology Center known as the Center for Microbial Oceanography - Research and Education (CMORE). As members of CMORE, we are working with an education and outreach team to help develop and implement a variety of outreach and education activities. A key educational goal is to work with CMORE's education office to increase diversity among under represented groups, particularly Native Hawaiians and Pacific Islanders (NHPI). C MORE has already established a strong, working partnership with (1) Ka Imi Ike ("Seekers of Knowledge"), aimed at recruiting and retaining NHPI undergraduates into the ocean and earth sciences, and (2) the Hawaiian Internship Program (HIP), a 10-week summer program that engages NHPI undergraduates and recent graduates in hands-on scientific research through close mentoring. The PI and post-doc have already hosted one NHPI student in their lab through the HIP program for the summer of 2007 with great success. This student plans to continue her work on viral diversity this fall and we anticipate that the results from her summer project will lead to a publication in a peer reviewed journal. We plan to participate in the HIP and Ka Imi Ike programs in future years and funds budgeted for undergraduate research in this proposal will be used to help support research by students identified through these programs.
