Deciphering the ecology of aquatic single-stranded DNA phages (Microviridae/Gokushovirinae)

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Phage (viruses that infect bacteria) are abundant and dynamic components of all ecosystems. Through infection of bacteria, phage can affect carbon and nutrient cycles, influence bacterial diversity, and play important roles in gene transfer. Phage come in two varieties - those with double stranded versus those with single-stranded DNA (ssDNA). It had been assumed that ssDNA phage were not important in aquatic ecosystems, but recent studies by this team suggest otherwise. As the first study of the role of ssDNA phage in inland waters, the proposed research will shed light on a previously overlooked component of aquatic ecosystems. Project results will provide the first insight regarding the abundance, diversity, and ecology of ssDNA phages in natural freshwater habitats. In addition to training graduate and undergraduate students, outreach will encourage teenage girls to pursue STEM fields through creating hands-on learning modules for an Oceanography Camp for 8th Grade Girls at the University of South Florida and through involvement with local Girl Scout troops. This proposal will study the diversity and ecological roles of the ssDNA phages known as gokushoviruses (family Microviridae, subfamily Gokushovirinae) by focusing on two ground-fed springs and associated aquatic ecosystems in Florida. Preliminary studies in one of these springs indicated low ssDNA phage diversity, simplifying evaluations of spatiotemporal variability and improving the likelihood of discriminating specific phage-host associations. This research combines quantitative and qualitative methods to analyze the distribution of gokushoviruses and their potential hosts, as well as lab-based microcosm studies to provide insight into the ecology of this unique viral group. Through diel, daily, and monthly sampling from three sites in each spring system, this project will determine the temporal and spatial patterns of gokushovirus abundance and diversity to test the hypothesis that the springs maintain a stable, low-diversity gokushovirus community, while gokushovirus diversity and abundance display seasonal fluctuations farther from the springs where environmental variation is more pronounced. Through simultaneous sequence-based analysis of host and phage communities, single-cell techniques, microcosm experiments, measurements of bacterial respiration and metabolic diversity, induction assays, and culturing attempts, this project will constrain the potential host range of gokushoviruses and determine their ecological roles in aquatic ecosystems. These experiments will test the hypothesis that ssDNA phages modify bacterial community composition and ecosystem function through infection of specific bacterial hosts, some of which they are able to lysogenize. Completion of these goals will transform aquatic microbial ecology by enabling the inclusion of ssDNA viruses as a well-understood, integral component of the aquatic virome.