Comparison of techniques used to count single-celled viable phytoplankton

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Journal of Applied Phycology 24 (2012): 751-758, doi:10.1007/s10811-011-9694-z.Four methods commonly used to count phytoplankton were evaluated based upon the precision of concentration estimates: Sedgewick Rafter and membrane filter direct counts, flow cytometry, and flow-based imaging cytometry (FlowCAM). Counting methods were all able to estimate the cell concentrations, categorize cells into size classes, and determine cell viability using fluorescent probes. These criteria are essential to determine whether discharged ballast water complies with international standards that limit the concentration of viable planktonic organisms based on size class. Samples containing unknown concentrations of live and UV-inactivated phytoflagellates (Tetraselmis impellucida) were formulated to have low concentrations (<100 ml-1) of viable phytoplankton. All count methods used chlorophyll a fluorescence to detect cells and SYTOX fluorescence to detect non-viable cells. With the exception of one sample, the methods generated live and non-viable cell counts that were significantly different from each other, although estimates were generally within 100% of the ensemble mean of all subsamples from all methods. Overall, percent coefficient of variation (CV) among sample replicates was lowest in membrane filtration sample replicates, and CVs for all four counting methods were usually lower than 30% (although instances of ~60% were observed). Since all four methods were generally appropriate for monitoring discharged ballast water, ancillary considerations (e.g., ease of analysis, sample processing rate, sample size, etc.) become critical factors for choosing the optimal phytoplankton counting method.This study was supported by the U.S. Coast Guard Research and Development Center under contract HSCG32-07- X-R00018. Partial research support to DMA and DMK was provided through NSF International Contract 03/06/394, and Environmental Protection Agency Grant RD-83382801-0

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms

Life-Cycle and Genome of OtV5, a Large DNA Virus of the Pelagic Marine Unicellular Green Alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon

Single Virus Genomics: A New Tool for Virus Discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable