In this study, grazing and virus-induced mortality of phytoplankton was investigated in a freshwater pond at the University of Toronto Mississauga, Canada, during September 2009. The modified dilution assay, which partitions phytoplankton mortality into virus and grazing-induced fractions, was used along with newly designed, taxon-specific quantitative polymerase chain reaction (qPCR) assays that target psbA gene fragments to estimate growth and mortality rates for both the entire phytoplankton community and four distinct phytoplankton populations. Community mortality was estimated via fluorometric determination of chlorophyll a (Chl a) concentrations, whereas the relative mortality of individual phytoplankton populations was estimated via qPCR. The sources and amounts of mortality for individual phytoplankton populations differed from those of the whole community, as well as from each other. Grazing was found to be the only significant source of mortality for the community (0.32 day(-1)), and the Prymnesiales (1.65 day(-1)) and Chroococcales (2.79 day(-1)) populations studied. On the other hand, the Chlamydomonadales population examined experienced both significant grazing (1.01 day(-1)) and viral lysis (0.96 day(-1)), while the Chlorellales population only experienced significant mortality as a result of viral lysis (1.38 day(-1)). Our results demonstrate that the combination of qPCR and the modified dilution method can be used to estimate both viral lysis and grazing pressure on several individual phytoplankton populations within a community simultaneously. Further, previously noted limitations of the modified dilution method associated with the dilution of specific phytoplankton populations at low abundances can be overcome with the qPCR-based approach. Most importantly, this study demonstrates that when used alone, whole community-based methods of assessing mortality can overlook valuable information about carbon flow in aquatic microbial food webs.

Extant and newly designed primers for the polymerase chain reaction (PCR) were used to amplify phycodnavirus DNA polymerase ( polB) gene fragments from numerous samples collected at different times of the year from 3 freshwater environments in Ontario, Canada. Overall, a total of 143 cloned PCR fragments were sequenced and 106 putative phycodnavirus polB gene fragments were identified. Although most of these 106 gene fragments were very closely related (i.e. > 97% identical) to polB sequences from chloroviruses, or environmental sequences related to prasinoviruses, 16 represented 2 new types of phycodnavirus polB genes. More specifically, polB fragments that formed a new clade of chloroviruses were amplified from Lake Ontario using newly designed Chlorovirus-specific PCR primers, and a polB sequence most closely related to genes from the prymnesioviruses PgV-03T and CbV-PW1 was amplified from a pond sample from Mississauga, Ontario, using the degenerate algal virus-specific PCR primers AVS1 and AVS2. Thus, the results of the present study provide evidence for a new type of Chlorovirus, and the first observation of polB sequences from freshwater phycodnaviruses that are presumed to infect algae other than chlorophytes.

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva׳s abundance in Lake Ontario was relatively stable, yet CpV-BQ1׳s abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history.

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Experimental determinations of phytoplankton growth and mortality were carried out in a freshwater pond in Mississauga, Canada, during May, July, and October 2011. Modified Landry-Hassett-type dilution assays were performed to assess grazing and virus-induced mortality for 4 distinct phytoplankton populations via taxon-specific quantitative polymerase chain reaction (qPCR) assays, and for the entire algal community via fluorometric determination of chlorophyll a (chl a) concentrations. The sources and amounts of mortality observed for the phytoplankton populations varied throughout the year, and often differed from those of the whole community and each other. In several cases, the effects of viruses in the system appeared to counteract the effects of grazing mortality on phytoplankton. In these instances, mortality was greater in the dilution series generated with filtrates that excluded grazers relative to the dilution series created with filtrates that excluded both viruses and grazers. Relative to the grazer-free dilution series, mortality in the virus-and grazer-free dilution series was reduced for the whole community in May by 0.29 d −1 , and in July for the Prymnesiales, Chlamydomonadales I, and Chlorellales populations by 0.93, 1.31, and 1.87 d −1 , respectively. Although grazing mortality was not observed for the Chlamydomonadales II population during the May experiment, the population's apparent growth rate increased in proportion with the presumed abundance of viruses in the incubation bottles. Our results reinforce the notion that individual phytoplankton taxa can have dynamic roles in aquatic food webs and suggest that viruses can potentially stimulate primary production, counteracting grazing mortality to the point of tipping a phytoplankton population's status from decline to growth.KEY WORDS: Algae · Viruses · Grazers · qPCR · Modified dilution assay · Seasonal · Growth enhancement Resale or republication not permitted without written consent of the publisher