Grantee Research Project Results
Final Report: Viruses as a Regulator of Harmful Algal Bloom Activity: Aureococcus anophagefferens as a Model System
EPA Grant Number: R829367Title: Viruses as a Regulator of Harmful Algal Bloom Activity: Aureococcus anophagefferens as a Model System
Investigators: Gastrich, Mary Downes , Anderson, O. Roger , Gobler, Christopher , Wilhelm, Steven W.
Institution: Columbia University in the City of New York , Long Island University - Southampton College , University of Tennessee
EPA Project Officer: Packard, Benjamin H
Project Period: January 15, 2002 through July 31, 2005 (Extended to January 14, 2006)
Project Amount: $210,232
RFA: Ecology and Oceanography of Harmful Algal Blooms (2001) RFA Text | Recipients Lists
Research Category: Water Quality , Water , Aquatic Ecosystems
Objective:
Recent studies have elucidated that viruses may play a role in control of harmful algal blooms, including a lytic virus specific to the brown tide organism, Aureococcus anophagefferens. Electron microscopy on field populations at the end of the bloom in l985 revealed the presence of intracellular, icosahedral virus‑like particles (VLPs) approximately 130‑150 nm in diameter in the newly described species, A. anophagefferens which indicated the possibility of viral control on “brown tide” population densities. During the summer of 1992, the brown tide returned to bloom levels in special experimental mesocosms at the University of Rhode Island, and virus infections again appeared to terminate the blooms.
In 1992, viral isolates were obtained from West Neck Bay on Shelter Island in the Peconic Bay system and Blue Point in Great South Bay, using methods similar to Suttle, et al. (1991). A “phage-like” virus, with a head (viral capsid) approximately 50-70 nm in diameter and a tail of 80-100 nm in length, lysed healthy cultures of A. anophagefferens. The potential for clones of other species to become infected (e.g., Thalassiosira pseudonana, Nannochloris spp.) was evaluated and results showed that only A. anophagefferens cultures were lysed by the viral isolate. Further characterization of this same lytic virus isolate indicated that the adsorption coefficient for the virus was 7.2 x 10-9 mL min-1 (90% of the viruses were adsorbed within 140 minutes). Complete lysis of inoculated cultures of A. anophagefferens (from 2.58 x 106 to 3.23 x 105 cells mL-1) occurred with a viral titer as low as 893 viruses mL-1 within 67 hours and the burst size was calculated to be 9.4 viruses per A. anophagefferens cell. Again, the same virus isolate, named BtV, caused a delay in lysis of clones of A. anophagefferens cultured in elevated iron and salinity levels, and the burst size was determined to be between 10-60 viral particles/host cell. Furthermore, ultrastructural analysis of healthy A. anophagefferens cultures inoculated with the same BtV indicated the presence of hexagonal (in cross section) intracellular viral capsids (indicating icosahedral geometry) approximately 140 nm in diameter, without tails, and similar to those found previously in Narragansett Bay. All cultures of A. anophagefferens, inoculated with BtV were lysed within 24-48 hours and infected cells often lacked an exocellular polysaccharide layer (EPS), but infected cells had an electron dense crenated plasmalemma, degraded organelles (plastids were the last to be degenerated) with up to 50 viral capsids in cross section. Intracellular VLPs were documented during 1999-2000 brown tide blooms in Little Egg Harbor, New Jersey, but it was not determined whether cells were infected during the termination of the bloom.
The objectives of this research project were to: (1) determine if the frequency that VLPs infect and lyse natural populations of A. anophagefferens in coastal bays of New York and New Jersey occurred with the same frequency as in 1999-2000, and especially at the termination of the bloom; (2) isolate viruses specific to A. anophagefferens, establish baseline information on the genetic diversity of viruses that infect A. anophagefferens, and determine the influence of viral activity on the proliferation of A. anophagefferens and bloom termination in situ; and (3) conduct field experiments to manipulate the natural concentrations of viruses in seawater to better understand the role of viruses in brown tide bloom dynamics.
Figure 1. Study Area For Objective 1 (the two sites (in red) in Little Egg Harbor sampled in the NJ study)
We studied the ecology of phytoplankton communities dominated by A. anophagefferens, especially the interaction and role of viruses, nutrients, and microzooplankton grazing. We also investigated the impact of viruses, nutrient loading, and microzooplankton grazing on phytoplankton communities in two New York estuaries that hosted blooms of the brown tide alga A. anophagefferens during 2000 and 2002. The absence of a bloom at one location during 2002 allowed for the fortuitous comparison of a bloom and nonbloom year at the same location, as well as a comparison of two sites experiencing bloom and nonbloom conditions during the same year. A map of the study areas can be found in Figures 1 and 2.
Figure 2. Study Area in New York for Objective 3
Summary/Accomplishments (Outputs/Outcomes):
Blooms of the brown tide organism A. anophagefferens have recurred in the coastal bays in New Jersey since 1995 and in the coastal bays of Long Island since 1985. Intracellular VLPs were documented during 1999-2000 brown tide blooms in Little Egg Harbor, New Jersey, but it was not determined whether cells were infected during the termination of the bloom. The objective of this study was to determine if VLPs infected and lysed natural populations of A. anophagefferens in coastal bays of New Jersey and New York in 2002 with the same frequency as in 1999-2000 and especially at the termination of the bloom. Our results confirmed that the highest percentage (37.5%) of VLP-infected cells occurred at the termination of the brown tide bloom in New Jersey in 2002. Intracellular VLPs were present throughout the bloom event. The percentage of visibly infected cells was higher at the beginning of the bloom than during the peak of the bloom. The intracellular VLPs in natural populations of A. anophagefferens were consistent in size and shape (approximately 140 nm in diameter) and comparable to those in previous studies. Concentrated viral isolates, prepared from waters during brown tide blooms in New York and New Jersey in 2002, infected healthy laboratory A. anophagefferens cultures in vitro. The viral isolates associated with the highest laboratory viral activity (lysis positive) were concentrated from water samples having the highest viral and bacteria concentrations. The intracellular viruses in these virally infected laboratory cultures of A. anophagefferens were similar in size and shape to those found in natural populations. The successful isolation of a virus specific to A. anophagefferens from a brown tide bloom in the field, the similarity of ultrastructure of VLPs infecting both natural populations and laboratory infected cultures, and the pattern of VLP infection during bloom activity in combination with the observed high percentage of VLP-infected cells during bloom termination supports the hypothesis that viruses may be a major source of mortality for brown tide blooms in regional coastal bays of New Jersey and New York (Gastrich, et al., 2004).
In addition, we investigated the impact of viruses, nutrient loading, and microzooplankton grazing on phytoplankton communities in two New York estuaries that hosted blooms of the brown tide alga Aureococcus anophagefferens during 2000 and 2002. The absence of a bloom at one location during 2002 allowed for the fortuitous comparison of a bloom and nonbloom year at the same location, as well as a comparison of two sites experiencing bloom and nonbloom conditions during the same year. During the study, blooms were found at locations with high levels of dissolved organic nitrogen and lower nitrate concentrations compared to a nonbloom location. Experimental additions of inorganic nitrogen and phosphorus yielded growth rates within the total phytoplankton community which significantly exceeded control treatments in 83 percent of experiments, whereas A. anophagefferens experienced significantly increased growth during only 20 percent of experimental nutrient additions. Consistent with prior research, these results suggest brown tides are not caused by eutrophication but instead are more likely to occur when sources of labile dissolved organic matter (DOM) are readily available. Microzooplankton grazing rates on the total phytoplankton community and on A. anophagefferens during a bloom were lower than grazing rates at a nonbloom site, suggesting that reduced grazing mortality also may promote brown tides. Mean densities of viruses during blooms (3 x 108 mL-1) were elevated compared to most estuarine environments and were twice the levels found at a nonbloom site during this study. Experimental enrichment of the natural viral densities yielded a significant increase in A. anophagefferens growth rates relative to control treatments when background levels of viruses were low (< 1.7 x 108 mL-1), suggesting that viruses may promote bloom occurrence by regenerating DOM or altering the composition of microbial communities (Gobler, et al., 2004).
The combined results of this study and previous studies clearly show continuing evidence of a persistent viral infection of natural populations of Aureococcus occurring within a regional geographic range over several bloom years. The sampling frequency in 2002 provided a better definition of the sequential stages of the bloom in Little Egg Harbor, New Jersey, and our transmission electron microscopy (TEM) results clearly characterized the percentage of VLP-infected Aureococcus in natural populations throughout the bloom period (e.g., elevated percentages at the beginning of the bloom, sharply decreased percentages during the peak of the bloom, and up to 60 percent of infected cells as the bloom subsided). Although these results also corroborated previous studies, our study provided additional evidence of an increased percentage of VLP-infection at the end of the bloom.
Our results showed that although we were able to readily amplify viruses in the laboratory on hosts, these virus particles did not survive standard cesium chloride (CsCl) density gradient purification. Similar results recently have been reported from other laboratories working with other virus/host systems. As such, to date we have been unable to confirm Koch’s postulates on these samples and have forgone isolating DNA from prominent bands in gels. Therefore, we have been unable to complete a restriction fragment length polymorphism (RFLP) assay. Because of this, we were unable to complete successfully the one-step growth curves (based on standard techniques) on the isolated viruses for several reasons. We outlined in previous reports that we are not able to recover the virus from CsCl gradients (although Gary, et al., have stated they can). During the course of our work, we isolated bacteria that lyse A. anophagefferens Therefore, we believe that the lytic bacteria we isolated may have been the agent responsible for the lytic activity that Gary, et al. (1998) observed (and hence why they saw phage-like viruses in their samples). Of course, without their samples (which would now be 8 years old), this is impossible to test.
As a result, our secondary approach was to look at the diversity of these viruses by employing poly chain reaction based amplification and sequencing of the DNA polymerase (pol) gene. Peer reviewed literature states that these viruses are Phycodnaviridae, and as such they should have DNA pol genes that are amplifiable using the standard ASV-1 and ASV-2 primer sets. Attempts in both the Suttle and Wilhelm laboratories have independently confirmed that this is not the case. Although outside the objectives of this proposal, the Wilhelm laboratory currently is designing new DNA pol gene probes as well as screening conditions for eukaryotic algal viruses, which we anticipate will be useful for the A. anophagefferens virus system. New approaches (i.e., different gradients, gel filtration) also are under consideration but may fall outside this proposal.
We can report our results of viral lysis of A. anophagefferens. Figure 3 shows typical results of “kill curves” generated during lysis experiments. To track the lysis of samples, regular fluorescence measurements were made throughout the procedure of all samples. Moreover, to avoid pseudoreplication, all samples collected were done so destructively (i.e., no subsampling occurred). The results indicated that a uniform, controlled lysis of cultures of about3.5 x 107 mL-1 occurred between the V7-9 samples that were taken. As such, all samples to this point should contain unlysed, infected A. anophagefferens cells.
Figure 3. Left: FSU Versus Treatment for all Samples Showing Lysis Occurred Approximately 42 hours After Viruses Were Added. Right: Graph of cell abundance versus FSU shows we are working at > 107 cells mL-1.
Our results of a microscopic examination of SYBR Green 1 aliquots of the V10 sample (42.2 h after inoculation) showed an initial accumulation of debris from cell lysis and the onset of bacterial proliferation and increased free virus particles (SYBR Green I stain was the first of the new generation of DNA stains introduced by Molecular Probes in 1995 and since then has been used in the detection of DNA in gels). A significant abundance (~ 106) of intact A. anophagefferens cells remained in the sample (detected via microscopic examination of chlorophyll a autofluorescence). After careful analysis of growth conditions, it was determined that cells do not grow with any regularity using fluorescence light only sources. Longer wavelength light (supplied in our case by Tungsten incandescent bulbs) seems important to getting consistent growth. Figure 4 shows the epifluorescent images of A. anophagefferens cultures exposed to virus-mediated lysates. Results from the examination of several cultures suggest that the A. anophagefferens virus can be distinguished from background phage because of its larger size. Figure 4 (right) shows autofluorescence of the same sample showing chlorophyll containing A. anophagefferens (Image by Rowe and Wilhelm; not for distribution).
Figure 4. Epifluorescent Images of A. anophagefferens Cultures Exposed to Virus-Mediated Lysates. Left: SYBR Green I stain of culture, showing distinct A. anophagefferens cells as well as fluorescent VLPs. Results from the examination of several cultures suggest that the A. Autofluorescence virus can be distinguished from background phage because of its larger size. Right: A. Autofluorescence of same sample showing chlorophyll containing A. anophagefferens. Image by Rowe and Wilhelm; not for distribution.
Figure 5 shows a scanning electron micrograph (SEM) image of viruses attached to the surface of A. anophagefferens cells. Viruses were isolated from cultures of A. anophagefferens after exposure the cells were exposed to virus concentrates from the 2002 and 2003 bloom events. Viruses were reinoculated into a fresh culture of A. anophagefferens, and samples were fixed after 30 minutes to determine whether particles were adhering to cells. SEM images demonstrate that particles are similar in diameter (~ 140-160 nm) and shape to intracellular VLPs previously observed (Gastrich, et al., 2004) (Image by Rowe, Dunlap, and Wilhelm).
Figure 5. SEM Image of Viruses Attached to the Surface of A. anophagefferens Cells
Negatively stained-TEM micrographs were completed showing that VLPs of the appropriate size and shape are abundant in lysates (Figure 6). Figure 6 shows a negatively stained TEM image of a typical acellular VLP after lysis of a brown tide culture. This image shows a tail-less VLP approximately the same size as the intracellular viruses found earlier.
Figure 6. Negatively Stained TEM Image of Typical Acellular VLP After Lysis of a Brown Tide Culture. Phage like particles (not shown) also are observed. Image by Rowe, Dunlap, and Wilhelm; not for distribution.
As part of our revised study that assessed the contribution of bacterial lysis to healthy cultures of A. anophagefferens, our significant findings show that six bacterial isolates (of which apparently five are unique), capable of consistently lysing A. anophagefferens 1784, have been brought into culture. Sequence analysis of a 16s rDNA amplicon places these bacteria in six unique clades. Cladistics or phylogenetic systematics is a branch of biology that determines the evolutionary relationships of living things based on derived similarities. It forms the basis for most modern systems of classification, which seek to group organisms by evolutionary relationships. Analysis of full 16s rDNA sequences (27F – 1522R) has validated each of the identifications so far.
These SEM micrographs are significant findings in that the viruses are the same size and shape (and without tails) as the intracellular viruses we have found in natural populations of A. anophagefferens, and these results are consistent with our published results of our field work (Gastrich, et al., 2004). In addition, A. anophagefferens cells not inoculated with viral isolate show no such viral particle attachments as the inoculated cells.
Negatively stained-TEM micrographs have been completed showing that virus-like particles of the appropriate size and shape are abundant in lysates (Figure 4).
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 8 publications | 4 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Gastrich MD, Leigh-Bell JA , Gobler CJ, Anderson OR, Wilhelm SW, Bryan M. Viruses as potential regulators of regional brown tide blooms caused by the alga, Aureococcus anophagefferens. Estuaries 2004;27(1):112-119. |
R829367 (2003) R829367 (Final) |
not available |
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Gobler CJ, Deonarine S, Leigh-Bell J, Gastrich MD, Anderson OR, Wilhelm SW. Ecology of phytoplankton communities dominated by Aureococcus anophagefferens: the role of viruses, nutrients, and microzooplankton grazing. Harmful Algae 2004;3(4):471-483 |
R829367 (2003) R829367 (Final) |
not available |
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Gobler CJ., Anderson OR, Gastrich MD, Wilhelm SW. Ecological aspects of viral infection and lysis in the harmful brown tide alga Aureococcus anophagefferens. AQUATIC MICROBIAL ECOLOGY 2007;47(1):25-36. |
R829367 (Final) |
Exit Exit |
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Gobler CJ., Anderson OR, Gastrich MD, Wilhelm SW. Ecological aspects of viral infection and lysis in the harmful brown tide alga Aureococcus anophagefferens. AQUATIC MICROBIAL ECOLOGY 2007;47(1):25-36. |
R829367 (Final) |
not available |
Supplemental Keywords:
viruses, brown tide blooms, Aureococcus anophagefferens, harmful algal, blooms, aquatic ecosystem,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Aquatic Ecosystems & Estuarine Research, Ecosystem/Assessment/Indicators, Ecosystem Protection, Chemistry, Environmental Microbiology, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, algal blooms, Biology, mortality rate, harmful algal blooms, control of algal blooms, Auerococcus, brown tide blooms, ECOHAB, water qualityProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.