Course:EOSC 475/ResearchProject/ViralAbundances

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High Abundance of Viruses Found in Aquatic Environments

Introduction

Viral counts that used bacterial hosts to count plaque-forming units found the viral abundance to be low in natural waters [1]. A new method measuring the abundance of virus particles in sea water samples saw the abundance of bacteriophages in natural aquatic environments to be 103 to 107 times higher than previous estimates [2]. Transmission electron microscopy showed that most heads on viruses were less than 60nm in diameter and the majority of the viral particles were free floating [2].

Instead of using culture methods to assess the abundance of viruses, Bergh et al used a new nonculture technique that was more able to accurately asses the true viral count [2]. While there was variation in the number of viral particles found in one milliliter of water depending on the source of the water, all the counts were still significantly higher than what was previously thought. For example, Table 1 in the paper showed the viral abundances to be an order of magnitude higher in open ocean such as the Atlantic Ocean with 14.9 x 106 ml -1 than compared to fresh water ecosystems like the Barents Sea (0.06 x 106 ml -1). Assuming that the amount of bacteria in one milliliter of natural seawater is 106 and the amount of bacteriophages in one milliliter of natural seawater is 107, Bergh et al calculated that one third of the bacterial population are attacked by bacteriophages each day [2]. If all the attacks result in infection and eventually lysis, then the high density of viruses is large enough to have a significant ecological impact in coastal, open ocean and freshwater ecosystems [2]. Therefore this paper proposed that it is very likely that the high mortality rate of bacteria in aquatic ecosystems that cannot be explained by protozoan grazing can be explained by viral infection.

Methods Used

The paper used transmission electron microscopy (TEM) to measure both the abundance, the morphology and distribution of the viral particles [2]. Unfortunately, TEM is associated with a heavy cost. To prepare the sample for TEM, the sea water sample must be concentrated, deposited on a supporting grid and then stained with an electron dense stain like uranyl acetate [3]. There are both advantages and disadvantages to using this technique. One major advantage is being able to see the morphology of the viruses. Disadvantages are that TEM has low throughput and that the abundance measurements can be highly variable and inaccurate due to the multiple steps required to go from sea water to TEM [3]. The more steps there are to a process, the more likely that there is error being made.

Relationship to Prior Literature

The research done on viruses before this paper was minimal because viruses were found to be in low abundance in marine environments - the viral counts were so low that it was presumed that viruses did not play a material role in oceanic ecosystems. There was growing speculation before this paper that viruses were important, but none of the research had found anything of significance. Therefore, the publication of this paper caused a "rebirth" in viral ecology research which caused researchers to reanalyze the impact of viral particles in the ocean. Some examples that showcase the progress made after the "rebirth" of viral ecology include the growing understanding of viral impact on bacterial mortality and the virus to bacteria ratio (VBR) - a pattern that is reflective of the differences in viral abundances as you move from offshore to the deep ocean [3]. The VBR ranges from less than 5:1 in lakes to over 100:1 in deep waters [3]. There is still ongoing research on the impact of virus population on bacterial communities, but there is a growing consensus that not only do viruses regulate bacterial growth but also their diversity. It has been almost three decades since the paper has been published, but as more research is conducted there is growing evidence that not only are viruses important - they are playing a big role in central processes in oceans around the world.

Carlucci et al.

A paper published by Carlucci et al. in the 1960's evaluated the factors that affected the survival of Escherichia coli (E.coli) in sea water. Before the publication of the Carlucci paper, there was an observation that bacteria mortality rates are much higher in natural sea water than filtered or sterilized sea water, but there was no insight into the identity of the factors that cause this phenomenon. Carlucci et al collected water samples during July and August of 1957 and measured the percentage of bacterial that survived in three different scenarios - natural, filtered and autoclaved [4]. The results showed that in four out of the six samples, E.coli had the lowest mortality rate in autoclaved water, with a close second being filtered and the highest mortality rate in untreated samples [4]. The paper speculated that predatory and competitive organisms were removed by filtration which caused the spike in survival, but suggested that the destruction of bacteriophages could be a factor contributing to the high survival rate in autoclaved water [4]. Although the Bergh et al paper does not reference the Carlucci et al paper, the Carlucci paper does show the beginning of the idea that viruses are important in bacterial production and mortality.

Torrella et al.

Almost twenty years after Carlucci et al discussed the possibility of viruses being a factor in causing bacterial mortality, there was an interest in studying viruses in water. However, the major emphasis was on quantifying viral abundances in sewage or sewage contaminated water because prior data in the literature showed insufficient concentrations of bacteriophages per milliliter in marine environments [5]. Due to the low abundances found in marine ecosystems, viruses were not considered to have had a significant role in bacterial population dynamics [5]. In 1979, ten years before the publication of the Bergh paper, Torella published a paper in Applied and Environmental Microbiology which showed bacteriophage concentration estimates of up to 104 per ml in marine environments [5]. Torrella et al gave a rough estimate that the bacteriophage abundance were at least 103 phages per ml in marine sea water and can be up to 104 per ml [5]. This paper had good evidence that showed that the abundance of viruses were in the tens of thousands per milliliter but it wasn't until the publication of Bergh's Nature paper that there was solid, quantitative estimates showing viral abundances to be in the tens of millions per mL [3].

Influence on the field of research downstream

A year after its publication, two papers published also in Nature confirmed the high abundance of viral particles and the speculation that viruses were regulating bacterial communities [6] [7]. Using electron microscopy, Suttle et al. showed that pathogenic viruses are able to infect important marine primary producers which could be a factor that regulates phytoplankton populations and primary productivity in marine oceans. Unlike the Bergh et al. paper, Suttle et al. used epifluorescence microscopy with a DAPI stain to quantify the number of presumed viruses based on size (less than 0.2um)[6]. These calculations confirmed the Bergh et al. count that viruses in sea water was in millions, ranging from 106 to 10 7 per mL. The research also showed that the particles that were assumed to be virual particles (0.002 - 0.2um) contained pathogens that infected phytoplankton and that it was noted that the primary production was decreased in 4 out of the five studies [6]. Going hand in hand with the Suttle findings, Proctor et al. showed convincing evidence that viruses were contributing to cyanobacteria mortality [7]. It was originally assumed that cyanobacteria and heterotroph death was due to protozoan grazing. This paper showed up to 7% of heterotrophic bacteria and 5% of cyanobacteria contained mature phage in the final irreversible stage of lysis, suggesting that viral infection and lysis could be a main contributor to microbial death [7]. There are thousands of papers that have cited the 1989 paper, and it is still referenced in recent publications. The Bergh et al. paper had such an immediate significant influence on research measuring viral abundance and the impact of viruses on bacterial communities that its impact can be seen even in the short span of a year. To this date, many papers have been published confirming or adding on to the crucial discovery made in 1989.

Evolving Methodology

Along with advancement in viral research comes an advancement in the technology used to quantify viruses. Due to the expense and inaccuracy of transmission electron microscopy, there was an effort to develop a methodology that was more accurate and had higher throughput[3]. Epifluorescence microscopy eventually came onto the scene as the most widely used method to measure viral abundances. In this method, viruses are concentrated on a membrane filter, and their nucleic acids are stained with an epifluorescent dye such as DAPI, YO-PRO or SYBR Green. It was reproducible and for the most part accurate, although the morphology of the viral particles cannot be seen. The most recent methodology to be used in the literature is flow cytometry. Flow cytometry is able to show different viral sub populations based on the way they scatter the light and how they fluoresce. This method has the highest throughput out of all three methods because it is able to process a large amount of data in a short amount of time.

New Areas of Research Opened

Although not the first paper to propose this idea, the Bergh et al paper did speculate that viral infection was an important factor in the ecological control of planktonic micro-organisms, and there were many studies published after this that looked at the role of viruses in regulating bacterial communities [2]. After its publication, it opened many new areas of research such as the investigation into the role viruses played in regulating bacterial mortality, viral impact on biogeochemical processes and further investigation into viral abundance.

Problems and Gaps in Research

Even though there were breakthroughs in viral research, there were still many problems and gaps to this knowledge. It is undoubted that the abundance of viruses is high in marine environments, but there is yet a high throughput technique used to reproduce viral abundance counts in marine sediments accurately [3]. Marine micobiologists have also been unable to connect the vast genetic diversity of viruses to their role in biological ecosystems and the effects of viruses on bacterial community structure and nutrient cycling [3].

Critical Reflection on the Importance and Impact of the paper

The Bergh et al paper was definitely high impact by reviving the investigation into the role of viruses in marine ecosystems. There are so many processes that happens in the world's oceans that are still unknown, and this paper shed some light onto a different variable that could be at play in these processes. It is the basis of many papers that has been published and there is still ongoing investigation into the subject. An example of the progress that has been made was the introduction of the virus to bacterium ratio after the publication of the Bergh et al paper to which a consensus has arrived in the scientific community that the VBR is 10:1 [8]. To exemplify the importance of this ratio, it became part of the BioNumbers database in 2010 [8]. BioNumbers is a database published by Harvard that gathers key numbers in molecular and cell biology in one place to make these widely used numbers highly accessible [8].

Becoming Part of the Ecological Food Chain

To show that viruses are being considered in all facets of marine research, a paper showed the difference between the classic food chain that is very established and the newer microbial food web that encompasses all known microbes [9]. The classic food chain only shows the energy flow from primary producers to higher trophic level organisms but it fails to encompass the microbial population that the new microbial food chain includes. The recognition that viruses are important to the world's oceans has been reshaping how we thought about some of the processes that happen in the oceans like the maintenance and generation of microbial diversity [9]. Similar to how the traditional food chain is being rethought, old ideas regarding our understanding of viruses will continually be replaced by newer ideas through the advancement of science.

Another Look at the 10:1 VBR

Another example of replacing old ideas with newer ideas is this recent paper published January 2016 in Nature Microbiology. This paper challenged the widely accepted notion that viral abundances are ten times higher than bacteria. In the paper titled "Re-examination of the relationship between marine virus and microbial cell abundances", Widington and his team "compiled 5,671 microbial cell and virus abundance estimates from 25 district marine surveys" [10] to find that there is variation to this ratio [10]. They found that the VBR ranges from 1.4 to 160 in the near surface ocean and 3.9 to 74 in the sub-surface; the ratio of 10: 1 is only a happy median between the numbers [10]. Furthermore, the paper goes on to propose that the relationship between viral and bacterial abundances is nonlinear and instead follows the power law [10]. This recently published paper goes to show that the Bergh et al paper published almost thirty years ago was able to lay the foundation for the current research that is being done on viral ecology, but new ideas are starting to either add on or entirely replace the numbers found in that paper. It has advanced our understanding of the central processes across global oceans and the viral abundance of 107 that the paper discovered is still a toolbox number that is being used today.

Bibliography

  1. ↑ Goyal, S. M., Gerba, C. P., & Bitton, G. (1987). Phage ecology. Wiley.
  2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Bergh, O., Børsheim, K. Y., Bratbak, G., & Heldal, M. (1989). High abundance of viruses found in aquatic environments. Nature, 340(6233), 467–468.
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Suttle, C. A. (2007). Marine viruses — major players in the global ecosystem. Nature Reviews Microbiology, 5(10), 801–812.
  4. ↑ 4.0 4.1 4.2 Carlucci, A. F., Scarpino, P. V., & Pramer, D. (1961). Evaluation of Factors Affecting Survival of Escherichia coli in Sea Water. Applied Microbiology, 9(5), 400–404.
  5. ↑ 5.0 5.1 5.2 5.3 Torrella, F., & Morita, R. Y. (1979). Evidence by electron micrographs for a high incidence of bacteriophage particles in the waters of Yaquina Bay, Oregon: ecological and taxonomical implications. Applied and Environmental Microbiology, 37(4), 774– 778.
  6. ↑ 6.0 6.1 6.2 Suttle, C. A., Chan, A. M., & Cottrell, M. T. (1990). Infection of phytoplankton by viruses and reduction of primary productivity. Nature, 347(6292), 467–469.
  7. ↑ 7.0 7.1 7.2 Proctor, L. M., & Fuhrman, J. A. (1990). Viral mortality of marine bacteria and cyanobacteria. Nature, 343(6253), 60–62.
  8. ↑ 8.0 8.1 8.2 Milo, R., Jorgensen, P., Moran, U., Weber, G., & Springer, M. (2010). BioNumbers—the database of key numbers in molecular and cell biology. Nucleic Acids Research, 38(Database issue), D750–D753.
  9. ↑ 9.0 9.1 DeLong, E. F., & Karl, D. M. (2005). Genomic perspectives in microbial oceanography. Nature, 437(7057), 336–342.
  10. ↑ 10.0 10.1 10.2 10.3 Wigington, C. H., Sonderegger, D., Brussaard, C. P. D., Buchan, A., Finke, J. F., Fuhrman, J. A., … Weitz, J. S. (2016). Re-examination of the relationship between marine virus and microbial cell abundances. Nature Microbiology, 15024.
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