Course:EOSC 475/ResearchProject/ViralMortalityOfEHux

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Figures 1,3,4,5 a-d of Bratbak et al. (1993) paper[1]. The rise in Large Virus-like Particles (LVLPs) following the crash of the Emiliania huxleyi population occurs in the mesocosm experiments replete in phosphate seemingly regardless of nitrate levels. The sharp decline of E. huxleyi population while nutrients are still available suggests the termination of a bloom is not due to nutrient limitation but rather caused by mass viral lysis. Algal blooms of other species can be seen succeeding the initial E. huxleyi bloom. E. huxleyi populations decline as expected in P limited conditions. (The entire 1991 experiment (Figure 4) and June 25 onwards in 1990 experiment can considered to be phosphate depleted because phosphate leakage from cells had increased dissolved phosphate concentrations during storage.)

Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms[1]

The role of viruses in controlling host population dynamics is explored in this 1993 publication. It is one of the many publications discussing the a series of 33 mesocosm experiments that discuss the effect of nutrient availability on phytoplankton population dynamics [2] [3]. The 11 mesocosms with silicate concentrations less than 0.2 µM produced E. huxleyi-dominated blooms, 8 of which are discussed in the paper. The authors used mesocosms stationed off the shores of Norway to create a controlled environment in which to study the effect of nutrient availability on the population dynamics of E. huxleyi and large virus-like particles (LVLPs) that infect it.

By producing a bloom of Emiliania huxleyi with nutrient-enriched mesocosms, a rise in a homogenous population of LVLPs was observed in those mesocosms with phosphate available in excess immediately following a collapse of the E. huxleyi population in non-nutrient-limiting conditions. Nitrogen limitation appears to have little effect on the change in LVLP abundance and both nitrogen and phosphate limitation does not appear to produce a significant rise in LVLP abundance nor a collapse in E. huxleyi collapse as observed in mesocosms high in phosphate. Instead, a gradual decrease in population likely attributed to nutrient limitation is observed. In those mesocosms where E. huxleyi populations collapsed, a second algal bloom would appear consisting of other algal species (dinoflagellates or Phaeocystis sp.). In addition to monitoring nutrient, LVLP, and algal concentrations, samples of the mesocosm contents were collected every two to three days during the collapse for thin-sectioning and transmission electron microscopy (TEM). Thin sectioning allowed the authors to determine burst size by counting LVLPs in clusters in, as well as determine the percentage of LVLP –containing cells. Mean burst size was 500 with a range of 350-700 and about 3 percent of E huxleyi cells observed contained LVLP. Burst size and free LVLP abundances were used to calculate the decay rates for the mesocosms exhibiting net algal mortality. The decrease in LVLP abundances were used to calculate a viral decay rates of 0.1-0.8 d-1, and viral mortality was calculated to account for between 25 and 100% of E. huxleyi mortality.

Bratbak et al. opened up the largely unexplored the field of E. huxleyi-virus interactions. Other virus-host interactions were studied in Micromonas pusilla[4] and the Chlorella-like alga NC64A [5], but E. huxleyi had yet to be studied. Aside from the initial observation of viral particles contained in E. huxleyi cells in 1972 by Manton & Leadbeater[6]., there was very little known about the relationship between the alga and its specific virus. The data presented validates the speculation that E. huxleyi blooms can be terminated by viral lysis, a hypothesis first confirmed by Castberg et al.[7]. The results of this study were necessary to demonstrate the viral influence in the boom-and-bust lifestyle of E. huxleyi; demonstrate the requirement for phosphate to viral production; and measure burst size, viral mortality rates and changes in viral and host abundance.

Background information

Emiliania huxleyi

A coccolithophore bloom south of England as seen from a satellite.

The coccolithophore Emiliania huxleyi is a bloom-forming phytoplankter with world-wide distribution and can turn sea water milky-white when a bloom experiences viral lysis and sheds its calcium carbonate shells, called coccoliths[8]. It has three main forms, a coccolith-bearing, diploid "C-cell", a non-motile "N-cell", and a flagellated, haploid "S-cell" [9]. Massive shedding of coccoliths can be visible from space and increase the albedo of surface waters, which in turn lower sea water temperatures as well as sequester carbon to the deep sea by sedimentation [10]. It is proposed that precipitation of calcium carbonate produces CO2 to enhance photosynthesis. E. huxleyi also affects the sulfur cycle in that it is a major producer of dimethylsulfionopropionate (DMSP) which breaks down into the volatile compound dimethyl sulfide (DMS). Aside from providing a highly labile source of organic sulfur to support the microbial community­[11], DMS acts as a cloud condensation nuclei and can increase cloud formation and in turn increase the albedo of the atmosphere[12]. As a primary producer, zooplankton graze on the phytoplankter and supports higher trophic levels when not being lysed en masse. Thus E. huxleyi is an extremely important species to consider when studying climate change, biogeochemical cycling, microbial, and macro ecology.

Marine viruses

Diagram showing how biomass is transfered and diverted from upper trophic levels to particulate and dissolved organic matter pools.[13]

Marine viruses contain genetic information in the form of RNA or DNA and require host cell machinery to replicate and spread. They fill crucial niches in sea water biology by specifically removing hosts organisms including phytoplankton and bacteria[14] [15]. Their effect on the entire biosphere is non-negligeable, considering up to 50% of bacteria are lysed every day, the high carbon turnover rate of the ocean is heavily impacted by viral activity in the sea.

Discussion of Methods

The floating laboratory with 11 m³ polyethylene enclosures attached to the raft.

Mesocosms: A floating laboratory was built in a bay by the Marine Biological Field station adjacent to Raunefjorden in Norway. Seawater is kept homogeneous by pumping seawater from the bottom of the mesocosm to the top. Unfiltered water from 1 m deep and nutrients in the form of concentrated NaNO3 and K2HPO4 enters the mesocosm from the top.

Phytoplankton enumeration: Live samples were counted using a hemacytometer and formaldehyde-preserved samples were concentrated by sedimentation (according to Ütermohl [16]) and counted.

Free VLP enumeration: Acid Lugol's solution was used to dissolve calcium carbonate debris. The procedure for virus counts using TEM are described by Børsheim et al. and Bratbak et al. [17][18]

Nutrient analysis: Whole samples preserved with chloroform were analyzed with a Chemlab Auto-analyzer according to Strickland & Parsons.

Thin sectioning: Thin sectioning was used to calculate the percentage of cells containing LVLP and to count viral burst size. 10 L was concentrated and processed to produce 100-300 cell sections per sample.

Critical review of methods

The authors state that analysis of unfiltered samples was carried out “within a few days or at the end of the experiments” when the procedure by Strickland & Parsons for using the Chemlab Auto-analyzer® advises against delaying analysis for more than 1-2 hours. In this case, it seems chloroform preservation was not adequate to produce accurate nutrient reading due to leakage of phosphate and the problem would have been avoided had the authors analyzed filtered seawater instead. If there was any advantage to using unfiltered water for nutrient analysis, it was not stated.

Counting viruses with TEM is advantageous because it allows identification based on morphology. LVLPs infecting were identified by size and morphology, therefore fluorescence microscopy counts would not have been appropriate for the purposes of this study. The more efficient, higher-throughput method of analytical flow cytometry (FCM) would not be employed to count viruses until 1999 [19] and the technique has since been improved upon to allow sorting of infected, healthy, naked, or coccolith-bearing cells [20][21] [22]. However, TEM counts have been shown to underestimate virus counts when compared to counting DAPI or Yo-Pro stained samples with fluorescence microscopy [23]. The use of TEM to estimate burst size and infected cells reduces accuracy of the method by introducing many confounding factors. The authors have argued that the numbers of virus-containing E. huxleyi cells could have been "severely underestimated" due to (1) lysing of delicate cells during centrifugation, (2) the difficulty of recognizing infected cells with very low numbers of virus particles, (3) exclusion of virus particles within the cell but not visible when thin-sectioned due to uneven spatial variation, and (4) the invisibility of virus particles during the eclipse period. Poor accuracy is likely to result without information on the spatial variation and eclipse period of the virus.

This paper provided a reliable way to induce an artificial bloom of phytoplankton to study native assemblages of species. It appears the more you oversaturate the mesocosms with N and P, the more homogenous and less diverse the bloom seems to be, since the more enriched 1988 produced a bloom of >99% ehux while the 1989 bloom produced 80% ehux algal bloom. The mesocosms have since been replicated (or re-used) to study phytoplankton blooms further(sources) to create a controlled environment to sample and use with other more novel methods such as flow cytometry [24], fluorescence-activated cell sorting [20] and transcriptome sequencing [25].

Article in relation to prior literature

The VLPs first observed in E. huxleyi cells were reported as having a diameter on 22nm [6]. The TEM imagery of LVLPs in infected cells are demonstratively about 200nm.

Hutchinson's paradox

The competitive exclusion principle states that two species occupying the same niche in the same geographical location will eventually compete with one another until one becomes extinct[26]. Thus Hutchinson asks, how is it possible for the phytoplankton, all competing for the same nutrients, to coexist without one or two key species driving the rest to extinction [27]? Hutchinson suggests that pelagic ocean could never be considered at equilibrium due to turbulence. Indeed, in highly competitive nutrient-enriched waters, one species may dominate up to 99 percent of the phytoplankton population as was the case with E. huxleyi but complete dominance of the microalga was prevented by the highly specific mechanism of viral infection [1]. Viral mortality acts as one of the many pressures keeping the system dynamic and preventing the system from reaching equilibrium. It contrasts the idea that primary producers in a traditional trophic system are controlled by pressures from grazers found above their trophic level ("top-down" control), as well as limited by the dissolved nutrients available to them ("bottom-up' control) [28], therefore adding a "side-in" species-specific control of a population in a given ecosystem [1].

Discussion of knowledge gaps

Effect of nutrient limitation

What is the effect of nitrogen limitation and why does it seem inconsequential to LVLP increase? The data suggests that phosphate depletion inhibits the development of LVLPs in E. huxleyi regardless of nitrogen depletion levels. The authors proposed that viruses require a higher P/N ratio than cells, thus phosphorus limitation would prevent or slow virus production[1].In 2002, Jacquet et al. corrects the initial claim that nitrogen limitation appeared to be "inconsequential" in viral infection by concluding that nitrogen limitation delays lysis while reconfirming the requirement for phosphate to produce viruses. Furthermore , it appears that phosphorus limitation can also lower viral burst size [24]. Presently, it is known that viral infection of E. huxleyi induces the the production of viral glycosphingolipids (vGSL) which are needed for viral release by budding and vGSL is proposed to act as a dose-dependent cue for lysis to occur [29]. The transcription of machinery needed to produce vGSL might become a bottleneck during times of nitrogen limitation and ultimately delay lysis until enough vGSL is produced.

Survival of lytic viruses while host population densities are low

How do lytic viruses survive during the unproductive time of year, considering the decay rate and low host population? The authors propose the observation can be explained if a select fraction of the virus population experienced a lower than average decay rate, which would be possible through genetic variation or environmental conditions. Microenvironments with lower UV exposure or lower temperature could act as refuge areas that lower the decay rate of a virus. In 2014, Frada et al. reported lowered viral decay rates in viruses ingested by copepods. Furthermore, defecation of EhV, acts as an amplified dispersion method that can be used to expand the reach of the viruses even when host abundance is low [30]

Genetic variation between EhV strains could also account for a difference in viral stability. The two virus strains EhV-86 and EhV-207 were used to infect E. huxleyi were compared, EhV-207 appeared to outcompete EhV-86 with increased speed in replication and increased lethality of the host, but whether or not the competitive advantage is offset by lowered viral stability is still a question to be answered [31].

The authors also propose two other hypotheses in attempt to answer the question, but so far no evidence has supported them:

  1. The virus is actually a temperate virus and lysis is induced only when infection of new cells is likely, i.e. when host abundance is high
  2. A temperate virus that mutates into a lytic virus could always exist in the host population during times of low abundance, and through mutation, could produce a mutated lytic version of the virus that only proliferates in the presence of high host concentrations.

Life cycle of the virus

A proposed working theory of how viral lysis and host resistance occurs[32]. The hexagons represent virus capsids and the black circles represent glycosphingolipid (GSL) rafts. Viruses enter the cell through the GSL rafts and go on to replicate and transcribe viral GSL. The infection produces reactive oxygen species (ROS) which can induce the transcription of caspases and metacaspases needed for programmed cell death (PCD). Emiliania huxleyi can control ROS by producing dimethylsulphoniopropionate (DMSP) and related compounds which seek out and deactivate ROS, thus preventing or postponing PCD and lysis. Other infochemicals may play a role in changing the behaviour of the surrounding population, such as differentiating into a resistant haploid phase[20].

The authors have argued that the numbers of virus-containing Ehux cells could have been "severely underestimated" due to (1) lysing of delicate cells during centrifugation, (2) the difficulty of recognizing infected cells with very low numbers of virus particles, (3) exclusion of virus particles within the cell but not visible when thin-sectioned due to uneven spatial variation, and (4) the invisibility of virus particles during the eclipse period. The life cycle of the virus had been yet to be elucidated at the time of the experiments. The authors recognize the gap in knowledge about the approximate timeline of the virus infecting Ehux.

Mechanism of viral infection

Mackinder et al. has described the EhV infection strategy as such: The virus enters the cell by the fusing of viral and host membranes, possibly by recognizing GSL rafts on the host membrane. The virus replicates its genome and viral proteins and transcription of viral GSL occurs, which in turn induces metacaspase activity required for programmed cell death (PCD). Meanwhile, virus assembly of genome and viral proteins occurs and controlled release of viruses through budding occurs [32]. Eventually the infected cell blebs and lyses. The timing of lysis may be controlled by vGSL concentrations in the cell which have been measured to be 80-300 fg/cell, or enough to initiate PCD [33].

Host resistance strategy

Evidently, E. huxleyi populations in the mesocosm experiments are not removed completely after viral lysis. Thus, it can safely assume E. huxleyi has a mechanism to develop resistance to infection in some way. The Cheshire Cat hypothesis proposes that that the survival of the population after an initial bloom collapse could be attributed to sexual differentiation of E. huxleyi cells causing uninfected cells to shift from the diploid phase to the haploid phase. The haploid phase has been shown to be resistant to infection, a mechanism most likely a resulting from a change in cell membrane markers that the virus must recognize to enter the cell. It is so named because E. huxleyi acts as if it hides from its virus. TEM micrographs of a declining E. huxleyi bloom from a mesocosm were first shown to shift from diploid to haploid phase in 1996[34]. The coccolith-less, naked, haploid cells were recognized based on descriptions given in 1972 [35]. The shift can also be recorded by FCM as naked cells show up with the same fluorescence patterns but with a lower right-angle light scatter signal [24].

In contrast, "Red Queen" mechanics produce constant co-evolution keeps the virus and its host locked in an "evolutionary arms race" of sorts. Susceptible diploid cells exhibit "Red Queen" mechanics when eactive oxygen species, ROS, are produced during the infection as part of PCD [36]. E.huxleyi produces DMSP and related compounds to inactivate ROSs and potentially stop the progress of PCD. DMSP does not decrease susceptibility to viral infection as diploid cells are equally susceptible to infection as mixed diploid and haploid cultures [20]

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 Bratbak, G., Egge, J. K., & Heldal, M. (1993). Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms. Marine Ecology Progress Series 93, 39-48.
  2. ↑ Egge, J. K., & Aksnes, D. L. (1992). Silicate as regulating nutrient in phytoplankton competition. Marine ecology progress series. Oldendorf, 83(2), 281-289
  3. ↑ Egge, J. K., & Heimdal, B. R. (1994). Blooms of phytoplankton including Emiliania huxleyi (Haptophyta). Effects of nutrient supply in different N: P ratios. Sarsia, 79(4), 333-348.
  4. ↑ Mayer, J. A., & Taylor, F. J. R. (1979). A virus which lyses the marine nanoflagellate Micromonas pusilla. Nature, 281(5729), 299-301.
  5. ↑ Van Etten, J. L., Burbank, D. E., Xia, Y., & Meints, R. H. (1983). Growth cycle of a virus, PBCV-1, that infects Chlorella-like algae. Virology, 126(1), 117-125.
  6. ↑ 6.0 6.1 Leadbeater, B. S. C. (1972). Fine structural observations in six new species of Chrysochromulina (Haptophyceae) from Norway with preliminary observations on scale production in C. microcylindra sp. Nov. Sarsia, 49(1), 65-80.
  7. ↑ Castberg, T., Larsen, A., Sandaa, R. A., Brussaard, C. P. D., Egge, J. K., Heldal, M., ... & Bratbak, G. (2001). Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta). Marine Ecology Progress Series, 221.
  8. ↑ Berge, G. (1962). Discoloration of the sea due to Coccolithus huxleyi “bloom”. Sarsia, 6(1), 27-40
  9. ↑ Green, J. C., Course, P. A., & Tarran, G. A. (1996). The life-cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry. Journal of marine systems, 9(1), 33-44.
  10. ↑ Honjo, S. (1976). Coccoliths: production, transportation and sedimentation. Marine Micropaleontology, 1, 65-79. Chicago
  11. ↑ Vila-Costa, M., Simó, R., Harada, H., Gasol, J. M., Slezak, D., & Kiene, R. P. (2006). Dimethylsulfoniopropionate uptake by marine phytoplankton. Science, 314(5799), 652-654.
  12. ↑ Charlson, R. J., Lovelock, J. E., Andreae, M. O., & Warren, S. G. (1987). Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature, 326(6114), 655-661
  13. ↑ Fuhrman, J. A. (1999). Marine viruses and their biogeochemical and ecological effects. Nature, 399(6736), 541-548.
  14. ↑ Suttle, C. A., Chan, A. M., & Cottrell, M. T. (1991). Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton. Applied and environmental microbiology, 57(3), 721-726.
  15. ↑ Proctor, L. M., & Fuhrman, J. A. (1990). Viral mortality of marine bacteria and cyanobacteria. Nature, 343, 60 - 62.
  16. ↑ Utermöhl, V. H. (1931). Neue Wege in der quantitativen Erfassung des Planktons. Verh. int. Verein. theor. angew. Limnol, 5, 567-596.
  17. ↑ Børsheim, K. Y., Bratbak, G., & Heldal, M. (1990). Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy. Applied and Environmental Microbiology, 56(2), 352-356.
  18. ↑ Bratbak, G., Heldal, M., Norland, S., & Thingstad, T. F. (1990). Viruses as partners in spring bloom microbial trophodynamics. Applied and Environmental Microbiology, 56(5), 1400-1405.
  19. ↑ Marie, D., Brussaard, C. P., Thyrhaug, R., Bratbak, G., & Vaulot, D. (1999). Enumeration of marine viruses in culture and natural samples by flow cytometry. Applied and environmental microbiology, 65(1), 45-52.
  20. ↑ 20.0 20.1 20.2 20.3 Frada, M., Probert, I., Allen, M. J., Wilson, W. H., & de Vargas, C. (2008). The “Cheshire Cat” escape strategy of the coccolithophore Emiliania huxleyi in response to viral infection. Proceedings of the National Academy of Sciences, 105(41), 15944-15949
  21. ↑ Brussaard, C. P. (2004). Optimization of procedures for counting viruses by flow cytometry. Applied and Environmental Microbiology, 70(3), 1506-1513.
  22. ↑ Martínez, J. M., Poulton, N. J., Stepanauskas, R., Sieracki, M. E., & Wilson, W. H. (2011). Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi. PloS one, 6(7), e22520.
  23. ↑ Weinbauer, M. G., & Suttle, C. A. (1997). Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Aquatic Microbial Ecology, 13(3), 225-232.
  24. ↑ 24.0 24.1 24.2 Jacquet, S., Heldal, M., Iglesias-Rodriguez, D., Larsen, A., Wilson, W., & Bratbak, G. (2002). Flow cytometric analysis of an Emiliana huxleyi bloom terminated by viral infection. Aquatic Microbial Ecology, 27(2), 111-124.
  25. ↑ Gilbert, J. A., Field, D., Huang, Y., Edwards, R., Li, W., Gilna, P., & Joint, I. (2008).Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities. PloS one, 3(8), e3042.
  26. ↑ Hardin, G. (1960). The competitive exclusion principle. Science, 131(3409), 1292-1297.
  27. ↑ Hutchinson, G. E. (1961). The paradox of the plankton. The American Naturalist, 95(882), 137-145.
  28. ↑ McQueen, D. J., Post, J. R., & Mills, E. L. (1986). Trophic relationships in freshwater pelagic ecosystems. Canadian Journal of Fisheries and Aquatic Sciences, 43(8), 1571-1581.
  29. ↑ Bidle, K. D., & Vardi, A. (2011). A chemical arms race at sea mediates algal host–virus interactions. Current opinion in microbiology, 14(4), 449-457.
  30. ↑ Frada, M. J., Schatz, D., Farstey, V., Ossolinski, J. E., Sabanay, H., Ben-Dor, S., ... & Vardi, A. (2014). Zooplankton may serve as transmission vectors for viruses infecting algal blooms in the ocean.Current Biology, 24(21), 2592-2597.
  31. ↑ Nissimov, J. I., Napier, J. A., Allen, M. J., & Kimmance, S. A. (2015). Intragenus competition between coccolithoviruses: an insight on how a select few can come to dominate many. Environmental microbiology, 18(1), 133-145.
  32. ↑ 32.0 32.1 Mackinder, L. C., Worthy, C. A., Biggi, G., Hall, M., Ryan, K. P., Varsani, A., ... & Schroeder, D. C. (2009). A unicellular algal virus, Emiliania huxleyi virus 86, exploits an animal-like infection strategy. Journal of General Virology, 90(9), 2306-2316.
  33. ↑ Bidle, K. D., & Vardi, A. (2011). A chemical arms race at sea mediates algal host–virus interactions. Current opinion in microbiology, 14(4), 449-457.
  34. ↑ Brussaard, C. P. D., Kempers, R. S., Kop, A. J., Riegman, R., & Heldal, M. (1996). Virus-like particles in a summer bloom of Emiliania huxleyi in the North Sea. Aquatic Microbial Ecology. 10, 105-113
  35. ↑ Klaveness, D. (1972). Coccolithus huxleyi (Lohm.) Kamptn II. The flagellate cell, aberrant cell types, vegetative propagation and life cycles. British phycological journal, 7(3), 309-318.
  36. ↑ Evans, Claire, et al. VIRAL INFECTION OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) LEADS TO ELEVATED PRODUCTION OF REACTIVE OXYGEN SPECIES. Journal of Phycology 42(5) (2006): 1040-1047.
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