Shunt, shuttle and link: how viruses provide a food source for marine life

It is estimated that there are around 1031 viruses in the world’s oceans accounting for ~94% of all biological entities (nucleic acid containing particles). Despite their small size, the amount of carbon they contain is estimated to be the equivalent of 75 million blue whales. This post highlights some recent findings about the role of viral particles as a direct source of nutrients for marine organisms.

KThe viral shunt. Since the 1990s, we have recognized the importance of viral infection of ocean microbes in nutrient cycling. The best known and most abundant viruses are those known as phages — these infect bacteria and archaea. Eukaryotic microbes (protists) are also attacked by a wide range of viruses. Viruses infecting microalgae — members of the autotrophic phytoplankton important as primary producers — have also been extensively characterized and studied. More recently, many heterotrophic protists have been shown to be hosts for specific viruses. The lysis of these diverse host microbes by viruses plays a major role in structuring microbial communities as well as releasing vast amounts of dissolved organic matter (DOM) and particulate organic matter (POM). DOM released from cells is rich in nitrogen and phosphorus as well as carbon, so it is rapidly absorbed by heterotrophic bacteria and archaea which use it for cell growth and multiplication — a recycling process termed the “viral shunt” by Wilhelm & Suttle (1999). Assessing the quantitative impact of this process has proved difficult, but Weitz et al. (2012) developed a model that successfully predicts the contribution of viral stimulation of organic matter cycling and stimulation of primary productivity.

The viral shuttle. Recently, it has been realized that viral lysis has more complex effects than first envisaged, due to release of cell contents containing large polymeric molecules, which form microgels that coalesce as transparent polymeric particles (TEPs). These aggregate with other cell debris, mucus and fecal pellets to form marine snow. This process accelerates the export of carbon to the ocean floor via the biological pump and has been termed the “viral shuttle” (Guidi et al., 2016). Its influence seems to be especially associated with viral lysis of certain microbial groups in oligotrophic ocean regions, such as the infection of massive blooms of the coccolithophoid Emiliania huxleyi due to viral infection. Viral lysis leads to stimulation of production of large amounts of TEPs.

Schematic diagram showing critical roles of viruses in ocean food webs and export of carbon and other nutrients to the deep ocean. Credit: Colin Munn, CC BY-NC 4.0.

The viral link – direct consumption by protists. Phagotrophic protists consume bacteria and archaea, as well as other protists smaller than themselves. This grazing enables the nutrients contained in very small microbes to become increasingly available to larger planktonic organisms, so they work their way up the food chain. Could virus particles provide a direct food source for protists, leading to a shortcut link to higher trophic levels? One of the conceptual difficulties in answering this question comes from the paradox that, because of their small size, virus particles constitute part of the DOM pool. Organic matter in the oceans is traditionally defined as “dissolved” or “particulate” organic matter (DOM and POM, respectively), but this is a purely operational distinction based on the use of filters (typically with a pore size of 0.2 – 1.0 µm) used in sample preparation. In fact, many bacteria and almost all viruses would pass through such filters and therefore appear in the DOM fraction, although they are clearly particles rather than truly dissolved or colloidal.  Suttle & Chen (1992) first suggested that ingestion by nanoflagellates (phagotrophic protists in the 2–20 µm size range) was one of the factors accounting for the decline of viruses in seawater. In subsequent experiments by Gonzalez & Suttle (1993), natural flagellate communities and enrichment cultures were supplied with fluorescently labelled viruses (FLVs) — mostly phages <100 nm diameter), bacteria, or polymer microspheres. Clearance rates were measured by counting particles under an epifluorescence microscope. At natural concentrations, the clearance rate of viruses was ~4% that of bacteria, with the smallest virus cleared at the lowest rate; however, due to the methods used the authors considered this to be an underestimate. Digestion times of viruses and bacteria was evaluated using a dilution technique with fluorecent microspheres as a control. Flagellate communities ingested different viruses at different rates, suggesting a selective grazing mechanism, although the basis of this is unknown. Gonzalez & Suttle (1993) concluded that nanoflagellates obtain significant nutritional benefit from the process, necessitating recognition of direct grazing of viruses as an important pathway for the cycling of carbon, nitrogen and phosphorus in marine systems. Only a few other studies of grazing of viruses by marine protists have been conducted until recently, but the concept was developed by Jover et al. (2014), using a biophysical scaling model to predict the elemental composition of virus particles, which they validated using sequence analysis. They concluded that when virus particles are released during the lysis of their host, it provides a burst of nutrients that is enriched with phosphorus. Only a few other studies of this phenomenon been conducted, but recent studies have provided further evidence that protists can remove viruses from the water column. In the modelling analysis mentioned above, Jover et al. (2014) suggested that intact virus particles could be a target for consumption by marine zooplankton. A recent study by Brown et al. (2020) has employed the latest single cell genomic methods to answer the question: what do viruses eat? Their method might be considered analogous to analyzing the diet of a predatory fish by examining its gut contents and assessing the number and types of tell-tale indicators like otoliths from fish prey, or the hard parts of invertebrate prey.  Brown and colleagues isolated individual cells of protists belonging to diverse groups from seawater at two sites, using a fluorescent activated cell-sorting technique (see video). The genomic DNA of single cells was amplified to generate numerous single amplified genomes (SAGs), which were then shotgun sequenced. The genetic sequences obtained consist of the protist’s own genome, plus any DNA that might be present from intracellular symbionts, ingested prey, and any bacteria or viruses that might be adhering to its exterior. The sequences were cross-matched for similarity to known sequences of protists, bacteria and viruses found in the genomic databases. Bacterial DNA was found in many of the protists, indicating that they are likely to be prey organisms consumed by the protists. Notably, 100% of the cells in two protistan groups — choanoflagellates (choanozaons) and picozoans — contained viral sequences, but most had few bacterial sequences. Obviously, some of the viral sequences detected could exist because the protist cells have a viral infection, but this seems unlikely as the same viral sequences and ssDNA sequences) were detected across highly diverse protistan species. Brown and colleagues evaluated a variety of non-specific associations arising from their analytical methods as possible explanations for the presence of viruses within the cells, but concluded that the most plausible explanation is that some of the protists, in particular, choanoflagellates and picozoans, are grazing and actively ingesting free virus particles. The authors consider the ecological importance of this and present the concept that consumption of intact virus particles provides a direct trophic link, in addition to the shunt and shuttle mechanisms previously described.

Viruses can provide nutrition directly to invertebrates. Sponges, tunicates, molluscs and other invertebrates feed by filtering large volumes of seawater and have long been known to trap and ingest picoplankton (bacteria, archaea and protists in the size range 0.2 – 3 µm) as a food source. This plays a major role in nutrient recycling, marine food webs and carbon export. One of the first hints that viruses might also contribute directly to marine food webs though ingestion and digestion by invertebrates came from the study by Hadas et al. (2006). Sponges feed by drawing in large quantities of water — a modest size sponge can filter tens of thousands of litres per day — through incurrent pores, connected by canals to filtering chambers. Here, food particles are removed by specialized cells (choanocytes), before water is ejected via an exhalent opening (see video). Notably, the feeding cells (choanocytes) of sponges have a similar structure to the choanoflagellates, mentioned above as active grazers of viruses. Hadas and colleagues maintained samples of the Red Sea coral reef sponge Negombata magnifica kept in aquaria supplied with reef water, and the concentration of virus particles found in the inflow and outflow water was measured by flow cytometry (see video), using the  green fluorescent dye SYBR Green 1 to stain the virus particles by binding to viral DNA).  The ambient reef water contained ~8.7 x 105 virus particles mL-1 which were removed with ~23% efficiency by the sponge’s filtering mechanism. By calculating the rates of filtration and respiration of the sponge, and estimating the amount of carbon and nitrogen contained in the viruses consumed, Hadas and colleagues conclude that the viral contribution to nutrition of a single sponge is probably low. However, they propose that the large biomass of sponges in some tropical areas could lead to a substantial flow of nutrients from the virioplankton to higher trophic levels, contributing to the concept of “sponge loop” to explain how reef ecosystems can maintain their high productivity and biodiversity in oligotrophic oceans: “surviving in a marine desert” (De Goeij et al., 2013).

Choanoflagellates and sponges are active grazers of pico- and nanoplankton. Left: 3D visualization of a clump of choanoflagellate cells. Credit: Davis Laundon. Right: Examples of erect sponges. Credit John Turnbull, CCBY-NC-SA 2.0, via flickr.

Recent experiments have shed further light on the role of viruses in invertebrate nutrition. One extensively studied organism is the appendicularian tunicate Oikopleura dioica, which is abundant and widely distributed in surface waters of the worlds’ oceans. Because they can consume particles many thousands of time smaller than themselves — “mammoth grazers on the ocean’s minuteness”: Conley et al. (2018) — pelagic tunicates such as this species play a major role in ocean food webs as a food source for other organisms and by influencing the export of carbon from surface to deep waters.  O. dioica has an ovoid body (0.5 – 1 mm) and a tail about four times the head length, resembling a tadpole.  O. dioica creates a mucus net structure (“house”) around its body and uses the beating of its tail to pump water into the house, trapping particles of different sizes through a series of mucus filter structures (see video). Previous modelling had suggested that the pores could be capable of trapping nanoparticles below 0.2 µm, so Lawrence et al. (2017) devised experiments to study the removal of E. huxleyi virus (EhV) from seawater. EhV is a large virus with a 160-180 nm diameter capsid structure.  Cultures of O. dioica were maintained in the lab by feeding with a mixture of plankton protists and cyanobacteria. Different numbers of animals in the developmental stages (1–5 days) were added to containers filled with 0.2 µm filtered seawater, with and without the presence of animals or viruses at a concentration of 106 EhV mL-1. Regular samples of the incubation mixture, the animal bodies and the separated houses were taken up to 21 h to determine the number of EhV virions. This was assessed by flow cytometry and by qPCR (detecting the DNA encoding the major capsid protein). The rate of clearance by O. dioica varied with developmental stage of the animals, incubation time, and extraction method; however, in all cases, it was similar to rates determined for larger particles such as microalgae. Lawrence and colleagues consider the ecological impact of their findings. Because virus particles are trapped in the houses (which appendicularians shed frequently) as well as ingested and excreted in the faeces, large amounts of carbon could be rapidly transferred from the surface to deep waters — both the houses and faecal pellets sink rapidly. The abundance and activity of appendicularians may also affect transmission of EhV and the dynamics of blooms. These experiments were not able to determine how much nutrition animals like this can obtain from ingesting viruses, but they do support previous suggestions that this could be significant. For example, Sutherland et al. (2010) concluded that the energetic needs of oceanic salps (another type of tunicate) might be fully satisfied by feeding on particles in this size range.

Welsh et al. (2020) devised a model experimental system to test the ability of a range of other marine invertebrates from temperate coastal water to remove the Phaeocystis globosa virus (PgV) from seawater. Like E. huxleyi, P. globosa is a marine bloom-forming microalga. Small pots containing test animals were filled with seawater containing seawater with 106 PgV mL-1, and removal of virus particles was detected by flow cytometry. Compared with no-animal controls, anemones (Actinia equina), cockles (Cerastoderma edule), crabs (Carcinus maenas), oysters (Magallana gigas), adult copepods (Acartia tonsa), polychaete larvae (a mix of species), and breadcrumb sponge (Halichondria panicea) all showed rapid and significant declines in the abundance of PgV. The breadcrumb sponge showed the most rapid removal of viruses (94% within 3 h, 98% within 24 h), and by periodic spiking of the experimental system, it was shown that the sponge showed a constant and continuous removal of viruses. The experiments of Lawrence et al. (2017) and Welsh et. al (2020) both used large viruses (150 – 200 nm particle size), but results of other studies suggest that much smaller viruses can be efficiently consumed as prey by sponges and other invertebrates.  Sponges are particularly effective at filtering and retaining small particles, as well as removing colloids and dissolved organic matter (e.g.  Thomassen et al., 1995; de Goeij et al., 2008). Further application of the latest techniques for genomic and elemental analysis will undoubtedly shed more light on this important phenomenon.

REFERENCES

Brown, J. M., Labonté, J. M., Brown, J., Record, N. R., Poulton, N. J., Sieracki, M. E., … & Stepanauskas, R. (2020). Single cell genomics reveals viruses consumed by marine protists. Frontiers in microbiology11, 2317. . https://doi.org/10.3389/fmicb.2020.524828

Conley, K. R., Lombard, F., & Sutherland, K. R. (2018). Mammoth grazers on the ocean’s minuteness: a review of selective feeding using mucous meshes. Proceedings of the Royal Society B: Biological Sciences285(1878), 20180056. https://royalsocietypublishing.org/doi/full/10.1098/rspb.2018.0056

De Goeij, J. M., Van Den Berg, H., van Oostveen, M. M., Epping, E. H., & Van Duyl, F. C. (2008). Major bulk dissolved organic carbon (DOC) removal by encrusting coral reef cavity sponges. Marine Ecology Progress Series357, 139-151. https://www.int-res.com/abstracts/meps/v357/p139-151/

De Goeij, J. M., Van Oevelen, D., Vermeij, M. J., Osinga, R., Middelburg, J. J., De Goeij, A. F., & Admiraal, W. (2013). Surviving in a marine desert: the sponge loop retains resources within coral reefs. Science342(6154), 108-110. https://science.sciencemag.org/content/342/6154/108.abstract

Deng, L., Krauss, S., Feichtmayer, J., Hofmann, R., Arndt, H., & Griebler, C. (2014). Grazing of heterotrophic flagellates on viruses is driven by feeding behaviour. Environmental Microbiology Reports6(4), 325-330. https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1758-2229.12119

González, J. M., & Suttle, C. A. (1993). Grazing by marine nanoflagellates on viruses and virus-sized particles: ingestion and digestion. Marine Ecology Progress Series, 1-10. https://www.int-res.com/articles/meps/94/m094p001.pdf

Guidi, L., Chaffron, S., Bittner, L., Eveillard, D., Larhlimi, A., Roux, S., … & Coelho, L. P. (2016). Plankton networks driving carbon export in the oligotrophic ocean. Nature532(7600), 465-470. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851848/

Hadas, E., Marie, D., Shpigel, M., & Ilan, M. (2006). Virus predation by sponges is a new nutrient‐flow pathway in coral reef food webs. Limnology and Oceanography51(3), 1548-1550. https://aslopubs.onlinelibrary.wiley.com/doi/abs/10.4319/lo.2006.51.3.1548

Jover, L. F., Effler, T. C., Buchan, A., Wilhelm, S. W., & Weitz, J. S. (2014). The elemental composition of virus particles: implications for marine biogeochemical cycles. Nature Reviews Microbiology12(7), 519-528. https://www.researchgate.net/publication/263167259_The_elemental_composition_of_virus_particles_Implications_for_marine_biogeochemical_cycles

Lawrence, J., Töpper, J., Petelenz‐Kurdziel, E., Bratbak, G., Larsen, A., Thompson, E., … & Ray, J. L. (2018). Viruses on the menu: the appendicularian Oikopleura dioica efficiently removes viruses from seawater. Limnology and Oceanography63(S1), S244-S253. https://www.pnas.org/content/107/34/15129

Sutherland, K. R., Madin, L. P., & Stocker, R. (2010). Filtration of submicrometer particles by pelagic tunicates. Proceedings of the National Academy of Sciences107(34), 15129-15134. https://www.pnas.org/content/107/34/15129

Suttle, C. A., & Chen, F. (1992). Mechanisms and rates of decay of marine viruses in seawater. Applied and Environmental Microbiology58(11), 3721-3729. https://aem.asm.org/content/aem/58/11/3721.full.pdf

Thomassen, S., & Riisgård, H. U. (1995). Growth and energetics of the sponge Halichondria paniceaMarine Ecology Progress Series128, 239-246. http://www.int-res.com/articles/meps/128/m128p239.pdf

Weitz, J. S., Stock, C. A., Wilhelm, S. W., Bourouiba, L., Coleman, M. L., Buchan, A., … & Middelboe, M. (2015). A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes. The ISME journal9(6), 1352-1364. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434959/

Welsh, J. E., Steenhuis, P., de Moraes, K. R., van der Meer, J., Thieltges, D. W., & Brussaard, C. P. (2020). Marine virus predation by non-host organisms. Scientific reports10(1), 1-9. https://www.nature.com/articles/s41598-020-61691-y

Wilhelm, S. W., & Suttle, C. A. (1999). Viruses and nutrient cycles in the sea: viruses play critical roles in the structure and function of aquatic food webs. Bioscience49(10), 781-788. https://academic.oup.com/bioscience/article/49/10/781/222807

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