Small but Not Safe. Phages Fall Prey to Protists

Viruses are the most abundant biological entities on the planet. They play a key role in regulating biogeochemical cycles and are thought to constitute large reserves of carbon in the marine environment. However viruses do not persist for ever and maybe removed by biotic and abiotic mechanisms. For example, adsorption onto surfaces and the action of bacterial extracellular nucleases and proteases. Protistan grazing also removes viruses, but this area is poorly studied. Deng et al. (2014) set out to investigate if protists with different feeding mechanisms differed in their ability to remove viruses.

Three different heterotrophic flagellates, isolated from ground water, were used in the study. The Choanoflagellate Salpingoeca sp., a filter feeder, Thaumatomonas coloniensis a Thaumatomonad, which uses pseudopodia to graze sedimentary particles and Goniomonas truncata, a raptorial feeder which actively searches for prey. The model RNA phage MS2 was used to measure grazing. Flagellates were incubated in the dark at 12^oC with local bacteria and viruses along with the phage MS2. The numbers of MS2 were enumerated regularly using the standard double-agar overlay technique. When incubated with T. coloniensis and Salpingoeca sp. the numbers of MS2 declined steadily from day 2 to undetectable levels by the end of the experiment, day 92. This affect was not seen for G. truncata, indicating raptorial feeders are unable to uptake or digest phages.

Ingestion was then examined in detail using T. coloniensis. Random absorption of the phages was ruled out by performing qPCR on subsamples of nucleic acid which confirmed the decline in MS2 was real. Ingestion of the phage was then examined by labelling with a protein dye and feeding them to the flagellates. Confocal microscopy showed the labelled phages were taken into the cells (see picture). The use of phages as a carbon source was then investigated by incubating T. coloniensis with a low concentration of bacteria and either a high or low concentration of MS2 for 28 days. When incubated with a low concentration of phages, flagellate numbers only increased up to day 14 and then dropped off. But for the high phage concentration, numbers continued to increase until day 28. This suggests phages can be used as a supplementary carbon source.

Although the species examined were from groundwater both Choanoflagellates and Thaumatomonads are represented in the marine environment (Munn 2011 & Ota et al. 2012). Considering a wide range of protists use similar feeding mechanisms it is likely that marine phages are also consumed in this manner. This has deep implications for marine ecology and biogeochemistry. It may be that viruses form another link in the microbial loop, as the carbon taken up via flagellates will feed through into higher trophic levels. Therefore the pool of viral carbon could be more accessible to the wider community than previously thought. Interestingly sponge choanocytes have a similar structure and feeding mechanism to Choanoflagellates. Is it possible that sponges feed directly on phages? In conclusion, the study highlights that such interactions must be investigated in the marine environment to fully understand the role that viruses play in marine food webs. 

Florescently labeled MS2 phage (green) after being ingested by T. coloniensis (Deng et al. 2014).

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 Reports, 6(4), 325-330. 

Munn, C.B. (2011). Marine Microbiology: Ecology and Applications, 2^nd ed. New York: Garland Science. 

Ota, S., Eikem, W. & Edvardsen, B (2012). Ultrastructure and Molecular Phylogeny of Thaumatomonads (Cercozoa) with Emphasis on Thaumatomastix salina from Oslofjorden Norway. Protist, 163(4), 560-573.