The “Cheshire Cat” escape strategy of the coccolithophore Emiliania huxleyi in response to viral infection Miguel Frada*†‡, Ian Probert*, Michael J. Allen§, William H. Wilson¶, and Colomban de Vargas* *Station Biologique, Equipe EPPO-Evolution du Plancton et Pale´oOce´ans, Centre National de la Recherche Scientifique et Universite´Pierre et Marie Curie (Unite´Mixte de Recherche 7144), Station Biologique, 29682 Roscoff, France; †Departamento de Geologia, Faculdade de Cieˆncias, Universidade de Lisboa, Edificio C6, Campo Grande, 1749-016 Lisbon, Portugal; ¶Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, United Kingdom; and §Bigelow Laboratory for Ocean Sciences, 180 McKown Point, P.O. Box 475, West Boothbay Harbor, ME 04575-0475 Communicated by Paul G. Falkowski, Rutgers, The State University of New Jersey, New Brunswick, NJ, August 6, 2008 (received for review June 9, 2008) The coccolithophore Emiliania huxleyi is one of the most successful and EhV populations have been shown to be genetically diverse eukaryotes in modern oceans. The two phases in its haplodiploid (9), with host succession suggested to follow ‘‘kill the winner’’ life cycle exhibit radically different phenotypes. The diploid calci- dynamics (10). However, recent observations show that the same fied phase forms extensive blooms, which profoundly impact E. huxleyi genotype blooms and is infected and decimated by the global biogeochemical equilibria. By contrast, the ecological role of same EhV genotype over multiannual time scales (11). the noncalcified haploid phase has been completely overlooked. What, then, is the selective advantage for the ‘‘winner’’ E. Giant phycodnaviruses (Emiliania huxleyi viruses, EhVs) have been huxleyi clone(s) to bloom? Is there intense and permanent shown to infect and lyse diploid-phase cells and to be heavily selection pressure for resistance to viral infection [‘‘Red Queen’’ implicated in the regulation of populations and the termination of (RQ) dynamics]? And how is the high lytic virulence of EhVs blooms. Here, we demonstrate that the haploid phase of E. huxleyi sustained? These questions are fundamental to understanding is unrecognizable and therefore resistant to EhVs that kill the the evolutionary ecology of this biogeochemically important diploid phase. We further show that exposure of diploid E. huxleyi species. The answers may be related to sex and life cycling, basic to EhVs induces transition to the haploid phase. Thus we have biologic features of unicellular eukaryotes that are typically clearly demonstrated a drastic difference in viral susceptibility ignored in oceanographic models addressing the ecology of between life cycle stages with different ploidy levels in a unicel- planktonic functional groups. The fitness of many eukaryotic lular eukaryote. Resistance of the haploid phase of E. huxleyi species may be based on their potential for alternation between provides an escape mechanism that involves separation of meiosis variable life cycle phases and adaptation of each phase to from sexual fusion in time, thus ensuring that genes of dominant different ecological niches (12, 13), including in terms of bio- diploid clones are passed on to the next generation in a virus-free logical interactions. Current evidence suggests that coccolith- environment. These ‘‘Cheshire Cat’’ ecological dynamics release ophore life cycles are characterized by independent haploid and host evolution from pathogen pressure and thus can be seen as an diploid phases displaying radically different morphologies (14, opposite force to a classic ‘‘Red Queen’’ coevolutionary arms race. 15) and distinct physiologies (refs. 16 and 17; our unpublished In E. huxleyi, this phenomenon can account for the fact that the data). In E. huxleyi, the life cycle comprises two main forms: the selective balance is tilted toward the boom-and-bust scenario of diploid (2N), nonmotile, coccolith-bearing phase that forms optimization of both growth rates of calcifying E. huxleyi cells and blooms, and the haploid (N) flagellated phase that possesses infectivity of EhVs. nonmineralized organic scales overlying the cell membrane (18, 19). This motile, noncalcifying, haploid stage is not easily eukaryotic life cycle ͉ haplo-diploidy ͉ marine viruses ͉ amenable to identification by conventional microscope tech- host-parasite interaction ͉ Red Queen niques and has been almost completely overlooked by biological oceanographers. Its ecological role and the importance of sexual he coccolithophore Emiliania huxleyi (Lohmann) Hay and cycling in E. huxleyi in the natural environment remain unknown. TMohler is one of the most abundant and widely distributed Flow cytometric surveys of mesocosm blooms of 2N E. huxleyi photosynthetic unicellular eukaryotes in modern oceans. Coc- have revealed the onset, after virus-mediated bloom demise, of colithophores (Calcihaptophycidae, Haptophyta) produce com- new active populations of cells with the same chlorophyll fluo- posite skeletons of minute calcite platelets (the coccoliths) and, rescence signature but lower light-scattering values than 2N consequently, have been key contributors to both the oceanic calcified cells (20, 21). We hypothesized that these new popu- lations consisted of noncalcifying N cells, and that their presence carbon pump and the counterpump, and thus to the flux of CO2 between atmosphere and oceans, since their origin in the Triassic reflected their resistance to infection by the viruses responsible (1). In this context, the impact of predicted anthropogenically for the decline of the 2N blooms. induced ocean acidification on calcifying plankton is a subject of In the present work, we explore the in vitro infectivity profiles intense debate (2, 3). Coccolith-bearing E. huxleyi cells period- of both the coccolith-bearing diploid and noncalcifying haploid ically develop extensive blooms covering wide coastal and mi- life cycle phases of E. huxleyi by using multiple host and viral doceanic areas at high latitudes in both the northern and southern hemispheres. Termination of these blooms is accom- Author contributions: M.F., I.P., and C.d.V. designed research; M.F. and I.P. performed panied by massive release of organic and inorganic matter to the research; I.P. and C.d.V. contributed new reagents/analytic tools; M.F., I.P., M.J.A., W.H.W., water column, including detached coccoliths that reflect sunlight and C.d.V. analyzed data; and M.F., I.P., and C.d.V. wrote the paper. and are readily detectable in satellite images (4). Over the last The authors declare no conflict of interest. decade, the role of large (Ϸ175 nm) lytic coccolithoviruses See Commentary on page 15639. (Phycodnaviridae), named E. huxleyi viruses (EhVs), in the ‡To whom correspondence should be addressed. E-mail: [email protected]. regulation and termination of massive E. huxleyi blooms has been This article contains supporting information online at www.pnas.org/cgi/content/full/ clearly established (5), to the extent that this system has become 0807707105/DCSupplemental. a case study in marine virology (e.g., refs. 6–8). Both E. huxleyi © 2008 by The National Academy of Sciences of the USA 15944–15949 ͉ PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807707105 Downloaded by guest on October 3, 2021 SEE COMMENTARY Fig. 1. The impact of EhV on the growth of diploid and haploid E. huxleyi. Growth curves of both life cycle stages of E. huxleyi (strain RCC1216) and the virus EhV201 are shown. The arrow indicates the day of virus addition (mul- tiplicity of infection 0.2). Standard deviation bars are generally too short to be visible. Fig. 2. Microscopy and genetic tests for the presence/absence of EhVs inside and/or on infected E. huxleyi haploid and diploid cells. (A) TEM: 1, healthy 2N strains and various experimental setups. The results show the cell before infection; 2, 2N cell at day 1 after infection, displaying newly critical significance of life cycling in the survival and ecological formed viral particles (dashed circle); 3, N cell from an infected culture; and 4, dynamics of E. huxleyi. They illustrate a previously unrecognized detail of the N cell periplast, showing the presence of organic scales attached ␮ type of ecological and evolutionary interaction that opposes to the membrane and the absence of coccoliths and viruses. (Scale bars: 1 m.) (B) PCR amplifications of the viral MCP gene at day 11 after infection (see Fig. classical RQ host–pathogen dynamics and is potentially a fun- 1). Agarose gel lanes: 1, positive control (EhV201 DNA extract); 2, N culture; 3, damental force for the maintenance of sex and life cycling and 2N culture; 4, N culture exposed to viruses; and 5, 2N culture exposed to for untying the constraints imposed by pathogens in the evolu- viruses. Cells were carefully filtered and washed several times to remove free tion of eukaryotic microbes. viral particles before DNA extractions. Results As a first approach, we qualitatively scanned the infectivity of the by haploid E. huxleyi suggested by flow cytometry. No viral 15 available EhV strains against both life cycle stages of three capsids were detected inside or adsorbed to the N cells from MICROBIOLOGY different strains of E. huxleyi (RCC1216, RCC1249, and virus-infected cultures in any of the multiple TEM preparations, RCC1213). All 2N E. huxleyi strains were sensitive to five of the whereas capsids were obvious in the cytoplasm of infected 2N viral strains (EhV201, EhV202, EhV205, EhV207, and EhV208), cells as early as 1 day after virus addition (Fig. 2A). Furthermore, whereas none of the N strains were affected. As no obvious the MCP gene was easily detected by PCR of DNA extracts from differences in infectivity were observed between the E. huxleyi filtered and washed infected 2N cells, whereas the N cells in contact with viruses never yielded positive amplifications and EhV strains tested, the host–virus combination (Fig. 2B). RCC1216:EhV201 was chosen for subsequent experiments. Finally, we set up a series of 50-day experiments to test We then monitored the growth (Fig. 1) and photosynthetic longer-term responses of 2N and mixed 2N–N E.