Detection of Phagotrophy in the Marine Phytoplankton Group of The
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J. Phycol. *, ***–*** (2020) © 2020 Phycological Society of America DOI: 10.1111/jpy.12997 NOTE DETECTION OF PHAGOTROPHY IN THE MARINE PHYTOPLANKTON GROUP OF THE COCCOLITHOPHORES (CALCIHAPTOPHYCIDAE, HAPTOPHYTA) DURING NUTRIENT- REPLETE AND PHOSPHATE-LIMITED GROWTH1 Yoav Avrahami , and Miguel J. Frada2 The Interuniversity Institute for Marine Sciences in Eilat, 88103 Eilat, Israel Department of Ecology, Evolution and Behavior, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel Mixotrophic algae that combine photoautotrophy Increasing evidence indicates that most phyto- with phagotrophy in a single cell are prevalent in plankton are mixotrophic, possessing both the capa- marine ecosystems. Here, we assessed the ability of bility for photoautotrophy but also, to varying food ingestion in coccolithophores, an important group extents, for ingesting prey items through phagotro- of calcifying haptophytes inhabiting the oceans. We phy (Sanders 1991, Jones 1997, Stoecker 1998, tested four species from different coccolithophore Flynn and Mitra 2009, Stoecker et al. 2017). This lineages (Emiliania huxleyi, Calcidiscus leptoporus, dual trophic strategy appears to be particularly ben- Coccolithus braarudii,andCalyptrosphaera sphaeroidea). eficial when light or inorganic nutrients are limiting For both E. huxleyi and C. leptoporus we included (Mitra et al. 2016). Moreover, recent studies indi- different life phases (haploid and diploid). For cate that mixotrophy is widespread and can be C. braarudii we only tested diploid hetero- responsible for the largest fraction of total bac- coccolithophore cells, while for C. sphaeroidea we only terivory in the oceans (Unrein et al. 2007, 2014, tested haploid holococcolithophore cells. Phagotrophy Zubkov and Tarran 2008, Hartmann et al. 2012, was assessed using fluorescently labeled bacteria (FLB) Sanders and Gast 2012). Thus, the functional role as model prey item, under nutrient-replete and of phytoplankton influencing the structure of the phosphate-limited conditions. We detected by marine food web and the flow of carbon to higher microscopy ingestion of FLB by all species, except the trophic levels and the deep ocean is more complex diploid C. braarudii strain. However, a previous study than usually depicted (Worden et al. 2015, Mitra detected ingestion by haploid cells of C. braarudii. et al. 2016, Stoecker et al. 2017). Mixotrophy These overall results indicate that mixotrophy and the appears to be important in several microalgal lin- ability to ingest prey is widespread in coccolithophores. eages, namely dinoflagellates, cryptophytes, chryso- Yet, in all tested species the ingestion of FLB was low phytes, and haptophytes that are widely abundant in (<1% of the population contained prey at all time marine ecosystems (Zubkov and Tarran 2008, points over 2 days), namely for E. huxleyi and the Unrein et al. 2014, Leles et al. 2017). However, the diploid cells from C. leptoporus where detection of prevalence of mixotrophy within some of these lin- ingestion was sporadic. Moreover, no clear differences eages has been less explored. In this context, we were detected between life phases in E. huxleyi and examined the occurrence of mixotrophy (i.e., com- C. leptoporus under equal circumstances, or between bined photoautotrophy and phagotrophy) in coccol- replete and limited growth conditions. ithophores that are a distinct subgroup of Key index words: haptophytes (Calcihaptophycidae, de Vargas et al. bacterivory; coccolithophores; life 2007), which typically produce composite exoskele- cycle; mixotrophy; phytoplankton tons composed of calcite scales called coccoliths, Abbreviations: DAPI, 40,6-Diamidino-2-Phenylindole, and thus play a central role in the global carbon Dihydrochloride; DTAF, 5-([4,6 dichlorotriazin-2-yl]- cycle (Rost and Riebesell 2004, Thierstein and amino)-fluorescein; FLB, fluorescently labeled bacte- Young 2013). Additionally, coccolithophores, like ria; HET, heterococcolithophore; HOL, holococcol- other haptophytes, display complex sexual hap- ithophore lodiplontic and heteromorphic life cycles (Billard 1994, Houdan et al. 2004, Cros and Estrada 2013, Frada et al. 2019). Diploid cells often produce coc- coliths made of interlocking calcite crystals with modified shapes and are denominated as heterococ- 1Received 2 January 2020. Accepted 12 March 2020. colithophores (HET). In contrast, haploid cells 2Author for correspondence: e-mail [email protected]. often possess lighter coccoliths composed of Editorial Responsibility: T. Mock (Associate Editor) 1 2 YOAV AVRAHAMI AND MIGUEL J. FRADA numerous euhedral calcite crystallites and are used to count the fraction of coccolithophores with denominated as holococcolithophores (HOL), but ingested FLB by epifluorescence microscopy (Eclipse in some species like Emiliania huxleyi haploid cells Ti-S, Nikon Instruments Inc., Melville, NY, USA). are noncalcified (Houdan et al. 2004, Frada et al. Uptake counts at t0 was considered artifactual and 2019). Broadly, HET are frequently associated with subtracted from the following time points. Additional nutrient rich conditions, while HOL are frequently samples of C. leptoporus and C. sphaeroidea were fixed found in higher light, nutrient poor conditions with 1% paraformaldehyde, counterstained with (Cros and Estrada 2013, Supraha et al. 2016, Frada DAPI (Invitrogen, Thermo Fisher Scientific, Wal- et al. 2019). Overall, given the affiliation to the hap- tham, MA, USA), and used for confocal microscopy tophytes it is expected that mixotrophy is inherent (FV-1200; Olympus, Tokyo, Japan). to coccolithophores. Yet, reports on coccol- We detected FLB ingestion by most coccol- ithophore mixotrophy are confined to single obser- ithophores used in this study and respective life vations of particle ingestion (synthetic micro-beads) phases over the 48 h time course, with the excep- by a haploid (1n) Coccolithus braarudii cell (Houdan tion of HET cells of Coccolithus braarudii (Fig. 1). et al. 2006) and a diploid (2n) E. huxleyi cell Yet, the fraction of cells with FLB was low and in (Rokitta et al. 2011). Thus, in order to further vali- some strains only recorded sporadically, namely in date and expand the current understanding of both life phases of Emiliania huxleyi although the mixotrophy in coccolithophores and to test the vari- tested cell densities and thus cell: FLB contact rates ability between life cycle phases that generally were the highest (Table S1). Higher fractions of occupy distinct niches, we monitored in culture four ingestion were detected in 1n Calcidiscus leptoporus species (including for two species both life cycle and in Calyptrosphaera sphaeroidea where up to ~1% phases): 2n (HET) and 1n noncalcified E. huxleyi of the cells detected over 48 h consistently con- (RCC 1216/RCC1217), 2n (HET) and 1n (HOL) tained FLB (Fig. 2). Further confirmation of FLB Calcidiscus leptoporus (RCC 1131/RCC 1130), 2n ingestion in these species was performed by confo- (HET) C. braarudii (RCC 3779) and the 1n (HOL) cal microscopy, clearly highlighting the presence of Calyptrosphaera sphaeroidea (RCC 1178). The cultures FLB within the cells’ cytoplasm (Fig. S2 in the Sup- were nonaxenic, containing variable densities of porting Information). However, no significant dif- cohabiting bacteria (Table S1 in the Supporting ferences were detected between nutrient regimes, Information). Cells were grown at 18°C, 80 lmol with the exception of C. sphaeroidea where at t48 h À À photons Á m 2 Á s 2 of irradiance, and 14:10 h light:- cells in K/5-P contained slightly more FLB than in dark photoperiod in both nutrient-replete (K/5; Kel- K/5 (~0.5% and ~0.2%, respectively, P < 0.05, ler et al. 1987) and phosphate-limited (K/5-P) media n = 3, t-test; Fig. 2A). Given higher ingestions in 1n to test the effect of P limitation that usually enhances C. leptoporus and C. sphaeroidea, we attempted to phagotrophy in other mixotrophs (Smalley et al. determine the FLB removal rates by assessing the 2003, Carvalho and Graneli 2010). P limitation was decline of FLB in the media over time. However, ensured by testing cells displaying for at least 24 h sta- the decay of FLB when grown without coccol- tionary growth in K/5-P (Fig. S1 in the Supporting ithophores was often higher than in the presence of Information). For comparative purposes, the cells in coccolithophore cells. We could not technically K/5 were diluted with fresh medium to the same den- solve this problem, which hampered accurate calcu- sities as K/5-P cultures prior to feeding assays lations of ingestion rates by coccolithophores. Yet, (Table S1). Phagotrophy was tested with fluores- assuming that within each experiment in the pres- cently labeled bacteria (FLB) as model prey item ence of algal cells the decay of FLB was identical, (FLB: coccolithophore ratio ~20:1) prepared with a we used regression analysis of FLB over time to Brevundimonas diminuta (Spanish Type Culture Col- comparatively determine the relative prey removal À lection, Burjassot, Valencia, Spain) heat-killed and rates (FLB Á h 1) and the relative prey removal rates À À stained with 100 pg Á mL 1 of the green fluo- per coccolithophore cell (FLB Á cell 1). Total prey rochrome DTAF (Invitrogen, Thermo Fisher Scien- removal rates in C. sphaeroidea in K/5, where cell tific, Waltham, MA, USA; Sherr et al. 1987). This density was also 39 higher than in K/5-P, was twice bacterial strain was selected as prey item because it as high than in K/5-P. However, no statistically sig- was shown to be grazed by haptophytes (Unrein et al. nificant differences were detected on a per cell basis 2007, 2014). Cells and FLB, as well as total living bac- (Fig. S3, A and B in the Supporting Information). teria were enumerated by flow cytometry (Attune In 1n C. leptoporus total prey removal was compara- NxT, Thermo Fisher Scientific, Waltham, MA, USA; ble in both media, but here prey removal rates per Unrein et al. 2007, Marie et al. 2014) at t0, t1 h, t2 h, cell were significantly higher (29 to 4x) in K/5-P t4 h, t24 h, and t48 h. Samples were also collected (Fig. S3, C and D).