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In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy

In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy
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  In situ   interactions between photosyntheticpicoeukaryotes and bacterioplankton in the AtlanticOcean: evidence for mixotrophy Manuela Hartmann, 1 Mikhail V. Zubkov, 1 Dave J. Scanlan 2 and Cécile Lepère 2 * † 1 Ocean Biogeochemistry and Ecosystems Research Group, National Oceanography Centre, Southampton SO14 3ZH, UK. 2 School of Life Sciences, University of Warwick,Coventry CV4 7AL, UK. SummaryHeterotrophic bacterioplankton, cyanobacteria andphototrophic picoeukaryotes ( <  5    m in size) numeri-cally dominate planktonic oceanic communities.While feeding on bacterioplankton is often attributedto aplastidic protists, recent evidence suggests thatphototrophic picoeukaryotes could be importantbacterivores. Here, we present direct visual evidencefrom the surface mixed layer of the Atlantic Oceanthat bacterioplankton are internalized by photo-trophic picoeukaryotes.  In situ   interactions ofphototrophic picoeukaryotes and bacterioplankton(specifically  Prochlorococcus   cyanobacteria and theSAR11 clade) were investigated using a combinationof flow cytometric cell sorting and dual tyramidesignal amplification fluorescence  in situ   hybridiza-tion. Using this method, we observed plastidicPrymnesiophyceae and Chrysophyceae cells con-taining  Prochlorococcus  , and to a lesser extentSAR11 cells. These microscopic observations of  in situ   microbial trophic interactions demonstrate thefrequency and likely selectivity of phototrophicpicoeukaryote bacterivory in the surface mixed layerof both the North and South Atlantic subtropicalgyres and adjacent equatorial region, broadening ourviews on the ecological role of the smallest oceanicplastidic protists. Planktonic protists have traditionally been characterizedbased on their modes of energy and carbon acquisition aseither phototrophic or heterotrophic. In marine ecosys-tems, phototrophic picoeukaryotes (or plastidic pico-eukaryotes,  <  5  μ m) are generally known only for theirsignificant contribution to phytoplankton biomass (Zubkov et al  ., 1998; Cuvelier  et al  ., 2010) and primary production(Li, 1994; Jardillier  et al  ., 2010). Bacterioplankton is gen-erally considered to be mainly consumed by specialistaplastidicprotistsinaquatichabitats(Christaki et al  .,2001;Ichinotsuka  et al  ., 2006). However, many aquatic protistscan also function as mixotrophs (i.e. combine both nutri-tionalmodes)(Harada et al  .,2007;WordenandNot,2008;Not  et al  ., 2009; Heywood  et al  ., 2011). Understanding themajor source of bacterioplankton mortality is central tounderstandingthestructureofthemicrobialfoodweb,andtheregulationofbothmarineprimaryproductionandnutri-ent cycling in the ocean.Planktonic protists have diverse evolutionary histories.During the last decade, molecular techniques, such ascloning and sequencing of nuclear (e.g. Moon-van derStaay  et al  ., 2001) and plastid (Rappé  et al  ., 1998) smallsubunit rRNA genes, have begun to provide insights intothe structure of phototrophic picoeukaryotes communitiesin various marine environments. These studies haveshown members of the Prymnesiophyceae, Chryso-phyceae, Prasinophyceae and Pelagophyceae to be keycomponentsofthephototrophicpicoeukaryotecommunityin open ocean waters (e.g. see Lepère  et al  ., 2009;Cuvelier  et al  ., 2010). Unfortunately, with few exceptions,many of the dominant clades within these groups remainuncultured, and hence little or no information is availableon their morphology and physiology (Vaulot  et al  ., 2008),and thus their nutritional modes, including mixotrophy.However, recently, there has been increasing evidencefrom studies of natural populations that phototrophicpicoeukaryotescanbeecologicallyimportantbacterivores(Zubkov and Tarran, 2008; Frias-Lopez  et al  ., 2009;SandersandGast,2011;Hartmann et al  .,2012).Nonethe-less, as a result of the techniques used, identification ofboth the phototrophic picoeukaryote ‘predator’ and itsnatural ‘prey’ under  in situ   conditions has been missing.Using a stable isotope technique, Frias-Lopez andcolleagues (2009) were able to taxonomically identifymixotrophic organisms but could not establish bacterivoryrates. The opposite holds true for the Hartmann and Received 31 January, 2013; revised 20 June, 2013; accepted 22June, 2013. *For correspondence. E-mail; Tel. 0033 473407460; Fax 0033 473407869.  † Presentaddress: Clermont Université, Université Blaise Pascal, Laboratoire‘Microorganismes: Génome et Environnement’, BP 10448, F-63000Clermont-Ferrand. bs_bs_banner Environmental Microbiology Reports (2013)  5 (6), 835–840 doi:10.1111/1758-2229.12084  © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology  colleagues’ study (2012), where bacterivory rates ofphototrophic picoeukaryotes and aplastidic protists werequantified using radio-labelling of the natural bacterio-plankton, but the taxonomic identity of the grazers was notevaluated.Although both studies tried to minimize the riskof label accumulation by other means than predation, theyare lacking crucial visual evidence of prey bacterial cellsinside the phototrophic picoeukaryotes.Aiming to close this gap, we investigated the  in situ  interactions of individual phototrophic picoeukaryote andbacterioplankton (i.e. the presence of picocyanobacteriaand heterotrophic bacterioplankton cells inside photo-trophic picoeukaryotes).Samples for this study were collected along theAtlanticMeridional Transect (AMT) carried out by the RoyalResearch Ship James Cook, cruise JC53-AMT20(Fig. S1) from 12 October to 25 November 2010. Fivestations encompassing the North and South subtropicalgyres and the equatorial region (NG  n   =  2, SG  n   =  2, EQ n   =  1) of theAtlantic Ocean were sampled. Samples werecollected at a depth of 20 m, which represents the surfacemixed layer unaffected by the presence of the researchship. We combined flow cytometric cell sorting anddual tyramide signal amplification fluorescence in situhybridization (TSA-FISH) to determine phototrophicpicoeukaryote/bacterioplankton interactions for two differ-ent picoeukaryote groups: Plast-S cells ( ∼ 2  μ m in size)and Plast-L cells ( ∼ 3  μ m in size) (populations were sortedusing the same parameters as Hartmann  et al  ., 2012).Additionally, in order to measure and quantify bacterivoryrates, the same water was sampled in the EQ and SGregions, and bacterivory experiments were performed(results presented in Hartmann  et al  ., 2012). Phototrophicpicoeukaryote cells were sorted using a flow cytometer(FACSort, Becton Dickinson, NJ, USA) from pre-concentrated seawater samples fixed with 1% (w/v) para-formaldehyde final concentration and amended with0.03% (v/v) non-ionic surfactant (Pluronic F-68) in order tominimize any influence of the pre-concentration step onbacterivory rates by inhibiting cell adhesion, a prerequisiteto prey ingestion (Winblade  et al  ., 2000; Roberts  et al  .,2006). Flow cytometric cell sorting not only provided accu-rate separation of algal cells from bacterial cells, butalso allowed extensive washing of sorted cells usingthe sheath fluid (1000 cells in 50 ml sheath fluid) inorder to dislodge any loosely associated bacterial cells.Moreover,sortedalgalcellswerecollectedon0.8  μ mporesize polycarbonate filters (Whatman, GE Healthcare LifeSciences, Buckinghamshire, UK) efficiently further sepa-ratingalgaefrompotentiallyby-sortedbacteria.Indeed,no‘free’ bacterial cells were seen by FISH analysis, confirm-ing that the sorting was very efficient, and that the likeli-hood of finding a free bacterial cell randomly associatedwith a photosynthetic picoeukaryote was remote.Previous work indicated that the phototrophic pico-eukaryote classes Prymnesiophyceae, ChrysophyceaeandPelagophyceaedominatedPlast-SandPlast-LcellsinAtlantic waters (Jardillier  et al  ., 2010; Grob  et al  ., 2011).Due to generally low cell abundances in these waters, weassumed a random mode of predation at the samplingsites,i.e.whenencounteringanypotentialpreyparticlethepredator will internalize it non-selectively. Hence, numeri-cally dominant  Prochlorococcus   cyanobacteria and lownucleic acid bacteria (LNA) (Morris  et al  ., 2002; Zubkov et al  .,2007)areconsideredthemostlikelypreyorganisms.Along theAMT-20 transect, flow cytometric analyses veri-fiedacontributionof73  ±  8%ofthesetwobacterialgroupsto the total bacterioplankton (Hartmann  et al  ., 2012).Earlier studies in the Atlantic Ocean identified thealphaproteobacteriaSAR11cladeasthemaingroupwithinLNA bacteria (Mary  et al  ., 2006; 2008; Gómez-Pereira et al  ., 2013). Therefore, we performed dual-TSA-FISH(see Supporting Information) using specific probes(CHRYSO1037,PRYM02andPELA01)targetingthethreedominant classes of phototrophic picoeukaryotes, in com-bination with probes targeting  Prochlorococcus   (PRO405)(Fig. 1A and B) or the SAR11 clade (SAR11_441R)(Fig. 1C and D). A  Synechococcus   probe (SYN405) wasalso used on some samples, but no association of Synechococcus   with eukaryotic cells was observed, inaccordance with their low abundance in these watersmaking predator–prey encounters less likely. Neverthe-less, using confocal laser scanning microscopy, weobserved prokaryotic cells (SAR11 and  Prochlorococcus  )inside eukaryotic cells. So far, neither SAR11 nor Prochlorococcus   have been found in symbiotic relation-shipwitheukaryotes;thus,weassumethatthepresenceofthese cells within eukaryotes indicates bacterivorousbehaviour.Prymnesiophyceae, Chrysophyceae and Pelago-phyceae were present in both Plast-S and Plast-L groups(Fig. 2Aand B). However, their contribution to each groupwas significantly different ( P   <  0.001) in agreement withprevious studies (Jardillier  et al  ., 2010; Grob  et al  ., 2011).In each sample, between 100 and 200 plastidic cells ofeither Plast-S or Plast-L groups were examined. Onaverage, 6% of Prymnesiophyceae cells, the numericallymost abundant class of the Plast-L population (48–75%),showed internalization with  Prochlorococcus   cells at allstudied stations (Fig. 1E). In the NG and EQ regions,SAR11 cells were also observed inside the largestPrymnesiophyceae(Plast-L)(Fig. 1F),withonaverage3%of Prymnesiophyceae cells labelled with the SAR11_441Rprobe. In comparison, Plast-L Chrysophyceae showed upto 10% (CTD 18, NG; average 4.3%) internalization with Prochlorococcus   and an average 2.4% internalization ofSAR11 cells (Fig. S2A). The Plast-S populations wereclearly dominated by Pelagophyceae (on average 51%) Bacterivory by marine phototrophic picoeukaryotes   836  © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology,  Environmental Microbiology Reports,  5 , 835–840  and showed on average 1.2% internalization with Prochlorococcus  , while no SAR11 cells could be observedinside these protists (Fig. S2B). Plast-S Prymne-siophyceae contained  Prochlorococcus   cells only at theequatorial station (3%).Acomparison of the percentage ofbacterial cells inside the three phototrophic picoeukaryoteclasses along the transect showed, in general, no signifi-cant difference between regions, with the exception of theChrysophyceae/SAR11 association, which was onlydetected in the NG, and the Prymnesiophyceae/  Prochlorococcus   association, which showed a significantdifference at the SG stations ( P   <  0.05) (Fig. 2). Slightlylower bacterivory rates in the SG were also measured in aprevious study (Hartmann  et al  ., 2012), possibly owing tothe seasonal difference between gyres (boreal autumn inthe NG and austral spring in the SG) during sampling.Bacterioplankton cells were not exclusively observedinside the phototrophic picoeukaryote cells but werealso seen attached to their cell surface, especially Prochlorococcus   cells attached to Prymnesiophyceae,although at a relatively low incidence (on average  <  1%Plast-S  +  Plast-L cells) (Fig. S3). This surface attachedassociation could be interpreted as a step prior to preyingestion. However, we cannot exclude other interpreta-tions, e.g. a beneficial bacterial association to acquirenutrientsproducedbythealgal‘host’(seeGengandBelas,2010). Of the classes studied, Prymnesiophyceae wereobserved most commonly in association (internaland surface) with bacterioplankton, especially  Prochlo- rococcus  , suggesting they are key bacterivores in theocean.Mixotrophy in natural populations of larger plastidicprotists (i.e.  >  5  μ m) has also been recently documented.For example, single cell sequencing has shown the pres-ence of mixotrophic Chrysophyceae within the  <  20  μ mplastidic protist size fraction in the North Atlantic coastalwaters (Martinez-Garcia  et al  ., 2012), while a stableisotope probing approach revealed Prymnesiophyceae asefficient grazers on picocyanobacteria (Frias-Lopez  et al  .,2009). Indeed, evidence for a global distribution of pico-sized Prymnesiophyceae now exists (Liu  et al  ., 2009;Cuvelier  et al  ., 2010; Jardillier  et al  ., 2010), and thissuccess in the open ocean might be explained by theirmixotrophic behaviour. The nutritional flexibility offered bymixotrophy potentially gives a significant competitiveadvantageoverbothpurelyphototrophicandheterotrophiccells under differing light and nutrient regimes. Forinstance,a Micromonas  -likepico-prasinophytewasshownto be an important bacterivore in the Arctic Ocean watersduring autumn, suggesting phagotrophy as an important A BC DE F Fig. 1.  Epifluorescence micrographs offree-living  Prochlorococcus   (A and B) andSAR11 clade (C and D) cells from theequatorial region after4 ′ ,6-diamidino-2-phenylindole (DAPI) staining(in blue), while the green colour shows thepositive signal of the horseradish peroxidase(HRP)-labelled probes. Lower panelepifluorescence images show the associationbetween a Prymnesiophyceae and Prochlorococcus   cell (E), andPrymnesiophyceae and SAR11 cell (F).Images (E) and (F) are an overlay of threepictures of the same cell observed under UVexcitation (showing the blue nucleus afterDAPI staining), green light excitation (redcytoplasm after TSA-FISH with the PRYM02probe) and blue light excitation (green cellafter TSA-FISH with prokaryote probe). Thescale bar is 5  μ m and applies to all figures. 837  M. Hartmann, M. V. Zubkov, D. J. Scanlan and C. Lepère   © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology,  Environmental Microbiology Reports,  5 , 835–840  survival attribute during winter darkness (Sanders andGast, 2011). However, in phototrophic picoeukaryotes,mixotrophy is not confined to cold, deep waters. Usingradio-labelled, pulse-chased bacterioplankton prey andsubsequent flow cytometric sorting, uptake of bacterialprey by plastidic protists was unequivocally demonstratedin surface waters of the oligotrophic Atlantic Ocean(Hartmann  et al  ., 2012), and reinforced using a TSA-FISHapproach in this study. On the basis of theoretical calcula-tions (see supplementary information), we compared ourmicroscopy observations with these radio-labelled meas-urements(Hartmann et al  .,2012).Onehastokeepinmindthat microscopy reveals the ingestion of prey by predator,whereas radio-labelling estimates the assimilation ofprey biomass by predator. The combined percentage of Prochlorococcus  andSAR11cellsobservedinsideallthreestudied phototrophic picoeukaryote classes was 17.2%,while the estimated ingestion of bacterioplankton cellsbased on radio-labelling was 9.2%. The difference ratiobetween the above two values (9.2%:17.2%  ×  100%  ∼ 50%) is an estimate of assimilation efficiency of bacterialprey by protists, and shows that phototrophicpicoeukaryotes might be as highly efficient in prey assimi-lation as specialized protistan predators, such asmicroflagellatesandplanktonicciliates(Caron et al  .,1988;Zubkov and Leakey, 2009).The significant difference ( P   <  0.05) in ingestion of Prochlorococcus   and SAR11 bacteria by phototrophicpicoeukaryote cells (on average 5.2% vs. 1.1% across thethreePhototrophicPicoEukaryotes(PPE)classesstudiedand the Plast-L and Plast-S size fractions) hints at selec-tive predation of  Prochlorococcus   by mixotrophic pico-eukaryotes. LNA cells (i.e. SAR11 cells) are significantlymore abundant than  Prochlorococcus   in the environmentsstudied here ( t  -test,  P   <  0.001, on average 46% vs. 27%,Fig. S4), but are also smaller and hence less nutritious. Fig. 2.  Taxonomic composition of small plastidic protists (Plast-S) (A) and large plastidic protists (Plast-L) (B), per region (Northern subtropicalgyre (NG), equatorial region (EQ) and Southern subtropical gyre (SG) as determined using fluorescence  in situ   hybridization (FISH).Percentage of internal association per region and per PPE class (Plast-S  +  Plast-L) for  Prochlorococcus   (C) and SAR11 (D) cells. Bacterivory by marine phototrophic picoeukaryotes   838  © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology,  Environmental Microbiology Reports,  5 , 835–840  In summary, we demonstrate here the utility of a dual-TSA-FISH approach to analyse the ecological interactionsbetween oceanic protistan and bacterioplankton popula-tions, opening up novel opportunities to examine  in situ  microbial trophic interactions, and demonstrating herethe frequency and possible selectivity of phototrophicpicoeukaryote bacterivory. Predation on bacterioplanktonby phototrophic picoeukaryotes reconfigures energy andnutrient flows within the marine microbial food webs ofopen ocean ecosystems. Acknowledgements The authors would like to thank the Captain and crew aboardthe RRS James Cook during AMT20. This study was sup-ported by the UK Natural Environment Research CouncilthroughResearchGrantsNE/E016138/1andNE/G005125/1,as well as the European Commission Seventh FrameworkProgramme through the GreenSeas Collaborative Project(FP7-ENV-2010Contract265294).C.L.wassupportedbytheFP7- IEF Marie Curie programme. This is Atlantic MeridionalTransect Publication No. 216. References Caron, D., Goldman, J., and Dennett, M. (1988) Experimentaldemonstration of the roles of bacteria and bacterivorousprotozoa in plankton nutrient cycles.  Hydrobiologia   159: 27–40.Christaki, U., Giannakourou, A., Wambeke, F.V., and Gregori,G. 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