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Ocean Acidification Reduces Growth and Calcification in a Marine DinoflagellateDedmer B. Van d e W aal1'2*, Uwe John3, Patrizia Ziveri4'5, Gert-Jan Reichart6'7, Mirja H oins1'6, Appy Sluijs6, Björn Rost11 M arine Biogeosciences, Alfred W egener Institu te for Polar and M arine Research, B rem erhaven, G erm any, 2 D epartm en t o f A quatic Ecology, N etherlands Institu te o f Ecology (NIOO-KNAW), W ageningen, The N etherlands, 3 Ecological Chem istry, Alfred W egener Institu te fo r Polar and M arine Research, B rem erhaven, Germ any, 4 Institu te o f Environm ental Science and T echnology (ICTA), Universität A utónom a d e Barcelona, Barcelona, Spain, 5 D epartm en t o f Earth Sciences, Vrije Universiteit A m sterdam ,

A m sterdam , The N etherlands, 6 D epartm en t o f Earth Sciences, U trecht University, U trecht, The N etherlands, 7 G eology D epartm ent, Royal N etherlands Institu te fo r Sea Research (NIOZ), Den H oorn (Texel), The N etherlands

AbstractO cean acidification is co n sid ered a m ajor th rea t to m arine eco sy stem s an d m ay particularly affect calcifying organ ism s such as corals, foram inifera an d co cco lithophores . Here w e in v es tig a te th e im pact o f e lev a ted p C 0 2 and low ered pH on g ro w th and calcification in th e co m m o n calcareous d inoflagellate Thoracosphaera heimii. We o b se rv e a su b stan tia l red uction in g ro w th rate, calcification an d cyst stability o f T. heim ii u n d e r e lev a ted p C 0 2. Furtherm ore, tran scrip tom ic analyses reveal C 0 2 sensitive regu la tion o f m any g en es , particularly th o se being asso c ia ted to inorgan ic carb o n acquisition and calcification. Stable carb o n iso to p e fractiona tion for o rg an ic carb o n p ro d u c tio n increased w ith increasing p C 0 2 w hereas it d ec reased for calcification, w hich su g g e s ts in te rd e p e n d e n c e b e tw ee n b o th processes. We also fo u n d a stro n g effect o f p C 0 2 on th e stab le oxygen iso top ic co m p o sitio n o f calcite, in line w ith earlier ob se rv a tio n s co n cern ing a n o th e r T. heim ii strain. The obse rv ed c h an g es in stab le oxygen an d ca rb o n iso to p e com p o sitio n of T. heim ii cysts m ay provide an ideal tool for reconstruc ting p ast se a w ate r c a rb o n a te chem istry, an d u ltim ately p ast p C 0 2. A lthough th e function o f calcification in T. heim ii rem ains unresolved, th is trait likely plays an im p o rtan t role in th e ecological and evo lu tionary success o f this species. Acting on calcification as well as g row th , o cean acidification m ay th e re fo re im p o se a g re a t th rea t for T. heimii.

C ita tio n : Van d e Waal DB, John U, Ziveri P, Reichart G-J, Hoins M, e t al. (2013) O cean Acidification Reduces G rowth and Calcification in a M arine Dinoflagellate. PLoS ONE 8(6): e65987. doi:10.1371/journal.pone.0065987

E d ito r: Howard I. Browman, Institu te o f M arine Research, Norway

R eceived March 27, 2013; A c cep ted April 30, 2013; P u b lish ed Ju n e 11, 2013

C o p y rig h t: © 2013 Van d e Waal e t al. This is an open-access article d istribu ted u n d er th e term s o f th e Creative C om m ons A ttribution License, w hich perm its unrestricted use, distribution , and reproduction in any m edium , provided th e original au th o r and source are credited .

F u n d in g : The w ork w as fu n d ed by BIOACID, financed by th e G erm an Ministry o f Education and Research. Furtherm ore, th is w ork was su p p o rted by th e European C om m unity 's Seventh Fram ew ork Programm e/ERC g ran t ag reem en ts # 2 0 5 1 5 0 and # 2 5 9627 , and con tribu te s to th e EC FP7 projects EPOCA, g ran t ag ree m en t #211 3 8 4 , and MedSeA, g ran t ag ree m en t #265 1 0 3 . T he funders had no role in s tudy design , d a ta collection and analysis, decision to publish, o r p repara tion o f th e m anuscript.

C o m p e tin g In te rests : The au tho rs have declared th a t no com peting in terests exist.

* E-mail: d .vandew aal@ nioo.knaw .nl.

Introduction

The oceans have taken up about one third of all C 0 2 emitted by anthropogenic activities since the onset of the industrial revolution [1—3]. This directly impacts seawater carbonate chemistry by increasing concentrations of C 0 2 and bicarbonate (H C 0 3_ ), decreasing concentrations of carbonate (C 0 3~ ) and a lowering of pH [4], The acidification of ocean waters might impact marine life, notably calcifying organisms that use inorganic carbon to produce a calcium carbonate (C aC 0 3) shell. Calcifying organisms play an important ecological and biogeochemical role in marine ecosystems, evident from extensive coral reefs and vast calcite deposits found in geological records. Ocean acidification has been shown to reduce calcification of various key calcifying organisms such as corals [5], foraminifera [6], and coccolithophores [7,8], Little is yet known about the general responses of calcareous dinoflagellates [9], and no study so far investigated the impact of ocean acidification on their calcification.

Dinoflagellates feature a complex life-cycle that often includes formation of cysts. In some species, these cysts are made of calcite and can contribute substantially to the ocean carbonate flux in certain regions [10-12]. Thoracosphaera heimii, the most common

calcareous dinoflagellate species in present-day ocean, is autotrophica and occurs typically in subtropical and tropical waters [ISIS]. The main life-cycle stage of T. heimii comprises coccoid vegetative cells with a calcium carbonate shell, so-called vegetative cysts [16,17]. Although the term cyst is most often used for longterm resting stages that are typically produced after sexual reproduction, in T. heimii this term is used for its coccoid vegetative stage. Cysts of T. heimii can be commonly found in the fossil record in sediments dating back to the Cretaceous [18], Therefore, T. heimii cysts may serve as potential proxy for reconstructing the past climate. For instance, S r/C a ratios have been shown to correlate well with sea surface temperatures [19], but also the oxygen and carbon isotopes trapped in the cysts could provide useful proxies.

The oxygen isotopic composition (S180 ) of calcite was found to be strongly controlled by the temperature and the S180 of the seawater in which the organism calcifies [20-22]. In abiotic precipitation experiments, the S180 of calcite is mainly a function of the S180 and spéciation of dissolved inorganic carbon (DIC), where dissolved C 0 2 is heavier with respect to 180 than H C 0 3- and C 0 3~- [23,24], Similarly, the carbon isotopic composition (S13C) of calcite is predominantly controlled by the S13C and spéciation of DIC, yet dissolved C 0 2 is depleted with respect to

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C 02 Effects on a Calcareous Dinoflagellate

13C relative to H C 0 3 and C 0 32 [21,25]. In unicellularcalcifiers like coccolithophores and T. heimii, calcification occurs intracellularly in specialized vesicles [16,26,27]. Therefore, the inorganic carbon used for calcification by these organisms must be derived from the intracellular inorganic carbon (C¡) pool. Consequendy, changes in S180 and S13C of calcite should resemble changes in the intracellular C; pool and may provide insights in the physiological processes underlying calcification and organic carbon production.

Comparable to coccolithophores, ocean acidification likely reduces calcification in T. heimii as well. Furthermore, increasing concentrations of C 0 2 are expected to alter the stable carbon and oxygen isotopic composition of T. heimii cysts. To test these hypotheses, we grew T. heimii at a range of C 0 2 levels and followed its responses in growth and calcification. Besides the assessment of S180 and S13C in T. heimii as a proxy, we use its isotopic composition as a tool to understand processes involved in organic carbon production and calcification. Transcriptomic analyses were applied to reveal mechanisms underlying the observed responses.

Materials and M ethods

Experimental Set-upCells of Thoracosphaera heimii RCC1512 (formerly AC214;

Roscoff Culture Collection) were grown as dilute batch cultures in 2.4 L air-tight borosilicate bottíes. Population densities were kept low at all times ( 3 cell divisions. Cell growth was monitored by means of triplicate cell counts daily or every other day with an inverted light microscope (Axiovert 40C, Zeiss, Germany), using 0.5-2 ml culture suspension fixed with Lugol’s solution (2% final concentration in mQ). Cell counts included determination of vegetative cysts, i.e. shells containing cell material, and empty shells. Because empty shells also contain inorganic carbon, the total number of cysts was used for estimating inorganic carbon quota, while only vegetative cysts were included in the growth rate estimations. From each biological replicate, growth rates were estimated by means of an exponential function fitted through the number of vegetative cysts over time, according to:

N t = N 0^ ‘ (1)

where N t refers to the population density at time t (in days), .No to

the population density at the start of the experiment, and ¡X to the growth rate (Fig. SI).

For total alkalinity (TA) analyses, 25 mL of culture suspension was filtered over glass-fibre filters (GF/F, —0.6 pm pore size, W hatman, Maidstone, UK) and stored in gas-tight borosilicate bottíes at 3°C. Duplicate samples were analysed by means of potentiometric titrations using an automated TitroLine burette system (SI Analytics, Mainz, Germany). pH was measured immediately after sampling with a pH electrode (Schott Instruments, Mainz, Germany), applying a two-point calibration on the NBS scale prior to each measurement. For DIC analyses, 4 mL culture suspension was filtered over 0.2 pm cellulose-acetate filters, and stored in headspace free gas-tight borosilicate bottles at 3°C. Duplicate samples of DIC were analysed colorimetrically with a QuAAtro autoanalyser (Seal Analytical, Mequon, USA). Carbonate chemistry (Table SI) was assessed by total alkalinity (TA) in combination with pH NBs, temperature and salinity, using the program C 0 2sys [28]. For the calculations, an average phosphate concentration of 6.4 pmol L _ was assumed, the dissociation constant of carbonic acid was based on M ehrbach et al. [29], refit by Dickson and Millero [30]. The dissociation constant of sulfuric acid was based on Dickson [31].

To determine the isotopic composition of DIC (8 13Cd ic) and the water (S18O water), 4 mL of culture suspension was sterile- filtered over 0.2 pm cellulose-acetate filters and stored at 3°C. Prior to analyses, 0.7 mL of sample was transferred to 8 mL vials. For determination of 8 Cdjc, the headspace was filled with helium and the sample was acidified with three drops of a 102% H 3P 0 4 solution. For determination of 818O water, the headspace was flushed with helium containing 2% C 0 2. C 0 2 and 0 2 isotopic composition in the headspace were measured after equilibration using a GasBench-II coupled to a Thermo Delta-V advantage isotope ratio mass spectrometer with a precision of


Isotopic FractionationIsotopic fractionation during organic carbon production and

calcification was calculated based on the carbon isotopic composition of the cellular organic carbon, cellular inorganic carbon and DIC, and the oxygen isotopic composition of the calcite and seawater, respectively. The carbon isotopic composition is reported relative to the PeeDee belemnite standard (PDB):

, i 3 r A 13c m ample \ W - ^ (13c / i2c)pdb x 1 ° (2)

The isotopic composition of C 0 2 (8 13CGo 2) was calculated from 8 13C Dic using a mass balance relation according to Zeebe and Wolf-Gladrow [24], applying fractionation factors between C 0 2 and H C 0 3_ from Mook et al. [33] and between H C 0 3_ and C 0 3 from Zhang et al. [34], The isotopic fractionation during PO C formation (Sp) was calculated relative to 8 13C Go 2 according to Freeman and Hayes [35]:

_ 8 13C c o 2 — S13Cpoc . .Sp_ l + S 13CpOCx l 0 - 3

The carbon isotopic fractionation during calcite formation (Sy) was calculated relative to 813CDIG:

_ 8 13C p i c — 8 13C p ic

Sk _ 1 + 8 13C p iC x 1 0 - 3

The oxygen isotopic composition in the calcite is also reported relative to the PDB standard:

c t8 0 _ /" (180 / 160 ) caicite l V , i n 3 / O

calc,te“ l ( i 80 / ^ 0 ) PDB - l ) x W (5)

The oxygen isotopic composition in DIC (S18O d ic) was determined using the oxygen fractionation factor between DIC, calculated after Zeebe and Wolf-Gladrow [24], and water (apic- H20))j calculated after Zeebe [36], with temperature corrected fractionation factors from Beck et al. [37]. The isotopic composition of DIC (8 O dic) was calculated according to:

a ü l í A . , « . (6)\ (̂DIC —H20) I

Transcriptomic AnalysesFor RNA extraction, 500 mL of culture suspension was

concentrated to 50 mL with a 10 pm mesh-sized sieve, and subsequendy centrifuged at 15°C for 15 min at 4000 g. Cell pellets were immediately mixed with 1 mL 60°C TriReagent (Sigma- Aldrich, Steinheim, Germany), frozen with liquid nitrogen and stored at — 80°C. Subsequently, cell suspensions were transferred to a 2 mL cryovial containing acid washed glass beads. Cells were lysed using a BIO 101 FastPrep instrument (Thermo Savant, Illkirch, France) at maximum speed (6.5 m s 1) for 2 x30 s, with

an additional incubation of 5 min at 60°C in between. For RNA isolation, 200 pL chloroform was added to each vial, vortexed for 20 s and incubated for 10 min at room temperature. The samples were subsequently centrifuged for 15 min at 4°C with 12,000 g. The upper aqueous phase was transferred to a new vial and 2 pL 5 M linear acrylamide, 10% volume fraction of 3 M sodium acetate, and an equal volume of 100% isopropanol were added. Mixtures were vortexed and subsequendy incubated overnight at — 20°C in order to precipitate the RNA. The RNA pellet was collected by 20 min centrifugation at 4°C and 12,000 g. The pellet was washed twice, first with 70% ethanol and afterwards with 96% ethanol, air-dried and dissolved with 100 pi RNase free water (Qiagen, Hilden, Germany). The RNA sample was further cleaned with the RNeasy Kit (Qiagen) according to manufacturer’s protocol for RNA clean-up including on-column DNA digestion. RNA quality check was performed using a NanoDrop ND-100 spectrometer (PeqLab, Erlangen, Germany) for purity, and the RNA Nano Chip Assay with a 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) was performed in order to examine the integrity of the extracted RNA. Only high quality RNAs (OD260/O D 280> 2 and O D 260/O D 230>1.8) as well as RNA with intact ribosomal peaks (obtained from the Bioanalyzer readings) were used for microarrays.

454-libraries were constructed by Vertís Biotechnologie AG (http://www.vertis-biotech.com /). From the total RNA samples poly(A)+ RNA was isolated, which was used for cDNA synthesis. First strand cDNA synthesis was primed with an N 6 randomized primer. Then 454 adapters were ligated to the 5' and 3 ' ends of the cDNA, and the cDNA was amplified with 19 PC R cycles using a proof reading polymerase. cDNA with a size range of 500- 800 bp was cut out and eluted from an agarose gel. The generated libraries were quantified with an RL-Standard using the Quanti- Fluor (Promega, Mannheim, Germany). The library qualities were assessed using the High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer (Agilent, W aldbronn, Germany). For all sequencing runs 20x10 molecules were used for the emulsion PC R that were carried out on a MasterCycler PCR cycler (Eppendorf, Hamburg, Germany). The following enrichment was performed according to the manufacturer’s instructions. Sequencing was performed with the GS Junior Titanium Sequencing K it under standard conditions. The 454 Sequencing System Software version 2.7 was used with default parameters, i.e., Signal Intensity filter calculation, Primer filter, Valley filter, and Base-call Quality Score filter were all enabled.

Statistical AnalysisNormality was confirmed using the Shapiro-Wilk. Variables

were log-transformed if this improved the homogeneity of variances, as tested by Levene’s test. Significance of relationships between variables and concentration of C 0 2 and C 0 32- were tested by means of linear regression. Significance treatments was tested using one-way ANOVA, followed by post hoc comparison of the means using Tukey’s HSD (a = 0.05) [38].

Results

Increasing concentrations of C 0 2 cause a strong decline in growth (Fig. 1A), which decreases by up to 53% over the investigated C 0 2 range (Table S2). Although the total carbon quota (TPC) is not affected by C 0 2 (Table S2), the organic carbon quota (POC) gradually increases while the inorganic carbon quota (PIC) shows a substantial decrease (Fig. IB). Consequently, the PIC:POC ratio strongly decreases with increasing concentrations


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Figure 2. Effect of elevated p C 0 2 on cyst m orphology. Cells grow n under (A-C) 150 patm C 0 2 and (D-H) 1400 patm C 02. Black arrows indicate cysts th a t are show n in detailed im ages, white arrows show collapsed cysts. doi:10.1371/journal.pone.0065987.g002

from the low to the high CI02 treatment (Fig. 4). Similarly, the relative expression of genes associated to carbonic anhydrases (CIA) and aquaporins decreases with increasing pC O i. In the present- day CI02 treatment, we observed expression of a gene associated to an SLCI4 family anion exchanger (AE), most likely responsible for the transport of H C I03_ into the cell (Fig. 4) [39]. This gene was expressed in neither the low nor the high CI02 treatment. An SLC26 family S0 4 3“ / HCI0 3“ / CI20 42“ anion exchanger (SAT-1) was yet another exclusive expression of a gene only found in the low CI02 treatment. The potential role of this anion exchanger in Cl; acquisition by phytoplankton remains to be elucidated.

Discussion

Growth and Carbon ProductionO ur results show considerable impacts of elevated pC O i on T.

heimii, with strong decreases in its growth rate and degree of calcification (Fig. 1, 2). Despite the increase in organic carbon quota (POCI), the overall biomass production decreases substantially with increasing jiiCOo (Table S2). Fligher availability of CI02 has been shown to promote phytoplankton growth and carbon production [40,41], Such CI02 responses are typically associated to the poor catalytic properties of RubisCIO, which is characterized by low affinities for its substrate CI02. Increasing concentrations of CI02 are however accompanied by a reduction in pH, which may have consequences for calcification. For the most common coccolithophore Emiliania huxleyi, lowered pH in fact hampers calcification while elevated p C 0 2 stimulates biomass production, causing a reallocation of carbon and energy between these key processes [42,43]. This flexibility may explain why growth in E. huxleyi is typically not affected by ocean acidification [44], In T. heimii, however, we observed a strong decrease in calcification, in biomass production as well as in growth. Apparently, T. heimii

lacks the ability to efficiently reallocate cellular carbon between pathways and maintain growth relatively unaffected. O ur data furthermore suggests that calcification plays a fundamental role in its growth, life cycle and hence survival. Recent findings have shown that growth and calcification by E. huxleyi may, at least partly, recover from ocean acidification as result of evolutionary adaptation [45]. W hether or not T. heimii exhibits such capabilities of adaptive evolution can only be answered from long-term incubations over hundreds of generations [46].

Transcriptomic analyses reveal a substantial regulation of genes in response to elevated p C 0 2. Even though no major shift in the relative distribution of expressed genes to the functional categories (KOGs) is induced by the treatments, T. heimii uses different sets of genes within these categories. There is a slight increase in the expression of genes associated to signal transduction and posttranslational modifications upon elevated />C02, and a decrease in the expression of genes involved in inorganic ion transport (Fig. S3), suggesting that T. heimii readjusts its transcriptome on several levels when grown under different pCO-2 ■ Many phytoplankton species have the ability to deal with changes in CI02 availability by regulating their so-called carbon concentrating mechanism (CICMs) [47-49], T. heimii also appears to regulate its proteome towards changes by down-regulating genes involved in CIA and aquaporins under elevated /)C 0 2, and by up-regulating these genes under lowered p C 0 2 (Fig. 4). CIA accelerates the equilibrium between CI02 and H C !03 , and can be located both intra- and extracellularly. From our results it remains unclear whether T. heimii expresses intra- or extracellular CIA. Yet, in both cases CIA plays a key role in the CICIM, as it replenishes the CI02 around RubisCIO (intracellular) or the carbon source being depleted in the boundary layer due to active uptake (extracellular) [49,50], Aquaporins have been suggested to play a role in CI02 transport [47,51], which is supported by the observed CI02-


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O

Oco

'cO

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20

15

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- O - s k= -0.15 [C 0 2] + 10.20-54

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5 180 = -0.024 [CC>32'] + 1.115 180 = -0.022 [C 0 32'] + 2.20

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Figure 3. Effect o f increasing C 0 2 concentrations on the stable isotope composition. (A) 13C fractionation of organic carbon (e„) and calcite (ek), (B) 180 com position of calcite (S18Oca|dte) and DIC (S1 0 D!C), and (C) relationship betw een the oxygen isotopic com position of calcite (S180 c a ic i te -w a te r ) in Thoracosphaera from this study (open diam onds) and from Ziveri e t al. [9] (grey diam onds). Horizontal lines in (B) indicate S180 values for HC03~ and C 032~, and dashed line indicates trend of curve. Solid lines indicate linear regressions (n = 12) with (A) ep: R2 = 0.75, PC0.001, and ek: R2 = 0.90, P


treatm ent was close to 7.6, the water still remained supersaturated with respect to calcite (i.e. an ñ calcite >1.2; Table S2), and calcite dissolution seem unlikely to have caused the incompletion and cavities in the calcite structure (Fig. 2). Thus, the large number of affected T. heimii cysts at elevated p G 0 2 seems mainly to be a result of reduced calcification rates by the cells.

Calcification in T. heimii likely takes place intracellularly in vesicles [16,26], comparable to coccolithophores [9,27]. Hence, the inorganic carbon needed for calcification is obtained from the intracellular inorganic carbon pool (C¡), which may deviate strongly from external conditions in terms of spéciation as well as isotopic composition. We observed an increase of carbon isotope fractionation for organic carbon production (sp), whereas it decreased for calcite formation (s )̂ in response to elevated p G 0 2 (Fig. 3A). W ith a higher availability of C 0 2, more of the intracellular C; pool may be replenished by C 0 2, which is depleted in 13C compared to H C 0 3 . Consequently, RubisCO can fractionate against an isotopically lighter C; pool and thus better express its preference for lighter 12C, which could explain the increasing Sp. As a consequence, the intracellular C; pool becomes enriched with 13C by so-called Rayleigh distillation, which a priori could explain the decrease in s^. However, increased C 0 2 availability in combination with a reduced organic carbon production should lead to a lowered Rayleigh distillation, and in fact decrease the enrichment of 13C within the cell. Also, Rayleigh distillation should always feedback on C 0 2 fixation as well as C a C 0 3 precipitation, and thus cannot explain the opposing trends of fractionation in those processes.

The opposing C 0 2 effects on Sp and s^ can thus only be explained if both processes use C; pools that are isotopically different. C 0 2 fixation uses the C¡ pool within the chloroplast, which is affected by the relative C 0 2 and H C 0 3 fluxes, the C 0 2 leakage as well as the intrinsic fractionation by RubisCO [53,54], The C; pool for calcification will mainly be controlled by the condition in the cytosol, which in turn is largely affected by the processes in the chloroplast. Discrimination of 13C during fixation will lead to 13C 0 2 efflux from the chloroplast, causing the cytosolic C; pool to be enriched with 13C 0 2. If this 13C 0 2 is prevented from fast conversion to H C 0 3 due to a lack of cytosolic CA activity, it could enter the calcifying vesicle by diffusion and be ‘trapped’ by the high pH resulting from proton pumping (Fig. 5). In fact, we do observe a higher Sp (i.e. more 13C 0 2 can accumulate) and lower overall CA activities under elevated p C 0 2 (i.e. 13C 0 2 is not rapidly converted to H C 0 3 ), which could have attributed to the opposing trends of 13C fractionation during organic and inorganic carbon production. To fully understand the intriguing interplay between these processes and their 13C fractionation, detailed measurements on the modes of C; acquisition in T. heimii are needed.

The oxygen isotopic composition (S180 ) of calcite was also strongly affected in T. heimii, and increased by almost 6 %o over the investigated C 0 2 range (Fig. 3B). Even though biologically mediated, precipitation of calcite is an abiogenic process, which does not directly involve enzymatic reactions and thus mainly depends on the carbonate chemistry at the calcification site. Assuming negligible fractionation during the transport into the calcification vesicle, S18O cajc;te should therefore predominantly reflect the S180 of the C¡ species used for calcification. C¡ species differ strongly in their S180 values, ranging from lower values for C 0 32- (—4.7 %o) and H C 0 3_ (2.1 %o) to much higher values for C 0 2 (11.2 %o) [24]. A previous study proposed a conceptual model to explain the S180 dependence of T. heimii calcite and other unicellular planktonic calcifiers on seawater C 0 32- concentration (Fig. 3C) [9], The authors attribute the negative slope between

S180 and [C 0 32 ] to an increased contribution of H C 0 3 to the calcification vesicle. Also in our data, S18O cajc;te increases with increasingp C 0 2, starting from values close to the S180 of C 0 32 towards those of H C 0 3 (Fig. 3B). As argued above, however, the C; pool in the calcifying vesicle may also be increasingly influenced by C 0 2, which is in line with the observed trends in S18O caicite. Such a shift in C; spéciation may be an indication for a lowered intracellular pH, which in fact could be the reason for the hampered calcification under elevated p G 0 2 [55,56].

Multiple genes associated to calcification have been described for E. huxleyi and include genes associated to the regulation of inorganic ions [39,55-59]. Here we show that the expression of genes in T. heimii being involved in inorganic ion transport, in particular C a2+ transport, decreased upon elevated p C 0 2 (Fig. 4; Fig. S3). This decrease in ion transport is in line with the observed decrease in calcification, which is comparable to observations in E. huxleyi [39,59]. We also observed a strong C 0 2 dependent regulation of the vacuolar H -ATPases (V-ATPase). These pumps play a key role in generating H + gradients and membrane voltage, which drive multiple transport processes [57,60]. As indicated from our data, H +-ATPases seem to play an im portant role in calcification in T. heimii, which is in agreement to observations for E. huxleyi and Pleurochiysis carterae [39,59,61]. Here we propose a conceptual model of calcification in T. heimii, which comprises some of the main processes described in this study (Fig. 5). Although many processes remain to be elucidated, this is a first step towards understanding the process of calcification in dinoflagellates.

Paleo ProxiesThe S180 isotopic composition of T. heimii cysts has been used

for the reconstruction of past temperatures [22,62]. Indeed, S180 changed linearly from about —1 to — 4 %o with an increase in temperature from about 12 to 30°C. At the same time, however, pH decreased from about 8.4 to 7.9 in this study [22]. Hence, the observed changes in S180 were most probably a result of both changes in temperature and seawater carbonate chemistry [see also 62]. Here we show remarkable changes in S180 from about 0 to —5 %o with an increase in [C 0 32 ] from 50 to 260 pmol L *, which is largely in agreement to an earlier study including a different T. heimii strain (Fig. 3C) [9], Interestingly, the observed slopes of S180 / [ C 0 32 ] in both T. heimii strains are up to 10-fold steeper compared the coccolithophora Calcidiscus leptoporus and different foraminifera species [9,25,63]. Thus, the apparent 180 fractionation during calcification in T. heimii is much more sensitive to changes in [C 0 32-] as compared to other key planktonic marine calcifiers. The steep slope and negative correlation between S180 and [C 0 32-] observed in both T. heimii strains suggests that the S180 in T. heimii cysts may be a good candidate to serve as a proxy for past C 0 32- concentrations in ocean waters. This relationship may provide an ideal asset, especially when combined with different S180 / [ C 0 32 ] slopes observed in for instance coccolithophores, which will exclude confounding effects of additional environmental parameters such as temperature. Ultimately, this proxy could be further developed for reconstructing past atmospheric p G 0 2.

ConclusionWe observed a strong reduction in growth rate and calcification

of T. heimii under elevated p G 0 2. Although the function of calcification in T. heimii remains unresolved, it likely plays an im portant role in its ecological and evolutionary success. Acting on calcification as well as growth, ocean acidification may impose a great threat for T. heimii. Furthermore, the strong correlations




Figure 5. Conceptual model of regulated proteins in a T. heim ii cell. The regulated proteins involved in ion transport and C, acquisition are show n on their putative locations [39,49,57]. Proteins involved in vacuolar Ca2+ and H+ transport include P-type Ca2+ ATPases (P-ATPase), Ca2+/N a+ exchangers (NCX), Ca2+/H+ antiporters (VCX), and vacuolar H+ ATPases (V-ATPase). Active uptake of HC03~ may occur via a SLC4 family anion exchanger (AE) or an SLC26 family S 043_/HC03_/C20 42_ anion exchanger (SAT-1). Carbonic anhydrases (CA) are located intracellularly or extracellularly and enhance the interconversion betw een C 02 and HC03A doi:10.1371/journal.pone.0065987.g005

between the stable isotope composition and carbonate chemistry suggest a great potential of T. heimii cysts to be used as paleo proxy for reconstructing seawater carbonate chemistry and ultimately past atmospheric p G 0 2.

Supporting Information

Figure SI Population growth dynamics. Population densities in each replicate over time in the (A) 150 patm, (B) 380 patm, (C) 750 patm, and (D) 1400 patm CI02 treatments. Lines indicate an exponential function fitted through the population densities (n = 8) of replicate 1 (black), 2 (grey) and 3 (white), with (A) 1 : R 2 = 0.98, pCO.0001, 2: R 2 = 0.97, pCO.0001, and 3: R 2 = 0.97, pCO.0001, (B) 1: R 2 = 0.97, pCO.0001, 2: R 2 = 0.97, pCO.0001, and 3: R 2 = 0.92, pCO.0001, (Cl) 1: R 2 = 0.92, p = 0.0007, 2: R 2 = 0.96, pCO.0001, and 3: R 2 = 0.97, pCO.0001, and (D) 1: R 2 = 0.96, pCO.0001, 2: R 2 = 0.95, pCO.0001, and 3: R 2 = 0.91,p = 0.0002. (EPS)

Figure S2 Number of expressed genes. Venn diagram of the number of expressed genes in the 150 patm, 380 patm, and 1400 patm CI02 treatments.(EPS)

Figure S3 Distribution of expressed genes grouped according to KOG. Values represent the number of genes expressed per K O G , relative to the total number of genes expressed in the respective treatment.(EPS)

Table SI Carbonate chemistry at the start and end of the experiment. Overview ofpGOn, pH NBs, dissolved inorganic carbon (DIC), CI02 concentration in the water, total alkalinity (TA), and the seawater calcite saturation state f í caicit c . Values indicate mean ± SD (n = 3).(DOCIX)

Table S2 Growth, elemental composition and calcification at the end of the experiment. Overview of growth rate, POCI production, carbon quota (TPCI, POCI, and PIG), PICI:POC ratio, and the number of completed cysts. Values indicate mean ± SD (n = 3).(DOCIX)

Table S3 Overview of all expressed genes grouped according to KOG.(XLSX)

Table S4 Overview of the number of readings for genes associated to ion transport and C¡ acquisition.(XLSX)

PLOS ONE I www.plosone.org June 2013 | Volume 8 | Issue 6 | e65987



A cknow ledgm entsT h e au tho rs like to th an k Y vette B ublitz for assistance w ith the experim ents an d F riedei H inz fo r assistance w ith the SE M pictures. T h e au tho rs thank Ian P ro b e r tfro m Station B iologique de R osco fffo r p rov id ing Thoracosphaera heimii R C C 1512.

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