A Nanoscale Secondary Ion Mass Spectrometry Study of Dinoflagellate

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Environmental Microbiology (2014) doi:10.1111/1462-2920.12518

A nanoscale secondary ion mass spectrometry study of dinoﬂagellate functional diversity in reef-building corals

Mathieu Pernice,1,2*† Simon R. Dunn,3† Linda Tonk,3 ability of the intact symbiosis between the reef- Sophie Dove,3 Isabelle Domart-Coulon,4 building coral Isopora palifera, and Symbiodinium C Peter Hoppe,5 Arno Schintlmeister,6 Michael Wagner6,7 or D types, to assimilate dissolved inorganic carbon 1,8 and Anders Meibom (via photosynthesis) and nitrogen (as ammonium). 1 Laboratory for Biological Geochemistry, School of Our results indicate that Symbiodinium types from Architecture, Civil and Environmental Engineering two clades naturally associated with I. palifera (ENAC), École Polytechnique Fédérale de Lausanne possess different metabolic capabilities. The (EPFL), Lausanne, Switzerland. Symbiodinium C type fixed and passed significantly 2 Plant Functional Biology and Climate Change Cluster more carbon and nitrogen to its coral host than the D (C3), Faculty of Science, University of Technology, type. This study provides further insights into the Sydney, NSW, Australia. metabolic plasticity among different Symbiodinium 3 ARC Centre of Excellence for Coral Reef Studies, types in hospite and strengthens the evidence that School of Biological Sciences, University of the more temperature-tolerant Symbiodinium D type Queensland, Brisbane, Qld, Australia. may be less metabolically beneficial for its coral host 4 UMR7245, Molécules de Communication et Adaptation under non-stressful conditions. des Microorganismes, Muséum National d’Histoire Naturelle, Paris, France. 5Particle Chemistry Department, Max Planck Institute for Introduction Chemistry, Mainz, Germany. 6Large-Instrument Facility for Advanced Isotope Coral reefs are among the most productive and biologically Research and 7Department of Microbiology and diverse ecosystems on Earth. The unicellular dinoflagel- Ecosystem Science, Division of Microbial Ecology, late algae (genus Symbiodinium), which live within coral University of Vienna, Vienna, Austria. tissue, play a fundamental role in the maintenance of 8Center for Advanced Surface Analysis, University of healthy reefs by providing corals with important nutrients Lausanne, Lausanne, Switzerland. (Muscatine, 1990). Thus, corals are ‘polytrophic’, being able to acquire nutrients from sunlight through their photosynthetic symbionts (‘autotrophy’), feeding on plankton Summary (‘heterotrophy’) and absorbing dissolved nutrients from the Nutritional interactions between corals and symbiotic surrounding seawater. These multiple strategies increase dinoflagellate algae lie at the heart of the structural the nutritional options for corals in the oligotrophic environ- foundation of coral reefs. Whilst the genetic diversity ment they commonly inhabit. of Symbiodinium has attracted particular interest Symbiotic dinoflagellates of the genus Symbiodinium because of its contribution to the sensitivity of corals are divided into nine broad genetic clades, (clades A–I) to environmental changes and bleaching (i.e. disrup- defined by rRNA gene sequences (Rowan and Powers, tion of coral–dinoflagellate symbiosis), very little is 1991a; Coffroth and Santos, 2005; Pochon and Gates, known about the in hospite metabolic capabilities of 2010). These clades contain various genetically distinct different Symbiodinium types. Using a combination of types according to comparative analyses of internal tran- stable isotopic labelling and nanoscale secondary ion scribed spacer (ITS) sequence regions (van Oppen et al., mass spectrometry (NanoSIMS), we investigated the 2001; LaJeunesse et al., 2004a,b; Pochon et al., 2007; Sampayo et al., 2007). Corals can simultaneously harbour dinoflagellates from different types in variable densities depending on coral species and environmental Received 8 August, 2013; accepted 25 May, 2014. *For correspond- ence. E-mail [email protected]; Tel. +6129514 4038; Fax conditions (Baker and Romanski, 2007). Differences in +6129514 4079. †These authors contributed equally to this work. physiological properties between Symbiodinium types

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd 2 M. Pernice et al. can influence photosynthetic efficiency, growth and 2013). Here, we use isotopic labelling in combination thermal tolerance of the host (Iglesias-Prieto et al., 2004; with single-cell NanoSIMS imaging of the intact coral Little et al., 2004; Berkelmans and van Oppen, 2006; symbiosis to offer an integrated view of the differential Sampayo et al., 2008). Although considered as a key ability of Symbiodinium types from different clades to factor that defines the health of coral reefs in a changing acquire dissolved inorganic carbon and nitrogen. climate, the functional diversity of most Symbiodinium In this study, we investigated (i) whether different types remains under-explored. Symbiodinium types that naturally associate with a single Assimilation of carbon and nitrogen is essential for coral species possess different metabolic capabilities and corals, and therefore, adequate supply of these two nutri- (ii) to what extent these genetically distinct symbiont types ents is critical to sustain optimal growth, development affect coral host metabolism. To address these questions, and response to a wide array of stresses. As yet, we compared the assimilation of dissolved inorganic however, most nutritional investigations of the functional carbon and nitrogen between colonies of the reef-building diversity of symbiotic dinoflagellates have focused on the coral Isopora palifera (Wallace et al., 2012) (Fig. 1A), metabolism of photosynthetically fixed carbon (Loram which associates with Symbiodinium types from clades C et al., 2007; Stat et al., 2008). Recently Baker and and D in its natural habitat. colleagues (2013) compared nitrate assimilation in juvenile corals infected with different Symbiodinium clades Results and discussion and suggested that under non-stressful conditions, Symbiodinium type C1 outcompetes an undefined D type Sixty-eight adult I. palifera colonies growing at different via enhanced nitrogen acquisition. Nevertheless, assimi- sites adjacent to Heron Island Research Station (HIRS, lation of ammonium, which is the preferred source of 231330S 1511540E) were assessed regarding the diver- inorganic dissolved nitrogen for coral dinoflagellates sity of Symbiodinium before November 2011, when corals (Grover et al., 2008), has so far never been compared were sampled for the labelling experiment. For each between Symbiodinium types from different clades. Fur- colony, Symbiodinium clades were identified by extracting thermore, most bulk isotope analysis studies measuring genomic DNA from a single branch and analysing the the relative assimilation of carbon and nitrogen by sym- pattern of PCR-restriction fragment length polymorphism biotic dinoflagellates suffer from potential cross- (PCR-RFLP) of the small subunit rRNA gene. Fifty-nine contamination of coral host and dinoflagellate fractions, I. palifera colonies harboured Symbiodinium clade C, as the intertwined nature of the coral–dinoflagellate while eight colonies were associated with Symbiodinium endosymbiosis makes the relative quantification of dino- clade D, and only one colony hosted members of both flagellate symbiont metabolism in hospite extremely dif- clades C and D. All Symbiodinium could be further ficult (Yellowlees et al., 2008). In this context, recent assigned to either type C3 or D1 by polymorphism screen- advances in nanoscale secondary ion mass spectrom- ing of the ITS1–ITS2 region using denaturing gradient gel etry (NanoSIMS) have the potential to provide direct electrophoresis (DGGE, Fig. 1B) and sequencing of imaging and precise quantification at the individual cell excised bands (data not shown). level of up to seven different isotopes or molecular Subsequently, a combination of isotopic labelling with species within an intact symbiosis (Musat et al., 2008; 15N-ammonium and 13C-bicarbonate and NanoSIMS Pernice et al., 2012; Berry et al., 2013; Kopp et al., 2013) imaging of tissue sections was used to quantify the (for review, see Orphan and House, 2009; Hoppe et al., assimilation of dissolved inorganic carbon and nitrogen in

Fig. 1. NanoSIMS quantification of 15N and 13C assimilation by two different Symbiodinium types naturally associated with the reef-building coral Isopora palifera. A. A colony of I. palifera collected on Heron Island in November 2011. B. Symbiodinium types were identified in 68 coral samples by amplification of the ITS1-ITS2 region followed by screening of polymorphisms using denaturing gradient gel electrophoresis (DGGE). In this panel DGGE data for four representative coral colonies are depicted. Selected 15 13 corals containing the different Symbiodinium types (C3 or D1) were incubated in ASW containing NH4Cl (5 μM) and NaH CO3 (2 mM) for 48 h. D–I. The distribution of 15N/14N and 13C/12C isotopic ratio was imaged directly in the coral tissue and quantified using NanoSIMS. The Hue Saturation Intensity images are mapping the 15N/14N (D, F, H) and 13C/12C (E, G, I) isotopic ratio. The rainbow scale corresponds to the isotopic ratios and ranges from blue, set to natural ratio (0.0037 for 15N/14N and 0.0110 for 13C/12C), to red, where the ratio is several fold above natural ratio (0.0370, 10 times the natural ratio for 15N/14N and 0.1100, 10 times the natural ratio for 13C/12C). Quantification of 15N and 13C enrichment of individual dinoflagellate cells and coral host tissue was obtained by selecting Regions of Interest (ROIs) that were defined in Open_MIMS by drawing directly on the NanoSIMS images (i) the contours of the dinoflagellate cells and (ii) circles of about 8–10 μm diameter, covering host tissue (as described in D to I). C. For each NanoSIMS image, the mean 15N/14N and 13C/12C ratio are given for each individual ROI corresponding to dinoflagellate symbiont (Zooxanthellae, Zx) or host tissue (Ht). Scale bars, 10 μm.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 4 M. Pernice et al. symbiotic dinoflagellate cells from colonies hosting either df = 65; P = 0.006). There was no significant difference in Symbiodinium type C3 or D1. NanoSIMS analyses the density of the symbionts per surface area of coral revealed substantial incorporation of the 13C and 15N iso- branch (Supporting Information Fig. S1; t-test: t = 1.501; topic tracers into both coral and dinoflagellate cells in df = 18; P = 0.151) or in their size distribution (Supporting corals incubated in isotope-enriched artificial sea water Information Fig. S2A and B; Kolmogorov–Smirnov test: (ASW) (Fig. 1D–G) in contrast to unlabelled control P = 0.769). Both these latter features suggest similar divi- samples (Fig. 1H and I). Quantitative analysis based on sion rates amongst these Symbiodinium types under the tissue sections from two different coral colonies for each imposed experimental conditions. Furthermore, additional Symbiodinium type indicates that, during the 48 h incuba- analyses showed that there was no significant correlation tion period, the incorporation of 15N-ammonium and between the apparent size of Symbiodinium cells ana- 13C-bicarbonate by individual dinoflagellate cells was sig- lysed by NanoSIMS and their incorporation of nitrogen nificantly higher in Symbiodinium type C3 than type D1 and carbon during the 48 h labelling experiment (15N, (δ15N: 6704 ± 438‰ for type C3 and 4159 ± 379‰ for type R2 = 0.02, P = 0.742, Supporting Information Fig. S3A; D1, t-test: final P-value = 0.006; δ13C: 3613 ± 134‰ for 13C, R2 < 0.001, P = 0.995; Supporting Information Fig. type C3 and 2773 ± 115‰ for type D1, final S3B). Taken together, these data suggest that greater P-value < 0.001; Fig. 2A and B). These results provide assimilation rates of 15N-ammonium and 13C-bicarbonate evidence that in hospite, within the same coral host by Symbiodinium type C3 over D1 symbionts did not species and under the same environmental conditions, enhance symbiont proliferation rates. In this respect, Symbiodinium type C3 assimilated more dissolved inor- future studies including longer experiments are needed in ganic carbon and nitrogen than Symbiodinium type D1. order to investigate the relationships between metabolism These significant differences observed in the metabolism and proliferation rates. of Symbiodinium type C3 and D1 in hospite could be On the other hand, D1 symbionts may have reduced explained by (i) substantial variability in their efficiency to concentrations of labelled carbon and nitrogen isotopes fix carbon via photosynthesis and provide carbon- because assimilated carbon and nitrogen are more skeleton for ammonia assimilation from seawater or by (ii) rapidly transferred to the host compared with C3 potential differences in the respiration rate of fixed carbon, symbionts. To clarify this issue, the incorporation of inor- but also in the translocation rate of both carbon (Cantin ganic carbon and nitrogen was quantified within the coral et al., 2009) and nitrogen compounds between symbiont host cells by measuring 15N and 13C-enrichment in and host. 8–10 μm diameter regions of interests (ROI) located in Differential assimilation rates of carbon and nitrogen direct proximity to the symbionts (Fig. 1D–I). Compared may reflect differential growth rates amongst taxonomi- with corals containing Symbiodinium type D1, the cally distinct intracellular Symbiodinium as suggested pre- I. palifera tissue associated with Symbiodinium type C3 viously (Fitt, 1985; Wooldridge, 2012). On this note, incorporated substantially more carbon (δ13C: 460 ± 37‰ Wooldridge (2012) predicted that the bleaching-tolerant for corals associated with type C3 and 177 ± 23‰ clade D symbionts would possess a slower division rate for corals associated with type D1, t-test: final and a larger cell size than clade C. Our data indicate that P-value < 0.0001) and nitrogen (δ15N: 1614 ± 160‰ for Symbiodinium types hosted by I. palifera displayed differ- corals associated with type C3 and 730 ± 112‰ for corals ences in relative size, with Symbiodinium type C3 being associated with type D1, t-test: final P-value = 0.022; on average smaller than type D1 (Supporting Information Fig. 2C and D). The results support the hypothesis that C3 Table S1; 7.1 ± 0.8 μm diameter for type C3 versus symbionts are more efficient at both assimilating carbon 8.2 ± 1.8 μm diameter for type D1; t-test: t =−2.831; and nitrogen from the environment and transferring

Fig. 2. Quantification of nitrogen and carbon fixation by host tissue and dinoflagellate symbionts within the reef-building coral Isopora palifera. Median enrichment measured in dinoflagellate cells by NanoSIMS, in I. palifera associated with Symbiodinium C3 (in black, n = 21) or Symbiodinium D1 (in white, n = 43): A. In 15N. B. In 13C. Median enrichment measured in the coral tissue by NanoSIMS in I. palifera associated with Symbiodinium C3 (in black, n = 19) and Symbiodinium D1 (in white, n = 35). C. In 15N. D. In 13C. The box-whisker plot separates the data into quartiles, with the top of the box defining the 75th percentile, the line within the box giving the median and the bottom of the box showing the 25th percentile. The upper ‘whisker’ defines the 95th percentile; the lower whisker, the 5th percentile. Individual values are given in Supporting Information Tables S1 and S2 for dinoflagellate symbiont and host tissue respectively. *Significant difference between corals associated with Symbiodinium type C3 and D1 (P < 0.05, P-value calculated as 95th percentile of the P-values obtained after 100 000 bootstrap-based resampling and t-tests).

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 6 M. Pernice et al. acquired carbon and nitrogen to their hosts, than D1 the symbionts possess nitrate and nitrite reductase symbionts. Previous studies have indicated that an unde- enzymes for assimilating nitrate into ammonium and sub- fined Symbiodinium type belonging to clade D might be sequently into organic products (Crossland and Barnes, considered less mutualistic than Symbiodinium type C1 1977). A first disadvantage of using nitrate as a substrate since it provides less energy to juvenile corals (Cantin for labelling experiments and for quantifying metabolic et al., 2009) and can result in reduced growth (Little et al., rates is that uptake rate by Symbiodinium cell is known to 2004), lipid storage and reproductive output for adult be significantly slower for nitrate than for ammonium corals (Jones and Berkelmans, 2011). Consistent with this (D’Elia et al., 1983). Second, nitrate transporters can gen- finding, our study shows a stronger capability of erally regulate ion uptake by switching from a low-affinity Symbiodinium type C3 compared with type D1 for fixing to a high-affinity mode depending on the availability of inorganic carbon and ammonium and for subsequent con- nitrate (Parker and Newstead, 2014) and, consequently, tribution of these newly assimilated compounds to their cellular uptake of nitrate would likely be variable, depend- coral host nutrition (including potential additional carbon ing on the nitrate concentration in which the symbiotic fixation via carboxylation reactions). cnidarian were maintained prior to the isotopic incubation In the following section, we would like to discuss experiments. Ammonium transport across biological several points that are of relevance for the interpretation membranes is known to be mediated by Amt proteins and of the data obtained in this study. First, sample prepara- several corresponding genes have been identified in tion for transmission electron microscopy (TEM) and Symbiodinium previously (Leggat et al., 2007). However, NanoSIMS analyses can cause biases. For example, the it is still unclear if these genes are subjected to nitrogen successive steps of sample rinsing, ethanol dehydration control or not in symbiotic dinoflagellate, and if cellular and acetone storage could have resulted in partial extrac- uptake is more or less variable for ammonium than for tion of sugars and other soluble molecules poor in amino nitrate. In the future, more detailed studies are clearly groups. Note that this extraction can be minimized by needed to (i) assess more precisely the advantages and rapid osmium post-fixation before rinsing and by avoiding disadvantages of using ammonium or nitrate for isotopic acetone storage. In addition, fixation and embedding of labelling experiments in coral symbiosis and to (ii) estab- the samples add additional 12C atoms and thus dilute the lish to what extent the rate of assimilation of these differ- 13C signal. Therefore the 13C concentrations reported in ent substrates is affected by different concentrations of our NanoSIMS analyses are underestimates, but the rela- nitrogen and by different feeding regime of the coral tive isotopic enrichment ratios remain valid. Second, hosts. corals are unusual animals because they can directly and The large standard deviation observed in our study for rapidly assimilate ammonium into amino acids using carbon and nitrogen enrichment ratios (i.e. high value of GS/GOGAT cycle (Wang and Douglas, 1999; Grover standard deviation indicated in Supporting Information et al., 2002; Pernice et al., 2012). Consequently, the pres- Tables S1 and S2 and illustrated by the difference in ence of 15N in either the host or the symbiont may origi- colour scale within individual cells in Fig. 1D–G), reflects a nate from two different sources of ammonium strong spatial heterogeneity of carbon and nitrogen assimilation: the animal host or the symbiont. However, enrichment. Such a spatial heterogeneity is likely to be because the coral colonies were collected from the same related either to variability in physiological activity reef site, and subsequently exposed to the same experi- between individual cells or to the thin (100 nm) section mental conditions, one would, unless host assimilation of orientation through specific subcellular structures. For ammonium is stimulated differently by the presence of instance, in symbiotic dinoflagellate, Kopp and colleagues different symbionts, not expect this unusual aspect of host (2013) previously demonstrated that nitrogen storage into physiology to effect the interpretation of the 15N data pro- cytosolic crystals of uric acid could largely explain the vided above. Third, coral symbiosis can assimilate inor- spatial heterogeneity of 15N-enrichment observed in ganic nitrogen dissolved in seawater as ammonium and Symbiodinium cells after incorporation of 15N-ammonium nitrate. In this study, 15N-ammonium was used as a sub- and 15N-nitrate. The substantial cell-to-cell variation strate for the labelling experiment because it is recog- observed in our study could also reflect the presence of nized as the preferred source of inorganic nitrogen for physiologically distinct populations of cells in the tissue of corals (Grover et al., 2002). However, it is important to I. palifera. In the case of symbiotic dinoflagellates, it is note that nitrate is also an important factor for growth of possible that the observed individual Symbiodinium cells symbiotic dinoflagellates (Fagoonee et al., 1999) and is were in different physiological state as a result of different generally more abundant on coral reefs than ammonium. life histories, including mitotic activity. However, additional Nitrate can be used as a substrate for isotopic labelling analyses failed to show any significant correlation experiments combined with NanoSIMS analysis (Kopp between the apparent size of Symbiodinium cells ana- et al., 2013), and offers some interesting insights as only lysed by NanoSIMS and their incorporation of nitrogen

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Functional diversity of dinoflagellate symbionts in corals 7 and carbon (15N, R2 = 0.02, P = 0.742, Supporting Infor- capability of Symbiodinium type C3 versus type D1, both mation Fig. S3A; 13C, R2 < 0.001, P = 0.995; Supporting naturally associated with I. palifera for fixing inorganic Information Fig. S3B). In order to more accurately reveal carbon and ammonium and passing these newly assimi- the mechanisms driving the differences in metabolic rates lated nutrients to their coral host cells at local ambient between Symbiodinium populations, future isotopic label- temperature (24°C). These findings strengthen the evi- ling studies in corals should also include determination of dence that, although more temperature-tolerant Symbiodinium growth rates and population doubling (LaJeunesse et al., 2009; Oliver and Palumbi, 2009), times. clade D symbionts may be less metabolically efficient Nevertheless, our results are consistent with a recent than clade C members under non-stressful conditions. study based on isotopic incubations and bulk analysis (Baker et al., 2013) as well as with previous data on coral Experimental procedures growth and reproductive outputs (Little et al., 2004; Jones Collection, Symbiodinium genotyping and maintenance and Berkelmans, 2011) linked to symbiont identity. of corals Indeed, although the substrates employed for the 15N labelling were different (nitrate versus ammonium in our Sixty-eight adult colonies of I. palifera were assessed at dif- study), it is remarkable to note that our results match ferent sites adjacent to Heron Island Research Station closely the differences between symbiont clades pub- (231330S 1511540E) before November 2011, when corals were sampled for the labelling experiment. For each of the 68 lished by Baker and colleagues (2013). More specifically, coral colonies, total genomic DNA was extracted from a in our study, Symbiodinium type C3 acquired 30% more single branch using the modified protocol of Wilson and 15N than type D1, while in Baker’s (2013), Symbiodinium colleagues’ (2002). Symbiodinium clades associated with the clade C incorporated 22% more 15N than clade D. In different coral colonies were first identified by PCR-RFLP of congruence with these previous studies, the work pre- the SSU rRNA gene, using a combination of the universal sented here advances the current understanding of eukaryotic ‘ss5’ and Symbiodinium-specific ‘ss3Z’ primers trophic relationships between corals and their symbionts (Rowan and Powers, 1991b) for PCR amplification, and a combination of TaqI and DpnII restriction enzymes for diges- by highlighting reduced fixation and transfer rates of tion of the PCR products (Bythell et al., 1997). The single carbon (fixed via photosynthesis) and nitrogen (fixed via colony hosting a mix of both clade C and clade D assimilation of ammonium dissolved in seawater) for Symbiodinium was excluded from the experiment. Symbiodinium type D1 compared with type C3. Individual coral branches (5–6 cm long) were carefully frag- There is increasing evidence that environmental condi- mented from six I. palifera colonies (three replicates hosting tions interact with symbiont diversity and host factors to C3 and three replicates hosting D1 type symbionts) using co-determine the delicate balance of compounds bone cutters and immediately transferred to flow-through aquaria at Heron Island Research Station. Coral fragments exchanged within the symbiosis, affecting its functioning were then attached to fine nylon fishing line and acclimatized and stability (Mieog et al., 2009; Wiedenmann et al., to the mean local ambient temperature (24°C) for a week in 2012; Beraud et al., 2013). Given the trophic role of the natural seawater in six independent flow-through aquarium coral symbionts, the intriguing question arises as to tanks (60 l; three tank replicates per treatment). Within the whether coral bleaching may in part reflect a shift in the period of the experiment, the sunrise time was 4:45 a.m. and balance of supply versus demand for nitrogen and carbon the sunset time was 6:28 p.m. The ambient light levels were within the host–symbiont interaction (Wooldridge, 2012). controlled throughout the experiment using shade cloths to mimic natural reef flat conditions [noon irradiance ranging In this respect, previous studies highlighted a strong cor- − − from 700 to 1000 μmol photons m 2 S 1 as measured by relation between heterotrophic feeding, nutrient enrich- Odyssey light logger (Dataflow, Christchurch, New Zealand]. ment and bleaching (Grottoli et al., 2006; Ferrier-Pages For each coral fragment used for the isotopic labelling et al., 2010; Wiedenmann et al., 2012). Whilst the genetic experiment and NanoSIMS analysis, a subsample was used diversity of Symbiodinium has attracted particular interest to confirm the identity of Symbiodinium types as C3 and D1 because of its contribution to the thermal tolerance of by amplification of the ITS1–ITS2 region using primers corals (Rowan, 2004; Berkelmans and van Oppen, 2006; ‘ITSintfor 2’ and ‘ITS 2 clamp’ (Sheffield et al., 1989; LaJeunesse and Trench, 2000), followed by screening of Jones et al., 2008) very little is known about the impact of polymorphisms using DGGE (Fig. 1B) on a C.B.S. Scientific different Symbiodinium types on the nutritional status of system (USA) (LaJeunesse, 2001). Subsequently, bands the cnidarian–dinoflagellate symbiosis. Studies based on were excised and sequenced. In addition, identification of the bulk analysis (Baker et al., 2013) recently indicated that, coral host was confirmed for each sample by analyses of the when exposed to seawater enriched in nitrate, skeleton morphology. Symbiodinium type C1 and an undefined Symbiodinium type from clade D displayed different patterns of nutrient Isotopic labelling experiment acquisition and these patterns could be reversed at In order to quantify the assimilation of dissolved inorganic elevated temperature. Our study demonstrates a stronger carbon and ammonium in symbiotic dinoflagellate cells, coral

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology 8 M. Pernice et al. fragments were exposed to isotopically enriched ASW (and the Core Facility of Advanced Isotope Research (University of control non-enriched ASW). Coral fragments were randomly Vienna) in order to quantify the distribution of newly fixed 13C distributed among the treatment and control aquaria (10 l; and 15N within I. palifera. Samples were bombarded with a closed-water system; continuously stirred using one power 16 keV primary ion beam of (1–3 pA) Cs+ focused to a spot head pump for each tank and aerated with air stones, previ- size of about 100–150 nm on the sample surface. Secondary ous experiments demonstrated that sufficient isotopic label- molecular ions 12C12C-, 12C13C-, 12C14N- and 12C15N- were simul- ling was obtained using this outdoor aquaria system). The taneously collected in electron multipliers at a mass resolution temperature of the tanks was maintained at 24°C by placing (ΔM/M, according to Cameca’s definition) of about 9000, experiment and control tanks in flow-through ambient seawa- enough to resolve the 13C14N- and 12C15N- ions from potentially ter. Isotopically enriched ASW was made in accordance with problematic interferences. Charge compensation was not 15 15 Dunn and colleagues’ (2012) with the addition of NH4Cl ( N necessary. Typical images of 35 × 35 μm with 256 × 256 13 13 12 12 - 12 13 - 12 14 - 12 15 - isotopic abundance of 98%, Sigma) and NaH CO3 ( C iso- pixels for C C , C C , C N and C N respectively were topic abundance of 99%, Sigma) to a final concentration of obtained by rastering the primary beam across the sample 5 μM and 2 mM respectively. Control non-enriched ASW con- with a dwell-time of 5 ms. After drift correction, the 13C/12Cor 14 12 15 14 tained NH4Cl and NaH CO3 in the same concentrations as N/ N maps were obtained by taking the ratio between the enriched media. Treatment and control media were changed 12C13C- and 12C12C images or the 12C15N- and 12C14N- images every 12 h throughout the experiment. A subset of coral respectively. Additional samples were analysed with the branches (n = 3) for each colony was randomly removed from NanoSIMS ion probe at the Max Planck Institute for Chemistry the treatment and control tanks at T = 0 and 48 h, respec- (Mainz) using the following secondary molecular ions: 12C-, tively. Each coral branch was chemically fixed for 24 h at 4°C 13C-, 12C14N- and 12C15N-. 13C and 15N enrichments were by immersion in a solution containing 2.5% glutaraldehyde expressed in the delta notation (δ13C and δ15N in ‰) as follows: and 1% formaldehyde in PBS-sucrose buffer (0.1 M phos- ⎛ C ⎞ δ 13C =−mes 110× 3 phate, 0.65 M sucrose, 2.5 mM CaCl2), pH 7.5. Samples ⎝ ⎠ Cnat were then stored in PBS-sucrose buffer (0.1 M phosphate,

0.65 M sucrose, 2.5 mM CaCl2), pH 7.5 at 4°C for 3 days until Where: 13 12 further processing for TEM and NanoSIMS analyses. Cmes is the measured C/ C ratio and Cnat is the natural 13C/12C ratio measured in non-labelled control coral samples.

⎛ N ⎞ Tissue preparation for TEM and NanoSIMS analyses δ 15=−mes × 3 N ⎝ 110⎠ Nnat Coral fragments were embedded in 1.5% agarose prior to 15 14 decalcification at 4°C and pH 7.5 in Sörensen 0.1 M buffer Where Nmes is the measured N/ N ratio and Nnat is the (Sörensen, 1909) containing 0.5 M EDTA, with solution natural 15N/14N ratio measured in non-labelled control coral changed every 12 h until the skeleton was completely dis- samples. Cnat and Nnat were quantified on unlabelled coral solved. After decalcification, coral samples were dissected tissue each day of the NanoSIMS analyses and served as under a stereomicroscope into small pieces containing two or internal standard. Under the analytical conditions applied, three individual polyps from the lateral part of the coral signal ranged from 113 to 829 counts per pixel for 12C12C-, 12 13 - branch, and post-fixed 1 h at RT in 1% OsO4 in Sörensen 10–62 counts per pixel for C C , 270–2195 counts per pixel phosphate buffer (0.1 M). Tissue samples were then dehy- for 12C14N-, and 1–56 counts per pixel for 12C15N-. Each coral drated in an increasing series of ethanol concentrations sample analysed by NanoSIMS was obtained from an indi- (50%, 70%, 90% and 100%) and stored in acetone until resin vidual coral branch (5–6 cm long) that was incubated with embedding. Samples were embedded in Spurr resin, cut to seawater–enriched, either (i) with unlabelled ammonium and 100–120 nm sections using an Ultracut E microtome (Leica bicarbonate (for unlabelled samples) or (ii) with seawater- Microsystems, Australia), mounted onto finder grids for TEM enriched in 15N-ammonium and 13C-bicarbonate (for labelled (ProsciTech, Australia) and counterstained with uranyl samples). acetate 2% (10 min) and Reynold’s lead citrate (10 min). For each Symbiodinium type, two branches, each one coming from a different colony, were analysed using NanoSIMS (as described above). Coral sections were TEM analyses prepared in an area corresponding to the polyp oral tissue. Levels of 15N and 13C enrichment were quantified for individual Regions of interest within the tissue sections were mapped dinoflagellate and host cells by using ROIs defined in and imaged at the Centre for Microscopy and Microanalysis Open_MIMS (http://nrims.harvard.edu/software) by drawing (University of Sydney, Sydney, Australia) using a JEOL the contours of (i) the dinoflagellate cells and (ii) circles of JEM1400 Transmission Electron Microscope (JEOL, Korea) about 8–10 μm diameter, covering host tissue directly on the operated at 80 kV accelerating voltage. Then the TEM grids NanoSIMS maps (as illustrated in Fig. 1D–I). For cell quanti- were mounted on 10 mm diameter aluminium stubs and gold- fication purpose, ROIs were drawn on the NanoSIMS maps of coated for subsequent NanoSIMS analyses. the 12C14N-ions, which allow finding the contours of the dinoflagellate cells without any 15N- or 13C-label present. The NanoSIMS analyses isotopic ratios and enrichments measured for each ROI are reported in Supporting Information TablesS1 and S2. For each The same regions of interest mapped with TEM within the ROI, the standard deviation given directly by the software tissue sections were imaged with the NanoSIMS ion probe at OpenMIMS is indicated.

Cell densities of Symbiodinium size of Symbiodinium cells analysed by NanoSIMS and their incorporation of nitrogen and carbon. Throughout the paper, Three coral branches were used per colony per results were considered significant at the 5% level. Symbiodinium type in order to assess differences in the density of endosymbiotic dinoflagellates within I. palifera. Coral tissue was removed from the samples previously Acknowledgements stored at −80°C by using an airbrush and phosphate buffer solution (0.06 M solution KH2PO4, pH 6.65) (Pernice et al., Isopora palifera samples were collected with permission of 2011). Symbiodinium cells were separated from coral tissue Great Barrier Reef Marine Park (Permit G11/34433.1). This by centrifugation (4000 g for 5 min at 4°C). The pellet con- work was supported in part by European Research Council taining Symbiodinium cells was then washed three times to advanced Grants 246749 (BioCarb) and 294343 (Nitricare), minimize tissue contamination and re-suspended in phos- Swiss National Science Foundation Grant CR23I2_141048 phate buffer solution. The population density of dinoflagel- and by Australian Research Council CE0561435 and lates was determined using a SEDGEWICK rafter cell 550 LP110100566 Grants. Dr. Christophe Kopp and Dr. Andrea haemocytometer (ProSciTech S8050) by counting and aver- Nicastro are thanked for insightful comments on the manu- aging the number of dinoflagellate cells present in 10 script and assistance in statistical analyses, respectively. The subsamples. Dinoflagellate density was then normalized to authors have no conflict of interest to declare. the skeletal surface area (cm2) of each coral branch by using the modified foil wrapping technique (Marsh, 1970). References

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Sampayo, E.M., Franceschinis, L., Hoegh-Guldberg, O.V.E., Fig. S3. Least square regressions between apparent diam- and Dove, S. (2007) Niche partitioning of closely related eter of dinoflagellate cells and 15N (A) and 13C (B) enrichment. symbiotic dinoflagellates. Mol Ecol 16: 3721–3733. The linear models were checked by an F-test and did not Sampayo, E.M., Ridgway, T., Bongaerts, P., and Hoegh- display any significant correlation (15N, R2 = 0.02, P = 0.742 Guldberg, O. (2008) Bleaching susceptibility and mortality Supporting Information Fig. S3A; 13C, R2 < 0.001, P = 0.995; of corals are determined by fine-scale differences in sym- Supporting Information Fig. S3B). The blue lines indicate biont type. Proc Natl Acad Sci USA 105: 10444–10449. 95% confidence interval predicting the distribution of esti- Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. mates of the present population of symbiotic dinoflagellate, (1989) Attachment of a 40-base-pair G + C-rich sequence the brown lines indicate prediction interval, which forecast the (GC-clamp) to genomic DNA fragments by the polymerase distribution of future observations. chain reaction results in improved detection of single-base Fig. S4. Frequency distribution of P-value for t-tests compar- changes. Proc Natl Acad Sci USA 86: 232–236. ing corals associated with Symbiodinium type C3 or D1. The Sörensen, S.P.L. (1909) EΓgänzung zu der Abhandlung: distribution of P-value for t-tests comparing the two groups of Enzymstudien II: Über die Messung und die Bedeutung corals is indicated after resampling for: der Wasserstoffionenkonzentration bei enzymatischen A. Enrichment in 15N in dinoflagellate cells. Prozessen. Biochem Zeit 22: 352–356. B. Enrichment in 13C in dinoflagellate cells. Stat, M., Morris, E., and Gates, R.D. (2008) Functional diver- C. Enrichment in 15N in coral tissue. sity in coral-dinoflagellate symbiosis. Proc Natl Acad Sci D. Enrichment in 13C in coral tissue. USA 105: 9256–9261. Resampling was done 100 000 times. Wallace, C.C., Done, B.J., and Muir, P.R. (2012) Revision Table S1. Increase in 15N/14N and 13C/12C ratio of individual and Catalogue of Worldwide Staghorn Corals Acropora dinoflagellate symbionts within the reef-building coral Isopora and Isopora (Scleractina: Acroporidae) in the Museum of palifera during the experiment. Regions of interest (ROI) refer Tropical Queensland. Brisbane, Australia: Queensland to cells analysed and marked in the respective figure. Museum. Example: Fig. 1H, ROI 1 is Fig. 1, panel H, dinoflagellate 1. Wang, J.T., and Douglas, A.E. (1999) Essential amino acid For each cell the standard deviation given directly by the synthesis and nitrogen recycling in an alga–invertebrate software OpenMIMS is indicated. The high values of standard symbiosis. Mar Biol 135: 219–222. deviations indicate a strong spatial heterogeneity of C and N Wiedenmann, J., D’Angelo, C., Smith, E.G., Hunt, A.N., enrichment, related to specific subcellular structures. *Cells Legiret, F.-E., Postle, A.D., and Achterberg, E.P. (2012) not shown in figures. Nutrient enrichment can increase the susceptibility of reef Table S2. Increase in 15N/14N and 13C/12C ratio in Isopora corals to bleaching. Nat Clim Change 3: 160–164. palifera tissue during the experiment. Regions of interest Wilson, K., Li, Y., Whan, V., Lehnert, S., Byrne, K., Moore, S., (ROI) refer to circles of about 8–10 μm diameter, covering the et al. (2002) Genetic mapping of the black tiger shrimp host tissue in the direct proximity of the symbionts, analysed Penaeus monodon with amplified fragment length polymor- and marked in the respective figure. Example: Fig. 1H, ROI 1 phism. Aquaculture 204: 297–309. is Fig. 1, panel H, circle 1. For each cell the standard devia- Wooldridge, S.A. (2012) Breakdown of the coral-algae sym- tion given directly by the software OpenMIMS is indicated. biosis: towards formalising a linkage between warm-water The high values of standard deviations indicate a strong bleaching thresholds and the growth rate of the intracellular spatial heterogeneity of C and N enrichment, related to spe- zooxanthellae. Biogeosci Discuss 9: 8111–8139. cific subcellular structures. *Cells not shown in figures.