EFFECT OF OCEAN ACIDIFICATION ON MARINE PHYTOPLANKTON Debora Iglesias-Rodriguez

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1 EFFECT OF OCEAN ACIDIFICATION ON MARINE PHYTOPLANKTON Debora Iglesias-Rodriguez

2 Changes in physiology biogeochemistry O 2 photosynthesis CO 2

3 OCEAN ACIDIFICATION Time series of atmospheric CO 2 Mauna Loa data: Dr. Pieter Tans, NOAA/ESRL ( HOTS/Aloha data: Dr. David Karl, University of Hawaii ( (modified after Feely, 2008).

Chemistry of ocean acidification (CO 2 + H 2 O H 2 CO 3 H + + HCO 3-2H + + CO 3 2- ) Photosynthesis CO 2 HCO 3 - Calcification CO 3 2-1.

4 Chemistry of ocean acidification (CO 2 + H 2 O H 2 CO 3 H + + HCO 3-2H + + CO 3 2- ) Photosynthesis CO 2 HCO 3 - Calcification CO [CO 2 ] increase increase in photosynthesis 2. [HCO 3- ] increase in photosynthesis increase in calcification 3. [CO 3 2 ] decrease in saturation state of CaCO 3 : = ([CO 3 2 ] [Ca +2 ])/K sp decrease in calcification 4. ph

5 Physiological Response Major group # species studied Response to increasing CO 2 a b c d References Calcification Photosynthesis 1 Coccolithophores Planktonic Foraminifera Nitrogen Fixation Molluscs Echinoderms Corals Coralline red algae Arthropods Coccolithophores 2 Prokaryotes Seagrasses Diatoms Cyanobacteria Reproduction, development, metabolic health Molluscs Echinoderms Arthropods * ,34,35,44,55,61,118,128,162/ 57,83,140/57,118,126/5,104,105 12,13,145/-/-/- 64,67,76,110,121/-/-/- 27,43,114/66,157/-/131 7,32,63,72,73,86,89,94,101,102, 107/-/132 /- 98/-/-/131 57/5,57,83,128,140/57/- 3,100/131/-/- -/59,80/59/- -/85,123,142,161/-/- -/148,150/-/- 33/4,59,80,97,109/-/- 1,37,76,113,124/-/78/- 6,43,99/-/-/- 112,154,158/-/3/- Iglesias-Rodriguez et al., 2011

6 Why is ph important? ph affects enzyme activity, substrate speciation, charge on macromolecules that control biomineralization, crystal size, structure and crystallography Need to control ph in intracellular compartments: - Seawater: Intracellular compartments: 7.1 and Calcification reservoir: >>8.0 Unknowns: Synergistic effects of CO 2 and temperature? Factors controling adaptation to ocean acidification (e.g. mineralogy, metabolic versatility, genotypic versatility)? Threshold levels (tipping points) at the organism and community levels? Socio-economics of ocean acidification?

Effect of OA on photosynthesis and calcification External medium (~8.1) H + Cytoplasm (7.1-7.8) H + H + Calcifying reservoir (>8.1) Ca 2+ Ca 2+ Ca2+ * CaCO 3 CO 2-3 CO 2-? CO 2-3? 3 H +?

7 Effect of OA on photosynthesis and calcification External medium (~8.1) H + Cytoplasm ( ) H + H + Calcifying reservoir (>8.1) Ca 2+ Ca 2+ Ca2+ * CaCO 3 CO 2-3 CO 2-? CO 2-3? 3 H +? HCO - 3 HCO - HCO H + 3 CO 2 CA? CA CO 2 CA? CA CO 2 + H 2 O Plastid (photosynthesis) CO 2 CO 2 CO 2 CH 2 O + O 2 H 2 O H 2 O Coupling between photosynthesis and calcification: (Buitenhuis et al., 1999, J. Phycol. 35, 949) Decoupling between photosynthesis and calcification: (Buitenhuis et al., 2001, GBC 15, 57722)

8 Sources and sinks of CO 2 Different methods to manipulate seawater ph (bubbling with CO 2 -enriched air, acid addition) yield different % of inorganic carbon species (Iglesias-Rodriguez et al., 2008a; Hurd et al., 2009; Gattuso et al., 2010) Comparison of these methods reveals comparable responses in some coccolithophore species (Shi et al., 2009; Hoppe et al., 2011). To what extent does a drop in ph affect the conditions in the calcification reservoir in intimate contact with the external medium (e.g., corals) or isolated from seawater in a compartment within the cytosol (e.g., coccolithophores, foraminifera)? Iglesias-Rodriguez et al., 2008, Science 322, 1466c. Most OA studies report calcification responses as a function of CO 2 levels in seawater (which are comparable across different ph manipulations) but not HCO 3-, a reported carbon source for calcification that can vary significantly between different manipulations of ph.

9 Calcification CO 3 2- is the carbon substrate for calcification (Ca 2+ + CO 3 2- CaCO 3 ) Increases of CO 2 and particularly HCO 3 - outside the calcification compartment are known to promote calcification in many calcifying organisms (e.g., Marubini and Thake, 1999; Bentov et al., 2009; Raven, 2011). Under current ocean conditions, the concentration of bicarbonate ions represents >85% of the total dissolved inorganic carbon and yet the effect of changes in HCO 3 - are ignored in ocean acidification studies with the focus remaining almost exclusively on ph, CO 2 and CO 3 2-.

10 Iglesias-Rodriguez et al. (in prep) Calcification

11 Calcification We need to know: What molecules are involved in calcification (e.g., chemistry of coccolithogenesis)? How is CaCO 3 formed, delivered and stored? What is the purpose of calcification? What is the source of carbon for calcification? Iglesias-Rodriguez et al. (in prep)

Homologues of the coccolithophore H + channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles

12 Adaptation to ocean acidification via transmembrane H + electrochemistry Taylor et al., PLoS Biology, 2011 Plasma membrane voltage-gated H + conductance in Coccolithus pelagicus and E. huxleyi similar to those in metazoans. Homologues of the coccolithophore H + channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the Hv1 class of proteins. Novel cellular role for voltage gated H + channels and provide mechanistic insight into biomineralisation by establishing a direct link between ph homeostasis and calcification.

Variable effect of ocean acidification on

Emiliania huxleyi Iglesias-Rodriguez et al.

huxleyi 13 10 24 nd 80 -cal 5.3 5.2 3.8 2.

13 Variable effect of ocean acidification on calcification Langer et al. 2006, Geochem. Geophys. Geosys Calcidiscus leptoporus Langer et al. 2009, Biogeosc Discus 6, Emiliania huxleyi Shi et al. 2009, Biogeosciences Discussions 6, Emiliania huxleyi Iglesias-Rodriguez et al. 2008, Science 320, Emiliania huxleyi nd 80 -cal Riebesell et al. 2000, Nature 407, Emiliania huxleyi Gephyrocapsa oceanica Present-day CO 2 Elevated CO 2

14 Biogeochemical implications PIC:POC > 1.5 source PIC:POC < 1.5 sink M. Frankignoulle, C. Canon, J.-P. Gattuso, Limnol Oceanogr 39, , (1994). Riebesell et al., Nature, Langer et al., et al., G 3, Iglesias-Rodriguez et al., Science, 2008.

15 Functional inter and intraspecific diversity Blanco-Ameijeiras, S., Lebrato, M., Stoll, H.M., Iglesias-Rodriguez, M.D., Mendez-Vicente, A., Sett, S., Müller, M.N., Oschlies, A., Schulz, K.G. (in review). Blanco-Ameijeiras, 2010.

10(10):R114. Proteomics data (Jones et al., 2010, Mar. Biotechnol.

16 Intraspecific diversity and life cycle Functional differences - transcriptomic data Von Dassow et al., 2009, Genome Biol. 10(10):R114. Proteomics data (Jones et al., 2010, Mar. Biotechnol.): Photosynthesis unaffected (no change in expression of photosystem II protein PsbQ)

17 Where do the uncertainties come from? Timing - life cycle Different life cycle stages and different stages of population/bloom development has different biogeochemical signatures m - PIC - POC Cell number m - PIC - POC m - PIC - POC Bloom progress (nutrients)

18 How genomic and post-genomic technologies can be utilized within ocean acidification research

19 Ontological analysis NZEH protein process NZEH protein location Metabolism N/A Organelle organisation Photosynthesis Stress response Translation Transport 8 29 Cytoplasm Cytoskeleton Membrane N/A Nucleus Plastid CCMP1516 protein process CCMP1516 protein location Metabolism N/A Photosynthesis Stress response Transport 2 7 Cytoplasm Cytoskeleton Golgi Membrane N/A Nucleus Plastid CCMP371 protein process CCMP371 protein location Metabolism N/A Organelle organisation Photosynthesis Signal transduction Stress response Translation Transport Cytoplasm Golgi Membrane N/A Nucleus Plastid Ribosome

20 385 or 1300 ppm CO 2 2.4L 14L 14L 2-3 days in exponential 5/6 generations 3/4 generations Sampling: Sampling: Sampling: Before adding culture Before transfer to 14L culture Before starting bubbling Before adding culture Before transfer to 14L culture Before starting bubbling Before adding culture Final harvest SEM Nutrients ph Salinity Temperature DIC/Alk Nutrient ph Salinity Temperature DIC/Alk Nutrient ph Salinity Temperature DIC/Alk Nutrient ph Salinity Temperature PIC POC SEM FRRF DIC/Alk Nutrient ph Salinity Temperature DIC/Alk Nutrient ph Salinity Temperature DIC/Alk Nutrient ph Salinity Temperature PIC POC SEM FRRF Proteins for itraq

Cells lysed Proteins Four current day samples Four 4x current day Proteolysis with site specific enzyme Peptides tagged with itraq reagents (8 tags) Samples combined and analysed

21 Cells lysed Proteins Four current day samples Four 4x current day Proteolysis with site specific enzyme Peptides tagged with itraq reagents (8 tags) Samples combined and analysed by 2D LC-MS/MS Tag free quantification using 2D LC-MS/MS and E MS approach What are cells doing under high CO 2 (low ph?) Analysis of trends using BUDAPEST EST and genome searches

22 Proteomic results Cells of Emiliania huxleyi (strain NZEH) incubated at ~1300 p.p.m.v. CO 2 possess more cellular particulate organic carbon (POC), organic nitrogen (PON) and inorganic carbon (PIC, CaCO 3 ) whilst simultaneously exhibiting a significantly lower growth rate compared to those grown under current pco 2 conditions. No difference in calcification rate was observed. Combining Jones et al 2011 with CO 2 experiment dataset: 184 experimentally validated proteins (using high-throughput MS/MS) within the E. huxleyi genome (BUDAPEST analysis using database of ~130,000 Ehux ESTs). When compiled by nomenclature: 44 proteins of which only three were down regulated: Histones H4, H2A and a chloroplastic 30S ribosomal protein showed reduced expression under high CO 2, which may reflect the lower growth rate exhibited by these cells. All quantified AND non-quantified proteins (44) (i.e. those that did not get an itraq tag): 110 identifications (tagging success ~40%) 21 identifications from the taxonomically restricted database search and 98 from EST analysis with only 8 overlaps by nomenclature between the two datasets: 37 of these identifications are no BLAST hits 9 are predicted This means that 46 are unidentifiable (41.8%) (calcification process?) Emiliania huxleyi NZEH may possess a plastic biochemical response to low ph

23 Where do uncertainties come from? Huge genetic (and functional) diversity within species (Iglesias- Rodriguez 2002, 2006) Unknown sources of carbon for calcification (HCO 3- /CO 3 2- ) Short term/abrupt manipulations versus long-term experiments: few studies involving long-term adaptation (Collins and Bell, 2004; Barrick et al., 2009) but these experiments do not address the evolutionary/adaptive mechanisms underlying adaptation over numbers of generations representative of decadal or centennial timescales Lack of knowledge of the calcification process Lab versus field results

24 Deep cold, high CO 2 Approaches

25 Ocean acidification monitoring sites Ocean acidification monitoring sites (coral reefs)

26 Vertical profile of carbon chemistry Photosynthesis (C-sink) Calcification (C-source) 100m 50m CO 3 2- CO m 500m CO 2 CO 3 2- CO 2 CO m Seabed

27 Artificially-induced upwelling in the PAP site: deep-sea fertilization of phytoplankton? Debora Iglesias-Rodriguez, Bethan M. Jones, Sonia Blanco Ameijeiras and Mario Lebrato

28 The Porcupine Abyssal Plain: 49 o N, 16 o W ~ 4840 m water depth

29 Natural upwelling events Questions: 1. How do deep (cold, high CO 2 ) waters affect the physiology of coccolithophores? 2. How does the -Cal (CaCO 3 calcite saturation state) affect growth and calcification in coccolithophores Distribution of the depths of the undersaturated water ( aragonite saturation < 1.0; ph <7.75) on the continental shelf of western North America from Queen Charlotte Sound, Canada to San Gregorio Baja California Sur, Mexico (Feely et al. 2008).

30 Vertical profile of -Cal Suarez Bosche et al., 2011 Vertical distribution of -Cal along the WOCE A16 (from the GLODAP atlas) transect line in the Atlantic Ocean from 0 to 1000 m (top plot) and from 0 to 6000 m (bottom plot) (from Suarez-Bosche, 2011).

NZEH 100m 50m CO 3 2-0 m CO 2 C R 1 R 2 R 3 1000m

31 Artificial upwelling at the PAP site Photosynthesis (C-sink) Calcification (C-source) NZEH 100m 50m CO m CO 2 C R 1 R 2 R m 500m RCC1212 CO 2 CO 3 2- C R 1 R 2 R 3 CO 2 CO m Seabed

32 Inorganic carbon chemistry parameters TA ( mol kg SW -1 ) DIC ( mol kg SW -1 ) ph [HCO 3 - ] ( mol L -1 ) [CO 3 2- ] ( mol L -1 ) [CO 2 ] ( mol L -1 ) -cal CO 2 (p.p.m.v) NZEH 4831m (1.46) (5.21) 7.89 (0.00) (7.56) (2.97) (0.53) 2.97 (0.07) (7.52) Blank NZEH 1010m (4.87) (5.07) 7.90 (0.01) (4.91) (0.13) (0.24) 2.95 (0.00) (16.76) Blank NZEH 508m (17.22) (0.13) (0.04) (8.71) (10.63) (1.79) (0.25) (53.30) Blank NZEH 38m (13.17) (17.99) (0.06) (34.12) (18.79) (2.39) (0.45) (69.79) Blank m (2.50) (2.17) 7.90 (0.01) (3.67) (2.24) (0.46) 2.99 (0.05) (12.75) Blank m (4.84) (3.00) (0.01) (2.46) (1.61) (0.28) (0.04) (9.27) Blank m (8.07) (2.33) (0.02) (8.23) (6.67) (0.77) (0.16) (22.44) Blank m (2.41) (0.96) (0.01) (2.08) (1.71) (0.19) (0.04) (6.88) Blank

33 Cellular growth rate and calcification *

NZEH Coccosphere Coccolith diameter dsl dsw

5000m avg 7,17 3,04 2,32 1,38 0,79 stdev

34 NZEH Coccosphere Coccolith diameter dsl dsw cal caw depth m m m m m Chlmax avg ,09 2,42 1,42 0,88 stdev ,44 0,42 0,24 0,22 n m avg 7,17 3,04 2,32 1,38 0,79 stdev 0,39 0,22 0,26 0,26 0,13 n NZEH 5000 m NZEH Chlorophyl max

Past observations of marine chemistry and calcification Ward, P., 2007.

calcifiers Difficult to distinguish signal from different climate-relevant

35 Past observations of marine chemistry and calcification Ward, P., Uncoupling between carbon chemistry and the abundance and diversity of calcifiers Difficult to distinguish signal from different climate-relevant variables (T, CO 2, CO 3 2-, Mg, Ca) Imperfect analogs for ocean acidification De Vargas et al., 2007

36 Field studies Beaufort et al 2011

37 In conclusion OA generally enhances photosynthetic carbon fixation but there are many calcification responses in marine phytoplankton. Sources of carbon in the calcification process may explain the inter and intraspecific diversity? Proteomic results revealed only three proteins downregulated under elevated CO 2 From the lab to the field: designing smart experiments for evolutionary studies Synergistic approaches are preferable to single process studies to elucidate causes of discrepancies in monofactorial laboratory experiments

38 Co-authors, NOC team and collaborators Univ. Southampton (UK) Bethan Jones Darryl Green Alex Poulton Sam Gibbs David O Connor (CPR, UoS) John Gittins Paul Skipp (CPR, UoS) IFM-GEOMAR Mario Lebrato Sonia Blanco-Ameijeiras University of Dundee John Raven Oxford University (UK) Paul Halloran Ros Rickaby NOAA (USA) Chris Brown Rutgers (USA) Oscar Schofield Paul Falkowski Earth, Ocean & Planetary Sciences University of Wales Cardiff (UK) Elena Colmenero-Hidalgo Karin Boessenkool Ian Hall

39 Emiliania huxleyi CCMP2090 (Non-C) 61/12/04 (Low-C) NZEH (Super-C) Iglesias-Rodriguez et al., Global Biogeochemical Cycles, 2002.

40 Biomineralization What we need to know: What molecules are involved in calcification (e.g., coccolithogenesis)? How is CaCO 3 formed, delivered and stored? What is the purpose of calcification? What is the source of carbon for calcification? Approaches: Gene identification Gene expression analysis Functional and structural protein analysis Protein localization Environmental sample analysis Schematic TEM section of Pleurochrysis carterae (from van der Wal et al. 1983).

41 Carbonic anhydrase? Carbonic anhydrase Ca +2 ATPase Carbonic anhydrase HCO 3 - CO 2 H 2 O

42 Methods Acid/base control Advantages: experimentally easier to handle, inexpensive Disadvantages: it reproduces ph but not the associated chemistry Iglesias-Rodriguez et al., 2008, Science 322, 1466c.

43 How long should my experiment be? How long is a long-term experiment? How many changes can be detected during the acclimation phase? Continuous versus batch approaches Different questions require different approaches Müller et al., 2008.

44 Where do the uncertainties come from? Huge genetic (and functional) diversity within species (Iglesias- Rodriguez 2002, 2006) Unknown sources of carbon for calcification (HCO 3- /CO 3 2- ) Short term/abrupt manipulations versus long-term experiments: few studies involving long-term adaptation (Collins and Bell, 2004; Barrick et al., 2009) but these experiments do not address the evolutionary/adaptive mechanisms underlying adaptation over numbers of generations representative of decadal or centennial timescales Lack of knowledge of the calcification process

45 Evolutionary questions - How will a change in elemental ratios (e.g. from increasing the C:N ratio) affect the biochemical composition of phytoplankton and thus grazing - Effect on size - more CO 2, bigger cells (Iglesias-Rodriguez et al., 2008; Irie et al., 2010) but slower growth - grazing? - Competition (exclusion): effect on relative % functional groups (e.g. calcifiers: silicifiers:nitrogen fixers) on elemental cycling - How fast do eukaryotic phytoplankton evolve in a fastchanging environment (time scales)? - epigenetic changes - gene additions, deletions, etc

46 Vision Continue and expand collaborations with geologists, geochemists, remote sensing experts, proteomics scientists and bioinformaticians. Build a program to study adaptation at different levels of organization (ecological, molecular; regional, global; shortterm, geological time scales) with ongoing monitoring programs (Ocean Sites). Explore creative ways (following proteomics) to relate biological diversity and ecosystem function (biogeochemistry)

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