Abstract
Marine organisms have to cope with increasing CO2 partial pressures and decreasing pH in the oceans. We elucidated the impacts of an 8-week acclimation period to four seawater pCO2 treatments (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 μatm) on mantle gene expression patterns in the blue mussel Mytilus edulis from the Baltic Sea. Based on the M. edulis mantle tissue transcriptome, the expression of several genes involved in metabolism, calcification and stress responses was assessed in the outer (marginal and pallial zone) and the inner mantle tissues (central zone) using quantitative real-time PCR. The expression of genes involved in energy and protein metabolism (F-ATPase, hexokinase and elongation factor alpha) was strongly affected by acclimation to moderately elevated CO2 partial pressures. Expression of a chitinase, potentially important for the calcification process, was strongly depressed (maximum ninefold), correlating with a linear decrease in shell growth observed in the experimental animals. Interestingly, shell matrix protein candidate genes were less affected by CO2 in both tissues. A compensatory process toward enhanced shell protection is indicated by a massive increase in the expression of tyrosinase, a gene involved in periostracum formation (maximum 220-fold). Using correlation matrices and a force-directed layout network graph, we were able to uncover possible underlying regulatory networks and the connections between different pathways, thereby providing a molecular basis of observed changes in animal physiology in response to ocean acidification.
Similar content being viewed by others
References
Addadi L, Joester D, Nudelman F, Weiner S (2006) Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chem Eur J 12:980–987
Addadi L, Politi Y, Nudelman F, Weiner S (2008) Biomineralization design strategies and mechanisms of mineral formation: operating at the edge of instability. In: Novoa JJ, Braga D, Addadi L (eds) Engineering of crystalline materials properties state-of-the-art in modeling, design and applications. NATO Science for peace and security series B: physics and biophysics, Springer, Berlin, pp 1–15
Almeida MJ, Machado J, Coelho MAV, Soares-da-Silva P, Coimbra J (1998) L-3,4-Dihydroxyphenylalanine (L-DOPA) secreted by oyster (Crassostrea gigas) mantle cells: function aspects. Comp Biochem Physiol B 120:709–713
Andersen SO (2010) Insect cuticular slerotization: a review. Insect Biochem Molec 40:166–178
Beniash E, Addadi L, Weiner S (1999) Cellular control over spicule formation in sea urchin embryos: a structural approach. J Struct Biol 125:50–62
Beniash E, Ivanina A, Lieb NS, Kurochkin I, Sokolova IM (2010) Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica. Mar Ecol Prog Ser 419:95–108
Bubel A (1973) An electron-microscope study of periostracum formation in some marine bivalves. II. The cells lining the periostracal groove. Mar Biol 20:222–234
Chapman RW, Mancia A, Beal M, Veloso A, Rathburn C, Blair A, Holland AF, Warr GW, Didinato G, Sokolova IM, Wirth EF, Duffy E, Sanger D (2011) The transcriptomic response of the eastern oyster, Crassostrea virginica, to environmental conditions. Mol Ecol doi. doi:10.1111/j.1365-294X.2011.05018.x
Charlet M, Chernysh S, Philippe H, Hetru C, Hoffmann JA, Bulet P (1996) Innate immunity—isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis. J Biol Chem 271(36):21808–21813
Clark GR II (1976) Shell growth in the marine environment: approaches to the problem of marginal calcification. Amer Zool 16:617–626
Cox AG, Winterbourn CC, Hampton MB (2010) Mitochondrial peroxiredoxin involvement in antioxidant defence and redox signalling. Biochem J 245:313–325
de la Fuente A, Bing N, Hoeschele I, Mendes P (2004) Discovery of meaningful associations in genomic data using partial correlation coefficients. Bioinformatics 20(18):3565–3574
Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO2 problem. Annu Rev Mar Sci 1:169–192
Ehrlich H (2010) Chitin and collagen as universal and alternative templates in biomineralization. Int Geol Rev 52(7–8):661–699
Ellington WR (1993) Studies of intracellular pH regulation in cardiac myocytes from the marine bivalve mollusk, Mercenaria campechiensis. Biol Bull 184:209–215
Endo H, Persson P, Watanabe T (2000) Molecular cloning of the Crustacean DD4 cDNA encoding a Ca2+ - binding protein. Biochem Biophys Res Commun 276:286–291
Falini G, Albeck S, Weiner S, Addadi L (1996) Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 271:67–69
Falini G, Weiner S, Addadi L (2003) Chitin-silk fibroin interactions: relevance to calcium carbonate formation in invertebrates. Calcif Tissue Int 72:548–554
Feely RA, Alin SR, Newton J, Sabine CL, Warner M, Devol A, Krembs C, Maloy C (2010) The combined effects of ocean acidification, mixing, and respiration on pH and carbonate saturation in an urbanized estuary. Estuar Coast Shelf S 88:442–449
Hand SC, Hardewig I (1996) Downregulation of cellular metabolism during environmental stress: mechanisms and implications. Annu Rev Physiol 58:539–563
Harper EM (1997) The molluscan periostracum: an important constraint in bivalve evolution. Palaeontology 40(1):71–97
Hattan SJ, Laue TM, Chasteen ND (2001) Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis. J Biol Chem 276(6):4461–4468
Heinemann A, Fietzke J, Melzner F, Böhm F, Thomsen J, Garbe-Schönberg D, Eisenhauer A (2011) Conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: acid-base status, trace elements and δ11B. Geochem Geophys Geosyst. doi:10.1029/2011GC003790
Itoi S, Kinoshita S, Kikuchi K, Watabe S (2003) Changes of carp FOF1-ATPase in association with temperature acclimation. Am J Physiol Integr Comp Physiol 248:R153–R163
Jacob DE, Wirth R, Soldati AL, Wehrmeister U, Schreiber A (2011) Amorphous calcium carbonate in the shells of adult Unionoida. J Struct Biol 173:241–249
Jankowsky E (2011) RNA helicases at work: binding and rearranging. Trend Biochem Sci 36(1):19–29
Kikuchi G, Motokawa Y, Yoshida T, Hiraga K (2008) Glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia. Proc Jpn Acad Ser B 84:246–263
Kramer KJ, Muthukrishnan S (2005) Chitin metabolism in insects. In: Gilbert LI, Iatrou K, Gill S (eds) Comprehensive molecular insect science. Vol. 4, biochemistry and molecular biology, chapter 3. Elsevier Press, Oxford, pp 111–144
Lannig G, Eilers S, Pörtner HO, Sokolova IM, Bock C (2010) Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas – changes in metabolic pathways and thermal response. Mar Drugs 8:2318–2339
Levi-Kalisman Y, Falini G, Addadi L, Weiner S (2001) Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM. J Struct Biol 135:8–17
Li S, Xie L, Zhang C, Zhang Y, Gu M, Zhang R (2004) Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata). Comp Biochem Physiol B 138:235–243
Lischka S, Büdenbender J, Boxhammer T, Riebesell U (2011) Impact of ocean acidifaction and elevated temperatures on early juveniles of the polar shelled pteropod Limacina helicina: mortality, shell degradation, and shell growth. Biogeosciences 8:919–932
Machado J, Coimbra J, Castilho F, Sã C (1990) Effects of diflubenzuron on shell formation of the freshwater clam, Anodonta cygnea. Arch Environ Contam Toxicol 19:35–39
Marie B, Zanella-Cléon I, Corneillat M, Becchi M, Alcaraz G, Plasseraud L, Luquet G, Marin F (2011a) Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus. FEBS J 278:2117–2130
Marie B, Le Roy N, Zanella-Cléon I, Becchi M, Marin F (2011b) Molecular evolution of mollusc shell proteins: insights from proteomic analysis of the edible mussel Mytilus. J Mol Evol doi: 10.1007/s00239-011-9451-6
Marin F, Luquet G, Marie B, Medakovic D (2008) Molluscan shell proteins: primary structure, origin and evolution. Curr Top Dev Biol 80:209–276
Masui DC, Furriel RPM, McNamara JC, Mantelatto FLM, Leone FA (2002) Modulation by ammonium ions of gill microsomal (Na+, K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion. Comp Biochem Physiol C 132:471–482
Masui DC, Mantelatto FLM, McNamara JC, Furriel RPM, Leone FA (2009) Na+, K+-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: novel perspectives on ammonia excretion. Comp Biochem Physiol A 153:141–148
Mayzaud P, Conover RJ (1988) O:N atomic ratio as a tool to describe zooplankton metabolism. Mar Ecol Prog Ser 45:289–302
Melzner F, Stange P, Trübenbach K, Thomsen J, Casties I, Panknin U, Gorb SN, Gutowska MA (2011) Food supply and seawater pCO2 impact calcification and internal shell dissolution in the blue mussel Mytilus edulis. PLoS ONE 6(9):e24223. doi:10.1371/journal.pone.0024223
Merzendorfer H, Zimoch L (2003) Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol 206:4393–4412
Michaelidis B, Ouzounis C, Paleras A, Pörtner HO (2005) Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis. Mar Ecol Prog Ser 293:109–118
Michaelidis B, Spring A, Pörtner HO (2007) Effects of long-term acclimation to environmental hypercapnia on extracellular acid-base status and metabolic capacity in Mediterranean fish Sparus aurata. Mar Biol 150:1417–1429
Mingliang Z, Jianguang F, Jihong Z, Bin L, Shengmin R, Yuze M, Yaping G (2011) Effect of marine acidification on calcification and respiration of Chlamys farreri. J Shellfish Res 30(2):267–271
Misogianes MJ, Chasteen ND (1979) A chemical and spectral characterization of the extrapallial fluid of Mytilus edulis. Anal Biochem 100:324–334
Mitta G, Vandenbulcke F, Hubert F, Salzet M, Roch P (2000) Involvement of mytilins in mussel antimicrobial defense. J Biol Chem 275(17):12954–12962
Nagai K, Yano M, Morimoto K, Miyamoto H (2007) Tyrosinase localization in mollusc shells. Comp Biochem Physiol B 146:207–214
Olivares C, Solano F (2009) New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins. Pigment Cell Melanoma Res 22(6):750–760
Palmer AR (1983) Relative cost of producing skeletal organic matrix versus calcification: evidence from marine gastropods. Mar Biol 75:287–292
Palmer AR (1992) Calcification in marine molluscs: How costly is it? Proc Natl Acad Sci USA 89:1379–1382
Philipp EER, Kraemer L, Melzner F, Poustka AJ, Thieme S, et al. (2012) Massively parallel RNA sequencing identifies a complex immune gene repertoire in the lophotrochozoan Mytilus edulis. PLoS ONE 7(3):e33091. doi:10.1371/journal.pone.0033091
Politi Y, Arad T, Klein E, Weiner S, Addadi L (2004) Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase. Science 306:1161–1164
Pörtner HO, Bock C, Reipschläger A (2000) Modulation of the cost of pHi regulation during metabolic depression: a 31P-NMR study in invertebrate (Sipunculus nudus) isolated muscle. J Exp Biol 203:2128–2417
Pörtner HO, Langenbuch M, Reipschläger A (2004) Biological impact of elevated ocean CO2 concentrations: lessons from animal physiology and earth history. J Oceanogr 60:705–718
Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology 37(12):1131–1134
Riis B, Rattan SI, Clark BF, Merrick WC (1990) Eukaryotic protein elongation factors. Trends Biochem Sci 11:420–424
Rodolfo-Metalpa R, Houlbrèque F, Tambutté É, Biosson F, Baggini C, Patti FP, Jeffree R, Fine M, Foggo A, Gattuso J-P, Hall-Spencer JM (2011) Coral and mollusc resistance to ocean acidification adversely affected by warming. Nature Clim Change 1:308–312
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425
Schönitzer V, Weiss IM (2007) The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z. BMC Struct Biol 7:71. doi:10.1186/1472-6807-7-71
Soares-da-Silva IM, Almeida MJ, Serrão PM, Coelho MA, Machado J (1998) L-3,4-dihydroxyphenylalanine (L-DOPA) in Anodonta cygnea: variation with acidosis. Comp Biochem Physiol A 120:463–468
Suzuki M, Sakuda S, Nagasawa H (2007) Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata. Biosci Biotechnol Biochem 71(7):1735–1744
Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, Nagasawa H (2009) An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–1390
Taylor JD, Kennedy WJ (1969) The influence of the periostracum on the shell structure of bivalve molluscs. Calc Tiss Res 3:274–283
R Development Core Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. URL http://www.R-project.org/
Thomsen J, Melzner F (2010) Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis. Mar Biol 157(12):2667–2676
Thomsen J, Gutowska MA, Saphörster J, Heinemann A, Trübenbach K, Fietzke J, Hiebenthal C, Eisenhauer A, Körtzinger A, Wahl M, Melzner F (2010) Calcifying invertebrates succeed in a naturally CO2-rich coastal habitat but are threatened by high levels of future acidification. Biogeosciences 7:3879–3891
Todgham AE, Hofmann GE (2009) Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification. J Exp Biol 212:2579–2594
Tomanek L, Zuzow MJ, Ivanina AV, Beniash E, Sokolova IM (2011) Proteomic response to elevated P CO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress. J Exp Biol 214:1836–1844
Tunnicliffe V, Davies KTA, Butterfield DA, Embley RW, Rose JM, Chadwick WW Jr (2009) Survival of mussels in extremely acidic waters on a submarine volcano. Nat Geosci 2(5):344–348
Uozumi S, Suzuki S (1979) “Organic membrane-shell” and initial calcification in shell regeneration. J Fac Sci Hokkaido Univ Ser IV 19(1–2):37–74
Waite JH (1983) Quinone-tanned scleroproteins. In: Hochachka PW (ed) The mollusca, Metabolic biochemistry and molecular biomechanics, vol 1. Academic Press, New York, pp 467–504
Waite JH, Andersen SO (1978) 3,4-Dihydroxyphenylalanine in an insoluble shell protein of Mytilus edulis. Biochim Biophys Acta 541:107–114
Waite JH, Wilbur KM (1976) Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn. J Exp Zool 195:359–368
Waite JH, Saleuddin ASM, Andersen SO (1979) Periostracin—a soluble precursor of sclerotized periostracum in Mytilus edulis L. J Comp Physiol 130:301–307
Waldbusser GG, Voight EP, Bergschneider H, Green MA, Newell RIE (2011) Biocalcification in the Eastern Oyster (Crassostrea virginica) in relation to long-term trends in Chesapeake Bay pH. Estuar Coast 34:221–231
Wang L, Liu Y, Wang W-N, Mai W-J, Xin Y, Zhou J, He W-Y, Wang A-L, Sun R-Y (2011) Molecular characterization and expression analysis of elongation factors 1A and 2 from the Pacific white shrimp Litopenaeus vannamei. Mol Biol Rep 38:2167–2178
Weihrauch D, Morris S, Towle DW (2004) Ammonia excretion in aquatic and terrestrial crabs. J Exp Biol 207:4491–4504
Weiner S, Addadi L (2011) Crystallization pathways in biomineralization. Annu Rev Mater Res 41:21–40
Weiss IM (2010) Jewels in the pearl. ChemBioChem 11:297–300
Weiss IM, Schönitzer V (2006) The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis. J Struct Biol 153:264–277
Weiss IM, Tuross N, Addadi L, Weiner S (2002) Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite. J Exp Zool 293:478–491
Yin Y, Huang J, Paine ML, Reinhold VN, Chasteen ND (2005) Structural characterization of the major extrapallial fluid protein of the mollusc Mytilus edulis: implications for function. Biochem 44:10720–10731
Yotsumoto N, Takeoka M, Yokoyama M (2010) Tail-suspended mice lacking calponin H1 experience decreased bone loss. Tohoku J Exp Med 221:221–227
Zaba BN (1981) Glycogenolytic pathways in the mantle tissue of Mytilus edulis L. Mar Biol Lett 2:67–74
Zange J, Pörtner HO, Jans AWH, Grieshaber MK (1990) The intracellular pH of a molluscan smooth muscle during a contraction-catch-relaxation cycle estimated by the distribution of [14C]DMO and by 31P-NMR spectroscopy. J Exp Biol 150:81–93
Zanker A (1984) Detection of outliers by means of Nalimov’s test. Chem Eng 91(16):74
Acknowledgments
We would like to thank Ulrike Panknin and Katja Trübenbach for tissue sampling, as well as UP for her help with RNA isolation and cDNA synthesis. This study is part of the German joint project “Biological Impact of Ocean Acidification (BIOACID)” (3.1.3) and funded by the Federal Ministry of Education and Research (BMBF, FKZ 03F0608B). It is a contribution to the PACES research program (work package 1.6) of the Alfred Wegener Institute funded by the Helmholtz Association. The Excellence Cluster ‘Future Ocean’ awarded grants to F.M., E.P., M.A.G. and P.R.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by S. Dupont.
Electronic supplementary material
Below is the link to the electronic supplementary material.
227_2012_1930_MOESM1_ESM.pdf
Fig. 1 supplement Correlation network of genes in a) inner mantle tissue and b) outer mantle tissue based on the R-script after removing pCO2 dependent correlations. Line width of vertices corresponds to correlation. Genes were set as correlated when Spearman’s |ρ| ≥ 0.5. (PDF 299 kb)
227_2012_1930_MOESM2_ESM.pdf
Fig. 2 supplement Comparison of Copper-binding site CuA from several tyrosinases and one hemocyanin. Essential histidines, responsible for copper-binding are denoted with arrows. MeTYRs: Mytilus edulis putative tyrosinases from our transcriptome, LgTYR1: Lottia gigantea tyrosinase (ID: 166196 on JGI), PmTYR: Pinctada maxima tyrosinase (GH280185), PfOT47: P. fucata tyrosinase (DQ112679), PfTYR2: P. fucata tyrosinase (AB254133), PfTYR1: P. fucata tyrosinase (AB254132), CfTYR: Clamys farreri tyrosinase (ACF25906.1), LgTYR2: L. gigantea tyrosinase (ID: 160808 on JGI), IaTYR1: Illex argentinus tyrosinase (AB107880.1), IaTYR2: I. argentinus tyrosinase (AB107881.1), SoTYR: Sepia officinalis tyrosinase (AJ297474.1), HtHem: Haliotis tuberculata hemocyanin (CAC82192.1), GgTYR: Gallus gallus tyrosinase (AAB36375.1), MmTYR: Mus musculus tyrosinase (P11344.3), HsTYR: Homo sapiens (P14679.3). (PDF 1116 kb)
227_2012_1930_MOESM3_ESM.pdf
Fig. 3 supplement Phylogenetic tree of protein sequences including CuA sites of several tyrosinases and one hemocyanin. The length of the used sequences varied between 77 and 111 amino acids. The tree was constructed with the neighbor-joining method (Saitou and Nei 1987). Distances between the roots were un-corrected. For abbreviations see Fig. 2 supplement. (PDF 418 kb)
Rights and permissions
About this article
Cite this article
Hüning, A.K., Melzner, F., Thomsen, J. et al. Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism. Mar Biol 160, 1845–1861 (2013). https://doi.org/10.1007/s00227-012-1930-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00227-012-1930-9