Andreas Anestis, Antigone Lazou, Hans O. Pörtner and Basile Michaelidis Am J Physiol Regulatory Integrative Comp Physiol 293:911-921, 2007. First published May 23, 2007; doi:10.1152/ajpregu.00124.2007 You might find this additional information useful... This article cites 38 articles, 12 of which you can access free at: http://ajpregu.physiology.org/cgi/content/full/293/2/R911#BIBL This article has been cited by 1 other HighWire hosted article: Metabolic and molecular stress responses of sublittoral bearded horse mussel Modiolus barbatus to warming sea water: implications for vertical zonation A. Anestis, H. O. Portner, A. Lazou and B. Michaelidis J. Exp. Biol., September 1, 2008; 211 (17): 2889-2898. [Abstract] [Full Text] [PDF] Updated information and services including high-resolution figures, can be found at: http://ajpregu.physiology.org/cgi/content/full/293/2/R911 Additional material and information about American Journal of Physiology - Regulatory, Integrative D o and Comparative Physiology can be found at: w n http://www.the-aps.org/publications/ajpregu lo a d e d T his information is current as of February 25, 2009 . from a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 The American Journal of Physiology - Regulatory, Integrative and Comparative Physiology publishes original investigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at http://www.the-aps.org/. AmJPhysiolRegulIntegrCompPhysiol293:R911–R921,2007. FirstpublishedMay23,2007;doi:10.1152/ajpregu.00124.2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature Andreas Anestis,1 Antigone Lazou,1 Hans O. Po¨rtner,2 and Basile Michaelidis1 1LaboratoryofAnimalPhysiology,DepartmentofZoology,SchoolofBiology,FacultyofScience,AristotleUniversityof Thessaloniki,Thessaloniki,Greece;and2Alfred-Wegener-Institutfu¨rPolar-undMeeresforschung,O¨kophysiologiemariner Tiere,Bremerhaven,Germany Submitted20February2007;acceptedinfinalform22May2007 Anestis A, Lazou A, Po¨rtner HO, Michaelidis B. Behavioral, cover an animal’s temperature-dependent oxygen demand. metabolic, and molecular stress responses of marine bivalve Thermal adaptation relies on the capacity of glycolytic and Mytilus galloprovincialisduring long-term acclimation at increas- mitochondrialmetabolismsuchastherespiratorychainandthe ing ambient temperature. Am J Physiol Regul Integr Comp tricarboxylicacidcycle.Thethermalresponsesofsuchfunda- Physiol 293: R911–R921, 2007. First published May 23, 2007; mentalbiochemicalmechanismscontributetodefiningperfor- D doi:10.1152/ajpregu.00124.2007.—Thepresentstudyaimedtodeter- mancelevels,whichareoptimalonlywithinalimitedwindow o w mine the thermal response of the Mediterranean mussel Mytilus ofthermaltolerance.Thelatterdefineshowanimalsexerttheir n galloprovincialis by integrating information from various levels of lo ecologicalfunctionsthroughtheirmodeoflifeandbehavioral a biological organization including behavior, metabolic adjustments, d traits(18–20).Itiswellknown,however,thatmoderatelevels e heatshockproteinexpression,andproteinkinaseactivity.Behavioral d responses were determined by examining the effect of warming on of a stressful factor may only become effective during long- fro valve closure and opening. Metabolic impacts were assessed by termexposure.Thus,long-termstudiesoftheeffectofambient m examiningtheactivityofthekeyglycolyticenzymepyruvatekinase temperatureonseverallevelsofbiologicalorganizationshould ajp (PK).Molecularresponseswereaddressedthroughtheexpressionof give a realistic picture of its impact on the biology of animals re g Hsp70andHsp90andthephosphorylationofstress-activatedprotein intheirhabitats.Overall,thephysiologicalmechanismsdefin- u kinases,p38mitogen-activatedproteinkinase(p38MAPK)andcJun- ing long-term tolerance and their interaction toward setting .p h N-terminal kinases (JNKs). Mussels increased the duration of valve thermal limits of the intact organism are not yet sufficiently ys closurebyaboutsixfoldwhenacclimatedto24°Cratherthanto17°C. understood,whereasindividualphysiologicalmechanismsthat iolo As indicated by the activity of PK, such behavior caused metabolic respond to elevated temperature have been identified (25). g y depressionandprobablyashiftfromaerobictoanaerobicmetabolism. Thermallimitsandadaptationhavebeenstudiedinbivalves .o Acclimation to temperatures higher than 24°C caused an increase in rg myloatritoanlityofanpd38indMucAePdKtheanedxpJreNsKsisoninodficHastped72.acItnicvraetaiosnedopfhoMspAhPoKr- frreolamtiornoschkiypsohforheesa.tTshheoscekinpvroetsetiigna(tHiosnps)heaxvpereressvieoanlewdiathclvoesre- on F tical zonation (33). The heat shock response is an energy- e signaling cascades. The potential involvement of MAPKs in the b induction of Hsp genes in the tissues of M. galloprovincialis is consuming process, during activation of transcription, synthe- rua discussed.Inconclusion,itseemsthatM.galloprovincialislivesclose sis of Hsps, and the ATP-dependent chaperoning by Hsps. ry to its acclimation limits and incipient lethal temperature and that a Hawkins (10) has estimated that the cost of protein synthesis 25 smalldegreeofwarmingwillelicitstressresponsesatwholeorganism constitutes20–25%oftheenergybudgetoftheNorthernblue , 2 andmolecularlevels. mussel, Mytilus edulis. A shift to anaerobiosis as a result of 00 9 thermallyinducedhypoxiainmarinebivalves,asittakesplace mussel;metabolism;Hsp;p38MAPK;JNK;pyruvatekinase inothermarineanimals(32,34),willcausemetabolicdepres- sion and consequently a reduction in ATP turnover. If this is IN VARIOUS PHYLA OF MARINE invertebrates, the transition to the case, there might be an effect on the expression of Hsp, internal (systemic) hypoxia was found to characterize the althoughthishasnotbeenexperimentallyshown.Furthermore, borders of the thermal tolerance window, in fully oxygenated changesintheperiodthatmusselskeeptheirvalvesopenedor waters. In general, anaerobic metabolism sets in at both cold closed will affect the amount of water they filter and conse- andwarmtemperatureextremes.Theseobservationsledtothe quently the amount of food they retain. It is not known how thermal stress affects valve opening vs. closure behaviors; development of the concept of oxygen- and capacity-limited however, exposure of blue mussel M. edulis to water temper- thermal tolerance in animals (19, 20, 23, 24). This concept atures higher than 25°C impairs their clearance rate (30) and suggests that, at the low and high borders of the thermal consequentlytheamountoffoodavailableforenergyturnover. envelope (defined as pejus thresholds, Tp), animals show a Thus, during prolonged thermal stress, bivalves may readjust reduction in aerobic capacity. This reduction is not caused by their metabolic profile and energy budget to meet the energy falling levels of ambient oxygen but through limited capacity demand for Hsp synthesis. It has been proposed (5) that of oxygen supply mechanisms (ventilation, circulation) to mitogen-activated protein kinase (MAPK) signaling might be Addressforreprintrequestsandothercorrespondence:B.Michaelidis,Labo- ratoryofAnimalPhysiology,Dept.ofZoology,SchoolofBiology,Facultyof Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment Science, Aristotle Univ. of Thessaloniki, Thessaloniki 54124, Greece(e-mail: ofpagecharges.Thearticlemustthereforebeherebymarked“advertisement” [email protected]). inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. http://www.ajpregu.org 0363-6119/07$8.00Copyright©2007theAmericanPhysiologicalSociety R911 R912 STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING involved in the regulation of Hsp expression in blue mussels. chambersweredividedintoeightgroupsoften.Theneachgroupof Evidencefrommammaliancellsystemsalsoindicatesthatthe chamberswasplacedintoeightseparateaquariacontainingseawater. activity of p38 MAPK and cJun-N-terminal kinases (JNKs) is Musselswereallowedtoacclimatefor10daystoeachtemperatureof 7°C,14°C,18°C,20°C,24°C,26°C,28°C,or30°C,respectively.The essential for Hsp expression during various cell stresses (26, periods and the mean fraction of time during which valves re- 31).However,toourknowledge,thereisnoevidencerelating mained open or closed at each temperature of acclimation were MAPK activation and Hsp expression in the tissues of marine estimated from recorded traces. The experiment was repeated five bivalves during prolonged thermal stress. times. Although major advances have been made to resolve the 3) Effects of water temperature on the expression of Hsp70 and mechanismsthroughwhichmarineanimalsrespondtothermal Hsp90 on the phosphorylation of p38 MAPK and JNKs and on the stress,littleisknownaboutthelong-termresponsetowarming activityofPK.ANIMALTREATMENTS.Thresholdtemperaturesforthe (33).Thisstudyattemptstointegrateinformationfromvarious expression of Hsp70 and Hsp90, for the phosphorylation of p38 levels of biological organization, including behavior (valve MAPK and JNKs and for changes in the activity of PK, were opening/closing), metabolic adjustments, heat shock protein determinedinmusselsplacedintofiveaquaria,wheretheywereleft expression,andproteinkinaseactivity,intoacoherentstoryof toacclimateto18°Cfor2wk.Thenwatertemperaturewasadjusted to20°C,24°C,26°C,or28°C,andmusselswerelefttoacclimatefor how heat stress may influence the Mediterranean mussel M. 30 days. Individuals were removed after 5, 10, 15, 20, or 30 days. galloprovincialis. The impact of temperature on behavior was Mantletissueandposterioradductormuscle(PAM)weredissected, analyzed by examining the frequency and duration (ml/h) of freeze-clampedbetweenaluminumtongscooledinliquidnitrogen, valve closure, which relate to the rate of filtration and food and ground under liquid nitrogen. Tissue powders were stored at assimilation, and thus the rate of energy (ATP) supply. More- "80°C. Animals kept at 18°C were used as controls. Do over,valveclosuremayinducetissuehypoxiaeveninoxygen- PREPARATIONOFTISSUESAMPLESFORSDS-PAGE.Tissuepowders wn ated water (16). As an aspect of regulation in aerobic and werehomogenizedin3ml/gofcoldlysisbuffer[20mM#-glycero- lo a anaerobic metabolism, we analyzed the effect of ambient phosphate, 50 mM NaF, 2 mM EDTA, 20 mM HEPES, 0.2 mM d e temperature on the activation of the key glycolytic enzyme Na VO , 10 mM benzamidine, pH 7, supplemented with 200 $M d pyruvate kinase (PK). PK controls the flux of phosphoenol- leu3pepti4n, 10 $M trans-epoxysuccinyl-L-leucylamido-(4-guanidi- fro pyruvate(PEP)tosuccinateduringanaerobiosis.Modification no)butane,5mMdithiothreitol(DTT),300$Mphenylmethylsulfonyl am of the enzyme to a less-active form contributes to metabolic jp depression (4, 35). In addition, we determined whether the re g Hsp70andHsp90responseandmetabolicreadjustmentsoccur u.p concomitantlyinthetissuesofM.galloprovincialis.Giventhat h y the MAPK family may play an important role in coordinating sio gene responses to various stresses, we also examined the lo g phosphorylation and hence activation of stress-activated pro- y .o tein kinases, p38 MAPK and JNKs, in the tissues of M. rg galloprovincialis during long-term acclimation at increasing o n temperature. F e b MATERIALS AND METHODS ru a ry Animals 2 5 AdultspecimensofM.galloprovincialis(75–80mmlength)used , 2 0 forthepresentstudywerecollectedduringspring(averageseawater 0 9 temperature17°C)intheareaofHalastraintheThermaikosGulf,and these were held in aquaria containing recirculating natural aerated seawater. Water temperature was controlled at 18 ! 0.5°C and salinity at 32 ! 3.5‰. Mussels were kept in aquaria under these conditions for 2 wk prior to experimentation. Seawater pH was 8.05 ! 0.02. ExperimentalProcedures 1)Effectofwatertemperatureonmortalityofmussels.After2wk of acclimation to 18°C, mussels (30–40 animals) were introduced into six aquaria and brought to 18°C, 20°C, 24°C, 26°C, 28°C, or 30°Cbywarmingofthewateratarateof0.1°Cperminute.Mussels werecheckedformortalityeverydayfor30days.Musselsfailingto closetheirshellsinresponsetoexternalstimuliwereconsidereddead. The number of dead animals in each experiment was expressed as percentofthetotalnumber. 2)Effectofwatertemperatureonvalveclosingbehavior.Individ- ualmusselswereplacedinoneopen500-mlglasschambereach.One ofthetwovalveswasgluedtothebottomofthechamber.Oneendof Fig. 1. A: effect of water temperature on the mortality of Mytilus gallopro- a thin wire was glued to the surface of the second valve while the vincialis during 30 days of acclimation to different temperatures. B: mean other end was attached to an isotonic transducer. This arrangement periods of valve closure or opening during acclimation to different water permittedtherecordingofvalvemovements(closureoropening).The temperatures.Valuesaremeans!SE;n%5. AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING R913 fluoride (PMSF), 120 $M pepstatin, 1% vol/vol Triton X-100] and homogenized(1:5,wt/vol)inice-cold50mMimidazole-HCl(pH7.0) extractedonicefor30min.Sampleswerecentrifuged(10,000g,10 containing100mMsodiumfluoride,10mMEDTA,10mMEGTA, min, 4°C), and the supernatants were boiled with 0.33 vol of SDS- 30 mM 2-mercaptoethanol, 40% glycerol (vol/vol), and 0.1 mM PAGE sample buffer (330 mM Tris!HCl, pH 6.8, 13% vol/vol PMSF added just prior to homogenization, using a Polytron PT10 glycerol,133mMDTT,10%wt/volSDS,0.2%wt/volbromophe- homogenizer(3periods,20seachtime).Aftercentrifugation(25,000 nol blue). Protein concentrations were determined using the Bio- g, 20 min, 4°C), the supernatant was removed and passed through a Rad protein assay (Bio-Rad, Hercules, CA). 5-mlcolumnofSephadexG-25equilibratedin40mMimidazole-HCl PREPARATIONOFTISSUESAMPLESFORSDS-PAGE.Equalamounts buffer(pH7.0)containing5mMEDTA,15mM2-mercaptoethanol, of proteins (100 $g) were separated on 10% (wt/vol) acrylamide, and20%glyceroltoremovemetabolitesoflowmolecularmass(11). 0.275% (wt/vol) bisacrylamide slab gels and transferred electro- Thecolumnwascentrifugedinadesktopcentrifugeat2,000gfor1 phoreticallyontonitrocellulosemembranes(0.45$m;Schleicherand min, and the supernatant was used for the determination of enzyme Schuell, Keene, NH). Nonspecific binding sites on the membranes activity. Standard assay conditions for PK were as follows: 50 mM were blocked with 5% (wt/vol) nonfat milk in TBST [20 mM imidazole-HClbuffer,2mMADP,0.15mMNADH,50mMKCl,5 Tris!HCl,pH7.5,137mMNaCl,0.1%(vol/vol)Tween20]for30min mM MgCl , 2 IU dialyzed lactate dehydrogenase and PEP, either 2 at room temperature. Subsequently, the membranes were incubated 2 mM for the determination of V (V ) or 0.05 mM for the overnight with the appropriate primary antibodies. Antibodies used max 2mM determination of V (V ). The ratio V /V , which reflects the wereasfollows:monoclonalmouseanti-heatshockprotein,70kDa, 0 0.05mM 0 max and monoclonal mouse anti-heat shock protein, 90 kDa (Sigma); relative activity of PK, was then calculated to determine metabolic monoclonal mouse anti-phospho-SAPK-JNK (Thr183-Tyr185) and depressionduringacclimationtohightemperature.TheratioV0/Vmax polyclonalrabbitanti-phospho-p38MAPkinase(Thr180-Tyr182)(Cell was found to decline in the adductor muscle and mantle of bivalve D Signaling). After washing in TBST (3 periods, 5 min each time) the mollusks during anoxia indicating a shift of PK toward a less-active o blots were incubated with horseradish peroxidase-linked secondary form during metabolic depression (15, 16). Assays for V0 and Vmax wn antibodies,washedagaininTBST(3periods,5mineachtime),and wereconductedat18°Candateachacclimationtemperature. lo a the bands were detected using enhanced chemiluminescence d e (qCuhanemtifiiceodnb)ywliathseerx-spcoasnunrientgodFeunjsiiMtoemdeictraylX(G-realPyrfiolmAsn.aFlyilzmersSwoefrte- Statistics d fro ware, GraphPad). Changesovertimeweretestedforsignificanceatthe5%levelby m PREPARATION OF TISSUE HOMOGENATES FOR THE DETERMINA- using one-way analysis of variance (ANOVA) and by performing ajp TIONOFPKACTIVITY.ForthedeterminationofPKactivity,samples Bonferronipost-hoctestsforgroupcomparisons.Valuesarepresented re g of frozen tissue powders (200–500 mg) were rapidly weighed and asmeans!SE. u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 Fig.2. LevelsofHsp70inthemantletissueofsubmersed M.galloprovincialisduringacclimationtodifferentwater temperatures. Tissue extracts were subjected to SDS- PAGEandimmunoblottedforHsp70.Representativeim- munoblots are shown for each acclimation temperature. Blotswerequantifiedbylaser-scanningdensitometry.Val- ues are means ! SE; n % 5 preparations from different animals.InducibleisoformHsp72,opencircles;constitu- tive isoform Hsp73, closed circles. *P & 0.05 compared withthecontrol(0days). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org R914 STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING RESULTS Effect of Acclimation Temperature on the Expression of Hsp70 and Hsp90 Effect of Water Temperature on Mussel Mortality TheexpressionofHspswasmonitoredinmantletissueand Figure 1A shows the development of mortality over time PAM. In the Hsp70 family two main bands were identified, during acclimation to different temperatures. Very few (about Hsp72 (the inducible form) and Hsp73 (the constant form) 1%) of the mussels died below 26°C, although some were during acclimation to any temperature. The levels of Hsp72 closing their shells progressively at 26°C. Animals started to showed a gradual increase within 30 days of acclimation of mussels to 26°C. Increased Hsp72 expression was also ob- dieaswatertemperaturereached26°C,leadingto5%mortality served during acclimation to 28°C (Fig. 2). A similar expres- within 5 days and 20% after 30 days. Mussel mortality sion profile was observed in the PAM (Fig. 3). increased significantly in animals acclimated to 28°C, when In response to acclimation to various temperatures, Hsp90 almost 20% of the mussels died within the first 5 days and displayed similar changes as Hsp72 (Figs. 4 and 5). 30% after 30 days. Mussel mortality increased further at 30°C, when 80% of the animals died within 15 days of Effect of Acclimation Temperature on the Phosphorylation of acclimation. the Kinases p38 MAPK and JNKs Effect of Water Temperature on Valve Opening vs. Closure ActivationofMAPKshasbeenimplicatedinthestimulation of Hsp expression (26, 31). Thus, phosphorylation and hence D The effect of acclimation temperature on the mean peri- activation of p38 MAPK and JNKs was determined in both ow ods of valve closure or opening is shown in Fig. 1B. The mantle and PAM at different acclimation temperatures. One nlo period of valve opening increased as the acclimation tem- form of JNK was detected, which corresponds to the 46-kDa a d perature increased from 7°C to 16°C. Thereafter, however, isoform of the mammalian enzyme (Figs. 6 and 7). Increased ed the mean period of valve opening declined, suggesting that acclimation temperatures caused significant changes in the fro further increases in acclimation temperature stimulated levels of the phosphorylated forms of JNK and p38 MAPK in m valve closure up to 24°C. Beyond 26°C, a trend to increase both mantle (Fig. 6) and PAM (Fig. 7). In the mantle, phos- ajp the period of valve opening was observed. phorylationlevelsofJNKdoubledafter10daysofacclimation re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 Fig.3. LevelsofHsp70intheposterioradductormuscle (PAM)ofsubmersedM.galloprovincialisduringacclima- tion to different water temperatures. Tissue extracts were subjected to SDS-PAGE and immunoblotted for Hsp70. Representative immunoblots are shown for each acclima- tion temperature. Blots were quantified by laser-scanning densitometry.Valuesaremeans!SE;n%5preparations from different animals. Inducible isoform Hsp72, open circles; constitutive isoform Hsp73, closed circles. *P & 0.05comparedwiththecontrol(0days). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING R915 Fig.4. LevelsofHsp90inthemantleofsubmersedM.gallo- provincialisduringacclimationtodifferentwatertemperatures. TissueextractsweresubjectedtoSDS-PAGEandimmunoblot- tedforHsp90.Representativeimmunoblotsareshownforeach acclimation temperature. Blots were quantified by laser-scan- ningdensitometry.Valuesaremeans!SE;n%5preparations fromdifferentanimals.*P&0.05comparedwiththecontrol(0 days). D o w n lo a d e d fro m a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 Fig. 5. Levels of Hsp90 in the PAM of submersed M. gallo- provincialisduringacclimationtodifferentwatertemperatures. TissueextractsweresubjectedtoSDS-PAGEandimmunoblot- tedforHsp90.Representativeimmunoblotsareshownforeach acclimation temperature. Blots were quantified by laser-scan- ningdensitometry.Valuesaremeans!SE;n%5preparations fromdifferentanimals.*P&0.05comparedwiththecontrol(0 days). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org R916 STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING Fig.6. PhosphorylationlevelsofcJun-N-terminal kinase(JNK)inthemantletissueofsubmersedM. galloprovincialis during acclimation to different water temperatures. Tissue extracts were sub- jectedtoSDS-PAGEandimmunoblottedforthe phosphorylatedformofJNKs.Representativeim- munoblots are shown for each acclimation tem- perature.Blotswerequantifiedbylaser-scanning densitometry. Values are means ! SE; n % 5 preparations from different animals. *P & 0.05 comparedwiththecontrol(0days). D o w n lo a d e d fro m a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 Fig.7. PhosphorylationlevelsofJNKinthe PAMofsubmersedM.galloprovincialisdur- ing acclimation to different water tempera- tures. Tissue extracts were subjected to SDS-PAGEandimmunoblottedforthephos- phorylatedformofJNKs.Representativeim- munoblots are shown for each acclimation temperature. Blots were quantified by laser- scanningdensitometry.Valuesaremeans! SE; n % 5 preparations from different ani- mals. *P & 0.05 compared with the control (0days). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING R917 Fig. 8. Phosphorylation levels of p38 mitogen- activatedproteinkinase(p38MAPK)inthemantle ofsubmersedM.galloprovincialisduringacclima- tiontodifferentwatertemperatures.Tissueextracts weresubjectedtoSDS-PAGEandimmunoblotted for the phosphorylated form of p38 MAPK. Rep- resentativeimmunoblotsareshownforeachaccli- mationtemperature.Blotswerequantifiedbylaser- scanning densitometry. Values are means ! SE; D n % 5 preparations from different animals. *P & o 0.05comparedwiththecontrol(0days). w n lo a d e d fro m a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 9 Fig.9. Phosphorylationlevelsofp38MAPKinthe PAMofsubmersedM.galloprovincialisduringac- climation to different water temperatures. Tissue extractsweresubjectedtoSDS-PAGEandimmuno- blottedforthephosphorylatedformofp38MAPK. Representative immunoblots are shown for each acclimation temperature. Blots were quantified by laser-scanning densitometry. Values are means ! SE;n%5preparationsfromdifferentanimals.*P& 0.05comparedwiththecontrol(0days). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org R918 STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING to24°Canddecreasedthereaftertocontrollevels.Warmingto to different water temperatures. No change in PK activity 26°Ccausedafivefoldincreaseinthelevelsofphosphorylated occurredattemperatureslowerthan24°C.However,warming JNK after 20 days. After 30 days, phosphorylated JNK had seemed to modify enzyme activity, with a decrease in the decreased slightly but levels remained higher than those of mantle at 24°C. The relative activity of the enzyme (V /V ) 0 max controls. During acclimation of mussels to 28°C, the levels of decreased from 0.95 to 0.57 within 15 days of acclimation, phosphorylated JNK increased progressively (more than indicatingashiftofPKtowardaless-activeform(Fig.10).A 4-fold) and remained elevated after 30 days. The level of similarpatternwasobservedinthePAM.Therelativeactivity phosphorylated p38 MAPK showed a significant increase in of PK from the PAM decreased within the first 20 days of themantleafter10daysofacclimationto24°Cand26°Cand acclimation to 24°C and remained reduced thereafter. Further remained elevated thereafter (Fig. 8). A more prominent in- warming, however, seemed to modulate PK activity from creaseinthephosphorylationofp38MAPKwasobservedafter mantle tissue or PAM in a way entirely different from the 5 days of acclimation to 28°C. Similar patterns of changes in patternobservedat24°C.Changesinenzymaticactivitywithin the phosphorylation levels of JNK and p38 MAPK were the first 10 days of exposure to either 26°C or 28°C were observed in the PAM (Figs.7and9)afteracclimationto20°C, similartothoseobservedwhenthemusselswereacclimatingto 24°C, and 26°C. A stronger response in the phosphorylation of 24°C. Thereafter, however, the activity and activity status p38MAPKwasobservedinthePAMthaninthemantleat28°C. (V /V ) of PK from mantle reached levels beyond control 0 max values, indicating activation of the enzyme (Fig. 10). Similar Effects of Acclimation Temperature on the Activity of PK increase in PK activity was observed for PAM at 26°C D Figures 10 and 11 summarize the patterns of PK activity in (Fig.11).However,at28°CPKactivityinthePAMrecovered o w the mantle tissue and the PAM during acclimation of mussels up to control levels. n lo a d e d fro m a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 Fig.10. Activityofpyruvatekinase(PK)fromthe 5 mantleofM.galloprovincialisduringacclimation , 2 0 to different water temperatures. Activity ($mol/ 0 minpergwetwt)wasdeterminedat2mM(Vmax) 9 and0.05mMofPEP(V0).Valuesaremean!SE; n%10preparationsfromdifferentanimals.Vmax (closedcircles)andV (opencircles)aregivenat 0 left,andtheratioV /V isgivenattheright. 0 max AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org STRESSRESPONSESINMytilusgalloprovincialisDURINGWARMING R919 Fig.11. ActivityofPKfromthePAMofM.gallo- provincialis during acclimation to different water temperatures.Activity($mol/minpergwetwt)was D o determined at 1 mM (Vmax) and 0.05 mM of PEP w (fVro0m). dViaffleureesntaraenimmeaalsn.!VmSaxE(;clnos%ed1c0ircplreesp)aarantdioVns0 nloa (open circles) are given at the left, and the ratio d e V /V isgivenattheright. d 0 max fro m a jp re g u .p h y s io lo g y .o rg o n F e b ru a ry 2 5 , 2 0 0 DISCUSSION night temperature may drop 0.5–1.5°C. According to unpub- 9 lishedobservations,30–55%ofmusselsdiewhendaytemper- The data obtained in the present study demonstrate that M. ature of surface sea water rises up to 28°C, indicating that galloprovincialiscannotsurviveseawatertemperaturesofand mussels live at the upper limit of their acclimation capacity. beyond 26°C over extended periods of time (Fig. 1A). The Similar to M. galloprovincialis, M. edulis displays an upper mortality of mussels increased most drastically during warm- lethaltemperaturebetween26°Cand28°C(27).Atfirstsight, ing to 30°C, and 45% of mussels died within the first 5 days. this appears puzzling since M. galloprovincialis displays a The increase in the mortality of mussels during exposure to more southern distribution pattern and experiences environ- temperatures higher than 24°C might be attributed to reduced mental temperatures that are on average warmer than for M. ability to assimilate food and associated energy. When tem- perature exceeds 25°C, filtration falls significantly in M. gal- edulisorathirdcongener,thePacificbluemussel,M.trossulus loprovincialis(AnestisA,PortnerHO,StaikouA,Michaelidis (3).However,theMediterraneanisabasin,whichhasnotides, B,unpublisheddata)andinM.edulis(1,2,9,30).Underthese suchthatMediterraneanmusselsliveinthesublittoralzoneand conditions mussels would rely on stored fuels to replenish the are not exposed to large diurnal temperature fluctuations. In consumedATP.Thelattermightcausedepletionofstoresand contrast, M. edulis and M. trossulus live in the intertidal zone mightcontributetocelldeath,explainingpartlytheincreasein and they are thereby potentially exposed to much higher mussel mortality in water temperatures beyond 24°C, which temperatureextremes.Thismayexplainthesimilarityinupper wouldonlysupporttime-limitedtolerance.Duringsummerand thermal limits despite different geographical distribution especially between the end of July and the middle of August, ranges. surface sea water temperatures in the habitat of M. gallopro- M.galloprovincialisacclimatedto24°Cdisplayedextended vincialis fluctuated between 26°C and 28°C (6). However, at periods of valve closure compared with controls (Fig. 1B). AJP-RegulIntegrCompPhysiol•VOL293•AUGUST2007•www.ajpregu.org