LJournal of Experimental Marine Biology and Ecology251 (2000) 205–225

www.elsevier.nl / locate / jembe

Effect of reproduction on escape responses and muscle¨metabolic capacities in the scallop Chlamys islandica Muller

1776

*Katherina B. Brokordt , John H. Himmelman, Helga E. Guderley´ ´Departement de Biologie and GIROQ (Groupe Interuniversitaire de Recherches Oceanographiques du

´ ´ ´ ´Quebec), Universite Laval, Cite Universitaire, Ste.-Foy, Quebec, Canada G1K 7P4

Received 15 January 2000; received in revised form 27 March 2000; accepted 15 April 2000

Abstract

In scallops, gametogenesis leads to mobilization of glycogen and proteins from the adductormuscle towards the gonad. This mobilization is likely to diminish the metabolic capacities of theadductor muscle and thereby the scallops’ escape response. We examined the escape response interms of number of valve claps until exhaustion, rate of clapping and the recovery during and aftervalve closure in adult scallops, Chlamys islandica, sampled at different stages in the reproductivecycle (immature, mature, before and after spawning). In parallel, we measured muscle glycogen,protein and phosphoarginine contents, the oxidative capacity of mitochondria isolated from theadductor muscle and levels of muscle enzymes which are active during exercise and recovery. The

21number of claps (24–26), rate of clapping ( | 13 claps min ) and phosphoarginine and argininekinase levels were similar during the different reproductive stages. All immature scallopsresponded to restimulation immediately after opening their valves, while only 62% of mature, 82%of prespawned and 38% of spawned scallops responded. Immature animals completely recoveredtheir initial swimming capacity within 4 h of opening their valves, but mature, prespawned andspawned scallops needed 18, 12 and 18 h, respectively. Overall phasic adductor muscle frommature, prespawned and spawned animals showed decreased glycogen phosphorylase, phospho-fructokinase, pyruvate kinase (except for prespawned), octopine dehydrogenase and citratesynthase levels, a deterioration of the oxidative capacity of mitochondria and a marked decrease inglycogen content compared to immature scallops. Therefore, during gonadal maturation andspawning, C. islandica did not change its clapping capacity, but slowed its recuperation fromexhausting burst exercise, both during and after valve closure, likely due to the decreasedmetabolic capacity of the adductor muscle. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Chlamys islandica; Escape response; Muscle; Reproduction; Scallop

*Corresponding author. Tel.: 11-418-656-2131; fax: 11-418-656-2339.E-mail address: katherina.brokordt@giroq.ulaval.ca (K.B. Brokordt).

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 00 )00215-X

206 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

1. Introduction

Gametogenesis represents a period of high energy demand, particularly for organismswith broadcast spawning, and when external food supplies are limited, gameteproduction occurs at the expense of biochemical components in somatic tissues (Calow,1985). In bivalves and fish, muscle is one of the tissues most affected during gonadalmaturation, with both protein and glycogen levels decreasing (Shevechenko, 1972;reviewed by Barber and Blake, 1991; Von der Decken, 1992). Such mobilization ofmacromolecules could decrease muscle metabolic capacities.

In scallops the adductor muscle is one of the largest soft tissues and while its primaryrole is the movement of the valves during escape responses or swimming, it also servesas a major site of reserve deposition. In the bay scallop Argopecten irradiansconcentricus, 63–99% of total energy gain by the gonad during gametogenesis isthought to come from the adductor muscle (Epp et al., 1988). In this species,gametogenesis occurs mainly at the expense of muscle protein, however, in otherscallops the substrate most mobilized is glycogen (reviewed by Barber and Blake, 1991;

´ ´Martınez, 1991; Martınez and Mettifogo, 1998).How far the reproductive cycle affects biochemical reserves may depend on the

timing of gonadal proliferation in relation to food availability (Shafee, 1981). In theblack scallop Chlamys varia, during the spring reproduction, when food is abundant,only carbohydrate reserves are used for gonadal development, while during the autumnreproduction, when food is less abundant, all reserves (protein, lipid and glycogen)decrease with gonadal development (Shafee, 1981). In fishes, such as the salmon,Oncorhynchus nerka and Salmo salar, not only are lipid reserves from muscle mobilizedduring gonadal maturation and their non-feeding spawning migration, but the levels ofenzymes and contractile proteins are also affected (Mommsen et al., 1980; Olin and vonder Decken, 1987). In the roach Rutilus rutilis, swimming activity is markedly reducedduring gonadal maturation (Koch and Wieser, 1983), either due to the metabolic costs ofgonad maturation or to decreases in muscle metabolic capacities. A decrease of musclemetabolic capacities after gonadal maturation and spawning could reduce an animal’slocomotor ability and thereby the capacity to escape predators.

Among bivalves, scallops are well known for their swimming ability (Brand, 1991).They swim by jet propulsion using a succession of claps consisting of alternateadduction and abduction of the valves (Olson and Marsh, 1993). Swimming is poweredby the adductor muscle, principally by the large phasic portion and to a lesser extent bythe smaller tonic (catch) muscle (de Zwaan et al., 1980).

The physiological and biochemical aspects of the escape response (valve clapping andvalve closure) are best understood in the giant scallop Placopecten magellanicus(Thompson et al., 1980; de Zwaan et al., 1980; Livingstone et al., 1981) and in the bayscallop Argopecten irradians concentricus (Chih and Ellington, 1983, 1986). InPlacopecten magellanicus, the main source of ATP during valve clapping is argininephosphate, and anaerobic glycolysis, supported by octopine accumulation, only makes asmall contribution (de Zwaan et al., 1980). Following intense valve clapping, partialrecuperation of arginine phosphate occurs during valve closure with further accumula-tion of octopine.

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Once the valves have opened, the full restoration of arginine phosphate pools andelimination of octopine is achieved aerobically (Livingstone et al., 1981). Similarly, inArgopecten irradians concentricus intense valve clapping is initially supported byarginine phosphate and only toward the end do anaerobic glycolysis and octopineaccumulation intervene (Chih and Ellington, 1983, 1986). Mitochondria isolated fromthe adductor muscle of the tropical scallop, Euvola ziczac, seem adapted for a role inrecovery metabolism given their increased affinity for pyruvate at the pH values likely tooccur in muscle fibers after intense clapping (Guderley et al., 1995). Oxidativecapacities and respiratory control ratios of mitochondria isolated from the adductormuscle are lower during the first of two spawnings than during other periods (Boadas etal., 1997), suggesting an impact of reproduction.

We reasoned that changes in muscle metabolic capacities or in the levels of energeticreserves during the reproductive cycle of adult scallops could modify the escaperesponse or recuperation from an exhausting escape response. We predicted that beforegonadal maturation, scallops would show a stronger escape response and wouldrecuperate faster from exhausting burst exercise given the higher muscle metaboliccapacities and energy reserves the scallops should have at this time.

To evaluate these hypotheses, we compared the escape response and capacity forrecuperation from exhausting exercise for adult Iceland scallops, Chlamys islandica,sampled at different stages in the annual reproductive cycle (immature, mature, beforeand after spawning). In parallel, we determined the effect of these reproductive stages onthe energetic reserves in the phasic adductor muscle (arginine phosphate, glycogen andproteins). Given that in fish muscle, starvation leads to more extensive mobilization ofsarcoplasmic than structural (insoluble) proteins (Beaulieu and Guderley, 1998), wequantified both fractions. To assess the impact of the reproductive cycle on musclemetabolic capacities, we measured muscle levels of the glycolytic enzymes, glycogenphosphorylase (GP), pyruvate kinase (PK), phosphofructokinase (PFK), octopinedehydrogenase (ODH) and arginine kinase (AK), as well as the mitochondrial enzyme,citrate synthase (CS). We measured the oxidative capacities, substrate preferences,respiratory control ratios and CS levels of mitochondria isolated from the phasicadductor muscle at different reproductive stages. By profiling these enzyme activitiesand mitochondrial capacities we sought to assess the capacity of enzymes likely tosupply ATP for contraction (AK and glycolytic enzymes (GP, PFK, PK and ODH)),recuperation during valve closure (glycolytic enzymes) and recuperation after reopeningof the valves (CS and mitochondrial capacities).

2. Materials and methods

2.1. Sampling and maintenance of scallops

ˆ ˆThe population studied was located at the southern side of Ile du Fantome in theMingan Islands, northern Gulf of St. Lawrence, eastern Canada. Samples of 78 adultscallops (80–95 mm shell height) were collected by scuba-diving at 37 m from Junethrough August 1996 at the following reproductive stages: immature, mature, before

208 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

spawning (prespawned) and after spawning (spawned). Reproductive stage was de-termined using qualitative and quantitative criteria. As qualitative criteria we used theshape of the gonad and the degree of vascularization by secondary genital ducts whichwere observable through the gonadal epithelium (David Arsenault, personal communica-tion). Histological classification used the gonadal developmental stages described for

´Chlamys islandica by Thorarinsdottir (1993). As quantitative criteria, we determined thegonadosomatic index corrected for the size of the scallop ((gonadal mass 3 total tissue

21 21mass ) 3 shell height ), as described by Bonardelli and Himmelman (1995). We alsoevaluated the lipid and protein content of the gonads on the different sampling dates.

At each sampling period, we determined the shell height and wet mass of the gonad,muscle and remaining soft tissues of 20 scallops. One part of the phasic muscle wasrapidly excised, quickly frozen ( | 1 min) using a freeze clamping press (Gagnon et al.,1998) prechilled in liquid nitrogen and stored at 2 708C for determination of enzymeactivities and arginine phosphate concentrations. The remainder was frozen at 2 208Cfor protein and glycogen determinations. Part of the gonad was frozen at 2 208C for

´lipid and protein determinations. Ten animals were transported live to Universite Lavalin Quebec City where they were maintained in sea water aquaria and fed with Isochrysisgalbana and Chaetoceros gracilis for at least one day, before determinations of therespiratory capacity of muscle mitochondria.

The remaining 48 scallops were acclimated during 2 days in holding tanks withrunning sea water pumped from 10 m in depth at Havre St. Pierre, and then used toevaluate escape responses. Despite the variations in ambient temperature, at the time ofmeasurements of escape responses in the laboratory water temperatures were con-sistently 5–68C.

2.2. Environmental conditions

Bottom temperature was recorded using a Sealog-T thermograph (Vemco Inc.,Halifax, N.S.), which was anchored on the bottom in the scallop bed. Phytoplanktonabundance was quantified periodically from water samples collected with a Niskin bottlefrom 1 m above the scallop bed. From each sample, chlorophyll a determinations weremade using the spectrophotometric method as described by Parsons et al. (1984).

2.3. Evaluation of escape responses

When the scallop, Chlamys islandica, is stimulated with its natural predator, theseastar Leptasterias polaris (Himmelman, 1991), the escape response is highlystereotyped and reproducible. It begins with a series of valve claps and if the stimulus ismaintained until fatigue, the scallop closes its valves firmly and remains closed for acertain period, after which the valves slowly reopen.

Based on this behavior, we carried out the following experiments: 48 scallops, eachplaced in different aquarium, were stimulated until fatigue, by repeatedly touching thetentacles and mantle with an arm tip of L. polaris. We counted the number and durationof claps, and measured the period that the scallops kept their valves closed. Afterfatigue, the scallops were separated into six groups of eight individuals each. The groups

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 209

were restimulated (in the same fashion as initially) at set times after reopening of thevalves. One group was restimulated after 2 min, and the others 2, 4, 6, 12 or 18 h afterreopening the valves. Again, the number of claps was determined for each animal.

2.4. Metabolite assays

2.4.1. Muscle phosphoarginineDeproteinization of the samples followed the method described by Lamprecht and

Trautschold (1974). The neutralized perchloric acid extracts were used immediately forthe determination of phosphoarginine, in an assay analogous to the phosphocreatineassay described by Lamprecht et al. (1974).

2.4.2. Muscle glycogenThe concentration of glycogen was determined by enzymatic hydrolysis with

amyloglucosidase as described by Keppler and Decker (1974).

2.4.3. Muscle and gonad proteinMuscle proteins were separated into sarcoplasmic and myofibrillar fractions according

to the method of Bates and Millard (1983) as modified by Somero and Childress (1990).Muscle and gonad protein concentrations were determined using the bicinchoninic acidmethod of Smith et al. (1985) with bovine serum albumin as the standard.

2.4.4. Gonadal lipidThe lipid concentration in the gonad was estimated gravimetrically after extraction

with chloroform–methanol according to the method of Folch et al. (1957), except thatafter the centrifugations the sediment was resuspended for an additional extraction.

2.5. Enzyme assays

Samples of phasic adductor muscle were homogenized on ice, using a Polytron(Brinkman Instruments; Rexdale, Ontario, Canada), in 10 vol of 50 mM imidazole–HCl,2 mM EDTA-Na (ethylene dinitrilotetraacetic acid), 5 mM EGTA (ethyleneglycol2

tetraacetic acid), 1 mM dithiothreitol, 0.1% Triton X-100, pH 6.6 for PK, ODH and AK,pH 7.2 for GP, PFK and CS. One part of the homogenates was centrifuged at 10 000 3 gat 48C for 15 min for the assay of GP and PFK, and another at 600 3 g at 48C for 15 minfor the assay of CS.

Assay temperature was controlled at 68C with a circulating refrigerating water bath(Haake D8). Enzyme activity was measured using a UV/VIS spectrophotometer(Beckman/DU 640) to follow the absorbance changes of NAD(P)H at 340 nm, with theexception of CS which was monitored at 412 nm to detect the transfer of sulfydrylgroups from CoASH to 5,59-dithio-bis(2-nitro)benzoic acid (DTNB). The molarextinction coefficients used for NAD(P)H and DTNB were 6.22 and 13.6, respectively.All assays were run in duplicate and the specific activities were expressed in

21 21international units (mmol of substrate converted to product min ) 3 g wet mass.

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Conditions for enzyme assays (except for CS) were adapted from conditions used by de21Zwaan et al. (1980) for P. magellanicus, as follows (all concentrations in mmol l ):

2.5.1. Glycogen phosphorylase (EC 2.4.1.1)21Imidazole–HCl 50, KH PO 80, Mg-acetate 5, EDTA 2.5, 10 mg ml glycogen2 4

(omitted for the control), AMP 0.8, AMP cyclic 0.5, NADP 0.6, glucose-1,6-diphosphate0.004, excess glucose-6-phosphate dehydrogenase and phosphoglucomutase, pH 7.5.

2.5.2. Phosphofructokinase (EC 2.7.1.11)Tris–HCl 50, KCl 50, Mg-acetate 5, fructose-6-phosphate 1 (omitted for the control),

fructose-2,6-diphosphate 0.08, ATP 1, AMP 0.8, NADH 0.2, excess aldolase, glycerol-3-phosphate dehydrogenase and triosephosphate isomerase, pH 7.5.

2.5.3. Pyruvate kinase (EC 2.7.1.40)Imidazole–HCl 50, MgSO 13, KCl 100, phosphoenolpyruvate 5 (omitted for the4

control), ADP 5, NADH 0.2, excess lactate dehydrogenase, pH 6.6.

2.5.4. Arginine kinase (EC 2.7.3.3)Imidazole–HCl 50, MgCl 5, phosphoarginine 5 (omitted for the control), glucose 10,2

ADP 0.4, NADP 0.6, excess hexokinase and glucose-6-phosphate dehydrogenase, pH6.6.

2.5.5. Octopine dehydrogenaseImidazole–HCl 50, EDTA-Na 2, EGTA 5, KCN 1, sodium pyruvate 5 (omitted for2

the control), arginine–HCl 6, NADH 0.2, pH 6.6.

2.5.6. Citrate synthase (EC 4.1.3.7)Tris–HCl 75, oxaloacetate 0.3 (omitted for the control), DTNB 0.1, acetyl CoA 0.2,

pH 8.0.

2.6. Mitochondrial assays

2.6.1. Mitochondrial isolation and polarographic measurementsAll procedures followed Guderley et al. (1995) as adapted from Ballantyne and Moon

(1985), except that media were prepared in nanopure water. Mitochondrial oxygenuptake was measured in a water-jacketed respiration cell (Cameron Instruments) using aClarke-type electrode (Yellow Springs Instrument Comp., Yellow Springs, OH). Theelectrode was connected to a chart recorder (Linear, model 0585, Canlab Comp.) by anoxymeter (Cameron Instruments, Port Aransas, TX). The respiration chamber wasmaintained at 68C by a circulating refrigerated water bath. The values of oxygenconcentration were calculated from the data of Graham (1987) for physiological buffersusing the atmospheric pressure on the day of assay. Around 1.3 mg mitochondrialprotein was added to 1 ml of assay medium. The reaction medium contained (in

21mmol l ): sucrose 480, Hepes 70, KCl 100, KH PO 10, taurine 50, b-alanine 50,2 4

0.5% bovine serum albumin (BSA, fraction V, fatty acid free), pH 7.0 at 68C.

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Preliminary studies indicated that glutamate, malate, pyruvate and succinate at final21concentrations of 30, 6, 0.9 and 24 mmol l , respectively, gave maximal rates. To attain

21maximal rates of pyruvate oxidation, ‘sparking’ levels of malate (0.6 mmol l ), whichby themselves did not support significant rates of oxygen uptake, were required. Tomeasure maximal rates of respiration we added ADP at a final concentration of 0.6

21mmol l . The respiratory control ratio (RCR) was calculated from the ratio of the state3 rate (in presence of ADP) to state 4 rate (when all ADP had been phosphorylated)(Estabrook, 1967).

To estimate the maximal aerobic capacity of the phasic adductor muscle, we measuredthe level of CS in the mitochondrial suspensions and, using the specific activity of CSdetermined for the same muscle, carried out the following conversion:

21 21 21 21 21(nmol O min ? CS U )CS U g 5 nmol O g min2 2

where the first factor is mitochondrial oxygen uptake expressed per unit CS in themitochondrial preparation and the second factor is the CS activity in the musclehomogenates.

2.6.2. Protein concentrationsMitochondrial protein concentrations were determined using the bicinchoninic acid

method of Smith et al. (1985) with BSA as the standard. The concentration of BSA inthe resuspension medium was subtracted to establish the concentration of mitochondrialprotein.

2.7. Chemicals

All biochemicals were from Boehringer Mannheim Co. (Montreal, Canada) or SigmaChemical Co. (St. Louis, MO). All other chemicals were analytical grade.

2.8. Statistical analysis

Data were analyzed using a one-way ANOVA to test the null hypotheses of nodifferences between reproductive stages (Sokal and Rohlf, 1981). Normality was testedusing a Shapiro–Wilk’s test (SAS, 1991) and homogeneity of variances using a Levenetest (Snedecor and Cochran, 1989). Multiple pairwise comparisons (Tukey) were used totest for specific differences when the ANOVAs indicated significant differences (SAS,1991). Comparisons between the proportions of scallops, for each reproductive stage,that responded to restimulation 2 min after opening their valves were made using

2x -tests (Sokal and Rohlf, 1981).

3. Results

3.1. Reproductive stages

Animals sampled in mid-June (1996) were immature (or maturing), as indicated by alow gonadal index and significantly lower gonadal lipid and protein content compared

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with animals sampled in mid-July and early August (Fig. 1). The scallops sampled inmid-July and early August did not differ in their gonadal indices or in the lipid andprotein contents of the gonads. As the latter sample was taken only one week beforespawning began, we identified the groups sampled in mid-July and those sampled inearly August as mature and prespawned scallops, respectively. The scallops sampled inmid-August showed a marked decrease of the gonadal index and of the lipid and proteincontents in their gonads, indicating that animals had spawned (Fig. 1). Our classificationof these reproductive stages was supported by histological observations of gonadalsections from the scallops sampled in the four periods.

3.2. Environmental conditions

Phytoplankton abundance fluctuated considerably during the study. In general, during21maturation scallops had more phytoplankton available (|0.5 mg l of chlorophyll a)

than before and after spawning, where phytoplankton availability decreased (|0.321

mg l , Fig. 2).During gonadal maturation, bottom temperature was higher and more variable than

during spawning (Fig. 2). During July and until early August, temperature fluctuatedaround 68C. During spawning, it dropped to |48C and remained low until the end ofAugust.

3.3. Escape response

When Chlamys islandica were stimulated with the seastar Leptasterias polaris, theyresponded with a series of |26 claps in a period of |2 min. There was no difference inthe mean number of claps performed by the scallops at different reproductive stages

21(Table 1). The clapping rate of 13 claps min remained relatively stable, except for21prespawning scallops for which the rate was 17 claps min (Table 1).

The clapping response was followed by a period of |30 min where the animalsremained closed and refractory to further stimulation. This period was similar betweenreproductive stages, with the exception of mature animals which spent less time closed(Table 1). To evaluate the extent of recovery during valve closure, scallops wererestimulated 2 min after they reopened their valves, to measure the proportion of initialclaps performed during restimulation and the proportion of animals that responded tostimulation. Spawned scallops could only do 11% of the initial claps, compared to theother reproductive stages which responded with 25–35% of their initial claps (Table 2).Interestingly, all immature animals responded after restimulation with the seastar,whereas after gonadal maturation only 62% of mature and 82% of prespawning animalsresponded (Fig. 3). Spawned animals markedly decreased their responsiveness as only38% reacted 2 min after reopening their valves.

Even after the scallops reopened their valves, the time required for full recovery oftheir initial clapping capacity varied markedly with reproductive stage (Table 2).Immature scallops needed only 4 h for complete recovery. In contrast, after gonadalmaturation 18 h were needed to regain initial capacity; 12 h were required beforespawning and 18 h after spawning. Therefore, during gonadal maturation and spawning,

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Fig. 1. Gonadosomatic index and total content of lipid and protein in gonads of female and male Chlamysislandica at different reproductive stages. Values represent means6S.E. (n520 for GSI; n58 for lipid andprotein content). Means sharing the same letters were not significantly different (P,0.01) as determined byTukey multiple comparisons.

214 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

21 ˆFig. 2. Variations in temperature (8C) and chlorophyll a (mg l ) 1 m above the Chlamys islandica bed, at Ileˆdu Fantome Mingan Islands from May to August 1996. Values represent means6S.E. Arrows indicates the

times when scallops were sampled. P-S5prespawned.

C. islandica did not change its clapping capacity, but markedly slowed its rate ofrecuperation from exhausting escape responses. Both the recuperation during valveclosure and that occurring once valves reopened were diminished.

Table 1Mean number (S.E.) of claps, clapping rate and time spent with the valves closed after exhaustive exercise forthe scallop Chlamys islandica at different reproductive stages

Reproductive Number Clapping rate Time spent n21stage of claps (claps min ) closed (min)

Immature 26 (1) 13 (1) 33.2 (2.7) 40Mature 25 (1) 13 (1) 23.2 (1.7)** 48Pre-spawned 30 (2) 17 (1)** 34.1 (3.4) 29Spawned 24 (1) 13 (1) 28.8 (3.3) 48

** P,0.01.

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Table 2Mean (S.E.) proportion (%) of initial claps recovered at different time intervals after exhausting escaperesponse for the scallop Chlamys islandica at different stages in gonadal development

Recuperation time Immature Mature Prespawned Spawnedwith valves open

a2 min 34.8 (2.7) 24.6 (3.6) 28.1 (6.7) 10.6 (5.8)*2 h 73.3 (5.5) 54.2 (7.1) 72.7 (3.4) 69.5 (11.5)4 h 95.2 (6.3)** 60.5 (6.3) 60.9 (9.8) 70.1 (8.9)6 h 103.0 (4.1) 63.0 (7.0) 78.1 (6.2) 70.8 (4.8)12 h 50.0 (10.7) 102.0 (3.9)** 81.9 (5.5)18 h 100.6 (4.7)** 105.2 (8.8)**

a Recovery efficiency during valve closure.* Comparisons between reproductive stages for 2 min (P,0.05); ** Statistically different from the earlier

test (P,0.05), indicate that recovery was completed.

3.4. Biochemical composition of muscle at different reproductive stages

The concentration of glycogen in the phasic muscle decreased markedly as the gonadmatured and remained low until after spawning (Fig. 4). The decrease in muscleglycogen coincided with a strong increase of total lipid and protein in the gonads ofmature and prespawning scallops (Fig. 1). Total protein concentration in the phasicmuscle remained stable during the course of gonadal maturation and spawning (Fig. 4).

Fig. 3. Proportion of Chlamys islandica at different reproductive stages that responded to restimulation withthe seastar, 2 min after the end of glycolytic recuperation (closed valves) (n58). Bars sharing the same letters

2were not significantly different (P,0.05) as determined by a x -test.

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21Fig. 4. Concentration of glycogen (mmol glucosyl U g wet mass), total, structural and sarcoplasmic proteins21 21(mg g wet mass) and of arginine phosphate (mmol g wet mass) in the phasic adductor muscle of the

scallop Chlamys islandica at different reproductive stages. Values represent means6S.E. (n59–14). Meanssharing the same letters were not significantly different (P,0.01) as determined by Tukey multiplecomparisons. In muscle protein, different kind of lettering indicate different comparisons.

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 217

However, structural protein oscillated and the level of sarcoplasmic protein fell slightlybefore spawning. Muscle arginine phosphate gradually increased during gonadalmaturation and was highest before and after spawning (Fig. 4).

3.5. Muscle enzyme levels at different reproductive stages

With the exception of AK, the enzymes showed similar fluctuations in activity duringgonadal maturation and spawning (Fig. 5). The glycolytic enzymes, GP, PFK, PK andODH, and the mitochondrial enzyme, CS, showed their highest levels in immaturescallops and levels declined with gonadal maturation. PK recovered its initial levelsbefore spawning, but showed its lowest level after spawning. Much like the levels ofarginine phosphate, AK activity increased during the course of gonadal maturation,reaching a maximum before spawning (Fig. 5).

Thus, the levels of enzymes that participate in the recovery of the adductor musclefrom exhausting escape responses decreased during gonadal maturation and spawning. Incontrast, the enzyme which generates most of the ATP required for the escape responsegradually increased its activity during gonadal maturation and spawning.

3.6. Oxidative capacities of mitochondria and adductor muscle during thereproductive cycle

Rates of mitochondrial oxygen uptake and respiratory coupling varied with substrateand reproductive stage (Fig. 6). Glutamate and succinate gave highest oxidation rates atmost times. Rates of glutamate and malate oxidation changed little during thereproductive cycle. In contrast, mitochondrial rates of pyruvate and succinate oxidationdecreased considerably after gonadal maturation and remained low after spawning (Fig.6). For all substrates, the respiratory control ratio (RCR) decreased during thereproductive cycle (Fig. 6). The RCR values for glutamate and pyruvate oxidation werelower in mitochondria from mature and spawned scallops, whereas those for theoxidation of malate and succinate declined only after spawning.

Total muscle oxidative capacities were calculated from mitochondrial rates ofsubstrate oxidation, mitochondrial CS levels and muscle CS levels. Reproductive statusmarkedly affected the muscle’s capacity for oxidizing pyruvate and succinate, withdecreases occurring with maturation and spawning (Fig. 6). As pyruvate generated fromoctopine is metabolized during recuperation, the total capacity for pyruvate oxidation ismost pertinent to evaluation of the capacity for recuperation from exhausting escaperesponses. Overall, the mitochondrial oxidative capacities, total muscle aerobic capacityand activities of enzymes involved in glycolytic and aerobic phases of recovery followedvirtually the same pattern, a decrease in capacity with maturation and spawning.

4. Discussion

In scallops, depletion of glycogen and protein from the adductor muscle duringgonadal maturation has been shown many times (reviewed by Barber and Blake, 1991).

218 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

21Fig. 5. Enzyme activities (U g wet mass) in the phasic adductor muscle of the scallop Chlamys islandica atdifferent reproductive stages. Values represent means6S.E. (n57–12). Means sharing the same letters werenot significantly different (P,0.01) as determined by Tukey multiple comparisons.

However, our study of the Iceland scallop, Chlamys islandica, is the first to show thatthis substrate mobilization has a detrimental effect upon muscle physiology. Effectivelysubstrate mobilization is paralleled by a reduction in the metabolic capacity of the

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 219

21 21Fig. 6. Maximal oxidation rates (nmol O min mg mitochondrial protein ), respiratory control ratios (state221 213 / state 4) and calculated maximal muscle aerobic capacity (mmol O g min ) for mitochondria isolated2

from the phasic muscle of the scallop Chlamys islandica at different reproductive stages, oxidizing glutamate,malate, pyruvate and succinate. Values represent means6S.E. (n53–9). Means sharing the same letters werenot significantly different (P,0.01) as determined by Tukey multiple comparisons.

220 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

adductor muscle and in the organismal capacity to recover from exhausting escaperesponses. Both gonadal maturation and spawning decreased these parameters.

The decrease of the recovery efficiency with gonadal maturation and spawningoccurred both during valve closure and after valves had reopened. The time scallopsspent with their valves closed after exhausting exercise was not markedly affected by thereproductive stage. Only mature scallops spent less time closed. Given that prolongedvalve closure is powered exclusively by the tonic (catch) part of the adductor muscle (deZwaan et al., 1980), the time scallops spent closed could be related to the metaboliccapacity of this section. However, the energetic cost of valve closure is only 1% of thatof valve claps (de Zwaan et al., 1980). Therefore, the metabolic capacities of the tonicmuscle should not strongly affect its capacity to maintain the valves closed andlimitations may arise from the requirements of other tissues including the phasicadductor muscle. Since we did not measure the energetic status and metabolic capacitiesof the tonic muscle, studies of this muscle are required to confirm this interpretation.

The extent of recovery during valve closure could have been affected by its duration.However, spawned scallops showed the least recovery, although they spent the sametime closed as immature and prespawned animals, which had the highest percentage ofrecovery (Table 2, Fig. 3). The negative impact of gonadal maturation and spawning onrecovery during valve closure may be due to the lower levels of the glycolytic enzymes.Reliance upon glycolysis is likely since, in analogy with the responses of P. magel-lanicus, the scallops probably consumed most of their oxygen reserves during valveclapping and took up no oxygen during valve closure (Thompson et al., 1980). GP andPFK are important sites of metabolic control in the adductor muscle of Argopectenirradians concentricus (Chih and Ellington, 1986) and ODH is used during therestoration of ATP and arginine phosphate pools when oxygen supplies are depleted orabsent as during valve closure (Livingstone et al., 1981). The fact that the activities ofthese enzymes are diminished after gametogenesis and spawning in C. islandica, couldexplain the reductions in glycolytic recovery.

In Placopecten magellanicus, the complete restoration of the phosphoarginine poolafter exhaustive swimming requires aerobic metabolism (Livingstone et al., 1981). Forthe phasic muscle of C. islandica, the levels of CS, the mitochondrial capacity (and RCRvalues) for pyruvate oxidation and the total muscle aerobic capacity decreased aftergonadal maturation and remained low after spawning. These reductions could explainwhy mature and spawned scallops needed 14 h more to recover their initial clappingcapacity than immature animals (Table 2). In the adductor muscle of the tropical scallop,Euvola ziczac, mitochondrial capacities for pyruvate oxidation were lower during thefirst spawning, which occurred after a period of low food availability, than during otherperiods (Boadas et al., 1997). As pyruvate is the principal substrate to be oxidized aftermuscular activity (Chih and Ellington, 1986), a reduced capacity for pyruvate oxidationcould slow the aerobic recovery of mature and spawned scallops from exhausting escaperesponses.

The impact of the reproductive cycle upon aerobic recovery could reflect theinstantaneous metabolic costs of gonadal maturation and feeding. In P. magellanicus,oxygen uptake rises during gonadal maturation and with increases in food availabilityand temperature (Shumway et al., 1988). If phytoplankton levels in the water pumpedinto our wet lab were similar to those near the scallop bed (Fig. 2), resting maturing

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 221

animals would have been feeding at higher levels than immature or prespawninganimals, thereby reducing the aerobic scope for recuperation. The metabolic costs ofgonadal maturation would have exacerbated this effect. In accordance with thisinterpretation, prespawning scallops partially regained the capacity to recover fromexercise, compared to mature animals (Table 2, Fig. 3). However, spawning decreasedthe percentage of aerobic recuperation even more than gonadal maturation (Table 2, Fig.3). As increased metabolic rates due to feeding and gonadal maturation would notexplain this decrease, reductions in muscle metabolic capacity are a more likelyexplanation for the slowed rates of recuperation. In Argopecten irradians concentricusspawning leads to a state of negative energy balance and generally poor physiologicalcondition, as indicated by a loss of carbon 14 from all body components (Barber andBlake, 1985). Assays of oxygen uptake at rest and during aerobic recovery wouldascertain the extent to which gonadal maturation and feeding reduce the aerobic scopefor recuperation.

Changes in the composition of body components should indicate which biochemicalcomponents contribute to energy metabolism during gametogenesis (Barber and Blake,1991). In C. islandica, even though food availability was high during the accumulationof gonadal lipid and protein (Fig. 2), muscle glycogen decreased strongly while muscleprotein changed little (Figs. 1 and 4). Therefore, muscle glycogen is likely a majorprecursor for lipid and protein synthesis in the gonad. This agrees with patterns foundfor C. islandica in Norway (Sundet and Vahl, 1981; Sundet and Lee, 1984); for Chlamysvaria (Shafee, 1981) and Placopecten magellanicus (Robinson et al., 1981). However,in some scallops, such as Argopecten irradians concentricus, muscle protein is also usedto support gonadal maturation after the depletion of glycogen (Barber and Blake, 1981).In this subspecies, physiological indices, such as O/NH and CO /O (respiratory3 2 2

quotient), indicate that gametogenesis is initially supported by lipid. Then, during thecytoplasmic growth of oocytes, gametogenesis is supported by muscle glycogen, whichis converted to lipid. Finally, near and after spawning, metabolism is fueled by protein(Barber and Blake, 1985). In contrast, in A. irradians irradians muscle carbohydratesmake a negligible contribution to gonadal development, rather gametogenesis occursmainly at the expense of adductor muscle protein, which accounts for 63–99% ofgonadal buildup in the spring (Epp et al., 1988). Total protein in the adductor muscle ofC. islandica did not change during gonadal maturation and spawning (Fig. 4). Asgonadal maturation is completed during spring and summer, when chlorophyll aconcentrations are the highest (Spence and Steven, 1974; Arsenault and Himmelman,1998), external food supplies may decrease the need to mobilize muscle protein.

As the protein content of the adductor muscle did not decrease during gonadalmaturation and spawning in C. islandica, the coincident decrease of glycolytic andmitochondrial enzymes during these processes was not due to generalized proteinmobilization. During our study, the water content of the phasic adductor muscleremained constant and changes in enzyme levels were the same when expressed permilligram of protein (data not shown). The changes in enzyme levels may reflect aspecific proteolysis. Due to the high energy demand during reproduction, proteinsynthesis in muscle could be reduced. Similarly, myosin synthesis was diminishedduring the intensive synthesis of vitellogenin in the liver of the salmon, Salmo salar(Olin and von der Decken, 1987). Moreover, during the spawning migration of sockeye

222 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

salmon, Oncorhynchus nerka, muscle enzymes show different rates of catabolism thanthe general categories of soluble and insoluble proteins (Mommsen et al., 1980).Whereas we do not know why scallops sacrifice the enzymes that facilitate recuperationof muscle performance, we suggest this may be a consequence of glycogen mobilization.It has been suggested that glycolytic enzymes are attached to glycogen particles(Hubbard and Cohen, 1989; Lam, 1990; Vardanis, 1990). In skeletal muscle of rabbit,GP accumulates concomitantly with glycogen (Francois et al., 1992). Thus, a decrease inglycogen levels could reduce binding sites for glycolytic enzymes. Once enzymes are insolution in the cytoplasm, they could be more susceptible to degradation. Theconcomitant decline of glycogen and glycolytic enzymes with gonadal maturationsupports such a mechanism.

Contrary to our initial premise, arginine phosphate, the principal fuel for musclecontraction during the escape response, increased after gonadal maturation and afterspawning (Fig. 4). Although the arginine phosphate levels we measured are lower thanthose previously obtained (possibly as our technique for sampling the muscle was slowerthan that of other workers), the relative levels should be reliable, as we applied the samesampling technique to each reproductive stage. Moreover, AK, the enzyme that convertsarginine phosphate to ATP and arginine, also increased after gametogenesis and itslevels remained high after spawning (Fig. 5). These results may partly explain why thenumber of claps did not change during reproduction of C. islandica (Table 1).

The fact that mature and spawned scallops need more time to recuperate afterexhausting burst exercise may have ecological significance. After gonadal maturationand spawning, scallops could be more susceptible to predation and to capture bycommercial fisheries. This is particularly true since many scallop species are captured bytrawling or dragging (Joll, 1989; C. islandica in the northern Gulf of St. Lawrence),where their vulnerability to capture will depend on their capacity to escape andrecuperate. This is particularly true since trawls or drags may be used repeatedly in thesame area of the scallop bed.

Interindividual differences in the locomotor performance of vertebrates can be due tophysiological changes or morphological variability (Garland, 1984; Walsberg et al.,1986; Bennett et al., 1989; Kolok, 1992); to size, age (Garenc et al., 1999), thermalacclimation (Elliott, 1991; Johnson and Bennett, 1995) or reproductive stage (Koch andWieser, 1983; Calow, 1985; James and Johnston, 1998). Many of these factors modify

¨muscle metabolic capacities (Sanger, 1993). In invertebrates, the evidence for inter-individual differences in locomotor performance is limited, nevertheless, there arestudies, particularly for scallops, that show that swimming performance changes withmorphology (Gould, 1971) and size or age (Winter and Hamilton, 1985; Joll, 1989;Manuel and Dadswell, 1991). Most of the differences have been attributed tohydrodynamic changes with size (Manuel and Dadswell, 1991). Our study demonstratesa marked effect of reproduction on the recuperation of clapping capacity after exhaustiveexercise and on muscle metabolic capacity in scallops.

Acknowledgements

We are particularly grateful to Carlos Gaymer for field, laboratory and statistical

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 223

support and valuable comments on the manuscript. We also thank Martin Lafrance andMartin Giasson for technical assistance. This work was supported by operating grantsfrom the NSERC to HEG and JHH. KBB was recipient of scholarships from the

´ ´ ´Gouvernement du Quebec, Departement de Biologie de l’Universite Laval andGIROQ. [SS]

References

¨Arsenault, D.J., Himmelman, J.H., 1998. Spawning of the Iceland scallop (Chlamys islandica Muller, 1776) inthe northern Gulf of St. Lawrence and its relationship to temperature and phytoplankton abundance. Veliger41, 180–185.

Ballantyne, J.S., Moon, T.W., 1985. Hepatopancreas mitochondria from Mytilus edulis: substrate preferencesand effects of pH and osmolarity. Mar. Biol. 87, 239–244.

Barber, B.J., Blake, N.J., 1981. Energy storage and utilization in relation to gametogenesis in Argopectenirradians concentricus (Say). J. Exp. Mar. Biol. Ecol. 52, 121–134.

Barber, B.J., Blake, N.J., 1985. Substrate catabolism related to reproduction in the bay scallop Argopectenirradians concentricus, as determined by O/N and RQ physiological indexes. Mar. Biol. 87, 13–18.

Barber, B.J., Blake, N.J., 1991. Reproductive physiology. In: Shumway, S.E. (Ed.), Scallops: Biology, Ecologyand Aquaculture. Elsevier, Amsterdam, pp. 377–428.

Bates, P.C., Milllard, D.J., 1983. Myofibrillar protein turnover: synthesis rates of myofibrillar and sarcoplasmicprotein fractions in different muscles and the changes observed during postnatal development and inresponse to feeding and starvation. Biochem. J. 214, 587–592.

Beaulieu, M.A., Guderley, H., 1998. Changes in qualitative composition of white muscle with nutritional statusof Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. A. 121, 135–141.

Bennett, A.F., Garland, T., Else, P.L., 1989. Individual correlation of morphology, muscle mechanics andlocomotion in a salamander. Am. J. Physiol. 256, R1200–R1208.

Boadas, M.A., Nusetti, O., Mundarain, F., Lodeiros, C., Guderley, H., 1997. Seasonal variation in theproperties of muscle mitochondria from the tropical scallop Euvola (Pecten) ziczac. Mar. Biol. 128,247–255.

Bonardelli, J.C., Himmelman, J.H., 1995. Examination of assumptions critical to body component indices:application to the giant scallop Placopecten magellanicus. Can. J. Fish. Aquat. Sci. 52, 2457–2469.

Brand, A.R., 1991. Scallop ecology: distribution and behaviour. In: Shumway, S.E. (Ed.), Scallops: Biology,Ecology and Aquaculture. Elsevier, Amsterdam, pp. 517–584.

Calow, P., 1985. Adaptive aspects of energy allocation. In: Tyler, P., Calow, P. (Eds.), Fish Energetics: NewPerspectives. The Johns Hopkins University Press, Maryland, pp. 13–32.

Chih, P.C., Ellington, W.S., 1983. Energy metabolism during contractile activity and environmental hypoxia inthe phasic adductor muscle of the bay scallop Argopecten irradians concentricus. Physiol. Zool. 56,623–631.

Chih, P.C., Ellington, W.S., 1986. Control of glycolysis during contractile activity in the phasic adductormuscle of the bay scallop, Argopecten irradians concentricus: identification of potential sites of regulationand a consideration of the control of octopine dehydrogenase activity. Physiol. Zool. 59, 563–573.

Elliott, J.M., 1991. Tolerance and resistance to thermal stress in juvenile Atlantic salmon, Salmo salar. Freshw.Biol. 25, 61–70.

Epp, J., Bricelj,V., Malouf, R., 1988. Seasonal partitioning and utilization of energy reserves in two age classesof the bay scallop Argopecten irradians irradians. J. Exp. Mar. Biol. Ecol. 121, 113–136.

Estabrook, R.W., 1967. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios.Methods Enzymol. 10, 41–47.

Folch, J.M., Lee, S.M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of totallipids from animal tissues. J. Biol. Chem. 226, 497–509.

Francois, J.M., Thompson-Jaeger, S., Skroch, J., Zellenka, U., Spevak, W., Tatchell, K., 1992. GAC1 mayencode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae. Eur. Mol. Biol.Org. J. 11, 87–96.

224 K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225

Gagnon, R., Lehoux, E., Guderley, H., 1998. Manually operated freeze clamping press. Anal. Biochem. 260,251–252.

Garenc, C., Couture, P., Laflamme, M.A., Guderley, H., 1999. Metabolic correlates of burst swimmingcapacity of juvenile and adult threespine stickleback (Gasterosteus aculeatus). J. Comp. Physiol. B 169,113–122.

Garland, Jr. T., 1984. Physiological correlates of locomotory performance in a lizard: an allometric approach.Am. J. Physiol. 247, R806–R815.

Gould, S.J., 1971. Muscular mechanics and the ontogeny of swimming in scallops. Paleontology 14, 61–94.Graham, M., 1987. The solubility of oxygen in physiological salines. Fish Physiol. Biochem. 4, 1–4.Guderley, H.E., Rojas, F.M., Nusetti, O.A., 1995. Metabolic specialization of mitochondria from scallop phasic

muscle. Mar. Biol. 122, 409–416.Himmelman, J.H., 1991. Diving observations of subtidal communities in the northern Gulf of St. Lawrence. In:

Therriault, J.C. (Ed.), The Gulf of St. Lawrence Small Ocean or Big Estuary. Can. Spec. Publ. Fish. Aquat.Sci, Vol. 113, pp. 319–332.

Hubbard, M.J., Cohen, P., 1989. Regulation of protein phosphatase 1-G from rabbit skeletal muscle: 1.Phosphorylation by cyclic AMP-dependent protein kinase at site 2 releases catalytic subunit fromglycogen-bound holoenzyme. Eur. J. Biochem. 186, 701–710.

James, R.S., Johnston, I.A., 1998. Influence of spawning on swimming performance and muscle contractileproperties in the short-horn sculpin. J. Fish Biol. 53, 485–501.

Johnson, T.P., Bennett, A.F., 1995. The thermal acclimation of burst escape performance in fish: an integratedstudy of molecular and cellular physiology and organismal performance. J. Exp. Biol. 198, 2165–2175.

Joll, L.M., 1989. Swimming behaviour of the saucer scallop Amusium balloti (Mollusca: Pectinidae). Mar.Biol. 102, 299–305.

Keppler, D., Decker, K., 1974. Glycogen determination with amyloglucosidase. In: Bergmeyer, H.U. (Ed.).Methods of Enzymatic Analysis, Vol. 3. Academic Press, New York, pp. 1127–1131.

Koch, F., Wieser, W., 1983. Partitioning of energy in fish: can reduction of swimming activity compensate forthe cost of production? J. Exp. Biol. 107, 141–146.

Kolok, A.S., 1992. Morphological and physiological correlates with swimming performance in juvenilelargemouth bass. Am. J. Physiol. 263, R1042–R1048.

Lam, R., 1990. The nature of cytoplasmic vacuoles in chordoma cells: a correlative enzyme and electronmicroscopic histochemical study. Pathol. Res. Pract. 186, 642–650.

Lamprecht, W., Trautschold, I., 1974. Adenosine triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer, H.U. (Ed.). Methods of Enzymatic Analysis, Vol. 4. AcademicPress, New York, pp. 2101–2110.

Lamprecht, W., Stein, P., Wieser, H., 1974. Creatine phosphate. In: Bergmeyer, H.U. (Ed.). Methods ofEnzymatic Analysis, Vol. 4. Academic Press, New York, pp. 1777–1781.

Livingstone, D.R., de Zwaan, A., Thompson, R.J., 1981. Aerobic metabolism, octopine production andphosphoarginine as sources of energy in the phasic and catch adductor muscles of the giant scallopPlacopecten magellanicus during swimming and the subsequent recovery period. Comp. Biochem. Physiol.70B, 35–44.

Manuel, J.L., Dadswell, M.L., 1991. Swimming behaviour of juvenile giant scallop Placopecten magellanicus,in relation to size and temperature. Can. J. Zool. 69, 2250–2254.

´Martınez, G., 1991. Seasonal variation in biochemical composition of three size classes of the Chilean scallop,Argopecten purpuratus Lamarck, 1819. Veliger 34, 335–343.

´Martınez, G., Mettifogo, L., 1998. Mobilization of energy from adductor muscle for gametogenesis of thescallop, Argopecten purpuratus Lamarck. J. Shellfish Res. 17, 113–116.

Mommsen, T.P., French, C.J., Hochachka, P.W., 1980. Sites and patterns of protein and amino acid utilizationduring the spawning migration of salmon. Can. J. Zool. 58, 1785–1799.

Olin, T., von der Decken, A., 1987. Estrogen treatment and its implication on vitellogenin and myosinsynthesis in salmon (Salmo salar). Physiol. Zool. 60, 346–351.

Olson, J.M., Marsh, R.L., 1993. Contractile properties of the striated adductor muscle in the bay scallopArgopecten irradians at several temperatures. J. Exp. Biol. 176, 175–193.

Parsons, T.R., Maita, Y., Lalli, C.M. (Eds.), 1984. A Manual of Chemical and Biological Method for SeawaterAnalysis. Pergamon Press, Toronto, p. 173.

K.B. Brokordt et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 205 –225 225

Robinson, W.E., Wehling, W.E., Morse, M.P., McLeod, G.C., 1981. Seasonal changes in soft-body componentindices and energy reserves in the Atlantic deep-sea scallop, Placopecten magellanicus. Fish. Bull. 79,449–458.

¨Sanger, A.M., 1993. Limits to the acclimation of fish muscle. Rev. Fish Biol. Fish. 3, 1–15.SAS, 1991. In: SAS/STAT User’s Guide, release 6.03 Edition. SAS Institute Press, Cary, NC, p. 1028.Shafee, M.S., 1981. Seasonal changes in the biochemical composition and calorific content of the black scallop

Chlamys varia (L.) from Lanvec, Bay of Brest. Oceanol. Acta 4, 331–341.Shevechenko, V.V., 1972. The dynamics of protein and fat content in the organs and tissues of the north sea

haddock Melanogrammus aeglefinus in the process of seasonal growth and gonad maturation. J. Ichthyol.12, 830–842.

Shumway, S.E., Barter, J., Stahlnecker, J., 1988. Seasonal changes in oxygen consumption of the giant scallop,Placopecten magellanicus (Gmelin). J. Shellfish Res. 7, 77–82.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Matha, A.K., Garther, F.H., Provenzano, M.D., Fujimoto, E.K.,Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurements of protein using bicinchoninic acid. Anal.Biochem. 150, 76–85.

Snedecor, G.W., Cochran, W.G. (Eds.), 1989. Statistical Methods. Iowa State University Press, Ames, Iowa, p.503.

Sokal, R.R., Rohlf, F.J. (Eds.), 1981. Biometry. W.H. Freeman, New York, p. 859.Somero, G.N., Childress, J.J., 1990. Scaling of ATP-supplying enzymes, myofibrillar proteins and buffering

capacity in fish muscle: relationship to locomotory habit. J. Exp. Biol. 149, 319–333.Spence, C., Steven, D.M., 1974. Seasonal variation of the chlorophyll a: pheopigment ratio in the Gulf of St.

Lawrence. J. Fish. Res. Bd Can. 31, 1263–1268.Sundet, J.H., Vahl, O., 1981. Seasonal changes in dry weight and biochemical composition of the tissues of

sexually mature and immature Iceland scallops Chlamys islandica. J. Mar. Biol. Assoc. UK 61, 1001–1010.Sundet, J.H., Lee, J.B., 1984. Seasonal variations in gamete development in the Iceland scallop Chlamys

islandica. J. Mar. Biol. Assoc. UK 64, 411–416.Thompson, R.J., Livingstone, D.R., de Zwaan, A., 1980. Physiological and biochemical aspects of valve snap

and valve closure responses in the giant scallop Placopecten magellanicus. I. Physiology. J. Comp. Physiol.137, 97–104.

´ ¨ ¨Thorarinsdottir, G.G., 1993. The Iceland scallop, Chlamys islandica (O.F. Muller), in Breidafjodur, westIceland. II. Gamete development and spawning. Aquaculture 110, 87–96.

Vardanis, A., 1990. Fractionation of particulate glycogen and bound enzymes using high-performance liquidchromatography. Anal. Biochem. 187, 115–119.

Von der Decken, A., 1992. Physiological changes in skeletal muscle by maturation-spawning of non-migratingfemale Atlantic salmon, Salmo salar. Comp. Biochem. Physiol. 101B, 299–301.

Walsberg, G.E., Lean, M.S., Hillman, S.S., 1986. Individual variation in maximum aerobic capacity:cardiovascular and enzymatic correlates in Rana catesbiana. J. Exp. Zool. 239, 1–5.

Winter, M.A., Hamilton, P.V., 1985. Factors influencing swimming in bay scallops, Argopecten irradians(Lamarck, 1819). J. Exp. Mar. Biol. Ecol. 88, 227–242.

de Zwaan, A., Thompson, R.J., Livingstone, D.R., 1980. Physiological and biochemical aspects of the valvesnap and valve closure responses in the giant scallop Placopecten magellanicus. II. Biochemistry. J. Comp.Physiol. 137, 105–114.