Effect of reproduction on escape responses and muscle metabolic capacities in the scallop Chlamys islandica Müller 1776

Journal of Experimental Marine Biology and Ecology 251 (2000) 205–225

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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 ´ ´ du Departement de Biologie and GIROQ ( Groupe Interuniversitaire de Recherches Oceanographiques ´ ), Universite´ Laval, Cite´ Universitaire, Ste.-Foy, Quebec ´ , Canada G1 K 7 P4 Quebec 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 adductor muscle towards the gonad. This mobilization is likely to diminish the metabolic capacities of the adductor muscle and thereby the scallops’ escape response. We examined the escape response in terms of number of valve claps until exhaustion, rate of clapping and the recovery during and after valve closure in adult scallops, Chlamys islandica, sampled at different stages in the reproductive cycle (immature, mature, before and after spawning). In parallel, we measured muscle glycogen, protein and phosphoarginine contents, the oxidative capacity of mitochondria isolated from the adductor muscle and levels of muscle enzymes which are active during exercise and recovery. The number of claps (24–26), rate of clapping ( | 13 claps min 21 ) and phosphoarginine and arginine kinase levels were similar during the different reproductive stages. All immature scallops responded to restimulation immediately after opening their valves, while only 62% of mature, 82% of prespawned and 38% of spawned scallops responded. Immature animals completely recovered their initial swimming capacity within 4 h of opening their valves, but mature, prespawned and spawned scallops needed 18, 12 and 18 h, respectively. Overall phasic adductor muscle from mature, prespawned and spawned animals showed decreased glycogen phosphorylase, phosphofructokinase, pyruvate kinase (except for prespawned), octopine dehydrogenase and citrate synthase levels, a deterioration of the oxidative capacity of mitochondria and a marked decrease in glycogen content compared to immature scallops. Therefore, during gonadal maturation and spawning, C. islandica did not change its clapping capacity, but slowed its recuperation from exhausting burst exercise, both during and after valve closure, likely due to the decreased metabolic 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: [email protected] (K.B. Brokordt). 0022-0981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 00 )00215-X

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1. Introduction Gametogenesis represents a period of high energy demand, particularly for organisms with broadcast spawning, and when external food supplies are limited, gamete production 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 gonadal maturation, with both protein and glycogen levels decreasing (Shevechenko, 1972; reviewed by Barber and Blake, 1991; Von der Decken, 1992). Such mobilization of macromolecules could decrease muscle metabolic capacities. In scallops the adductor muscle is one of the largest soft tissues and while its primary role is the movement of the valves during escape responses or swimming, it also serves as a major site of reserve deposition. In the bay scallop Argopecten irradians concentricus, 63–99% of total energy gain by the gonad during gametogenesis is thought to come from the adductor muscle (Epp et al., 1988). In this species, gametogenesis occurs mainly at the expense of muscle protein, however, in other scallops 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 the black scallop Chlamys varia, during the spring reproduction, when food is abundant, only carbohydrate reserves are used for gonadal development, while during the autumn reproduction, 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 mobilized during gonadal maturation and their non-feeding spawning migration, but the levels of enzymes and contractile proteins are also affected (Mommsen et al., 1980; Olin and von der Decken, 1987). In the roach Rutilus rutilis, swimming activity is markedly reduced during gonadal maturation (Koch and Wieser, 1983), either due to the metabolic costs of gonad maturation or to decreases in muscle metabolic capacities. A decrease of muscle metabolic capacities after gonadal maturation and spawning could reduce an animal’s locomotor 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 alternate adduction and abduction of the valves (Olson and Marsh, 1993). Swimming is powered by the adductor muscle, principally by the large phasic portion and to a lesser extent by the smaller tonic (catch) muscle (de Zwaan et al., 1980). The physiological and biochemical aspects of the escape response (valve clapping and valve 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 bay scallop Argopecten irradians concentricus (Chih and Ellington, 1983, 1986). In Placopecten magellanicus, the main source of ATP during valve clapping is arginine phosphate, and anaerobic glycolysis, supported by octopine accumulation, only makes a small contribution (de Zwaan et al., 1980). Following intense valve clapping, partial recuperation of arginine phosphate occurs during valve closure with further accumulation of octopine.

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Once the valves have opened, the full restoration of arginine phosphate pools and elimination of octopine is achieved aerobically (Livingstone et al., 1981). Similarly, in Argopecten irradians concentricus intense valve clapping is initially supported by arginine phosphate and only toward the end do anaerobic glycolysis and octopine accumulation intervene (Chih and Ellington, 1983, 1986). Mitochondria isolated from the adductor muscle of the tropical scallop, Euvola ziczac, seem adapted for a role in recovery metabolism given their increased affinity for pyruvate at the pH values likely to occur in muscle fibers after intense clapping (Guderley et al., 1995). Oxidative capacities and respiratory control ratios of mitochondria isolated from the adductor muscle are lower during the first of two spawnings than during other periods (Boadas et al., 1997), suggesting an impact of reproduction. We reasoned that changes in muscle metabolic capacities or in the levels of energetic reserves during the reproductive cycle of adult scallops could modify the escape response or recuperation from an exhausting escape response. We predicted that before gonadal maturation, scallops would show a stronger escape response and would recuperate faster from exhausting burst exercise given the higher muscle metabolic capacities and energy reserves the scallops should have at this time. To evaluate these hypotheses, we compared the escape response and capacity for recuperation from exhausting exercise for adult Iceland scallops, Chlamys islandica, sampled at different stages in the annual reproductive cycle (immature, mature, before and after spawning). In parallel, we determined the effect of these reproductive stages on the energetic reserves in the phasic adductor muscle (arginine phosphate, glycogen and proteins). Given that in fish muscle, starvation leads to more extensive mobilization of sarcoplasmic than structural (insoluble) proteins (Beaulieu and Guderley, 1998), we quantified both fractions. To assess the impact of the reproductive cycle on muscle metabolic capacities, we measured muscle levels of the glycolytic enzymes, glycogen phosphorylase (GP), pyruvate kinase (PK), phosphofructokinase (PFK), octopine dehydrogenase (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 phasic adductor muscle at different reproductive stages. By profiling these enzyme activities and mitochondrial capacities we sought to assess the capacity of enzymes likely to supply ATP for contraction (AK and glycolytic enzymes (GP, PFK, PK and ODH)), recuperation during valve closure (glycolytic enzymes) and recuperation after reopening of the valves (CS and mitochondrial capacities).

2. Materials and methods

2.1. Sampling and maintenance of scallops ˆ The population studied was located at the southern side of ˆIle du Fantome in the Mingan Islands, northern Gulf of St. Lawrence, eastern Canada. Samples of 78 adult scallops (80–95 mm shell height) were collected by scuba-diving at 37 m from June through August 1996 at the following reproductive stages: immature, mature, before

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spawning (prespawned) and after spawning (spawned). Reproductive stage was determined using qualitative and quantitative criteria. As qualitative criteria we used the shape of the gonad and the degree of vascularization by secondary genital ducts which were observable through the gonadal epithelium (David Arsenault, personal communication). Histological classification used the gonadal developmental stages described for ´ Chlamys islandica by Thorarinsdottir (1993). As quantitative criteria, we determined the gonadosomatic index corrected for the size of the scallop ((gonadal mass 3 total tissue mass 21 ) 3 shell height 21 ), as described by Bonardelli and Himmelman (1995). We also evaluated 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 was rapidly 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 enzyme activities and arginine phosphate concentrations. The remainder was frozen at 2 208C for 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´ Laval in Quebec City where they were maintained in sea water aquaria and fed with Isochrysis galbana and Chaetoceros gracilis for at least one day, before determinations of the respiratory capacity of muscle mitochondria. The remaining 48 scallops were acclimated during 2 days in holding tanks with running sea water pumped from 10 m in depth at Havre St. Pierre, and then used to evaluate escape responses. Despite the variations in ambient temperature, at the time of measurements of escape responses in the laboratory water temperatures were consistently 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. Phytoplankton abundance was quantified periodically from water samples collected with a Niskin bottle from 1 m above the scallop bed. From each sample, chlorophyll a determinations were made 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, the seastar Leptasterias polaris (Himmelman, 1991), the escape response is highly stereotyped and reproducible. It begins with a series of valve claps and if the stimulus is maintained until fatigue, the scallop closes its valves firmly and remains closed for a certain period, after which the valves slowly reopen. Based on this behavior, we carried out the following experiments: 48 scallops, each placed in different aquarium, were stimulated until fatigue, by repeatedly touching the tentacles and mantle with an arm tip of L. polaris. We counted the number and duration of claps, and measured the period that the scallops kept their valves closed. After fatigue, the scallops were separated into six groups of eight individuals each. The groups

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

2.4. Metabolite assays 2.4.1. Muscle phosphoarginine Deproteinization of the samples followed the method described by Lamprecht and Trautschold (1974). The neutralized perchloric acid extracts were used immediately for the determination of phosphoarginine, in an assay analogous to the phosphocreatine assay described by Lamprecht et al. (1974). 2.4.2. Muscle glycogen The concentration of glycogen was determined by enzymatic hydrolysis with amyloglucosidase as described by Keppler and Decker (1974). 2.4.3. Muscle and gonad protein Muscle 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 acid method of Smith et al. (1985) with bovine serum albumin as the standard. 2.4.4. Gonadal lipid The lipid concentration in the gonad was estimated gravimetrically after extraction with chloroform–methanol according to the method of Folch et al. (1957), except that after 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 2 (ethylene dinitrilotetraacetic acid), 5 mM EGTA (ethyleneglycol 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 g at 48C for 15 min for the assay of GP and PFK, and another at 600 3 g at 48C for 15 min for 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 the exception of CS which was monitored at 412 nm to detect the transfer of sulfydryl groups from CoASH to 5,59-dithio-bis(2-nitro)benzoic acid (DTNB). The molar extinction 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 international units (mmol of substrate converted to product min 21 ) 3 g 21 wet mass.

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Conditions for enzyme assays (except for CS) were adapted from conditions used by de Zwaan et al. (1980) for P. magellanicus, as follows (all concentrations in mmol l 21 ):

2.5.1. Glycogen phosphorylase ( EC 2.4.1.1) Imidazole–HCl 50, KH 2 PO 4 80, Mg-acetate 5, EDTA 2.5, 10 mg ml 21 glycogen (omitted for the control), AMP 0.8, AMP cyclic 0.5, NADP 0.6, glucose-1,6-diphosphate 0.004, 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-3phosphate dehydrogenase and triosephosphate isomerase, pH 7.5. 2.5.3. Pyruvate kinase ( EC 2.7.1.40) Imidazole–HCl 50, MgSO 4 13, KCl 100, phosphoenolpyruvate 5 (omitted for the 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 2 5, phosphoarginine 5 (omitted for the control), glucose 10, ADP 0.4, NADP 0.6, excess hexokinase and glucose-6-phosphate dehydrogenase, pH 6.6. 2.5.5. Octopine dehydrogenase Imidazole–HCl 50, EDTA-Na 2 2, EGTA 5, KCN 1, sodium pyruvate 5 (omitted for 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 measurements All procedures followed Guderley et al. (1995) as adapted from Ballantyne and Moon (1985), except that media were prepared in nanopure water. Mitochondrial oxygen uptake was measured in a water-jacketed respiration cell (Cameron Instruments) using a Clarke-type electrode (Yellow Springs Instrument Comp., Yellow Springs, OH). The electrode was connected to a chart recorder (Linear, model 0585, Canlab Comp.) by an oxymeter (Cameron Instruments, Port Aransas, TX). The respiration chamber was maintained at 68C by a circulating refrigerated water bath. The values of oxygen concentration were calculated from the data of Graham (1987) for physiological buffers using the atmospheric pressure on the day of assay. Around 1.3 mg mitochondrial protein was added to 1 ml of assay medium. The reaction medium contained (in mmol l 21 ): sucrose 480, Hepes 70, KCl 100, KH 2 PO 4 10, taurine 50, b-alanine 50, 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 final concentrations of 30, 6, 0.9 and 24 mmol l 21 , respectively, gave maximal rates. To attain maximal rates of pyruvate oxidation, ‘sparking’ levels of malate (0.6 mmol l 21 ), which by themselves did not support significant rates of oxygen uptake, were required. To measure maximal rates of respiration we added ADP at a final concentration of 0.6 mmol l 21 . The respiratory control ratio (RCR) was calculated from the ratio of the state 3 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 measured the level of CS in the mitochondrial suspensions and, using the specific activity of CS determined for the same muscle, carried out the following conversion: (nmol O 2 min 21 ? CS U 21 )CS U g 21 5 nmol O 2 g 21 min 21 where the first factor is mitochondrial oxygen uptake expressed per unit CS in the mitochondrial preparation and the second factor is the CS activity in the muscle homogenates.

2.6.2. Protein concentrations Mitochondrial protein concentrations were determined using the bicinchoninic acid method of Smith et al. (1985) with BSA as the standard. The concentration of BSA in the resuspension medium was subtracted to establish the concentration of mitochondrial protein. 2.7. Chemicals All biochemicals were from Boehringer Mannheim Co. (Montreal, Canada) or Sigma Chemical 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 no differences between reproductive stages (Sokal and Rohlf, 1981). Normality was tested using a Shapiro–Wilk’s test (SAS, 1991) and homogeneity of variances using a Levene test (Snedecor and Cochran, 1989). Multiple pairwise comparisons (Tukey) were used to test 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 x 2 -tests (Sokal and Rohlf, 1981). 3. Results

3.1. Reproductive stages Animals sampled in mid-June (1996) were immature (or maturing), as indicated by a low 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 in mid-July and early August did not differ in their gonadal indices or in the lipid and protein contents of the gonads. As the latter sample was taken only one week before spawning began, we identified the groups sampled in mid-July and those sampled in early August as mature and prespawned scallops, respectively. The scallops sampled in mid-August showed a marked decrease of the gonadal index and of the lipid and protein contents in their gonads, indicating that animals had spawned (Fig. 1). Our classification of these reproductive stages was supported by histological observations of gonadal sections from the scallops sampled in the four periods.

3.2. Environmental conditions Phytoplankton abundance fluctuated considerably during the study. In general, during maturation scallops had more phytoplankton available (|0.5 mg l 21 of chlorophyll a) than before and after spawning, where phytoplankton availability decreased (|0.3 mg l 21 , Fig. 2). During gonadal maturation, bottom temperature was higher and more variable than during spawning (Fig. 2). During July and until early August, temperature fluctuated around 68C. During spawning, it dropped to |48C and remained low until the end of August.

3.3. Escape response When Chlamys islandica were stimulated with the seastar Leptasterias polaris, they responded with a series of |26 claps in a period of |2 min. There was no difference in the mean number of claps performed by the scallops at different reproductive stages (Table 1). The clapping rate of 13 claps min 21 remained relatively stable, except for prespawning scallops for which the rate was 17 claps min 21 (Table 1). The clapping response was followed by a period of |30 min where the animals remained closed and refractory to further stimulation. This period was similar between reproductive stages, with the exception of mature animals which spent less time closed (Table 1). To evaluate the extent of recovery during valve closure, scallops were restimulated 2 min after they reopened their valves, to measure the proportion of initial claps performed during restimulation and the proportion of animals that responded to stimulation. Spawned scallops could only do 11% of the initial claps, compared to the other 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 animals responded (Fig. 3). Spawned animals markedly decreased their responsiveness as only 38% reacted 2 min after reopening their valves. Even after the scallops reopened their valves, the time required for full recovery of their initial clapping capacity varied markedly with reproductive stage (Table 2). Immature scallops needed only 4 h for complete recovery. In contrast, after gonadal maturation 18 h were needed to regain initial capacity; 12 h were required before spawning 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 Chlamys islandica at different reproductive stages. Values represent means6S.E. (n520 for GSI; n58 for lipid and protein content). Means sharing the same letters were not significantly different (P,0.01) as determined by Tukey multiple comparisons.

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Fig. 2. Variations in temperature (8C) and chlorophyll a (mg l 21 ) 1 m above the Chlamys islandica bed, at ˆIle ˆ du 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 of recuperation from exhausting escape responses. Both the recuperation during valve closure and that occurring once valves reopened were diminished.

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

Number of claps

Clapping rate (claps min 21 )

Time spent closed (min)

n

Immature Mature Pre-spawned Spawned

26 25 30 24

13 13 17 13

33.2 (2.7) 23.2 (1.7)** 34.1 (3.4) 28.8 (3.3)

40 48 29 48

** P,0.01.

(1) (1) (2) (1)

(1) (1) (1)** (1)

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

Immature

Mature

Prespawned

Spawned

2 min a 2h 4h 6h 12 h 18 h

34.8 (2.7) 73.3 (5.5) 95.2 (6.3)** 103.0 (4.1)

24.6 (3.6) 54.2 (7.1) 60.5 (6.3) 63.0 (7.0) 50.0 (10.7) 100.6 (4.7)**

28.1 (6.7) 72.7 (3.4) 60.9 (9.8) 78.1 (6.2) 102.0 (3.9)**

10.6 (5.8)* 69.5 (11.5) 70.1 (8.9) 70.8 (4.8) 81.9 (5.5) 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 gonad matured and remained low until after spawning (Fig. 4). The decrease in muscle glycogen coincided with a strong increase of total lipid and protein in the gonads of mature and prespawning scallops (Fig. 1). Total protein concentration in the phasic muscle 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 with the seastar, 2 min after the end of glycolytic recuperation (closed valves) (n58). Bars sharing the same letters were not significantly different (P,0.05) as determined by a x 2 -test.

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Fig. 4. Concentration of glycogen (mmol glucosyl U g 21 wet mass), total, structural and sarcoplasmic proteins (mg g 21 wet mass) and of arginine phosphate (mmol g 21 wet mass) in the phasic adductor muscle of the scallop Chlamys islandica at different reproductive stages. Values represent means6S.E. (n59–14). Means sharing the same letters were not significantly different (P,0.01) as determined by Tukey multiple comparisons. In muscle protein, different kind of lettering indicate different comparisons.

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However, structural protein oscillated and the level of sarcoplasmic protein fell slightly before spawning. Muscle arginine phosphate gradually increased during gonadal maturation 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 during gonadal maturation and spawning (Fig. 5). The glycolytic enzymes, GP, PFK, PK and ODH, and the mitochondrial enzyme, CS, showed their highest levels in immature scallops and levels declined with gonadal maturation. PK recovered its initial levels before spawning, but showed its lowest level after spawning. Much like the levels of arginine 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 muscle from exhausting escape responses decreased during gonadal maturation and spawning. In contrast, the enzyme which generates most of the ATP required for the escape response gradually increased its activity during gonadal maturation and spawning.

3.6. Oxidative capacities of mitochondria and adductor muscle during the reproductive cycle Rates of mitochondrial oxygen uptake and respiratory coupling varied with substrate and reproductive stage (Fig. 6). Glutamate and succinate gave highest oxidation rates at most times. Rates of glutamate and malate oxidation changed little during the reproductive cycle. In contrast, mitochondrial rates of pyruvate and succinate oxidation decreased considerably after gonadal maturation and remained low after spawning (Fig. 6). For all substrates, the respiratory control ratio (RCR) decreased during the reproductive cycle (Fig. 6). The RCR values for glutamate and pyruvate oxidation were lower in mitochondria from mature and spawned scallops, whereas those for the oxidation of malate and succinate declined only after spawning. Total muscle oxidative capacities were calculated from mitochondrial rates of substrate oxidation, mitochondrial CS levels and muscle CS levels. Reproductive status markedly affected the muscle’s capacity for oxidizing pyruvate and succinate, with decreases occurring with maturation and spawning (Fig. 6). As pyruvate generated from octopine is metabolized during recuperation, the total capacity for pyruvate oxidation is most pertinent to evaluation of the capacity for recuperation from exhausting escape responses. Overall, the mitochondrial oxidative capacities, total muscle aerobic capacity and activities of enzymes involved in glycolytic and aerobic phases of recovery followed virtually the same pattern, a decrease in capacity with maturation and spawning.

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

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Fig. 5. Enzyme activities (U g 21 wet mass) in the phasic adductor muscle of the scallop Chlamys islandica at different reproductive stages. Values represent means6S.E. (n57–12). Means sharing the same letters were not 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 that this substrate mobilization has a detrimental effect upon muscle physiology. Effectively substrate mobilization is paralleled by a reduction in the metabolic capacity of the

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Fig. 6. Maximal oxidation rates (nmol O 2 min 21 mg mitochondrial protein 21 ), respiratory control ratios (state 3 / state 4) and calculated maximal muscle aerobic capacity (mmol O 2 g 21 min 21 ) for mitochondria isolated 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 were not significantly different (P,0.01) as determined by Tukey multiple comparisons.

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adductor muscle and in the organismal capacity to recover from exhausting escape responses. Both gonadal maturation and spawning decreased these parameters. The decrease of the recovery efficiency with gonadal maturation and spawning occurred both during valve closure and after valves had reopened. The time scallops spent with their valves closed after exhausting exercise was not markedly affected by the reproductive stage. Only mature scallops spent less time closed. Given that prolonged valve closure is powered exclusively by the tonic (catch) part of the adductor muscle (de Zwaan et al., 1980), the time scallops spent closed could be related to the metabolic capacity of this section. However, the energetic cost of valve closure is only 1% of that of valve claps (de Zwaan et al., 1980). Therefore, the metabolic capacities of the tonic muscle should not strongly affect its capacity to maintain the valves closed and limitations may arise from the requirements of other tissues including the phasic adductor muscle. Since we did not measure the energetic status and metabolic capacities of 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 same time closed as immature and prespawned animals, which had the highest percentage of recovery (Table 2, Fig. 3). The negative impact of gonadal maturation and spawning on recovery 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. magellanicus, the scallops probably consumed most of their oxygen reserves during valve clapping and took up no oxygen during valve closure (Thompson et al., 1980). GP and PFK are important sites of metabolic control in the adductor muscle of Argopecten irradians concentricus (Chih and Ellington, 1986) and ODH is used during the restoration of ATP and arginine phosphate pools when oxygen supplies are depleted or absent as during valve closure (Livingstone et al., 1981). The fact that the activities of these enzymes are diminished after gametogenesis and spawning in C. islandica, could explain the reductions in glycolytic recovery. In Placopecten magellanicus, the complete restoration of the phosphoarginine pool after exhaustive swimming requires aerobic metabolism (Livingstone et al., 1981). For the phasic muscle of C. islandica, the levels of CS, the mitochondrial capacity (and RCR values) for pyruvate oxidation and the total muscle aerobic capacity decreased after gonadal maturation and remained low after spawning. These reductions could explain why mature and spawned scallops needed 14 h more to recover their initial clapping capacity than immature animals (Table 2). In the adductor muscle of the tropical scallop, Euvola ziczac, mitochondrial capacities for pyruvate oxidation were lower during the first spawning, which occurred after a period of low food availability, than during other periods (Boadas et al., 1997). As pyruvate is the principal substrate to be oxidized after muscular activity (Chih and Ellington, 1986), a reduced capacity for pyruvate oxidation could slow the aerobic recovery of mature and spawned scallops from exhausting escape responses. The impact of the reproductive cycle upon aerobic recovery could reflect the instantaneous metabolic costs of gonadal maturation and feeding. In P. magellanicus, oxygen uptake rises during gonadal maturation and with increases in food availability and temperature (Shumway et al., 1988). If phytoplankton levels in the water pumped into our wet lab were similar to those near the scallop bed (Fig. 2), resting maturing

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animals would have been feeding at higher levels than immature or prespawning animals, thereby reducing the aerobic scope for recuperation. The metabolic costs of gonadal maturation would have exacerbated this effect. In accordance with this interpretation, prespawning scallops partially regained the capacity to recover from exercise, compared to mature animals (Table 2, Fig. 3). However, spawning decreased the percentage of aerobic recuperation even more than gonadal maturation (Table 2, Fig. 3). As increased metabolic rates due to feeding and gonadal maturation would not explain this decrease, reductions in muscle metabolic capacity are a more likely explanation for the slowed rates of recuperation. In Argopecten irradians concentricus spawning leads to a state of negative energy balance and generally poor physiological condition, as indicated by a loss of carbon 14 from all body components (Barber and Blake, 1985). Assays of oxygen uptake at rest and during aerobic recovery would ascertain the extent to which gonadal maturation and feeding reduce the aerobic scope for recuperation. Changes in the composition of body components should indicate which biochemical components contribute to energy metabolism during gametogenesis (Barber and Blake, 1991). In C. islandica, even though food availability was high during the accumulation of gonadal lipid and protein (Fig. 2), muscle glycogen decreased strongly while muscle protein changed little (Figs. 1 and 4). Therefore, muscle glycogen is likely a major precursor for lipid and protein synthesis in the gonad. This agrees with patterns found for C. islandica in Norway (Sundet and Vahl, 1981; Sundet and Lee, 1984); for Chlamys varia (Shafee, 1981) and Placopecten magellanicus (Robinson et al., 1981). However, in some scallops, such as Argopecten irradians concentricus, muscle protein is also used to support gonadal maturation after the depletion of glycogen (Barber and Blake, 1981). In this subspecies, physiological indices, such as O / NH 3 and CO 2 / O 2 (respiratory quotient), indicate that gametogenesis is initially supported by lipid. Then, during the cytoplasmic growth of oocytes, gametogenesis is supported by muscle glycogen, which is converted to lipid. Finally, near and after spawning, metabolism is fueled by protein (Barber and Blake, 1985). In contrast, in A. irradians irradians muscle carbohydrates make a negligible contribution to gonadal development, rather gametogenesis occurs mainly at the expense of adductor muscle protein, which accounts for 63–99% of gonadal buildup in the spring (Epp et al., 1988). Total protein in the adductor muscle of C. islandica did not change during gonadal maturation and spawning (Fig. 4). As gonadal maturation is completed during spring and summer, when chlorophyll a concentrations 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 gonadal maturation and spawning in C. islandica, the coincident decrease of glycolytic and mitochondrial enzymes during these processes was not due to generalized protein mobilization. During our study, the water content of the phasic adductor muscle remained constant and changes in enzyme levels were the same when expressed per milligram of protein (data not shown). The changes in enzyme levels may reflect a specific proteolysis. Due to the high energy demand during reproduction, protein synthesis in muscle could be reduced. Similarly, myosin synthesis was diminished during 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

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salmon, Oncorhynchus nerka, muscle enzymes show different rates of catabolism than the general categories of soluble and insoluble proteins (Mommsen et al., 1980). Whereas we do not know why scallops sacrifice the enzymes that facilitate recuperation of 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 in glycogen levels could reduce binding sites for glycolytic enzymes. Once enzymes are in solution in the cytoplasm, they could be more susceptible to degradation. The concomitant decline of glycogen and glycolytic enzymes with gonadal maturation supports such a mechanism. Contrary to our initial premise, arginine phosphate, the principal fuel for muscle contraction during the escape response, increased after gonadal maturation and after spawning (Fig. 4). Although the arginine phosphate levels we measured are lower than those previously obtained (possibly as our technique for sampling the muscle was slower than that of other workers), the relative levels should be reliable, as we applied the same sampling technique to each reproductive stage. Moreover, AK, the enzyme that converts arginine phosphate to ATP and arginine, also increased after gametogenesis and its levels remained high after spawning (Fig. 5). These results may partly explain why the number of claps did not change during reproduction of C. islandica (Table 1). The fact that mature and spawned scallops need more time to recuperate after exhausting burst exercise may have ecological significance. After gonadal maturation and spawning, scallops could be more susceptible to predation and to capture by commercial fisheries. This is particularly true since many scallop species are captured by trawling 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 and recuperate. This is particularly true since trawls or drags may be used repeatedly in the same area of the scallop bed. Interindividual differences in the locomotor performance of vertebrates can be due to physiological changes or morphological variability (Garland, 1984; Walsberg et al., 1986; Bennett et al., 1989; Kolok, 1992); to size, age (Garenc et al., 1999), thermal acclimation (Elliott, 1991; Johnson and Bennett, 1995) or reproductive stage (Koch and Wieser, 1983; Calow, 1985; James and Johnston, 1998). Many of these factors modify ¨ muscle metabolic capacities (Sanger, 1993). In invertebrates, the evidence for interindividual differences in locomotor performance is limited, nevertheless, there are studies, particularly for scallops, that show that swimming performance changes with morphology (Gould, 1971) and size or age (Winter and Hamilton, 1985; Joll, 1989; Manuel and Dadswell, 1991). Most of the differences have been attributed to hydrodynamic changes with size (Manuel and Dadswell, 1991). Our study demonstrates a marked effect of reproduction on the recuperation of clapping capacity after exhaustive exercise and on muscle metabolic capacity in scallops.

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

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support and valuable comments on the manuscript. We also thank Martin Lafrance and Martin Giasson for technical assistance. This work was supported by operating grants from the NSERC to HEG and JHH. KBB was recipient of scholarships from the ´ ´ Gouvernement du Quebec, Departement de Biologie de l’Universite´ Laval and GIROQ. [SS]

References ¨ Arsenault, D.J., Himmelman, J.H., 1998. Spawning of the Iceland scallop (Chlamys islandica Muller, 1776) in the northern Gulf of St. Lawrence and its relationship to temperature and phytoplankton abundance. Veliger 41, 180–185. Ballantyne, J.S., Moon, T.W., 1985. Hepatopancreas mitochondria from Mytilus edulis: substrate preferences and 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 Argopecten irradians 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 Argopecten irradians 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, Ecology and Aquaculture. Elsevier, Amsterdam, pp. 377–428. Bates, P.C., Milllard, D.J., 1983. Myofibrillar protein turnover: synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation. Biochem. J. 214, 587–592. Beaulieu, M.A., Guderley, H., 1998. Changes in qualitative composition of white muscle with nutritional status of 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 and locomotion in a salamander. Am. J. Physiol. 256, R1200–R1208. Boadas, M.A., Nusetti, O., Mundarain, F., Lodeiros, C., Guderley, H., 1997. Seasonal variation in the properties 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: New Perspectives. The Johns Hopkins University Press, Maryland, pp. 13–32. Chih, P.C., Ellington, W.S., 1983. Energy metabolism during contractile activity and environmental hypoxia in the 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 adductor muscle of the bay scallop, Argopecten irradians concentricus: identification of potential sites of regulation and 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 classes of 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 total lipids 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 may encode 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 swimming capacity 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 from glycogen-bound holoenzyme. Eur. J. Biochem. 186, 701–710. James, R.S., Johnston, I.A., 1998. Influence of spawning on swimming performance and muscle contractile properties 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 integrated study 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 for the cost of production? J. Exp. Biol. 107, 141–146. Kolok, A.S., 1992. Morphological and physiological correlates with swimming performance in juvenile largemouth bass. Am. J. Physiol. 263, R1042–R1048. Lam, R., 1990. The nature of cytoplasmic vacuoles in chordoma cells: a correlative enzyme and electron microscopic histochemical study. Pathol. Res. Pract. 186, 642–650. Lamprecht, W., Trautschold, I., 1974. Adenosine triphosphate. Determination with hexokinase and glucose-6phosphate dehydrogenase. In: Bergmeyer, H.U. (Ed.). Methods of Enzymatic Analysis, Vol. 4. Academic Press, New York, pp. 2101–2110. Lamprecht, W., Stein, P., Wieser, H., 1974. Creatine phosphate. In: Bergmeyer, H.U. (Ed.). Methods of Enzymatic Analysis, Vol. 4. Academic Press, New York, pp. 1777–1781. Livingstone, D.R., de Zwaan, A., Thompson, R.J., 1981. Aerobic metabolism, octopine production and phosphoarginine as sources of energy in the phasic and catch adductor muscles of the giant scallop Placopecten 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 the scallop, 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 utilization during 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 myosin synthesis 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 scallop Argopecten 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 Seawater Analysis. 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 component indices 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, west Iceland. II. Gamete development and spawning. Aquaculture 110, 87–96. Vardanis, A., 1990. Fractionation of particulate glycogen and bound enzymes using high-performance liquid chromatography. Anal. Biochem. 187, 115–119. Von der Decken, A., 1992. Physiological changes in skeletal muscle by maturation-spawning of non-migrating female 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 valve snap and valve closure responses in the giant scallop Placopecten magellanicus. II. Biochemistry. J. Comp. Physiol. 137, 105–114.