K .B. Brokordt et al. J. Exp. Mar. Biol. Ecol. 251 2000 205 –225
207
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
ˆ The population studied was located at the southern side of Ile 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 de- termined 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 communica-
tion. 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
21 21
mass 3 shell height
, 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 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. 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
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 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
ethylene dinitrilotetraacetic acid, 5 mM EGTA ethyleneglycol
2
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 NADPH 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-bis2-nitrobenzoic acid DTNB. The molar extinction coefficients used for NADPH and DTNB were 6.22 and 13.6, respectively.
All assays were run in duplicate and the specific activities were expressed in
21 21
international units mmol of substrate converted to product min 3 g
wet mass.
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.B. Brokordt et al. J. Exp. Mar. Biol. Ecol. 251 2000 205 –225
Conditions for enzyme assays except for CS were adapted from conditions used by de
21
Zwaan et al. 1980 for P . magellanicus, as follows all concentrations in mmol l
: 2.5.1. Glycogen phosphorylase EC 2.4.1.1
21
Imidazole–HCl 50, KH PO 80, Mg-acetate 5, EDTA 2.5, 10 mg ml
glycogen
2 4
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-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 the
4
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, pH 6.6.
2.5.5. Octopine dehydrogenase Imidazole–HCl 50, EDTA-Na 2, EGTA 5, KCN 1, sodium pyruvate 5 omitted for
2
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
21
mmol 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.
K .B. Brokordt et al. J. Exp. Mar. Biol. Ecol. 251 2000 205 –225
211
Preliminary studies indicated that glutamate, malate, pyruvate and succinate at final
21
concentrations of 30, 6, 0.9 and 24 mmol l , respectively, gave maximal rates. To attain
21
maximal rates of pyruvate oxidation, ‘sparking’ levels of malate 0.6 mmol l , 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
21
mmol l . 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:
21 21
21 21
21
nmol O min ? CS U
CS U g 5 nmol O g
min
2 2
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
2
x -tests Sokal and Rohlf, 1981.
3. Results
