´ 32
G . Martınez et al. J. Exp. Mar. Biol. Ecol. 247 2000 29 –49
mobilization of muscle reserves, given its role in liberating glucose equivalents from glycogen. We reasoned that the localization of CS in the mitochondrial matrix would
protect it during reserve mobilization.
2. Materials and methods
2.1. Feeding and sampling of scallops Scallops experiment 1: shell height of 80–90 mm; experiment 2: shell height of
70–80 mm just obtained in mature stage from Culture Centers in Tongoy Bay 308 S were induced to spawn by increasing the temperature and adding excess microalgae
consecutive years. The spent animals were distributed among nine tanks 34 animals per tank at 168C and nine tanks at 208C. Three groups at each temperature were fed with a
mixture of microalgae 50 of Isochrysis galbana 1 50 of Chaetoceros gracilis, three other groups received 70 of the same mixture of algae plus 30 carbohydrates
commercial potato starch, and the other tanks received 70 microalgae and 30 of a lipid emulsion provided by Artemia Reference Center in Belgium, corresponded to the
EmDHA whose composition is described in Caers et al., 1999. The gross biochemical composition of these diets is given in Table 1. More details about diets and their
absorption efficiency are described in Navarro et al. 2000. The daily food ration, supplied continuously by a peristaltic pump, amounted to 3 of the animals’ dry mass
during the first experiment and to 6 during the second experiment. Both experiments were run from January to March in consecutive years 1998 and 1999.
Before beginning conditioning, five spent scallops were sampled to evaluate their biochemical status this sample was called ‘control’. Periodically, similar analyses were
performed for three scallops from each experimental treatment each animal from a different tank to follow the course of gonadal recovery and the levels of the biochemical
components. For this, the scallops were sorted into three groups according to their visually determined degree of maturation and one scallop from the largest group in each
tank was sampled. The duration of the experiments was determined according to the progress of gonadal ripening; when about 40 of the scallops appeared mature in any of
the tanks, the entire experiment was ended to avoid spontaneous spawning. When the first experiment 3 ration was ended, the gonadal indices in the groups with the best
performance approached 10 whereas in the second experiment 6 ration, the
´ gonadal indices of the best groups were greater than 13 Martınez et al., 2000.
Table 1
a
Biochemical composition of the three experimental diets used for conditioning A . purpuratus
Proteins Lipids
Carbohydrates Microalgae
212.99611.02 75.0566.69
62.9564.16 Microalgae 1 lipids
191.9568.45 108.8762.92
69.8267.07 Microalgae 1 carbohydrates
200.00615.43 80.00616.11
215.00626.26
a 21
The concentrations are expressed in mg g . Values are the mean6S.E. N 5 3.
´ G
. Martınez et al. J. Exp. Mar. Biol. Ecol. 247 2000 29 –49 33
2.2. Biochemical and enzymatic analyses The scallops were sacrificed and their gonads and muscles were weighed before
freezing and storage in liquid nitrogen for subsequent biochemical determinations. The muscle samples used for enzymatic measurements were transported on dry ice to
´ Universite Laval where they were stored at 2 708C before analysis. The levels of
proteins, total carbohydrates and lipids in muscle and gonad were analysed following the
21
´ methods of Martınez 1991. Muscle levels were presented as concentrations mg g
dry mass, whereas gonadal components were presented as total gonadal contents for the female and male portions respectively given the marked changes in gonadal size and
no significant changes in muscle size during conditioning gonadal size increase was more than 300
For measurements of PK and ODH, portions of the adductor muscle were homogen- ized in 50 mM imidazole–HCl, 2 mM Na -EDTA, 5 mM EGTA, 1 mM dithiothreitol
2
and 0.1 Triton X-100, pH 6.6. For GP and CS measurements, samples of adductor muscle were homogenized in this buffer at pH 7.2 supplemented with 150 mM KCl. The
pH of all solutions was adjusted at room temperature. Homogenisation of the muscle samples 1:10 m v was carried out on ice using a Polytron Brinkmann, Rexdale,
Ontario at 50 maximal speed for three, 30-s periods separated by 1-min rest periods.
Spectrophotometric measurements were carried out using a Beckman DU-640 UV-Vis spectrophotometer. The cuvette temperature was controlled at 168C by a Haake G8
refrigerating circulator. Measurements were made at 340 nm for PK, ODH and GP following the changes in absorbance of NADPH, whereas measurements of CS
followed the absorbance of 5,59dithio-bis2-nitrobenzoic acid DTNB at 412 nm. Enzyme activities were measured using the following conditions:
Pyruvate kinase: 50 mM imidazole–HCl, 13 mM MgSO , 100 mM KCl, 5 mM ADP,
4
0.2 mM NADH, pH 6.6, 5 U lactate dehydrogenase and 5 mM phosphoenolpyruvate omitted for the control.
Octopine dehydrogenase: 50 mM imidazole–HCl, 2 mM Na EDTA, 5 mM EGTA, 1
2
mM K-cyanide, 0.2 mM NADH, 6 mM Na-pyruvate and 6 mM arginine omitted for the control. The control activity, which represents that of lactate dehydrogenase, was
virtually nil. Glycogen phosphorylase: 50 mM imidazole–HCl, 80 mM KH PO , 5 mM Mg-
2 4
acetate, 2.5 mM Na EDTA, 0.8 mM AMP, 0.5 mM cyclic AMP, 0.6 mM NADP, 0.004
2
mM glucose-1,6-diphosphate, 2 U glucose-6-phosphate dehydrogenase, 2.5 U phosphog-
21
lucomutase, 10 mg ml glycogen, pH 7.5.
Citrate synthase: 75 mM Tris–HCl, 0.25 mM DTNB, 0.35 mM acetyl CoA, 0.05 mM oxaloacetate, pH 8.0. Oxaloacetate was omitted for the control.
The mmolar extinction coefficients were 6.22 and 13.6 for NADPH and DTNB, respectively. Enzyme activities are expressed as international units mmol of substrate
21 21
converted to product min 3 g
wet mass. 2.3. Calculations and statistical comparisons
The gonadal contents of biochemical components were established by measuring the
´ 34
G . Martınez et al. J. Exp. Mar. Biol. Ecol. 247 2000 29 –49
concentration of the component in a sample of the female and male gonad, and then extrapolated to the total content of that section by assuming that the gonad was equally
divided into male and female sections sections never differed by more than 10 and histological analysis showed a clear separation between them. The effect of diet and
duration of conditioning upon the levels of biochemical components in gonad and muscle, upon enzyme levels in muscle and upon muscle mass and water content was
examined using two-way ANOVAs which considered diet and duration of conditioning as the two factors Systat. When no interactions were observed between the factors,
one-way ANOVAs considering only the effect of duration of conditioning were run for each diet. When significant effects of duration of conditioning were observed, Tukey a
posteriori tests were used to identify significant differences P , 0.05. For each experiment, the data obtained under the two thermal regimes were analysed separately.
3. Results
