E . Mayrand et al. J. Exp. Mar. Biol. Ecol. 255 2000 37 –49
39
term changes in the nutritional status of Carcinus maenas and Pacifastacus leniusculus. On the other hand, Anger and Hirche 1990 did not detect a significant correlation
between growth and the RNA:DNA ratio measured in whole early juvenile spider crabs. To our knowledge, the present study is the first to investigate the relationship between
growth rate, on one hand, and the RNA:DNA ratio and metabolic enzyme levels in crustacean muscle, on the other.
The energetic status of an animal can be assessed by indices such as the condition factor and the liver somatic index Lambert and Dutil, 1997. Somatic growth and
condition indices often covary Dutil et al., 1998, although this depends on the exact nutritional history. For example, a positive growth rate in muscle of snow crab can be
accompanied by a low or high value of digestive gland dry mass, depending on the food ration which the animals were fed Mayrand et al., 2000. It is thus important to
discriminate the effect of growth rate from that of the nutritional status on the variates which are tested as potential indicators of growth.
The aims of this study were to examine how the metabolic capacity of muscle in snow crab changes during growth, and to assess the usefulness of glycolytic and mitochondrial
enzyme activity and of the RNA:DNA ratio as biochemical indicators of muscle growth. From a mechanical point of view, it is interesting to examine cell content and the
metabolic capacity of muscle in the whole merus. Indeed, the degree of merus ‘filling’ and the metabolic capacity of the whole merus muscles are likely to affect the locomotor
ability of the animal. In order to correct for the slight differences among the mean merus volume calculated for the various experimental groups, the DNA content mg and the
enzyme activity international units: mmole of substrate converted to product per minute were expressed per ml of merus. The DNA content per ml of merus is related to the
density of muscle cells in the merus, without discriminating between differentiated cells which are fused into multinuclear fibers and, on the other hand, undifferentiated and
unfused myosatellite cells. Muscle cell size was evaluated by the protein:DNA ratio which has been shown to reflect the ratio of sarcoplasm to nucleus volume in fish muscle
Koumans et al., 1993.
2. Materials and methods
2.1. Animals In September 1993, male snow crabs were captured by beam trawl in the St.
´ Lawrence Estuary, near Mont-Joli, Quebec, Canada. Eighty-seven crabs of similar
carapace width 54.163.2 mm were selected and brought back to the laboratory. The animals were held in 1.2 3 1.8 m tanks, with a water depth of 0.35 m at drain level. A
flow-through circulation system was used, with water temperature maintained at 2.060.38C and salinity at 29.160.1‰. The animals were fed ad libitum with frozen
capelin and shrimp. Moulting began on December 10, 1993 and ended on February 21, 1994, with a survival rate of 85. Fifty-four crabs underwent a terminal moult, as
40 E
. Mayrand et al. J. Exp. Mar. Biol. Ecol. 255 2000 37 –49
assessed by the ratio of chela height to carapace width Conan and Comeau, 1986 and were used in the subsequent experiment.
2.2. Feeding conditions The experimental conditions were designed to obtain a wide range of muscle growth
rates. Newly moulted mature crabs were transferred to tanks similar to those described above, with water temperature at 2.960.38C in two tanks and 3.060.28C in the third
one. Salinity was 29.160.1‰ in all tanks. The mean carapace width was 66.263.1 mm and the mean volume of the merus of the first left walking leg was 4.560.7 ml n 5 54.
Ten unfed crabs were sampled 5 days after they had moulted, to assess their initial status. The remaining 44 animals were assigned to three groups, one being starved,
another being fed twice a week to obtain an average ration of 0.4 g of frozen capelin and shrimp per animal per day, and the last one being fed twice a week to obtain an average
ration of 2.0 g per animal per day. Twenty-one crabs which had been fed with the various rations were sampled 25 days after moulting, and 23 more animals were sampled
60 days after moulting. The number of crabs selected from the various combinations of food rations and time post-moult varied from 6 to 10. More detailed information on the
experimental conditions, measurements and tissue sampling is given in Mayrand et al. 2000.
2.3. Biochemical analyses The total protein concentration per g of muscle was measured by the method of
Bradford 1976. The nucleic acid concentration was assessed by fluorimetry, following the method of Karsten and Wollenberger 1972, except that ethidium bromide was
replaced by thiazole orange courtesy of Molecular Probes, Eugene, Oregon, USA, as the latter has been shown to be a more sensitive marker of nucleic acids Berdalet and
Dortch, 1991. Enzymatic activities were measured at 108C, using the assay conditions described by Pelletier et al. 1993a,b. A Beckman DU-600 spectrophotometer coupled
with a circulating refrigerated water bath was used. The following enzymes were measured: citrate synthase CS, E.C.4.1.3.7, cytochrome c oxidase CCO, E.C.1.9.3.1,
phosphofructokinase
PFK, E.C.2.7.1.11
and lactate
dehydrogenase LDH,
E.C.1.1.1.27. Detailed methods are given in Mayrand et al. 1998. 2.4. Growth rates
Muscle content in merus MC was estimated as the dry mass mg of total muscle tissue in the merus of the first left walking leg divided by the volume of this merus ml.
Muscle growth rate G was determined for each crab by one of the following equations, depending on the time they were sacrificed after moulting:
Individual MC 2 mean MC
day 25 at ration X day 5 at ration 0
]]]]]]]]]]]]]]] G
5
day 25 – day 5
Number of days
E . Mayrand et al. J. Exp. Mar. Biol. Ecol. 255 2000 37 –49
41
Individual MC 2 mean MC
day 60 at ration X day 25 at ration X
]]]]]]]]]]]]]]] G
5
day 60 – day 25
Number of days The variation in protein:DNA ratio and DNA content per ml of merus as well as growth
rates for the whole dry digestive gland were calculated in the same way. 2.5. Statistics
The number of observations was 44 for all variables except proteins n 543. Statistical tests were conducted with the following nine variables: muscle growth rate,
temporal variation in protein:DNA ratio, DNA content per ml of merus and the whole dry mass of digestive gland, RNA:DNA ratio, PFK, LDH, CS, and CCO activity per ml
of merus. Normality was tested using Lilliefors’ test. To attain normality, the RNA:DNA ratio was log transformed. The other variates were untransformed. The relationships
between the variates were assessed by listwise Pearson’s simple correlation. Partial correlations coefficients were also computed to evaluate the relationship between two
variables when the seven others were held constant. Partial F-test was used to determine if the coefficients were significantly different from 0. We used this approach to
discriminate between the respective effects of muscle growth rate and nutritional status on the variates which were tested as potential indicators of growth. To allow comparison
with the literature, simple and partial correlation coefficients between muscle growth rate and the enzyme activity expressed per g of protein, mg of DNA and g of dry muscle
were calculated.
3. Results
