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255 (2000) 37–49

www.elsevier.nl / locate / jembe

Biochemical indicators of muscle growth in the snow crab

Chionoecetes opilio (O. Fabricius)

a ,_* a b

Elise Mayrand , Helga Guderley , Jean-Denis Dutil

´ ´ ´

Departement de Biologie, Universite Laval, Quebec, Canada G1K 7P4

` ˆ ´

Ministere des Peches et des Oceans, Institut Maurice-Lamontagne, 850 route de la Mer, C.P. 1000, ´

Mont-Joli, Quebec, Canada G5H 3Z4

Received 7 April 2000; received in revised form 27 June 2000; accepted 27 August 2000 Abstract

This study examined the relationships between muscle growth rate, the activity of metabolic enzymes and the RNA:DNA ratio, in adult snow crabs Chionoecetes opilio. After moulting, crabs were assigned to three feeding rations to attain a range of tissue growth rates. Muscle growth rate, estimated by the variation in dry tissue content per ml of merus of the first walking leg, was positively correlated with changes in muscle cell number, as evaluated by the DNA content per ml of merus. However, no significant correlation was detected between growth rate and the variation in muscle cell size, the latter being estimated by the change in the protein:DNA ratio. This is due to the fact that, in starved crabs, a reduction in the number of cells is partly compensated by a size increment of the remaining ones. This phenomenon also weakened the overall relationship between muscle growth rate and the phosphofructokinase (PFK) capacity per ml of merus. The simple correlation between those two variables was significantly positive for animals which increased their mass of muscle but insignificant for those which were loosing muscle mass. The lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome c oxidase (CCO) capacity per ml of merus did not match growth rate. The significant simple correlations that were detected between growth rate and the various enzyme activity expressed per g of protein, permg of DNA and per g of dry mass did not hold when partial correlations were computed. Variations in muscle cell size were related to adjustments in the quantity of RNA per cell, as depicted by the RNA:DNA ratio. Since muscle growth was not correlated with the variation in muscle cell size, it was not correlated with the RNA:DNA ratio either.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Snow crab; Chionoecetes opilio; Growth; Muscle; Nucleic acids; Enzymes

*Corresponding author. Present address: Universite de Moncton, Campus de Shippagan, 218 Boul. J.D. Gauthier, Shippagan, Nouveau-Brunswick, Canada E8S 1P6. Tel.: 11-506-336-3425; fax: 1 1-506-336-3477.

E-mail address: [email protected] (E. Mayrand).

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1. Introduction

In crustaceans, moulting leads to carapace growth. In contrast to the rapid shift in carapace size, flesh growth takes place gradually during the interval between successive moults (Passano, 1960; Stevenson, 1972). Anger and Darwis (1981) noted that the rate of tissue growth is affected by the feeding status of Hyas araneus, while the length of the moult cycle is independent of food ration once the animals have attained a certain threshold of reserves content. In the snow crab Chionoecetes opilio, muscle growth rate is strongly affected by food ration and by the time elapsed since moulting (Mayrand et al., 2000). Crustaceans of similar size and moult stage can exhibit different degrees of carapace ‘filling’, according to the conditions to which they have been previously exposed. Starved crabs have a lower muscle dry mass per merus, a higher tissular water content and fewer muscle cells per merus than well-fed ones (Mayrand et al., 2000). Since muscle content in the walking legs is likely to alter the animals’ locomotory capacity and thus their ability to forage and migrate, it is likely to affect their fitness. Estimates of muscle growth are important when studying the performance of intermoult crustaceans under field conditions.

Muscle growth rate cannot be estimated by temporal changes in crustacean body mass, since growth is constrained by the carapace and because tissue water is gradually replaced by proteins during growth (Passano, 1960) with no appreciable change in density. Biochemical indicators reflecting muscle growth could be an interesting alternative to the classical methods of evaluating growth rates. An advantage of such indicators is that they do not require repeated measures on the same animal. The relationships between somatic growth and biochemical variables have been studied in fish and crustaceans. Promising biochemical indicators are the activity of metabolic enzymes and the RNA:DNA ratio. The rationale of testing these potential indicators is that growth depends on ATP production, which in turn depends on the activity of glycolytic and mitochondrial enzymes. Growth also requires the cellular machinery for protein synthesis, i.e. the ribosomal RNA, which represents 85–94% of cellular RNA (McMillan and Houlihan, 1988). Growth rate is positively correlated with the specific activity of glycolytic and mitochondrial enzymes in muscle and gastrointestinal tract of saithe (Mathers et al., 1992), cod (Pelletier et al., 1993a,b, 1995; Dutil et al., 1998; Lemieux et al., 1999), largemouth bass (Goolish and Adelman, 1987) and threespine stickleback (Guderley et al., 1994). The RNA:DNA ratio has been shown to be a good indicator of growth in fish (e.g. Bulow, 1970; Kearns and Atchison, 1979; Goolish et al., 1983; Buckley, 1984; Barron and Adelman, 1984; Mathers et al., 1992). Nevertheless, the correlation between somatic growth and the RNA:DNA ratio does not hold in all fish

species (Dagg and Littlepage, 1972; Jurss et al., 1987; Mathers et al., 1994; Pelletier et al., 1994; Dutil et al., 1998). In comparison with fish, crustaceans have received little attention. Positive relationships between the RNA:DNA ratio and growth rate or feeding level have been reported for the blue crab Callinectes sapidus (Wang and Stickle, 1986, 1988) and postlarval American lobster (Juinio and Cobb, 1994). While the these authors worked with whole animals, Houlihan et al. (1990) and Edsman et al. (1994) examined changes in muscle and found that the RNA concentration in this tissue matches the short

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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 1.231.8 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

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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 (n554). 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 MCday 25 at ration X2mean MCday 5 at ration 0% ]]]]]]]]]]]]]]]

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Individual MCday 60 at ration X2mean MCday 25 at ration X ]]]]]]]]]]]]]]]

Gday 60 – day 255 _{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 (n543). 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

Muscle growth rates (G) ranged from 20.732 to 0.915 mg dry muscle ml merus

day . Changes in muscle cell size (Dprotein:DNA), as estimated by the change in protein:DNA ratio per day, varied from 214.586 to 15.772. Variation in muscle cell

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number (DDNA) was estimated by the change in mg of DNA ml merus day and ranged from 20.500 to 0.750. The growth rate of the digestive gland varied from

21 20.050 to 0.052 g dry tissue day .

Muscle growth rate was based on changes in the number of muscle cells, as indicated by the positive simple and partial correlation between G andDDNA (Table 1 and Fig. 1). An increase in muscle cell size was accompanied by higher levels of cellular machinery available for protein synthesis, as shown by the significant positive correlation between Dprotein:DNA and log RNA:DNA ratio (Table 1 and Fig. 2).

Muscle growth rate was positively correlated with the activity of PFK per ml of merus, while the variation in muscle cell size matched the PFK and CS activity per ml of merus (Table 1 partial correlations, Fig. 3). Significant negative simple correlations were noted between muscle growth rate and the enzyme activity expressed per g of protein (PFK, LDH, CS, CCO), permg of DNA (CCO), and per g of dry muscle (PFK, LDH, CS, CCO), as shown in Table 2. However, none of these relationships was significant

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Table 1

Pearson’s correlation between the physiological variables measured in male snow crabs 25 and 60 days after

moult

G DDNA DP:D Ddig. gl. R:D PFK LDH CCO CS G 0.645*** 0.661*** 0.709*** 0.377* 0.400** 0.660***

DDNA 0.465*** 0.399** 20.401** 0.506*** 0.503*** 0.469*** 0.618***

DP:D 0.681*** 0.362*

Ddig.gl. 0.556*** 0.396** 0.311*

R:D 0.508***

PFK 0.335* 0.388* 0.483*** 0.600*** 0.675***

LDH 0.374* 0.627***

CCO 0.468*** 0.617***

CS 0.347* 0.491*** 0.425***

Upper right: r values for simple correlation, df541. Lower left: (in bold) r values for partial correlation, df534. Only significant r values are given. * P,0.05, ** P,0.01, *** P,0.001. G: muscle growth rate in

21 21 21 21

mg dry tissue ml merus day ;DDNA: variation in muscle cell number, inmg DNA ml merus day ; 21

DP:D: variation in muscle cell size, in protein:DNA day ;Ddig. gl.: digestive gland growth rate, in g dry

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tissue day ; R:D: log RNA:DNA ratio. Enzyme activities are expressed in Units ml of merus . The RNA:DNA ratio is log transformed, the other variates are untransformed.

Fig. 1. Muscle growth rate versus variation in the number of muscle cells as estimated by changes in the DNA content per ml of merus per day in C. opilio.

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Fig. 2. Variation in muscle cell size versus the RNA:DNA ratio in muscle of C. opilio.

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Table 2

Pearson’s correlation coefficients between muscle growth rate (G) and the enzyme activity

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Enzymes Activity g protein Activitymg DNA Activity g dry mass

Simple Partial Simple Partial Simple Partial

PFK 20.310* 0.068 20.051 20.030 20.341* 20.273

LDH 20.361* 20.071 20.247 20.026 20.395** 20.034

CCO 20.317* 20.144 20.326* 20.126 20.508*** 20.131

CS 20.317* 0.009 20.031 20.076 20.293* 20.265

Simple and partial coefficients are given. Partial correlation coefficients are computed between muscle growth rate and a given enzyme activity with the other seven variates kept constant. * P,0.05, ** P,0.01, *** P,0.001. n543, df541 for simple correlations and 34 for partial correlations.

when partial correlation coefficients were computed, holding the seven other variates constant.

Somatic growth and the nutritional status, as represented by changes in the digestive gland dry mass, varied together as shown by the positive simple and partial correlations between G and Ddigestive gland (Table 1 and Fig. 4). It is noteworthy that the significant relationships between Ddigestive gland and the PFK and CS activity per ml of merus did not hold when partial correlations were computed.

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4. Discussion

Our results suggest that both hyperplasia and hypertrophy are important in muscle growth in intermoult snow crab. A positive relationship between muscle growth and the number of muscle cell is clearly shown by our results (Table 1, Fig. 1). A gain in DNA content per ml of merus probably reflects the proliferation of myosatellite cells, as mitotic activity of the latter precedes the incorporation of daughter myosatellite cells into myofibers (Campion, 1984; Koumans et al., 1993, 1994). Nathanailides et al. (1996) reported that more myofiber nuclei were added to the post-anal caudal region in juvenile sea bass (Dicentrarchus labrax) undergoing fast somatic growth than in those with low growth rates. A loss in DNA content per ml of merus may represent the death of muscle fibers, unfused myosatellite cells or both. Myofiber death in response to starvation is generally not considered plausible, the generally accepted idea being that cells with slow turnover rates are conserved to allow rapid recovery when food becomes available. Nonetheless, the existence of programmed myofiber death has been reported for the snapping shrimp Alpheus heterochelis by Quigley and Mellon (1986). Moreover, snow crabs lost about 30% of their DNA mass per ml of merus after 55 days of starvation (Mayrand et al., 2000). Considering that the percentage of myosatellite cell nuclei varies between 0 and 15% in carp muscle (Koumans et al., 1994), and between 1.7 and 10.7% in various rat muscles (reviewed by Campion, 1984), death of myofibers and myosatel-lite cells is likely to account for the decrease in DNA content in starved snow crabs.

On the other hand, no significant correlation was detected between muscle growth and the size of muscle cells, as represented by the protein:DNA ratio (Table 1). This does not mean that muscle growth was achieved solely through the addition of differentiated myosatellite cells. In a previous study, we have proposed that starved snow crabs could transfer materials from sacrificed muscle cells to the preserved ones (Mayrand et al., 2000). As a result, an increase in the protein:DNA ratio was noted in muscle tissue of starved as well as well fed animals. This could account for the absence of a positive correlation between muscle growth rate and the protein:DNA ratio, although a positive muscle growth rate is probably based on both hyperplasia and hypertrophy.

Muscle growth positively affects the total glycolytic capacity in the merus muscle of snow crabs. Indeed, G and the PFK activity per ml of merus are significantly correlated (Table 1, Fig. 3). It is interesting to note that the simple correlation between these variables is stronger for growth rates higher than 0 (r50.646, P,0.001, n527) than for those smaller than 0 (r50.192, P.0.05, n516). Once again, the nutrients transfer strategy would account for this. Animals with negative growth rate lose muscle cells (Fig. 1) and this negatively affects the total PFK activity. On the other hand, the remaining cells increase in size, enhancing their PFK capacity. This is supported by the positive partial correlation betweenDprotein:DNA and the PFK activity permg of DNA (R12.3 – 950.561, P,0.001, n543). The net result is an absence of correlation between negative muscle growth and the total PFK activity, as it can be seen on the bottom of Fig. 3. This weakens the overall relationship so that the PFK capacity per ml of merus muscle explains only 50% of the variability of muscle growth making the partial correlation coefficient barely significant. The PFK total activity in merus muscle may be a useful indicator of growth in snow crabs, as both simple and partial correlation

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coefficients are significant. However, at least another variable, related to muscle growth but not to muscle cell number and size, should be identified in order to predict growth rate more accurately. Metabolic enzyme activity in other tissues such as the intestine would be good candidates. Goolish and Adelman (1987), Pelletier et al. (1994) and Dutil et al. (1998) have shown that the intestinal CCO activity per g of wet mass varies with the growth rate of Atlantic cod, reflecting the capacity of fish to assimilate nutrients which in turn allows an increased growth rate. As far as we know, no study has examined the relationship between growth rate and enzyme activities in crustacean tissues.

According to our results, the total activity of LDH, CS and CCO in merus muscle cannot be used as predictors of muscle growth, since the significant simple correlations detected between each of them and muscle growth do not hold when partial correlations are computed (Table 1). Significant simple correlation or regression coefficients between growth and other variables must be considered with prudence, as they do not necessarily represent robust relationships. Differences in statistical treatment and in physiological strategies may explain discrepancies between our conclusions and those from studies on fish. The strategy of sacrificing muscle cells and transferring their nutrients to the remaining ones is unlikely to exist in fish. Indeed, somatic growth and the protein:DNA ratio in muscle cells vary in the same direction in fish while growth rate is independent of the DNA concentration per mg of dry mass (Goolish et al., 1983; Goolish and Adelman, 1987). Pelletier et al. (1995), using linear regression, found a positive relationship between somatic growth rate and the activity of PFK and LDH measured in white muscle of Atlantic cod, regardless whether the enzyme activity was expressed per

mg DNA, per g protein or per g dry mass. Our results also show significant simple correlations between muscle growth and enzyme activity when expressed per g protein (PFK, LDH, CS and CCO), per mg DNA (CCO), or per g of dry muscle (PFK, LDH, CS, CCO), as shown in Table 2, although all the coefficients were negative. None of these relationships held when partial correlations coefficients were computed. The coefficient values may be negative because of a dilution effect due to the synthesis of larger quantities of total proteins in muscle of crabs with positive growth rate than in muscle of crabs with negative growth rate.

Variation in muscle cell size results from adjustments in the quantity of cellular machinery for protein synthesis, asDP:D and the log of RNA:DNA ratio were positively correlated (Table 1, Fig. 2). A higher content of RNA per cell allows the synthesis of more proteins and thus leads to a greater increase in cell size. Since muscle growth is not correlated with DP:D, which depends on the RNA:DNA ratio, it is not correlated with the RNA:DNA ratio either. This is in agreement with other studies conducted on fish, in which no relationship was detected between somatic growth and the RNA:DNA ratio

(Dagg and Littlepage, 1972; Jurss et al., 1987; Mathers et al., 1994; Dutil et al., 1998). This absence of correlation is generally attributed to the fact that the rate of protein synthesis can be adjusted not only by modifications in the number of RNA molecules but also by variations in the synthetic activity of the RNA molecules already present in the cells. Adjustments in RNA activity rather than in RNA quantity may be partly responsible for the absence of correlations between G and the log of RNA:DNA ratio, but the strategy of nutrient transfer certainly played a major part, as the protein:DNA

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ratio, which covaries with the RNA:DNA ratio, increases in starved as well as in well fed crabs (Mayrand et al., 2000). It is difficult to compare our results with those from other studies on crustaceans since, in most of them, nucleic acids have been measured in homogenates of whole animals. In some cases, the RNA:DNA ratio has been found to be related with growth rate (Wang and Stickle, 1988; Juinio and Cobb, 1994), in others it was independent of growth (Anger and Hirche, 1990). To our knowledge, only Houlihan et al. (1990) and Edsman et al. (1994) measured the RNA concentration in specific tissues of crustaceans, including muscle. Houlihan et al. (1990) noted a short term effect of starvation and refeeding on the RNA activity in all tissues but leg muscle, in Carcinus

maenas. Edsman et al. (1994) reported a significant decrease of RNA content per g of

crayfish tail muscle after 2 days of starvation. They also noted a positive correlation between the number of days of refeeding and the RNA content. Whether these relationships were caused by changes in muscle cell number or by changes in RNA cell content is impossible to say, since the DNA concentration was not measured.

Acknowledgements

We are grateful to Mario Peloquin and Claudie Vigneault for their technical help. This ˆ

´ `

work was supported by funds from the Universite Laval, from the Ministere des Peches ´

et des Oceans, Canada, and from the Natural Sciences and Engineering Research Council, Canada. [SS]

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Table 2

Pearson’s correlation coefficients between muscle growth rate (G) and the enzyme activity

21 21 21

Enzymes Activity g protein Activitymg DNA Activity g dry mass

Simple Partial Simple Partial Simple Partial

PFK 20.310* 0.068 20.051 20.030 20.341* 20.273

LDH 20.361* 20.071 20.247 20.026 20.395** 20.034

CCO 20.317* 20.144 20.326* 20.126 20.508*** 20.131

CS 20.317* 0.009 20.031 20.076 20.293* 20.265

Simple and partial coefficients are given. Partial correlation coefficients are computed between muscle growth rate and a given enzyme activity with the other seven variates kept constant. * P,0.05, ** P,0.01, *** P,0.001. n543, df541 for simple correlations and 34 for partial correlations.

when partial correlation coefficients were computed, holding the seven other variates constant.

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4. Discussion

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maenas. Edsman et al. (1994) reported a significant decrease of RNA content per g of

Acknowledgements

We are grateful to Mario Peloquin and Claudie Vigneault for their technical help. This ˆ

´ `

work was supported by funds from the Universite Laval, from the Ministere des Peches ´

et des Oceans, Canada, and from the Natural Sciences and Engineering Research Council, Canada. [SS]

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