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Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua)
Comparative Biochemistry and Physiology, Part A 146 (2007) 174 – 179 www.elsevier.com/locate/cbpa
Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua) P.U. Blier a,⁎, J.-D. Dutil c , H. Lemieux a , F. Bélanger a , L. Bitetera b a
Laboratoire de Biologie Intégrative, Département de Biologie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1 b Département d'Océanographie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1 c Institut Maurice-Lamontagne, Ministère des Pêches et des Océans, 850 route de la Mer, Mont-Joli, Québec, Canada G5H 3Z4 Received 10 July 2006; received in revised form 3 October 2006; accepted 8 October 2006 Available online 13 October 2006
Abstract This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases. © 2006 Elsevier Inc. All rights reserved. Keywords: Citrate synthase; Cytochrome c oxidase; Gadid; Gastrointestinal tract; Metabolic enzymes; Trypsin
1. Introduction Many animal species experience long episodes of food deprivation during their life history. In Atlantic cod (Gadus morhua Linnaeus), these periods of fasting result in marked fluctuations in condition factor and energy reserves (Lambert and Dutil, 1997; Schwalme and Chouinard, 1999). During those episodes of low food availability, animals are expected to minimize the costs of maintenance and activity in order to enhance survival. Studies in mammals and birds have shown that the maintenance of a functional intestinal epithelium is costly, mainly because of the high cellular turnover rate in this tissue (Johnson, 1987; Starck, 1996). Given the benefits of a reduced rate of energy expenditure during long periods of low food availability, evolution should have favoured the capacity
⁎ Corresponding author. Tel.: +1 418 723 1986x1852; fax: +1 418 724 1849. E-mail address: [email protected] (P.U. Blier). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.10.012
to significantly downregulate the digestive performance during long episodes of fasting, and the subsequent capacity to rapidly upregulate it with feeding (Secor, 2005, 2001). For example, pythons, vipers and rattlesnakes can feed at intervals varying from one to several months and consume preys that can even exceed their own body mass (Greene, 1992; Pope, 1961; Secor and Diamond, 2000). These scarce meals are accompanied with a 2-fold increase in intestinal mass, 5- to 20-fold increases in intestinal nutrient transport capacity (Secor and Diamond, 2000) and 4.8-fold increases in the length of enterocyte microvilli (Lignot et al., 2005). Those postprandial responses were reversed following the completion of digestion (Secor and Diamond, 2000; Lignot et al., 2005). In food-deprived Atlantic cod, pyloric caeca and intestine mass increased over the values of controls (without food deprivation) during refeeding (Bélanger et al., 2002). In mammals and birds (Wilson and Osbourn, 1960) as well as in fish (review of Ali et al., 2003; Jobling, 1994), the period of feeding occurring after a period of food deprivation or
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malnutrition is usually accompanied with a burst of growth, a phenomenon known as compensatory growth. In cod, the activity of metabolic enzymes in the intestine (cytochrome c oxidase, CCO and citrate synthase, CS) is correlated with growth rate (Pelletier et al., 1994; Bélanger et al., 2002) and the activity of the same enzymes in the pyloric caeca even exceed values of control (without food deprivation) during compensatory growth (Bélanger et al., 2002). The metabolic capacity of digestive tissues is also sensitive to variations in nutritional status. Proteolytic enzyme (trypsin) activity may be positively correlated with growth rate and/or conversion efficiency (Bélanger et al., 2002; Lemieux et al., 1999) and the expression of different isozymes of trypsin in Atlantic salmon (Salmo salar L.) appears to be related to growth rate (RungruangsakTorrissen et al., 1999). Long-term fasting in Atlantic salmon has been associated with a decrease in carbohydrate digestive enzymes, whereas upon refeeding, enzyme capacities were regenerated after only 1 week (Krogdahl and Bakke-McKellep, 2005). For fast growth periods to occur, physiological adjustments may need to take place before or during the recovery growth phase. The aim of our study was to assess physiological factors suspected to partly modulate growth capacity following a period of food deprivation. We postulated that any factor determining growth performance after fasting should recover or increase its activity before a burst of body mass growth can take place. Fish were sampled at different intervals after a food deprivation period and the growth of different tissues and activity of metabolic and digestive enzymes were measured.
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Forty-eight hours after the last meal, cod were killed by a blow to the head. They were weighed, measured (fork length) and dissected. The total wet mass of the liver, pyloric caeca (rinsed with NaCl 0.7%), empty intestine (from pyloric caeca to the anus), carcass (eviscerated and head-off) were taken. A
2. Materials and methods Atlantic cod (G. morhua, Gadidae) were trawled in the St. Lawrence estuary near Matane (Québec, Canada) in June 1997. They were held under natural photoperiod and ambient salinity and temperature at the Maurice-Lamontagne Institute (MontJoli, Québec, Canada). The fish were fed a maintenance ration of frozen capelin (Mallotus villosus Müller) from June 1997 to May 1998 until the experiment started in May 1998. One hundred cod were anaesthetised with metomidate hydrochloride (5 mg·l− 1), weighed (± 0.1 g) and their fork length measured (±1 mm). They were then tagged with visible implant tags (Northwest Marine Technology, Shaw Island, Wash.) in the first dorsal fin. The fish were transferred to a 7 m3 fibreglass tank and underwent food deprivation for 68 days. After food deprivation, 10 fish were sampled (day 0) and 40 others were anaesthetised, measured (weight and length) and randomly transferred to eight 1.5 m3 fibreglass tanks (two tanks of five fish for each treatment) equipped with partially recirculating and temperature-controlled systems. The four treatments involved feeding periods of 2, 6, 14 or 28 days during which capelin was offered in excess for 1 hour on alternate days. Water temperature was kept at 10 °C and oxygen saturation was above 80%. All the procedures were approved by the University committee of animal welfare (CPA-UQAR 07– 11) according to the recommendations of the Canadian Council of Animal Care.
Fig. 1. Relative gains in mass (RGM), and pyloric caeca (PCSI), intestine (ISI) and liver (HSI) as an index of fish length in Atlantic cod refed after a deprivation period of 68 days. Differences between treatment were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate) for relative fish growth in mass (A) and with an ANOVA (factors tank nested in treatment) for individual morphometric indices (B: pyloric caeca, C: intestine, D: liver). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Means followed by one similar letter were not statistically different (post hoc Tukey's multiple comparison test).
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Table 1 Cod wet tissue masses (g) at days 0, 2, 6, 14 and 28 of refeeding after a food deprivation of 68 days Day Liver 0 2 6 14 28
Pyloric caeca
8.58 ± 4.94a 6.44 ± 1.55a a 7.89 ± 3.35 7.09 ± 1.93a 11.52 ± 3.46a 11.00 ± 3.11b 27.03 ± 9.83b 15.09 ± 4.58c 64.55 ± 21.96c 20.59 ± 6.12d
Intestine
Gonads
Carcass
4.13 ± 0.67a 21.30 ± 23.38 509.3 ± 106.6 5.23 ± 1.38ab 6.68 ± 3.56 515.4 ± 149.4 6.14 ± 1.88b 12.46 ± 21.43 552.6 ± 175.4 7.96 ± 1.69c 9.73 ± 9.86 556.6 ± 148.7 8.16 ± 2.12c 6.65 ± 2.47 623.7 ± 142.4
Differences were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different (post hoc Tukey's multiple comparison test).
sample of ∼ 10 g of white muscle was taken under the first dorsal fin and divided into two sub-samples. The pyloric caeca were finely chopped with a razor blade and divided into three sub-samples. Samples were handled on ice and tissues for enzymatic assays (sub-samples of muscle, pyloric caeca and whole intestine) were immediately frozen in liquid nitrogen and kept at − 80 °C. For each individual, initial (before feeding) or final (after feeding) Fulton condition factor, relative gain in mass (% total mass), and pyloric caeca (PCSI), intestine (ISI), carcass (CSI) and liver (HSI) somatic index (relative to fish length3) were calculated as follows:
CT, USA) equipped with a thermostated cell holder and a circulating refrigerated water bath. All assays were run in duplicate and enzymatic activity was expressed in U·g tissue− 1 (U is the amount of the enzyme that catalyzes the conversion of 1 μmol of substrate per minute). Conditions for enzyme assays followed those of Lemieux et al. (1999) for trypsin and those of Thibault et al. (1997) for CCO and CS. Statistical analyses were done with Statistica for Windows, release 5.1 (1997 edition; Statsoft, Tulsa, USA). Shapiro– Wilk's test was used to verify normality. Homogeneity of variance was tested with Levene's test. Differences in relative gain in mass and enzymatic activities were tested using ANCOVAs. We used the mixed model with treatment as a fixed effect (5 levels) and tank as a random effect (2 tanks per treatment). Fish length was used as the covariate. For morphometric indices, we tested treatment effect with a 2
Initial Fulton condition factor ðKi Þ ¼ 100ðMd L−3 d Þ Final Fulton condition factor ðKf Þ ¼ 100ððMf SCf ÞL3 f Þ Relative gain in mass ¼ 100½ððMf SCf Þ Md ÞMd1 Tissue somatic index ¼ 100ð Tissue massðL3 f ÞÞ where Md is the total mass (g) before feeding, Mf is the total mass (g) after feeding, SCf is the mass of the stomach content (g) after feeding, Ld is fork length (cm) before feeding, Lf is fork length (cm) after feeding, tissue mass is the wet total mass of the pyloric caeca, intestine, carcass or liver. To assay CCO and CS activities, samples of white muscle, intestine and pyloric caeca were homogenised in imidazole buffer (50 mM imidazole; 2 mM MgCl2·6H2O, 5 mM EDTA, 0.1% Triton X-100, and 1 mM reduced glutathione at pH 7.5). For the intestine and pyloric caeca, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) was added to the imidazole buffer. Tissues were disrupted in three 20-s bursts of homogenisation (Tekmar tissue grinder, Cincinnati, OH, USA). Intestine and pyloric caeca homogenates were centrifuged for 3 min at 1000 g (4 °C) (Centrifuge 22, Heraeus Sepatech, Osterode/Harz, Germany) and the supernatant taken for the running analysis. A second sample of pyloric caeca was diluted with Tris buffer (46 mM Tris, and 11.5 mM CaCl2, at pH 8.1), and subjected to three periods of 20 s of homogenisation and centrifugation for 15 min at 13 000 g (4 °C) to obtain the supernatant for the trypsin assay. Enzymatic assays were carried out at 20 °C for trypsin and 10 °C for CCO and CS activities. Readings were made using a UV/VIS spectrophotometer (Perkin Elmer Lambda 11, Shelton,
Fig. 2. Activities of trypsin (A), cytochrome c oxidase (CCO, B) and citrate synthase (CS, C) in pyloric caeca of Atlantic cod refed after a deprivation period of 68 days. Differences between treatment were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different (post hoc Tukey's multiple comparison test).
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Fig. 3. Intestinal activities of cytochrome c oxidase (CCO, A) and citrate synthase (CS, B) of Atlantic cod refed after a deprivation period of 68 days. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different. Statistical details are as presented in Fig. 2.
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among individuals within each treatment and no significant difference was observed between treatments (P = 0.06) (Table 1). The relative mass of the pyloric caeca and intestine was the same for the unfed fish as for fed fish after 2 days of feeding, but increased significantly between day 2 and day 28 (Fig. 1B). The relative mass of intestine increased significantly between days 2 and 14 and then plateaued (Fig. 1C). By days 14 and 28 of refeeding, the mass of the liver relative to fish length (liver mass in g/fish length in cm3) was much higher (0.024 ± 0.005 and 0.058 ± 0.017, for 14 and 28 days, respectively) than that of deprived fish at day 0 (0.008 ± 0.004) (Fig. 1D). No statistical differences in the relative mass of the carcass were found over the feeding period (0.48 ± 0.05 at day 0, 0.45 ± 0.08 at day 2, 0.50 ± 0.08 at day 6, 0.49 ± 0.06 at day 14, and 0.57 ± 0.11 at day 28). In the pyloric caeca (Fig. 2), trypsin activity increased soon after feeding started and levelled off after day 14 of refeeding. CS activity followed a similar pattern, but this was not the case for CCO, which showed no significant differences among treatments. In contrast, both CS and CCO activity increased over time in the intestine (Fig. 3). CS activity significantly increased from unfed level after 6 days of refeeding and no further changes were observed thereafter. CCO activity increased progressively from day 2 to day 28 of refeeding. CS activity in the intestine was approximately three times lower than in the pyloric caeca, but CCO activity in the intestine was similar to that in the pyloric caeca. In the white muscle (Fig. 4), CS and CCO activities did not show any clear response to refeeding. The enzymatic activities
way-ANOVA (tank nested in treatment). When treatment was found to have a significant effect, Tukey a posteriori pairwise comparisons were done. The factor ‘tank’ was never found to have a significant effect. PCSI, trypsin, CCO and CS activities in pyloric caeca were log transformed for the statistical analyses, but untransformed data are presented as mean ± SD for 8–10 individuals (pooled tanks) in the table and figures. Results were considered significant at P b 0.05. 3. Results Total mass averaged 1016 ± 220 and 772 ± 195 g before or after food deprivation, respectively, length 480 ± 49 and 474 ± 33 mm and condition factor 0.93 ± 0.15 and 0.71 ± 0.09. No mortality was observed during the deprivation and feeding period. The 68-day starved cod lost on average 26.4 ± 6.9% of their body mass (252 ± 70 g) with no significant differences among treatments. When feeding was resumed, relative gain in mass significantly increased between day 14 and day 28 (Fig. 1A). Tukey test showed that fish on day 28 were heavier than all other treatments. From day 0 to 28, liver mass increased at a high rate, pyloric caeca and intestine mass increased more slowly while gonads mass decreased. On average, carcass mass (adjusted for differences in length between treatments, not shown) increased by 20 g over the first 14 days and 68 g over the last 14 days of the experiment, suggesting a greater increase in axial muscle mass during the second period, but carcass mass varied markedly
Fig. 4. White muscle activities of cytochrome c oxidase (CCO, A) and citrate synthase (CS, B) of Atlantic cod refed after a deprivation period of 68 days. Means are represented by the middle point and standard deviation, by the bars. Values are mean ± standard deviations for pooled tanks (N = 8–10). Data showed no significant differences between treatments. Statistical details are as presented in Fig. 2.
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were on average lower in white muscle than in other tissues: CCO activity was nearly three times lower than in the pyloric caeca and intestine. CS activity was also one third that in the pyloric caeca, but at the same level as in the intestine. 4. Discussion During refeeding, the relative gain in mass averaged 5.1 ± 5.8% body mass for the first 2 weeks and 34.9 ± 9.7 % for the whole feeding period. Thus 14 days were required after feeding was resumed before fish weight increased markedly. Liver growth contributed to a significant portion of the gain in mass, particularly during the last 2 weeks of the experiment, which suggests that energy storage in the liver for later use is an important aspect of the cod strategy to face periodic variations in food abundance. Fish in our experiment were of similar size and mass and followed a very similar experimental design as those of Bélanger et al. (2002) who concluded that compensatory growth had occurred after 10 weeks of food deprivation and 24 days of refeeding. Interestingly, the burst in relative gain in mass observed after day 14 of refeeding in the present study occurred after PCSI growth slowed down and ISI had increased to a maximum level. During the first 2 weeks of refeeding, PCSI doubled and ISI almost doubled. Further changes in these indices were not significant over the last 2 weeks of the experiment, precisely in the period when the bulk of growth occurred. Similarly, other studies have shown a correlation between PCSI and/or ISI index and growth rate or feeding status in fish. PCSI was 1.2 times higher in rainbow trout fed ad libitum, compared to trout fed a maintenance ration (Stevens and McLeese, 1988). In cod (Linares et al., 1996; Lemieux et al., 1999) and rainbow trout (Weatherley and Gill, 1983), PCSI and/or ISI were found to correlate positively with growth rate. Transgenic GH salmon, which grow 1.6-fold faster than non-transgenic, also have larger pyloric caeca (Stevens and Devlin, 2005) and a larger pyloric caeca exchange area (1.2 times the control) (Stevens et al., 1999). In European plaice (Pleuronectes platessa Linnaeus) (Jobling, 1980) and Atlantic salmon (Krogdahl and BakkeMcKellep, 2005), gut size decreased during a food-deprivation period of 35–40 days, and in 13-week starved and refed rainbow trout, gut mass surpassed that of control (without food deprivation) (Weatherley and Gill, 1981). Studies in growth hormone (GH) transgenic coho (Oncorhynchus kisutch Walbaum) and Atlantic salmon (S. salar Linnaeus) show that once the relative mass of the intestine has increased, the number of intestinal folds may increase, leading to a larger exchange area and faster body growth (Stevens et al., 1999; Stevens and Devlin, 2000). The increase in PCSI and ISI, as observed in our experiment before the surge in growth actually took place, suggests that body growth capacity might be limited by the extent of the digestive system development and by the time needed for increasing the digestive capacity to its optimal level following a period of fasting. As pyloric caeca are responsible for the synthesis and secretion of a wide range of digestive enzymes including trypsin (Raae and Walther, 1989), a larger tissue mass may allow a greater production (synthesis and
secretion) of trypsin and would therefore enhance the digestion of dietary protein. This interpretation is reinforced by the marked increase in trypsin activity observed through day 14, followed by a plateau during the last 2 weeks. The combined effect of trypsin activity and size of pyloric caeca resulted in a 29-fold increase in digestive capacity of the fish during the refeeding period. Trypsin has a pervasive function in digestion by making amino acids available for protein synthesis (Torrissen et al., 1994). This is clearly shown by the impact of trypsin inhibitors on growth rate in rainbow trout (Dabrowski et al., 1989). In contrast, liver growth mainly took place during the last 2 weeks of the experiment and it occurred concurrently and not before a significant gain in mass was observed. Thus liver size does not appear to limit growth upon refeeding although it may still play an important role in setting survival and growth capacity in the longer term. We suggest that energy production capacity as indicated by CS activity may also modulate growth capacity by limiting enzyme synthesis and nutrient absorption. Enzyme synthesis is an energy demanding process and energy production in the pyloric caeca may be enhanced through elevations in tissue aerobic capacity. CS activity in the pyloric caeca and intestine and CCO activity in the intestine followed a similar pattern as PCSI, ISI and trypsin activity. CS activity per fish increased 7fold during the refeeding period. The response of CCO activity was not as clear as that of CS activity however. Intestinal CCO was linearly related to the length of the refeeding period in juvenile cod deprived for 35 days (Foster et al., 1993) and was found to differ between starved and fed cod (Guderley et al., 1996; Dutil et al., 1998). CCO in the pyloric caeca increased soon after feeding was resumed in cod showing compensatory growth response (Bélanger et al., 2002). In contrast, white muscle CCO and CS activities did not significantly change upon refeeding and cannot be considered as being good indicators of growth compensation in cod. Similar results were obtained previously: white muscle CCO and CS activity did not increase to match growth rate (Pelletier et al., 1995) and CCO was not found to be a sensitive index of shortterm response to refeeding in juvenile and fast growing cod (Foster et al., 1993; Bélanger et al., 2002). Given the lack of a short-term response of enzymes involved in the energetic metabolism and the lowest enzymatic activities among the three tissues assayed, we conclude that white muscle growth is unlikely to be limited by aerobic capacity (Blier et al., 1997) and that the restoration of this aerobic capacity should be subsequent to the restoration of size and capacity of digestive tissues. In summary, our results are consistent with the concept of a biphasic mechanism during growth recovery: optimising digestive protease synthesis would be the first phase and restoring the growth trajectory and increasing body mass, the second phase. During the first phase (first 14 days in our experiment), ISI, PCSI and activity of trypsin and CS in the pyloric caeca and intestine reached their maximum level. After a deprivation period of 68 days, a time interval of 14 days was required for the digestive system to become fully functional again. At the start of the second phase, size and CS activity of the digestive tissue are maximum and catch up growth in body
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mass proceeds. The lack of response of white muscle upon refeeding strengthens the idea of digestive capacity setting a limitation on growth capacity. Previous studies on phenotypic flexibility of the digestive tract have focused on morphological indices and transport capacity (Secor and Diamond, 2000; Lignot et al., 2005; Krogdahl and Bakke-McKellep, 2005) or carbohydrate digestive capacities (Krogdahl and BakkeMcKellep, 2005). Krogdahl and Bakke-McKellep (2005) reported the effect of starvation and refeeding on a marker of protein digestive capacity (intestinal leucine aminopeptidase). In the latter study, however, the increase in digestive tissue mass was monitored, but not growth rate. To our knowledge, our study is the first one to make a parallel between an increase in body mass and the recovery of protein digestive capacity during a long refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases. Acknowledgments We thank 3 anonymous referees for comments on the manuscript. The authors are grateful to the technical staff of the Maurice-Lamontagne Institute who was involved in sampling and dissecting the fish in the field and laboratory. Special thanks to Mario Péloquin for his consistent work in following laboratory experiments from tagging to dissections. This study was made possible through funds provided by the Department of Fisheries and Oceans Canada (Science Strategic Funds) to J.-D. Dutil and by NSERC to P. U. Blier and J.-D. Dutil. References Ali, M., Nicieza, A., Wootton, R.J., 2003. Compensatory growth in fishes: a response to growth depression. Fish Fish. 4, 147–190. Bélanger, F., Blier, P.U., Dutil, J.-D., 2002. Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua). Fish Physiol. Biochem. 26, 121–128. Blier, P.U., Pelletier, D., Dutil, J.-D., 1997. Does aerobic capacity set a limit on fish growth rate? Rev. Fish. Sci. 5, 323–340. Dabrowski, K., Poczyczynski, P., Koch, G., Berger, B., 1989. Effect of partially or totally replacing fish meal protein by soybean meal protein on growth, food utilization and proteolytic enzyme activities in rainbow trout. New in vivo test for exocrine pancreatic secretion. Aquaculture 77, 29–49. Foster, A.R., Houlihan, D.F., Hall, S.J., 1993. Effects of nutritional regime on correlates of growth rate in juvenile Atlantic cod: comparison of morphological and biochemical measurements. Can. J. Fish. Aquat. Sci. 50, 502–512. Greene, H.W., 1992. The ecological and behavioural context for pitviper evolution. In: Campbell, J.A., Brodie Jr., E.D. (Eds.), Biology of the Pitviper. Selva press, Tyler, pp. 107–117. Jobling, M., 1980. Effects of starvation on proximate chemical composition and energy utilization of plaice, Pleuronectes platessa. J. Fish Biol. 17, 325–334. Jobling, M., 1994. Fish bioenergetics. Chapman and Hall, London.
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Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua) P.U. Blier a,⁎, J.-D. Dutil c , H. Lemieux a , F. Bélanger a , L. Bitetera b a
Laboratoire de Biologie Intégrative, Département de Biologie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1 b Département d'Océanographie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1 c Institut Maurice-Lamontagne, Ministère des Pêches et des Océans, 850 route de la Mer, Mont-Joli, Québec, Canada G5H 3Z4 Received 10 July 2006; received in revised form 3 October 2006; accepted 8 October 2006 Available online 13 October 2006
Abstract This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases. © 2006 Elsevier Inc. All rights reserved. Keywords: Citrate synthase; Cytochrome c oxidase; Gadid; Gastrointestinal tract; Metabolic enzymes; Trypsin
1. Introduction Many animal species experience long episodes of food deprivation during their life history. In Atlantic cod (Gadus morhua Linnaeus), these periods of fasting result in marked fluctuations in condition factor and energy reserves (Lambert and Dutil, 1997; Schwalme and Chouinard, 1999). During those episodes of low food availability, animals are expected to minimize the costs of maintenance and activity in order to enhance survival. Studies in mammals and birds have shown that the maintenance of a functional intestinal epithelium is costly, mainly because of the high cellular turnover rate in this tissue (Johnson, 1987; Starck, 1996). Given the benefits of a reduced rate of energy expenditure during long periods of low food availability, evolution should have favoured the capacity
⁎ Corresponding author. Tel.: +1 418 723 1986x1852; fax: +1 418 724 1849. E-mail address: [email protected] (P.U. Blier). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.10.012
to significantly downregulate the digestive performance during long episodes of fasting, and the subsequent capacity to rapidly upregulate it with feeding (Secor, 2005, 2001). For example, pythons, vipers and rattlesnakes can feed at intervals varying from one to several months and consume preys that can even exceed their own body mass (Greene, 1992; Pope, 1961; Secor and Diamond, 2000). These scarce meals are accompanied with a 2-fold increase in intestinal mass, 5- to 20-fold increases in intestinal nutrient transport capacity (Secor and Diamond, 2000) and 4.8-fold increases in the length of enterocyte microvilli (Lignot et al., 2005). Those postprandial responses were reversed following the completion of digestion (Secor and Diamond, 2000; Lignot et al., 2005). In food-deprived Atlantic cod, pyloric caeca and intestine mass increased over the values of controls (without food deprivation) during refeeding (Bélanger et al., 2002). In mammals and birds (Wilson and Osbourn, 1960) as well as in fish (review of Ali et al., 2003; Jobling, 1994), the period of feeding occurring after a period of food deprivation or
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malnutrition is usually accompanied with a burst of growth, a phenomenon known as compensatory growth. In cod, the activity of metabolic enzymes in the intestine (cytochrome c oxidase, CCO and citrate synthase, CS) is correlated with growth rate (Pelletier et al., 1994; Bélanger et al., 2002) and the activity of the same enzymes in the pyloric caeca even exceed values of control (without food deprivation) during compensatory growth (Bélanger et al., 2002). The metabolic capacity of digestive tissues is also sensitive to variations in nutritional status. Proteolytic enzyme (trypsin) activity may be positively correlated with growth rate and/or conversion efficiency (Bélanger et al., 2002; Lemieux et al., 1999) and the expression of different isozymes of trypsin in Atlantic salmon (Salmo salar L.) appears to be related to growth rate (RungruangsakTorrissen et al., 1999). Long-term fasting in Atlantic salmon has been associated with a decrease in carbohydrate digestive enzymes, whereas upon refeeding, enzyme capacities were regenerated after only 1 week (Krogdahl and Bakke-McKellep, 2005). For fast growth periods to occur, physiological adjustments may need to take place before or during the recovery growth phase. The aim of our study was to assess physiological factors suspected to partly modulate growth capacity following a period of food deprivation. We postulated that any factor determining growth performance after fasting should recover or increase its activity before a burst of body mass growth can take place. Fish were sampled at different intervals after a food deprivation period and the growth of different tissues and activity of metabolic and digestive enzymes were measured.
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Forty-eight hours after the last meal, cod were killed by a blow to the head. They were weighed, measured (fork length) and dissected. The total wet mass of the liver, pyloric caeca (rinsed with NaCl 0.7%), empty intestine (from pyloric caeca to the anus), carcass (eviscerated and head-off) were taken. A
2. Materials and methods Atlantic cod (G. morhua, Gadidae) were trawled in the St. Lawrence estuary near Matane (Québec, Canada) in June 1997. They were held under natural photoperiod and ambient salinity and temperature at the Maurice-Lamontagne Institute (MontJoli, Québec, Canada). The fish were fed a maintenance ration of frozen capelin (Mallotus villosus Müller) from June 1997 to May 1998 until the experiment started in May 1998. One hundred cod were anaesthetised with metomidate hydrochloride (5 mg·l− 1), weighed (± 0.1 g) and their fork length measured (±1 mm). They were then tagged with visible implant tags (Northwest Marine Technology, Shaw Island, Wash.) in the first dorsal fin. The fish were transferred to a 7 m3 fibreglass tank and underwent food deprivation for 68 days. After food deprivation, 10 fish were sampled (day 0) and 40 others were anaesthetised, measured (weight and length) and randomly transferred to eight 1.5 m3 fibreglass tanks (two tanks of five fish for each treatment) equipped with partially recirculating and temperature-controlled systems. The four treatments involved feeding periods of 2, 6, 14 or 28 days during which capelin was offered in excess for 1 hour on alternate days. Water temperature was kept at 10 °C and oxygen saturation was above 80%. All the procedures were approved by the University committee of animal welfare (CPA-UQAR 07– 11) according to the recommendations of the Canadian Council of Animal Care.
Fig. 1. Relative gains in mass (RGM), and pyloric caeca (PCSI), intestine (ISI) and liver (HSI) as an index of fish length in Atlantic cod refed after a deprivation period of 68 days. Differences between treatment were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate) for relative fish growth in mass (A) and with an ANOVA (factors tank nested in treatment) for individual morphometric indices (B: pyloric caeca, C: intestine, D: liver). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Means followed by one similar letter were not statistically different (post hoc Tukey's multiple comparison test).
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Table 1 Cod wet tissue masses (g) at days 0, 2, 6, 14 and 28 of refeeding after a food deprivation of 68 days Day Liver 0 2 6 14 28
Pyloric caeca
8.58 ± 4.94a 6.44 ± 1.55a a 7.89 ± 3.35 7.09 ± 1.93a 11.52 ± 3.46a 11.00 ± 3.11b 27.03 ± 9.83b 15.09 ± 4.58c 64.55 ± 21.96c 20.59 ± 6.12d
Intestine
Gonads
Carcass
4.13 ± 0.67a 21.30 ± 23.38 509.3 ± 106.6 5.23 ± 1.38ab 6.68 ± 3.56 515.4 ± 149.4 6.14 ± 1.88b 12.46 ± 21.43 552.6 ± 175.4 7.96 ± 1.69c 9.73 ± 9.86 556.6 ± 148.7 8.16 ± 2.12c 6.65 ± 2.47 623.7 ± 142.4
Differences were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different (post hoc Tukey's multiple comparison test).
sample of ∼ 10 g of white muscle was taken under the first dorsal fin and divided into two sub-samples. The pyloric caeca were finely chopped with a razor blade and divided into three sub-samples. Samples were handled on ice and tissues for enzymatic assays (sub-samples of muscle, pyloric caeca and whole intestine) were immediately frozen in liquid nitrogen and kept at − 80 °C. For each individual, initial (before feeding) or final (after feeding) Fulton condition factor, relative gain in mass (% total mass), and pyloric caeca (PCSI), intestine (ISI), carcass (CSI) and liver (HSI) somatic index (relative to fish length3) were calculated as follows:
CT, USA) equipped with a thermostated cell holder and a circulating refrigerated water bath. All assays were run in duplicate and enzymatic activity was expressed in U·g tissue− 1 (U is the amount of the enzyme that catalyzes the conversion of 1 μmol of substrate per minute). Conditions for enzyme assays followed those of Lemieux et al. (1999) for trypsin and those of Thibault et al. (1997) for CCO and CS. Statistical analyses were done with Statistica for Windows, release 5.1 (1997 edition; Statsoft, Tulsa, USA). Shapiro– Wilk's test was used to verify normality. Homogeneity of variance was tested with Levene's test. Differences in relative gain in mass and enzymatic activities were tested using ANCOVAs. We used the mixed model with treatment as a fixed effect (5 levels) and tank as a random effect (2 tanks per treatment). Fish length was used as the covariate. For morphometric indices, we tested treatment effect with a 2
Initial Fulton condition factor ðKi Þ ¼ 100ðMd L−3 d Þ Final Fulton condition factor ðKf Þ ¼ 100ððMf SCf ÞL3 f Þ Relative gain in mass ¼ 100½ððMf SCf Þ Md ÞMd1 Tissue somatic index ¼ 100ð Tissue massðL3 f ÞÞ where Md is the total mass (g) before feeding, Mf is the total mass (g) after feeding, SCf is the mass of the stomach content (g) after feeding, Ld is fork length (cm) before feeding, Lf is fork length (cm) after feeding, tissue mass is the wet total mass of the pyloric caeca, intestine, carcass or liver. To assay CCO and CS activities, samples of white muscle, intestine and pyloric caeca were homogenised in imidazole buffer (50 mM imidazole; 2 mM MgCl2·6H2O, 5 mM EDTA, 0.1% Triton X-100, and 1 mM reduced glutathione at pH 7.5). For the intestine and pyloric caeca, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) was added to the imidazole buffer. Tissues were disrupted in three 20-s bursts of homogenisation (Tekmar tissue grinder, Cincinnati, OH, USA). Intestine and pyloric caeca homogenates were centrifuged for 3 min at 1000 g (4 °C) (Centrifuge 22, Heraeus Sepatech, Osterode/Harz, Germany) and the supernatant taken for the running analysis. A second sample of pyloric caeca was diluted with Tris buffer (46 mM Tris, and 11.5 mM CaCl2, at pH 8.1), and subjected to three periods of 20 s of homogenisation and centrifugation for 15 min at 13 000 g (4 °C) to obtain the supernatant for the trypsin assay. Enzymatic assays were carried out at 20 °C for trypsin and 10 °C for CCO and CS activities. Readings were made using a UV/VIS spectrophotometer (Perkin Elmer Lambda 11, Shelton,
Fig. 2. Activities of trypsin (A), cytochrome c oxidase (CCO, B) and citrate synthase (CS, C) in pyloric caeca of Atlantic cod refed after a deprivation period of 68 days. Differences between treatment were tested with an ANCOVA (factors tank nested in treatment and fish length as a covariate). The factor tank was never found to have a significant effect. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different (post hoc Tukey's multiple comparison test).
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Fig. 3. Intestinal activities of cytochrome c oxidase (CCO, A) and citrate synthase (CS, B) of Atlantic cod refed after a deprivation period of 68 days. Values are mean ± standard deviations for pooled tanks (N = 8–10). Mean followed by one similar letter was not statistically different. Statistical details are as presented in Fig. 2.
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among individuals within each treatment and no significant difference was observed between treatments (P = 0.06) (Table 1). The relative mass of the pyloric caeca and intestine was the same for the unfed fish as for fed fish after 2 days of feeding, but increased significantly between day 2 and day 28 (Fig. 1B). The relative mass of intestine increased significantly between days 2 and 14 and then plateaued (Fig. 1C). By days 14 and 28 of refeeding, the mass of the liver relative to fish length (liver mass in g/fish length in cm3) was much higher (0.024 ± 0.005 and 0.058 ± 0.017, for 14 and 28 days, respectively) than that of deprived fish at day 0 (0.008 ± 0.004) (Fig. 1D). No statistical differences in the relative mass of the carcass were found over the feeding period (0.48 ± 0.05 at day 0, 0.45 ± 0.08 at day 2, 0.50 ± 0.08 at day 6, 0.49 ± 0.06 at day 14, and 0.57 ± 0.11 at day 28). In the pyloric caeca (Fig. 2), trypsin activity increased soon after feeding started and levelled off after day 14 of refeeding. CS activity followed a similar pattern, but this was not the case for CCO, which showed no significant differences among treatments. In contrast, both CS and CCO activity increased over time in the intestine (Fig. 3). CS activity significantly increased from unfed level after 6 days of refeeding and no further changes were observed thereafter. CCO activity increased progressively from day 2 to day 28 of refeeding. CS activity in the intestine was approximately three times lower than in the pyloric caeca, but CCO activity in the intestine was similar to that in the pyloric caeca. In the white muscle (Fig. 4), CS and CCO activities did not show any clear response to refeeding. The enzymatic activities
way-ANOVA (tank nested in treatment). When treatment was found to have a significant effect, Tukey a posteriori pairwise comparisons were done. The factor ‘tank’ was never found to have a significant effect. PCSI, trypsin, CCO and CS activities in pyloric caeca were log transformed for the statistical analyses, but untransformed data are presented as mean ± SD for 8–10 individuals (pooled tanks) in the table and figures. Results were considered significant at P b 0.05. 3. Results Total mass averaged 1016 ± 220 and 772 ± 195 g before or after food deprivation, respectively, length 480 ± 49 and 474 ± 33 mm and condition factor 0.93 ± 0.15 and 0.71 ± 0.09. No mortality was observed during the deprivation and feeding period. The 68-day starved cod lost on average 26.4 ± 6.9% of their body mass (252 ± 70 g) with no significant differences among treatments. When feeding was resumed, relative gain in mass significantly increased between day 14 and day 28 (Fig. 1A). Tukey test showed that fish on day 28 were heavier than all other treatments. From day 0 to 28, liver mass increased at a high rate, pyloric caeca and intestine mass increased more slowly while gonads mass decreased. On average, carcass mass (adjusted for differences in length between treatments, not shown) increased by 20 g over the first 14 days and 68 g over the last 14 days of the experiment, suggesting a greater increase in axial muscle mass during the second period, but carcass mass varied markedly
Fig. 4. White muscle activities of cytochrome c oxidase (CCO, A) and citrate synthase (CS, B) of Atlantic cod refed after a deprivation period of 68 days. Means are represented by the middle point and standard deviation, by the bars. Values are mean ± standard deviations for pooled tanks (N = 8–10). Data showed no significant differences between treatments. Statistical details are as presented in Fig. 2.
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were on average lower in white muscle than in other tissues: CCO activity was nearly three times lower than in the pyloric caeca and intestine. CS activity was also one third that in the pyloric caeca, but at the same level as in the intestine. 4. Discussion During refeeding, the relative gain in mass averaged 5.1 ± 5.8% body mass for the first 2 weeks and 34.9 ± 9.7 % for the whole feeding period. Thus 14 days were required after feeding was resumed before fish weight increased markedly. Liver growth contributed to a significant portion of the gain in mass, particularly during the last 2 weeks of the experiment, which suggests that energy storage in the liver for later use is an important aspect of the cod strategy to face periodic variations in food abundance. Fish in our experiment were of similar size and mass and followed a very similar experimental design as those of Bélanger et al. (2002) who concluded that compensatory growth had occurred after 10 weeks of food deprivation and 24 days of refeeding. Interestingly, the burst in relative gain in mass observed after day 14 of refeeding in the present study occurred after PCSI growth slowed down and ISI had increased to a maximum level. During the first 2 weeks of refeeding, PCSI doubled and ISI almost doubled. Further changes in these indices were not significant over the last 2 weeks of the experiment, precisely in the period when the bulk of growth occurred. Similarly, other studies have shown a correlation between PCSI and/or ISI index and growth rate or feeding status in fish. PCSI was 1.2 times higher in rainbow trout fed ad libitum, compared to trout fed a maintenance ration (Stevens and McLeese, 1988). In cod (Linares et al., 1996; Lemieux et al., 1999) and rainbow trout (Weatherley and Gill, 1983), PCSI and/or ISI were found to correlate positively with growth rate. Transgenic GH salmon, which grow 1.6-fold faster than non-transgenic, also have larger pyloric caeca (Stevens and Devlin, 2005) and a larger pyloric caeca exchange area (1.2 times the control) (Stevens et al., 1999). In European plaice (Pleuronectes platessa Linnaeus) (Jobling, 1980) and Atlantic salmon (Krogdahl and BakkeMcKellep, 2005), gut size decreased during a food-deprivation period of 35–40 days, and in 13-week starved and refed rainbow trout, gut mass surpassed that of control (without food deprivation) (Weatherley and Gill, 1981). Studies in growth hormone (GH) transgenic coho (Oncorhynchus kisutch Walbaum) and Atlantic salmon (S. salar Linnaeus) show that once the relative mass of the intestine has increased, the number of intestinal folds may increase, leading to a larger exchange area and faster body growth (Stevens et al., 1999; Stevens and Devlin, 2000). The increase in PCSI and ISI, as observed in our experiment before the surge in growth actually took place, suggests that body growth capacity might be limited by the extent of the digestive system development and by the time needed for increasing the digestive capacity to its optimal level following a period of fasting. As pyloric caeca are responsible for the synthesis and secretion of a wide range of digestive enzymes including trypsin (Raae and Walther, 1989), a larger tissue mass may allow a greater production (synthesis and
secretion) of trypsin and would therefore enhance the digestion of dietary protein. This interpretation is reinforced by the marked increase in trypsin activity observed through day 14, followed by a plateau during the last 2 weeks. The combined effect of trypsin activity and size of pyloric caeca resulted in a 29-fold increase in digestive capacity of the fish during the refeeding period. Trypsin has a pervasive function in digestion by making amino acids available for protein synthesis (Torrissen et al., 1994). This is clearly shown by the impact of trypsin inhibitors on growth rate in rainbow trout (Dabrowski et al., 1989). In contrast, liver growth mainly took place during the last 2 weeks of the experiment and it occurred concurrently and not before a significant gain in mass was observed. Thus liver size does not appear to limit growth upon refeeding although it may still play an important role in setting survival and growth capacity in the longer term. We suggest that energy production capacity as indicated by CS activity may also modulate growth capacity by limiting enzyme synthesis and nutrient absorption. Enzyme synthesis is an energy demanding process and energy production in the pyloric caeca may be enhanced through elevations in tissue aerobic capacity. CS activity in the pyloric caeca and intestine and CCO activity in the intestine followed a similar pattern as PCSI, ISI and trypsin activity. CS activity per fish increased 7fold during the refeeding period. The response of CCO activity was not as clear as that of CS activity however. Intestinal CCO was linearly related to the length of the refeeding period in juvenile cod deprived for 35 days (Foster et al., 1993) and was found to differ between starved and fed cod (Guderley et al., 1996; Dutil et al., 1998). CCO in the pyloric caeca increased soon after feeding was resumed in cod showing compensatory growth response (Bélanger et al., 2002). In contrast, white muscle CCO and CS activities did not significantly change upon refeeding and cannot be considered as being good indicators of growth compensation in cod. Similar results were obtained previously: white muscle CCO and CS activity did not increase to match growth rate (Pelletier et al., 1995) and CCO was not found to be a sensitive index of shortterm response to refeeding in juvenile and fast growing cod (Foster et al., 1993; Bélanger et al., 2002). Given the lack of a short-term response of enzymes involved in the energetic metabolism and the lowest enzymatic activities among the three tissues assayed, we conclude that white muscle growth is unlikely to be limited by aerobic capacity (Blier et al., 1997) and that the restoration of this aerobic capacity should be subsequent to the restoration of size and capacity of digestive tissues. In summary, our results are consistent with the concept of a biphasic mechanism during growth recovery: optimising digestive protease synthesis would be the first phase and restoring the growth trajectory and increasing body mass, the second phase. During the first phase (first 14 days in our experiment), ISI, PCSI and activity of trypsin and CS in the pyloric caeca and intestine reached their maximum level. After a deprivation period of 68 days, a time interval of 14 days was required for the digestive system to become fully functional again. At the start of the second phase, size and CS activity of the digestive tissue are maximum and catch up growth in body
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mass proceeds. The lack of response of white muscle upon refeeding strengthens the idea of digestive capacity setting a limitation on growth capacity. Previous studies on phenotypic flexibility of the digestive tract have focused on morphological indices and transport capacity (Secor and Diamond, 2000; Lignot et al., 2005; Krogdahl and Bakke-McKellep, 2005) or carbohydrate digestive capacities (Krogdahl and BakkeMcKellep, 2005). Krogdahl and Bakke-McKellep (2005) reported the effect of starvation and refeeding on a marker of protein digestive capacity (intestinal leucine aminopeptidase). In the latter study, however, the increase in digestive tissue mass was monitored, but not growth rate. To our knowledge, our study is the first one to make a parallel between an increase in body mass and the recovery of protein digestive capacity during a long refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases. Acknowledgments We thank 3 anonymous referees for comments on the manuscript. The authors are grateful to the technical staff of the Maurice-Lamontagne Institute who was involved in sampling and dissecting the fish in the field and laboratory. Special thanks to Mario Péloquin for his consistent work in following laboratory experiments from tagging to dissections. This study was made possible through funds provided by the Department of Fisheries and Oceans Canada (Science Strategic Funds) to J.-D. Dutil and by NSERC to P. U. Blier and J.-D. Dutil. References Ali, M., Nicieza, A., Wootton, R.J., 2003. Compensatory growth in fishes: a response to growth depression. Fish Fish. 4, 147–190. Bélanger, F., Blier, P.U., Dutil, J.-D., 2002. Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua). Fish Physiol. Biochem. 26, 121–128. Blier, P.U., Pelletier, D., Dutil, J.-D., 1997. Does aerobic capacity set a limit on fish growth rate? Rev. Fish. Sci. 5, 323–340. Dabrowski, K., Poczyczynski, P., Koch, G., Berger, B., 1989. Effect of partially or totally replacing fish meal protein by soybean meal protein on growth, food utilization and proteolytic enzyme activities in rainbow trout. New in vivo test for exocrine pancreatic secretion. Aquaculture 77, 29–49. Foster, A.R., Houlihan, D.F., Hall, S.J., 1993. Effects of nutritional regime on correlates of growth rate in juvenile Atlantic cod: comparison of morphological and biochemical measurements. Can. J. Fish. Aquat. Sci. 50, 502–512. Greene, H.W., 1992. The ecological and behavioural context for pitviper evolution. In: Campbell, J.A., Brodie Jr., E.D. (Eds.), Biology of the Pitviper. Selva press, Tyler, pp. 107–117. Jobling, M., 1980. Effects of starvation on proximate chemical composition and energy utilization of plaice, Pleuronectes platessa. J. Fish Biol. 17, 325–334. Jobling, M., 1994. Fish bioenergetics. Chapman and Hall, London.
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