Muscle Enzyme Activities in a Deep-Sea Squaloid Shark, Centroscyllium Fabricii, Compared with Its Shallow-Living Relative, Squalus Acanthias JASON R

JOURNAL OF EXPERIMENTAL ZOOLOGY 300A:133–139 (2003)

Muscle Enzyme Activities in a Deep-Sea Squaloid Shark, Centroscyllium fabricii, Compared With Its Shallow-Living Relative, Squalus acanthias JASON R. TREBERG1n, R. AIDAN MARTIN2, and WILLIAM R. DRIEDZIC1 1Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, A1C 5S7, Canada 2ReefQuest Centre for Shark Research, Vancouver, BC V7X 1A3, Canada

ABSTRACT The activities of several enzymes of energy metabolism were measured in the heart, red muscle, and white muscle of a deep and a shallow living squaloid shark, Centroscyllium fabricii and Squalus acanthias, respectively. The phylogenetic closeness of these species, combined with their active predatory nature, similar body form, and size makes them well matched for comparison. This is the ﬁrst time such a comparison has been made involving a deep-sea elasmobranch. Enzyme activities were similar in the heart, but generally lower in the red muscle of C. fabricii. Paralleling the trend seen in deep-sea teleosts, the white muscle of C. fabricii had substantially lower activities of key glycolytic enzymes, pyruvate kinase and lactate dehydrogenase, relative to S. acanthias or other shallow living elasmobranchs. Unexpectedly, between the squaloid sharks examined, creatine phosphokinase activity was higher in all tissues of the deep living C. fabricii. Low white muscle glycolytic enzyme activities in the deep-sea species coupled with high creatine phosphokinase activity suggests that the capacity for short burst swimming is likely limited once creatine phosphate supplies have been exhausted. J. Exp. Zool. 300A:133–139, 2003. r 2003 Wiley-Liss, Inc.

INTRODUCTION to the deep-sea and not some other factor. For example, Sullivan and Somero (’80), as well as Aspects of the metabolic and biochemical adap- Siebenaller et al. (’82), have suggested interspe- tations found in deep-sea fish have received cific differences in white muscle enzyme activities significant study (see Somero, ’92; Childress, ’95; from shallow and deep living fish may be explained Gibbs, ’97 for recent reviews). While numerous in part by the feeding and locomotory habits of data on differences between deep and shallow each species. As explained by Gibbs (’97), these living teleosts are available, to our knowledge data are further confounded by the potential similar data on elasmobranchs are entirely lack- influence of phylogenetic differences. ing. Deep-sea teleosts generally have reduced To eliminate as many confounding factors as metabolic rates and low levels of key enzymes of possible, some investigators have made efforts to energy metabolism in white muscle compared to utilise species of close phylogeny, with similar life- shallow living species. No trend between enzyme styles, but inhabiting different depths. This is activity and depth has been observed in the heart exemplified by the work on Sebastolobus alasca- or brain of teleosts. nus and S. altivelis, the latter of which inhabits With no data available on deep-sea elasmo- deeper water (for example, Siebenaller and branchs, we test the hypothesis that the activities Somero, ’78, ’79, ’82). of several enzymes of energy metabolism in the Although C. fabricii and S. acanthias belong to heart, red muscle, and white muscle of the deep different families (Etmopteridae and Squalidae, living Centroscyllium fabricii differ from that of respectively) they are both basal squaloids in the the shallow living Squalus acanthias as well as n literature values for other elasmobranch species. Correspondence to: Jason Treberg, Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NL, A1C 5S7, Interpretation of data on the adaptations of Canada. E-mail: [email protected] deep-sea fish is often difficult due to the inability Received 24 March 2003; Accepted 15 July 2003 Published online in Wiley InterScience (www.interscience.wiley. to conclude that differences are due to adaptation com). DOI: 10.1002/jez.a.10318. r 2003 WILEY-LISS, INC. 134 J.R. TREBERG ET AL. order Squaliformes (de Carvalho, ’96; Compagno, nitrogen. Red muscle was taken from the lateral ’99). Furthermore, sharks feature low genetic region between the second dorsal fin and the diversity (Smith, ’86) and slow rates of genetic caudal peduncal, and white muscle was sampled change across familial and ordinal taxa (Martin from the deep dorsal region below the first dorsal et al., ’92). Morphologically, squaloids are ‘extre- fin. In the case of C. fabricii, only animals that mely homogeneous’ (Bigelow and Schroeder, ’57). were still responsive to handling were sampled. The chosen species share the same basic body form Tissues were stored at or below 601C until and swimming style (Thompson and Simanek, ’77) analysis (between 20 and 30 months). Although and are of similar size, having many body and morphological characteristics in common (Bigelow 1 1 and Schroeder, ’48; Bass et al., ’76; Compagno, TA BL E 1. Ac t iv i t i e s ( mmol min g wet tissue ) of heart enzymes in Squalus acanthias and Centroscyllium fabricii ’84). The general prey categories, and trophic level, Squalus Centroscyllium are very similar between these species (Cortez, Enzyme acanthias fabricii ’99) with S. acanthias known to be an active Oxidative predator of fast-swimming prey (Bigelow and CS 5.1270.81 3.7271.27 Schroeder, ’48, ’53; Compagno, ’84). The non- MDH 54.976.00 59.3713.2 overlapping tricuspidate teeth of C. fabricii sug- gest grasping fast-moving prey, an interpretation Amino acid AlaAT 0.5670.17 0.3970.28 supported by stomach contents (Bigelow and n Schroeder, ’53; Compagno et al., ’89; Ebert et al., AspAT 24.975.41 12.074.58 7 7 ’92). Both species appear to be active predators, GDH 0.60 0.23 0.64 0.23 and they are among the few shark species known Ketone body to school (Bigelow and Schroeder, ’53; Temple- bHBDH 0.3170.29 0.2070.10 man, ’63; Compagno, ’84). While there are many similarities between these Glycolytic/anaerobic n species, C. fabricii is abundant at 550–1010m PK 34.375.39 23.976.01 (Bigelow and Schroeder, ’57) or 400–1340m LDH 48.975.44 40.078.19 7 7 n (Templeman, ’63), depending on region, whereas CPK 2.14 0.89 7.23 2.43 S. acanthias is uncommon below 180m Values are means7SD with n¼5forall.n - sig. di¡erence between spe- (Templeman, ’63). cies (po0.05, Student’s t-test). Abbreviations: MDH, malate dehydrogenase; AlaAT, alanine aminotransferase; GDH, glutamate dehydrogenase In addition to being the first data of this kind on otherwise see text for full enzyme names. a deep-sea elasmobranch, we measured a greater variety of enzymes than in previous studies on 1 1 teleosts and for the first time include red muscle in TA BL E 2. Ac t iv i t i e s ( mmol min g wet tissue )ofredmuscle enzymes in Squalus acanthias and Centroscyllium fabricii the tissues surveyed for enzyme activities involved with energy metabolism in a deep-sea fish. Enzyme S. acanthias C. fabricii MATERIALS AND METHODS Oxidative CS 9.9271.48 5.8470.78n Animals MDH 51.075.95 75.179.70n

Specimens of C. fabricii were captured by otter Amino acid trawl on the CCGS Teleost in November, 1999 off AlaAT 2.1070.19 0.5970.25n AspAT 29.475.86 18.373.84n the coast of Newfoundland where bottom tem- n perature was between 3.5 and 51C. C. fabricii were GDH 5.9770.85 1.4170.23 caught in water with a bottom depth below 1000 m Ketone body and ranged from approximately 300 to 900 grams. bHBDH 2.4970.61 1.1170.63n S. acanthias were caught by otter trawl in Passamaquoddy Bay, New Brunswick, in less than Glycolytic/anaerobic 100 m of water at approximately 10 to 121Cin PK 29.675.26 26.479.04 n August, 2000, and weighed between approxi- LDH 20.174.98 33.577.79 7 7 n mately 400 and 1150 grams. All animals were CPK 32.8 6.15 56.7 17.1 killed by a blow to the head and tissues were Values are means7SD with n¼5 for all. n - sig. di¡erence between dissected out, blotted dry, and frozen with liquid species (po0.05, Student’s t-test). See Table 1 for abbreviations. DEEP-SEA SHARK MUSCLE ENZYMES 135

TA BL E 3. Ac t iv i t i e s ( mmol min1 g wet tissue1) of white muscle enzymes in Squalus acanthias and Centroscyllium fabricii S. acanthias

Enzyme S. acanthias C. fabricii C. fabricii Shallow elasmobranchs Oxidative 7 7 n CS 0.54 0.18 0.24 0.04 024681012 7 7 n MDH 2.39 0.27 4.05 1.03 (a) Citrate synthase (µmol min-1 g-1)

Amino acid n AspAT 2.0970.24 0.8270.39 S. acanthias

C. fabricii Glycolytic/anaerobic n PK 101717.3 18.475.25 Shallow elasmobranchs LDH 160725.0 35.979.75n 7 7 n CPK 94.0 4.43 107 8.95 0 1020304050 (b) Pyruvate kinase (µmol min-1 g-1) Values are means7SD with n¼5forall.n - sig. di¡erence between species (po0.05, Student’s t-test). See Table 1 for abbreviations. S. acanthias

C. fabricii

Shallow elasmobranchs S. acanthias 020406080 C. fabricii (c) Lactate dehydrogenase (µmol min-1 g-1) Shallow elasmobranchs Fig. 2. Red muscle enzyme activities for (a) citrate synthase, (b) pyruvate kinase and (c) lactate dehydrogenase 02468 10 12 in S. acanthias and C. fabricii compared to shallow living (a) Citrate synthase (µmol min-1 g-1) elasmobranchs. Shallow living elasmobranchs, values are means7SD, n¼5 for all enzymes. Data for shallow living elasmobranchs: Crabtree and Newsholme, 1972; Alp et al., S. acanthias 1976; Zammit et al., 1978; Moon and Mommsen, 1987; C. fabricii Dickson et al., 1993. Where necessary, activities adjusted to 51C using a Q of 2. Shallow elasmobranchs 10

Deep living pelagic teleosts 0 1020304050 this storage period was quite long, all samples (b) µ -1 -1 Pyruvate kinase ( mol min g ) were stored for similar duration until analysis allowing comparisons between species and tissues. S. acanthias Furthermore, the observed activities of several C. fabricii enzymes in S. acanthias are similar to values from the literature (see Figs. 1–3). Shallow elasmobranchs

Deep living pelagic teleosts Enzyme assays 0 20 40 60 80 100 120 140 Samples were weighed and homogenised in nine (c) Lactate dehydrogenase (µmol min-1 g-1) volumes of ice-cold buffer (50 mM imidazole at pH Fig. 1. Heart enzyme activities for (a) citrate synthase, (b) 7.4) with a Polytron homogenizer. Homogenates pyruvate kinase, and (c) lactate dehydrogenase in S. acanthias were centrifuged at 10 000 g for ﬁve minutes at and C. fabricii compared to shallow living elasmobranchs and 1 deep living pelagic teleosts, values are means7SD. Shallow 4–8 C and the supernatants used directly for living elasmobranchs, n ¼ 6 with duplicate values for Raja enzyme assays. All enzyme activities were deter- erinacae for CS and PK averaged, n¼5 with duplicate values mined spectrophotometrically at 51C 7 0.11Cona for Raja erinacea for LDH averaged. Deep living teleost, n¼13 Beckman DU640 spectrophotometer equipped for all enzymes. Data for shallow living elasmobranchs: Alp with a jacketed cell holder connected to a thermo- et al., 1976; Zammit et al., 1978; Moon and Mommsen, 1987; Sidell et al., 1987; Dickson et al., 1993. Deep living teleosts stated water chiller. This temperature was chosen data from Childress and Somero (1979). Where necessary, because it is near, or within, the published activities adjusted to 51C using a Q10 of 2. temperature ranges for both species, 1–4.51C for 136 J.R. TREBERG ET AL.

chemicals were purchased from Sigma Chemical S. acanthias Company. C. fabricii Assay conditions were as follows: Citrate

Shallow elasmobranchs synthase (E.C. 4.1.3.7). 50 mM imidazole (pH 8.0), 0.1 mM DTNB and 0.3 mM acetyl CoA. Deep-sea demersal teleosts The reaction was initiated with 0.5 mM oxaloace- 0.0 0.2 0.4 0.6 0.8 tate. Malate dehydrogenase (E.C 1.1.1.37). 50 mM (a) Citrate synthase (µmol min-1 g-1) imidazole (pH 7.4) and 0.2 mM NADH. The reaction was initiated with 1.0 mM oxaloacetate. Alanine aminotransferase (E.C. 2.6.1.2). 50 mM S. acanthias imidazole (pH 7.4), 200 mM L-alanine, 0.2 mM C. fabricii NADH, 0.05 mM pyridoxal–5–phosphate and Shallow elasmobranchs 5.0 IU/ml lactate dehydrogenase. The reaction

Deep-sea demersal was initiated with 11.5 mM a-ketoglutarate (aKG). teleosts Aspartate aminotransferase (E.C. 2.6.1.1). 50 mM 0 20 40 60 80 100 120 140 imidazole (pH 7.4), 30 mM aspartate, 0.2 mM (b) Pyruvate kinase (µmol min-1 g-1) NADH, 0.05 mM pyridoxal–5–phosphate and 3.0 IU/ml malate dehydrogenase. The reaction S. acanthias was initiated with 7.0 mM aKG. Glutamate C. fabricii dehydrogenase (E.C. 1.4.1.3). 50 mM imidazole

Shallow elasmobranchs (pH 7.4), 250 mM ammonium acetate, 0.25 mM EDTA, 0.1 mM NADH and 0.1 mM ADP. The Deep-sea demersal reaction was initiated with 14 mM aKG. teleosts 0 50 100 150 200 250 300 b-Hydroxybutyrate dehydrogenase (E.C. 1.1.1.30). (c) Lactate dehydrogenase (µmol min-1 g-1) 50 mM Imidazole (pH 8.0), 11.25 mM NAD, 50.0 mM nicotinamide, and 2.0 mM dithiothreitol. The Fig. 3. White muscle enzyme activities for (a) citrate synthase, (b) pyruvate kinase and (c) lactate dehydrogenase reaction was initiated with 25 mM DL-b-hydro- in S. acanthias and C. fabricii compared to shallow living xybutyrate. Pyruvate kinase (E.C. 2.7.1.40). 50 elasmobranchs and deep-sea demersal teleosts, values are mM imidazole (pH 7.4), 10.0 mM MgCl2, 50.0 mM means7SD. Shallow living elasmobranchs, n¼10 species with KCl, 0.15 mM NADH, 5.0 mM ADP, 0.1 mM duplicate values for Squalus acanthias for CS and PK fructose–1,6–bisphosphate and 5.0 IU/ml lactate averaged, n¼11 for lactate dehydrogenase. Deep-sea demersal teleosts, n¼8; with duplicate values for 6 species averaged for dehydrogenase. Reaction was initiated by the all enzymes. Data for shallow living elasmobranchs: Alp et al., addition of 5.0 mM phosphoenolpyruvate. Lactate 1976; Zammit et al., 1978; Sullivan and Somero, 1980; Moon dehydrogenase (E.C. 1.1.1.27). 50 mM imidazole and Mommsen, 1987; Dickson et al., 1993; Battersby et al., (pH 7.4) and 0.2 mM NADH. The reaction was 1996. Data for deep-sea demersal teleosts from Sullivan and initiated by the addition of 1.0 mM pyruvate. Somero, 1980; Siebenaller and Somero, 1982; Siebenaller et al., Creatine phosphokinase (E.C. 2.7.3.2). 50 mM 1982. Where necessary, activities adjusted to 51C using a Q10 of 2. Imidazole (pH 7.4), 5.0 mM MgCl2, 1.0 mM ADP, 0.4 mM NADP, 1.0 mM glucose, 10.0 mM AMP, 2.0 IU/ml hexokinase, and 2.0 IU/ml glucose–6– C. fabricii (Templeman, ’63) and 3–151C for phosphate dehydrogenase. The reaction was in- S. acanthias (Scott and Scott, ’88) and thus is itiated by the addition of 50.0 mM phosphocrea- unlikely to cause thermal instability in the tine. enzymes. Assays were based on established proto- cols on fish muscle enzymes (Suarez et al., ’86; Analysis Moon and Mommsen, ’87; Sidell et al., ’87) and All data were compared by Student’s t-test with were measured by monitoring the oxidation or po0.05 being considered significant. reduction of pyridine nucleotides at 340 nm with the exception of citrate synthase which followed RESULTS AND DISCUSSION the production of free CoA with 5,5’–dithiobis (2–nitrobenzoic acid), (DTNB) at 412 nm. Control With enzyme activities measured in only two rates were determined in the absence of substrate, species, our results allow for comparison between and preliminary studies confirmed reaction rates these particular sharks, but do not allow for broad were linear with time and homogenate added. All ‘generalisations’ to be made about deep-sea elas- DEEP-SEA SHARK MUSCLE ENZYMES 137 mobranch metabolism. Further, the possibility Comparison with shallow living that the ambient temperature to which each elasmobranchs and deep living teleosts species was adapted may have an effect on enzyme levels cannot be eliminated from this study. Data on muscle CS, PK, and LDH activities are However, as explained previously, despite not available for a sufficient number of shallow living being of as close phylogeny as the Sebastolobus elasmobranchs and deep-sea teleosts to allow species referred to above, the species used are comparison with our data. To accommodate for reasonable for comparison and similar data on differences in assay temperature, literature values deep-sea elasmobranchs are entirely lacking. The have been adjusted to 51C using a Q10 of 2 where present study, in conjunction with literature necessary. Despite the long storage period, our values for elasmobranch enzyme activities, pro- enzyme values for S. acanthias generally agree vides a first snapshot into the metabolism of a well with literature values (see references listed in deep-sea elasmobranch. Figs. 1–3). Furthermore, in a study of several elasmobranch species, Dickson et al. (’93) found that storage from 1.5 to 44 months under similar conditions as the present study did not affect the Enzyme activities in muscle tissues of activity of CS, PK, or LDH when expressed on a C. fabricii and S. acanthias wet weight basis. Therefore, tissue dehydration Overall, the enzyme activity profiles in the heart was not considered to be a critical factor. Shallow of both species are quite similar (Table 1). living teleosts have not been included in figures Although aspartate aminotransferase (AspAT) 1–3 as Dickson et al. (’93) found that elasmo- and pyruvate kinase (PK) activities are about half branchs and teleosts of similar locomotory habits those in S. acanthias, creatine phosphokinase have comparable muscle enzyme activities. (CPK) activity in C. fabricii is higher. This Both species in the present study have similar elevated CPK activity may be representative of heart CS, PK, and LDH activity as other elasmo- how C. fabricii deals with burst activity, as branchs (Fig. 1). Although the LDH activity outlined below. appears lower in the present study, the high In contrast to the heart, all measured red degree of variability in literature values makes muscle enzyme activities, with the exception of our numbers not sufficiently low to be considered PK, differ between S. acanthias and C. fabricii exceptional. This similarity is consistent with the (Table 2). Low citrate synthase (CS) in the red notion that heart does not show a decrease in muscle of C. fabricii indicates reduced overall enzyme activity with increasing depth (Childress oxidative metabolism, and implies lower rates of and Somero, ’79); however, heart PK and LDH mitochondrial fuel utilisation compared to activity in C. fabricii are much higher than the S. acanthias. Consistent with this, enzymes deep living pelagic teleosts examined by Childress associated with providing oxidative substrates, and Somero (’79). The pelagic species used by amino acid metabolising enzymes, and b-hydroxy- Childress and Somero were substantially smaller, butyrate dehydrogenase (bHBDH), are lower in up to three orders of magnitude, than the speci- C. fabricii. Red muscle lactate dehydrogenase mens used in the present study. LDH has been (LDH) activity is higher in C. fabricii, whereas shown to be positively scaled with increasing body PK is similar between species. CPK activity in the mass while PK is negatively scaled in a teleost red muscle of C. fabricii was nearly double that of heart (Ewart et al., ’88). Thus, higher heart LDH S. acanthias, similar to the case in heart. in C. fabricii compared to much smaller deep-sea The white muscle of C. fabricii has much lower teleosts may be explained by scaling effects, but activities for CS, AspAT, PK, and LDH than this does not explain the high PK values. S. acanthias (Table 3). As in red muscle, C. fabricii Red muscle CS, PK, and LDH activities show had higher white muscle CPK activity; however, the same trend as heart (Fig. 2), with neither this difference between species is much less than species appearing exceptional to shallow living in the heart or red muscle. Several white muscle elasmobranchs measured in previous studies. To enzymes, alanine aminotransferase (AlaAT), our knowledge, no comparable deep-sea teleost glutamate dehydrogenase (GDH), and bHBDH, data are available. With respect to white muscle had activities below 0.01 mmol min1 g wet enzymes, deep-sea elasmobranchs may show the tissue1 for both species and were not included same trend of reduced enzyme activity found in in the analysis. deep-sea teleosts (Fig. 3). White muscle CS activity 138 J.R. TREBERG ET AL. is similar in all groups, though C. fabricii is on the also Childress, ’95). Our results are consistent low end of the spectrum of the data. The enzymes with this, and we add that a predator’s loss of PK and LDH have parallel trends, with activity in visual cues may result in a similar ‘laxness’ in the C. fabricii being much lower than in the shallow selection for extended burst capacity, thus limiting elasmobranchs, yet very similar to the deep-sea the ability for prolonged rapid and metabolically demersal teleosts (Fig. 3b,c). costly pursuit of prey. Unlike in heart, white muscle PK and LDH In summary, we have shown that the trend of activity are positively scaled with increasing body greatly decreased activities of PK and LDH in the mass in teleosts (Childress and Somero, ’90), and white muscle of deep-sea teleosts also occurs in a presumably in elasmobranchs. Thus, higher white deep-sea elasmobranch. With respect to the aero- muscle activities of PK and LDH in shallow living bic red muscle and heart, the deep-sea species elasmobranchs relative to the generally smaller had mostly lower red muscle enzyme activities deep-sea demersal teleosts could be partially whereas the heart showed little difference be- explained by scaling effects but this does not tween species. The shallow living S. acanthias explain the substantial difference between values displays a white muscle enzyme profile typical of for shallow living elasmobranchs and C. fabricii. the utilisation of phosphocreatine stores with Of note, the deep-sea demersal teleosts in figure 3 subsequent upregulation of anaerobic glycolysis are generally smaller than the C. fabricii used in to fuel burst swimming; however, the very low the present work, but the size ranges have anaerobic glycolytic capacity in conjunction with substantial overlap (teleosts ranged from approxi- high CPK activity in the white muscle of C. fabricii mately 50 to 800 g). suggests that phosphocreatine stores alone may account for the majority of burst swimming capacity in C. fabricii. Relevance of elevated CPK activity in C. fabricii ACKNOWLEDGEMENTS The consistently higher CPK activity in C. fabricii may reflect interspecies differences in The authors thank the crew of CCGS Teleost as the dependence on phosphocreatine to supplement well as the Department of Fisheries and Oceans, ATP during ‘burst’ activity, although the possibi- Canada, for assistance in obtaining deep-sea lity that these results are due to thermal compen- samples and Dr. J. S. Ballantyne for shallow water sation cannot be ruled out. Newsholme et al. (’78) samples. J. E. Stacey is also thanked for insightful have demonstrated that CPK activity in fish comments on early drafts of this manuscript. This muscle is highest in white muscle, which has a work was funded by an NSERC grant to WRD. greater dependence on glycolysis than tricar- boxylic acid cycle for energy metabolism. LITERATURE CITED While higher CPK activity in the deeper living Alp PR, Newsholme EA, Zammit VA. 1976. 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