The metabolism of tissues of thermally acclimated trout (Salmo gairdneri)

Comp. Bioehem. Physiol., 1969, Vol. 29, pp. 185 to 196. Pergamon Press. Printed in Great Britain

T H E METABOLISM OF T I S S U E S OF T H E R M A L L Y A C C L I M A T E D T R O U T (SALMO GAIRDNER1)* JOHN M A R K DEAN Biology Depai-tment, Battelle Memorial Institute, Pacific Northwest Laboratory, Richland, Washington (Received 13 September 1968)

Abstract--1. Tissues from cold-acclimated fish oxidized acetate and palmitate

more rapidly than tissues from warm-acclimated fish. 2. Red muscle converted acetate and palmitate to CO2 at a faster rate than did white muscle. 3. The rate of conversion of acetate to 14COz increased with incubation temperature, up to temperatures as high as 38°C. INTRODUCTION TEMPERATURE is a critical factor in the existence of aquatic poikilotherms. Fresh-

water fish are absolutely dependent upon the temperature of the environment and their biogeographical distribution is limited by their physiological capabilities, which determine their tolerances to temperature. We are interested in their compensatory responses to temperature at the tissue level. Studies of tissue respiration of fish by Fuhrman et al. (1944) and Peiss & Field (1950) seemed to indicate that rate changes in metabolic pathways were responsible for the changes in the level of respiration of acclimated tissues. The work of Ekberg (1962) showed shifts in the sensitivity of warm- and cold-acclimated gill tissues to metabolic inhibitors. Warm-acclimated gill tissues were inhibited more by iodo-acetate, whereas cold-acclimated tissues were more sensitive to cyanide. This was the first clear indication that there was a possible shift during thermal acclimation in the relative participation of different metabolic pathways of glucose metabolism. Kanungo & Prosser (1959), also using metabolic inhibitors, found that stimulation of respiration during cold adaptation of goldfish was largely due to increased pentose cycle activity. The work of Hochachka & Hayes (1962) with the eastern brook trout, S a l v e l i n u s fontinalis, demonstrated directly a shift in enzymic pathways with temperature acclimation. They showed that trout tissues have predominantly an Embden-Meyerhof (EM) and Krebs cycle carbon flow, with a shift toward a higher level of pentose cycle participation accompanying acclimation at low temperatures. Their data suggest that possibility of altered acetate metabolism with low-temperature acclimation. Acetate, as acetyl-CoA, can be used by * This paper is based on work performed under United States Atomic Energy Commission Contract AT(45-1)-1830. 185

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JOHN MARK DEAN

t h e cell in t h e K r e b s cycle or, a l t e r n a t i v e l y , as a p r e c u r s o r to lipids. A n i m p o r t a n t control feature of intermediary metabolism may be the regulation by citrate synthetase and acetyl-CoA-carboxylase, respectively, catalyzing the entry of acetylC o A into t h e K r e b s o r into f a t t y acid s y n t h e s i s ( A t k i n s o n , 1966).

MATERIALS AND METHODS Rainbow trout were acclimated at 5 and 18°C for 3 weeks prior to killing for biochemical studies. T h e y were fed a diet of Clark's pellets and were held without food for 24 hr prior to sacrifice. Their light cycle was that of natural daylight, which, during the course of this experiment, consisted of 16 hr of light and 8 hr of darkness. The fish were of the same age, their average weight was 200 g and they were sexually immature. T h e fish were stunned by a blow on the head, tissues were quickly removed and a 2"0-g sample homogenized in a glass tube containing 15 ml of chilled fish saline. T h e fish saline consisted of the following reagents (pH of 7"3 and concentrations in g/liter) : NaC1, 9"12; KC1, 0"37; CaC12, 0"33; MgCI~, 0"14; NaHPO4, 0.17; N a H , P O , , 0'04. T h e homogenized tissue was centrifuged at 120 g to remove the cellular debris. Three ml of the supernatant fraction were incubated with 0'5/zc of acetate-l-l*C (specific activity 2 me/m-mole, New England Nuclear Co.) and palmitate-l-1*C (10 mc/ m-mole, New England Nuclear Co.) in 2% bovine serum albumin. T h e tissues and substrates were added to a flask, closed with a rubber vial stopper, which held 0'5 ml of 2-amino°ethanol as a trap for the respired '4CO~. Tissues were incubated at the desired temperature for 3 hr. Lipids were extracted from the incubated homogenate with two 10-ml vol. of chloroformmethanol ( 2 : 1 ) . T h e chloroform fraction was washed with distilled water, dried with sodium sulfate and taken to dryness in a vacuum oven at 45°C. The residue was extracted with anhydrous methanol, the solution evaporated to dryness in vacuo and the residue dissolved in 5 ml of toluene. The activity of the trapped '*CO, and lipid fraction was counted by standard methods in a liquid scintillation counter. Oxygen consumption of tissue homogenates was determined on a Gilson Respirometer by standard methods. Biochemical determinations included total lipids and glycogens (Bragdon, 1951; Montogmery, 1957), and total protein was determined by the method of Lowry et al. (1951). Tissues for electron microscopy were fixed in 1% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon. T h e tissues were sectioned and strained with lead hydroxide for glycogen (Watson, 1958). After an appropriate analysis of variance (Snedecor, 1956), Duncan's (1955) multiplerange test was routinely used to judge the significance of mean differences. In some cases, the Student t-test was employed. Logarithmic transformation resulted in stabilization of values but did not appreciably affect mean differences previously judged statistically significant without transformation. RESULTS Morphology

T i s s u e s w e r e e x a m i n e d b y light a n d e l e c t r o n m i c r o s c o p y to d e t e r m i n e i f previously observed functional changes could be correlated with observed alterat i o n s in t h e m o r p h o l o g y o f t h e tissue. T h u s , r e d a n d w h i t e m u s c l e f r o m w a r m a n d c o l d - a c c l i m a t e d fish w e r e c o m p a r e d . N o m o r p h o l o g i c a l difference c o u l d b e o b s e r v e d at t h e light m i c r o s c o p y level in t h e s e tissues.

187

METABOLISM OF TISSUES OF THERMALLY ACCLIMATED TROUT

Morphological observations at the subcellular level by electron microscopy may be seen in Fig. 1. T h e red muscle (Fig. 1A) had large numbers of mitochondria and lipid bodies, whereas the white muscle (Fig. 1B) had few and scattered mitochondria and very low lipid levels. Glycogen was found between the fibers in both tissues. It is known that differences in subcellular morphology of liver tissue in fish can be induced by temperature acclimation (Berlin & Dean, 1967). However, we could not observe, at the fine structural level, any morphological differences in red or white muscle after thermal acclimation.

Tissue levels of lipid and glycogen Histochemical examination revealed a pattern in the distribution of metabolites in the tissues of warm- and cold-acclimated trout. We therefore measured the content of lipid and glycogen in the tissues of each. T h e results of this examination are detailed in Table 1. These results confirmed that quantitative, as well as T A B L E 1 - - E N E R G Y RESERVES I N TISSUES OF W A R M - AND COLD-ACCLIMATED TROUT

Temperature of acclimation Calculated t-value

d.f.

35"9+ 5"1 40"7,+12"5 23-9 + 5"6 9"2 -+ 1-9 41"5_+ 4-5 26'8 + 4"3 299"9 + 35"6 152"2+ 26"6

0"13 1"67 2"13" 2"73"

15 16 15 16

417'3 + 50"6 292"1+ 43'4 43"5 + 8"4 34-2+ 6'0 182"2 + 13"4 109"2+ 12"0 92"9 + 7"8 74"2+ 7"6

1"75 0"88 4"07t 1"68

18 19 21 19

5°C

18°C

Glycogen (t~g/mgprotein) Red muscle White muscle Cardiac muscle Liver

Total lipids (ftg/mg protein) Red muscle White muscle Cardiac muscle Liver

Values are means + the standard error of the mean. * Significant at P < 0"05. Significant at P<0"01. qualitative, differences are present in the tissues. T h e r e was an overall trend toward higher levels of lipid and glycogen in the tissues of cold-acclimated fish. T h e exception to this was white muscle, where no difference was measurable. T h e s e results suggested that the metabolism of red and white muscle should be examined in more detail. As an initial step, the oxygen consumption of these tissues was determined. T h e red muscle has a QO2N of 1.24+_0.11 as compared with 0.84+_ 0.15 for white muscle, both measured at 18°C. T h e r e is more glycogen in the red muscle than in the white muscle of both warm- and cold-acclimated fish (Table 1). Cardiac muscle is very similar, morphologically, to red muscle, and there is more glycogen in the cardiac muscle of cold-acclimated fish than in warm. Therefore, a tendency to higher levels of glycogen is indicated in the tissues of the cold-acclimated fish.

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T h e r e is a larger total lipid level in red muscle than in white at both 5 and 18°C, and a tendency toward high lipid levels in the cold-acclimated fish red muscle and liver than in the warm-acclimated fish tissues. T h e r e is no real difference between the total lipids in the white muscle of the cold fish as compared with that of the w a r m fish. T h e cardiac muscle was significantly different, as the cold-acclimated cardiac muscle has almost twice as high a total lipid level as did the w a r m acclimated cardiac muscle.

Acetate metabolism in muscle T h e results of the in vitro studies for the conversion of acetate-l-14C to 14CO2 b y red muscle and white muscle are shown in Fig. 2. T h e r e was a higher turnover of acetate by the tissues of the 5°C fish at higher incubation temperatures.

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FIc. 2. Conversion of acetate-l-14C to 14CO~ by red and white muscle tissue of warm- and cold-acclimated trout. Values shown are the mean + the standard error of the mean. Number of samples (n) for © was 11, 11 and 12; for Q, n was 11, 11 and 11 ; for [], n was 6, 16 and 7; and for I , n was 6, 9 and 7 at 5, 11.5 and 18°C, respectively.

Palmitate metabolism in muscle Palmitate was actively metabolized by red muscle, and there was a higher conversion of palmitate-l-t4C to 14CO~ by the cold-acclimated white and red muscle than the w a r m tissue at the high incubation temperatures (Fig. 3).

METABOLISM OF TISSUES OF THERMALLY ACCLIMATED TROUT

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Fxo. 3. C o n v e r s i o n of palmitate-l-14C to 14CO~ b y muscle tissue of w a r m - a n d c o l d - a c c l i m a t e d trout. Values s h o w n are t h e m e a n + t h e s t a n d a r d e r r o r o f the m e a n . F o r &, n was 4, 5 a n d 3 ; for e , n was 6, 5 a n d 4; for A, n was 3, 6 a n d 4; a n d for O, n was 5, 5 a n d 5 at 5, 11.5 a n d 18°C, respectively.

It is interesting to note the seeming temperature dependence of the 5°C acclimated tissues as compared with the independent response of the 18°C acclimated tissues.

Oxidation and lipogenesis in the liver Generally, liver tissue of 5°C fish at incubation temperatures of 5, 11.5 and 18°C had a higher oxidation of acetate than did the 18°C fish (Fig. 4). Liver tissue of the cold-acclimated fish showed a significantly higher incorporation of labeled acetate in the total lipids at the intermediate incubation temperature of 11.5°C than did warm-acclimated tissues (Fig. 5). It is apparent that when the conversion of acetate to CO2 was down, at 11.5°C, there was a correspondingly higher level of incorporation of labeled acetate into the total lipid fraction.

Incubation of lethal temperature In this experiment, tissue preparations, obtained as previously described, were incubated at 4°C increments from 18 to 38°C. Temperatures above 26°C are lethal for the intact fish. As can be seen in Fig. 6, all the tissues showed increased conversion of acetate to CO 2 at the higher temperatures.

JOHN MARK DEAN

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FIG. 4. Conversion of acetate-1-14C to 1~COs by liver tissue of warm- and coldacclimated trout. Values shown are the mean _+the standard error of the mean. For × , n was 22, 22 and 22; and for O, it was 23, 17 and 20 at 5, 11-5 and 18°C, respectively. DISCUSSION

Salmonids maintain their position in a desired habitat in a stream by constant swimming. During migration, they cover long distances with prolonged periods of sustained swimming and short intervals of extremely vigorous swimming activity. While migrating they are subjected to many environmental stresses; among these, temperature change is one of the most critical. For species such as steelhead and salmon, this change is marked, since they migrate from the low temperature e n v i r o n m e n t o f t h e ocean, at 6 - 9 ° C , to an a r e a o f h i g h e r t e m p e r a t u r e , 16-22°C, w h i c h is t y p i c a l l y f o u n d in r i v e r s a n d s t r e a m s in t h e fall o f t h e year.

Characteristics of red and white muscle F i s h p o s s e s s s o m e r e d m u s c l e fibers in a d d i t i o n to t h e d o m i n a n t m a s s o f w h i t e fibers. B o t h t y p e s o f fiber m a y b e r e a d i l y i d e n t i f i e d a n d d i f f e r e n t i a t e d

METABOLISM

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FIG. 5. Acetate-1-14C incorporated into total lipids after incubation with liver tissue of warm- and cold-acclimated trout. Values shown are the mean_ the standard error of the mean. For x, n was 20, 22 and 20; and for ©, n was 22, 15 and 21 at 5, 11.5 and 18°C, respectively. morphologically, histochemically and biochemically. The red muscle fibers are particularly well developed in the Salmonidae and Scombridae fishes. An excellent and extensive review of the history and current status of studies on red and white muscle in fish is given by Bone (1966). This study on the electrophysiology of the dogfish mytome conclusively shows a functional difference between the two muscle tissues. The biochemical differences in the concentrations of lipids, glycogens, lipase and succinic dehydrogenase of the red and white muscle fiber have been demonstrated by George (1962) in the mackerel, and by George & Bokdawala (1964) in labeo rohita. Bilinski (1963) has shown that red muscle will metabolize fatty acids faster than will white muscle. Some workers had concluded that the red muscle does not function as a muscle but, instead, either may function as a metabolic organ by acting as an energy reserve or for transport of a metabolic material to a desired site, or may serve as a vitamin and co-factor reserve, e.g. a peripheral liver (Braekkan, 1956, 1959; Buttkus, 1963; Wittenberger & Diaciuc, 1965). The evidence that has accumulated in this and other laboratories (Boddeke et al., 1959; Andersen et at, 1963; Bone, 1966; Rayner & Keenean, 1967) shows that this hypothesis is not reasonable. The general characteristics of muscles are that red muscle, as contrasted with white muscle, has high numbers of mitochondria, high lipid levels and a high rate of oxygen consumption. These results (Fig. 1A, B, Table 1) support the conclusion that red muscle is highly aerobic and is metabolically and morphologically suited for long-term, low-level, sustained activity. White muscle,

192

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FIG. 6. Conversion of acetate-l-14C to 14CO2 by tissues of warm (18°C) acclimated trout. Values shown are the mean _+the standard error of the mean. For 0 , n was 11, 16, 10, 12, 14 and 14; for A, n was 8, 13, 14, 16, 16 and 14; and for El, n was 9, 15, 15, 15, 13 and 12 at the incubation temperatures of 18, 22, 26, 30, 34 and 38°C, respectively.

metabolically and morphologically, functions for bursts of energy and can operate under more anaerobic conditions.

Muscle metabolism Unlike that of red muscle, the oxidative ability of the 5°C fish white muscle showed a direct temperature dependence (Fig. 2). It should be noted that there was an enhanced level of oxidative activity in the 5°C as compared with the 18°C acclimated tissues. The rate/temperature curves can be interpreted as translation and rotation (Prosser, 1958) of type IV C. Translation or a change in the position of the rate/ temperature curve could be due to a change in the amount of enzyme present in pH or in ionic strength, all of which could be reflected in a change in enzyme activity. However, rotation, or a change in the slope (QlO), suggests a qualitative change in enzymic patterns which might result from a shift in metabolic pathways. Palmitate was oxidized more rapidly by 5°C red muscle than by 18°C tissues and also at a significantly higher rate than in white muscle. It is interesting that

M E T A B O L I S M O F TISSUES O F T H E R M A L L Y A C C L I M A T E D T R O U T

193

the 18°C red and white muscle was independent of temperature while there was evidence of acclimation in the 5°C tissues (Fig. 3). These curves again represent a rate/temperature curve of type IV C. Red muscle has a higher turnover of palmitate than white muscle at environmental temperatures. These results compare favorably with and substantiate those of Bilinski (1963) obtained at higher incubation temperatures. The higher turnover values of labeled acetate and palmitate observed in the 5°C acclimated tissues, as compared with the 18°C acclimated tissues at other incubation temperatures, can be correlated with classical respiration studies. This is not, in itself, direct evidence of increased activity of the Krebs cycle, but it does indicate that changes in enzymic pathways are occurring in the animal. Liver metabolism The overall oxidative ability of the cold-acclimated liver tissue was higher than that of the warm tissue (Fig. 4). At the intermediate temperature, however, there was no significant difference between the tissues. The general response would be classed as type IV A with translation and clockwise rotation. When acetate incorporation into total lipids was measured, the response of the cold-acclimated fish was temperature-dependent (Fig. 5). The warm trout appeared to be temperature-independent at lower temperatures since it had the same level of incorporation at 5 and 11-5°C. There was no significant difference between the amount of acetate converted to 14CO2 by a 5 and 18°C trout at 11.5°C (Fig. 4), but there was a marked difference in incorporation of acetate into lipids 11.5°C. These results are precisely what one might expect for branching pathways competing for a common metabolite. The response of the liver tissue to an acute temperature exposure, according to the interpretation of Hochachka & Hayes (1962), would indicate an enhanced level of lipogenesis at 11.5°C (Fig. 4). However, in their experiment, the coldacclimated fish had a higher level of incorporation of acetate lipids at the highest temperature (20°C) while this was not observed in these experiments. We observed a greater pool of lipid reserve in the cold-acclimated fish (Table 1). The possibility exists that the 14C-acetate could be diluted upon incorporation, and rates of synthesis could actually be higher under these conditions, while the specific activity might be lower. This may either reduce or eliminate the apparent discrepancy between these results and those of Hochachka & Hayes (1962). Incubation of tissues at high temperatures The increased levels of labeled acetate converted to labeled CO~ by the tissues (Fig. 6) may indicate that the production of energy from metabolites is not a limiting factor for these fish during acute exposures at higher temperatures. These results correlate well with those of Fuhrman et al. (1944), who found that oxygen consumption of brain minces of bass did not fall off until temperatures were reached which were well above the lethal limits of the fish.

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JOHN MARKDEAN

Acclimatory response We can interpret these results (Figs. 2-6) in terms of the classical concepts of temperature adaptation. If the observed changes in acetate oxidation rates are strictly a quantitative response to change in the enzyme systems, one would expect only translation of the curves to take place (that is, a shifting up and to the left) but no rotation. Rotation implies a change in Qx0, and therefore a qualitative change in the enzymic pathways utilized by tissues at different temperatures. The shift may not be an absolute switching to different pathways, but a percentage of utilization. Following acetylation of CoA, there are two major pathways for the further metabolism of acetyl-CoA, the Krebs cycle and lipid synthesis. These data suggest that the channeling of acetate carbon between these two pathways is dependent upon the thermal history of the tissue. For example, in the cold-adapted trout, acetate flow through the Krebs cycle is held constant between 5 and 11.5°C. Presumably, the competitive ability of the pathway to lipid metabolism remains high and contributes to holding the Krebs cycle temperature-independent between 5 and 11.5°C (Hochachka, 1967). This does not appear to be the case for warmacclimated preparations. It is evident, therefore, that in addition to changes in Krebs cycle activity, some basic changes in metabolic regulation occur during temperature acclimation. Presumably, the control mechanisms for channeling carbon between the Krebs cycle and fatty acid synthesis are fundamentally different or are operating with different efficiency in the warm and cold fishes (Hochachka, 1968). That the responses in muscle and liver seem quantitatively different implies adaptive significance, but the data allow only qualitative statements. Thus, capacities for energy production and for energy storage appear to be higher in cold-adapted fishes and presumably compensate to some extent for the tendency of low temperature to depress metabolism and activity. It has recently been shown that a small number of basic mechanisms may contribute to the control of carbon flow in multi-enzyme systems in poikilotherms (Hochachka, 1968).

Environmental considerations A fish living at low temperature is in a rigorous physical environment: food is at a minimum and more effort is necessary to obtain food than at higher temperatures. This strain is compounded in the Columbia River drainage by the fact that the trout spawn during the coldest part of the annual temperature cycle. In this experiment, cold-acclimated fish had faster metabolism of intermediary metabolites in liver and in red and white muscle tissue. Other species of fish, as well as trout, have higher energy reserves and increased performance at lower temperatures (Dean & Goodnight, 1964; Wendt, 1965). Thus, cold-acclimatized fish such as migrating salmonids entering warmer water would be better able to utilize energy reserves of lipids and carbohydrates. These results suggest that an adaptive mechanism of fish is an enhanced utilization of metabolic intermediates after acclimation at low temperatures. Also, during the low-temperature period of

METABOLISMOF TISSUESOF THERMALLYACCLIMATEDTROUT

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the temperature cycle, the fish would be able to function at a higher metabolic rate than if acclimation had not occurred. T r o u t cannot exist for extended periods of time at temperatures above 25°C, even t h o u g h the tissues are able to convert metabolites into energy up to 38°C. These metabolic compensations make it possible for the fish to perform well in the habitat, which is necessary for successful existence. Modification of the habitat due to increasing environmental temperatures may remove the species, as they appear to be cold-adapted and have limited physiological capabilities for acclimation tO warmer environments.

Aeknowledgements--I would like to thank Mrs. D. G. Bauer and Mrs. A. M. Russell for their excellent technical assistance and Dr. J. M. Thomas for his statistical analysis. My sincere thanks are expressed to Dr, J. D. Berlin, who prepared the electron micrographs, and to my colleagues who have read the manuscript. Special thanks go to Dr. P. W. Hochachka for the many exchanges and criticisms that have aided the research and this manuscript. REFERENCES ANDERSEN P., JANSEN J. K. S. & LYNING Y. (1963) Slow and fast muscle fibers in the Atlantic hagfish (MyMne glutlnosa). Acta physiol, scand. 57, 167-179. ATKINSOND. E. (1966) Regulation of enzyme activity. A. Rev. Biochem. 35, 167-179. BERLINJ. D. & DEANJ. M. (1967) Temperature-induced alterations in hepatocyte structure of rainbow trout (Salmo gairdneri)..7, exp. Zool. 164, 117-132. BILINSKIE. (1963) Utilization of fipids by fish--I. Fatty acid oxidation by tissue slices from dark and white muscle of rainbow trout (Salmo galrdneri). Can. j~. Biochem. Physiol. 41, 107-112. BODDEK~ R., SLIJER E. J. & VAN DER STELT A. (1959) Histological characteristics of the body-musculature of fishes in connection with their mode of life. Proc. K. Ned. Akad. Wet. C. 62, 321-349. BONE Q. (1966) On the function of the two types of myotomal muscle fiber in elasmobranch fish. 3. mar. Biol. Ass. U.K. 46, 321-349. BRAEKKANO. R. (1956) Function of the red muscle in fish. Nature, Lond. 178, 747-748. BRAEKKANO. R. (1959) A comparative study of vitamins in the trunk muscles of fishes. FisMr. Skr. Tekn. Undersok. 3, 1-42. BRAGDON J. H. (1951) Colorimetric determination of blood lipids. J. biol. Chem. 190, 513-517. BROWN W. D. (1960) Glucose metabolism in carp. ft. cell. comp. Physiol. 55, 81-85. BUTTKUS H. (1963) Red and white muscle of fish in relation to rigor morris. ~. Fish. Res. Bd Can. 20, 45-58. DEAN J. M. & GOODNIGHTC. J. (1964) A comparative study of carbohydrate metabolism in fish as affected by temperature and exercise. Physiol. Zool. 27, 280-299. DUNCAN D. R. (1955) Multiple range and multiple F tests. Biometrics 11, 1-42. EKBERGD. R. (1962) Anerobic and aerobic metabolism in gills of the crucian carp adapted to high and low temperatures. Comp. Biochem. Physiol. 5, 123-128. FUHRMANF. A., HOLLINGERN., CRIMSONJ. M., FIELDJ., II & WEYMOUTHF. W. (1944) The metabolism of the excised brain of the largemouthed bass (Huro saloides) at graded temperature levels. Physiol. Zool. 17, 42-50. GEORGEJ. C. (1962) A histophysiological study of the red and white muscles of the mackerel. Am. midl. Nat. 68, 478--494. GEORGE J. C. & BOKDAWALAF. D. (1964) Cellular organization and fat utilization in fish muscle..7. Anim. Morph. Physiol. 11, 124-132.

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HOCHACHKAP. W. (1965) Isoenzymes in metabolic adaptation of a poikilotherm: subunit relationships in lactic dehydrogenases of goldfish. Archs Biochem. Biophys. 111, 96-103. HOCHACHKAP. W. (1967) Organization of metabolism during temperature compensation. In Molecular Mechanisms of Temperature Adaptation (Edited by PROSSER C. L.), Publication 84 of Am. Ass. Adv. Sci., pp. 117-203. HOCHACHgAP. W. (1968) Action of temperature on branch points in glucose and acetate metabolism. Comp. Biochem. Physiol. 25, 107-118. HOCX~ACHV~P. W. & HAYESF. R. (1962) The effect of temperature acclimation pathways of glucose metabolism in the trout. Can.J. Zool. 40, 261-270. KaNUNOO M. S. & PROSSERC. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures--II. Oxygen consumption of liver homogenate; oxygen consumption and oxidative phosphorylation of liver mitochondria. J. cell. comp. Physiol. 54(3), 265-274. LOWRYO. H., ROSEBROUGHN. J., FA~a A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. MONTGOMERYR. (1957) The determination of glycogen. Archs Biochem. Biophys. 57, 378386. PEISS C. N. & FIELDJ. (1950) The respiratory metabolism of excised tissues of warm and cold adapted fishes. Biol. Bull., Woods Hole 99, 213-224. PROSSER C. L. (1958) General summary: The nature of physiological adaptation. In Physiological Adaptation (Edited by PROSS~R C. L.), pp. 167-180. American Physiological Society, Washington, D.C. RAYNERM. D. & KXE~mANJ. J. (1967) Role of red and white muscles in the swimming of the skipjack tuna. Nature, Lond. 214, 392-393. SNEDECOR G. W. (1956) Statistical Methods (Sth edn). Iowa State College Press, Ames, Iowa. WATSON M. L. (1958) Staining of tissue sections for EM with heavy metals. ~. Biophys. Biochem. Cytol. 4(6), 727-730. WENDT C. (1965) Liver and muscle glycogen and blood lactate in hatchery-reared Salmo salar I,. following exercise in winter and summer. Rep. Inst. Freshw. Res. Drottningholm 46, 167-184. WITTI~BRROER C. & DIACIUC I. V. (1965) Effort metabolism of lateral muscles in carp. ft. Fish. Res. Bd Can. 22, 1397-1406.

Key Word Index---Temperature adaptation; trout; Salmo gairdneri; metabolism; muscle metabolism; red muscle; white muscle; acetate; palmitate.