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Metabolic rate of the hagfish, Eptatretus stoutii (Lockington) 1878
Comp. Biochem. Phydoi., 1965, Iiol. 16, pp. 1 to 6. Pergamon Press Ltd. P ~ t e d in Great Britain
METABOLIC RATE OF THE HAGFISH, E P T . 4 T R E T U S S T O U T I I (LOCKINGTON) I878" FREDERICK W. M U N Z and ROBERT W. MORRIS Department of Biology, University of Oregon, Eugene, Oregon
(Received 26 January 1965; revised 23 March 1965) Abstract--1. The hagfish, Eptatretus stoutii, has a very low metabolic rate, amounting to only about 0.008-0.010 ml Oi consumed/g wet wt/hr at 10°C. 2. We found no relationship between body weight and metabolic rate, within the limits of available sizes. 3. Under the conditions of these experiments there was no significant metabolic adjustment that could be ascribed to thermal history. 4. There is a significant decrease in percentage of body water with increase in size, INTRODUCTION IN THE recent book of Brodal & F~inge (1963), contributions of many distinguished authors treat the biology of myxinoid chordates quite comprehensively, but there are no data on general metabolic rate of any of the several species discussed. The absence of such information is probably not due to oversight. Certain habits of these animals would tend to discourage one from seeking to make metabolic measurements, on technical grounds alone. Their prodigious secretion of slime would appear to have a potential for disrupting almost any concdvable respirometric routine. However, we have found that the species considered in the present study lends itself to one respirometric technique very well. This paper reports measurements of respiration rate of Eptatretus stoutii (Lockington) 1878. Such data are frequently found to be of rather general interest in several phases of ecology and physiology. MATERIALS AND METHODS The hagtish used in this study were from a collection that was trapped at a depth of approximately 70 m near Coos Bay, Oregon, on 11 August 1964. They were transported to the laboratory in plastic containers of seawater in which ice was suspended in polyethylene bags. The fish were maintained in laboratory cold rooms in aerated seawater. Some of the animals were maintained at approximately 4°C during their laboratory life. Others were maintained at I0°C. None of them was fed. The water in the holding * This study was collateral to other researches which are supported by the National Institutes of Health (A--6222) and the National Science Foundation (G-23319).
VOL. I-A
FREnm~ICK W . MUNZ AND ROBERT W . Mom~is
aquaria was changed once each week and antibiotics (streptomycin sulfate and penicillin-G, approximately 50 ppm each) were added to suppress bacterial activity. Salinity of the sea-water used throughout this study was approximately 34~oo. Respiration experiments were begun after the fish had been held in the laboratory for 21 days. On each of 13 consecutive days, parallel experiments were conducted, one with a fish having a 4°C laboratory history and the other with a fish having a 10°C laboratory history. Hence, the period of controlled laboratory life of the experimental fish ranged from 3 to 5 weeks. Two sets of apparatus of a type described by Morris (1963) were used for the experiments. This apparatus is a modified Barcroft respirometer utilizing principles described by Dixon (1951) for making direct measurements of gas volume changes. In its present form it was first used by Morris (1961) and has since been used in a number of other studies here and at other laboratories. With the experimental routine followed in the present work it gives results that are highly reproducible. It has distilling flasks of 1 1. capacity for respiration and compensation vessels. These flasks have two or three ground (standard taper) necks, the largest of which is No. 34/45. In this apparatus the volume of oxygen is measured directly and later the measurement is appropriately corrected for temperature and vapor pressure. The volumes discussed in this paper have been corrected to standard temperature and pressure. The routine of each experiment was as follows. Approximately 500 ml of water were placed in the compensation vessel of the respirometer. The respiration vessel was placed in the holding tank with the fish and filled with seawater. The water in the tank was stirred gently, inducing the fish to swim about. After a fish swam in, the vessel was quickly raised and approximately half of its contents poured off. When manipulated in this way, the animals did not secrete slime. The vessel was then fixed in the respriometric apparatus. The alkali container was charged with 10% potassium hydroxide solution and the stoppers were seated. The respirometer was then immersed in a water bath having a temperature of approximately 5°C. The respiration vessel was flushed with oxygen and the apparatus was allowed to stand overnight. The following morning the stopcocks were closed and a ½ hr period was allowed for equilibration. Immediately following was a 2 hr period for the respiration measurement. The stopcocks were then opened and the temperature of the bath was raised to 10°C. A ½ hr period for equilibration and a 2 hr period for measurement were again allowed. This procedure was repeated at 15°C. Thus, measurements were made on each fish at 5, 10 and 15°C. Temperature of the water baths was changed with hot water or ice. The experiments were carried out in a cold room and constant temperature of the baths was maintained by 300 W heaters operating on the demand of Fenwal thermoswitches through "hot-wire" relays. This arrangement gives good results in so far as tolerance (+ 0-1°C) is concerned. However, in practice it is difficult to make the initial setting of a given switch for the precise temperature desired. Hence, in any given experiment the temperatures may have only approximated 5, 10 or 15°C (Fig. 1). In recording and treating the data, the actual observed values (to the closest 0"I°C) were always used.
M E T A B O L I C RATE O F H A G F I S H
At the conclusion of the respiration run at 15°C the apparatus was opened, the fish was removed and dried with towelling, and its wet weight was determined. It was then tagged and placed in 10% formalin. At the conclusion of the experiments the fish were removed from formalin, cut open and placed individually in weighed aluminium foil trays. The trays were stacked in a vacuum oven and dried at 80°C until they were found to have come to constant weight.
x
0"02
es / k...J
E
.-. .- -
5 y
"~ o.ol
x Z 0
~ x
x
~
oX
x
0.003
DEGREES C
FIG. 1. Hagfish metabolic rate-temperature plot. Measurements made of fish acclimated to 4°C represented by ©; measurements of 10°C acclimated fish represented by x . Regression lines fitted by the method of least squares. T h e solid line is fitted to the data on 4 ° fish. T h e interrupted line is fitted to the data
on 10° fish. Twenty-six fish were considered in this study. Thirteen had a 10°C laboratory history and the other 13, a 4°C history. The weights of those animals having a 4°C thermal history ranged from 28.1-105.4 g (mean = 60-20 g). Those having the 10°C thermal history ranged from 34-0 to 106.8 g (mean = 61.83 g). The respirometric data were organized in two groups according to thermal history and each of the groups was treated with a multiple regression equation, Y = a+blXl+b2X2,
in which Y is the log of the estimated metabolic rate (ml 02 consumed/g/hr), a is the Y intercept, and bl and bs are the partial regression coefficients of X 1 and X 2 respectively. Xa represents the temperature in deg C and Xx represents the log of the weight in g.
4
FREDERICK W. MUNZ AND ROBBRT W. MORRIS
RESULTS AND DISCUSSION The data yielded the equations given below and the respective multiple regression coefficients (R) are also given. Fish acclimated to 4°C: Y-- -3-2608+0.0437Xl+0.1707X s R = 0.8742. Fish acclimated to 10°C. Y-- - 3 . 7 7 1 7 + 0 . 0 3 8 2 X 1 - 0 . 1 2 6 2 X 2 R = 0.7440. Tests by analysis of variance showed that each of the coefficients, R, was highly significant with P less than 0-001 in each case. It was found by t tests that the temperature coefficient (bl) of each equation was highly significant (P<0.001), but neither of the size coefficients (b2) was significant (Table 1). An analysis of variance indicated no significant difference between the two coefficients, R (P > 0.05 with 3 and 72 deg of freedom). Hence, under the conditions of this study this species shows no statistically significant ability to acclimate to temperature. Additional evidence that this species does not metabolically adjust to temperature is indicated by the fact that a t test comparing the temperature coefficients of the two groups showed no significant difference between them ( P > 0.50). Bullock (1955) made a strong point of the fact that thermal acclimation results not only in displacement of the rate-temperature curve but in a change in its slope as well. The acclimation temperatures (4 ° and 10°C) appear to be ecologically realistic for this species. This magnitude of difference in thermal history has been found sufficient to elicit a significant metabolic adjustment in a variety of bony fishes we have investigated. It seems probable that this apparent lack of ability in thermal acclimation can be related to the thermally stable natural habitat, where such an ability would confer little selective advantage. The lack of a significant relationship between size and metabolic rate is a further indication that this species is unable to acclimate to temperature. The early work of Krogh (1916) showed that, generally speaking, one could expect the metabolic rate of fishes to reflect size by a coefficient of about - 0 . 1 5 log wt. Rao & Bullock (1954), however, brought out the fact that in poikilotherms the Q10 of metabolic rate is directly related to size. Hence, the relationship between size and metabolic rate will vary, depending on the temperature at which measurements are made. It was recently reported by Morris (1962) that there appears to be a developmental sequence in acquisition of mechanisms for thermal acclimation which gives rise to the relationship between size and Qx0. The fact that in the hagfish we found no relationship between size and metabolic rate is indeed a negative result, and could have been caused by some defect in the experimental routine, but this seems improbable since a similar routine has yielded positive results in numerous other species we have investigated. Hence, we believe that this apparent lack of relationship between size and metabolic rate is real, within the size limits considered. The general levels of metabolic rate are conspicuously low. The rate-temperature curves were calculated from individual measurements of metabolic rate
4 10
Thermal history of experimental group (°C)
0"8742 0.7740
0.1054 < 0.1503 <
Multiple regression Standard coefficient error of R R
TABLE 1--INFORMATION
0.001 0-001
Probability of R 0.0437 0"0382
Temperature coefficient bl 0.0041 < 0"0058 <
Standard error of bx 0.001 0.001
Probability of bl
0-1707 --0-1262
0.1121 0.2055
Weight Standard coefficient error of b2 b2
> >
0-20 0.50
Probability of bj
ON THE REGRESSION EQUATIONS OF THE T W O EXPERIMENTAL GROUPS OF HAGFISH
Z/I
6
FREDERICK W . MUNZ AND ROBERT W. MORRIS
(Fig. 1) according to the equation, Y = a+bX,
i.e. body size has been dropped from consideration, X representing temperature in deg C, and b is the temperature coefficient. The slopes of the regression curves yield Q10 values of 2"8 in the fish with the 4°C thermal history and 2.4 in the 10°C fish. The animals were usually agitated each time the temperature of the water bath was increased. However, at such times they were active for only a few minutes. T h e y remained quietly coiled throughout the periods of measurement. Hence, the rates we have measured are of resting metabolism. From the regression curves we see that one could expect oxygen consumption to be 0.008-0.010 ml/g/hr at 10°C. This amounts to only about 0.1 the rate of oxygen consumption of a number of species of bony fishes measured at ecologically realistic temperatures (cf. Morris, 1961, 1962) and compares favourably with metabolic rate of the Antarctic zoarcid, Rhigophila dearborni (Wohlschlag, 1963). Determinations of dry weight demonstrated that, on the average, we can expect the water content of these animals to amount to approximately 78 per cent (S.D. = 3"01 per cent). Hence, at 10°C the average Qo2 would amount to approximately 0.035 or 0-040 ml O~/g dry wt/hr. The low metabolic level measured in terms of wet weight cannot be ascribed to an unusually high water content. T h e water content of the twenty-six animals used in this study ranged from 71.3 to 83.1 per cent. Calculation of the zero-order correlation coefficient, r, to determine the possible relationship between body size (wet wt. in g) and water content (per cent) showed this coefficient to be - 0.65. A t test of this coefficient showed it to be highly significant (P < 0.001). Thus, increase in size is accompanied by a decrease in percentage of water content. REFERENCES BRODALA. & FANCER. (1963) The Biology ofMyxine. Universitets-forlaget, Oslo, Norway. BULLOCKT. H. (1955) Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30, 311-342. DIXON M. (1951) Manometric Methods. Cambridge University Press, London. KROC;H A. (1916) Respiratory Exchange of Animals and Man. Longmans, Green & Co., London. MORRIS R. W. (1961) Distribution and temperature sensitivity of some eastern Pacific cottid fishes. Physiol. Zob'l. 34, 217-227. MORRIS R. W. (1962) Body size and temperature sensitivity in the cichlid fish, Aequidens portalegrensis (Hensel). Amer. Nat. 96, 35-50. MORRIS R. W. (1963) A modified Barcroft respirometer for study of aquatic animals. Turtox News 41, 22-23. RAO K. P. & BULLOCKT. H. (1954) Q~0 as a function of size and habitat temperature in poildlotherms. Amer. Nat. 88, 33 A.A. WOHLSCHLAGD. E. (1963) An Antarctic fish with unusually low metabolism. Ecology 44, 557-564.
METABOLIC RATE OF THE HAGFISH, E P T . 4 T R E T U S S T O U T I I (LOCKINGTON) I878" FREDERICK W. M U N Z and ROBERT W. MORRIS Department of Biology, University of Oregon, Eugene, Oregon
(Received 26 January 1965; revised 23 March 1965) Abstract--1. The hagfish, Eptatretus stoutii, has a very low metabolic rate, amounting to only about 0.008-0.010 ml Oi consumed/g wet wt/hr at 10°C. 2. We found no relationship between body weight and metabolic rate, within the limits of available sizes. 3. Under the conditions of these experiments there was no significant metabolic adjustment that could be ascribed to thermal history. 4. There is a significant decrease in percentage of body water with increase in size, INTRODUCTION IN THE recent book of Brodal & F~inge (1963), contributions of many distinguished authors treat the biology of myxinoid chordates quite comprehensively, but there are no data on general metabolic rate of any of the several species discussed. The absence of such information is probably not due to oversight. Certain habits of these animals would tend to discourage one from seeking to make metabolic measurements, on technical grounds alone. Their prodigious secretion of slime would appear to have a potential for disrupting almost any concdvable respirometric routine. However, we have found that the species considered in the present study lends itself to one respirometric technique very well. This paper reports measurements of respiration rate of Eptatretus stoutii (Lockington) 1878. Such data are frequently found to be of rather general interest in several phases of ecology and physiology. MATERIALS AND METHODS The hagtish used in this study were from a collection that was trapped at a depth of approximately 70 m near Coos Bay, Oregon, on 11 August 1964. They were transported to the laboratory in plastic containers of seawater in which ice was suspended in polyethylene bags. The fish were maintained in laboratory cold rooms in aerated seawater. Some of the animals were maintained at approximately 4°C during their laboratory life. Others were maintained at I0°C. None of them was fed. The water in the holding * This study was collateral to other researches which are supported by the National Institutes of Health (A--6222) and the National Science Foundation (G-23319).
VOL. I-A
FREnm~ICK W . MUNZ AND ROBERT W . Mom~is
aquaria was changed once each week and antibiotics (streptomycin sulfate and penicillin-G, approximately 50 ppm each) were added to suppress bacterial activity. Salinity of the sea-water used throughout this study was approximately 34~oo. Respiration experiments were begun after the fish had been held in the laboratory for 21 days. On each of 13 consecutive days, parallel experiments were conducted, one with a fish having a 4°C laboratory history and the other with a fish having a 10°C laboratory history. Hence, the period of controlled laboratory life of the experimental fish ranged from 3 to 5 weeks. Two sets of apparatus of a type described by Morris (1963) were used for the experiments. This apparatus is a modified Barcroft respirometer utilizing principles described by Dixon (1951) for making direct measurements of gas volume changes. In its present form it was first used by Morris (1961) and has since been used in a number of other studies here and at other laboratories. With the experimental routine followed in the present work it gives results that are highly reproducible. It has distilling flasks of 1 1. capacity for respiration and compensation vessels. These flasks have two or three ground (standard taper) necks, the largest of which is No. 34/45. In this apparatus the volume of oxygen is measured directly and later the measurement is appropriately corrected for temperature and vapor pressure. The volumes discussed in this paper have been corrected to standard temperature and pressure. The routine of each experiment was as follows. Approximately 500 ml of water were placed in the compensation vessel of the respirometer. The respiration vessel was placed in the holding tank with the fish and filled with seawater. The water in the tank was stirred gently, inducing the fish to swim about. After a fish swam in, the vessel was quickly raised and approximately half of its contents poured off. When manipulated in this way, the animals did not secrete slime. The vessel was then fixed in the respriometric apparatus. The alkali container was charged with 10% potassium hydroxide solution and the stoppers were seated. The respirometer was then immersed in a water bath having a temperature of approximately 5°C. The respiration vessel was flushed with oxygen and the apparatus was allowed to stand overnight. The following morning the stopcocks were closed and a ½ hr period was allowed for equilibration. Immediately following was a 2 hr period for the respiration measurement. The stopcocks were then opened and the temperature of the bath was raised to 10°C. A ½ hr period for equilibration and a 2 hr period for measurement were again allowed. This procedure was repeated at 15°C. Thus, measurements were made on each fish at 5, 10 and 15°C. Temperature of the water baths was changed with hot water or ice. The experiments were carried out in a cold room and constant temperature of the baths was maintained by 300 W heaters operating on the demand of Fenwal thermoswitches through "hot-wire" relays. This arrangement gives good results in so far as tolerance (+ 0-1°C) is concerned. However, in practice it is difficult to make the initial setting of a given switch for the precise temperature desired. Hence, in any given experiment the temperatures may have only approximated 5, 10 or 15°C (Fig. 1). In recording and treating the data, the actual observed values (to the closest 0"I°C) were always used.
M E T A B O L I C RATE O F H A G F I S H
At the conclusion of the respiration run at 15°C the apparatus was opened, the fish was removed and dried with towelling, and its wet weight was determined. It was then tagged and placed in 10% formalin. At the conclusion of the experiments the fish were removed from formalin, cut open and placed individually in weighed aluminium foil trays. The trays were stacked in a vacuum oven and dried at 80°C until they were found to have come to constant weight.
x
0"02
es / k...J
E
.-. .- -
5 y
"~ o.ol
x Z 0
~ x
x
~
oX
x
0.003
DEGREES C
FIG. 1. Hagfish metabolic rate-temperature plot. Measurements made of fish acclimated to 4°C represented by ©; measurements of 10°C acclimated fish represented by x . Regression lines fitted by the method of least squares. T h e solid line is fitted to the data on 4 ° fish. T h e interrupted line is fitted to the data
on 10° fish. Twenty-six fish were considered in this study. Thirteen had a 10°C laboratory history and the other 13, a 4°C history. The weights of those animals having a 4°C thermal history ranged from 28.1-105.4 g (mean = 60-20 g). Those having the 10°C thermal history ranged from 34-0 to 106.8 g (mean = 61.83 g). The respirometric data were organized in two groups according to thermal history and each of the groups was treated with a multiple regression equation, Y = a+blXl+b2X2,
in which Y is the log of the estimated metabolic rate (ml 02 consumed/g/hr), a is the Y intercept, and bl and bs are the partial regression coefficients of X 1 and X 2 respectively. Xa represents the temperature in deg C and Xx represents the log of the weight in g.
4
FREDERICK W. MUNZ AND ROBBRT W. MORRIS
RESULTS AND DISCUSSION The data yielded the equations given below and the respective multiple regression coefficients (R) are also given. Fish acclimated to 4°C: Y-- -3-2608+0.0437Xl+0.1707X s R = 0.8742. Fish acclimated to 10°C. Y-- - 3 . 7 7 1 7 + 0 . 0 3 8 2 X 1 - 0 . 1 2 6 2 X 2 R = 0.7440. Tests by analysis of variance showed that each of the coefficients, R, was highly significant with P less than 0-001 in each case. It was found by t tests that the temperature coefficient (bl) of each equation was highly significant (P<0.001), but neither of the size coefficients (b2) was significant (Table 1). An analysis of variance indicated no significant difference between the two coefficients, R (P > 0.05 with 3 and 72 deg of freedom). Hence, under the conditions of this study this species shows no statistically significant ability to acclimate to temperature. Additional evidence that this species does not metabolically adjust to temperature is indicated by the fact that a t test comparing the temperature coefficients of the two groups showed no significant difference between them ( P > 0.50). Bullock (1955) made a strong point of the fact that thermal acclimation results not only in displacement of the rate-temperature curve but in a change in its slope as well. The acclimation temperatures (4 ° and 10°C) appear to be ecologically realistic for this species. This magnitude of difference in thermal history has been found sufficient to elicit a significant metabolic adjustment in a variety of bony fishes we have investigated. It seems probable that this apparent lack of ability in thermal acclimation can be related to the thermally stable natural habitat, where such an ability would confer little selective advantage. The lack of a significant relationship between size and metabolic rate is a further indication that this species is unable to acclimate to temperature. The early work of Krogh (1916) showed that, generally speaking, one could expect the metabolic rate of fishes to reflect size by a coefficient of about - 0 . 1 5 log wt. Rao & Bullock (1954), however, brought out the fact that in poikilotherms the Q10 of metabolic rate is directly related to size. Hence, the relationship between size and metabolic rate will vary, depending on the temperature at which measurements are made. It was recently reported by Morris (1962) that there appears to be a developmental sequence in acquisition of mechanisms for thermal acclimation which gives rise to the relationship between size and Qx0. The fact that in the hagfish we found no relationship between size and metabolic rate is indeed a negative result, and could have been caused by some defect in the experimental routine, but this seems improbable since a similar routine has yielded positive results in numerous other species we have investigated. Hence, we believe that this apparent lack of relationship between size and metabolic rate is real, within the size limits considered. The general levels of metabolic rate are conspicuously low. The rate-temperature curves were calculated from individual measurements of metabolic rate
4 10
Thermal history of experimental group (°C)
0"8742 0.7740
0.1054 < 0.1503 <
Multiple regression Standard coefficient error of R R
TABLE 1--INFORMATION
0.001 0-001
Probability of R 0.0437 0"0382
Temperature coefficient bl 0.0041 < 0"0058 <
Standard error of bx 0.001 0.001
Probability of bl
0-1707 --0-1262
0.1121 0.2055
Weight Standard coefficient error of b2 b2
> >
0-20 0.50
Probability of bj
ON THE REGRESSION EQUATIONS OF THE T W O EXPERIMENTAL GROUPS OF HAGFISH
Z/I
6
FREDERICK W . MUNZ AND ROBERT W. MORRIS
(Fig. 1) according to the equation, Y = a+bX,
i.e. body size has been dropped from consideration, X representing temperature in deg C, and b is the temperature coefficient. The slopes of the regression curves yield Q10 values of 2"8 in the fish with the 4°C thermal history and 2.4 in the 10°C fish. The animals were usually agitated each time the temperature of the water bath was increased. However, at such times they were active for only a few minutes. T h e y remained quietly coiled throughout the periods of measurement. Hence, the rates we have measured are of resting metabolism. From the regression curves we see that one could expect oxygen consumption to be 0.008-0.010 ml/g/hr at 10°C. This amounts to only about 0.1 the rate of oxygen consumption of a number of species of bony fishes measured at ecologically realistic temperatures (cf. Morris, 1961, 1962) and compares favourably with metabolic rate of the Antarctic zoarcid, Rhigophila dearborni (Wohlschlag, 1963). Determinations of dry weight demonstrated that, on the average, we can expect the water content of these animals to amount to approximately 78 per cent (S.D. = 3"01 per cent). Hence, at 10°C the average Qo2 would amount to approximately 0.035 or 0-040 ml O~/g dry wt/hr. The low metabolic level measured in terms of wet weight cannot be ascribed to an unusually high water content. T h e water content of the twenty-six animals used in this study ranged from 71.3 to 83.1 per cent. Calculation of the zero-order correlation coefficient, r, to determine the possible relationship between body size (wet wt. in g) and water content (per cent) showed this coefficient to be - 0.65. A t test of this coefficient showed it to be highly significant (P < 0.001). Thus, increase in size is accompanied by a decrease in percentage of water content. REFERENCES BRODALA. & FANCER. (1963) The Biology ofMyxine. Universitets-forlaget, Oslo, Norway. BULLOCKT. H. (1955) Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30, 311-342. DIXON M. (1951) Manometric Methods. Cambridge University Press, London. KROC;H A. (1916) Respiratory Exchange of Animals and Man. Longmans, Green & Co., London. MORRIS R. W. (1961) Distribution and temperature sensitivity of some eastern Pacific cottid fishes. Physiol. Zob'l. 34, 217-227. MORRIS R. W. (1962) Body size and temperature sensitivity in the cichlid fish, Aequidens portalegrensis (Hensel). Amer. Nat. 96, 35-50. MORRIS R. W. (1963) A modified Barcroft respirometer for study of aquatic animals. Turtox News 41, 22-23. RAO K. P. & BULLOCKT. H. (1954) Q~0 as a function of size and habitat temperature in poildlotherms. Amer. Nat. 88, 33 A.A. WOHLSCHLAGD. E. (1963) An Antarctic fish with unusually low metabolism. Ecology 44, 557-564.