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Effects of fish growth rate, acclimation temperature and incubation temperature on in vitro glycine uptake by fish scales
Camp. Biochem. Phvsiol. Vol. 76A, No. I, pp. 121 to 134, 1983 Printed
in Great
Britain
c
0300-9629183 $3.00+0.00 1983 Pergamon Press Ltd.
EFFECTS OF FISH GROWTH RATE, ACCLIMATION TEMPERATURE AND INCUBATION TEMPERATURE ON IN VITRO GLYCINE UPTAKE BY FISH SCALES* EDWARD M. GOOLISH and Department
of Entomology.
Fisheries
and Wildlife, (Receiaed
IRA
University
R. ADELMAN of Minnesota,
St. Paul, MN 55108, USA
I 1 January 1983)
Abstract-l. For acclimation temperatures of 12, 18, 24 and 30 C. the incubation temperatures for maximum uptake of glycine by fish scales were approximately 28, 29, 30 and 32 C, respectively. 2. Cold acclimation resulted in a positive translational increase in the rate of glycine uptake. Increased fish growth rate caused both a translational increase and a rotational change in the Arrhenius-plot description of the data. 3. Arrhenius activation energies (E,) calculated over the linear range of incubation temperatures were most sensitive to growth rate for the 12”C-acclimated fish and least for the 30”C-acclimated fish.
4. A strong correlation was observed between the rate of glycine uptake and the growth rate of individual fish. This correlation was higher when uptake was assayed at the acclimation temperature of the fish than at the incubation temperature of maximum glycine uptake. 5. The confounding of growth rate and acclimation effects has usually not been considered in acclimation
studies
associated
with anabolic
processes,
Metabolic and biochemical compensation to changes in ambient temperature is required by temperate fish if they are to retain homeostatic capabilities. A large number of studies have begun to explain the mechanisms of this acclimation process (Hazel and Prosser, 1974; Smith, 1976; Alexandrov, 1977; Shaklee et al., 1977; Somero, 1978; Cossins et al., 1981) and they have also shown that the thermo-acclimatory response is often tissue and enzyme specific. The influence of thermal history on the rate of protein synthesis in fish has received considerable interest (Das, 1967; Haschemeyer, 1973; McCarthy et al., 1976) since quantitative and qualitative alterations in enzyme levels have been proposed as mechanisms for temperature acclimation. Dean and Berlin (1969) observed a higher level of [‘4C]leucine incorporation into the total liver protein of coldacclimated trout, Sulmo gairdneri, compared to warm-acclimated fish. Using a similar procedure, Das and Prosser (1967) also found the rate of maximal net protein synthesis in the tissue of cold-acclimated fish to be lOCrSOO% higher than in warm-acclimated fish when assayed at the temperature of the coldacclimated fish. The Q,,, of the incorporation rate was lower for 5°C fish than for 25°C fish in the lower range of temperatures; however, this relationship was reversed at higher temperatures. A recent study on temperature acclimation in eels, Anguilfa unguilla, showed Arrhenius plots of protein labeling which exhibited a discontinuity for fish acclimated to both 10 and 20°C (Jankowsky et al., 1981). Kent and Prosser (1980) found no discontinuity in U*Paper Number 13,244, Scientific Journal Series, Minnesota
(‘HP76114
Experiment
Station,
St. Paul,
MN 55108, 127
I
to be an important
influence.
[‘4C]glycine incorporation into protein due to incubation temperature, but higher Q,, values were reported (15-25°C measurement range) from warmer-acclimated fish. Investigations which use changes in amino acid uptake or incorporation as a measure of acclimation in protein synthesis could often be in error since uptake and incorporation can be influenced by other factors, both physical and physiological, which may be confounded with temperature. For example, Ottaway and Simkiss (1979) were able to show that in vitro glycine uptake by the scales of fish was correlated with “back-calculated” growth rate, and decreased by the stress of handling or starvation (Ottaway and Simkiss, 1977a). Similarly, the rate of [‘4C]leucine incorporation into the muscle of Fundulus heteroclitus was much reduced in fish starved for 3-7 days or subjected to various stresses (Jackim and LaRoche, 1973). The effect of cold temperature in decreasing food consumption and the potential for growth in well documented in fish (Brett, 1979). In many cases then, effects attributed to acclimation temperature may be more directly the result of nutritional status and only indirectly the result of temperature. In a series of acclimation experiments, Adelman (1980) was able to correlate glycine uptake by the scales of fish to the growth of individual fish. However, it was not possible to separate pure temperature effects from those due to growth because of a narrow range of growth rates within each acclimation temperature. Smagula and Adelman (1982), also studying glycine uptake by scales, reported that scales from fish acclimated to warmer temperatures exhibited a higher optimum incubation temperature for glycine uptake, although here again, the fish from each acclimation group possessed unique growth rates and the influence of acclimation temperature alone could not be completely isolated. Practical
INTRODUCTION
Agricultural USA.
yet it appears
E. M. GCIOLISHand I. R. ADELMAN
128
applications using glycine uptake by scales as an index of fish growth have the additional problem of defining the appropriate temperature for incubation when comparing fish acclimated to different temperatures. Smagula and Adelman (1982) recommended that, for an unbiased comparison, incubations be done at the optimum temperature for each acclimation group. However, empirical evidence to verify this is still lacking. The objectives of the present study were to (1) describe the effects and interactions of acclimation temperature and growth rate on glycine uptake by fish scales incubated at various temperatures, (2) determine the relationship of glycine uptake to individual fish growth rate and statistically remove this growth effect to examine the temperature acclimation phenomenon in isolation, (3) determine at what incubation temperature glycine uptake best reflects the rate of tish growth, i.e. the acclimation temperature of the fish (ambient) vs the optimum incubation temperature, and (4) characterize the die1 response of glycine uptake by the scales of fish acclimated to a cyclic temperature regime to determine if temperature compensation can occur within this time interval. METHODS
Within each acclimation temperature, fish were partitioned with screens so that three rations of commercial trout pellets could be provided to achieve low, medium. and high growth rates. The three ration sizes were provided so that fish growing at similar rates could be compared across acclimation temperatures. Fish were not fed the day prior to sampling for glycine uptake. After 4 weeks of temperature acclimation and feeding at different ration sizes, IO scales were removed from each fish and incubated over the temperature range of 12-39 ‘C at 3’ C intervals, one scale per incubation temperature. At the start of the experiment all tish were uniquely marked with fin clips so that growth rates could be measured for individual fish over the last 2-week period. Growth was expressed as an instantaneous daily rate (Ricker, 1975): Growth
rate = log,(Final
weight) - log,(Initial Number
weight)
x ,oo,
of days
Measurement of growth rates from changes in length were also attempted, however changes were too small over this time interval for a precise estimate. For its possible implications in the geometry of scale growth, Hile’s (1936) coefficient of condition (K) was also calculated for each fish. This coefficient has been described as an index of “well being” on the belief that heavier fish of a given length are in better condition. It was calculated as: lOO[weight(g)]/length (cm)‘.
Determination qf the rate CI/gl.vcine uptake
l@tct
The rate of glycine uptake was measured by a method similar to that used by Adelman (1980). Scales were re-
In a separate experiment, 56 bluegill sunfish (Lepomis macrochirus) were subjected to a daily cycle of ambient water temperature (22.8-28.O’C) for a period of 3 weeks. Three scales were then sampled from each of seven hsh every 3 hr for a 24-hr period for glycine uptake measurement. Food was made available continuously with automatic feeders. Scales were incubated at a constant intermediate temperature (26.o’C) and glycine uptake for each fish expressed as the mean of three scales. Individual growth rates, as defined above, were also measured for each fish over a 2-week interval.
moved from the fish with a fine forceps and transferred to a vial containing 0.2ml of teleost saline, modified from Shuttleworth (1972) as follows; NaCl 14 mM, KCI 2.68 mM, CaClz I.51 mM, NaHCO, 15.0 mM, HEPES 25 mM, and HCI to a pH of 7.5. To this buffer was added 0.4pCi/ml of [‘%]glycine (New England Nuclear) with a specific activity of 47.30 mCi/mmol to a final concentration of 8.46pM. The pH of this medium was relatively stable (7.5-7.8) over a 2-hr incubation period. Scales were taken from the same area of each fish to minimize the variability in uptake due to allometric growth (Ottaway and Simkiss. 1977b). After 2 hr of incubation the scales were removed. rinsed briefly in saline, and oven dried at 55‘ C to a constant weight. In previous studies the scales were rinsed in saline for 2 hr after incubation, however preliminary experiments have shown no consistent difference in the rate of uptake for scales rinsed briefly or for 2 hr. Each scale was then weighed to the nearest pg and solubilized in Soluene (Packard Instruments) before adding Aquasol(New England Nuclear) and heating for 5 hr at 55°C to eliminate chemiluminescence. Samples were then read on a Beckman LC100 scintillation counter using an external standard to estimate quench. Glycine uptake was expressed as pmol/mm’/2 hr using an empirically derived scale weight:area relationship to correct for bias introduced by scales of unequal size (Smagula and Adelman, 1982). Experimental fish and sampling design Age 0 common carp, Cyprinus carpio, were seined from Cedar Lake (Rice Co., MN) in September 1981, transferred to the laboratory, treated with tetracycline and elevated temperature (33’C) for prophylactic purposes and held in flowing water at 21’ C for 4 weeks. Thirty experimental fish, weighing between 5 and IO g, were placed in each of four rectangular 90 I. tanks with a flow rate of 6 l/min regulated to each of four acclimation temperatures; 12, 18, 24 and 30 +_ l.O’C. A l2L: l2D photoperiod was provided with incandescent lights. Dissolved oxygen was maintained above 6.0mgil and pH ranged between 8.0 and 8.3.
uf die1 temperature cycles on glycine uptake
Statistic& analysis A split-plot design. with fish growth rate as a covariate, was used to statistically analyze the acclimation-incubation temperature study described above. The whole-plot factor was the acclimation temperature of the fish and the sub-plot variable was the temperature of scale incubation. The following statistical linear model, with growth as a covariate, was used to test for effects and to derive fitted values for various combinations of factor levels: Y,,, = ji + A, + B, X,, + e,, + r, + AT,, + B, X,, + ellk where: Y,( fi A, B,
= = = =
I’,, = e, = T, = AT,k =
Bk= e,,L=
log, of glycine uptake overall mean effect of acclimation temperature (i) on uptake regression coefficient of growth effect for acclimation temperature (i) growth rate of fish (ii) whole-plot error effect of incubation temperature (k) on uptake interaction term between acclimation (i) and incubation temperature (k ) regression coefficient of growth effect for incubation temperature (k) sub-plot error.
When reporting the significance of each of these terms, they will be referred to as given here. In the study to examine the effects of acclimation to a diel cycle in ambient water
In virro glycine
uptake
temperature on glycine uptake, a one-way analysis of covariance, with growth rate as the covariate, was used to test for a significant time effect on the rate of glycine uptake. RESULTS
Eflects ofJish acclimation temperature andgrowth rate on glycine uptake at various incubation temperatures
Arrhenius plots for each acclimation temperature are presented in Fig. 1, with the data from low, medium, and high rations graphed separately. If all rations are considered, the optimum incubation temperature for glycine uptake seems to increase with warmer acclimation temperature. For acclimation temperatures of 12, 18, 24 and 3O”C, the highest rates of glycine uptake occurred at incubation temperatures of approximately 28, 29, 30 and 32°C respectively (P < 0.001 for A TJ. The response of the various acclimation groups to incubation temperature also differed in that scales from warmeracclimated fish exhibited a higher tolerance to very warm incubation temperatures. For example, the scales from 30”Cacchmated fish had similar uptake when the incubation temperature was raised from 12 to 39°C whereas scales from the 12”C-acclimated fish showed an almost 70% reduction. In addition, higher rates of growth, i.e. the higher levels of ration, consistently resulted in increased overall rates of uptake (P < 0.001 for B,). Arrhenius activation energies (E,), calculated for the linear portion of each curve through least squares regression, were always lowest for the smallest ration size and generally highest at the intermediate ration (P < 0.001 for &X,,). Acclimation temperature did not seem to have
by fish scales
any consistent influence on the E, other than manifested through its influence on growth However, a clear separation of the growth and perature acclimation effects is not possible until statistical effects are examined individually. Relationship
30
24
la
of glycine uptake to fish growth rate
12
36
( ’ C) 30
24
18
12
2.5 2.0 1.5 I y"?
1.0
I.
0.5
2
0.0
y
-0.5
2
24”
4 Y
2.0
u ?I
1.5
that rate. temtheir
Similar growth rates, including low, medium and high rates, were successfully achieved across most acclimation temperatures (Fig. 2). Growth rates ranged from near zero for all acclimation groups up to 4.2, 3.8, 3.0 and l.O%/day for fish acclimated to 30, 24. 18 and 12”C, respectively. To understand further the relationship between individual fish growth rate and the rate of glycine uptake by its scales, a mean uptake representative of each fish was calculated as follows. A regression equation was derived for the linear portion of each of the Arrhenius plots in Fig. 1, i.e. for each combination of acclimation temperature and ration. This equation was used to adjust the uptake values for the scales of each fish, incubated over that range of temperatures, to an incubation temperature equal to the fish’s acclimation temperature. A mean of these values represented the rate of glycine uptake for the fish in vivo, i.e. at ambient temperature. This mean value was used to increase precision since only one scale from each fish was incubated at the temperature. The resulting fish’s acclimation growth: uptake relationships were linear over the range of growth rates observed with r* values between 0.692 and 0.891 (Fig. 2, Table 1). The slopes of the regressions for fish acclimated to 30, 24 and 18°C were not significantly different; while the slope
INCUBATION TEMPERATURE 36
129
30
0 1.0 0.5 0.0
INCUBATION TEMPERATURE
( lo'x'i' 1
Fig. 1.Arrhenius plots for the rate of glycine uptake by scales of carp fed a low, medium and high ration at each of four acclimation temperatures. Each point is the mean of 10 scales, one from each of 10 fish. The number adjacent to each curve is the Arrhenius activation energy (cal/mol) for that line calculated over its linear range.
130
E. M.
GIRLISHand I. R.
0.75
1.63
INSTANTANEOUS
ADELMAN
2.51
GROWTH
RATE
3.39 (%
da?
4.27
1
Fig. 2. The relationship between the instantaneous growth rate of individual carp over a 2-week period and the rate of glycine uptake by their scales for fish acclimated to 12 (+), 18 (0). 24 (0) and 30 (A)’ C. Glycine uptake values are for scale incubation at the fish’s acclimation temperature.
for the 12%acclimated fish was significantly different from the other three (P < 0.001, Table 1). The uptake representative of each fish was also estimated as the mean uptake for the scales incubated at 27, 30 and 33”C, i.e. near optimum incubation temperature. These uptake values allow examination of the effect of acclimation temperature on the growth rate:glycine uptake relationship when the scales of fish from each acclimation group are incubated at a similar temperature. Within each acclimation group, the r* values for the growth:uptake relationship remained high (Table 1). However, for a given growth rate, comparisons of glycine uptake across acclimation groups showed a much larger disparity than for the comparison of scales incubated at the acclimation temperature of the fish. The r * for all acclimation groups was 0.300 for incubation at optimum, compared to 0.664 for incubation at the acclimation temperature. This result seems to be due to a trend of increasing slope with colder acclimation which is more apparent when uptake is expressed at the optimum incubation temperature than when it is expressed at the acclimation temperature. The re-
gression lines which describe the relationship between the coefficient of condition (K) and growth rate for each fish at each acclimation temperature are presented in Fig. 3. The slope for the 12”-acclimated fish was significantly different from the other three (P < 0.01) which were not significantly different from each other. Growth rate and ucclimation eflbets on glycine uptuke ut each incubation femperature fitted with stntisticul model
When various growth rates are fitted to the statistical model previously described, a more precise representation is obtained for the effect of growth on the rate of glycine uptake at various incubation temperatures for each acclimation temperature (Fig. 4). As higher growth rates are fitted, increased rates of glycine uptake are evident for all acclimation temperatures, and in addition, higher rates of growth resulted in elevated values for the E,. This influence on the slope was smallest for the 30”C-acclimated fish and largest for the 12’C-acclimated fish where the E,, more than tripled with a change in growth rate from
Table I. Regression coefficients for the growth rate:glycine uptake relationship at each acclimation temperature when glycine uptake is expressed at the fish’s acclimation temperature and at the optimum temperature for uptake Incubation temperature
Acclimation temperature (‘C)
Acclimation
Optimum
*Significantly ISignificantly fSignilicantly gsignificantly
different from different from different from different from
Intercept
Slope
r?
12 18 24 30
2.03* 2.761‘ 1.23 0.50
4.12 2.02 1.99 2.04
0.692 0.729 0.829 0.89 I
12
2.61:
7.48
0.713
18 24 30
4.405 1.31 0.53
2.61 2.42 1.91
0.496 0.837 0.841
3O’C 24’C 18 and 24’C
(P < 0.001). (P < 0.01) and 30 C (P < 0.001). 24“C (P < 0.05) and from 30 C at P < 0.001. (P < 0.01) and from 30 ‘C at P < 0.001,
In t+tro glycine uptake by fish scales
131
suggested by its higher slope in the rate:glycine uptake regression (Table 1).
growth
Response to a daily cycle in water temperature
The statistical analysis of the rate of glycine uptake throughout the 24-hr sampling period indicated that individual fish growth rate was a significant covariate of glycine uptake (P < 0.001) and explained 72% of the variability. The regression equation was:
180
24” 30”
Glycine uptake = -0.044 + 2.70 (Growth rate)
0
1
2
INSTANTANEOUS
3
4
GROWTH
After removing the influence of growth rate, there was no significant difference in the rate of glycine uptake among fish sampled throughout the 24-hr period of cycled temperature.
5
RATE (%,day’
)
Fig. 3. Relationship between the coefficient of condition (K) and growth rate of fish acclimated to 12, 18, 24 and 30°C.
DISCUSSION
0.0 to 1.O%/day. Similarly,’ it is possible to observe the effect of acclimation temperature alone on the incubation response for a variety of fitted growth rates (Fig. 5). The response here is higher rates of glycine uptake by colder-acclimated fish at each of the projected growth rates but without a consistent influence of acclimation temperature on the E,. Also apparent over this range of growth rates is the larger influence of higher growth on the uptake process for the fish acclimated to 12°C. This effect was previously
While the rate of glycine uptake is inffuenced by both the temperature of fish acclimation and scale incubation, the largest influence, in absolute terms, appears to be the growth rate of the fish. The acclimation response seen here was, in the terminology of Precht (1958), one of overcompensation and, according to the approach of Prosser (1973), primarily a ~anslational increase (Type II). If Presser’s concept can be applied more generally, the growth effect observed here can be considered both a translational and a counter-clockwise rotational response (Type IV). The link between the glycine uptake
12
18
24
30
12
36
INC~ATION
BERATE
24
16
30
36
(‘C)
Fig. 4. Predicted glycine uptake by scales at various incubation temperatures for fitted growth rates of fish held at each of four acclimation temperatures. Predicted glycine uptake is based on the statistical model described in the text (P < 0.001 for r,). The number beneath each curve is the E, calculated for that curve over its linear range in units of cal/mol. The temperature (“C) in the upper left of each graph is the acclimation temperature of the fish.
132
E. M. GCMILISHand I. R. AUELMAN
INC~ATION
TMAPERATURE
f”C)
Fig. 5. Predicted glycine uptake by scales at various incubation temperatures for fitted acclimation temperatures (“C) at each of three growth rates c/$day), Predicted rates of uptake are based on the statistical model described in the text (P < 0.001 for A,). The number beneath each curve is the E, for that curve calculated over its linear range (cal/mol).
process and protein synthesis is further strengthened by the close agreement with the E, values seen here and others reported for various steps of protein synthesis. The elongation of polypeptide chains in the liver of the toadfish, Opsanus tau, had an E, of 16 kcal/mol in the 17-30°C range and 26 kcal/mol in the 7-17°C range (Mathews and Haschemeyer, 1978). SimiIarIy, a value of 22 kcal/moi was observed by Lajtha and Sershen (1976) in studies of amino acid incorporation in the brain of goldfish. It is not clear why in the present study the E, increases with higher rates of growth, but perhaps it is the result of higher
intracellular concentrations of glycine that must be maintained at high anabohc rates while at growth rates near zero, a less energy demanding mechanism can operate. Had the fish in our acclimation groups been fed the same ration, or been fed ad ~ibitum, the disparity in growth that would have resulted would likely have masked the temperature acclimation response. This result may partly explain why some studies have not observed an acclimation response or why contrary reports of acclimation effects exist. For example, Kent and Prosser (1980) found a decreasing Q10from
In vitro glycine uptake colder acclimated fish for the rate of glycine incorporation into fish hepatocytes. Although fed to satiation, it is probable that the cold-acclimated fish were growing more slowly and, as seen here, this could account for the altered Q,, values. Also, in the European eel higher E, values were observed for warmer-acclimated animals (Jankowsky et al., 198 1). Here again, it is possible that growth rate, if not the same in each acclimation group, is having an influence on the response to incubation temperature. Studies have been done in which the experimental fish were not fed during the acclimation period. In these instances, because of a slower metabolic rate, coldacclimated fish would appear to be in a better nutritional state, and this positive influence on the rate of protein synthesis could also be misinterpreted as an acclimation effect to cold temperature. In general, cold-acclimated fish will exhibit a slower rate of growth, and therefore, reports of translational increases in protein synthesis for these fish are probably underestimated. After finding higher rates of glycine incorporation in cold-acclimated fish, Kent and Prosser (1980) suggested increased potential for transport as a possible mechanism. Evidence presented here would support this idea, although it could also occur by a variety of processes. Firstly, the strong growth rate:glycine uptake relationship (Fig. 2), and S&90% inhibition of glycine uptake in the presence of cycloheximide (Goolish, 1982) suggest that a rapid feedback mechanism exists between the transport system and the protein synthesis process. However, cold-acclimated fish transported higher quantities of glycine than could be accounted for by the requirements of body growth (Fig. 2). The additional uptake may be increased passive or active diffusion across a more permeable membrane. This could occur as a result of higher levels of unsaturated fatty acids which are known to occur in the membranes of cold-acclimated fish (Cossins et a/., 1981) and which have been implicated in changes in membrane permeability. An additional explanation may be that increased protein synthesis is indeed occurring and is accounted for by quantitative changes in enzyme levels as a compensation for a lower thermodynamic state. Finally, Somero and Doyle (1973) have demonstrated that the rate of protein degradation is more rapid in the muscle of 8’C-acclimated fish than in that of 26’C-acclimated fish. If this were also true for the scales of fish, then a higher rate of protein synthesis and glycine uptake might be expected from these colder-acclimated fish to balance this increased loss. The increase in optimum incubation temperature with warmer acclimation temperature does suggest, however, that some conformational or isozymic change is occurring in the enzymes of the transport system possibly resulting in a shift in K,,,. Further information is provided by the inability of these fish to compensate for an exposure to dielly cyclic temperatures, which suggests that the mechanism of acclimation seen here can not occur over a short interval of time (24 hr). It is of interest to note that the optimum incubation temperature for the 30‘C-acclimated fish, approximately 32°C is the same temperature reported to be the final preferendum for this species (Pitt et al., 1956). Often, the final
by fish scales
133
preferendum is the temperature at or near which fish have the maximum potential for growth. This same result has been recognized by Smagula and Adelman (1982) for the largemouth bass, Micropterus salmoides, and for several other temperate fish (Adelman, 1980). The anomalous way in which the 12”C-acclimated fish responded, taking up much more glycine than would be expected for a given growth rate, indicates still a further complexity. It is thought that the glycine transported by these scales is destined for incorporation into collagen, the primary structural protein in fish scales. There is also evidence that cold adaptation can result in changes in the amino acid composition of collagen between various species (Fischman and Levy, 1964; Rigby, 197 I ). Specifically, a decreased proportion of proline and hydroxyproline has been noted in temperate fish relative to fish from tropical regions. Perhaps then, at temperatures of 12°C and lower a type of collagenous material is being synthesized by the scale which contains a higher proportion of glycine. This would explain the unique slope of these fish in the growth rate:glycine uptake relationship. Since as growth rate increases, the higher proportion of glycine in the collagen of 12”C-acclimated fish would result in a higher rate of glycine uptake than that of warmeracclimated fish. In the absence of growth, and collagen synthesis, higher glycine uptake would not be expected and was not observed. This subject deserves further investigation, possibly by examining the ratio of uptake for various amino acids by fish acclimated to different temperatures. A final explanation for the response of the 12”C-acclimated fish may be that the geometry of scale growth is not constant with various acclimation temperatures. These fish had a much higher coefficient of condition than the other three groups (Fig. 3). As a result of this, additional scale material may have to be deposited to account for an increased girth, or perhaps, with “stunted” longitudinal growth a thicker scale is being laid down. The relationship between glycine uptake and growth at various acclimation temperatures has been previously addressed (Smagula and Adelman, 1982) where it was suggested that, for largemouth bass, the rate of uptake should be measured at the optimum incubation temperature. Rates of glycine uptake would be obtained then, that would not have been limited by incubation temperature for any of the acclimation groups, as would occur if incubation were done at the ambient temperature of the fish. It appears now that the compensatory response to an acclimation exposure, in the common carp at least, is sufficient to make uptake observations made at ambient temperature the most accurate index of growth across acclimation groups. It also makes intuitive sense that, while optimum incubation for 30’C-acclimated fish would be equivalent to that at ambient, fish acclimated to colder temperatures would require a biased positive adjustment that would increase in magnitude with colder acclimation. This phenomenon partly explains the more divergent slopes that result in the growth rate:glycine uptake relationship when uptake is measured at an optimum temperature (Table I). A more suitable expression of glycine uptake, than that at ambient incubation,
134
E. M.
GWLISH
and I. R. ADELUAN
would be a value adjusted mathematically using a model derived from the kind of information obtained here for the common carp. This would then account for the interactions discussed above and make comparisons across acclimation groups less biased. The present study has demonstrated the marked effect that growth rate can have on the results of a temperature acclimation study in which the processes of protein synthesis are measured. In future studies, more consideration should be given to the physiological state, past and present, of the organisms being used to minimize conclusions which may be misleading and only the result of a manipulated system. This concern is likely to extend beyond temperature acclimation studies pertaining just to protein synthesis since nutritional state is likely to manifest itself throughout the metabolism of the animal.
artedi (LeSuer), in the lakes of the northeastern highlands, Wisconsin. U.S. Bur. Fish. Bull. 48, 21 l-317.
Jackim E. and LaRoche G. (1973) Protein synthesis in Fundulus heteroclitus muscle. Comp. Biochem. Physiol. 44A, 851-866.
Jankowsky H. D., Hotopp W. and Vsiansky P. (1981) Effects of assay and acclimation tem~ratures on incorporation of amino acids into protein of isolated hepatocytes from the European eel, Anguilia anguiliu L. .I. Therm. Biol. 6, 201-208. Kent J. and Presser C. L. (1980) Effects of incubation and acclimation temperature on incorporation of Uf’4C]glycine into mitochondrial protein of liver cells and slices from green sunfish, Lfpomis c~uneIfus. Physiof. Zool. 53, 293-304.
Lajtha A. and Sershen H. (1976) Changes in the rates of protein synthesis in the brain of goldfish at various temperatures. L&e Sci. 17, 1861--1868. Mathews R. W. and Haschemeyer A. E. V. (1978) Temperature dependency of protein synthesis in toadfish liver in ciao. Camp. Biochem. Physiol. 61B, 479484.
Acknowledgements-Financial support for this study was provided by the University of Minnesota Agricultural Experiment Station and the University of Minnesota Computer Center. We would also like to thank K. Larntz for statistical advice, G. P. Busacker for helpful comments on the manuscript and Joe Nicolette for assistance in conducting the sampling.
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Hile R. (1936) Age and growth of the ciscoe. Leucichthvs
McCarthy J. F., Sastry A. N. and Trembiay G. C. (1976) Thermal compensation in protein and RNA synthesis during the intermolt cycle of the American lobster, Homarus americanus. Biol. Bull. 151, 538-547.
Ottaway E. M. and Simkiss K. (1977a) A method for assessing factors influencing ‘false check’ formation in fish scales. J. Fish Bioi. 11, 681-687. Ottaway E. M. and Simkiss K. (1977b) “Instantaneous” growth rates of fish scales and their use in studies of fish populations. /. Zoo!., Land. 181, 407-419. Ottaway E. M. and Simkiss K. (1979) A comparison of traditional and novel ways of estimating growth rates from scales of natural populations of voune; bass IDicentrarchus labrax). J. ma;. biol. Ass. 6.K. 59, 49-i9.
Pitt T. K.. Garside E. T. and Heuburn R. L. (1956\ Temperature selection of the carp icyprinus carp& L.): Can. J. Zoo/. 34, 555-557. Precht H. (1958) Concepts of the temperature adaptation of unchanging reactions of cold-blooded animals. In Physiological Adaptation (Edited by Prosser C. L.), pp. 50-78. Am. Physiol. Sot. Prosser C. I,. (1973) Comparative Animal Physiology, 3rd edn, pp. 362-428. W. B. Saunders, Philadelphia. Ricker W. E. ( 1975) Computation and Interpretati~~n oj the ~ioiog~c(ll Statistirs qf Fish Populations, pp. l-27. Fish. Res. Board Can. Bull. 191. Rigby B. J. (1971) The thermal stability of collagen: its significance in biology and physiology. In Chemical Dvnamics (Edited by Hirschfelder J. 0. and Henderson D.), pp. 537-555. John Wiley, Sydney. Shaklee J. B.. Christiansen J. A.. Side11B. D., Prosser C. L. and Whitt G. S. (1977) Molecular aspects of temperature acclimation in fish: contributions of changes in enzyme activities and isozyme patterns to metabolic reorganization in the green sunfish. J. e.wp. Zool. 201, I-20. Shuttleworth T. J. (1972) A new isolated perfused gill preparation for the study of the mechanisms of ionic regulation in teleosts. Camp. Biochrm. Physiol. 43.4, 59-64.
Smagula C. and Adelman I. R. (1982) Temperature and scale size errors in the use of [%II]glycine uptake by scales as a growth index. Can. J. Fish. Aquat. Sri. 39, 1366-1372. Smith M. W. (1976) Temperature adaptation in fish. Riothem. Sor. Symp. 41, 43-60.
Somero G. N. (1978) Temperature adaptation of enzymes: Biological optimization through structure-function compromises. A. Ret>. Ecoi. Syst. 9, l--29. Somero G, N. and Doyle D. (1973) Temperature and rates of protein degradation in the fish Gil1ichthy.s mirabiks. Camp. Biochem. Physiol. 468, 463474.
in Great
Britain
c
0300-9629183 $3.00+0.00 1983 Pergamon Press Ltd.
EFFECTS OF FISH GROWTH RATE, ACCLIMATION TEMPERATURE AND INCUBATION TEMPERATURE ON IN VITRO GLYCINE UPTAKE BY FISH SCALES* EDWARD M. GOOLISH and Department
of Entomology.
Fisheries
and Wildlife, (Receiaed
IRA
University
R. ADELMAN of Minnesota,
St. Paul, MN 55108, USA
I 1 January 1983)
Abstract-l. For acclimation temperatures of 12, 18, 24 and 30 C. the incubation temperatures for maximum uptake of glycine by fish scales were approximately 28, 29, 30 and 32 C, respectively. 2. Cold acclimation resulted in a positive translational increase in the rate of glycine uptake. Increased fish growth rate caused both a translational increase and a rotational change in the Arrhenius-plot description of the data. 3. Arrhenius activation energies (E,) calculated over the linear range of incubation temperatures were most sensitive to growth rate for the 12”C-acclimated fish and least for the 30”C-acclimated fish.
4. A strong correlation was observed between the rate of glycine uptake and the growth rate of individual fish. This correlation was higher when uptake was assayed at the acclimation temperature of the fish than at the incubation temperature of maximum glycine uptake. 5. The confounding of growth rate and acclimation effects has usually not been considered in acclimation
studies
associated
with anabolic
processes,
Metabolic and biochemical compensation to changes in ambient temperature is required by temperate fish if they are to retain homeostatic capabilities. A large number of studies have begun to explain the mechanisms of this acclimation process (Hazel and Prosser, 1974; Smith, 1976; Alexandrov, 1977; Shaklee et al., 1977; Somero, 1978; Cossins et al., 1981) and they have also shown that the thermo-acclimatory response is often tissue and enzyme specific. The influence of thermal history on the rate of protein synthesis in fish has received considerable interest (Das, 1967; Haschemeyer, 1973; McCarthy et al., 1976) since quantitative and qualitative alterations in enzyme levels have been proposed as mechanisms for temperature acclimation. Dean and Berlin (1969) observed a higher level of [‘4C]leucine incorporation into the total liver protein of coldacclimated trout, Sulmo gairdneri, compared to warm-acclimated fish. Using a similar procedure, Das and Prosser (1967) also found the rate of maximal net protein synthesis in the tissue of cold-acclimated fish to be lOCrSOO% higher than in warm-acclimated fish when assayed at the temperature of the coldacclimated fish. The Q,,, of the incorporation rate was lower for 5°C fish than for 25°C fish in the lower range of temperatures; however, this relationship was reversed at higher temperatures. A recent study on temperature acclimation in eels, Anguilfa unguilla, showed Arrhenius plots of protein labeling which exhibited a discontinuity for fish acclimated to both 10 and 20°C (Jankowsky et al., 1981). Kent and Prosser (1980) found no discontinuity in U*Paper Number 13,244, Scientific Journal Series, Minnesota
(‘HP76114
Experiment
Station,
St. Paul,
MN 55108, 127
I
to be an important
influence.
[‘4C]glycine incorporation into protein due to incubation temperature, but higher Q,, values were reported (15-25°C measurement range) from warmer-acclimated fish. Investigations which use changes in amino acid uptake or incorporation as a measure of acclimation in protein synthesis could often be in error since uptake and incorporation can be influenced by other factors, both physical and physiological, which may be confounded with temperature. For example, Ottaway and Simkiss (1979) were able to show that in vitro glycine uptake by the scales of fish was correlated with “back-calculated” growth rate, and decreased by the stress of handling or starvation (Ottaway and Simkiss, 1977a). Similarly, the rate of [‘4C]leucine incorporation into the muscle of Fundulus heteroclitus was much reduced in fish starved for 3-7 days or subjected to various stresses (Jackim and LaRoche, 1973). The effect of cold temperature in decreasing food consumption and the potential for growth in well documented in fish (Brett, 1979). In many cases then, effects attributed to acclimation temperature may be more directly the result of nutritional status and only indirectly the result of temperature. In a series of acclimation experiments, Adelman (1980) was able to correlate glycine uptake by the scales of fish to the growth of individual fish. However, it was not possible to separate pure temperature effects from those due to growth because of a narrow range of growth rates within each acclimation temperature. Smagula and Adelman (1982), also studying glycine uptake by scales, reported that scales from fish acclimated to warmer temperatures exhibited a higher optimum incubation temperature for glycine uptake, although here again, the fish from each acclimation group possessed unique growth rates and the influence of acclimation temperature alone could not be completely isolated. Practical
INTRODUCTION
Agricultural USA.
yet it appears
E. M. GCIOLISHand I. R. ADELMAN
128
applications using glycine uptake by scales as an index of fish growth have the additional problem of defining the appropriate temperature for incubation when comparing fish acclimated to different temperatures. Smagula and Adelman (1982) recommended that, for an unbiased comparison, incubations be done at the optimum temperature for each acclimation group. However, empirical evidence to verify this is still lacking. The objectives of the present study were to (1) describe the effects and interactions of acclimation temperature and growth rate on glycine uptake by fish scales incubated at various temperatures, (2) determine the relationship of glycine uptake to individual fish growth rate and statistically remove this growth effect to examine the temperature acclimation phenomenon in isolation, (3) determine at what incubation temperature glycine uptake best reflects the rate of tish growth, i.e. the acclimation temperature of the fish (ambient) vs the optimum incubation temperature, and (4) characterize the die1 response of glycine uptake by the scales of fish acclimated to a cyclic temperature regime to determine if temperature compensation can occur within this time interval. METHODS
Within each acclimation temperature, fish were partitioned with screens so that three rations of commercial trout pellets could be provided to achieve low, medium. and high growth rates. The three ration sizes were provided so that fish growing at similar rates could be compared across acclimation temperatures. Fish were not fed the day prior to sampling for glycine uptake. After 4 weeks of temperature acclimation and feeding at different ration sizes, IO scales were removed from each fish and incubated over the temperature range of 12-39 ‘C at 3’ C intervals, one scale per incubation temperature. At the start of the experiment all tish were uniquely marked with fin clips so that growth rates could be measured for individual fish over the last 2-week period. Growth was expressed as an instantaneous daily rate (Ricker, 1975): Growth
rate = log,(Final
weight) - log,(Initial Number
weight)
x ,oo,
of days
Measurement of growth rates from changes in length were also attempted, however changes were too small over this time interval for a precise estimate. For its possible implications in the geometry of scale growth, Hile’s (1936) coefficient of condition (K) was also calculated for each fish. This coefficient has been described as an index of “well being” on the belief that heavier fish of a given length are in better condition. It was calculated as: lOO[weight(g)]/length (cm)‘.
Determination qf the rate CI/gl.vcine uptake
l@tct
The rate of glycine uptake was measured by a method similar to that used by Adelman (1980). Scales were re-
In a separate experiment, 56 bluegill sunfish (Lepomis macrochirus) were subjected to a daily cycle of ambient water temperature (22.8-28.O’C) for a period of 3 weeks. Three scales were then sampled from each of seven hsh every 3 hr for a 24-hr period for glycine uptake measurement. Food was made available continuously with automatic feeders. Scales were incubated at a constant intermediate temperature (26.o’C) and glycine uptake for each fish expressed as the mean of three scales. Individual growth rates, as defined above, were also measured for each fish over a 2-week interval.
moved from the fish with a fine forceps and transferred to a vial containing 0.2ml of teleost saline, modified from Shuttleworth (1972) as follows; NaCl 14 mM, KCI 2.68 mM, CaClz I.51 mM, NaHCO, 15.0 mM, HEPES 25 mM, and HCI to a pH of 7.5. To this buffer was added 0.4pCi/ml of [‘%]glycine (New England Nuclear) with a specific activity of 47.30 mCi/mmol to a final concentration of 8.46pM. The pH of this medium was relatively stable (7.5-7.8) over a 2-hr incubation period. Scales were taken from the same area of each fish to minimize the variability in uptake due to allometric growth (Ottaway and Simkiss. 1977b). After 2 hr of incubation the scales were removed. rinsed briefly in saline, and oven dried at 55‘ C to a constant weight. In previous studies the scales were rinsed in saline for 2 hr after incubation, however preliminary experiments have shown no consistent difference in the rate of uptake for scales rinsed briefly or for 2 hr. Each scale was then weighed to the nearest pg and solubilized in Soluene (Packard Instruments) before adding Aquasol(New England Nuclear) and heating for 5 hr at 55°C to eliminate chemiluminescence. Samples were then read on a Beckman LC100 scintillation counter using an external standard to estimate quench. Glycine uptake was expressed as pmol/mm’/2 hr using an empirically derived scale weight:area relationship to correct for bias introduced by scales of unequal size (Smagula and Adelman, 1982). Experimental fish and sampling design Age 0 common carp, Cyprinus carpio, were seined from Cedar Lake (Rice Co., MN) in September 1981, transferred to the laboratory, treated with tetracycline and elevated temperature (33’C) for prophylactic purposes and held in flowing water at 21’ C for 4 weeks. Thirty experimental fish, weighing between 5 and IO g, were placed in each of four rectangular 90 I. tanks with a flow rate of 6 l/min regulated to each of four acclimation temperatures; 12, 18, 24 and 30 +_ l.O’C. A l2L: l2D photoperiod was provided with incandescent lights. Dissolved oxygen was maintained above 6.0mgil and pH ranged between 8.0 and 8.3.
uf die1 temperature cycles on glycine uptake
Statistic& analysis A split-plot design. with fish growth rate as a covariate, was used to statistically analyze the acclimation-incubation temperature study described above. The whole-plot factor was the acclimation temperature of the fish and the sub-plot variable was the temperature of scale incubation. The following statistical linear model, with growth as a covariate, was used to test for effects and to derive fitted values for various combinations of factor levels: Y,,, = ji + A, + B, X,, + e,, + r, + AT,, + B, X,, + ellk where: Y,( fi A, B,
= = = =
I’,, = e, = T, = AT,k =
Bk= e,,L=
log, of glycine uptake overall mean effect of acclimation temperature (i) on uptake regression coefficient of growth effect for acclimation temperature (i) growth rate of fish (ii) whole-plot error effect of incubation temperature (k) on uptake interaction term between acclimation (i) and incubation temperature (k ) regression coefficient of growth effect for incubation temperature (k) sub-plot error.
When reporting the significance of each of these terms, they will be referred to as given here. In the study to examine the effects of acclimation to a diel cycle in ambient water
In virro glycine
uptake
temperature on glycine uptake, a one-way analysis of covariance, with growth rate as the covariate, was used to test for a significant time effect on the rate of glycine uptake. RESULTS
Eflects ofJish acclimation temperature andgrowth rate on glycine uptake at various incubation temperatures
Arrhenius plots for each acclimation temperature are presented in Fig. 1, with the data from low, medium, and high rations graphed separately. If all rations are considered, the optimum incubation temperature for glycine uptake seems to increase with warmer acclimation temperature. For acclimation temperatures of 12, 18, 24 and 3O”C, the highest rates of glycine uptake occurred at incubation temperatures of approximately 28, 29, 30 and 32°C respectively (P < 0.001 for A TJ. The response of the various acclimation groups to incubation temperature also differed in that scales from warmeracclimated fish exhibited a higher tolerance to very warm incubation temperatures. For example, the scales from 30”Cacchmated fish had similar uptake when the incubation temperature was raised from 12 to 39°C whereas scales from the 12”C-acclimated fish showed an almost 70% reduction. In addition, higher rates of growth, i.e. the higher levels of ration, consistently resulted in increased overall rates of uptake (P < 0.001 for B,). Arrhenius activation energies (E,), calculated for the linear portion of each curve through least squares regression, were always lowest for the smallest ration size and generally highest at the intermediate ration (P < 0.001 for &X,,). Acclimation temperature did not seem to have
by fish scales
any consistent influence on the E, other than manifested through its influence on growth However, a clear separation of the growth and perature acclimation effects is not possible until statistical effects are examined individually. Relationship
30
24
la
of glycine uptake to fish growth rate
12
36
( ’ C) 30
24
18
12
2.5 2.0 1.5 I y"?
1.0
I.
0.5
2
0.0
y
-0.5
2
24”
4 Y
2.0
u ?I
1.5
that rate. temtheir
Similar growth rates, including low, medium and high rates, were successfully achieved across most acclimation temperatures (Fig. 2). Growth rates ranged from near zero for all acclimation groups up to 4.2, 3.8, 3.0 and l.O%/day for fish acclimated to 30, 24. 18 and 12”C, respectively. To understand further the relationship between individual fish growth rate and the rate of glycine uptake by its scales, a mean uptake representative of each fish was calculated as follows. A regression equation was derived for the linear portion of each of the Arrhenius plots in Fig. 1, i.e. for each combination of acclimation temperature and ration. This equation was used to adjust the uptake values for the scales of each fish, incubated over that range of temperatures, to an incubation temperature equal to the fish’s acclimation temperature. A mean of these values represented the rate of glycine uptake for the fish in vivo, i.e. at ambient temperature. This mean value was used to increase precision since only one scale from each fish was incubated at the temperature. The resulting fish’s acclimation growth: uptake relationships were linear over the range of growth rates observed with r* values between 0.692 and 0.891 (Fig. 2, Table 1). The slopes of the regressions for fish acclimated to 30, 24 and 18°C were not significantly different; while the slope
INCUBATION TEMPERATURE 36
129
30
0 1.0 0.5 0.0
INCUBATION TEMPERATURE
( lo'x'i' 1
Fig. 1.Arrhenius plots for the rate of glycine uptake by scales of carp fed a low, medium and high ration at each of four acclimation temperatures. Each point is the mean of 10 scales, one from each of 10 fish. The number adjacent to each curve is the Arrhenius activation energy (cal/mol) for that line calculated over its linear range.
130
E. M.
GIRLISHand I. R.
0.75
1.63
INSTANTANEOUS
ADELMAN
2.51
GROWTH
RATE
3.39 (%
da?
4.27
1
Fig. 2. The relationship between the instantaneous growth rate of individual carp over a 2-week period and the rate of glycine uptake by their scales for fish acclimated to 12 (+), 18 (0). 24 (0) and 30 (A)’ C. Glycine uptake values are for scale incubation at the fish’s acclimation temperature.
for the 12%acclimated fish was significantly different from the other three (P < 0.001, Table 1). The uptake representative of each fish was also estimated as the mean uptake for the scales incubated at 27, 30 and 33”C, i.e. near optimum incubation temperature. These uptake values allow examination of the effect of acclimation temperature on the growth rate:glycine uptake relationship when the scales of fish from each acclimation group are incubated at a similar temperature. Within each acclimation group, the r* values for the growth:uptake relationship remained high (Table 1). However, for a given growth rate, comparisons of glycine uptake across acclimation groups showed a much larger disparity than for the comparison of scales incubated at the acclimation temperature of the fish. The r * for all acclimation groups was 0.300 for incubation at optimum, compared to 0.664 for incubation at the acclimation temperature. This result seems to be due to a trend of increasing slope with colder acclimation which is more apparent when uptake is expressed at the optimum incubation temperature than when it is expressed at the acclimation temperature. The re-
gression lines which describe the relationship between the coefficient of condition (K) and growth rate for each fish at each acclimation temperature are presented in Fig. 3. The slope for the 12”-acclimated fish was significantly different from the other three (P < 0.01) which were not significantly different from each other. Growth rate and ucclimation eflbets on glycine uptuke ut each incubation femperature fitted with stntisticul model
When various growth rates are fitted to the statistical model previously described, a more precise representation is obtained for the effect of growth on the rate of glycine uptake at various incubation temperatures for each acclimation temperature (Fig. 4). As higher growth rates are fitted, increased rates of glycine uptake are evident for all acclimation temperatures, and in addition, higher rates of growth resulted in elevated values for the E,. This influence on the slope was smallest for the 30”C-acclimated fish and largest for the 12’C-acclimated fish where the E,, more than tripled with a change in growth rate from
Table I. Regression coefficients for the growth rate:glycine uptake relationship at each acclimation temperature when glycine uptake is expressed at the fish’s acclimation temperature and at the optimum temperature for uptake Incubation temperature
Acclimation temperature (‘C)
Acclimation
Optimum
*Significantly ISignificantly fSignilicantly gsignificantly
different from different from different from different from
Intercept
Slope
r?
12 18 24 30
2.03* 2.761‘ 1.23 0.50
4.12 2.02 1.99 2.04
0.692 0.729 0.829 0.89 I
12
2.61:
7.48
0.713
18 24 30
4.405 1.31 0.53
2.61 2.42 1.91
0.496 0.837 0.841
3O’C 24’C 18 and 24’C
(P < 0.001). (P < 0.01) and 30 C (P < 0.001). 24“C (P < 0.05) and from 30 C at P < 0.001. (P < 0.01) and from 30 ‘C at P < 0.001,
In t+tro glycine uptake by fish scales
131
suggested by its higher slope in the rate:glycine uptake regression (Table 1).
growth
Response to a daily cycle in water temperature
The statistical analysis of the rate of glycine uptake throughout the 24-hr sampling period indicated that individual fish growth rate was a significant covariate of glycine uptake (P < 0.001) and explained 72% of the variability. The regression equation was:
180
24” 30”
Glycine uptake = -0.044 + 2.70 (Growth rate)
0
1
2
INSTANTANEOUS
3
4
GROWTH
After removing the influence of growth rate, there was no significant difference in the rate of glycine uptake among fish sampled throughout the 24-hr period of cycled temperature.
5
RATE (%,day’
)
Fig. 3. Relationship between the coefficient of condition (K) and growth rate of fish acclimated to 12, 18, 24 and 30°C.
DISCUSSION
0.0 to 1.O%/day. Similarly,’ it is possible to observe the effect of acclimation temperature alone on the incubation response for a variety of fitted growth rates (Fig. 5). The response here is higher rates of glycine uptake by colder-acclimated fish at each of the projected growth rates but without a consistent influence of acclimation temperature on the E,. Also apparent over this range of growth rates is the larger influence of higher growth on the uptake process for the fish acclimated to 12°C. This effect was previously
While the rate of glycine uptake is inffuenced by both the temperature of fish acclimation and scale incubation, the largest influence, in absolute terms, appears to be the growth rate of the fish. The acclimation response seen here was, in the terminology of Precht (1958), one of overcompensation and, according to the approach of Prosser (1973), primarily a ~anslational increase (Type II). If Presser’s concept can be applied more generally, the growth effect observed here can be considered both a translational and a counter-clockwise rotational response (Type IV). The link between the glycine uptake
12
18
24
30
12
36
INC~ATION
BERATE
24
16
30
36
(‘C)
Fig. 4. Predicted glycine uptake by scales at various incubation temperatures for fitted growth rates of fish held at each of four acclimation temperatures. Predicted glycine uptake is based on the statistical model described in the text (P < 0.001 for r,). The number beneath each curve is the E, calculated for that curve over its linear range in units of cal/mol. The temperature (“C) in the upper left of each graph is the acclimation temperature of the fish.
132
E. M. GCMILISHand I. R. AUELMAN
INC~ATION
TMAPERATURE
f”C)
Fig. 5. Predicted glycine uptake by scales at various incubation temperatures for fitted acclimation temperatures (“C) at each of three growth rates c/$day), Predicted rates of uptake are based on the statistical model described in the text (P < 0.001 for A,). The number beneath each curve is the E, for that curve calculated over its linear range (cal/mol).
process and protein synthesis is further strengthened by the close agreement with the E, values seen here and others reported for various steps of protein synthesis. The elongation of polypeptide chains in the liver of the toadfish, Opsanus tau, had an E, of 16 kcal/mol in the 17-30°C range and 26 kcal/mol in the 7-17°C range (Mathews and Haschemeyer, 1978). SimiIarIy, a value of 22 kcal/moi was observed by Lajtha and Sershen (1976) in studies of amino acid incorporation in the brain of goldfish. It is not clear why in the present study the E, increases with higher rates of growth, but perhaps it is the result of higher
intracellular concentrations of glycine that must be maintained at high anabohc rates while at growth rates near zero, a less energy demanding mechanism can operate. Had the fish in our acclimation groups been fed the same ration, or been fed ad ~ibitum, the disparity in growth that would have resulted would likely have masked the temperature acclimation response. This result may partly explain why some studies have not observed an acclimation response or why contrary reports of acclimation effects exist. For example, Kent and Prosser (1980) found a decreasing Q10from
In vitro glycine uptake colder acclimated fish for the rate of glycine incorporation into fish hepatocytes. Although fed to satiation, it is probable that the cold-acclimated fish were growing more slowly and, as seen here, this could account for the altered Q,, values. Also, in the European eel higher E, values were observed for warmer-acclimated animals (Jankowsky et al., 198 1). Here again, it is possible that growth rate, if not the same in each acclimation group, is having an influence on the response to incubation temperature. Studies have been done in which the experimental fish were not fed during the acclimation period. In these instances, because of a slower metabolic rate, coldacclimated fish would appear to be in a better nutritional state, and this positive influence on the rate of protein synthesis could also be misinterpreted as an acclimation effect to cold temperature. In general, cold-acclimated fish will exhibit a slower rate of growth, and therefore, reports of translational increases in protein synthesis for these fish are probably underestimated. After finding higher rates of glycine incorporation in cold-acclimated fish, Kent and Prosser (1980) suggested increased potential for transport as a possible mechanism. Evidence presented here would support this idea, although it could also occur by a variety of processes. Firstly, the strong growth rate:glycine uptake relationship (Fig. 2), and S&90% inhibition of glycine uptake in the presence of cycloheximide (Goolish, 1982) suggest that a rapid feedback mechanism exists between the transport system and the protein synthesis process. However, cold-acclimated fish transported higher quantities of glycine than could be accounted for by the requirements of body growth (Fig. 2). The additional uptake may be increased passive or active diffusion across a more permeable membrane. This could occur as a result of higher levels of unsaturated fatty acids which are known to occur in the membranes of cold-acclimated fish (Cossins et a/., 1981) and which have been implicated in changes in membrane permeability. An additional explanation may be that increased protein synthesis is indeed occurring and is accounted for by quantitative changes in enzyme levels as a compensation for a lower thermodynamic state. Finally, Somero and Doyle (1973) have demonstrated that the rate of protein degradation is more rapid in the muscle of 8’C-acclimated fish than in that of 26’C-acclimated fish. If this were also true for the scales of fish, then a higher rate of protein synthesis and glycine uptake might be expected from these colder-acclimated fish to balance this increased loss. The increase in optimum incubation temperature with warmer acclimation temperature does suggest, however, that some conformational or isozymic change is occurring in the enzymes of the transport system possibly resulting in a shift in K,,,. Further information is provided by the inability of these fish to compensate for an exposure to dielly cyclic temperatures, which suggests that the mechanism of acclimation seen here can not occur over a short interval of time (24 hr). It is of interest to note that the optimum incubation temperature for the 30‘C-acclimated fish, approximately 32°C is the same temperature reported to be the final preferendum for this species (Pitt et al., 1956). Often, the final
by fish scales
133
preferendum is the temperature at or near which fish have the maximum potential for growth. This same result has been recognized by Smagula and Adelman (1982) for the largemouth bass, Micropterus salmoides, and for several other temperate fish (Adelman, 1980). The anomalous way in which the 12”C-acclimated fish responded, taking up much more glycine than would be expected for a given growth rate, indicates still a further complexity. It is thought that the glycine transported by these scales is destined for incorporation into collagen, the primary structural protein in fish scales. There is also evidence that cold adaptation can result in changes in the amino acid composition of collagen between various species (Fischman and Levy, 1964; Rigby, 197 I ). Specifically, a decreased proportion of proline and hydroxyproline has been noted in temperate fish relative to fish from tropical regions. Perhaps then, at temperatures of 12°C and lower a type of collagenous material is being synthesized by the scale which contains a higher proportion of glycine. This would explain the unique slope of these fish in the growth rate:glycine uptake relationship. Since as growth rate increases, the higher proportion of glycine in the collagen of 12”C-acclimated fish would result in a higher rate of glycine uptake than that of warmeracclimated fish. In the absence of growth, and collagen synthesis, higher glycine uptake would not be expected and was not observed. This subject deserves further investigation, possibly by examining the ratio of uptake for various amino acids by fish acclimated to different temperatures. A final explanation for the response of the 12”C-acclimated fish may be that the geometry of scale growth is not constant with various acclimation temperatures. These fish had a much higher coefficient of condition than the other three groups (Fig. 3). As a result of this, additional scale material may have to be deposited to account for an increased girth, or perhaps, with “stunted” longitudinal growth a thicker scale is being laid down. The relationship between glycine uptake and growth at various acclimation temperatures has been previously addressed (Smagula and Adelman, 1982) where it was suggested that, for largemouth bass, the rate of uptake should be measured at the optimum incubation temperature. Rates of glycine uptake would be obtained then, that would not have been limited by incubation temperature for any of the acclimation groups, as would occur if incubation were done at the ambient temperature of the fish. It appears now that the compensatory response to an acclimation exposure, in the common carp at least, is sufficient to make uptake observations made at ambient temperature the most accurate index of growth across acclimation groups. It also makes intuitive sense that, while optimum incubation for 30’C-acclimated fish would be equivalent to that at ambient, fish acclimated to colder temperatures would require a biased positive adjustment that would increase in magnitude with colder acclimation. This phenomenon partly explains the more divergent slopes that result in the growth rate:glycine uptake relationship when uptake is measured at an optimum temperature (Table I). A more suitable expression of glycine uptake, than that at ambient incubation,
134
E. M.
GWLISH
and I. R. ADELUAN
would be a value adjusted mathematically using a model derived from the kind of information obtained here for the common carp. This would then account for the interactions discussed above and make comparisons across acclimation groups less biased. The present study has demonstrated the marked effect that growth rate can have on the results of a temperature acclimation study in which the processes of protein synthesis are measured. In future studies, more consideration should be given to the physiological state, past and present, of the organisms being used to minimize conclusions which may be misleading and only the result of a manipulated system. This concern is likely to extend beyond temperature acclimation studies pertaining just to protein synthesis since nutritional state is likely to manifest itself throughout the metabolism of the animal.
artedi (LeSuer), in the lakes of the northeastern highlands, Wisconsin. U.S. Bur. Fish. Bull. 48, 21 l-317.
Jackim E. and LaRoche G. (1973) Protein synthesis in Fundulus heteroclitus muscle. Comp. Biochem. Physiol. 44A, 851-866.
Jankowsky H. D., Hotopp W. and Vsiansky P. (1981) Effects of assay and acclimation tem~ratures on incorporation of amino acids into protein of isolated hepatocytes from the European eel, Anguilia anguiliu L. .I. Therm. Biol. 6, 201-208. Kent J. and Presser C. L. (1980) Effects of incubation and acclimation temperature on incorporation of Uf’4C]glycine into mitochondrial protein of liver cells and slices from green sunfish, Lfpomis c~uneIfus. Physiof. Zool. 53, 293-304.
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Acknowledgements-Financial support for this study was provided by the University of Minnesota Agricultural Experiment Station and the University of Minnesota Computer Center. We would also like to thank K. Larntz for statistical advice, G. P. Busacker for helpful comments on the manuscript and Joe Nicolette for assistance in conducting the sampling.
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