The Thermal Acclimation of Burst Escape Performance in Fish: An Integrated Study of Molecular and Cellular Physiology and Organismal Performance

Many teleost fish species are often exposed to changes in temperature. These changes may be relatively acute, for example during migration, flooding, tidal changes, etc, or may be seasonal or long-term in nature. Following acute exposure to a decline in temperature, muscle power output (see Rome, 1990) and consequently locomotor performance (both sustained and burst-speed swimming) are severely compromised (reviewed in Beamish, 1978; Guderley and Blier, 1988; Sidell and Moerland, 1989; Bennett, 1990; Johnston et al. 1991).

Long-term exposure to cold temperature induces acclimatory changes in muscle contractile properties and enzyme activities, and these have been interpreted as enhancing locomotor performance at low temperatures (Loughna et al. 1983). Long-term acclimatory responses in red (slow twitch) muscle and sustained swimming behaviour during cold acclimation are well known and documented in several species of fish (see Johnston and Dunn, 1987). However, temperature acclimation of white (fast twitch) muscle fibres has been found only in a few species of cyprinid fish (Johnston et al. 1975; Heap et al. 1985, 1986; Crockford and Johnston, 1990; Fleming et al. 1990; Gerlach et al. 1990; Hwang et al. 1990; Goldspink et al. 1992) and has been demonstrated to be absent in others (Penney and Goldspink, 1981; Sidell et al. 1983; Heap et al. 1985; Sidell and Johnston, 1985; Johnston and Wokoma, 1986; Moerland and Sidell, 1986). Since the contractile properties of fast twitch muscle are thought to limit maximum locomotor speed at the organismal level (Wardle, 1975; Marsh and Bennett, 1985, 1986; Marsh, 1990; Swoap et al. 1993), the absence of acclimation at the muscle level suggests that organismal escape performance may not acclimate at cold temperatures. In any event, the acclimation of burst escape speed in fish has not been investigated. It was therefore the aim of this investigation to determine to what degree thermal acclimatory responses of muscle at the molecular and cellular levels are capable of ameliorating the detrimental effects of exposure to cold temperature on burst escape locomotor performance.

In this paper, we examine the effects of acute temperature exposure and thermal acclimation on the C-start, an escape response in teleost fish initiated by the Mauthner cell (Zottoli, 1977). This locomotor behaviour is ideal for this investigation as it is highly stereotyped and involves the simultaneous recruitment of all motor units, including all the fast twitch fibres. The C-start consists of two stages (Eaton et al. 1977). In stage 1, the fish bends into a ‘C’ as a result of the simultaneous contraction of all muscle fibres along one side of the body; stage 2 consists of an S-shaped propulsive stroke (see Fig. 1A; Eaton et al. 1977; Nissanov and Eaton, 1989). In this study, the thermal dependence of C-start performance and the underlying physiology of fast muscle function (myosin isoform expression, myofibrillar ATPase activity, twitch contraction kinetics) were investigated in one species known to demonstrate significant thermal acclimatory ability in these physiological parameters (goldfish) and compared with another species (killifish), previously shown to lack similar responses.

Goldfish (Family Cyprinidae, Carassius auratus) were obtained from a local fish farm (Bayou Aquatics, Ontario, CA, USA) and killifish (Family Cyprinodontidae, Fundulus heteroclitus) from Woods Hole Marine Biological Laboratory (MA, USA). All animals were of similar size, with a mean total length of 6.4±0.8 cm (S.D., N=75) for goldfish and 6.8±0.8 cm (N=64) for killifish. Goldfish were maintained in fresh water and killifish in 100 % sea water under ambient light conditions (16 h:8 h light:dark). Fish were divided into three groups and kept at 10, 20 and 35 °C (±0.5 °C) for at least 4 weeks prior to experimentation. All animals were fed ad libitum with fish flake and frozen bloodworms. For experimental procedures, fish were rapidly killed by a blow to the head followed by immediate decapitation.

Muscle samples from 10 °C- and 35 °C-acclimated fish were analysed using electrophoretic techniques. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was performed using small vertical slab gels (80 mm×73 mm×0.5 mm; Bio-Rad Mini-PROTEAN II). SDS–PAGE gels were run using separating gels containing a range of acrylamide concentrations of 10 % to 20 %, with a stacking gel of 4 %. Alkali urea gels were run to characterise the myosin light chains (MLCs) of actomyosin prepared from myofibril samples. MLCs were separated on gels containing 8 mol l⁻¹ urea, 10 % acrylamide at pH 8.6 (Perrie and Perry, 1970). Actomyosin was prepared from myofibril samples according to Huriaux et al. (1990). For SDS–PAGE and alkali urea gels, protein samples were prepared for electrophoresis by boiling for 3 min in a solution containing 60 mmol l^-1 Tris–HCl, pH 6.76, 2 % SDS, 10 % glycerol, 1 % mercaptoethanol and 0.002 % Bromophenol Blue (Crockford and Johnston, 1990).

Myofibril samples were also characterised using isoelectric focusing on 125 mm×65 mm×0.4 mm gels (Bio-Rad model 111 Mini IEF Cell), containing Bio-lyte carrier ampholytes (pH range 3.5–5.2, 2.25 %, pH range 3.5–9.5, 0.75 %) (Bio-Rad), 2 % Nonidet P-40, 1 % mercaptoethanol, 4.85 % acrylamide and 0.15 % N,N-methylene-bis-acrylamide (Crockford and Johnston, 1990). Samples were prepared by dissolving myofibrils in 8 mol l⁻¹ urea, 2 % Nonidet P-40 and 1 % mercaptoethanol.

Peptide mapping was used to characterise myosin heavy chains (MHCs). SDS–PAGE gels (7.5 %) of myofibril samples (approximately 5 mg ml⁻¹) were run according to the methodology described above, stained briefly in Coomassie Blue (30 s) and washed with distilled water. MHC bands were cut from the gel, chopped finely and homogenised with a glass pestle, in 400 μl of a medium containing 50 mmol l⁻¹ Tris–HCl (pH 7.4) and 0.5 % SDS, and left to stand for 60 min (Crockford et al. 1991). The samples were spun in an Eppendorf bench-top centrifuge for 15 min and the top 100–200 μl of buffer was carefully removed, avoiding contamination with gel fragments. MHCs purified in this way ran as single bands on re-electrophoresis. Samples of MHCs were digested with Type 1-S chymotrypsin (from bovine pancreas, Sigma; 200 ng of enzyme per 50 μl sample) at 20 °C for 30 min. Digestion was stopped by the addition of 2-mercaptoethanol (2 %) and SDS (2 %) and heating to 100 °C for 3 min (Crockford et al. 1991). The resulting peptides were resolved using 18 % to 22 % SDS–PAGE gels and stained using a Bio-Rad silver stain plus kit.

After the fish had been killed, the fast (white) epaxial muscle was rapidly removed from the entire length of the fish, taking care to avoid slow twitch muscle fibres along the lateral line. Myofibrils were prepared according to Johnston et al. (1975) in a solution containing 1 % (w/v) Triton X-100, 100 mmol l⁻¹ NaCl, 5 mmol l⁻¹ EDTA, 10 mmol l⁻¹ Tris–HCl, pH 7.2 at 0 °C. The protein concentration of the final sample was determined using a Bio-Rad detergent-compatible protein assay kit. A sample of myofibrils was suspended in glycerol and stored at −80 °C for electrophoretic analysis (described above). Myofibrils (approximately 0.2 mg ml⁻¹) were pre-incubated at 10, 20 and 35 °C for 2 min in a solution containing 30 mmol l⁻¹ Tris–HCl, 1.17 mmol l⁻¹ EGTA, 1.17 mmol l⁻¹ MgSO₄, 2.8 mmol l⁻¹ CaCl₂, pH 7.4 at assay temperature. Following the addition of 6 mmol l⁻¹ ATP, the activity of myofibrillar ATPase was assayed by measuring the amount of phosphate produced in 2 min. The reaction was terminated by the addition of 33 % (v/v) of 10 % trichloroacetic acid at 0 °C. The final concentration of inorganic phosphate was determined by the method of Fiske and Subbarrow (1925). To determine the thermal stability of the myofibrillar ATPase, enzyme activities were monitored following pre-incubation at high temperature (40 °C) for between 5 min and 1 h. ATPase activity was then determined at 40 °C according to the methods described above.

Fast muscle twitch contraction kinetics were measured in situ in freshly killed fish using a modification of the method described by Archer et al. (1990). The experiments were performed in a Perspex temperature-controlled (±0.1 °C) chamber with a base of Sylgard elastomer on which to secure the specimen with dissection pins. Experimental preparations of goldfish were continually immersed in Ringer’s solution containing 119 mmol l⁻¹ NaCl, 2.7 mmol l⁻¹ KCl, 1 mmol l⁻¹ MgCl₂, 1.8 mmol l⁻¹ CaCl₂, 20 mmol l⁻¹ NaHCO₃ and 10 mmol l⁻¹ sodium pyruvate (Johnson et al. 1994). Preparations of killifish were maintained in Ringer’s solution designed for marine teleosts (Altringham and Johnston, 1988) and containing 132.5 mmol l⁻¹ NaCl, 2.6 mmol l⁻¹ KCl, 1 mmol l⁻¹ MgCl₂, 2.7 mmol l⁻¹ CaCl₂, 18.5 mmol l⁻¹ NaHCO₃, 3.2 mmol l⁻¹ NaH₂PO₄ and 10 mmol l⁻¹ sodium pyruvate. The pH of the Ringer’s solution was maintained at 7.4 by bubbling with 95 % O₂:5 % CO₂. The abdominal cavity was exposed with a lateral incision, the viscera were carefully removed and the abdominal myotomes pinned back. Abdominal membranes were removed to expose the fast muscle myotomes and a suction electrode was placed on the fourth or fifth spinal nerve close to its origin from the spinal column. During the contraction of the appropriate myotome, force was monitored using a force transducer made from two Entran ESU-060-1000 strain gauges bonded to a spring steel bar. A pin, bonded to the surface of the transducer, was inserted into the adjacent myotome next to the posterior myoseptum. Output signals from the transducer were modified using a custom-designed bridge amplifier and displayed and stored using a Metrabyte A/D converter interfaced to an IBM-PC. Single, supramaximal stimuli (typically 0.1–0.5 ms duration, 20–30 V) were administered via the suction electrode using a Grass S48 stimulator connected to a current amplifier. The preparation was allowed to recover for at least 10 min between stimuli. Twitch contraction time (T_c, the time from the onset of force production to 50 % relaxation) was measured in preparations from all acclimation groups and both species at temperatures encompassing the acclimation regimes.

Fish were videotaped performing C-starts (escape responses) in an area 25 cm×20 cm×15 cm using a NAC HSV-400 high-speed video system at 400 frames s⁻¹. Experimental trials were performed in an insulated glass aquarium marked on the base with a 2 cm×2 cm×2 cm grid. Fish were filmed ventrally by placing a front-surface mirror at 45 ° to the horizontal below the tank. Illumination was provided by a combination of 500 W photofloods (Smith Victor) and synchronized strobes (NAC). Temperature was controlled (±0.5 °C) using submersible aquarium heaters and Neslab cryocool immersion coolers. C-starts were elicited either by striking the side of the tank or by dropping a small weight into the tank above the fish. Only C-starts performed from a stationary position were recorded and analyzed. Trials were performed over a broad range of temperatures within the critical thermal limits for the different acclimation groups and species. Critical thermal maximum was defined as the temperature at which fish lost equilibrium and critical thermal minimum as the temperature at which fish failed to respond to tactile stimulation. Fish recovered completely when returned to more moderate temperatures. Water temperature was changed at a rate not exceeding 10 °C h^-1. Animals were placed in the experimental arena at the acclimation temperature and allowed to adjust to their new surroundings for no less than 1 h. After recording C-starts at this temperature, the water was either cooled or heated to the next trial temperature. Within acclimation groups, the order in which trials were performed (i.e. above or below acclimation temperature first) was randomized from fish to fish. C-starts at acclimation temperature were recorded again at the end of the experiment to determine repeatability. Up to three C-starts were recorded per fish at each temperature.

C-start sequences were downloaded from the video and stored as digital images on an IBM-PC installed with a PC-VISIONplus framegrabber card (Imaging Technology Inc., MA, USA). Video frames were downloaded from the first detectable signs of movement by the fish following the stimulus, until the animal moved beyond the field of view or slowed significantly; only sequences in which both stage 1 and stage 2 of the C-start were fully visible were used in the analyses. Individual video images were digitized using customized software [‘Measurement Television’ (MTV), Garr Updegraff, Data Crunch, San Clemente, CA, USA]. The coordinates of ten evenly spaced points along the midline of the fish from the nose to the tip of the caudal fin were digitized for each image (Fig. 1A). The coordinates for images of each trial were downloaded to Microsoft Excel (version 5.0 for Windows) and plotted (Fig. 1B). During the initial stages of the C-start (stage 1), fish bend about a point known as the stretched body centre of mass (SBCM); this region of the body is particularly important as it is believed to be the target site for predatory strikes (Nissanov and Eaton, 1989). Thus, we chose the point along the midline closest to the SBCM for our kinematic measurements. Three kinematic parameters were measured using a customized macro function in Microsoft Excel. Maximum angular velocity during stage 1 (degrees s^-1; MaxANG), measured over 5 ms (2 frames), represents the maximum rate of change of angle between the tip of the head and the SBCM. Maximum velocity (m s^-1; MaxVEL) represents the maximum forward velocity attained by the SBCM, measured over 5 ms. The third kinematic parameter measured was the total distance moved by the SBCM in 40 ms (DIST_40ms), which represents the approximate time for the completion of both stage 1 and stage 2 of a C-start performed at 35 °C. The temperature at which maximum values for each of the kinematic parameters were obtained was noted and defined as the ‘optimum temperature’ (cf. Huey and Stevenson, 1979) for that variable.

For all experimental variables, the effects of acute temperature change and acclimation were determined using a two-way analysis of variance (ANOVA). The effect of acclimation temperature on the experimental parameters at individual test temperatures was subsequently determined using a one-way ANOVA nested within individuals. The ANOVA was followed by a Tukey’s multiple comparison test when it was necessary to isolate specific differences between the values for different acclimation groups. With kinematic data, a Mann–Whitney (non-parametric) U-test was used to incorporate data for warm-acclimated individuals that failed to elicit an escape response at low temperature (scored as zero). A Wilcoxon two-sample test (non-parametric) was used to examine differences in the optimum temperature for kinematic parameters between acclimation groups. The Tukey’s jackknife method was used to test for differences in the magnitude of the acclimation response between species for each of the experimental parameters (all tests are described in Sokal and Rohlf, 1981).

SDS–PAGE gels (10 % to 20 %) of fast muscle myofibrils isolated from animals acclimated to 10 and 35 °C were identical when compared within species. There was no apparent difference in the mobility of myosin heavy and light chains, actins, tropomyosins or troponins. Acclimation temperature also appeared to have no effect on the expression of myosin light chains, visualised using alkali urea gels and isoelectric focusing (IEF), in either goldfish or killifish. However, according to peptide maps obtained for MHCs isolated from 10 °C- and 35 °C-acclimated fish, there was a consistent and significant shift in the expression of MHCs in the goldfish (N=4) but not in the killifish (N=4). Fig. 2 shows peptide maps obtained for 10 °C- and 35 °C-acclimated goldfish and killifish (22 % SDS–PAGE).

The activity of myofibrillar ATPase was expressed as μmoles of phosphate (P_i) produced per milligram of myofibrillar protein per minute. Both acute temperature exposure and a change in acclimation temperature had a significant effect on myofibrillar ATPase activity in both species (P<0.01, two-way ANOVA). Measured at 10 °C, myofibrillar ATPase activity was affected by acclimation temperature for both goldfish (P<0.0001, ANOVA) and killifish (P=0.001, ANOVA) (Fig. 3). However, the magnitude of the effect was much greater in the goldfish (P<0.001, Tukey’s jackknife test), ATPase activity measured at 10 °C increasing by 530 % in the goldfish and by only 55 % in the killifish, in 35 °C-acclimated compared with 10 °C-acclimated fish. Acclimation temperature had no significant effect on myofibrillar ATPase activity measured at 35 °C (P=0.48, nested ANOVA). Assayed at 35 °C, the acclimation of killifish to 35 °C did result in a small decrease in ATPase activity compared with those of the 10 °C- and 20 °C-acclimated groups (P<0.01 ANOVA, P<0.01 Tukey’s multiple-comparison test) (Fig. 3).

By pre-incubating myofibrils isolated from goldfish and killifish at 40 °C for up to 1 h, we examined the thermal stability of the myofibrillar ATPase. In both species, the ATPase activity of myofibrils isolated from 35 °C-acclimated animals was stable throughout the full 60 min exposure to 40 °C (Fig. 4). In contrast, ATPase activity declined rapidly in muscle from 10 °C-acclimated animals; the responses were almost identical in the goldfish and killifish (Fig. 4), an observation in contrast to previous observations on the latter species (Sidell et al. 1983). This shift in thermal stability clearly represents a resistance adaptation and appears to be a widespread phenomenon in teleosts in response to temperature changes over evolutionary as well as seasonal time scales.

Twitch contraction time (T_c) was significantly affected by acute temperature changes and acclimation temperature in both species (P<0.001, two-way ANOVA; Fig. 5). For goldfish, twitch contraction at 10 °C was 100 % faster in 10 °C-acclimated compared with 35 °C-acclimated animals (P<0.001, ANOVA) and 55 % faster than that at 20 °C (P<0.001, ANOVA; Fig. 5). There was also a small, but significant, effect of acclimation temperature on T_c measured at 35 °C (P<0.01, ANOVA); T_c was 13.4 ms for 10 °C-acclimated animals and approximately 10 ms for 20 °C- and 35 °C-acclimated goldfish (Fig. 5).

The results for the acclimation of killifish followed an almost identical pattern, although the magnitude of the and response of T_c was much smaller (P<0.001, Tukey’s jackknife test on values obtained at 10 °C; Fig. 5). At 10 °C, T_c decreased by 35 % from 50 ms to 37 ms in killifish acclimated to 35 °C 10 °C respectively (P<0.001, ANOVA; Fig. 5). The T_c of animals acclimated to 10 °C was significantly slower at 20 °C by around 24 % (P<0.001 ANOVA) when compared with those acclimated to 20 and 35 °C (P<0.001, Tukey’s multiple comparison test). Acclimation temperature had no significant effect on T_c measured at 35 °C in killifish (P=0.69, ANOVA; Fig. 5).

Acute temperature exposure had a significant effect on all kinematic variables measured for both species and acclimation groups (P<0.05, two-way ANOVA).

Analysing the data with a two-way analysis of variance, all kinematic parameters were highly significantly affected by acclimation temperature in the goldfish (P<0.005). For goldfish acclimated to 35 °C, seven out of 10 individuals failed to respond to stimuli and elicit a C-start response at 10 °C. All goldfish acclimated to 10 °C responded with a C-start response at 10 °C. Using the available data at 10 °C for C-starts of 10 °C- and 35 °C-acclimated goldfish (N=10 and N=3, respectively), MaxVEL increased by 35 % (P=0.05, ANOVA; Fig. 6), DIST_40ms by 52 % (P=0.01, ANOVA; Fig. 7) and MaxANG by 42 % (P=0.07, ANOVA; Fig. 8) with a decrease in acclimation temperature from 35 to 10 °C. Alternatively, by scoring zero for 35 °C-acclimated individuals that would not C-start at 10 °C, a Mann–Whitney U-test resulted in a highly significant effect of acclimation temperature for all three kinematic variables measured at 10 °C.

For killifish, acclimation temperature had a significant effect on MaxANG only (P<0.01, two-way ANOVA), although the interaction between acute temperature exposure and acclimation temperature was significant for MaxVEL (P=0.02, two-way ANOVA). All killifish acclimated to 10 and 35 °C (N=5 at both temperatures) elicited C-starts at 10 °C. Measured at 10 °C, MaxVEL and MaxANG were significantly greater in 10 °C-acclimated compared with 35 °C-acclimated killifish (P=0.009 and P=0.004 respectively, ANOVA; Figs 6, 8). Acclimation temperature had no significant effect on DIST_40ms measured at 10 °C (P=0.22, ANOVA; Fig. 7).

In Figs 6–8, the three kinematic variables are plotted against temperature for goldfish and killifish acclimated to 10 and 35 °C. Note the shift of the curves to the right with an increase in acclimation temperature (i.e. an increase in optimum temperature). In goldfish, this increase in optimum temperature was significant for all kinematic parameters; optimum temperature increased by 21 °C for MaxVEL (P=0.004, Wilcoxon), 13 °C for MaxANG (P=0.01, Wilcoxon) and 14 °C for DIST_40ms (P=0.004, Wilcoxon; Figs 6–8). For killifish, acclimation temperature had a significant effect on the optimum temperature for MaxANG (P=0.04, Wilcoxon), increasing it from 22 to 35 °C. The changes in optimum temperature were only 8 °C for MaxVEL and 4 °C for DIST_40ms with an increase in acclimation temperature from 10 to 35 °C, which are not significant (P=0.197 and P=0.102 respectively, Wilcoxon).

The biochemical and contractile properties of fast muscle of goldfish and killifish are profoundly affected by acute changes in temperature (Table 1). This thermal dependence of muscle function agrees with many previous observations (Bennett, 1990; Johnston et al. 1990). Despite the significant thermal dependence of isolated muscle function, very little is known about the thermal dependence of C-start escape behaviour at the organismal level. Some earlier studies reported burst speed to be relatively independent of temperature (Beamish, 1966; Brett, 1967, 1970), but more recent investigations have found a significant thermal effect (Rulifson, 1977; Webb, 1978; Fuiman, 1986; Batty and Blaxter, 1992). The present investigation is the first strictly integrative study to examine the thermal dependence of C-start performance at all levels of organisation from molecular to organismal. For both species (goldfish and killifish), the thermal dependence of myofibrillar ATPase activity and twitch contraction kinetics is reflected in the thermal dependence for all organismal kinematic parameters measured for C-starts (Table 1). The thermal dependence of myofibrillar ATPase and twitch contraction kinetics are thus potentially important determinants of in vivo muscle function and C-start performance.

Both goldfish and killifish are known to be relatively eurythermal species, capable of tolerating a wide seasonal range of temperatures (Elliot, 1981; Sidell et al. 1983). However, killifish, being an estuarine species, are commonly subjected to large and rapid reductions in temperature of up to 10–15 °C in less than an hour (Sidell et al. 1983). Consistent with previous observations, myofibrillar ATPase activity is comparatively insensitive to changes in temperature in killifish (Q₁₀=1.4–1.5) (Table 1; Sidell et al. 1983). From observations in the wild (Sidell et al. 1983) and in the laboratory on C-start performance (this study; Figs 6–8), locomotor capability is not substantially impaired by the acute changes in temperature commonly experienced in the summer months (12–35 °C). However, a significant effect of acclimation temperature on myofibrillar ATPase activity was found in this study across a broader acclimation regime (10–35 °C) than previously studied (5–25 °C for Sidell et al. 1983). Killifish acclimated to 35 °C, compared with 10 °C- and 20 °C-acclimated fish, apparently have a significantly lower ATPase activity. No apparent changes in the expression of myofibrillar protein isoforms were found, so the response of ATPase to acclimation appears to be achieved through a mechanism other than differential isoform expression (reviewed in Johnston and Dunn, 1987). A small acclimatory response was also demonstrated for twitch contraction kinetics (Fig. 5). Apparently there are some compensatory mechanisms to maintain locomotor ability in this species at low temperature. In comparison with goldfish, however, acclimatory mechanisms are clearly different and the resulting compensation in locomotor performance is substantially less.

In addition to the acute effects of temperature, prolonged exposure to low temperature results in a significant increase in myofibrillar ATPase activity and twitch contraction speed in at least in some teleost species, notably the cyprinids (see Figs 3, 5). In relation to organismal performance, goldfish acclimated to 35 °C can swim at approximately 1.0 m s⁻¹ and yet may achieve only 0.2 m s⁻¹ at 10 °C, if they swim at all (a majority of our 35 °C-acclimated fish did not; Fig. 6). However, following a period of acclimation to 10 °C for 4 weeks, animals may swim at speeds up to 0.9 m s⁻¹, demonstrating almost perfect temperature compensation (Fig. 6). The results of this study are unique in that they corroborate the hypothesis that the thermal acclimatory responses in the cyprinids previously demonstrated at the biochemical and tissue level represent a mechanism of maintaining locomotor performance at low temperature.

The unique thermal acclimatory ability of carp and goldfish to undergo significant physiological changes at biochemical and cellular levels of organisation has received considerable attention (reviewed in Johnston and Dunn, 1987: Johnston et al. 1990). This ability has been attributed to their polyploid genetic structure (Sidell and Johnston, 1985; Goldspink et al. 1992). Specifically, the multiple gene copies afforded by chromosomal duplication are presumed to permit the evolution of alleles appropriate to different thermal environments, as demonstrated in the studies of Baldwin and Hochachka (1970) and Somero (1975). Differential expression of temperature-specific isoforms is, of course, not the only mechanism by which enzymatic changes in response to thermal acclimation may be mediated (for discussion, see Johnston and Dunn, 1987). However, the thermal acclimation of fast twitch muscle myofibrillar ATPase and twitch contraction speed in carp and goldfish appear to be mediated through the differential expression of temperature-specific isoforms of myosin heavy chains (Gerlach et al. 1990; Hwang et al. 1990; Goldspink et al. 1992; Fig. 2 this study) and myosin light chains (Crockford and Johnston, 1990), consistent with the hypothesis that polyploidy is important for the formation of temperature-specific isoforms in response to thermal acclimation in these fish.

The authors would like to thank Alistair Cullum for his technical support in the analysis of the data. We would also like to thank Berenice Perez, Matthew Corse, Micheal Bye and Kevin Hsu for their assistance in animal husbandry and in the analysis of video sequences. We are also grateful to Matthew Corse for his experimental work on the measurement of muscle contractile performance. This work was supported by a grant from the National Science Foundation (IBN-9118346) awarded to A.F.B.