The Role of Behavior in Temperature Acclimation and Tolerance in Ectotherms

AMER. ZOOL, 19:367-384 (1979). The Role of Behavior in Temperature Acclimation and Tolerance in Ectotherms VICTOR H. HUTCHISON AND JOSEPH D. MANESS Department of Zoology, University of Oklahoma, Norman, Oklahoma 73109 SYNOPSIS. A review of field and laboratory investigations suggests that many ectothermic vertebrates can exploit the spatiotemporal distribution of environmental temperatures to maximize energy utilization and to enhance survivability. Diel and seasonal cycles in thermal preference, acclimation rate, thermal tolerance and heat-hardening may well be adapted to temporal variations in environmental temperature. In addition, many ectoiherms behaviorally exploit thermal heterogeneity in the environment. Such behavioral adaptations are synergistic with various degrees of physiological regulation. Voluntary brief exposures to temperatures that would be lethal upon prolonged exposure can result in heat-hardening. Heat-hardening, distinct from acclimation to high temperature, is a short-term increase in thermal tolerance while tolerance acclimation is a longer lasting response within normal ranges of environmental temperatures; both are reversible nongenetic responses. The physiological and ecological significance of behaviorally mediated heat-hardening may be greater than previously realized and suggest new approaches for future study. INTRODUCTION Although temperature is but one of many physical factors in an organism's holocoenotic environment, it is the most pervasive; this fact, combined with ease of measurement, has led to a multitude of studies in thermal biology. Ectothermic vertebrates have received much of this attention, but only in the past three decades has the role of behavior received much attention. The rather precise behavioral thermoregulation of many ectothermic vertebrates is well documented. However, we are just beginning to understand the corn- Some of our own investigations cited in this paper were supported by the following grants (to V.H.H.): National Institutes of Health GM 10156, Biomedical Sciences Support Grants through the University of Oklahoma (NIH 507 RR07078), the University of Rhode Island Research Committee, the University of Oklahoma Research Council and the Society of Sigma Xi. We thank Thomas L. Beitinger, Bayard H. Brattstrom and William W. Reynolds for review of the manuscript. This symposium was supported by National Science Foundation Grant PCM 78-05691 to W. W. Reynolds. plexities involved in the interrelations between thermoregulatory behavior and diel and seasonal cycles (which may be endogenous), thermal history, nutritional status, photoperiod, life history stage, learning and memory, etc. A revie,w of field and laboratory investigations suggests that ectotherms may increase their thermal tolerances through behavioral exploitation of temperatures available to them in a normally cycling or heterogenous thermal environment, which accelerates the rate of acclimation, or by voluntary brief exposures to what would otherwise be lethal temperatures. The physiological and ecological significances of these thermoregulatory behaviors, which may be greater than previously realized, suggest some new avenues for future study. TEMPERATURE ACCLIMATION AND THERMAL TOLERANCE Definitions Terminology used to describe responses 367

368 V. H. HUTCHISON AND J. D. MANESS FIG. 1. Rates of acclimation of the red-spotted newt (Notophthalmus vindescens) transferred between different acclimation temperatures. Each point is the mean of 6 to 10 animals. (From Hutchison, 1961, Physiological Zoology, with permission of the University of Chicago Press) of organisms to various thermal histories has yet to be standardized, despite attempts by several authors (Hutchison, 1976). The term "acclimatization" is sometimes used to describe changes in organisms produced by responses to natural climatic conditions and "acclimation" to describe responses to experimental conditions (Prosser, 1973; Schmidt-Nielson, 1975). On the basis of priority, frequency, and authoritative uses, we follow the suggestions of Folk (1974). Acclimation is the compensation made to a single environmental factor. Animals held at a stated temperature regime before measurement or observation of experimentally produced changes have been acclimated to the given condition. The time course for completion of acclimation to a new temperature ranges from several hours to a few weeks. Acclimatization is an adjustment to two or more environmental factors. An organism in its natural environment has been acclimatized to a complex of factors, but the term is more frequently used for laboratory conditions where two or more factors are varied and other environmental factors are held constant. Two basic methods have been used for determinations of upper lethal temperatures in ectotherms: the lethal temperature method and the critical thermal maximum (CTM). The two methods have been compared by Fry (1967, 1971) and Hutchison (1976) and are defined by an earlier paper in this symposium (Reynolds and Casterlin, 1979a). Acclimation rates The rate of tolerance acclimation to high temperatures is usually significantly faster than that to low temperatures (Fig. 1). Exposure to temperature cycles usually results in acclimation to the highest temperature of the cycle, rather than to the mean value, and the rate of acclimation to cycling temperatures is often faster than the rate when an animal is transferred from the lowest temperature (held constant) to the highest temperature of the cycle. Leopard frogs (Rana pipiens) after acclimation to a constant 15 C had a faster rate of acclimation to the highest temperature of a 15 to 25 C daily cycle than did animals transferred from acclimation at a constant 15 C to constant 25 C (Hutchison and Ferrance, 1970). The CTM of animals exposed to only one 24-hour thermoperiod was significantly higher than in those exposed to constant acclimation temperatures (Fig. 2 >- There is evidence that animals adapt best to thermoperiods most closely approaching those of the natural environment (Heath, 1963). When cutthroat salmon (Salmo clarki) were exposed to square wave temperature cycles (thermoperiods) from 0.25 to 2.0 times the natural 24-hour time cycle, the highest thermal tolerance was at the 24-hour thermoperiod (Fig. 3). Similar increased scopes for thermal tolerance after acclimation to cyclic temperatures have also been reported in the Mojave Desert pupfish {Cyprinodon nevadensis amargosae) (Feldmeth et «/., 1974), the green-throat darter (Etheo.stoma lepidum) (Hubbs, 1964) and the Sonoran Des-

BEHAVIOR AND THERMAL TOLERANCE 369 36 1200 1800 2400 TIME (1ST) O.24 uuunnnr rr 8«J". j 2 * 3 * * A Acclimation Tim* (Days) 3».S 39.0 38.5 39 0 37.5 37.0 36.5 _ r f - '' -o' - o ( 1 1 1 1 1»4 4t 71 120 144! It2 o - _ - - - 30 o 29 U28 27 Cycltd (10* to 20*1 _ 10 19 20 D Acclimation T«mp«rotur«(*C) FIG. 2. Upper portion: Pattern of programmed temperature cycle of 15 to 25 C and an LD 12:12 photoperiod; black bars indicate the scotophase. Lower portion: The rate of acclimation of leopard frogs (Rana pipiens) transferred from acclimation at 15±1 C to the diel cycle of 15 to 25 C (solid circles) shown in the upper portion of the figure; each point is the mean of 14 to 19 animals. (From Hutchison and Ferrance, 1970, Herpetologica, published by the Herpetologists' League) ert pupfish (Cyprinodon macularius) (Lowe and Heath, 1969). Feldmeth et al. (1974) also found that pupfish had a scope for thermal tolerance of 39 C between the critical thermal maxima and minima when acclimated to constant temperatures of 15, 25 and 35 C. After acclimation to a daily cycle of 15 to 25 C, the scope increased to about 41 C, demonstrating that these fish can acclimate to both high and low temperatures concurrently when exposed to a thermal cycle by an increase in the critical thermal maximum and a decrease in the critical thermal minimum. An increase in the thermal tolerance o 30 r O69 P«.00l r«-.229 P«.0l 6 12 24 36 4* Vj Cycle Lengih (Hour*) FIG. 3. Acclimation to square-waved thermoperiods and critical thermal maxima (CTM) in the sea-run cutthroat salmon (Salmo clarki). Upper position: Patterns of thermoperiods; cycles are from 10 to 20 C. Middle portion: The regression of CTM on acclimation to constant temperature (solid squares) and to 24 hour thermoperiod (open squares). Lower portion: Relation of CTM to thermoperiod cycle length. Short horizontal line shows means; rectangular boxes, two standard errors on each side of the mean; vertical line, range; r and p are regression and probability, respectively, of the slopes of the lines on each side of the 24-hour cycle length. (From Heath, 1963, with permission; copyright 1963 by the American Association for the Advancement of Science)

370 V. H. HUTCHISON AND J. D. MANESS range is not the only adaptive value of exposure to temperature cycles. Lizards (Anolis carolinensis) exposed to warmer thermoperiods for4 or 8 hours at various times of day every 24 hours developed differences in weight, lipid stores, and reproductive status depending upon the time of day the warm thermoperiod was given (Noeske and Meier, 1977). A similar study with goldfish (Carassius auratus) suggested that testicular growth and weight gain was significantly influenced by the time of day at which the fish were exposed to a temperature increase from 15 to 24 for a 4-hour period (Spieler et al., 1977a). Studies on effects of thermoperiods show that time of day can affect the rate of acclimation of thermal tolerance. Similarly, there may well be seasonal cycles which influence the acclimatory process, but little data exists on seasonal differences in the rate of acclimation for ectothermic vertebrates, although numerous studies have shown seasonal differences in thermal tolerance. 2O*C I6L IO*C I6L o 2O*C 8L O 0*C 8L 0400 0800 1200 TIME - E.S.T. 2000 2400 FIG. 4. Daily cycles in the critical thermal maximum of painted turtles (Chrysemys picta) acclimatized to the four acclimations of photoperiod and temperature shown. Each point represents the mean of 19 to 20 animals. (Modified from Kosh and Hutchison, 1968, Copeia, Journal of the American Society of Ichthyologists and Herpetologists) Diel and seasonal cycles of thermal tolerance 4) (Kosh and Hutchison, 1968). Similar daily cycles of thermal tolerance have been In the lethal temperature (resistance reported in fishes (Johnson, 1976; Spieler time) method of determining thermal tolerance the time required to measure the Mahoney and Hutchison, 1969; Seibel, et al, 19776), amphibians (Dunlap, 1969; resistance time to death may extend over 1970; Johnson, 1971, 1972a,6)and reptiles several days, thus masking daily cycles in (Spellerberg and Hoffman, 1972). thermal sensitivity (Hutchison, 1976). With Methods used to test for thermal tolerance prevent the determination of whether the CTM method, where relatively short exposure to the test conditions are used, or not these diel cycles are truly circadian significantly diel cycles of thermal tolerance are commonly observed, and may be "free-running" conditions of constant light (endogenous cycles that persist under strongly influenced by combinations of acclimation temperature and photoperiod. of other circadian rhythms and the fre- or constant darkness), but the prevalence In the painted turtle (Chrysemys picta), a quently observed influence of photoperiod diurnal basking species, the CTM is highest at 1400 hr when the animals are accli- mechanism exists. on thermal tolerance, suggest that such a matized and tested under a photoperiod at Seasonal cycles in thermal tolerance LD 16:8 at 20 C and LD 8:16 at 10 C. measured under controlled photoperiods When "abnormal" combinations of high and constant acclimation temperatures temperature with short-day photoperiods have been observed in goldfish (Hoar, (LD 6:18, 20 C) or low temperature with 1955; Hoar and Robertson, 1959), three long-day photoperiods (LD 16:8, 10 C) stream fishes (Notropis stramineus, N. cornutus, Etheostoma nigrum) (Kowalski et al, acclimatizations were used, the time of day at which the maximum tolerance (CTM) occurred in the daily cycle was phase 1978), salamanders (Feder and Pough, shifted +4 and 8, hours, respectively (Fig. 1975) and anuran tadpoles (Lucas and Reynolds, 1967) (Fig. 5).

Examples of ectothermic vertebrates which exhibit diel cycles of preferred temperature in experimental gradients or shuttleboxes are bowfin, Amia calva (Reynolds et al., 19786); estuarine goby, Gillichthys mirabilis (DeVlaming, 1971); smallmouth blackbass, Micropterus dolmieui, and largemouth blackbass, M. salmoides (Reynolds and Casterlin, 1976, 1978c); brown trout, Salmo trutta (Reynolds and BEHAVIOR AND THERMAL TOLERANCE 371 i i i i i t Casterlin, 1979ft); goldfish, Carassius auratus (Reynolds et al., 1978a); mudpuppy, Not r opt s stramineus Necturus maculosus (Hutchison and Black, (10) _^ December 1974 unpublished data) (Fig. 6); leopard frog (13) tadpoles, Rana pipiens (Casterlin and January 1975 Reynolds, 1978); island night lizard, (10), Klauberina riversiana, Colorado Desert March 1975 fringe-toed lizard, Uma notata and Texas (18) March 1976 horned lizard, Phrynosoma cornutum (Regal, 1967); green anole, Anolis carolinensis (Hutchison and Kosh, 1974); desert spiny i i i t i i lizard, Sceloporus magister (Engbretson and 32 0 33 0 34 0 Hutchison, 1976); collared lizard, Crotaphytus collaris (Cothran and Hutchison, un- CTM, c FIG. 5. Seasonal variation in the critical thermal published data); plain-bellied water snake, maximum (CTM) of a minnow acclimatized to Natrix erythrogaster (Gehrman, 1971). 15±1 C and a photoperiod of LD 12:12. Means are Some species do not show significant diel shown as vertical line on black rectangles; the latter cycles in thermal selection of differences represents one standard error on each side of the mean; horizontal line bounded by short vertical lines, between photophase and scotophase when range; numbers in parentheses, sample size. (From tested in the laboratory: bluegill sunfish, Kowalski et al., 1978, J. Thermal Biology, with permission of Pergamon Press) Reynolds and Casterlin, 1976); green sun- Lepomis macrochirus (Beitinger, 1975; fish, Lepomis cyanellus (Beitinger et al., 1975); rock bass, Ambloplites rupestns BEHAVIORAL THERMOREGULATION (Reynolds and Casterlin, 1978a 1 ); white Definitions sucker, Catostomus commersoni Reynolds and Casterlin, 1978«) and yellow bullhead, Ictalurus natalis (Reynolds and Casterlin, Elsewhere in this symposium Reynolds and Casterlin (1979a) have discussed the 1978ft); painted turtle, Chrysemys picta, terminology used for descriptions of behavioral thermoregulation and the many pot, Sternotherus odoratus (Graham and spotted turtle, Clemmys giittata, and stink- factors which can influence thermal preferenda (also variously referred to as eccritic species have significant cycles of locomotor Hutchison, 1979). Since most of these temperature, thermal optimum, thermal activity, the failure to find similar cycles in preferendum, selected temperature, and preferred temperatures may result from preferred temperature). We wish to emphasize the temporal changes, in thermal straints placed on the animals by the ap- inadequate test conditions such as con- selection which result from normal behavior in a spatially heterogeneous or temperimental photoperiod, etc. paratus, thermal acclimation regime, exporally varying thermal environment. Seasonal cycles in thermal preference in the laboratory are illustrated by the speck- trout, Salvelinus fontinalu (Sullivan Diel and seasonal cycles of thermal preferenceled and Fisher, 1953) where the seasonal change of selected temperature was not caused by changes in acclimation temperature (Fig. 7). The preferred temperature increased in late winter, although the temperature at which the trout were maintained was at its lowest point (3-5 C). In the plaice, Pleuronectes platessa, and bitterling, Rhodevs sericeus, the effect of acclimation temperature varies with season (Zahn, 1963). In lizards (Sceloporus undulatus) acclimated to

372 V. H. HUTCHISON AND J. D. MANESS 16 1 1 1 1 I I 1 I I 15 IT O 14 o - 13 I 1 1 * * 12 - - 16 21 02 07 12 17 22 03 08 13 HOUR FIC. 6. Diel cycle of preferred temperatures in the mudpuppy Necturus maculosus acclimatized to 15±1 C and a photoperiod of LD 12:12 and placed in a linear thermal gradient (5 to 40 C). Each circle is the mean 20 C under photoperiods of LD 12:12 and ticulatm) by Zahn (1963) or in the coldtemperature rainbow trout (Salmo gaird- LD 6:18, the longer photoperiod increased the preferred temperature in animals collected in May and the shorter photoperiod ample. neri) by Garside and Tait (1958), for ex- decreased the preferred temperature of Excellent examples of field studies of lizards collected in July. However, short seasonal variation in behavioral thermoregulation are those of Case (1976) on photoperiods in May did not decrease the preferred temperature, nor did longer the chuckawalla, Sauromnlus obesiis, and of photoperiods in July increase the temperatures selected (Ballingerrfa/., 1969). This Kalahari Desert. Huey etal. (1977) on diurnal lizards of the refractoriness of thermal preferences to Laboratory studies on thermal preference sometimes predict temperature re- certain experimental photoperiods at different seasons and sensitivity at other times sponses in the field. Brown bullheads, Ictalurus nebulosus, acclimated at 3.5 to 28 C of the year is highly suggestive of an endogenous circannual rhythm. and tested in a thermal gradient preferred Although the majority of organisms temperatures higher than those at which tested show seasonal differences in thermal they were acclimated and tended to gravitate toward higher temperatures the preference when tested under constant photoperiod-temperature acclimatization, other animals do not; no seasonal and Ibara, 1978). These experimental re- longer they were in the gradient (Richards cycles in preferred temperature were observed in two tropical species (a barb, Punserved in autumn as the bullheads moved sults were consistent with movements obtiu\ rnnrhnnins, and the gupp), Li-bistc, re- into the thermal discharge canal of a of 10 animals; vertical line, one standard error of the mean; black bars, scotophase. (Hutchison and Black, unpublished data)

4 12 0 8 6 4 2 22 29 NOV PT ^\ 7 DEC BEHAVIOR AND THERMAL TOLERANCE 373 PT f AT 18 24 MAR FIG. 7. Seasonal variation in the temperature selection of speckled trout (Salvelinus fontinalis). Upper curves, temperature selected by fish; lower curves, acclimation temperatures. (Modified from Sullivan and Fisher, 1953) power plant on the Connecticut River; a "temperature trap" keeps the fish in the warmer thermal effluent in the winter. However, the bullheads leave the warmer waters of the discharge canal in the spring sooner than the experimental data on thermal selection would predict. Another example of the inadequacy of simple laboratory determinations of preferred temperatures in the prediction of thermoregulatory behavior in nature is provided by McDonald (1973) for the sockeye salmon, Oncorhynchus nerka. On a typical day in late summer in Babine Lake, British Columbia, young sockeye spend the daylight hours in the hypolimnion at 4 to 6 C, rise rapidly at dusk to feed near the surface at 16 to 18 C, descend in the middle of the night to the upper portion of the thermocline at about 15 C, rise slowly to feed near the surface at dawn, and descend rapidly back to the hypolimnion in early morning (Fig. 12). Perhaps the vertical migration of young sockeye results from diel cycles in the preferred temperature. Laboratory determinations of thermal selection over short periods, or in which diel changes in preferred temperature are not recorded may lead to faulty conclusions. Significance of preferred temperatures Many biochemical and physiological processes in ectotherms are optimal at or near the thermal preferendum. Documented examples include active metabolic rate, metabolic scope, oxygen debt load, maximum sustained speed, maximum volitional speed, growth rate, food conversion efficiency, resting and active blood pressure, active cardiac work, cardiac scope, elimination of anaerobically produced lactate, learning and memory, auditory sensitivity, appetite, digestion, egestion, immune response, renal function, hormone secretion and action, reproductive functions, and enzyme activity (Brett, 1971; Dawson, 1975; Precht, 1973; Beitinger and Fitzpatrick, 1979; Brattstrom, I979a,b). There are important exceptions to the generalization that physiological processes have optima near the preferred temperature. The significance of these exceptions, such as the temperature dependence of aerobic metabolic scope in some lizards and turtles (Dawson, 1975), are not yet known. Such exceptions may, however, be the clues to important future discoveries; an example, the shunting of the thermal optimum for growth efficiency in sockeye salmon, is discussed below. Another exception is the behavioral fever shown by ectotherms infected with pathogenic bacteria or bacterial endotoxin (Kluger, 1978). "Despite these exceptions, instances are now documented in which maintenance of individuals and survival of the species clearly depend on periodic attainment of temperatures within the preferred range. Such dependence more than justifies the often elaborate behavioral and physiological mechanisms leading to control of body temperature..." (Dawson, 1975). Although this statement was applied to reptiles, it can clearly be extended to all ectotherms (Hutchison, 1976). PHYSIOLOGICAL THERMOREGULATION Although in this paper we address the role of behavior in thermal relations, physiological regulation must not be overlooked. The separation of endothermy from ectothermy has now been somewhat blurred with the knowledge that many

374 V. H. HUTCHISON AND J. D. MANESS species of mammals and birds are heterothermic at different stages of development, times of day or seasons and that lower vertebrates show various degrees of physiological control over body temperature. The most prevalent physiological mechanism for thermoregulatory adjustments in ectothermic vertebrates is cardiovascular change which allows an increase in heat gain from the environment and a minimization of heat loss. Among aquatic species the best examples are some large, fastswimming scombrid fishes (Scombroidei) and lamnid sharks (Lamnidae) which achieve a high degree of homeothermy in limited parts of the body by the strategic location of efficient heat exchangers (retia mirabilia). These retain heat produced by the powerful red muscles used for sustained swimming and by the liver and digestive organs. The high temperature of the muscles increases power output, which requires a high rate of energy supplied by digestive processes; the digestive organs, in turn, are also maintained at a higher temperature (Carey et al., 1971; Dizon and Brill, 1979). Physiological thermoregulation among amphibians has received little attention compared with reptiles. In addition to some minor vasomotor changes to control heating and cooling rates, the major physiological control in terrestrial anurans involves evaporative cooling from pulsatile releases of fluid onto the skin with some central nervous control rather than just the passive transpiration of water (Brattstrom, 1970, 1979a; Lillywhite and Licht, 1975; McClanahan et al., 1978). This evaporative cooling is a supplement to behavior, the primary means of thermoregulation in amphibians. Reptiles not only show what is perhaps the highest development of behavioral thermoregulation among ectotherms, but also display some examples of welldeveloped physiological regulation. These include major changes in heart rate and peripheral circulation to control heating and cooling rates, (Bartholomew and Lasiewski, 1965; Morgareidge and White, 1969; Kour and Hutchison. 1970; Brattstrom, 1973), jugular shunts to produce head-body temperature differences (Heath, 1966), and panting (Dawson and Templeton, 1963; Firth and Heatwole, 1975). Although large monitor lizards, such as Varanus gouldii, have resting metabolic rates equal to those of other saurians of comparable size, during activity the metabolic rate exceeds resting levels of endotherms of equivalent size (Bartholomew and Tucker, 1964; Bennett, 1972). The female Indian python {Python molurus) consumes 9.3 times as much oxygen when brooding eggs as when not brooding, thus becoming essentially endothermic. This increased heat production results from spasmodic muscular contractions reminiscent of shivering in true endotherms and produces a temperature excess of 4.7 C over the environment (Hutchison et al., 1966). This physiological production of heat, however, is expensive; after 30 days of incubation a brooding python decreased from 14.3 Kg to 10.3 Kg, a loss of almost 30%, a high energy cost needed to fuel the greatly elevated metabolic rate. Such a mechanism may have evolved to ensure reproduction, particularly in temperate regions of the species range where environmental temperatures during the brooding season may drop well below the minimum required for egg development and hatching (Vinegar et al., 1970; Vinegar, 1973). The facultative endothermy of brooding pythons and active monitor lizards demonstrates a significant degree of physiological control of thermoregulation in reptiles. This group of vertebrates thus shows a parallel development of behavioral and physiological mechanisms to control body temperatures. "That the two should proceed together is perhaps as we should expect, for it seems appropriate that physiological abilities should be geared to the requirements imposed by particular behavior patterns" (Richards, 1973, p. 127). Definition TEMPERATURE HARDENING The concept of temperature hardening

was first applied to plants, in which exposure to heat or cold near the limits of thermal tolerance increased resistance to subsequent exposures to thermal extremes. The concept has been applied to animal and plant cells by Alexandrov (1964) and co-workers, who found that the thermal limits for the cessation of protoplasmic streaming can be shifted by hardening an adaptive process by which sensitive cells are transformed into tolerant ("hardy") cells. Precht (1973, p. 419) has extended the concept of temperature hardening to whole animals and defined it as "a quick, usually transitory, adaptation to high and low temperatures." Hardening is distinct from the "resistance adaptation" produced by acclimation; hardening is a brief exposure to extreme temperatures in the lethal or near-lethal range; acclimation is a longer-lasting response to temperatures within normal ranges. We will consider only heat-hardening here, since cold-hardening has not been extensively investigated in vertebrates. It is not always easy to differentiate between heat-hardening and the fast development of resistance adaptation due to acclimation to increased temperatures. Transitory heat-hardening may result from physiological stress as a consequence of exposure to a high sublethal temperature. Heat-hardening may be reasonable or paradoxical (Precht, 1967); increased tolerance results from the ability to resist further damage, or decreased tolerance results from the stress (Precht, 1973). Some fairly good examples of heathardening, as defined by Precht, have been found among ectothermic vertebrates. Laboratory studies Hutchison (1961) observed a phenomenon in newts, S'otophthalmus viridescens, which fits Precht's (1973) definition of hardening. Newts were heated to their CTM, immediately returned to their acclimation temperature to recover for various time periods, and then reheated to the CTM. The second CTM was significantly greater than the first at the shorter recovery intervals, but the CTM has returned to BEHAVIOR AND THERMAL TOLERANCE 375 the initial level after recovery intervals of several hours. Basedow (1969), working in Precht's lab, observed similar results with Triturus vulgaris using Hutchison's (1961) method. In recent experiments (Maness and Hutchison, unpublished data) we have observed heat-hardening in a frog, Rana berlandieri, and in two species of fish, Pimephales promelas and Notropis lutrensis, in addition to the newt, Notophthalmus viridescens (Fig. 8). In all species we found a significant increase in CTM within 2 hr of exposure to the initial CTM. In all animals the CTM had returned to initial levels within 24 hr. This phenomenon is more rapid than acclimation both in onset and in return to the original state. The most rapid rate of acclimation to higher temperatures (23 to 38 C) in anurans is about 24 hr (Brattstrom and Lawrence, 1962). Basedow (1969) found rapid increases in heat resistance due to "shock transfer" (acclimation) requiring about 8 to 12 hr in the fishes Idus idus and Anguilla vulgaris. However, the most distinct difference between our heat-hardening results and acclimation is that our animals had returned to the initial levels of tolerance within 24 hr after the initial CTM. The rate of acclimation of amphibians to lower temperatures or the rate of reacclimation to lower temperatures after acclimation to high temperatures is slower than acclimation to high temperatures and in all cases requires more than 24 hr (Brattstram and Lawrence, 1962; Hutchison and Ferrance, 1970). The rates of hardening and the published rates of acclimation are thus strikingly different. However, both Precht (1973) and Alexandrov (1964) stated that the rate of acclimation is often dependent on the direction of temperature change during acclimation. Therefore, heat-hardening could simply be rapid acclimation to extreme temperatures. If heat-hardening is real, animals heated to temperatures just below lethal levels should not have significantly increased CTM. In most cases, we found that preheating to temperatures just below the CTM had no effect on the CTM determined shortly after the preheating. In those cases where preheating was followed

376 V. H. HUTCHISON AND J. D. MANESS Notroon hitrwa ivc LOBE *p<.09 39.0 38.0 1! 1 1 2 J 2 0 20 B*C LD a:a 19 *p<.09 r9 9 I G 33 j/ ( ^. 37.0-3& - * - I. I. I I. I., 1.. 1. 1 I i 1 ^ -* «. 1 0 I 2 3 4 9 6 7 8 2 4 4 S 7 2 0 1 2 3 4 5 6 7 8 24 48 72 2 I5*C LD KG p<.05 19 H9 9 \ HOURS FIG. 8. Heat hardening in two species of fishes and two amphibians acclimatized to the conditions shown. At time 0 animals were heated to the critical thermal maximum (CTM) to 1 C per minute and then returned immediately to the acclimation temperature where they were allowed to recover before they were exposed a second time to the CTM; the elapsed time between the initial and second exposure is shown on 5 6 7 B 24 48 72 HOURS the abscissa. An asterisk indicates a significant difference between an initial and repeated CTM as determined by a one-way analysis of variance (Sokal and Rohlf, 1969) and Duncan (1955) multiple range test. The horizontal line is the mean; vertical line, the range; rectangles, two standard errors of the mean; numbers at top, sample size. (Maness and Hutchison, unpublished data) by a slightly higher CTM, the increase was not as great as that due to a previous exposure to the CTM (Maness and Hutchison, unpublished data). Hutchison (1961) also found no effect of preheating on the CTM and Basedow (1969) found that preheating increased heat resistance less than exposure to the resistance temperature in newts. Alexandrov (1964) found no increase in heat resistance of cells until lethal temperatures were reached. Thus, we conclude that heat-hardening requires an exposure to temperatures in the lethal range. If a single exposure to the CTM will increase tolerance to a subsequent exposure to the CTM, successive exposures might increase tolerance further with each exposure. However, this is not the case. Multiple exposures to the CTM do not increase tolerance beyond the increase due to one exposure (Fig. 9). In fact, successive exposures may increase mortality such that the ability of the animal to recover decreases with each test (Maness and Hutchison, unpublished data). Similar results were found with Notophthalmus viridescens (Hutchison, 1961). This is consistent with the idea that heat resistance can be increased by acclimation only to some upper limit or plateau (Fry 1958, 1967; Hutchison, 1976). Because the CTM temperature is the highest temperature tolerated by an animal, even though the animal is exposed only briefly, it is reasonable to expect that if this expo-

BEHAVIOR AND THERMAL TOLERANCE 377 sure produces a change in tolerance, this change must approach a limit beyond which no further change could take place. Since thermal tolerance varies diurnally and seasonally, one would expect that heat-hardening might also vary diurnally and seasonally. While the initial CTM may not vary diurnally, there appear to be times of the day when the animal is able to increase its tolerance via hardening and times when it is not able to do so (Fig. 10). Hardening ability varies seasonally as well, being most evident in spring and early summer (Fig. 11). Evidence from laboratory data indicates that heat-hardening may be widespread among other animals; observations in the literature indicate that its existence in nature is an adaptive mechanism. Field observations Juvenile spotted salamanders, Ambystoma maculatum, sheltered under rocks near a pond increased their CTM from a low of 38.6 C in early morning to a high of 39.7 C in the afternoon on sunny days (Pough and Wilson, 1970), this phenomenon was termed "natural daily acclimation" because laboratory tests under constant conditions showed no daily cycle in CTM. However, other data suggest that what Pough and Wilson observed was heat-hardening. They found no salamaders under rocks at temperatures greater than 32 C, but in the laboratory they found that salamanders did not leave the rocks until under-rock temperatures reached as high as 36 C. Salamanders leaving the rock shelter 39 i i r i r i r i r i i i Notoptholmus vindescens 15 C LD I2II2 *p <.O5 38 O o _ 37 -if / \ 36 i i i i i i i i i I I I I I I I I I 0 12 3 4 0 15 3 4.5 6 0 8 16 24 32 0 24 48 72 96 HOURS FIG. 9. Effect of multiple exposure of red-spotted newts to the critical thermal maximum (CTM) on successive exposures. Animals were heated to the CTM five times in succession at the time intervals shown on the abscissa and were allowed to recover at the acclimation temperature (15 C) between determinations. Manner of presentation, same as in Figure 8. (Maness and Hutchison, unpublished data)

378 V. H. HUTCHISON AND J. D. MANESS 39 38 i i i I I I I r Notopthalmus viridescens I5 C LD I2M2 *p<.05 moved into a sand temperature of 38 C, a temperature very close to the morning CTM. If this situation is an accurate representation of what occurs in nature, the salamanders could very well be behaviorally utilizing heat-hardening to increase their tolerance to temperatures they might encounter if all under-rock temperatures exceed 32 C. Eastern red eftsnotophthalmus v. viridescens, occupied the warmest regions under bark even though cooler regions were readily available (Pough, 1974). The CTMs of desert pupfish, Cyprinodon macularis, tested immediately after removal from the field, were much higher than the highest CTMs attained in the laboratory after acclimation to the highest possible constant temperatures (Lowe and Heath, 1969). This difference was attributed to the cyclical nature of the natural thermal regime, and to the possibility that the fish voluntarily exposed themselves to near lethal temperatures. Fish were voluntarily active at 40-41 C, a point 2-3 C below their highest laboratory lethal temperature. This temperature is also 4-5 C higher than the highest constant temperature (36 C) at which the fish can survive long enough to become acclimated. Heat-hardening may account for these results. Lowe and Heath (1969) suggested that the fish spend brief amounts of time at the highest temperatures available and return to cooler waters for periods of recovery and repair. Otto (1974) observed western mosquitofish, Gambusia qffinis, in water at 42 C, a temperature the fish were incapable of surviving for extended periods in a laboratory study of thermal tolerance where the ultimate upper incipient lethal limit was n 1 1 1 1 r 1 1 1 1 1 1 r 36 0000 0400 0800 1200 1600 Time(CST) 2000 2400 FIG. 10. Diel variation in the critical thermal maximum (CTM) and heat hardening of red-spotted newts. Animals were tested at four-hour intervals during each 24-hour cycle. Each group was heated to the CTM, allowed to recover at the acclimation temperature (15 C) for one hour and then reheated to the CTM. Manner of presentation, same as in Figure 8. The solid line connects initial CTM values; the dashed line, repeat CTM values. (Maness and Hutchison, unpublished data)

BEHAVIOR AND THERMAL TOLERANCE 379 I5"C NotfOPiS (y_ Natural P lofopcf EOO *P< os Y1 O 60 O 60 a I 0 60 FIG. 11. Seasonal variation in the critical thermal maximum (CTM) and heat-hardening ability in a minnow. Fish were collected from a single population and maintained throughout the year under the acclimatization regime shown. At the time of year shown on the abcissa samples were heated to the CTM, allowed to recover at the acclimation temperature for one hour and then reheated to the CTM. Each individual used only once. Manner of presentation, same as in Figure 8. (Maness and Hutchison, unpublished data) 38.3 C (Otto, 1973). He suggested that this increased tolerance might be due to brief exposures to temperatures above the lethal limit. Otto (1974) exposed fish in the laboratory to various cycles and found that brief sublethal exposures to temperatures above the upper lethal limit increased the temperature tolerance of fish substantially above the levels attained by fish acclimated to both constant and cyclic temperatures below the upper lethal limit. In laboratory studies on temperature selection, fish enter water exceeding their tolerance limits for short intervals (Bacon et nl., 1967; Reynolds and Thompson, 1974); these brief forays may result in increased tolerance (Bacon et «/., 1967). In addition, exposure to these extreme temperatures may also increase upper and lower avoidance temperatures (Beitinger, 1974). Heat-hardening may be utilized by many sepcies to increase their thermal range of activity and tolerance. Habitats with extreme daily fluctuations of temperature may well contain several species which use the heat-hardening strategy (through behavioral adjustment) to increase survivability and to enhance exploitation of the environment. ENERGETICS Biological success of a species is measured by survival and continued reproduction. Sufficient energy to fuel the entropy generators required for this success is the ultimate limiting factor; efficiency of energy conversion is often the "bottom line" in the adaptation of an organism to its environment and is ultimately the major determinant of the ecological niche. The predominantly optimum temperature for physiological functions and thermal preference in the sockeye salmon is about 15 C (Brett, 1971), although the diel cycles in vertical migration (discussed above) result in the fish spending 60% of each day near the bottom of lakes at 4 to 6 C (Fig. 12). One major exception to the approximate 15 C physiological optimum for this species was a "shunt" in the optimum temperature for growth rate from 15 C on excess rations to5 Con restricted rations (Fig. 12, K->K'). These observations led Brett (1971) to hypothesize that sockeye have evolved a thermoregulatory pattern to maximize growth through the selective pressure of bioenergetic efficiency. Brett compares this "metabolic pacing" of an aquatic ectotherm (which is denied the direct source of radiation exploited by many terrestrial vertebrates) to the heterothermy of hummingbirds and bats. This "bioenergetic model" of thermoregulatory behavior favorably balances daily metabolic expense to conserve energy when food supplies are limiting. Although selection of lower temperatures by animals on restricted diets ("starvation effect," Reynolds and Casterlin, 1979«) is common, such a response does not always occur (Beitinger and Fitzpatrick, 1979; Carey, 1978). Differences in behavioral thermoregulation may aid resource partitioning in the environments of closely related species. Largemouth (Micropterus salmoides) and smallmouth blackbass (M. dolomieui) often occur together in the same bodies of water. These two congeners have diel cycles of preferred temperature such that the former species has a minimum (27.1 C) during late photophase while the latter species is at the maximum (30.1 C) of its

380 V. H. HUTCHISON AND J. D. MANESS 18 16 25' 25 22 21 DUSK FEEDING DAWN FEEDING A SURFACE. 0 5 10 14 12 10 15 CO cc LJ h- UJ I I r- Q. Ul Q 20 1\\\\W\\W\\\W\\\\\\\\\\\\\\WWW1 1200 1600 2000 2400 0400 NOON MIDNIGHT HOUR 0800 1200 NOON 25 35 45 FIG. 12. Pattern of diurnal vertical migration of sockeye salmon (Oncorhynchus nerka) in mid summer in Babine Lake, British Columbia and optimum temperatures for physiological functions. Feeding (hatched areas) near the surface occurs over two-hour periods. Dotted lines indicate general limits of the temperatures experienced by the fish. The optimum temperatures (A-F) are shown with the horizontal lines as the mean and the vertical line, ±10% response: A, thermal preference; B, active metabolic rate; C, metabolic scope; D, maximum sustained speed; E, maximum volitional speed; F, voluntary food intake (appetite); G, active cardiac work; H, resting and active blood pressures; I, growth rate (excess ration); J, conversion efficiency (all rations); K, growth rate (restricted ration) at 15 C; K 1, growth rate (restricted ration) at 5 C. (Modified from Brett, 1971, after data from McDonald published in 1973) cycle. "This thermotemporal complementarity... suggests an aspect of niche segregation" which would reduce competition for energy sources between these largely sympatric species (Reynolds and Casterlin, 1978c). The ability of many ectotherms to select low environmental temperatures during inactive periods may be highly adaptive; the maintenance of temperatures near the physiological optimum throughout each diel period with the concomitant higher metabolism and elevated requirement for energy sources would be wasteful, especially if the temperatures encountered during normal activity periods permitted sufficient digestive, reproductive, and other essential functions. Behavioral thermoregulation thus plays an essential part in the coupling of many species to their environment through exchange of energy. PROBLEMS FOR FUTURE STUDIES Of the major topics we have reviewed, perhaps the phenomenon of heat hardening is the least understood. The most basic questions still to be resolved are: (1) Can hardening be distinguished from swiftly developing resistance adaptation

BEHAVIOR AND THERMAL TOLERANCE 381 (acclimation)? (2) Can hardening be nothing more than an adaptation to shock produced by heat stress? Both of these questions are discussed by Precht (1973). Definitive answers must await detailed information on the biochemical basis for the observed phenomenon, but experiments at the organismic level could provide valuable clues. Some of the questions which suggest such experiments are: Do animals increase the rate of brief exposures to temperatures in the range of resistance as the maximum temperature of the environment increases? Does this frequency with which an animal makes brief forays into the hot end of a laboratory gradient increase as the maximum temperature or the thermal range of the gradient is increased? In a thermally cycling laboratory environment do animals display increased brief voluntary exposures to high temperatures in the "lethal" range as the mean, mode or maximum temperatures of the gradient increase, and fewer such exposures as temperatures decline? Are seasonal differences and diel cycles in heat-hardening common? Is heathardening more common and better developed in thermophilic species from hot environments? Is heat-hardening enhanced by acclimation to high temperatures? Do other environmental stressors (low O 2, for example) influence the development of heat-hardening? Perhaps most importantly, do animals in the field display behavior which clearly results in heathardening? In many cases the behavior involved in the thermal relations of ectothermic vertebrates is cyclical. These temporal variations often match the natural thermoperiods and photoperiods, but "anticipatory" responses (displays of a behavior in advance of what would be expected if the organism were responding directly to the environmental stimuli) suggest that some of these behavior patterns have an endogenous component. Carefully controlled experiments under free-running conditions may well determine if these periodic oscillations are truly circadian or circannual and would provide additional information on photoperiod, temperature and other environmental factors which serve as the Zeitgebers. Do the sensitivities or setpoints of thermoregulatory centers in the hypothalamus or elsewhere change to produce diel or seasonal cycles in thermal selection? Indeed, very little is known about the central control of physiological thermoregulation in ectotherms; much less is known about control mechanisms involved in behavioral thermoregulation. Clues to the evolution of behavioral patterns which allow ectotherms to expand their thermal ranges of activity will come from studies on additional systematic groups. There are several major groups within each class of ectothermic vertebrates where the paucity of information prevents significant generalizations about behavioral roles in the thermal relations of these animals. The most serious deterrent to a better understanding of how behavior influences the thermal ecology and physiology of these animals is the paucity of information from field studies. The value of an integration of laboratory and field studies in understanding the complex relationships between the behavior, physiology and ecology of an organism is demonstrated by the work of Brett (1971) on the sockeye salmon discussed above and that of Carey (1978) on the boreal toad, Bufo boreas. Since all ectothermic vertebrates have both behavioral and physiological means for thermoregulation, future studies should include the recognition that experimental results or field observations may result from combinations of both methods. The synergistic actions of behavioral and physiological mechanisms need further study. For example, what role does cardiovascular change play in diel cycles of thermal selection and thermal tolerance? How important are head-body vascular shunts in various types of behavioral thermoregulation? How does increased metabolic heat generated by intense activity of sustained locomotion influence thermoregulatory behavior? The complexity of interactions of behavior with abiotic and biotic factors suggests that more multifactorial experiments are required to predict the behavior of an organism in nature. In addition, thespatio-

.S82 V. H. HUTCHISON AND J. D. MANESS temporal nature of the distribution of heat and other sources of energy in the environment will require a multidimensional synthesis of laboratory and field data for a clear understanding of the thermal biology of any species. The attainment of such an objective may require many years, but the tremendous growth of knowledge in this field during the past 10 years is promising. CONCLUDING REMARKS Ectothermic vertebrates, like most organisms, inhabit environments which are heterothermal in both space and time. This spatiotemporal characteristic allows ectothcrms to exploit the environment through behavioral responses. Such behavioral regulation, aided in various degrees by physiological regulation, in turn allows organisms to extend their range of thermal tolerance and increase the environmental space within which the utilization of energy can be maximally efficient, thus increasing the odds for survival of the species. REFERENCES Alexandrov, V. 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