Preservation of reproductive behaviors during modest cooling: rapid cold-hardening fine-tunes organismal response
1 Department of Zoology, Miami University, Oxford, OH 45056, USA
2 Department of Biology, 230A Brooks Hall, Central Michigan University, Mt
Pleasant, MI 48859, USA
* Author for correspondence (e-mail: leere{at}muohio.edu)
Accepted 17 February 2004
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Summary |
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Key words: rapid cold-hardening, temperature, courtship, reproductive behavior, mating performance, metabolism, Drosophila
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Introduction |
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Originally, Lee et al.
(1987) hypothesized that RCH
allows insects to `instantaneously' enhance their cold tolerance in a
thermally variable environment. Later investigations sought to test this
hypothesis and elucidate the ecological relevance of RCH by employing more
natural temperature regimes and moderate rates of cooling
(Coulson and Bale, 1990
). For
example, Kelty and Lee (1999
)
found that cooling adult Drosophila melanogaster at low rates induced
RCH, causing flies to enter a state of chill-coma at temperatures lower than
those cooled more rapidly. Similar results were obtained using ecologically
based thermoperiodic cycles (Kelty and
Lee, 2001
). In addition, Koveos
(2001
) induced RCH in the
olive fruit fly, Bactrocera oleae, by maintaining them in outdoor
field cages overnight. Flies tested at the coolest time of the day were more
cold tolerant, based on survival rates after a 2-h exposure to 7°C,
than flies tested at the warmest part. Recently, Bale
(2002
) re-stated the ecological
relevance hypothesis, suggesting that the RCH response functions by
`resetting' the thermal thresholds for behavioral characteristics such as
flight or critical thermal minimum (CTmin), the
temperature at which insects enter a state of chill-coma.
Several investigators have argued that survival to reproduction or
post-treatment fecundity, rather than immediate survival, provides more
ecologically relevant measures of cold tolerance and RCH
(Baust and Rojas, 1985;
Coulson and Bale, 1992
;
Kelty and Lee, 1999
). In
addition to its long-term effects on female egg production
(Coulson and Bale, 1992
;
Kelty and Lee, 1999
) and on
the rate of egg fertilization (Rinehart et
al., 2000
), RCH may also have a more immediate effect on
reproductive success. Courtship and reproduction in Drosophila, as in
other insects, require a complex series of reciprocal behaviors prior to
successful mating (Spieth,
1974
). RCH is known to preserve gross neuromuscular function at
low temperature, as demonstrated by its lowering of the chill-coma temperature
(Kelty and Lee, 1999
,
2001
). Additionally, RCH
prevents decreases in the resting membrane potential, reductions in neural
conduction velocity and impairment of neuromuscular coordination that would
otherwise occur as a result of chilling
(Kelty et al., 1996
).
Therefore, it seems likely that RCH may also act to protect complex courtship
behaviors, which require a fine degree of neuromuscular control.
Despite advances in understanding the ecological relevance of RCH, the
physiological mechanism behind the process remains poorly understood
(Kelty and Lee, 1999).
Although Chen et al. (1987
)
documented modest levels of glycerol production during RCH in the flesh fly
Sarcophaga crassipalpis, Kelty and Lee
(1999
) found no changes in
glycerol, or any other sugar or polyol, levels in D. melanogaster
during RCH. Thus, the elevation of sugars or polyols is not consistently
associated with RCH, and so other factors must also play a role. Adjustments
in metabolic rate may provide clues to the underlying mechanisms responsible
for RCH. Coulson and Bale
(1990
) speculated that
compensatory shifts in the metabolic rate might account, at least in part, for
the observed effects of RCH. If so, the time required to reach a stable
metabolic rate at a lower temperature after transfer from the rearing
temperature should be less in RCH flies since the compensatory changes are
already in place. In addition, if RCH is an energy-requiring process, then RCH
flies should exhibit a higher metabolic rate at low temperatures than control
flies.
In the present study, we determined whether the RCH response preserved reproductive behaviors and courtship success of D. melanogaster during brief periods of modest cooling that would be expected to occur frequently in nature. We also tested whether RCH came at the cost of reduced reproductive performance at a higher temperature. Lastly, we determined whether RCH was associated with an elevation in metabolic rate, a response that might provide clues as to the underlying mechanism of this response.
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Materials and methods |
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Mating trials
Since a mated female often exhibits decreased receptivity to subsequent
matings (Spieth, 1974), we
used virgin flies for the mating trials. We separated male and female adults
within 56 h after pupal eclosion, before they reached sexual maturity
(Spieth, 1974
). The mating
chambers for single pairs of flies were 60 mmx15 mm petri dishes with a
layer of medium (
2 mm) that was sprinkled with a few grains of yeast. We
used a video camera placed in an incubator to observe four chambers at a
time.
In the first experiment, we compared the reproductive behavior of male and female pairs from the control and RCH groups. Control flies were transferred directly from 23°C to 16°C and their reproductive behaviors recorded for 1 himmediately after transfer. The RCH flies were given a 2-h acclimation period at 16°C, permitting them to rapidly cold-harden, before we recorded their reproductive activity for 1 h at 16°C. The males and females of the RCH group were kept segregated during this pre-treatment to be sure that they were still virgins during the mating trial.
We also performed a reciprocal study in order to determine whether the increased reproductive success exhibited by RCH flies at lower temperatures was gained at the cost of reduced mating performance and success at a higher temperature (23°C). Control flies were held continuously at 23°C. RCH flies were subjected to the cold-hardening treatment of 16°C for 2 h with the sexes separated before they were paired and returned to 23°C for a 1-h mating trial. For both experiments, we recorded whether the pair courted, the duration of each courtship event, whether the pair ultimately mated, and the courtship index, defined as the percentage of time a pair spent in courtship or mating behaviors.
Respirometry
We used carbon dioxide production as a measure of metabolic rate in D.
melanogaster (Lee and Baust,
1982; Berrigan and Partridge,
1997
). A Sable Systems (Las Vegas, NV, USA) flow-through
respirometer with a Li-Cor CO2/H2O gas analyzer
(LI-6262) was used to measure CO2 production in the flies
(Lighton, 1988
;
Berrigan and Partridge, 1997
).
The Datacan V software from Sable Systems was used to collect and analyze the
data. We were unable to achieve the sensitivity needed to discern differences
in metabolic rate at 23°C versus 16°C in individual flies.
Therefore, 10 adult flies per replicate were placed in a small plastic chamber
set inside a refrigerated bath (NESLAB RTE-8). Temperatures during all the
respirometry experiments were recorded using a copperconstantan
thermocouple inserted directly into the chamber.
The CO2 production of the control flies was measured at room
temperature (23±0.5°C) until a stable respiratory rate was
observed, typically after 30 min. To determine the effect of direct
chilling to 16°C in the control group, we then placed the chamber directly
in the cold bath, allowed CO2 production to reach a new stable rate
at 16°C, and recorded this rate and the time needed to reach a stable rate
of CO2 production. The air temperature within the chamber reached
16°C in less than 5 min after transfer. To determine the effect of RCH,
flies from the RCH group were placed in the 16°C cold bath for 2 h. Next,
we removed the chamber from the bath and held it at room temperature for
25 min, allowing it to increase to 23°C, and then returned it to
16°C. Because temperature transfer was achieved by simply immersing the
chamber in a cold bath, the respirometry system maintained a stable baseline
throughout this experiment. The flies remained rapidly cold-hardened despite
the brief exposure to 23°C (Kelty and
Lee, 2001
). We allowed CO2 production to stabilize and
then measured the metabolic rate and the time required to attain the stable
rate.
During preliminary runs, flies often lost 15% or more of their body mass
due to water loss over a span of 3 h in the dry airstream. In order to avoid
excessive dehydration in all experimental trials, we rehydrated the air by
bubbling it through a solution of 15% potassium hydroxide to humidify the
airstream while keeping the CO2 dissolved in the water in solution.
The air then passed through a condensing chamber on a thermoelectric cold
plate (TCP-2) to remove excess moisture that would form droplets on the inside
of the tubing. This also had the effect of pre-cooling the airstream during
the RCH trials. However, the thermocouple inside the chamber showed that this
did not affect the temperature during the control trials. Despite these
measures, male D. melanogaster still lost 10% of their mass
during the experiments. Therefore, female flies, which only lost about 3% of
their mass, were used in the respirometry experiments.
Statistics
All values are given as means ± S.E.M. When comparing
courtship indices, the percentage data was first transformed by taking the
arcsine of the square root of the observed index. The numbers of control and
RCH pairs that courted or mated were compared using a chi-squared analysis.
Comparison of the acclimation time in the respiration experiments was
performed using an unpaired t-test. We used analysis of variance
(ANOVA) with Bonferroni post-hoc tests to compare all other
parametric data in both the mating trials and respiration experiments.
Non-parametric data were compared using KruskalWallis with multiple
comparisons (Gibbons, 1997).
Significance for all tests was determined at P<0.05. All
statistical analyses were performed using StatView 5.0.
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Results |
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In addition, RCH pairs engaged in courtship for longer continuous periods
and spent more time overall in reproductive activities at 16°C. The mean
duration of individual courtship events of the RCH flies was significantly
longer than that of flies in the control group
(Table 1), suggesting that the
RCH pairs had greater endurance during courtship. They also had a
significantly greater courtship index than control pairs. Flies in the RCH
group spent more than half of the 1-h trial period (53.3±8.4%) either
courting or mating, whereas control flies exhibited reproductive behaviors for
only 12 min (20.6±5.7%) (Table
1).
|
We hypothesized that enhancement of courtship success at 16°C by RCH would diminish the rate of success at 23°C. To discern if RCH diminished the flies' ability to mate at a higher temperature, we tested control and RCH groups at 23°C. Pairs from the RCH group did not appear to exhibit impaired courtship abilities at 23°C, as there was no difference in the number of pairs courting or mating between the two groups (Fig. 2). Nor were there differences in event duration and courtship index (Table 1).
|
Respirometry
There was no difference in the metabolic rate at 16°C between control
D. melanogaster that did not have any prior exposure to 16°C and
flies that had rapidly cold-hardened to 16°C. In the control group
(N=5 groups of 10 flies), the rate of CO2 production was
greater at 23°C than at 16°C (P<0.0001). The respiratory
rate of the RCH group (N=6 groups of 10 flies) at 16°C after the
brief exposure (5 min) to 23°C was also lower than that of the
control flies at 23°C (P<0.0001) but was not significantly
different from the control flies at 16°C (P=0.64)
(Fig. 3). We hypothesized that
the time to reach a stable metabolic rate after the temperature transfer was
longer for control flies (51.8±11.3 min) with no prior low temperature
exposure than for flies rapidly cold-hardened to 16°C (37.6±6.0
min). However, the difference was not statistically significant
(t=1.167, P=0.27).
|
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Discussion |
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The adaptive significance of compensatory responses is an important issue
in the evolution of physiological acclimation
(Woods and Harrison, 2002).
Leroi et al. (1994
) formulated
the beneficial acclimation hypothesis (BAH), which states
"acclimation to a particular environment gives an organism a
performance advantage in that environment over another organism that has not
had the opportunity to acclimate to that particular environment."
Our results support the BAH; flies that were acclimated to 16°C had a
reproductive advantage over flies that were not given the chance to acclimate
to the lower temperature. Many other researchers, however, have found that the
BAH is not well-supported (see references in
Woods and Harrison, 2002
).
These studies have tended to subject organisms to chronic, relatively severe
stresses, whereas the flies in our experiments had a single comparatively
brief exposure to a mild temperature. The differing results of the tests of
the BAH illustrate the differences between acclimation and hardening.
Acclimation is commonly referred to as a longer-term process that prepares the
organism for more severe conditions (Menke
and Claussen, 1982
; Hoffmann
et al., 2003
). Acclimatory processes tend not to follow the BAH
since they function not to increase fitness at the acclimation temperature but
rather to increase fitness at an even lower (or higher) temperature.
Hardening, on the other hand, supports the BAH because it is a shorter-term
phenomenon that increases fitness during a usually less severe stress
(Hoffmann et al., 2003
).
When the RCH response resets the lower thermal limit for a behavioral or
physiological parameter (Bale,
2002), it may come at the cost of a corresponding decrease in the
upper thermal limit. As the sheepshead minnow, Cyprinodon variegatus,
is acclimated to temperatures from 5°C to 40°C, its
CTmax increases; concomitantly, its
CTmin increases, so that the overall thermal range of
activity remains more or less constant
(Beitinger et al., 2000
). In
addition, Layne et al. (1987
)
found that crayfish acclimated to 5°C had lower CTmin
and CTmax than crayfish acclimated to 25°C. However,
the data of Klok and Chown
(2003
) indicate that
CTmin and CTmax are decoupled in
several species of weevils. The protective effects of RCH have been
demonstrated in numerous insect species; however, few studies have
investigated its potential costs. Kelty and Lee
(1999
) found that RCH D.
melanogaster females possessed the same early fecundity as control flies.
However, RCH resulted in shorter adult life spans in the housefly, Musca
domestica (Coulson and Bale,
1992
). We hypothesized that although RCH enabled courtship and
mating performance at a lower temperature, it would come at the cost of
decreased performance at higher temperatures. Although we did not observe any
evidence of impaired reproductive activity at 23°C in flies that had been
rapidly cold-hardened to 16°C, it is possible that the temperature range
used in our experiments was too small to detect such trade-offs. Perhaps if
flies had been tested at a higher temperature, e.g. 27°C, then we might
have observed such costs.
Changes in the metabolic rate of ectotherms that are observed during
cooling are two-fold: (1) immediate, direct Q10 effects of
temperature on chemical reactions and (2) biological, compensatory adjustments
that appear over time after the change in temperature
(Bullock, 1955;
Keister and Buck, 1974
;
Clarke, 1980
). Like the
compensatory adjustments of metabolic rate that occur during temperature
acclimation, the protective effects of RCH, and therefore the underlying
physiological mechanisms, also require time to develop
(Lee et al., 1987
;
Coulson and Bale, 1990
). We
hypothesized that the metabolic rate would be elevated during RCH if energy
were required for this response. We compared the metabolic rate at 16°C of
flies with and without a 2-h pre-treatment at 16°C that would induce RCH
and the hypothesized elevation of metabolic rate. However, no differences that
would suggest biological, compensatory adjustments were found
(Fig. 3), and the times
required to reach a stable metabolic rate did not differ between the control
and RCH groups. These results suggest that RCH is not associated with a
notable increase in the metabolic rate. Our results are consistent with the
findings of Misener et al.
(2001
), which suggest that the
RCH response does not require the synthesis of a new suite of proteins. They
found that the inhibition of protein synthesis by cycloheximide did not
inhibit RCH. The precise physiological mechanism of RCH remains unknown,
although these results suggest that it does not require a substantial energy
input for its induction or maintenance.
It is becoming increasingly evident that the RCH response represents a
constant fine-tuning of the physiological function of an insect to match
environmental conditions. Previously, investigators hypothesized that RCH
allows an insect's overall cold tolerance to track rapid environmental
temperature shifts, especially during the spring and autumn months when
diurnal temperature extremes are most dramatic
(Lee et al., 1987;
Coulson and Bale, 1990
;
Kelty and Lee, 1999
). However,
Kelty and Lee (2001
) showed
that cooling D. melanogaster from 23°C to 16°C induces an RCH
response that increases sub-zero survival. Finally, in the present study we
demonstrated that RCH preserved reproductive behaviors during a decrease of
only 7°C to 16°C, which represents the highest reported temperature at
which the protective effects of RCH are evident. As such, this strongly
suggests that the RCH response is much more pervasive and subtle than
previously thought. In addition to operating over diurnal patterns of warming
and cooling (Kelty and Lee,
2001
), RCH may also be induced as an insect experiences slight
variations in temperature that occur during a single afternoon or while moving
from sunlight to shade.
Minute organisms, including small insects, experience the external
environment on a very fine scale, spatially and temporally. Because of their
small size and correspondingly large surface area to volume ratio, the
internal body temperature closely tracks environmental temperature. Since they
lack the thermal inertia of large animals, their body temperature changes
rapidly in response to small, momentary variations in the environmental
temperature. For example, Willmer
(1986) found that the
temperature on the upper surface of a leaf can decrease from 23°C to
16°C in 2 h during a summer afternoon. In addition, temperatures just 3 cm
above a leaf may be as much as 7°C higher than those at the leaf surface
(Willmer, 1982
). Thus, insects
in nature routinely experience the temperature change used in our experiments
during a summer afternoon, or even while landing on a leaf.
The RCH response probably permits small insects like D.
melanogaster to quickly improve their behavioral performance in response
to even slight changes in environmental temperature. This distinguishes it
from the acclimation responses that do not necessarily benefit the organism at
the acclimation temperature but instead prepare it for more severe
temperatures (Hoffmann et al.,
2003). Consequently, the term rapid cold-hardening is, in a sense,
a misnomer because it is too restrictive to encompass the wide range of
temperature changes that elicit this rapid acclimation response. Insects
probably rapidly cold-harden in response to thermal variations in the
mid-summer that can hardly be considered `cold' in the same way that
5°C is `cold'. The RCH response is not a mechanism that is used
only on an occasional basis, during a sudden cold snap, but rather a much more
common process that occurs at relatively high temperatures and in response to
slight thermal changes in active insects throughout the year. The term rapid
cold-hardening places the conceptual emphasis on the prevention of cold injury
rather than on a continual fine-tuning of physiological function to match
small changes in environmental temperature. Our data further support the idea
that insects continuously make rapid, subtle acclimatory adjustments to very
minor thermal changes (Lee et al.,
1987
; Kelty and Lee,
2001
).
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Acknowledgments |
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References |
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---|
Bale, J. S. (2002). Insects and low temperatures: from molecular biology to distributions and abundance. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357,849 -862.[CrossRef][Medline]
Baust, J. G. and Rojas, R. R. (1985). Insect cold hardiness: facts and fancy. J. Insect Physiol. 31,755 -759.[CrossRef]
Beitinger, T. L., Bennett, W. A. and McCauley, R. W. (2000). Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature. Env. Biol. Fishes 58,237 -275.[CrossRef]
Berrigan, D. and Partridge, L. (1997). Influence of temperature and activity on the metabolic rate of adult Drosophila melanogaster. Comp. Biochem. Physiol. A 118,1301 -1307.[CrossRef]
Bullock, T. H. (1955). Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30,311 -341.
Burks, C. S. and Hagstrum, D. W. (1999). Rapid cold-hardening capacity in five species of coleopteran pests of stored grain. J. Stored Prod. Res. 35,65 -75.[CrossRef]
Chen, C.-P., Denlinger, D. L. and Lee, R. E. (1987). Cold-shock injury and rapid cold-hardening in the flesh fly Sarcophaga crassipalpis. Physiol. Zool. 60,297 -304.
Clarke, A. (1980). A reappraisal of the concept of metabolic cold adaptation in polar marine invertebrates. In Ecology in the Antarctic (ed. W. N. Bonner and R. J. Berry), pp. 77-92. London: Academic Press.
Coulson, S. J. and Bale, J. S. (1990). Characterization and limitation of the rapid cold-hardening response in the house fly Musca domestica (Diptera: Muscidae). J. Insect Physiol. 36,207 -211.[CrossRef]
Coulson, S. J. and Bale, J. S. (1992). Effect of rapid cold-hardening on reproduction and survival of offspring in the house fly Musca domestica. J. Insect Physiol. 38,421 -424.[CrossRef]
Czajka, M. C. and Lee, R. E. (1990). A rapid cold-hardening response protecting against cold shock injury in Drosophila melanogaster. J. Exp. Biol. 148,245 -254.[Abstract]
Gibbons, J. D. (1997). Nonparametric Methods for Quantitative Analysis. Third edition. Columbus: American Sciences Press, Inc.
Hoffmann, A. A., Sørensen, J. G. and Loeschcke, V. (2003). Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. J. Therm. Biol. 28,175 -216.[CrossRef]
Keister, M. and Buck, J. (1974). Respiration: some exogenous and endogenous effects on rate of respiration. In Physiology of the Insecta, vol.6 , second edition (ed. M. Rockstein), pp.469 -509. New York: Academic Press.
Kelty, J. D. and Lee, R. E. (1999). Induction of rapid cold-hardening by cooling at ecologically relevant rates in Drosophila melanogaster. J. Insect Physiol. 45,719 -726.[CrossRef][Medline]
Kelty, J. D. and Lee, R. E. (2001). Rapid
cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae)
during ecologically based thermoperiodic cycles. J. Exp.
Biol. 204,1659
-1666.
Kelty, J. D., Killian, K. A. and Lee, R. E. (1996). Cold shock and rapid cold-hardening of pharate adult flesh flies (Sarcophaga crassipalpis): effects on behavior and neuromuscular function following eclosion. Physiol. Entomol. 21,283 -288.
Klok, C. J. and Chown, S. L. (2003). Resistance to temperature extremes in sub-Antarctic weevils: interspecific variation, population differentiation and acclimation. Biol. J. Linn. Soc. 78,401 -414.[CrossRef]
Koveos, D. S. (2001). Rapid cold-hardening in the olive fruit fly Bactrocera oleae under laboratory and field conditions. Entomol. Exp. Appl. 101,257 -263.
Layne, J. R., Claussen, D. L. and Manis, M. L. (1987). Effects of acclimation temperature, season, and time of day on the critical thermal maxima and minima of the crayfish Orconectes rusticus. J. Therm. Biol. 12,183 -187.[CrossRef]
Lee, R. E. and Baust, J. G. (1982). Respiratory metabolism of the Antarctic tick, Ixodes uriae. Comp. Biochem. Physiol. A 72,167 -171.[CrossRef]
Lee, R. E., Chen, C.-P. and Denlinger, D. L. (1987). A rapid cold-hardening process in insects. Science 238,1415 -1417.
Leroi, A. M., Bennett, A. F. and Lenski, R. E. (1994). Temperature acclimation and competitive fitness: an experimental test of the beneficial acclimation assumption. Proc. Natl. Acad. Sci. USA 91,1917 -1921.[Abstract]
Lighton, J. R. B. (1988). Discontinuous CO2 emission in a small insect, the formicine ant Camponotus vicinus. J. Exp. Biol. 134,363 -376.
Menke, M. and Claussen, D. (1982). Thermal acclimation and hardening in tadpoles of the bullfrog Rana catesbeiana.J. Therm. Biol. 7,215 -219.[CrossRef]
Misener, S. R., Chen, C.-P. and Walker, V. K. (2001). Cold tolerance and proline metabolic gene expression in Drosophila melanogaster. J. Insect Physiol. 47,393 -400.[CrossRef][Medline]
Rinehart, J. P., Yocum, G. D. and Denlinger, D. L. (2000). Thermotolerance and rapid cold-hardening ameliorate the negative effects of brief exposures to high or low temperatures on fecundity in the flesh fly, Sarcophaga crassipalpis. Physiol. Entomol. 25,330 -336.[CrossRef]
Spieth, H. T. (1974). Courtship behavior in Drosophila. Annu. Rev. Entomol. 19,385 -405.[CrossRef][Medline]
Willmer, P. G. (1982). Microclimate and the environmental physiology of insects. Adv. Insect Physiol. 16,1 -57.
Willmer, P. G. (1986). Microclimatic effects on insects at the plant surface. In Insects and the Plant Surface (ed. B. Juniper and R. Southwood), pp.65 -80. Oxford: Edward Arnold.
Woods, H. A. and Harrison, J. F. (2002). Interpreting rejections of the beneficial acclimation hypothesis: when is physiological plasticity adaptive? Evolution 56,1863 -1866.[Medline]
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