Am J Physiol Regul Integr Comp Physiol 301: R1R14, 2011.

First published March 23, 2010; doi:10.1152/ajpregu.00719.2010. Review

Comparative physiology: a crystal ball for predicting consequences

of global change
George N. Somero
Hopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, California
Submitted 1 November 2010; accepted in final form 22 March 2011

Somero GN. Comparative physiology: a crystal ball for predicting conse-

quences of global change. Am J Physiol Regul Integr Comp Physiol 301: R1R14,
2011. First published March 23, 2010; doi:10.1152/ajpregu.00719.2010.Com-
parative physiology offers powerful approaches for developing causal, mechanistic
explanations of shifts in biogeographic patterning occurring in concert with global

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change. These analyses can identify the cellular loci and intensities of stress-
induced perturbation and generate predictions about ecosystem alterations in a
changing world. Congeneric species adapted to different abiotic conditions offer
excellent study systems for these purposes. Several findings have emerged from
such comparative studies: 1) In aquatic and terrestrial habitats, the most heat-
tolerant ectotherms may be most threatened by further increases in temperature, due
to proximity of these species thermal optima and tolerance limits to current
maximal ambient temperatures and limited capacities for acclimatization to higher
temperatures. 2) Cardiac function is a weak link in acute thermal tolerance.
3) Stress-induced changes in gene expression comprise a graded response involving
genes linked to damage repair, lysis of irreversibly damaged molecules, and
downregulation of cell proliferation. Transcriptomic and proteomic analyses pro-
vide biomarkers for diagnosing degrees of stress. 4) Different abiotic stresses
may have synergistic or opposing effects on gene expression, a complexity needing
consideration when developing integrated pictures of effects of global change.
5) Adaptation of proteins can result from one to a few amino acid substitutions,
which can occur at many sites in a protein, a discovery with implications for rates
of adaptive evolution. 6) Greater thermal tolerance of invasive species may favor
their replacement of natives. 7) Losses of protein-coding genes and temperature-
responsive gene regulatory abilities in stenothermal ectotherms of the Southern
Ocean may lead to broad extinctions.
adaptation; climate change; gene expression; invasive species; transcriptomics

COMPARATIVE PHYSIOLOGY HAS a long and distinguished history,
which reaches back to the foundational contributions of schol-
ars like Claude Bernard, Christian Bohr, and August Krogh.
Knut Schmidt-Nielsen (59) may have provided the most suc-
cinct statement of the overarching goals of our field, which are
to elucidate the fundamental mechanisms that explain how
animals work and to reveal how the workings of these
physiological systems have evolved to adapt organisms to a
wide range of environmental conditions (31). These two con-
tributions of our field are coming to be increasingly appreciated
as offering a relevant means for understanding how global
change is likely to affect different species and, thereby, the
structures and interactions of the ecosystems of which they are
members. Through understanding the underlying mechanistic
bases of sublethal and lethal stress and the differences that exist
among species in capacities to respond to these stresses, a
foundation can be constructed for developing predictions about
the probable success or failure of organisms to cope with
changes in physiologically significant abiotic factors like tem-
Address for reprint requests and other correspondence: G. N. Somero, 120
Ocean View Blvd., Hopkins Marine Station, Dept. of Biology, Stanford Univ.,
Pacific Grove, CA 93950-3094 (e-mail: somero@stanford.edu).
http://www.ajpregu.org 0363-6119/11 Copyright 2011 the American Physiological Society
perature, salinity, oxygen availability, and pH, all of which are
aspects of global change.
Comprehensive analyses designed to provide a realistic
assessment of the effects of global change require examination
of a broad set of phenomena, ranging from studies of behav-
ioral regulation of body temperature to molecular level pro-
cesses, such as the gene regulatory events that comprise the
cellular stress response (CSR) (39) and modulate many of the
processes that underlie adaptive phenotypic plasticity. In this
review, I take a reductionist approach and focus largely on
cellular- and molecular-level processes, primarily those asso-
ciated with the CSR, which have received less discussion in the
context of global change than have processes at higher levels of
biological organization (20, 24, 5254). The primary rationale
for this presentational focus, however, is based on a wish to
provide the most general treatment possible of how cellular-
and molecular-level aspects of physiology play out in the arena
of global change. Thus, a focus on the widely occurring CSR
is appropriate. Here, wide refers to two very different aspects
of the CSR. First, many different types of cellular stress,
including stress from temperature, oxygen availability [and
production of reactive oxygen species (ROS)], osmolality, and
acidity, lead to common types of cellular damage and, there-
R1
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R2 COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
fore, trigger a common set of stress-related responses, the CSR
(39). If successful, the CSR can sufficiently offset the damag-
ing effects of stress to allow other processes to restore cellular
homeostasis (the cellular homeostasis response, or CHR) (39).
The second wide aspect of the CSR relates to its breadth of
occurrence among organisms. The CSR is observed across all
taxa (39); it is a fundamental property of almost all cells that
has been strongly conserved during evolution. However, as
discussed in the context of extreme stenotherms, long-term
evolution in stable habitats may lead to loss of key elements of
the CSR, which places these organisms in extreme jeopardy
from global change (10).
I hope that this review will serve to complement other recent
analyses of physiological aspects of global change biology (20,
24, 5254, 68) and help to further establish a pivotal role of
reductionist, mechanistic analysis in guiding our understanding
of one of the greatest challenges ever to confront our planets
ecosystems.
Congeners: A Kroghian Crystal Ball for Studying
Adaptive Physiological Variation and Global Change
As is commonly the case in comparative physiology, anal-
yses of the physiological aspects of global change biology
benefit from adherence to the well-known August Krogh Prin-
ciple, here as stated succinctly by Sir Hans Krebs: For many
problems there is an animal on which it can be most conve-
niently studied. (38). Comparative analyses of environmental
sensitivities and capacities for erecting adaptive responses,
either through genetic change or by altered use of existing
genetic information, have been especially effective when
closely related congeneric species adapted to different abiotic
environmental conditions have been examined. Congeners af-
ford several key experimental advantages (31). They allow
adaptive variation to be clearly distinguished from the effects
of phylogeny per se. They provide insights into how much
environmental change is sufficient to favor adaptive change in
diverse physiological systems. Congeners relatively low lev-
els of evolutionary divergence facilitate analysis of how much
genetic variation is needed to alter environmental optima and
tolerance limits, including variation in gene regulatory capac-
ities and protein amino acid sequence. The existence of suites
of congeners with different environmental optima enables
predictions to be made about local extinctions of species and
their potential replacement with a congener with different and
more appropriate environmental tolerances. In some cases, this
replacement may represent the introduction of an invasive
congener, with potentially large effects on the ecosystem
overall. Thus, comparative studies of congeners have enabled
an integrated analysis that spans all levels of biological orga-
nization, from the population to the level of fine-scale adaptive
molecular variation.
Below, I illustrate the power of such comparative analyses to
shed light on several key questions related to the impacts of
global change on the biosphere. Studies performed with the
comparative approach, including experiments that exploit new
omics technology imported from biomedical science, can
indeed provide biologists (and policy makers as well) with an
effective crystal ball for predicting the effects of global
change on individual organisms and ecosystems.
AJP-Regul Integr Comp Physiol VOL
Biogeographic Patterning Correlated with Global Change: A
Challenge for a Cause-Effect Physiological Analysis
Changes in biogeographic patterning, putatively caused by
global change, have been documented in dozens of studies
during the past two decades (for reviews, see Refs. 48, 57, and
76). These pronounced changes in organisms latitudinal and
vertical distribution patterns are widespread in both terrestrial
and marine environments and involve a wide array of taxa,
both ectotherms and endotherms. In terrestrial ecosystems,
range expansions or contractions of 6 25 km per decade are
common (48). Recent studies of lizards have revealed dramatic
shifts in distribution and extremely high probabilities of ex-
tinction (35, 63). For example, it is estimated that by 2080,
local extinctions of lizards could reach 39% worldwide, and
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species extinctions may reach 20% (63). In marine rocky
intertidal habitats, which figure importantly in the discussion to
follow, especially rapid shifts have been observed, between 49
and 540 km per decade (37). For one dominant rocky intertidal
species in the Western Atlantic, the mussel Mytilus edulis, the
southern range has contracted by 350 km over the past 47
years (7.5 km per year) as air- and water temperatures have
risen (37). In the Eastern Pacific, the rapid changes observed in
the biogeographic limits of native and invasive blue mussels,
Mytilus trossulus and Mytilus galloprovincialis, respectively,
are correlated with shifts in ocean temperature (30). Fortu-
nately for investigators attempting to explain such trends at the
physiological level, the genus Mytilus has been a popular study
system, and it affords excellent insights into causal mecha-
nisms behind biogeographic change, as illustrated later in this
review. Many biogeographic changes also have been found in
pelagic marine habitats. Ranges of North Atlantic plankton
species have shifted northward by 10 of latitude since the
1960s (2). In the North Sea, 87% of demersal fish species
studied extended their northern range limits and 50% of north-
ern species either contracted their southern distribution limits
or migrated to greater depths (49).
These and many other examples of shifts in biogeographic
ranges in a changing world challenge comparative physiolo-
gists to provide a mechanistic explanation that can help to
account for these observed changes and serve as a strong
foundation for developing predictions about future shifts in
distribution patterns and ecosystem structure. Such explanatory
and predictive analyses need to examine several related ques-
tions. First, what are the physiological mechanisms that are
responsible for acute lethality, as might occur during periods of
one to a few days of extreme heating? Second, what types of
physiological effects of sublethal thermal stress might so un-
dermine an organisms physiological condition as to preclude
its continued existence in a habitat? Do the effects of sublethal
stress occur in a graded manner that physiological methods can
identify, thereby enabling physiologists to generate a useful
suite of diagnostic biomarkers for assessing the level of
stress an organism is confronting? Third, how do species differ
in potential for acclimatizing to global change? Do some
species have more complete genetic tool kits for effecting
these phenotypic responses? Fourth, what types of changes in
the genomein both protein-coding genes and gene regulatory
elementsare needed to evolve enhanced physiological toler-
ance to abiotic stress? Do these evolutionary changes in the
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COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
genome appear feasible, in light of the rapidity of global
change?
Organisms Differ in Susceptibility to Global Warming:
Patterns and Causal Mechanisms
The primary focus of this review is on the effects of rising
temperatures (global warming), although the interactions of
temperature with other abiotic factors likely to vary with global
change, notably salinity and oxygen concentration, will be
considered briefly as well. Emphasis will be placed primarily
on ectothermic species, which have received the greatest
amount of attention by biogeographers and physiologists in-
terested in effects of global warming. However, its pertinent to
note that recent analyses and modeling efforts predict severe
effects of global warming on endothermic homeotherms, mam-
mals, and birds. Thus, for mammals, including many human
populations, an increasing fraction of the globe is predicted to
become inimical to habitation as the planet continues to warm
(45, 61). It is estimated that when wet-bulb temperatures reach
or exceed 35C, many low- to mid-latitude regions that cur-
rently support a large fraction of the human population will no
longer be hospitable to humans and other mammals.
Studies of ectotherms also have pointed to high probabilities
of risk of extinction at low latitudes (14, 7275), and also to
varying threats from warming among congeners from a single
latitude that have different vertical zonation patterns (74).
Latitudinal and vertical comparisons have led to an important
generalization: the most warm-adapted (heat-tolerant) species
may be in greatest danger from further increases in environ-
mental temperature. This seemingly paradoxical conclusion
has been reached in broad surveys of terrestrial species from
different latitudes (14, 75) and in analyses of congeners of
marine invertebrates found at different vertical positions in the
intertidal zone and at different latitudes (7174). The surveys
of terrestrial species point to a much higher susceptibility to
extinction in tropical regions than in higher-latitude habitats
because tropical ectotherms currently live much closer to
temperatures that exceed their thermal optima than related
species from higher latitudes. Warm-adapted species also may
have lesser abilities to acclimate to increases in temperature,
relative to more cold-adapted species. Furthermore, because
warm-adapted species may have higher metabolic rates, ther-
mal acceleration of metabolism by rising temperatures has a
higher absolute effect on rates of metabolic turnover than in
cold-adapted species with intrinsically lower rates of metabo-
lism (16). Some implications of these differential Q10 effects
are discussed later.
These same conclusions about relative susceptibilities of
different species to global warming have been reached in
studies of congeneric marine arthropods and mollusks, where
the underlying mechanistic bases of these differential thermal
sensitivities have been examined in some detail. The most
extensive data set on thermal tolerance differences among
congeners has come from studies of marine porcelain crabs
(genus Petrolisthes) from subtidal and intertidal habitats at
different latitudes (7274). Congeners of Petrolisthes manifest
a classical pattern of adaptive variation in heat tolerance.
Congeners from low-latitude (tropical) habitats and those oc-
curring at higher sites along the subtidal-to-intertidal vertical
gradient at a single latitude exhibit the greatest tolerance of
AJP-Regul Integr Comp Physiol VOL
Review
R3
high temperatures. However, the most heat-tolerant porcelain
crabs are more likely than their less heat-tolerant congeners to
experience maximal habitat temperatures that equal or exceed
their thermal tolerance limits. In addition, the most warm-
adapted congeners exhibited the least ability to increase heat
tolerance through acclimation (73).
Studies of marine turban snails of the genus Chlorostoma
(formerly Tegula) show additional aspects of the types of
variation in thermal responses that occur among congeners
with different distribution patterns (Table 1). Three congeners
of Chlorostoma are common in rocky shore habitats along the
midlatitude coastline of the Eastern Pacific Ocean (77, 79). The
black turban snail (C. funebralis) is found in the low- to
mid-intertidal zone from Vancouver Island, Canada (48N) to
central Baja California, Mexico (28N). Chlorostoma brunnea
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occurs in subtidal to low-intertidal zones from Cape Arago, OR
(43N) to the Santa Barbara Channel Islands (34N). Chloros-
toma montereyi occurs almost exclusively in subtidal habitats
and ranges from Sonoma County, CA (38N) to the Santa
Barbara Channel Islands. The three congeners differ signifi-
cantly in upper lethal temperature (64, 71, 77), in accord with
their different vertical positions and biogeographic ranges; C.
funebralis withstands body temperatures 5 6C higher than
the two lower-occurring congeners.
Studies of cardiac function at different temperatures have
provided evidence for one proximate cause of these differences
in thermal tolerance limits and distribution ranges. As in the
case of porcelain crabs (72), congeners of Chlorostoma differ
substantially in tolerance of cardiac function to both high and
low extremes of temperature (71). Warm- and cold-adapted
congeners of these genera also differ in the proximity of
contemporary body temperatures to temperatures at which
heart failure occurs and in the capacities for increasing heat
tolerance of heart function through warm acclimation. These
relationships are illustrated for congeners of Chlorostoma in
Table 1.
Considering first the thermal tolerances of heart function in
field-acclimatized specimens, there is a 6 7C difference
among species in CTmax, the temperature at which a sharp
break (change in sign of the slope) occurs in an Arrhenius plot
[ln heart rate in beats per minute (bpm) vs. reciprocal of
Table 1. CTmax and CTmin (C) of hearts of congeners of
Chlorostoma: effects of acclimation and acclimatization
C. funebralis C. brunnea C. montereyi
CTmax
Field-acclimatized specimens
31.0 0.7 25.0 0.5 24.2 0.7
Laboratory-acclimated specimens14C
28.5 0.5 20.2 0.8 21.7 0.8
Laboratory-acclimated specimens22C
30.1 0.7 26.8 0.7 25.7 0.6
CTmin
2.1 0.2 3.5 0.2 4.8 0.5
Snails were laboratory acclimated for 15-19 days. CTmax is the temperature
at which a break in the slope of an Arrhenius plot (ln heart rate in beats per
minute (bpm) vs. reciprocal of temperature in Kelvins) occurred during the
heat ramp. CTmin is the temperature at which cessation of heart beat occurred
during a decrease in temperature. Data are given as means SE. Data are from
Stenseng et al. (71).
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R4 COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
temperature in Kelvins]. For the two lower occurring conge-
ners, the CTmax occurs at temperatures well above the maximal
temperatures that the species are likely to encounter in their
low-intertidal and subtidal habitats, 22C (79). However, for
C. funebralis, which has a CTmax near 31C in field-acclima-
tized specimens, field body temperatures as high as 34.5C
have been reported (78), and temperatures between 27C and
33C are common during low tides (79). Thus, unlike its two
lower-occurring congeners, in its normal habitat, C. funebralis
faces challenges to cardiac function because of a rapid fall in
heart rate above CTmax.
Acclimation studies revealed additional differences between
C. funebralis and the two lower-occurring congeners. Whereas
all three congeners exhibited an ability to increase CTmax when
acclimation temperature was increased from 14C (a common
ambient water temperature) to 22C (the highest temperature
recorded for C. brunnea), the increase in CTmax differed
significantly among species. C. funebralis was able to increase
its CTmax by only 1.6C, but C. brunnea and C. montereyi
increased CTmax by 6.6C and 4C, respectively (71). Thus, as
in the case of congeners of Petrolisthes, the most warm-
adapted species not only have CTmax values close to current
peak body temperatures, but also have a relatively limited
ability to further increase CTmax through acclimation. These
findings on two phylogenetically distantly related sets of con-
geners suggest an important conclusion: acclimation (acclima-
tization) to rising temperatures is not likely to ameliorate
substantially the effects of global warming in those species
most threatened by further increase in body temperature.
Further aspects of the studies of congeners of Chlorostoma
merit comment. First, as shown in Table 1, the field-acclima-
tized CTmax values for the two lower-occurring congeners are
more similar to the 22C values than the 14C ( ambient
water temperature) values. This suggests that even limited-
duration exposures during low tides led to an acclimatization
response that resembles the warm-acclimation response. Sec-
ond, as in the case of porcelain crabs (72), the low- to
mid-intertidal species has a greater tolerance of low as well as
high temperatures; it is more eurythermal than its lower-
occurring relatives (Table 1). This difference likely reflects the
colder temperatures experienced during prolonged emersion in
winter. Third, as shown by Tomanek and Sanford (78), levels
of heat-shock protein 70 (HSP70) in the lower-occurring con-
geners are significantly lower than those in C. funebralis in
field-acclimatized specimens. This finding suggests that field
body temperatures for the most heat tolerant of the three
congeners exceed the threshold induction temperature for
genes encoding HSP70 but that this may not be the case for C.
brunnea or C. montereyi. This conclusion is supported by
metabolic labeling studies with the three species, which
showed induction temperatures for HSP70 synthesis near 24C
for the two lower-occurring congeners, but near 27C for C.
funebralis (79). The metabolic labeling studies showed signif-
icant interspecific differences in the thermal optima and max-
ima for protein synthesis as well. Incorporation of 35S methi-
onine cysteine into newly synthesized proteins peaked near
27C in 13C-acclimated C. brunnea and C. montereyi and
near 33C in C. funebralis (79). Cessation of protein synthesis
occurred near 33C in the two lower-occurring congeners and
near 38C in C. funebralis. Thus, at field temperatures com-
monly encountered by C. funebralis, neither of the other
AJP-Regul Integr Comp Physiol VOL
congeners would be capable of synthesizing proteins or sus-
taining optimal cardiac function. In fact, when heated at rates
that simulate the rise in body temperature that occurs during
emersion (4C/h), the two lower-occurring congeners died
near 33C. Nonetheless, because of their occurrence in habitats
where temperatures rarely exceed 20C, C. brunnea and C.
montereyi are much less threatened by elevated temperatures
than C. funebralis.
A final difference worth noting between C. funebralis and
the other two congeners is a difference in heart rate (bpm) at a
common temperature of measurement (13C) (71). Hearts of
C. funebralis, C. brunnea, and C. montereyi of similar body
mass had heart rates of 6.8 1.3 bpm, 2.0 0.3 bpm, and 3.6
0.4 bpm, respectively. Because rates of oxygen consumption in
marine mollusks correlate with heart rate (58), the higher heart
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rate of C. funebralis has been conjectured to reflect higher
energy demands, relative to those of its congeners, resulting
from the abiotic conditions it encounters in its low- to mid-
intertidal habitat (64). And, as emphasized by Dillon et al. (16),
because effects of temperature on metabolism are exponential,
rising temperatures will have a greater absolute effect on
metabolic rates in species like C. funebralis that already have
relatively high rates of metabolism.
Differences in cardiac responses to change in temperature
may also play a role in governing the success of biological
invasions. In the case of native (Mytilus trossulus) and invasive
(Mytilus galloprovincialis) blue mussels on the coast of Cali-
fornia, the CTmax of heart function is 23.70 0.80C for the
native species and 28.30 1.08C for the invasive (specimens
common-gardened acclimated to 14C and 28 ppt salinity) (5).
Acclimation to 21C led to increases in CTmax to 26.00
0.59C and 30.70 1.04C in the native and invasive species,
respectively. Difference in the heat tolerance of cardiac func-
tion is but one of the physiological differences between these
two blue mussel congeners that may help to explain the marked
success of the invasive in replacing the native species over the
southern portion of its former biogeographic range (5). The
competitive success of the invasive M. galloprovincialis at
high temperatures (60) is an example of what has been pro-
posed to be a common pattern wherein elevated temperatures
enhance species invasions due to a suite of physiological and
ecological factors (69, 70).
A species of blue mussel native to the East Coast of the
United States, M edulis, exhibited a sensitivity of cardiac
function to heat stress that was intermediate between those of
the two California blue mussels (5). For 14C-acclimated M.
edulis, CTmax was 25.50 0.99C; for 21C-acclimated spec-
imens, CTmax rose to 28.50 0.51C. The high rates of
mortality of M. edulis observed in the field following expo-
sures to temperatures of 32C and the associated rapid con-
traction of the southern distribution limit of this species
biogeographic range as air and water temperatures have in-
creased during the past few decades (37), may find at least a
partial explanation in the thermal sensitivity of this species
cardiac function, which, even in warm-acclimated specimens,
is compromised at temperatures above 28 29C (5).
Another significant difference in cardiac performance among
the three congeners of blue mussels is in the resting heart rates of
the species. For M. trossulus, M. edulis, and M. galloprovin-
cialis, resting heart rates (bpm) at 14C were 25.91 1.17,
21.30 0.87, and 17.53 0.85 bpm, respectively (5). These
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COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
differences in heart rate may reflect a temperature-compensa-
tory adaptation in that rates are highest in the most cold-
adapted species and lowest in the most warm-adapted conge-
ner, the invasive. Activities of enzymes associated with aerobic
and anaerobic supply of ATP are higher in tissues of M.
trossulus than in M. galloprovincialis (41), consistent with
temperature compensation. The higher intrinsic metabolism of
the native species may impose challenges at high temperatures
if metabolism rises to values that cannot be supported by
substrate supply or end-product removal. Also, because of the
exponential rise in physiological rates with increasing temper-
ature, thermal acceleration of metabolism will be greater in an
absolute sense for M. trossulus because of its higher intrinsic
metabolic rate relative to its congeners. Suffice it to say,
cardiac function and metabolic capacities of differently ther-
mally adapted congeners may play important roles in estab-
lishing environmental optima, tolerance limits, and, thereby,
biogeographic patterning. The findings that recent cooling of
the Eastern Pacific has been accompanied by a southward shift
in the northern distribution limit of M. galloprovincialis (30)
and that warming in the Western Atlantic has led to a north-
ward shift in the southern limit of M. edulis (37) provide
support to this conjecture.
Sublethal Thermal Stress and Its Consequences for Aerobic
Metabolic Processes
The most prevalent threats from global change, especially
from rising temperatures, are likely to be sublethal effects that,
while not proving lethal to the individual in the short run, as
acute cardiac failure might be, nonetheless significantly com-
promise the organisms performance in such critical processes
as locomotory activity, growth, and reproduction. These com-
promised abilities to sustain a healthy population at a site may
lead to local extinctions and to latitudinal range shifts.
Shortfalls in oxygen delivery have been emphasized as a
critical element in sublethal thermal stress (20, 5254), as well
as in the context of acute death at thermal extremes due to
cardiac failure, as discussed above. There is a wealth of
evidence showing that reductions in oxygen delivery capacity
at thermal extremes may so impair an animals aerobic scope
(difference between minimal and maximal rates of oxygen
consumption) as to reduce its chances for persistence in a
habitat (5254). Reduced locomotory performance, which is
key in predator-prey interactions, may be a major physiological
consequence of heat stress. And, as in the case of acute cardiac
failure that occurs when temperatures exceed the CTmax of
heart function, species may differ in terms of the proximity of
current habitat temperatures to the temperatures at which
aerobic scope is compromised. In both instances, tropical
species appear to be more threatened by global warming than
species from higher latitudes. For example, Nilsson and col-
leagues (47) reported that tropical cardinal fishes lost almost
50% of their aerobic scope when water temperatures were
raised only 2C above current summer average temperatures.
Similarly, tropical terrestrial ectotherms may be living closer to
their thermal limits for optimal metabolic performance than
species from higher latitudes (14, 75).
In the broad context of temperature-induced changes in
metabolic rate and in organisms capacities for sustaining
adequate aerobic production of ATP for different physiological
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Review
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activities, it is important to consider the fact that allocation of
available ATP production among different processes may be
temperature dependent. Temperature changes thus affect me-
tabolism both quantitatively (Q10 effects on total rates) and
qualitatively (relative energy needs of different processes). For
example, if cellular damage at higher temperatures increases
needs for aerobic ATP turnover, the combination of reduced
oxygen solubility for aquatic species (solubility falls by 2%
per degree Celsius rise in temperature), reduced potential for
oxygen delivery, and rising oxygen demands for processes
linked to the CSR may confront an organism with a combina-
tion of problems that lead to suboptimal physiological perfor-
mance and, at the extreme, to local extinction of a species.
Furthermore, increased oxygen consumption is likely to ele-
vate production of reactive oxygen species (ROS), which are
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damaging to the cell and lead to rising energy demands for
repair or replacement of damaged cellular constituents like
proteins and removal of irreversibly damaged cells through
apoptosis (39). Elevated temperature per se may also increase
ROS production, independently of effects arising from in-
creased respiration (1). Rising temperatures thus confront ec-
totherms with a complex set of physiological challenges that
may demand a large modification in the flow of cellular energy
among competing processes linked to damage-repair, growth,
cell proliferation, and reproduction.
There are many studies at the whole organism level that
suggest this sort of redistribution of cellular energy. Some of
these effects have been observed with only modest increases in
temperature. For example, Munday et al. (46) found that
growth of a damselfish was significantly reduced by a 3C rise
in temperature. The classic studies of sockeye salmon by Brett
(6) show how strongly temperature, food availability, and
growth are inter-related. For mussels (Mytilus californianus) in
rocky intertidal habitats, abiotic stress has been shown to alter
the allocation of metabolic resources, with conspecifics occur-
ring high in the intertidal zone having relatively less energy to
support reproduction (50). Below, I consider what recent stud-
ies of changes in gene expression are revealing about the
cellular and subcellular processesand the potential energy
costsassociated with different intensities of sublethal stress.
Sublethal Stress at the Cellular Level: A Graded Series of
Repair, Demolition, and Reconstruction Functions
Physiologists studying environmental stress at the cellular
level are beginning to elucidate how the intensity of stress
confronting an organism affects the types of compensatory
responses made by the cell and how these responses might
affect the cells overall energy budget and its allocation of
energy for different processes. Important new insights into
these issues are emerging from two types of omic experi-
mentation: transcriptomic studies of gene expression, which
employ DNA microarrays (gene chips) to survey tempera-
ture-dependent transcriptional activities of hundreds to thou-
sands of different genes (9, 25, 26, 40, 43, 44), and proteomic
studies, which provide a more direct picture of changes in
composition of the cellular protein pool (proteome) and the
states of post-translational modification of the proteome (80).
These studies are revealing new aspects of temporal patterning
of transcriptional and translational changes, respectively, dur-
ing exposures to increasingly stressful temperatures. Studies of
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differently temperature-adapted congeners are beginning to
elucidate the types of adaptive changes in gene expression that
distinguish transcriptomic (40) and proteomic (80) responses
of species adapted to different temperatures.
Here, I review some of the relevant recent findings from
microarray studies that shed light on the following questions:
1) When does rising temperature first elicit expression of genes
associated with the CSR? 2) How do the recruitment patterns
of different types of CSR-related genes change as the intensity
of thermal stress increases? 3) When do repair processes give
way to demolition events such as proteolysis? 4) When is
cell proliferation inhibited to allow repair of DNA and protein
damage to be achieved and to maximize the amount of energy
that remains available for ATP-demanding processes of repair
and demolition? 5) How do the transcriptional responses of
congeners evolutionarily adapted to different temperature con-
ditions vary in the face of thermal stress? 6) Is wholesale
modification of gene expression needed, or are only a small
number of gene regulatory events different between cold- and
warm-adapted species? 7) Are transcriptional responses to
temperature indicative of the responses to other forms of
abiotic stress such as changes in salinity? 8) Does simultaneous
exposure to two or more forms of abiotic stress lead to
synergistic or antagonistic shifts in transcriptional responses?
Studies of gene expression in the eurythermal goby fish
Gillichthys mirabilis (9, 43, 44) have shown that rising tem-
peratures induce shifts in expression of hundreds of different
genes that encode proteins involved in protein refolding, pro-
teolysis, biosynthesis, cell signaling, and cell proliferation. For
the purposes of this review, the most relevant of these changes
in gene expression are those that 1) provide insights into the
threshold temperatures at which heat stress is first evident,
2) reveal a graded response to temperature in which different
categories of genes are expressed as heat stress increases, and
3) offer insights into the types of changes in energy allocation
strategies that might be necessitated by a rising need for
ATP-costly stress responses, which may demand suppression
of processes like cell proliferation. Some of the transcriptional
changes associated with the CSR in G. mirabilis are tissue
specific (9). The discussion below focuses on gill tissue, which
shows a robust transcriptomic change under heat stress and
salinity stress as well (18).
The threshold temperatures at which genes associated with
the CSR first show increased expression differ among genes
associated with different elements of the stress response and
with acclimation histories of specimens (8, 15, 44). For fish
acclimated to 9C, 19C, and 28C, threshold temperatures for
Fig. 1. Transcriptional responses (mRNA expres-
sion) of genes encoding two heat-shock proteins,
HSP70 and HSPA9, in gill tissue of Gillichthys
mirabilis acclimated for 4 wk to three tempera-
tures (9C, 19C, and 28C) and then subjected to
a heat ramp of 4C/h (44). mRNA expression is
normalized to a reference pool of mRNA, accord-
ing to methods in Logan and Somero (43, 44).
Solid bars and asterisks indicate a statistically
significant increase in mRNA relative to values in
prestressed specimens. [Modified after Logan and
Somero (44).]
AJP-Regul Integr Comp Physiol VOL
induction of stress-related genes increased 2C for each
9 10C rise in acclimation temperature (44). Although many
genes change expression as part of the CSR, increased tran-
scription of genes encoding HSPs is commonly regarded as the
canonical indicator of onset of heat stress (21). However, HSPs
comprise several protein families that, in addition to their
protein-repair activities, play multiple roles in such critical
stress-related processes as regulation of programmed cell death
(apoptosis) and cell proliferation (3). Thus, it is essential to
examine differences in expression among classes of HSPs to
elucidate how the diverse cellular processes linked to these
proteins are affected as thermal stress increases. Different
types of HSPs potentially can serve as biomarkers of differ-
ent degrees of stress-induced cellular damage.
In gills of G. mirabilis, different classes of heat-shock
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proteins show different temperatures for onset of increased
expression and different levels of upregulation (Fig. 1), con-
sistent with the heterogeneity in function of this broad set of
proteins (44). As shown in Fig. 1A, genes encoding HSP70 and
HSPA9, the two genes showing the highest levels of upregu-
lation in heat-stressed fish, differed in their temperatures of
increased expression. In 9, 19, and 28-acclimated fish,
increased expression of HSPA9 occurred at temperatures 4C
higher than those at which increased expression of HSP70 was
observed. The sequence of activation of these two genes
reflects a broader pattern of sequential upregulation of genes
involved in repair events and higher-level control of cellular
proliferation and degradation. Thus, upregulation of HSP70 is
taken as an indication that thermal stress has reached an
intensity at which damage to cellular proteins can no longer be
reversed by constitutively expressed molecular chaperones,
thereby necessitating production of HSPs for adequate protein-
refolding capacity. HSPA9, encoded by the second-most up-
regulated gene, is a mitochondrial paralog of the 70-kDa class
of molecular chaperones. It performs a number of functions in
addition to molecular chaperoning. HSPA9 is involved in the
inhibition of two cellular processes that are of pivotal impor-
tance in cellular stress and restoration of cellular homeostasis:
cell proliferation and apoptosis. Stress-related regulation of
these two processes appears to be necessitated only at slightly
higher temperatures than those at which repair of heat-dam-
aged proteins is initiated.
Another illustration of the variation in induction tempera-
tures among genes associated with different components of the
CSR is shown in Fig. 2. Here, genes associated with protein
folding (HSP70), ubiquitin-based proteasomal degradation of
proteins (UBIQ), and cell cycle arrest/apoptosis (CDKN1B) are
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COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
seen to show differences in onset temperatures of increased
expression (44). Increased expression of genes encoding pro-
teins involved in ubiquitin-mediated proteasomal degradation
of proteins is indicative of an incomplete rescue of heat-
damaged proteins by molecular chaperones like HSP70. Sig-
nificantly increased expression of the UBIQ gene occurred at
36C, a temperature 7C greater than the onset temperature of
increased expression of HSP70. Thus, onset of protein rescue
may ensue well before the cell reaches a temperature at which
protein degradation is activated. Increased expression of UBIQ
was found in studies of steady-state acclimated G. mirabilis;
levels of expression were directly correlated with acclimation
temperature (43). Thus, whereas expression of HSP70 did not
vary with acclimation temperature, genes linked to proteolysis
were increasingly upregulated as acclimation temperature in-
creased. This observation speaks to the issue of energy costs at
different temperatures, specifically the temperature-depen-
dence of costs of protein turnover and homeostasis (43).
Increased expression of the gene encoding cyclin-dependent
kinase inhibitor 1B (CDKN1B) is associated with late-stage
inhibition of the cell cycle (11). The upregulation of this gene
may be an indication that cell proliferation is blocked after a
certain level of cellular stress has been reached, thereby allow-
ing increased allocation of ATP to repair processes and pre-
venting cells with damage to DNA from proliferating.
Changed expression of genes associated with either inhibi-
tory or stimulatory effects on apoptosis was also observed in
this study. Whereas CDKN1B has been linked to induction of
apoptosis, several heat-shock proteins have been shown to
inhibit this process (3). Experiments that directly measure
apoptotic activity, e.g., measurements of DNA damage and
activation of caspase systems, are thus needed to resolve the
issue of how the balance between proapoptotic and antiapop-
totic factors changes with temperature stress. The lack of an
unambiguous signal in these gene expression data that apopto-
sis is increased following heat stress may reflect the time
course over which apoptosis occurs in heat-stressed cells.
Induction of apoptosis may be delayed until the cell has had
adequate time to try to repair stress-induced damage, especially
to DNA. And, if this repair cannot be achieved, there may be
a further delay in onset of apoptosis until sufficient energy is
AJP-Regul Integr Comp Physiol VOL
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Fig. 2. Transcriptional responses of genes encod-
ing proteins indicative of three aspects of the
cellular stress response: protein folding (HSP70),
proteolysis (ubiquitin-based proteasomal degrada-
tion of proteins, UBIQ), and cell cycle arrest/
apoptosis (CDKN1B), in gills of heat-stressed G.
mirabilis acclimated to 9C. mRNA expression is
normalized to a reference pool of mRNA, accord-
ing to methods in Logan and Somero (44). Solid
bars and asterisks indicate a statistically signifi-
cant increase in mRNA relative to values in pre-
stressed specimens. [Modified after Logan and
Somero (44).]
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available to drive the energy-demanding events associated with
programmed cell death. The rapid upregulation of genes en-
coding proteins involved in ATP-synthesizing pathways fol-
lowing heat-stress of G. mirabilis (9) suggests that increased
ATP production and turnover may be an important element in
the stress response.
Microarray studies of congeners of Mytilus have provided
further information on temperature-dependent patterning of
gene expression and initial insights into the ways in which
these gene regulatory responses evolve in congeners from
different abiotic environments. Comparisons of the transcrip-
tional (40) and proteomic (80) responses of M. trossulus and
M. galloprovincialis have shown that the different evolutionary
histories of these congeners have led to a number of species-
specific gene regulatory responses, although most stress-in-
duced transcriptional changes were similar in the two species.
Numerous genes encoding HSPs showed the canonical re-
sponse to rising temperature seen in other studies of sublethal
heat stress (Fig. 3A). Above a certain threshold temperature
(24C), production of message for commonly occurring
HSPs like HSP70 rose sharply (Fig. 3A). For HSP70, there was
no significant difference in response between the congeners, an
observation that applies as well for almost all of the HSPs that
were detected (Fig. 3B). In fact, the only large difference in
expression between the native and invasive species was found
for HSP24, a member of the -crystallin family of small heat
shock proteins.
The similarities, as well as the single difference, in expres-
sion of HSPs between the congeners raise two issues. First, the
similarities in responses of the two species for most HSPs
suggest that evolution of gene regulatory capacities for acti-
vating the heat-shock response has not been of a wholesale
nature, but rather has been restricted to a subset of HSP-
encoding genes. Second, the strong upregulation of message
for HSP24 seen in the more warm-adapted Mediterranean
mussel provides insights into the nature of thermal stress and
its downstream consequences in cellular regulation. Induction
of high levels of expression of HSP24 may be an indication
that the cytoskeleton is of particular sensitivity to heat stress,
because this small heat-shock protein is known to be involved
in refolding of cytoskeletal proteins (40). In fact, this conclu-
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Fig. 3. Gene expression in gill tissue of blue mussels, Mytilus trossulus and Mytilus galloprovincialis. 13C-acclimated specimens of both species were subjected
to a thermal ramp, and sampling was done at 24C, 28C, and 32C. A: cross-plot of expression levels of all genes encoding molecular chaperones. Except for
small heat-shock proteins (HSP24), the transcriptional changes of chaperones were similar in the two species. B: transcription of the genes encoding HSP70s.
mRNA for HSP70 showed a similar response to heating in both species, as indicated in Fig. 3A. C: transcription of genes encoding proteins of the proteasomal
apparatus. Induction occurred at a lower temperature in M. trossulus. [Modified after Lockwood et al. (40).]

sion has been reached in other studies of gene expression under
heat stress and osmotic stress, which have shown that proteins
associated with the cytoskeleton appear especially prone to
damage from heat- (9) and osmotic shock (18). In addition,
small heat-shock proteins are believed to play a role in sup-
pressing apoptosis (3). Thus, an alternative (or additional)
explanation for the rise in HSP24 message during heat stress is
that apoptosis is blocked or delayed during the period of heat
stress, as discussed earlier for G. mirabilis. If this is the case,
M. galloprovincialis appears to be more effective than M.
trossulus in inhibiting programmed cell death under heat stress.
Transcriptional studies of blue mussels also have revealed a
relationship between intensity of stress and recruitment of
different types of repair and demolition processes (40). As
shown in Fig. 3B, HSP70 is induced in both congeners of blue
mussels near 24C. This initial phase of the CSR thus involves
an emphasis on refolding of proteins that have incurred a
reversible loss of conformation at high temperature. The in-
duction of genes encoding subunits of the proteasome, which
conducts nonlysosomal protein degradation, occurs only at
higher temperatures and shows a species-specific pattern (Fig.
3C): M. trossulus exhibited a much higher level of expression
of proteasome-encoding genes than M. galloprovincialis, and
induction occurred at a lower temperature in the more cold-
adapted mussel. This difference in expression of genes encod-
ing proteasomal proteins, which mirrors the results of the
proteomic analysis of these two species (80), suggests that
greater damage was done to proteins in M. trossulus by heat
stress. This conjecture is corroborated by the discovery of
higher levels of ubiquitinated proteins in M. trossulus relative
to M. galloprovincialis in common-gardened specimens (33).
A further illustration of how increasing heat stress shifts the
pattern of stress-related gene expression is found in a field
study of another species of Mytilus, the ribbed mussel, M.
californianus (25). In field-acclimatized specimens collected
over several tidal cycles from sites differing in thermal stress,
induction of message for HSP70 occurred at lower tempera-
tures than those necessary for increased transcription of genes
associated with proteasomal function. Moreover, in the more
heat-stressed population of M. californianus, there was tempo-
ral separation between maximal transcription of genes associ-
ated with cell proliferation and genes associated with aerobic
metabolism, consistent with studies of yeast and other model
organisms that have revealed temporal separation of oxidative
and reductive phases in the cell cycle (81, 82). This striking
periodicity in expression of genes under extreme heat stress
illustrates a further, and heretofore unappreciated, aspect of
thermal stress.
In summary, studies of temperature effects on gene expres-
sion in controlled laboratory studies and in a limited number of
field studies have begun to reveal the graded nature of gene
regulatory responses to heat stress. Activation of transcription
of genes related to the CSR is not an all or nothing response
to stress. Rather, as stress intensity increases, new classes of
genes are activated that encode proteins playing a variety of
functions in repair of cellular damage, removal of irreversibly
damaged cellular constituents (and, most likely, of entire cells
that are damaged beyond repair, notably in DNA fidelity), and

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COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
regulation of cellular proliferation and life span. These tran-
scriptomic analyses not only are revealing the mechanisms of
cellular damage and repair, but in so doing are providing
physiologists with molecular biomarkers for characterizing
the degree of stress a cell has experienced and the differences
among species in sensitivity to damage of the cellular appara-
tus. These insights into the fundamental aspects of cellular
stress are starting to provide important knowledge concerning
how sublethal stress might impact energy budgets and, ulti-
mately, the abilities of ectotherms to persist in warming hab-
itats.
Responses to Multiple Stresses: Gene Expression and
Post-Translational Modifications of Proteins
Global change confronts organisms with multiple and often
cooccurring forms of abiotic stress. A comprehensive analysis
of the effects of a changing environment on organisms must
take this complexity into account. In the case of many marine
species from intertidal and estuarine habitats, changes in pre-
cipitation and storm runoff can lead to stress from osmoregu-
latory challenges. It is pertinent, then, to examine how changes
in ambient salinity alter gene expression in these species and
how these changes compare with transcriptional responses to
changing temperature.
A study of blue mussels sheds light on these issues and
reveals as well the degree to which congeneric species differ in
their transcriptional responses to osmotic stress (42). Compar-
isons of transcriptional responses of native (M. trossulus) and
invasive (M. galloprovincialis) blue mussels to hypoosmotic
stress showed changes in expression of a variety of genes, but
relatively few differences between species. The interspecific
differences in response to sudden reductions in salinity that
characterize the two blue mussels may include rapid behavioral
changes [valve closure under salinity stress (5)] and post-
translational modification of proteins (19), as well as somewhat
slower changes in gene expression.
Comparisons of shifts in gene expression that occurred
during either hypo-osmotic- or heat stress revealed 45 genes
that changed expression in response to both stresses (42).
However, almost all of the genes that were strongly upregu-
lated by heat stress were strongly downregulated by osmotic
stress. Among these oppositely responding genes were several
that encode proteins involved in transmembrane movement of
ions. Transport-related genes that exhibited strong upregula-
tion under heat stress were strongly downregulated under
hypoosmotic stress. Other transcriptomic studies of heat stress
have shown strong upregulation of genes associated with
membrane transport, a finding that may relate to disruption of
membrane structure and barrier functions under acute heat
stress (9).
A further aspect of the responses to reduced salinity by
native and invasive blue mussels was revealed by comparisons
of rapid post-translational modifications of gill proteins during
hypo-osmotic stress (19). Compared with its congener, M.
trossulus exhibited significantly greater amounts of protein
phosphorylation by MAPKs during hypoosmotic stress. Be-
cause these signal transduction proteins are pivotal in mounting
osmoregulatory responses and in suppressing apoptosis, the
enhanced ability of M. trossulus to cope with sudden reduc-
tions in salinity may be more a consequence of rapid changes
AJP-Regul Integr Comp Physiol VOL
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in protein phosphorylation than shifts in gene expression. The
native and invasive congeners also displayed differences in
protein phosphorylation under thermal stress (19), revealing a
further level of regulatory complexity and an additional inter-
specific difference between the two species.
In summary, transcriptional responses and post-translational
modifications of proteins show both similarities and differ-
ences under heat and osmotic stress. In some cases, these two
stresses may be synergistic and create a higher level of cellular
damage than found with either stress alone. Disruption of the
cytoskeleton, seen in osmotic and heat stress studies of gills, is
one possible example. These initial studies of transcriptional
responses and rapid post-translational modifications of proteins
in response to different abiotic stresses illustrate the complex-
ities of the challenges to organisms arising from multi-faceted
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global change.
Orthologous Enzymes of Congeners: a Window into Protein
Structure-Function Relationships and Evolutionary
Adaptation of Proteins
The well-characterized high sensitivities of protein structure
and function to alterations in temperature (22, 64, 65) indicate
that climate change is likely to exert many of its effects through
perturbation of enzymatic and structural proteins. These per-
turbations comprise impaired enzyme function, when temper-
atures reach levels at which kinetic properties like ligand
binding depart from their optimal ranges; molecular chaperon-
ing costs, which are associated with refolding of reversibly
denatured proteins; and costs linked to proteolysis and protein
synthesis, which arise when irreversibly damaged proteins are
removed from the cell and replaced with newly synthesized
proteins, through processes that have high ATP requirements.
Thus, the effects of rising cell temperatures on enzymes can
lead not only to metabolic impairment but also to increased
costs for sustaining protein homeostasis. To the extent that
protein evolution can modify protein stability and function in
temperature-adaptive manners, these metabolic perturbations
and energy costs could be substantially reduced.
Comparative studies of orthologous proteins from species
adapted to different temperatures have addressed several ques-
tions related to global change. One of these questionsper-
haps the most critical one of all concerns the amount of
change in ambient temperature that is sufficient to favor adap-
tive variation in proteins, to allow optimal function and stabil-
ity to be maintained. A second question asks how much change
in protein amino acid sequence is needed to achieve such
adaptations. A follow-up question is where in the structure of
the protein are adaptive changes possible and how many of
these adaptable sites are available for change.
Protein orthologs of congeners adapted to different environ-
mental temperatures have proven to be excellent study systems
for elucidating these issues. For example, studies of orthologs
of lactate dehydrogenase-A (LDH-A) in barracuda fishes (ge-
nus Sphyraena) with different latitudinal distributions (temper-
ate, subtropical, and tropical) have shown that differences in
habitat temperature of 35C are sufficient to favor adaptive
variation in sequence and function (34). Whether even smaller
differences in average or maximal habitat temperature can
favor protein evolution remains to be investigated. Suffice it to
say, however, that predicted increases in global temperature are
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R10 COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE

likely to be sufficient to favor adaptive evolution of proteins, if
species are to sustain their current biogeographic ranges and
maintain optimal metabolic capacities and acceptable costs of
protein homeostasis. Conversely, the observed shifts in bio-
geographic ranges of many ectothermic species, including
many pelagic fishes (2, 49) may be a reflection, at least in part,
of suboptimal performance of proteins under newly elevated
habitat temperatures.
Thermal perturbation of proteins can be separated into two
categories, function and structural stability, which in fact are
tightly linked (22, 64, 65). One of the most studied aspects of
temperature-protein interactions in the context of function is
the influence of changes in temperature on two kinetic prop-
erties of enzymes: enzyme-ligand interactions, which are often
quantified by measuring Michaelis-Menten constants (Km val-
(31). Both of these kinetic properties are at once strongly
affected by temperature and highly conserved among species at
their normal body temperatures (22, 31). An illustration of this
relationship is given in Fig. 4, A and B, which shows how Km
of pyruvate (KmPYR) and kcat differ between orthologous
LDH-As of temperate (Chromis punctipinnis) and tropical
(Chromis caudalis and C. xanthochira) damselfishes (36). In
agreement with the results of broader phylogenetic surveys that
examined orthologous LDH-As from vertebrates adapted to
body temperatures between approximately 1.9C (Antarctic
notothenioid fishes) and 45C (a thermophilic reptile) (22, 31),
at a common temperature of measurement, the values of Km
and kcat are higher for cold-adapted orthologs than those from
warm-adapted species. These intrinsic differences in binding
and catalytic power yield high degrees of conservation in these

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ues), and catalytic rate constants (kcat), which provide an index kinetic properties at species normal body temperatures (values
of the rate at which an enzyme can convert substrate to product enclosed by ovals in Fig. 4, A and B).

Fig. 4. Differences in kinetic and structural characteristics of lactate dehydrogenase-A (LDH-A) orthologs in tropical (Chromis caudalis and Chromis
xanthochira) and temperate (Chromis punctipinnis) congeners of damselfish. A: effects of evolutionary- and measurement temperatures on the Michaelis-Menten
constant for pyruvate (Kmpyr). B: effects of evolutionary and measurement temperatures on the catalytic rate constant (kcat). C: three-dimensional model of one
subunit of LDH-A showing the site (219) at which an alanine residue in the tropical congeners is replaced with a threonine residue in the temperate species.
[Modified after Johns and Somero (36).]

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COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
A key advantage of studying temperature-protein interac-
tions with congeneric species is illustrated by the study of
damselfish LDH-A orthologs. Because of the high similarity in
nucleotide and amino acid sequences observed in these closely
related species, identifying the amino acid substitution(s) re-
sponsible for adaptive change in kinetic properties is relatively
straightforward. In the case of the damselfish LDH-As, four
differences in amino acid sequence distinguished the temperate
species from the two tropical congeners (36). Using site-
directed mutagenesis to test the effects of these amino acid
differences on function, we showed that a single substitution at
site 219 in the sequence (alanine in the two tropical congeners;
threonine in the temperate congener) was sufficient to account
for the observed differences in KPYR
m . This amino acid substi-
tution resulted from a single nucleotide change (transition), the
replacement of the adenine found at position 658 in the ldh-a
gene of the temperate species with a guanine in the two tropical
species. Thus, in a gene of 1,000 nucleotides, only a 0.1%
change in sequence was sufficient to effect adaptive change in
function.
Comparisons of a sister enzyme of LDH-A, cytosolic
malate dehydrogenase (cMDH), another member of the Ross-
mann-fold family of dehydrogenases, have provided additional
examples of how a single amino acid substitution can lead to
temperature-adaptive modifications of Km and kcat (17, 23).
Differences in kinetic properties of cMDH orthologs of native
and invasive blue mussels, which are due to a single amino
acid substitution, may help to explain the higher thermal
tolerance and, therefore, the invasive success of the warm-
adapted species, M. galloprovincialis. A similar biogeographic
shift involving a northward range extension of a warm-adapted
limpet, Lottia austrodigitalis, and a contraction of the southern
distribution limit of a cold-adapted congener, L. digitalis, is
also marked by adaptive variations in cMDH function and
stability that arise from a single amino acid substitution (17).
The locations and the number of sites within an enzyme
where this minimal change in sequence is adequate to foster
adaptive change are also being revealed by comparisons of
orthologous enzymes of congeners (17, 22, 23, 34, 36). As
would be expected, sites in the catalytic vacuole of the enzyme,
where ligands interact directly with amino acid side chains, are
conserved among orthologs. The sites showing the highest
levels of variation among orthologs typically are within regions
of the protein that govern the energy changes that accompany
conformational alterations essential for binding and catalysis.
For LDH-A, these highly mobile and variable regions comprise
the 1G- 2G-helix, the BJ- 1G loop, and the H helix (Fig.
4C). Movement of these regions during function affects cata-
lytic rate (kcat) because the energy requirements needed to
drive catalytically essential conformational changes establish
the rate of enzyme function (22, 34). Higher rigidity of these
mobile regions leads to lower kcat values. Conversely, when
these mobile regions have greater intrinsic flexibility, the
reaction faces lower energy barriers. However, as a conse-
quence of these more flexible structures the binding of ligands
is less probable (Km values are higher). This results from
differences in the probability of the enzyme existing in a
binding-competent (ligand-recognizing) conformation. Mobile
regions with higher stability will more likely be in a ligand-
recognizing geometry.
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Because the structural flexibilities of several regions of a
protein have effects on Km and kcat, there are a number of
targets for adaptive evolution. Comparisons of several sets of
orthologous LDH-As (34, 36) and cMDHs (17, 23) have shown
that multiple sites of adaptive change underlie the variation in
kinetic properties that have been observed. This finding, in
concert with the discovery that a single nucleotide substitu-
tion is sufficient for temperature-adaptive variation, provides
an initial basis for conjectures about the rate of protein evolu-
tion, the question of how likely it is that adaptive protein
evolution will keep up with global warming. Whereas the
minimal number of substitutions needed for adaptive change
and the availability of numerous sites for this change to occur
support a model of rapid change, a full analysis of this issue
demands answers to several additional questions. These in-
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clude the importantand essentially unanswerable at pres-
ent question about how many proteins need to adapt when
temperature rises by a few degrees. The seminal work of
Dennis Powers et al. (55) on allelic variation of enzymes in the
teleost Fundulus heteroclitus along the East Coast of North
America has shown that not all proteins show latitudinal
variation in isozyme patterning (55). This suggests that some
classes of proteins are more likely than others to benefit from
adaptive variation in the face of small changes in temperature.
This conclusion is supported by analyses such as that of Ream
et al. (56), who found that the sequence of skeletal muscle actin
was conserved among animals with body temperatures span-
ning a range of almost 40C (Antarctic fishes to a desert
reptile), whereas for LDH-A, sequence variation and concom-
itant adaptive change in kinetic properties was prevalent in this
same suite of species. The rapid advances in sequencing
technology and bioinformatic methods for sequence analysis, if
coupled with appropriate examination of protein functional
responses to temperature, may allow rapid increases in our
appreciation of how many types of proteins are apt to benefit
from fine-tuning of function and stability in the face of the
increases in temperature expected from global change.
DNA Decay and Loss of Thermal Tolerance
The abilities of ectotherms to cope successfully with
changes in temperature depend strongly on two different as-
pects of genome content. First, the variety of protein-coding
genes in the genome may govern the ability of a species to
withstand changes in temperature and other abiotic factors.
Second, species abilities to regulate the expression of this
genetic information to acclimatize to changes in temperature
may be critically important in determining how well they
succeed in a warming world, at least in the short run before
adaptive genetic variation can help to blunt the effects of stress.
Elucidation of the differences that may exist in gene content
and gene regulatory capacities among species may provide
important insights into the fundamental genetic determinants of
stenothermy and eurythermy.
A very Kroghian set of species for examining these
questions are the cold-adapted stenothermal animals of the
Southern Ocean (54). These species, for example, fish of the
teleost suborder Notothenioidei, have evolved in extremely
cold and thermally stable waters for 14 million years (54). As
a consequence of this long evolutionary history under environ-
mental conditions that did not require acclimatization to vary-
301 JULY 2011 www.ajpregu.org
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R12 COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
ing temperatures or salinities and that afforded the fish access
to abundant dissolved oxygen, a number of types of DNA
decay, the loss of genetic information, have taken place. These
genetic lesions portend significant problems for these fish (and
similarly genetically depauperate invertebrates) as the South-
ern Ocean increases in temperature.
One form of DNA decay is the loss of protein-coding
elements in the genome. These losses may result from point
mutations that disrupt coding regions or disappearance from
the genome of all or part of a protein-coding gene. Perhaps the
most striking illustration of DNA decay in protein-coding
regions of the genome is the loss of hemoglobin (Hb) genes in
notothenioids of the Family Channichthyidae (13), the white-
blooded icefish. All members of this family have lost the
entire gene encoding the -chain of Hb and a fraction of the 5=
region of the -chain as well. In addition to complete loss of
circulating Hb, the expression of myoglobin (Mb) has been lost
among some members of this family. All icefish so examined
were found to possess the Mb-encoding gene, but expression of
the gene has been lost in at least three lineages due to disrup-
tion of the reading frame of the gene or failure of the Mb
message to be translated into protein (62). These genetic
lesions poise the Channichthyidae in particular jeopardy from
oceanic warming because this process will reduce oxygen
concentration of the seawater and body fluids and elevate rates
of oxygen consumption. In fact, studies of thermal tolerance of
white-blooded- (Channichthyidae) and red-blooded notothe-
nioids demonstrated that the former group has significantly
lower tolerance of elevated temperatures than their Hb-con-
taining relatives (4).
Another form of DNA decay identified in Antarctic fishes is
the loss of gene regulatory capacities for initiating upregulation
of heat-shock protein synthesis (10, 32). Although notothe-
nioids possess genes for molecular chaperones, which in all
organisms serve fundamental roles in the processes of protein
folding and transport in the cell, there is an absence of capacity
to upregulate any of these chaperone-encoding genes in re-
sponse to heat stress. In a study using cDNA microarray
analysis of transcriptional changes during heat stress in the
notothenioid species Trematomus bernacchii, the only gene
related to molecular chaperone function that exhibited upregu-
lation was the gene encoding HSP40, which is a cochaperone
that modulates activity of heat-shock proteins (10). Overall,
even though a number of genes in T. bernacchii exhibited
heat-induced upregulation, the responses were very muted
compared with gene regulatory responses seen in temperate
fish.
The loss of the ability to upregulate heat shock proteins may
have several ramifications for fish experiencing rising temper-
atures. Not only may the processes of molecular chaperoning
be impaired, but because of the role of HSPs in such important
cellular processes as apoptosis, broader regulatory dysfunction
may ensue. For example, HSP70 is important in suppressing
apoptosis (3). Without sufficient controls on programmed cell
death, stress could lead to an unregulated course of cellular
destruction. Genes encoding proteins that upregulate apoptosis
increased expression during heat stress in T. bernacchii, so a
potential for imbalance in this aspect of cellular regulation
seems possible (10).
Subsequent to the initial discovery of loss of the heat-shock
response in notothenioid fishes, a number of Antarctic inver-
AJP-Regul Integr Comp Physiol VOL
tebrate species have been shown to lack this process as well
(12). The loss of an ability to upregulate synthesis of molecular
chaperones and the overall muted response of gene regulatory
systems to increases in temperature seen in some Antarctic fish
(10) may help to explain why these species are so stenother-
mal. Many Antarctic ectotherms show behavioral impairment
at temperatures of only 13C and upper lethal temperatures
commonly are below 5 6C (51, 54, 67).
In summary, a variety of physiological and genetic studies
have begun to reveal some of the mechanisms underlying the
extreme stenothermy of ectotherms of the Southern Ocean.
Genetic lesions that disrupt or eliminate protein-coding regions
and impede gene-regulatory functions would seem to put these
species in extremely high risk from the effects of global
warming. Reversal of these genetic lesions, notably those that
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on March 13, 2017
involve loss of entire coding regions, to restore physiological
function for life in a warming world seems highly improbable
vis a` vis the pace of warming of the Southern Ocean. Further
studies of these cold-adapted stenotherms will provide added
insights into this risk and help us to characterize the funda-
mental genetic differences that underlie the wide variation in
environmental tolerance ranges of eury- and steno-tolerant
species.
Perspectives and Significance
In preparing this review, I was again struck by the advances
that have been made in comparative physiology conceptually
and technicallyas well as in the massive amounts of data that
are now availablesince I entered this field almost 50 years
ago. Had someone predicted in 1963, when I began my work
in Antarctica with notothenioid fishes, that during my career
there would appear technology that would allow global gene
expression to be followed in these (or any!) organism; that the
amino acid sequences of enzymes could be tinkered with,
residue by residue, to test hypotheses about adaptation; and
that the physiological studies we were doing on cold-adapted
ectotherms from the Southern Ocean and elsewhere would
contribute to study of a problem as broad as climate changea
concept that was first enunciated by Broecker (7) in 1975I
would have been a bit skeptical in all cases. What lies ahead for
our field in terms of what we will be able to do and what the
importance of our discoveries mean to our peers and to broader
societal issues may be as difficult to foresee as the five-
decades-off future was for me back in the early 1960s. I think,
however, that with the advent of so many omics and bio-
computational tools, in particular, comparative physiologists
are on the threshold of seeing in remarkably fine detail how
organisms work, from the level of contents of genetic tool
kits to the many ways in which this information shapes the
phenotype and confers upon it a species-specific level of
phenotypic plasticity. The window on organismal function
our field now possesses will allow continued growth of our
understanding on these fronts and, in the process, our approach
to physiology, ecology, and evolution will indeed continue to
offer a powerful crystal ball for predicting the consequences
of global change.
ACKNOWLEDGMENTS
This review has benefited greatly from discussions with many colleagues,
especially Dr. Bruce D. Sidell, who helped to make our field one that is both
intellectually vibrant and collegial, in the best sense of both of those terms. His
301 JULY 2011 www.ajpregu.org

COMPARATIVE PHYSIOLOGY AND GLOBAL CHANGE
work was an exemplar of how the comparative approach can be used most
creatively and productively to address the issues that I have attempted to cover
in this review.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
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