To freeze or not to freeze: adaptations for overwintering by hatchlings of the North American painted turtle
Department of Biology, Colorado State University, Fort Collins, CO 80523-1878, USA
* Author for correspondence (e-mail: packard{at}lamar.colostate.edu)
Accepted 2 June 2004
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Summary |
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Key words: turtle, Chrysemys picta, hibernation, freezing, supercooling
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Introduction |
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How do neonatal painted turtles survive in the cold? Early research on this
subject revealed that hatchlings are able to withstand freezing by as much as
50% of their bodily water, and this discovery led in turn to the proposition
that turtles spend some part of the northern winter in a frozen state
(Storey et al., 1988;
Churchill and Storey, 1992
).
The case was quite convincing (Storey,
1990
; Storey and Storey,
1992
,
1996
), and the concept of
`natural freeze-tolerance' in hatchling painted turtles quickly found its way
into textbooks of animal physiology (e.g.
Schmidt-Nielsen, 1997
). Work
performed more recently, however, indicates that the correlation between
overwintering by hatchlings in their nest and the indisputable ability of the
animals to recover from limited freezing (see
Rubinsky et al., 1994
) is
probably spurious and that a tolerance for freezing is not a general means by
which hatchling painted turtles withstand exposure to low temperatures in the
field (Packard and Packard,
2003a
; Packard et al.,
1997b
,
1999a
).
The preceding contention is based on numerous reports that turtles are able
to withstand freezing - but only when said freezing occurs under a set of very
restrictive conditions that are likely to have limited relevance to the
natural history of the animals. For example, if hatchlings are to survive
freezing, they must begin to freeze at a temperature that is only slightly
below the equilibrium freezing point for their bodily fluids
(Packard et al., 1999b), that
is, at a temperature only marginally lower than -0.7°C
(Storey et al., 1991
;
Packard and Packard, 1995
;
Costanzo et al., 2000b
). The
initiation of freezing at such a high subzero temperature apparently protects
turtles from the osmotic shock to cells that accompanies the `flash freezing'
of more deeply supercooled animals
(Claussen et al., 1990
;
Storey and Storey, 1996
;
Lee and Costanzo, 1998
).
Osmotic shock associated with flash freezing is usually fatal
(Claussen et al., 1990
), even
to hatchling painted turtles (see Packard
et al., 1999b
).
Additionally, hatchling painted turtles recover from freezing only in the
event that their exposure to cold is relatively brief and that temperature
does not go below -4°C (Churchill and
Storey, 1992; Costanzo et al.,
1995
; Attaway et al.,
1998
; Packard et al.,
1999b
; Packard and Packard, in
press
). Frozen turtles typically withstand exposure to -2°C
for as long as 4 days, but most animals that are held frozen at this
temperature have died from unknown causes by the end of 6 days and none
survives for as long as 8 days (Fig.
2; also Churchill and Storey,
1992
). Moreover, when hatchlings are frozen over 24 h to a thermal
equilibrium at -2°C and then exposed to slightly lower subzero
temperatures, the animals recover from a very brief exposure to -3°C, but
mortality increases rapidly thereafter and is nearly complete after 24 h
(Fig. 2; also
Costanzo et al., 1995
).
Finally, frozen hatchlings typically are unable to withstand even brief
exposures to -4°C, which represents the absolute lower limit of tolerance
(Fig. 2; also
Churchill and Storey, 1992
;
Costanzo et al., 1995
).
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Two exceptions to the preceding generalizations merit brief mention. First,
Churchill and Storey (1992)
reported that two hatchlings studied at the end of hibernation (April)
survived freezing at -2.5°C for 11 days; that is, the turtles survived in
a frozen state for longer than has been reported in any other investigation
(see Fig. 2). This finding,
which is unusual even in comparison with other results from the same
investigation, could not be confirmed by research performed more recently
(Packard and Packard, in
press
). Second, Costanzo et al.
(2004
) reported that animals
collected during winter from nests in the field withstood freezing at
-3.0°C for 3 days, which again is a survival time that exceeds expectation
(Fig. 2). We suspect, however,
that many of the turtles studied by Costanzo et al. did not freeze in the
intended way at the outset of study and that most of these turtles remained
unfrozen for the duration of their exposure to subzero temperature (see
Packard and Packard, 1993a
).
Thus, while we acknowledge the potential importance of these deviant findings,
we do not attach significance to them at this time.
Accordingly, we take the weight of evidence to indicate that hatchling
painted turtles may survive freezing in nature, but only in the event that (1)
they begin to freeze at a temperature near the equilibrium freezing point for
their bodily fluids (i.e. without supercooling appreciably) and (2) the
exposure is limited in duration and to temperatures above -4°C. However,
turtles that freeze in the laboratory under conditions like those they
encounter in natural nests usually are supercooled by several degrees at the
instant of nucleation and the experience is usually fatal (Packard and
Packard, 1993b,
1995
; Packard et al.,
1997b
,
1999a
). Moreover, temperatures
in natural nests at northern localities generally go below -2°C for longer
than 4 days, and they commonly go below the minimum tolerable temperature of
-4°C at some point during the winter
(Woolverton, 1963
;
DePari, 1996
;
Packard, 1997
;
Packard et al., 1997a
;
Weisrock and Janzen, 1999
;
Nagle et al., 2000
;
Costanzo et al., 2004
). These
considerations alone render it highly unlikely that hatchlings overwintering
in the field typically survive until spring by withstanding the physiological
challenges of freezing. Moreover, even when temperatures in nests are
sufficiently benign that hatchlings could conceivably survive a short bout of
freezing (e.g. Nagle et al.,
2000
; Costanzo et al.,
2004
), the turtles are unlikely to be frozen (Packard and Packard,
1995
,
2003a
,b
;
Willard et al., 2000
) for
reasons detailed below.
The physiology of hatchlings also has bearing on the question of natural
freeze-tolerance in painted turtles. For example, neonates do not produce any
of the nucleating agents, thermal hysteresis factors or cryoprotectants that
enable some arthropods and anurans to withstand freezing by water in the
extracellular space (Storey et al.,
1988,
1991
;
Churchill and Storey, 1991
;
Costanzo et al., 2000b
), and
acclimation of hatchling painted turtles to low (but non-freezing)
temperatures elicits a suite of physiological and behavioral responses that
actually are appropriate to an alternative adaptive strategy for withstanding
exposure to ice and cold (see below). Such findings, when taken together with
the ecological factors mentioned earlier, indicate that tolerance of
hatchlings for freezing simply reflects a limited capacity to withstand the
physiological stress associated with the formation of ice in bodily fluids
(Baust, 1991
). This ability to
survive the stress of limited freezing is not unique to hatchling painted
turtles but instead is a trait that is shared with neonates of several other
species of turtle, most of which have distributions or life histories that
effectively prevent neonates from being exposed to the threat of freezing
(Costanzo et al., 1995
;
Packard et al., 1997c
,
1999b
,
2000b
). Consequently, the
ability of hatchling painted turtles to withstand limited freezing probably is
a correlate of some as-yet unidentified process that is widespread in turtles,
and it is no more an adaptation to cold than the limited capacity for humans
to recover from serious burns is an adaptation to mishaps involving fire (see
Baust, 1991
). This is not to
say that hatchlings in the field never recover from shallow freezing
(Costanzo et al., 2004
); it is
simply to say that recovery from freezing probably is not a phenomenon of
general importance to the species and that the capacity is not an outcome of
natural selection enhancing the ability for turtles to recover from freezing
by water in the extracellular compartment.
A more likely explanation for how neonatal painted turtles typically
withstand the rigors of winter in the northern United States and southern
Canada is that they remain unfrozen and that they do so without the benefit of
an antifreeze (Costanzo et al.,
2000b; Packard and Packard,
2003a
; Packard et al.,
1997b
,
1999a
). The secret to such an
adaptive strategy is to remove nucleating agents from all body compartments
and simultaneously to prevent ice crystals from entering the body from frozen
soil (Packard and Packard,
2003a
). In the absence of an organizing site to facilitate the
change in phase from liquid to solid, bodily fluids cannot freeze at high
subzero temperatures (Franks,
1985
; Wilson et al.,
2003
). Hatchling map turtles [Graptemys geographica (Le
Sueur 1817)] probably manifest the same adaptive strategy for overwintering
that we advance here for neonatal painted turtles
(Baker et al., 2003
).
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The innate capacity for supercooling |
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Key to understanding this process is an appreciation of one of the many
peculiarities of turtles. As baby turtles are breaking out of their egg, they
typically ingest quantities of soil and fragments of eggshell (Packard et al.,
2000a,
2001
;
Costanzo et al., 2003
;
Packard and Packard, 2003c
).
Such geophagy is of uncertain function in any animal
(Dominy et al., 2004
), but the
behavior has important consequences for creatures such as hatchling painted
turtles. Natural soils typically contain potent nucleating agents - probably
bacteria such as Pseudomonas syringae
(Cochet and Widehem, 2000
) -
capable of initiating the formation of ice at relatively high temperatures
(Costanzo et al., 1998b
,
2000a
,
2001c
). If water in the gut
begins to freeze, however, the resulting crystals of ice penetrate the wall of
the intestine and initiate (`seed') freezing by water in the extracellular
compartment, in much the same manner that ice forming in the gut of insects
propagates across the wall of that organ and into the hemolymph
(Salt, 1966
;
Shimada, 1989
;
Block, 1990
). Thus, the
nucleating agents in the gut of newly hatched painted turtles set the limit of
supercooling at temperatures between approximately -5 and -6°C
(Costanzo et al., 2003
).
Baby painted turtles purge their gut of particulate matter before the start
of winter (Packard et al.,
2001; Costanzo et al.,
2003
; Packard and Packard,
2003c
), and the purging is accompanied by a reduction in the
population of bacterial nucleators (Fig.
3). However, emptying the gut of particulate matter is not, by
itself, sufficient to remove the nucleators. Turtles that are held at a
relatively high ambient temperature for 6 weeks following hatching purge most
or all of the particulate matter from their gut, but this purging does not
result in a major reduction in the limit for supercooling, and the apparent
concentration of nucleating agents remains high (as indicated by the low
variability for data in column 2 of Fig.
4). By contrast, turtles that are acclimated to approximately
3°C over the same 6-week period purge all particulate material from their
gastrointestinal (GI) tract, yet the animals also achieve a substantial
reduction in the apparent concentrations of nucleators (note the generally
lower values and increased variability for data in column 3 of
Fig. 4). Consequently, the
limit of supercooling for acclimated turtles is considerably lower than that
of recent hatchlings as well as that of turtles held at high ambient
temperature (Fig. 4). Full
development of the capacity for supercooling seems, therefore, to require that
hatchlings be exposed to declining temperatures like those to which animals in
natural nests are exposed as autumn turns to winter
(Costanzo et al., 2000b
;
Packard and Packard,
2003c
).
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It is unclear exactly how acclimation to low temperature elicits a
reduction in the populations of bacterial nucleators in the guts of hatchling
painted turtles, but the findings for turtles have parallels among
freeze-intolerant terrestrial insects, many of which also purge their gut of
food and nucleating agents before the start of winter
(Lee et al., 1996). Such
purging of the gut of particulate material seldom enables the insects in
question to remove all the nucleating agents, which persist in variable
numbers in an otherwise empty GI tract
(Strong-Gunderson et al.,
1990
; Costanzo et al.,
1998a
; Castrillo et al.,
2001
) and which elicit spontaneous freezing over relatively wide
ranges of temperature (Salt,
1970
; Strong-Gunderson et al.,
1990
).
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The role of the integument |
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It is noteworthy, however, that the integument affords little resistance to
the penetration of ice into turtles in the days immediately after hatching
(Costanzo et al., 2000b;
Packard and Packard, 2003b
),
at which time neonates are highly susceptible to freezing by inoculation
(Fig. 5). The cutaneous barrier
to penetration of ice - like the capacity for supercooling-is enhanced in the
weeks leading up to winter, and the process again requires that hatchlings be
exposed to low temperatures like those to which they would be exposed in
natural nests (Fig. 5). The
specific changes leading to enhancement of the cutaneous barrier are currently
unknown, but an explanation probably lies in a better understanding of the
epidermis, which is the site of barrier functions in amniotes generally
(Alibardi, 2003
).
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Two kinds of epidermis cover the body of neonatal painted turtles.
Epidermis overlying the head, neck and limbs (and including that in the
axillary and inguinal pockets) is limited externally by an -keratin
layer comprised of distinct cells and intercellular spaces
(Landmann, 1986
; Alibardi,
1999
,
2002
). By contrast, epidermis
covering the shell (i.e. the carapace, plastron and bridges) is bounded on the
external surface by a compact ß-keratin layer in which intercellular
spaces are reduced or absent (Alibardi and
Thompson, 1999
; Alibardi,
2002
; Alibardi and Sawyer,
2002
). The outermost cells of the epidermis are flattened, dead
structures containing filaments of either
-keratin or ß-keratin,
as is implied by the names assigned to the respective layers. The
-keratin layer is what confers flexibility to skin on the head, neck
and limbs, while the ß-keratin layer causes scutes on the shell to be
hard and inflexible.
The -keratin layer of epidermis from flexible skin is similar in its
general appearance to the stratum corneum of mammalian integument, which has
been likened to a wall formed from `bricks and mortar'. The `bricks' in the
stratum corneum of mammalian epidermis are the corneocytes, and the `mortar'
is the lipid occupying the intercellular domain
(Elias, 1983
;
Elias and Menon, 1991
;
Menon and Ghadially, 1997
).
Moreover, lipids in the extracellular matrix of mammalian stratum corneum
limit the loss of water from body compartments to the surrounding atmosphere
(Elias and Friend, 1975
;
Grubauer et al., 1989
;
Elias and Feingold, 1992
),
whereas lipids in the
-keratin layer of flexible skin from exposed
surfaces of neonatal painted turtles seemingly resist the penetration of ice
crystals into body compartments from frozen soil
(Willard et al., 2000
). The
parallels in structure and function between flexible skin of turtles and the
integument of mammals are apparent.
Flexible skin from cold-acclimated hatchlings of the painted turtle varies
from place to place over the surface of the body in the amount of lipid that
is present in the -keratin layer, and this variation also seems to be
correlated with regional variation in effectiveness of the barrier to
inoculation. The
-keratin layer of skin from exposed surfaces of the
forelimbs, for instance, has dense deposits of lipid in the inner domain
whereas the
-keratin layer of integument from more protected sites at
the base of the neck lacks such deposits
(Willard et al., 2000
). The
exposed sites on the forelimbs are suspected to be resistant to inoculation
while skin at the base of the neck and limbs is thought to be penetrated more
readily by crystals of ice (Packard and
Packard, 1995
). Thus, resistance of flexible skin to inoculation
is positively correlated with lipid in the
-keratin layer. Parallels
with mammals again are apparent, because mammalian skin exhibits regional
variation in lipid content and in efficacy of the integument in resisting the
passage of water (Elias et al.,
1981
; Lampe et al.,
1983
; Law et al.,
1995
).
The similarities between the stratum corneum of mammals and the
-keratin layer of flexible integument from hatchling painted turtles
also afford a plausible explanation for how the cutaneous barrier of neonates
becomes enhanced by cold acclimation. For example, the stress of desiccation
elicits an increase in formation and deposition of lipid (especially
ceramides) in the stratum corneum of mammalian skin
(Denda et al., 1998
;
Kömüves et al.,
1999
). We speculate, therefore, that acclimation of hatchling
painted turtles to cold entails an increase in the amount of lipid in the
-keratin layer of epidermis overlying exposed surfaces of the head and
limbs and that this accounts for the increase in effectiveness of the
cutaneous barrier to inoculation (Fig.
5).
However, a substantial (arguably the largest) fraction of the epidermis
making contact with frozen soil is that of the shell. Certainly this is so
when hatchlings withdraw their head and limbs into the shell, as we have
observed them to do when they are exposed to low temperatures (also
Storey et al., 1988). The
resistance of the shell to penetration by growing crystals of ice has not been
studied, but we predict that ß-epidermis is an effective barrier to
inoculation right from the time of hatching. This prediction is based on
studies indicating (1) that scutes covering the shell are largely impermeable
to water (Rose, 1969
), (2)
that water traverses the epidermis of mammalian integument via
intercellular spaces (Elias and Friend,
1975
; Nemanic and Elias,
1980
; Simonetti et al.,
1995
; Meuwissen et al.,
1998
) and (3) that intercellular spaces are absent from the
ß-keratin layer overlying the shell of turtles
(Alibardi and Thompson, 1999
;
Alibardi, 2002
). Assuming that
crystals of ice grow through the same channels that are followed by water, we
hypothesize that ice is unable to penetrate integument overlying the shell of
hatchling painted turtles owing to the absence of pathways through which the
crystals might grow.
In summary, we believe that ß-epidermis overlying the shell is
impermeable to ice from the moment of hatching, and that the -epidermis
on exposed surfaces of head and limbs has a low permeability to ice in
acclimated hatchlings (but not in unacclimated ones). The part of the
-epidermis that is in more concealed sites at the base of the neck and
limbs apparently affords less protection against inoculation, even in
acclimated turtles, but these sites are also shielded somewhat from exposure
to ice in the soil, thereby reducing the risk that animals will be inoculated
across these surfaces.
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Physiological consequences of supercooling |
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Of more immediate concern, however, is the likelihood that losses of water
across the integument actually increase the risk to overwintering hatchlings
of freezing by inoculation (Packard and
Packard, 1993b). By this hypothesis, vapor escaping across the
skin (or from a bodily orifice) condenses on the nearest crystal of ice in the
environment, thereby causing the crystal to grow toward the source of the
vapor (Salt, 1963
). If
temperature in the nest is low enough (thereby increasing the transcutaneous
gradient in vapor pressure), or if the exposure to subzero temperature is long
enough, the crystal penetrates into and through the integument and seeds the
formation of ice in the extracellular space
(Packard and Packard, 1993b
).
Such delayed inoculation usually results in the death of the hatchling
(Packard and Packard,
1993a
,b
,
1995
; Packard et al.,
1997b
,
1999a
).
Circulation, metabolism and acid/base balance
Total metabolic activity has yet to be measured in supercooled hatchlings,
but it seems reasonable at this juncture to assume that metabolism and
temperature are positively correlated. However, the oxygen-dependent component
of metabolism seems to be affected more than total metabolic activity by
declining temperature, owing to the influence of temperature on cardiac
activity. The heart of a hatchling painted turtle contracts about once each
minute at 0°C, but heart rate declines rapidly as temperature goes lower
and reaches zero between -9 and -10°C
(Birchard and Packard, 1997).
The resultant reduction in delivery of oxygen to peripheral tissues means that
those tissues have to increase their reliance on anaerobic pathways to
generate ATP, despite the fact that overall demand for ATP is also lower
(Hartley et al., 2000
;
Costanzo et al., 2001a
).
Patterns of accumulation of lactic acid in bodies of hatchling painted turtles are shown in Fig. 6, where the slopes of the lines reflect rates of accumulation of lactate in bodies of supercooled animals held at different temperatures. Turtles at 0°C did not accumulate lactate during 25 days of exposure, indicating that these animals were able to meet requirements for ATP by aerobic respiration; this means, in turn, that the already low rate of circulation was still sufficient to supply peripheral tissues with oxygen. Neonates at -4°C and -8°C, however, were unable to support peripheral tissues by oxidative respiration alone, and lactate consequently accumulated. The rate of accumulation of lactate was higher in hatchlings held at -8°C than in those held at -4°C.
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All the turtles that were held in a supercooled state at -8°C had died by day 25. The amount of lactate in bodies of these animals was only slightly higher than the quantity of lactate recovered from hatchlings held at the same temperature for 15 days. We take this finding to mean that turtles survived in a supercooled state for 15 days at -8°C but that they died soon after - perhaps as a result of shifts in pH resulting from the presence of the lactic acid.
The concentration of lactic acid in the dead hatchlings was only about half
to two-thirds that which can be tolerated by adult painted turtles held in
cold, anoxic water (Hartley et al.,
2000). Why might a hatchling succumb to a lactic acidosis that can
be readily tolerated by an adult animal? We propose that the difference in
levels of tolerance stems, in part, from differences in the perfusion of
peripheral tissues with blood and, in part, from differences in the degree of
calcification of the bony shell.
Adult painted turtles subjected to anoxic water buffer the lactic acid by
sequestering a portion of the acid in the shell itself and by mobilizing
calcium carbonate from the shell to buffer lactate in other parts of the body
(Jackson, 2000). These
processes depend on functioning of the circulatory system
(Herbert and Jackson, 1985
),
which is impaired in supercooled neonates and which ceases to function
altogether as temperature approaches -10°C. Additionally, the shell of
hatchlings is not fully calcified
(Zangerl, 1969
;
Ewert, 1985
), so the animals
do not have as large a reserve of mineral as adults to buffer the lactate.
With a more limited supply of mineral to buffer acid, and with an impaired (or
non-functioning) circulation to move around both the buffer and the acid,
hatchlings apparently are more susceptible to a fatal lactic acidosis.
Even when lactate does not accumulate to levels that are life-threatening,
the accompanying impairment of acid/base status may have important
consequences. Supercooled hatchlings that have survived long exposures at low
temperatures in laboratory tests commonly exhibit a prolonged lethargy after
they have been rewarmed to room temperature
(Hartley et al., 2000;
Costanzo et al., 2001a
). If
the lethargic state is a result of the lactic acidosis, it is easy to imagine
that hatchlings might delay their emergence from their nest in the spring
until the lactic acid has been metabolized or otherwise removed from the
system (Jackson et al., 1996
).
Moreover, if the lactic acid debt has not been repaid fully by the time
hatchlings dig their way out of their nest, their behavior might be affected
adversely during the trek to water.
The interactions among temperature, circulatory function, metabolism,
acid/base balance and behavior are an area of research that promises to yield
new insights into the adaptive strategy for overwintering by these animals.
One of the more intriguing of the many unanswered questions has to do with the
possibility that the stagnant hypoxia induced by exposure of hatchlings to
subzero temperature elicits the same kind of metabolic depression that has
been reported for adult painted turtles subjected to anoxia
(Jackson, 2000). Such a
depression would help to conserve the reserves of glycogen that are needed to
fuel metabolism of such critical organs as heart and brain, and it would also
serve to minimize the disruption of acid/base balance resulting from the
formation of lactic acid.
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Evolutionary considerations |
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Another outstanding question has to do with factors that underlie the
acquisition of traits enabling hatchling painted turtles to undergo
supercooling while they are overwintering in the frost zone of the soil
(Packard et al., 2002). One
possibility is that the cutaneous barrier to penetration of ice, as well as
the physiological or behavioral means for inactivating nucleating agents in
bodily fluids, was acquired as a result of directional (natural) selection
exerted by ice and cold during the northward expansion of the species at the
end of Pleistocene glaciation. Alternatively, these attributes of morphology,
physiology and behavior may have been acquired before the end of the
Pleistocene in response to different selection pressures altogether, in which
case the role of the characters in promoting supercooling was entirely
serendipitous. Phylogeographic analysis has the potential to distinguish
between these competing hypotheses, albeit the one such analysis that has been
performed to date failed to resolve the issue
(Starkey et al., 2003
).
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Acknowledgments |
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References |
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Alibardi, L. (1999). Differentiation of the epidermis of neck, tail and limbs in the embryo of the turtle Emydura macquarii (Gray, 1830). Belgian J. Zool. 129,391 -404.
Alibardi, L. (2002). Immunocytochemical observations on the cornification of soft and hard epidermis in the turtle Chrysemys picta. Zoology 105, 31-44.
Alibardi, L. (2003). Adaptation to the land: the skin of reptiles in comparison to that of amphibians and endotherm amniotes. J. Exp. Zool. 298B,12 -41.[CrossRef]
Alibardi, L. and Sawyer, R. H. (2002). Immunocytochemical analysis of beta (ß) keratins in the epidermis of chelonians, lepidosaurians, and archosaurians. J. Exp. Zool. 293,27 -38.[CrossRef][Medline]
Alibardi, L. and Thompson, M. B. (1999). Epidermal differentiation during carapace and plastron formation in the embryonic turtle Emydura macquarii. J. Anat. 194,531 -545.[CrossRef][Medline]
Attaway, M. B., Packard, G. C. and Packard, M. J. (1998). Hatchling painted turtles (Chrysemys picta) survive only brief freezing of their bodily fluids. Comp. Biochem. Physiol. A 120,405 -408.
Baker, P. J., Costanzo, J. P., Iverson, J. B. and Lee, R. E., Jr (2003). Adaptations to terrestrial overwintering of hatchling northern map turtles, Graptemys geographica. J. Comp. Physiol. B 173,643 -651.[Medline]
Baust, J. G. (1991). The freeze tolerance oxymoron. Cryo-Lett. 12,1 -2.
Birchard, G. F. and Packard, G. C. (1997). Cardiac activity in supercooled hatchlings of the painted turtle (Chrysemys picta). J. Herpetol. 31,166 -169.
Block, W. (1990). Cold tolerance of insects and other arthropods. Philos. Trans. R. Soc. Lond. B 326,613 -631.
Castrillo, L. A., Lee, R. E., Jr, Wyman, J. A., Lee, M. R. and Rutherford, S. T. (2001). Field persistence of ice-nucleating bacteria in overwintering Colorado potato beetles. Biol. Control 21,11 -18.[CrossRef]
Churchill, T. A. and Storey, K. B. (1991). Metabolic responses to freezing by organs of hatchling painted turtles Chrysemys picta marginata and C. p. bellii. Can. J. Zool. 69,2978 -2984.
Churchill, T. A. and Storey, K. B. (1992). Natural freezing survival by painted turtles Chrysemys picta marginata and C. picta bellii. Am. J. Physiol. 262,R530 -R537.[Medline]
Claussen, D. L. and Zani, P. A. (1991). Allometry of cooling, supercooling, and freezing in the freeze-tolerant turtle Chrysemys picta. Am. J. Physiol. 261,R626 -R632.[Medline]
Claussen, D. L., Townsley, M. D. and Bausch, R. G. (1990). Supercooling and freeze-tolerance in the European wall lizard, Podarcis muralis, with a revisional history of the discovery of freeze-tolerance in vertebrates. J. Comp. Physiol. B 160,137 -143.
Cochet, N. and Widehem, P. (2000). Ice crystallization by Pseudomonas syringae. Appl. Microbiol. Biotechnol. 54,153 -161.[CrossRef][Medline]
Costanzo, J. P., Iverson, J. B., Wright, M. F. and Lee, R. E., Jr (1995). Cold hardiness and overwintering strategies of hatchlings in an assemblage of northern turtles. Ecology 76,1772 -1785.
Costanzo, J. P., Humphreys, T. L., Lee, R. E., Jr, Moore, J. B., Lee, M. R. and Wyman, J. A. (1998a). Long-term reduction of cold hardiness following ingestion of ice-nucleating bacteria in the Colorado potato beetle, Leptinotars decemlineata. J. Insect Physiol. 44,1173 -1180.[CrossRef][Medline]
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E.,
Jr (1998b). Soil hydric characteristics and environmental ice
nuclei influence supercooling capacity of hatchling painted turtles
Chrysemys picta. J. Exp. Biol.
201,3105
-3112.
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E., Jr (2000a). Ice nuclei in soil compromise cold hardiness of hatchling painted turtles (Chrysemys picta). Ecology 81,346 -360.
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E.,
Jr (2000b). Seasonal changes in physiology and development of
cold hardiness in the hatchling painted turtle Chrysemys picta.
J. Exp. Biol. 203,3459
-3470.
Costanzo, J. P., Jones, E. E. and Lee, R. E., Jr (2001a). Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta. J. Comp. Physiol. B 171,335 -340.[Medline]
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E., Jr (2001b). Cold-hardiness and evaporative water loss in hatchling turtles. Physiol. Biochem. Zool. 74,510 -519.[CrossRef][Medline]
Costanzo, J. P., Litzgus, J. D., Larson, J. L., Iverson, J. B. and Lee, R. E., Jr (2001c). Characteristics of nest soil, but not geographic origin, influence cold hardiness of hatchling painted turtles. J. Therm. Biol. 26, 65-73.[CrossRef][Medline]
Costanzo, J. P., Baker, P. J., Dinkelacker, S. A. and Lee, R.
E., Jr (2003). Endogenous and exogenous ice-nucleating agents
constrain supercooling in the hatchling painted turtle. J. Exp.
Biol. 206,477
-485.
Costanzo, J. P., Dinkelacker, S. A., Iverson, J. B. and Lee, R. E., Jr (2004). Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress. Physiol. Biochem. Zool. 77, 74-99.[CrossRef][Medline]
Denda, M., Sato, J., Masuda, Y., Tsuchiya, T., Koyama, J., Kuramoto, M., Elias, P. M. and Feingold, K. R. (1998). Exposure to a dry environment enhances epidermal permeability barrier function. J. Invest. Dermatol. 111,858 -863.[Abstract]
DePari, J. A. (1996). Overwintering in the nest chamber by hatchling painted turtles, Chrysemys picta, in northern New Jersey. Chelon. Conserv. Biol. 2, 5-12.
Dominy, N. J., Davoust, E. and Minekus, M.
(2004). Adaptive function of soil consumption: an in
vitro study modeling the human stomach and small intestine. J.
Exp. Biol. 207,319
-324.
Elias, P. M. (1983). Epidermal lipids, barrier function, and desquamation. J. Invest. Dermatol. 80,44s -49s.[Medline]
Elias, P. M. and Feingold, K. R. (1992). Lipids and the epidermal water barrier: metabolism, regulation, and pathophysiology. Sem. Dermatol. 11,176 -182.
Elias, P. M. and Friend, D. S. (1975). The permeability barrier in mammalian epidermis. J. Cell Biol. 65,180 -191.[Abstract]
Elias, P. M. and Menon, G. K. (1991). Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv. Lipid Res. 24, 1-26.[Medline]
Elias, P. M., Cooper, E. R., Korc, A. and Brown, B. E. (1981). Percutaneous transport in relation to stratum corneum structure and lipid composition. J. Invest. Dermatol. 76,297 -301.[Abstract]
Ernst, C. H., Barbour, R. W. and Lovich, J. E. (1994). Turtles of the United States and Canada. Washington, DC: Smithsonian Institution Press.
Ewert, M. A. (1985). Embryology of turtles. In Biology of the Reptilia, vol. 14, Development A (ed. C. Gans, F. Billett and P. F. A. Maderson), pp.75 -267. New York: Wiley.
Finkler, M. S. (1999). Influence of water availability during incubation on hatchling size, body composition, desiccation tolerance, and terrestrial locomotor performance in the snapping turtle Chelydra serpentina. Physiol. Biochem. Zool. 72,714 -722.[CrossRef][Medline]
Franks, F. (1985). Biophysics and Biochemistry at Low Temperatures. Cambridge: Cambridge University Press.
Gibbons, J. W. and Nelson, D. H. (1978). The evolutionary significance of delayed emergence from the nest by hatchling turtles. Evolution 32,297 -303.
Grubauer, G., Feingold, K. R., Harris, R. M. and Elias, P. M. (1989). Lipid content and lipid type as determinants of the epidermal permeability barrier. J. Lipid Res. 30, 89-96.[Abstract]
Hartley, L. M., Packard, M. J. and Packard, G. C. (2000). Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival. J. Comp. Physiol. B 170, 45-50.[Medline]
Herbert, C. V. and Jackson, D. C. (1985). Temperature effects on the responses to prolonged submergence in the turtle Chrysemys picta bellii. II. Metabolic rate, blood acid-base and ionic changes, and cardiovascular function in aerated and anoxic water. Physiol. Zool. 58,670 -681.
Jackson, D. C. (2000). Living without oxygen: lessons from the freshwater turtle. Comp. Biochem. Physiol. A 125,299 -315.
Jackson, D. C., Toney, V. I. and Okamoto, S. (1996). Lactate distribution and metabolism during and after anoxia in the turtle, Chrysemys picta bellii. Am. J. Physiol. 271,R409 -R416.[Medline]
Kömüves, L. G., Hanley, K., Jiang, Y., Katagiri, C.,
Elias, P. M., Williams, M. L. and Feingold, K. R.
(1999). Induction of selected lipid metabolic enzymes and
differentiation-linked structural proteins by air exposure in fetal rat skin
explants. J. Invest. Dermatol.
112,303
-309.
Lampe, M. A., Burlingame, A. L., Whitney, J., Williams, M. L., Brown, B. E., Roitman, E. and Elias, P. M. (1983). Human stratum corneum lipids: characterization and regional variations. J. Lipid Res. 24,120 -130.[Abstract]
Landmann, L. (1986). The skin of reptiles/epidermis and dermis. In Biology of the Integument/ 2 Vertebrates (ed. J. Bereiter-Hahn, A. G. Matoltsy and K. Sylvia Richards), pp. 150-187. Berlin: Springer.
Law, S., Wertz, P. W., Swartzendruber, D. C. and Squier, C. A. (1995). Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy. Arch. Oral Biol. 40,1085 -1091.[CrossRef][Medline]
Lee, R. E., Jr and Costanzo, J. P. (1998). Biological ice nucleation and ice distribution in cold-hardy ectothermic animals. Annu. Rev. Physiol. 60, 55-72.[CrossRef][Medline]
Lee, R. E., Jr, Costanzo, J. P. and Mugnano, J. A. (1996). Regulation of supercooling and ice nucleation in insects. Eur. J. Entomol. 93,405 -418.
Lundheim, R. and Zachariassen, K. E. (1993). Water balance of over-wintering beetles in relation to strategies for cold tolerance. J. Comp. Physiol. B 163, 1-4.
Menon, G. and Ghadially, R. (1997). Morphology of lipid alterations in the epidermis: a review. Microsc. Res. Tech. 37,180 -192.[CrossRef][Medline]
Meuwissen, M. E. M. J., Janssen, J., Cullander, C., Junginger, H. E. and Bouwstra, J. A. (1998). A cross-section device to improve visualization of fluorescent probe penetration into the skin by confocal laser scanning microscopy. Pharmaceut. Res. 15,352 -356.[CrossRef][Medline]
Miller, K., Packard, G. C. and Packard, M. J. (1987). Hydric conditions during incubation influence locomotor performance of hatchling snapping turtles. J. Exp. Biol. 127,401 -412.
Nagle, R. D., Kinney, O. M., Congdon, J. D. and Beck, C. W. (2000). Winter survivorship of hatchling painted turtles (Chrysemys picta) in Michigan. Can. J. Zool. 78,226 -233.[CrossRef]
Nemanic, M. K. and Elias, P. M. (1980). In situ precipitation: a novel cyotchemical [sic.] technique for visualization of permeaability pathways in mammalian stratum corneum. J. Histochem. Cytochem. 28,573 -578.[Medline]
Packard, G. C. (1997). Temperatures during winter in nests with hatchling painted turtles (Chrysemys picta). Herpetologica 53,89 -95.
Packard, G. C. and Packard, M. J. (1993a). Hatchling painted turtles (Chrysemys picta) survive exposure to subzero temperatures during hibernation by avoiding freezing. J. Comp. Physiol. B 163,147 -152.
Packard, G. C. and Packard, M. J. (1993b). Delayed inoculative freezing is fatal to hatchling painted turtles (Chrysemys picta). Cryo-Lett. 14,273 -284.
Packard, G. C. and Packard, M. J. (1995). The basis for cold tolerance in hatchling painted turtles (Chrysemys picta). Physiol. Zool. 68,129 -148.
Packard, G. C. and Packard, M. J. (2001). The overwintering strategy of hatchling painted turtles, or how to survive in the cold without freezing. BioScience 51,199 -207.
Packard, G. C. and Packard, M. J. (2003a). Natural freeze-tolerance in hatchling painted turtles? Comp. Biochem. Physiol. A 134,233 -246.
Packard, G. C. and Packard, M. J. (2003b). Cold acclimation enhances cutaneous resistance to inoculative freezing in hatchling painted turtles, Chrysemys picta. Funct. Ecol. 17,94 -100.[CrossRef]
Packard, G. C. and Packard, M. J. (2003c). Influence of acclimation and incubation medium on supercooling by hatchling painted turtles, Chrysemys picta. Funct. Ecol. 17,611 -618.
Packard, G. C., Fasano, S. L., Attaway, M. B., Lohmiller, L. D. and Lynch, T. L. (1997a). Thermal environment for overwintering hatchlings of the painted turtle (Chrysemys picta). Can. J. Zool. 75,401 -406.
Packard, G. C., Lang, J. W., Lohmiller, L. D. and Packard, M. J. (1997b). Cold tolerance in hatchling painted turtles (Chrysemys picta): supercooling or tolerance for freezing? Physiol. Zool. 70,670 -678.[Medline]
Packard, G. C., Tucker, J. K., Nicholson, D. and Packard, M. J. (1997c). Cold tolerance in hatchling slider turtles (Trachemys scripta). Copeia 1997,339 -345.
Packard, G. C., Lang, J. W., Lohmiller, L. D. and Packard, M. J. (1999a). Resistance to freezing in hatchling painted turtles (Chrysemys picta). Can. J. Zool. 77,795 -801.[CrossRef]
Packard, G. C., Packard, M. J., Lang, J. W. and Tucker, J. K. (1999b). Tolerance for freezing in hatchling turtles. J. Herpetol. 33,536 -543.
Packard, G. C., Packard, M. J. and Birchard, G. F. (2000a). Availability of water affects organ growth in prenatal and neonatal snapping turtles (Chelydra serpentina). J. Comp. Physiol. B 170,69 -74.[CrossRef][Medline]
Packard, G. C., Packard, M. J. and Lang, J. W. (2000b). Why hatchling Blanding's turtles don't overwinter inside their nest. Herpetologica 56,367 -374.
Packard, G. C., Packard, M. J. and McDaniel, L. L.
(2001). Seasonal change in the capacity for supercooling by
neonatal painted turtles. J. Exp. Biol.
204,1667
-1672.
Packard, G. C., Packard, M. J., Morjan, C. L. and Janzen, F. J. (2002). Cold-tolerance of hatchling painted turtles (Chrysemys picta bellii) from the southern limit of distribution. J. Herpetol. 36,300 -304.
Packard, M. J. and Packard, G. C. (in press). Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance. Physiol. Biochem. Zool.
Rose, F. L. (1969). Desiccation rates and temperature relationships of Terrapene ornata following scute removal. Southwest. Nat. 14, 67-72.
Rubinsky, B., Hong, J. and Storey, K. B. (1994). Freeze tolerance in turtles: visual analysis by microscopy and magnetic resonance imaging. Am. J. Physiol. 267,R1078 -R1088.[Medline]
Salt, R. W. (1963). Delayed inoculative freezing of insects. Can. Entomol. 95,1190 -1202.
Salt, R. W. (1966). Factors influencing nucleation in supercooled insects. Can. J. Zool. 44,117 -133.
Salt, R. W. (1970). Analysis of insect freezing temperature distributions. Can. J. Zool. 48,205 -208.
Schmidt-Nielsen, K. (1997). Animal Physiology/Adaptation and Environment. 5th edition. Cambridge: Cambridge University Press.
Shimada, K. (1989). Ice-nucleating activity in the alimentary canal of the freezing-tolerant prepupae of Trichiocampus populi (Hymenoptera: Tenthredinidae). J. Insect Physiol. 35,113 -120.[CrossRef]
Simonetti, O., Hoogstraate, A. J., Bialik, W., Kempenaar, J. A., Schrijvers, A. H. G. J., Boddé, H. E. and Ponec, M. (1995). Visualization of diffusion pathways across the stratum corneum of native and in-vitro-reconstructed epidermis by confocal laser scanning microscopy. Arch. Dermatol. Res. 287,465 -473.[Medline]
Starkey, D. E., Shaffer, H. B., Burke, R. L., Forstner, M. R. J., Iverson, J. B., Janzen, F. J., Rhodin, A. G. J. and Ultsch, G. R. (2003). Molecular systematics, phylogeography, and the effects of Pleistocene glaciation in the painted turtle (Chrysemys picta) complex. Evolution 57,119 -128.[Medline]
Storey, K. B. (1990). Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am. J. Physiol. 258,R559 -R568.[Medline]
Storey, K. B. and Storey, J. M. (1992). Natural freeze tolerance in ectothermic vertebrates. Annu. Rev. Physiol. 54,619 -637.[CrossRef][Medline]
Storey, K. B. and Storey, J. M. (1996). Natural freezing survival in animals. Annu. Rev. Ecol. Syst. 27,365 -386.[CrossRef]
Storey, K. B., Storey, J. M., Brooks, S. P. J., Churchill, T. A. and Brooks, R. J. (1988). Hatchling turtles survive freezing during winter hibernation. Proc. Natl. Acad. Sci. USA 85,8350 -8354.[Abstract]
Storey, K. B., McDonald, D. G., Duman, J. G. and Storey, J. M. (1991). Blood chemistry and ice nucleating activity in hatchling painted turtles. Cryo-Lett. 12,351 -358.
Strong-Gunderson, J. M., Lee, R. E., Jr, Lee, M. R. and Riga, T. J. (1990). Ingestion of ice-nucleating active bacteria increases the supercooling point of the lady beetle Hippodamia convergens. J. Insect Physiol. 36,153 -157.[CrossRef]
Ultsch, G. R. (1989). Ecology and physiology of hibernation and overwintering among freshwater fishes, turtles, and snakes. Biol. Rev. 64,435 -516.
Weisrock, D. W. and Janzen, F. J. (1999). Thermal and fitness-related consequences of nest location in painted turtles (Chrysemys picta). Funct. Ecol. 13, 94-101.[CrossRef]
Willard, R., Packard, G. C., Packard, M. J. and Tucker, J. K. (2000). The role of the integument as a barrier to penetration of ice into overwintering hatchlings of the painted turtle (Chrysemys picta). J. Morphol. 246,150 -159.[CrossRef][Medline]
Wilson, P. W., Heneghan, A. F. and Haymet, A. D. J. (2003). Ice nucleation in nature: supercooling point (SCP) measurements and the role of heterogeneous nucleation. Cryobiology 46,88 -98.[CrossRef][Medline]
Woolverton, E. (1963). Winter survival of hatchling painted turtles in northern Minnesota. Copeia 1963,569 -570.
Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature (ed. R. E. Lee, Jr and D. L. Denlinger), pp.47 -63. New York: Chapman and Hall.
Zangerl, R. (1969). The turtle shell. In Biology of the Reptilia, vol. 1, Morphology A (ed. C. Gans), pp. 311-339. London: Academic Press.