Endogenous and exogenous ice-nucleating agents constrain supercooling in the hatchling painted turtle
Department of Zoology, Miami University, Oxford, OH 45056, USA
* Author for correspondence (e-mail: costanjp{at}muohio.edu)
Accepted 28 October 2002
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Summary |
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Key words: painted turtle, Chrysemys picta, cold hardiness, hibernation, acclimation, supercooling, ice nucleation, gut, yolk
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Introduction |
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Under idealized laboratory conditions, fully cold-hardened hatchlings can
supercool to remarkably low temperatures (e.g. -20°C), and, in fact, their
supercooling capacity is on par with that of a droplet of water
(Lee and Costanzo, 1998).
However, the hypothesis that these hatchlings survive every chilling episode
by virtue of their innate supercooling capacity (e.g.
Packard et al., 1997
;
Packard and Packard, 2001
) is
at odds with recent findings (Costanzo et al.,
1998
,
2000a
,
2001
) that the turtles are
susceptible to inoculation by ice or ice-nucleating agents (INA) in the winter
microenvironment. Environmental INA include particulates, such as sand grains,
dust and other motes, and various organic entities, including certain
microorganisms and amino acids. Even brief contact with nesting soil harboring
such agents can markedly constrain the supercooling capacity of hatchling
C. picta (Costanzo et al.,
2000a
).
A recent study of the seasonal development of cold hardiness in C.
picta showed that, whereas fully cold-hardened turtles supercool
extensively, recently hatched turtles spontaneously freeze at relatively high
temperatures (Costanzo et al.,
2000b). Packard and colleagues
(2001
) confirmed this result
and also reported that the guts of recently hatched turtles contained bits of
eggshell and soil ingested during pipping and hatching. Finding that winter
turtles had empty guts, these authors surmised that seasonal development of
supercooling capacity requires elimination of ingested matter from the
gastrointestinal tract. However, they did not determine whether the substrata
and eggshell ingested by hatchlings actually expressed ice-nucleating
activity, so this association remains conjectural. In addition, the
possibility that hatchlings harbor INA of endogenous origin, as was earlier
suggested by Costanzo et al.
(2000b
), remains untested.
Our goal was to rigorously test the hypothesis that seasonal development of supercooling capacity in hatchling C. picta is the result of attenuation or elimination of INA, of either endogenous or exogenous origin, that is present shortly after hatching. We also characterized the ice-nucleating activity of experimental substrata and eggshell in order to elucidate their influence on the supercooling capacity of hatchlings. Finally, by feeding INA commonly found in nesting soil to winter turtles, we directly evaluated the effect of these agents on hatchling cold hardiness.
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Materials and methods |
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Hatchlings were kept in darkness, denied food and free water, and
progressively acclimated to temperatures corresponding to those occurring in
C. picta nests during late summer, autumn and winter (see
Costanzo et al., 1995). Upon
hatching (or manual extrication from the shell), in mid-August, they were
exposed to 22°C, but on 1 October they were placed in an environmental
chamber that was set initially at 15°C and then changed to 10°C on 1
November. Turtles were exposed to 4°C on 1 December and were thereafter
held at this temperature. We remoistened substrata as necessary to prevent
turtles from desiccating.
We also examined C. picta that had hatched and overwintered inside natural nests located near Gimlet Lake. Nests were found by observing the nesting forays of females and were protected from predators by installing a piece of hardware cloth over each one. We excavated these nests on 6 April 2002, at which time 4-5 hatchlings per nest were briefly rinsed with water, blotted dry with paper towel, placed in plastic bags and shipped under refrigeration to Miami University, where they were kept chilled (4°C) for 1-2 days before being used in supercooling trials. Upon completion of the trials, we dissected the turtles and isolated the gut, which was later examined for the presence of endogenous and exogenous matter (see below). Temperatures experienced by these turtles during winter were recorded by miniature data loggers (Onset Computer, Tidbit; Pocasset, MA, USA) placed in the soil column adjacent to the nest cavity.
Supercooling capacity of summer and winter turtles
The primary purpose of this experiment was to elucidate the association
between the anticipated increase in supercooling capacity with cold
acclimation and elimination of ingested matter in hatchling C. picta.
Our experimental approach was to determine the supercooling capacity of
hatchlings reared on paper, vermiculite or nesting soil, both shortly after
hatching (summer) and after cold acclimation (winter). Trials with summer
turtles were conducted in late August, approximately two weeks after turtles
had emerged from their eggs and were transferred to 22°C; trials with
winter turtles were conducted in late January, after hatchlings had been
exposed to 4°C for eight weeks.
Following Costanzo et al.
(1998), we determined
supercooling capacity by progressively cooling hatchlings until they
spontaneously froze. Turtles were prepared for testing by gently brushing away
any adherent vermiculite or soil and then holding them in a sheltered box for
24 h. This procedure, performed in darkness at the prevailing acclimation
temperature, permitted evaporation of any surface moisture that otherwise
might freeze and inoculate the tissues. Turtles were placed separately in 50
ml plastic tubes and covered with a piece of plastic foam, which insulated the
hatchling and anchored a 30-gauge thermocouple (copper/constantan) in
position, with the sensing junction nearly touching the carapace. The tubes
were suspended in a refrigerated ethanol bath (Neslab, model RTE 140;
Portsmouth, NH, USA) programed to cool turtles from the acclimation
temperature to -0.4°C, at which temperature they were held for 1 h before
being further cooled (3°C h-1) until each produced a freezing
exotherm. During cooling, turtle temperature, as registered by the
thermocouple, was logged at 30-s intervals on a data logger (Omega, model
RD3752; Stamford, CT, USA). We then determined the temperature of
crystallization (Tc) from the recording and took this
value to represent the supercooling limit.
We investigated somatic compartmentalization of INA by measuring the Tc of the isolated gut, internalized yolk sac (which, in summer turtles, contained residual yolk and hereafter is termed `yolk') and carcass. Turtles used in the supercooling trials described above were removed from the bath upon appearance of the exotherm, thawed briefly on ice, euthanized by decapitation and dissected under virtually aseptic conditions. The gastrointestinal tract, from esophagus to rectum, and the yolk sac were removed and placed in tared, 0.5 ml microcentrifuge tubes, which were then weighed to the nearest 0.01 g. Samples were coated with mineral oil (in order to prevent them from dehydrating) and instrumented with a thermocouple, whose tip was placed next to, but not touching, the tissue. We chilled the tubes in a refrigerated bath, measuring the Tc of the samples as we did for intact turtles. The guts were reserved for examination of their contents (see below).
An important assumption inherent in our experimental design was that ice-nucleating activity of turtle tissues was unaltered by the nucleation event and brief freezing associated with the initial supercooling trial. Reasoning that an increase in ice-nucleating activity in any compartment necessarily diminishes the supercooling capacity of the entire turtle, we validated our assumption by comparing the Tc values determined for turtles (hatched and reared on vermiculite) subjected to two successive supercooling trials. After the first trial, conducted as described above, turtles were euthanized and left intact (rather than being dissected), coated with oil and used in a second supercooling trial. Because the mean difference between the pair of Tc values did not differ from zero (paired t-test: t=0.977, d.f.=4, P=0.384, N=5), we concluded that brief freezing, in the context of our experimental protocol, did not alter ice-nucleating activity of the tissues.
Identification of gut contents
Guts isolated from lab-reared and field-collected turtles were placed on
translucent dissecting trays and examined with a dissecting scope (Olympus
America, SZH10 Research Stereo; Melville, NY, USA). Viewing the gut under low
magnification, we mapped areas of concentration of matter on an enlarged
diagram of the digestive tract. Having been familiarized with the magnified
image of reference samples of nesting soil, vermiculite and eggshell, we then
characterized any matter as being of exogenous (substratum and/or eggshell) or
endogenous origin.
Ice-nucleating activity in experimental substrata, eggshell and
feces
We established indices of ice-nucleating activity associated with materials
that may occur in turtle gastrointestinal tracts. Samples of vermiculite and
nesting soil were collected, in August, from the boxes housing the hatchlings
and kept refrigerated until analyzed. Eggshells of turtles reared on paper
were stored at -20°C for this purpose. Eggshells were thawed, rinsed with
water, dried at 65°C and pulverized with a glass rod before use in the
assay.
We measured the ice-nucleating activity expressed in bulk samples of these
materials, and also in washings prepared from them, because water-soluble INA
may be retained in the body even after solids have been eliminated. In
addition, we characterized the INA as to whether the agents were derived from
organic matter. Interpretation of the assay results was predicated on the
facts that small volumes of pure (i.e. INA-free) water supercool extensively
and that organic INA are deactivated by high heat and pressure
(Vali, 1995). Water used in
the assays was obtained from a reverse-osmosis ultrapurification system (0.2
µm filter, Dayton Water Systems; Dayton, OH, USA) and sterilized in an
autoclave. To guard against contaminating samples with ambient INA, all
vessels and utensils used in the assays were thoroughly cleaned and autoclaved
before use. Potency of constituent INA was gauged relative to the
Tc of samples of water.
Following Costanzo et al.
(1998), we measured
ice-nucleating activity expressed in natural (non-autoclaved) and autoclaved
samples of vermiculite, nesting soil and eggshell. A 100 mm3 sample
of air-dried material was placed in a 0.5 ml polypropylene microcentrifuge
tube to which we added 12.5 µl of water. The contents were mixed by
vortexing and then consolidated by gentle centrifugation
(180g, 3 min). We taped the sensing junction of a 36-gauge
copper/constantan thermocouple to the exterior of each tube, which was then
inserted into a dry, 20 ml test tube. Samples (N=5 replicates) of
each material were chilled to 0°C in a refrigerated ethanol bath and then
further cooled (1.5°C min-1) until the water within them froze.
The Tc of each sample was read from the output of a data
logger to which the thermocouples were connected.
We also measured ice-nucleating activity expressed in washings prepared from natural and autoclaved samples of vermiculite, nesting soil and eggshell (N=5 replicates of each material). Washings were prepared by adding water to a 100 mm3 sample of each material, vortexing the mixture for 60s and isolating the particulates by centrifugation (2000g, 5 min). To avoid excessive dilution of INA, samples were washed with the smallest volume of water, which, beyond that absorbed by the sample, yielded the minimum volume of washing needed for assay (150-300 µl). The supernatant was expressed through a 5 µm disk filter to remove any fine particulates, and a 10 µl aliquot of the filtrate was drawn into the center of a 20 µl glass microcapillary tube, such that the fluid column was bounded by equal volumes of air. The ends of the tube were sealed with clay and the sensing junction of a 36-gauge copper/constantan thermocouple was taped to its center. Five tubes prepared from each washing were chilled, as described above, until each exhibited a freezing exotherm. We took the average of the five Tc values to represent each sample.
During their first month of life, hatchlings reared on paper produced feces that we weighed, placed individually in a microcentrifuge tube and stored at -20°C for subsequent analysis of ice-nucleating activity. No droppings were found after 10 September. Feces may have been produced by turtles reared on vermiculite or soil, but we could not confirm their presence. Ice-nucleating activity associated with the feces was determined as described for other materials.
Experimental ingestion of INA
We inoculated the guts of live hatchlings with two distinct types of INA
commonly found in nesting soil in order to compare their effects on the
supercooling capacity of winter turtles. Turtles were administered autoclaved
nesting soil, which contained soil particles but no organic INA, or a soil
washing, which contained organic INA but no soil particles. Control turtles
ingested only the water vehicle, or were sham treated (i.e. they ingested
nothing). We conducted the experiments in mid-winter, using cold-acclimated
turtles reared on vermiculite; hatchlings cultured under these conditions have
an extensive capacity for supercooling
(Costanzo et al., 2000b;
Packard et al., 2001
).
We prepared for the experiment by cleaning and air drying the turtles (as described above). We filled a 25 µl syringe (Hamilton; Reno, NV, USA) with water obtained from a reverse-osmosis ultrapurification system and sterilized in an autoclave, expelling 15 µl of this volume into a length of polyethylene tubing (PE50) attached to the hub. An aliquot of soil slurry (1.0 g autoclaved nesting soil mixed with 300 µl water), soil washing (prepared by vortexing 1.0 g nonautoclaved soil with 500 µl water, centrifuging the mixture and passing the supernatant through a 0.5 µm filter) or water was then drawn into the tube. Using broad forceps, we withdrew the head from the shell, stabilizing the extended neck by placing the thumb and forefinger behind the skull. The blade of a second pair of forceps was inserted between the maxilla and mandible, and, by applying gentle downward pressure, the mandible yielded to expose the buccal cavity. We inserted the tubing and dispensed 10 µl of the prescribed material (air, in the case of sham-treated animals) into the pharynx. Turtles were returned to their cages and used in supercooling trials, in general the following day. However, one group was tested for supercooling capacity 33 days after turtles were fed soil washing. We observed no ill effects of forced ingestion on any of the turtles used in this experiment.
Statistical inferences
Sample means were compared using one- or two-factor analysis of variance
(ANOVA) followed by appropriate multiple comparisons tests. Significance of
statistical analyses was accepted at P0.05. Mean values are
reported ± S.E.M.
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Results |
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Experiments in which we measured the Tc of the isolated
yolk and gut, and the carcass were intended to elucidate compartmentalization
of INA within the live turtle. However, because the ice-nucleating activity
expressed in the carcass and gut was generally higher than that found in the
intact turtle (Table 1), these
samples must have acquired INA that were not present in life. This result is
not readily explained, but perhaps the novel activity is a consequence of
tissue damage or contamination with ambient INA sustained during the
dissection (e.g. Baust and Zachariassen,
1983). Furthermore, we cannot exclude the possibility that samples
of yolk also expressed unnaturally high levels of ice-nucleating activity.
These cautions aside, the experimental results yielded some useful
information. For instance, the greater supercooling capacity in winter
turtles, as compared with summer turtles, was invariably reflected in the
results for yolk, gut and carcass (Table
1). In addition, because the Tc values
determined for yolk corresponded with those determined for live turtles,
ice-nucleating activity associated with this compartment may effectively set
the limit of supercooling. However, ice-nucleating activity associated with
the gut and carcass more closely matched the Tc of the
summer turtles reared on nesting soil
(Table 1). These qualitative
assessments were corroborated by the results of multiple regression analyses
testing the effects of three independent variables (yolk
Tc, gut Tc and carcass
Tc) on the Tc of live turtles. This
model was significant for turtles reared on paper
(F3,14=5.37, P=0.011,
r2=0.535), vermiculite (F3,16=3.59,
P=0.037, r2=0.402) and nesting soil
(F3,16=19.68, P<0.0001,
r2=0.787). Yolk Tc was the best
predictor of live turtle Tc for hatchlings reared on paper
or vermiculite; however, gut Tc was the most important
factor for turtles reared on nesting soil.
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Morphometric variables
Because turtles assigned to the summer and winter groups did not differ in
carapace length (F1,54=0.43, P=0.52) or plastron
length (F1,54=0.08, P=0.78), any difference in
response variables can be ascribed to a particular experimental treatment,
rather than the influence of body size
(Table 2). Comparing values for
summer turtles and winter turtles indicated that body mass decreased
(F1,54=26.74, P<0.0001) by 15-24% during cold
acclimation, and that this change was, in part, due to yolk consumption
(F1,54=47.23, P<0.0001) and a decrease in the
mass of the gastrointestinal tract (F1,54=32.06,
P<0.0001). The latter, which chiefly reflected elimination of
ingested matter, was significant only for the turtles reared on vermiculite or
nesting soil (Table 2).
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Identification of substances in turtle guts
Examination of the dissected guts, isolated from the specimens used in the
supercooling trials, showed that the summer turtles reared on vermiculite or
nesting soil had ingested large amounts of substrata
(Table 3). Approximately 50% of
them also had ingested eggshell fragments. Hatchlings purged their guts during
cold acclimation, as neither substratum nor eggshell was found in the guts of
winter turtles.
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Except for one summer turtle, which had ingested a few paper fibers, the guts of turtles reared on paper were devoid of exogenous matter (Table 3). The intestines of eight of the 10 hatchlings in this group contained a small quantity of a yellowish-green, amorphous substance that may have been derived from yolk. We found this material, in trace amounts, in only three of the 10 hatchlings examined in winter.
Ice-nucleating activity of experimental substrates, eggshell and
turtle feces
Bulk samples of nesting soil catalyzed the freezing of water at -5°C,
indicating that they contained potent INA
(Table 4). By contrast, samples
of ultrapurified water supercooled to approximately -18°C. Ice-nucleating
activity was also expressed in vermiculite and eggshell, but the activity
temperature was lower than that determined for soil. Autoclaving, which
destroys organic INA, reduced the activity in soil (t=3.08,
P=0.015, N=10) but not in vermiculite (t=1.15,
P=0.28, N=10) or eggshell (t=0.46, P=0.66,
N=10). Washings prepared from nesting soil contained potent INA,
whereas washings of vermiculite or eggshell supercooled extensively and
therefore lacked INA (Table 4).
Autoclaving the nesting soil greatly diminished (t=10.39,
P<0.0001, N=10) the ice-nucleating activity expressed in
washings prepared therewith, suggesting that the water-soluble INA in nesting
soil was organic.
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Feces produced by the summer turtles reared on paper contained weak INA that were sensitive to autoclaving (t=3.82, P=0.005, N=10; Table 4). Given that the washings prepared from the feces expressed little ice-nucleating activity, the constituent INA were probably insoluble.
Supercooling capacity of hatchlings ingesting INA in nesting
soil
Experimental ingestion of INA strongly influenced the supercooling capacity
of winter turtles (F4,24=208.50, P<0.0001;
Fig. 2). Whereas the
sham-treated turtles and the turtles ingesting water supercooled extensively
(as did other winter turtles reared on vermiculite;
Fig. 1), turtles ingesting a
small quantity of autoclaved soil, or washing prepared from unadulterated
soil, froze at relatively high temperatures. Soil washing was the more potent
INA (Fig. 2). The mean
Tc of turtles tested 33 days after ingesting soil washing
(-7.5±0.1°C) was similar to that determined for turtles tested 24h
afterwards (-6.8±0.04°C).
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Supercooling capacity of hatchlings collected from natural nests
We determined the Tc for 2-4 live turtles collected
from each of nine nests on 6 April 2002. Tc values
averaged for turtles in each nest ranged from -6.4°C to -9.1°C, and
the mean Tc for all 32 turtles was -7.5°C. The
intestines of 22 (69%) of the turtles contained a white, caseous material,
apparently of endogenous origin, that was not observed in our
laboratory-reared animals. This material did not influence the supercooling
limit, as the Tc of the affected turtles was identical
(t=0.007, d.f.=30, P=0.99) to that of the turtles lacking
it. The mean mass of the gut (101 mg) and yolk sac (40 mg) of the
field-collected turtles was comparable with values determined for the
laboratory-reared winter turtles (Table
2). The thermal minimum recorded in each nest ranged from
-6.1°C to -15.0°C (mean, -9.5°C); mortality ranged from 0 to 33.3%
(mean, 12.6%).
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Discussion |
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Changes in supercooling capacity
Our finding that supercooling capacity was greater in winter turtles than
in summer turtles concurs with a previous report
(Costanzo et al., 2000b). In
addition, our finding that the guts of nearly all turtles reared on
vermiculite or nesting soil were distended and packed with these materials
accords with the observations of Packard et al.
(2001
), who found that C.
picta commonly ingested substratum during hatching or shortly thereafter.
On the other hand, relatively few of our turtles ingested eggshell, which
purportedly provides a supplemental source of calcium needed for ossification
of the maturing hatchling (Packard et al.,
2000
,
2001
). The contention that
recently hatched C. picta consume eggshell for this reason is
inconsistent with the appearance of the ingested eggshell fragments, which
were dense and sharp-edged, rather than eroded from mineral leaching.
Furthermore, eggshell constituted a small proportion of the volume of the
matter ingested, and the guts of some turtles contained substratum but no
eggshell (Table 3). We believe
that hatchlings intend to ingest primarily substratum (i.e. nesting soil),
rather than eggshell, although the adaptive value of doing so is unclear.
Perhaps the passage of soil aids in the elimination of feces or otherwise
promotes proper gut function. In addition, ingesting soil (and eggshell) may
serve to inoculate hatchlings with normal intestinal flora. Turtles urinate on
their nests after ovipositing (see Carr,
1952
), possibly introducing beneficial microbes, flushed from the
cloaca, into the nest chamber. Coprophagy permits some neonatal reptiles to
inoculate themselves with the symbiotic gut flora required for proper
digestive efficiency (Troyer,
1983
; Lance and Morafka,
2001
).
Among the hatchlings reared on vermiculite, greater supercooling capacity
in winter turtles, as compared with summer turtles, was strongly associated
with elimination of ingested substratum and eggshell. Packard et al.
(2001) also found this
association, reporting that recently hatched turtles harboring these materials
in the gut froze at -7.1°C, whereas cohorts having empty guts supercooled
to -12.9°C. Nevertheless, our finding that hatchlings reared on
vermiculite supercooled to the same degree as the hatchlings reared on paper,
which consumed nothing, argues that the presence of vermiculite and eggshell
in the gut has no effect on supercooling capacity. This conclusion is
bolstered by results of a post-hoc analysis of the data for
vermiculite-reared turtles: there was no difference (t=0.85, d.f.=8,
P=0.38) in Tc between the hatchlings that had
ingested eggshell and the hatchlings that had not. Vermiculite and eggshell
fragments are moderately active INA (Table
4); however, these materials did not limit supercooling in summer
hatchlings.
Evidence for endogenous INA in summer turtles
Our finding that supercooling capacity improved with cold acclimation in
the paper-reared turtles revealed that seasonal development of cold hardiness
derives from elimination of endogenous INA that are present in summer turtles.
The exact identity of the INA is unknown, although our results suggest they
are associated with residual yolk. We found good accord between ice-nucleating
activity in isolated yolk and the Tc values determined for
live turtles reared on paper or vermiculite. Also, egested feces, probably
derived from yolk, which is heavily mobilized after hatching
(Table 2; see
Filoramo and Janzen, 1999;
Lance and Morafka, 2001
)
contained INA whose activity also matched the supercooling limit of these
summer turtles (Tables 1,
4). Given their apparent
insolubility, defecation should eliminate these INA from the body, and,
indeed, supercooling capacity was markedly improved in the winter turtles,
which had voided their guts. Therefore, seasonal development of cold hardiness
in hatchling C. picta apparently depends on the elimination of INA
that would otherwise constrain supercooling capacity rather than production of
cryoprotective solutes or specialized proteins (see
Costanzo et al., 2000b
).
Our results provide few clues about the factors triggering gut evacuation
in recently hatched C. picta, although some evidence suggests that
gut clearance may be stimulated by cold exposure. For example, hatchlings
reared on vermiculite and subjected to a naturalistic cold-acclimation regimen
apparently developed their full capacity for supercooling in late November,
after a four-week exposure to 10°C, because the Tc of
4°C-acclimated turtles, tested in mid-winter, was virtually unchanged
(Costanzo et al., 2000b). On
the other hand, gut evacuation might be promoted by maturational changes, as
at least some of our turtles defecated within two weeks of hatching, even
though ambient temperature remained high.
Effect of ingested INA on supercooling capacity
Our results suggest that ingestion of exogenous matter may or may not
influence the supercooling capacity of hatchling C. picta. The effect
may be negligible if, as was the case with vermiculite and eggshell, the
ice-nucleating activity of the ingesta is similar to, or less than, the
activity of any endogenous INA. However, in the case of nesting soil, which
commonly harbors a host of relatively potent INA (Costanzo et al.,
1998,
2000a
,
2001
), the effect may strongly
influence winter survival. Purging the gut of nesting soil during cold
acclimation may improve supercooling capacity modestly, but ultimately the
degree of cold hardiness the hatchlings attain is inferior to that achieved by
turtles that do not ingest soil.
Our study confirmed previous findings
(Costanzo et al., 2000a;
Packard et al., 2001
) that
C. picta hatched and reared on nesting soil supercool modestly as
compared with turtles kept on vermiculite. Turtles are readily contaminated
with environmental INA, as immersing vermiculite-reared hatchlings in nesting
soil for as little as 48 h markedly reduces their supercooling capacity
(Costanzo et al., 2000a
).
Ingestion is probably an important means by which INA gain access to the body
fluids of turtles; however, transmission through non-oral apertures (cloaca,
nares, ocular openings) or the integument may occur in recently hatched and
older hatchlings (Costanzo et al.,
2000a
). Our results for turtles reared on nesting soil, showing
limited supercooling in both the isolated gut and the carcass
(Table 1), imply that
contamination with exogenous INA occurred via multiple routes.
Nesting soil contains at least two classes of INA that demonstrably impact
cold hardiness in hatchling turtles. These include various mineral
particulates, which are water insoluble and are largely unaffected by high
temperature and pressure, as well as water-soluble, autoclave-sensitive
agents, which may be derived from ice-nucleating microorganisms
(Costanzo et al., 2000a).
Experimental ingestion of either type dramatically constrained supercooling,
although the effect of the organic INA was more pronounced, consistent with
its greater ice-nucleating activity
(Tanaka, 1994
). Hatchlings
reared on nesting soil eliminated particulate INA through defecation, and,
apparently, this process improved their supercooling capacity. However, they
probably retained at least some organic INA, because the winter turtles were
unable to supercool to the same extent as turtles in the other groups. Their
solubility and small size (see Costanzo et
al., 2000a
) may render these agents highly vagile and, thus,
especially difficult to purge from the body. Indeed, the relatively high
Tc determined for the yolk isolated from soil-reared
turtles (Table 1) suggests that
this organ was contaminated with INA, which may have dispersed from the gut
via the yolk stalk, Meckel's diverticulum
(Ewert, 1985
;
Lance and Morafka, 2001
).
Furthermore, results of our experimental ingestion trials indicate that
organic INA found in nesting soil can long retain their ice-nucleating
activity in the internal milieu.
Ecological implications of contamination by soil INA
In reviewing the available literature, Costanzo et al.
(2000a) identified a
dichotomous pattern in which C. picta hatching under natural
conditions (or those otherwise exposed to nesting soil) supercooled much less
extensively than laboratory-reared turtles hatched on vermiculite, an
essentially INA-free, artificial substratum, attributing the variation to
contamination of the field-collected turtles with INA present in the nesting
soil. Our present work not only bolsters this explanation but also confirms
ingestion of nesting soil as a primary avenue of INA contamination.
Furthermore, our finding that hatchlings purge particulate INA from their
guts, as well as the similarity between the Tc of turtles
experimentally ingesting organic INA and the hatchlings extricated from
natural nests in winter (Packard et al.,
2001
; present study), may indicate that the organic class of soil
INA is of greater importance to the cold hardiness of hatchling C.
picta.
Some of the results for naturally hibernating turtles were perplexing. For
example, we cannot explain why the average Tc for these
animals was approximately 2°C higher than the average minimum temperature
recorded within their nests. In addition, unexpectedly, we found that
supercooling capacity differed between the field-collected turtles
(Tc-7.5°C) and the winter turtles reared on
nesting soil (Tc
-10°C). Packard and colleagues
(2001
) obtained the same
result, speculating that their field-collected turtles, sampled in November,
had not yet experienced a level of cold sufficient to induce full development
of supercooling capacity. However, our field-collected turtles, sampled at
winter's end, had experienced temperatures much lower than those encountered
by our lab-reared turtles. An alternative explanation is that the organic INA
influencing turtle Tc, being sensitive to changes in their
thermal environment (Costanzo et al.,
2000a
), lost potency under static laboratory conditions, but
retained, or even increased, their ice-nucleating activity under the more
favorable thermal and trophic conditions in natural nests
(Lindow et al., 1982
;
Nemecek-Marshall et al., 1993
;
Fall and Fall, 1998
).
Our conclusion that supercooling is constrained by INA that persist in the
body, even after ingested soil has been eliminated from the gut, raises new
questions about the efficacy of supercooling as a cold-hardiness strategy in
hatchling C. picta. Whereas some authors (e.g.
Packard and Packard, 2001)
have maintained that their supercooling capacity meets or exceeds the demands
imposed by the winter thermal environment, this argument derives from studies
of hatchlings reared on a vermiculite substratum, and it is now clear that
such experiments grossly overestimate the supercooling capacity of this
species. Furthermore, recent work has established that, under certain
environmental conditions, hatchling C. picta are highly susceptible
to inoculation by ice or INA within the nest microenvironment (Costanzo et
al., 1998
,
2000a
,
2001
). Considering the high
efficacy of endogenous and exogenous INA, and the fact that the lower limit
for freeze tolerance is approximately -4°C, it is remarkable that these
turtles can survive winter inside nests that attain minimum temperatures below
-12°C (Packard et al.,
1997
; Packard and Packard,
2001
) and even -15°C (present study).
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References |
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