Lactate accumulation, glycogen depletion, and shell composition of hatchling turtles during simulated aquatic hibernation
1 Department of Molecular Pharmacology, Physiology and Biotechnology, Brown
University, Providence, Rhode Island, 02912, USA
2 Department of Biological Sciences, University of Alabama, Tuscaloosa,
Alabama, 35487, USA
* Author for correspondence at address 1 (e-mail: scott_reese{at}brown.edu)
Accepted 7 June 2004
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Summary |
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Key words: hatchling turtle, lactate, buffering, hibernation physiology, anoxia, shell composition
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Introduction |
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Differences in anoxia tolerance can affect habitat selection in adult
turtles and may influence life history traits of hatchling conspecifics.
Turtles lay eggs in shallow nests on land, which hatch in late summer or early
fall. Snapping turtle hatchlings emerge immediately after hatching and move to
water (Congdon et al., 1987;
Costanzo et al., 1995
,
1999
), where they spend their
first winter submerged. In contrast, map and painted turtle hatchlings remain
in the nest, surviving the freezing temperatures of winter via
supercooling or freeze-tolerance, and emerge from the nest the following
spring (map turtles: Costanzo et al.,
2001b
; Pappas et al.,
2000
; Baker et al.,
2003
; painted turtles:
Packard, 1997
;
Weisrock and Janzen 1999
).
Delayed emergence by hatchlings has been viewed as an advantage, allowing
young animals to minimize exposure to predators during late summer and early
autumn, when resources are in decline. The hatchlings then enter the water in
spring, when resources are increasing in quality and abundance, allowing the
turtles to grow rapidly (Wilbur,
1975
; Gibbons and Nelson,
1978
).
We hypothesized that hatchling turtles spending their first winter aquatically possess similar abilities for dealing with potential hypoxia/anoxia as previously described for adults. We also hypothesized that overwintering in the nest may be an evolutionary response to the relatively low anoxia tolerance of hatchlings, and that those species that do not overwinter terrestrially are either at risk of death during the first winter, or must hibernate in a different microenvironment than adults.
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Materials and methods |
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There were three experimental treatments, all at 3°C, for hatchling turtles. Containers of similar dimensions were used for each treatment and all treatments were run concurrently. The first group was submerged, without access to air, in air-equilibrated water (PO2=158 mmHg). The second group was forcibly submerged in nitrogen-equilibrated water, also without access to air (PO2<5 mmHg). The final group was placed in sealed containers (except for inlet and outlet ports) and humidified nitrogen was passed through the containers to maintain a gaseous anoxia. Turtles were checked daily to determine survival. Animals were removed on days 5, 10, 15, 20, 25, 30 and 40 (N=515) of aquatic or gaseous anoxic exposure for sampling, as described below. Animals submerged in normoxic water were sampled on days 10, 25, 50, 75, 100, 125 and 150 (N=515).
Lactate and glycogen
Animals used for the determinations of whole-body concentrations of lactate
and glycogen were removed from the experimental treatment, immediately frozen
in liquid nitrogen, and stored at 80°C until analysis. To measure
whole body lactate concentrations, animals were homogenized (Virtis, Gardiner,
NY, USA) in 0.6 mol l1 perchloric acid (5x volume to
mass) and a 0.2 ml sample of the homogenate was saved for glycogen analysis
(see below). The remainder of the homogenate was centrifuged at 4°C (3000
g for 10 min, Beckman GS-15R, Fullerton, CA, USA), the
supernatant decanted and filtered, and the filtrate centrifuged at 4°C (12
000 g for 10 min, Beckman GS-15R). The supernatant from the
second centrifugation was decanted and used for the determination of lactate
and free glucose concentrations (YSI 2300 Stat Plus-D lactate/glucose
analyzer, Yellow Springs, OH, USA).
Glycogen was determined with amyloglucosidase as described by Keppler and
Decker (1974). The sample of
homogenate for glycogen analysis was incubated (2 h at 40°C with
continuous shaking in stoppered test tubes) with 0.1 ml of a 1 mol
l1 KHCO3 and 2.0 ml of a 10 mg
ml1 amyloglucosidase (Sigma #A-3514, St Louis, MO, USA)
solution. The amyloglucosidase was suspended in a 0.2 mol l1
acetate buffer, pH 4.8. The incubation was stopped by adding 1.0 ml of cold
0.6 mol l1 perchloric acid. The incubated solution was
centrifuged (10 000 g for 15 min) and the supernatant analyzed
for glucose (YSI 2300 Stat Plus-D lactate/glucose analyzer). Glycogen content
was calculated as the glucose concentration measured by this method minus the
free glucose concentration measured as above.
Shell analyses
Turtles used for shell analyses were removed from the treatment, patted dry
and weighed. Animals were then killed by decapitation and pithing, and the
plastron and carapace were dissected out and weighed. The shells were placed
in a drying oven and dried to constant mass. Dried shells were randomly
assigned to carbonate analysis or ion analysis. Dried shell was powdered at
liquid nitrogen temperatures (SPEX 6700 freezer mill, Metuchen, NJ, USA) and
carbonate content was measured using an incubation system previously described
(Jackson et al., 1999).
Pre-weighed shell powder was placed in a custom sample chamber that had two
compartments, the contents of which could be combined without breaking a seal.
Powder was added to 15 ml of2 mol l1 HCl without opening the
chamber, liberating any carbonate as CO2 gas. Nitrogen gas was
humidified, passed through the chamber, dried, and then analyzed for
CO2 content (Applied Electrochemistry CD-3A CO2
analyzer, Naperville, IL, USA). The CO2 analyzer was calibrated
with a precision gas (verified by analysis with a Scholander 0.5 cc analyzer),
the signal digitized (Biopac MP100, Goleta, CA, USA), and the resultant trace
integrated (Acknowledge 3.7) to determine total CO2 content of the
gas passing through the system. Flow rate through the system was measured
using a Brooks Vol-U-Meter (Hatfield, PA, USA).
For ion analyses, dried shell was ashed (24 h at 450°C, Thermolyne 1300 furnace, Dubuque, IA, USA) and weighed. The ash was dissolved in 1 mol l1 HCl (20x volume to mass). The resultant solution was used to measure [Na+] and [K+] (Instrumentation Laboratory 943 flame photometer, Lexington, MA, USA). Total magnesium was measured following a further dilution to 60x (Perkin-Elmer 280 atomic absorption spectrophotometer, Boston, MA, USA). Total calcium (Perkin-Elmer 280 atomic absorption spectrophotometer) and inorganic phosphates (Sigma kit 670-A with a Milton Roy Spectronic 601 spectrophotometer) were measured following a dilution to 1800x.
Statistics
Data were analyzed with STATISTICA'99 edition (Statsoft Inc., Tulsa, OK,
USA). Analysis of covariance (ANCOVA) was used to compare lactate accumulation
and glycogen depletion among species with time and mass as covariates.
KruskalWallis analysis of variance (ANOVA) or MannWhitney
U tests (where appropriate) were used to test end-point
determinations in [lactate], [glycogen], energy utilization, shell
characteristics and shell composition. Post-hoc comparisons used
Tukey's HSD for unequal N values. Significance was accepted at the
P<0.05 level and all values are mean ± S.E.M.
The University of Alabama and the Brown University Internal Animal Care and
Use Committees (IACUC #26-03) approved this study.
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Results |
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Hatchling Chrysemys picta survived 40 days submerged in anoxic water, Chelydra serpentina survived 30 days and Graptemys geographica survived 15 days. All of the animals submerged in anoxic water accumulated lactate, and at similar rates (Fig. 1A), but to different levels by the survival limit (3.5±0.1 mg g1 in G. geographica, 5.5±0.1 mg g1 in C. serpentina, and 6.7±0.2 in C. picta) (15, 30 and 40 days, respectively; Fig. 1A). When held in gaseous nitrogen, lactate accumulated more rapidly than during anoxic submergence (0.36±0.03 vs. 0.24±0.01 mg g1 day1 in C. picta, 0.39±0.01 vs. 0.28±0.02 mg g1 day1 in G. geographica, and 0.43±0.02 vs. 0.33±0.01 mg g1 day1 in C. serpentina after 10 days of anoxic exposure) and survivability decreased (maximum survival was 10 days for G. geographica, 20 days for C. serpentina, and 30 days for C. picta in gaseous anoxia). Anoxic glycogen utilization rates were 10x faster than normoxic rates (Fig. 1B). Graptemys geographica had used 48%, Chelydra serpentina used 67%, and Chrysemys picta used 66% of their glycogen by the limits of survival. Utilization rates were slower in C. picta than in C. serpentina or G. geographica.
Shell
The amount of shell (as % of wet body mass) differed among species with
G. geographica (23.3±0.4% shell) higher than C.
serpentina (18.3±0.3%) or C. picta (19.4±0.4%;
Fig. 2). There were some
differences in the composition of shell among species of hatchling turtles.
Chelydra serpentina had higher water content (80.0±0.3%) and
lower organic content (16.9±0.4%) than C. picta
(74.3±0.7% and 22.9±0.9%, respectively), which was higher and
lower, respectively, than in G. geographica (69.4±0.4% and
27.2±0.6%, respectively; Fig.
2). However, ash content was the same among hatchlings (3.0%;
Fig. 2).
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Shell [Na+], [K+] and total Mg were at similar levels in control animals, but G. geographica had higher total Ca and [Pi] (Tables 1 and 2). Total CO2 was highest in control C. serpentina. Submergence in normoxic water for 7581 days had little impact on shell composition. [Pi] tended to increase in all three species, C. picta had elevated [Na+] and [K+], and C. serpentina had elevated total Ca. Shell CO2 was unaffected by normoxic submergence.
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Nitrogen exposure did not affect shell composition any differently than submergence in anoxic water, except for [K+] in C. serpentina, which was elevated after anoxic submergence (Table 1). Anoxic treatment had no dramatic effects on shell ionic composition. Chelydra serpentina had elevated total Ca and [Pi] after 31 days of anoxia and Chrysemys picta had elevated [Pi]. Total CO2 decreased to similar levels in all three species after anoxia treatment.
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Discussion |
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Hatchling turtles placed in nitrogen accumulated lactate and lost glycogen
faster than their conspecifics submerged in anoxic water, indicating a
reduction in metabolism due to submergence. A dramatic depression in
metabolism (10 000x below that of a similarly sized, euthermic, resting
mammal; Jackson, 2002) is a
mechanism central for extended survival in adult painted turtles
(Jackson, 2002
) and has been
attributed to the inherent lowering of metabolism in all ectotherms
(Bennett and Ruben, 1979
), the
Q10 effect of temperature
(Bennett and Dawson, 1976
;
Herbert and Jackson, 1985
),
and the reduction induced by anoxia
(Jackson, 1968
;
Buck et al., 1993
). The reduced
metabolism of hatchling turtles due to submergence, independent of
[O2] or temperature, may also be applicable to adult turtle
hibernation, and thus would be another factor influencing whole-animal
metabolic reduction.
Compared to adults of the same species, hatchling turtles in this study had
significantly shorter survival times when submerged in anoxic water. Adult
Chrysemys picta bellii can survive submerged in anoxic water for 150
days (Ultsch and Jackson,
1982; S. A. Reese, C. E. Crocker, D. C. Jackson and G. R. Ultsch,
unpublished data) while conspecific hatchlings survive only 40 days. Similar
reductions of survival time were seen in hatchling turtles of other species
(50 days vs. 15 days in Graptemys geographica,
Reese et al., 2001
; and 100
days vs. 30 days in Chelydra serpentina,
Reese et al., 2002
). This
represents a little over a 3x shorter survival time of hatchling turtles
whether they are anoxia-tolerant (e.g. C. picta and C.
serpentina) or anoxia-intolerant (e.g. G. geographica) as adults
and whether they spend their first winter aquatically (e.g. C.
serpentina) or terrestrially (e.g. C. picta and G.
geographica) as hatchlings. Two integrative physiological mechanisms are
central to the anoxia tolerance of adult painted turtles. First, mentioned
above, is a dramatic reduction in metabolic rate
(Jackson, 1968
;
Jackson and Heisler, 1982
;
Jackson et al., 2000
) and
second, the effective buffering of lactic acid (Jackson et al.,
1999
,
2000
;
Jackson, 2000
). The lower
anoxia tolerance of hatchling turtles may be attributed to inferior
effectiveness of these mechanisms.
Anoxia tolerance
Measurements of whole-body [lactate] in the present study permit us to
calculate anaerobic metabolic rate directly. Anoxic metabolism, in the form of
adult whole-body [lactate], has not been measured in hibernating turtles;
however, Gatten (1981)
measured whole-body [lactate] after 16 days of cold, anoxic submergence in
painted turtles and estimates of whole-body [lactate] for cold, anoxic painted
turtles have been made from plasma [lactate] using water content of various
body compartments (Jackson,
1997
). We assume that C. serpentina and G.
geographica are similar to C. picta in the relative amount of
lactate distributed in various body compartments. After 10 days of anoxic
submergence, adult C. picta have whole-body [lactate] of 2.25 mg
g1 (Gatten,
1981
) while hatchling [lactate] range from 2.363.27 mg
g1, indicating very little difference in metabolic rate.
Adult C. picta have accumulated 4.7 mg g1 of
lactate after 40 days of anoxic submergence, while hatchlings have accumulated
6.7 mg g1 (40 days is the limit of hatchling C.
picta survival). A similar comparison for C. serpentina at 30
days yields 4.2 mg g1 for adults and 5.5 mg
g1 for hatchlings (30 days is the limit of hatchling C.
serpentina survival), while for G. geographica adults accumulate
3.7 mg g1 after 15 days and hatchlings 3.4 mg
g1 (15 days is the limit of hatchling G.
geographica survival). Although the coarse nature of the calculation for
adult [lactate] prevents us from concluding a dramatic difference between
adults and hatchlings, taking the greatest difference (e.g. C. picta)
there is still only
1.4-fold increase in metabolic rate, which cannot
account for the threefold reduction in survival time seen in hatchlings from
all three species.
Lactic acid buffering
Adult painted turtles can maintain a viable pH by the release of calcium
and magnesium carbonates from the bone into the extracellular fluid (ECF), and
by sequestration of lactate within bone, buffering it in situ and
removing it from the ECF (Jackson et al.,
2000,
1999
;
Jackson, 2000
). In adult
animals, the first part of this mechanism is readily apparent as a loss of
Mg2+ from the shell (Jackson et
al., 2000
) and accumulation of Ca2+ and Mg2+
in vivo to similar levels as seen in bone incubated at the same pH
in vitro (Jackson et al.,
1999
). In addition, loss of CO2 from the shell during
incubation is indicative of mobilization of carbonates
(Jackson et al., 1999
) and is
readily apparent during anoxic submergence in intact animals
(Warburton and Jackson, 1995
).
Although there is no loss of calcium or magnesium from the shells of anoxic
hatchlings, there is a decrease in CO2 content
(Table 2). However, when
compared to the initial shell [CO2] in adult painted turtles (1.325
mmol g1; Warburton and
Jackson, 1995
) and the magnitude of CO2 loss in adult
painted turtles for a given amount of lactate accumulated
(Fig. 3), hatchling turtles do
not appear to have adequate buffer stores.
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Two aspects of hatchling turtles account for their inability to tolerate
anoxic submergence as well as their conspecific adults. The first is the
reduced content of bone, particularly in the shell. Bone ossification in
turtles occurs over the first year of the animal's life after emerging from
the nest (Ewert, 1985), thus
hatchling turtles do not have substantial amounts of bone to utilize as an
ionic buffer reserve. This is readily apparent when comparing adult and
hatchling shell ash content even though the relative amount of shell on the
hatchlings is similar to that of adults
(Fig. 2). Secondly, the
buffering content of the bone that is available in hatchling turtles is
reduced when compared to adult turtles. The Ca:P ratio in adult turtles is 2:1
while in hatchling turtles it ranges from 1.41.5:1. This difference in
Ca:P ratios is attributable to reduced calcium carbonates within the structure
of the bone, apparent from the reduced CO2 content of hatchling
bone vs. adult bone. Thus the small amount of bone that is present in
hatchling turtles is very low in the available buffer stores that adults
normally utilize for dealing with lactate accumulation.
Ecological considerations
Delayed emergence has been viewed as a strategy that allows hatchling
turtles to enter an environment expanding in resources
(Wilbur, 1975;
Gibbons and Nelson, 1978
).
However, since adult painted turtles routinely inhabit aquatic systems that
are likely to become hypoxic or anoxic during winter months
(Ultsch, 1989
), evolution of
delayed emergence may have been influenced by the hatchling turtle's inability
to tolerate long-term anoxia.
Map turtles are anoxia-intolerant as adults, and thus do not inhabit
aquatic systems that are likely to become hypoxic
(Crocker et al., 2000;
Reese et al., 2001
). Hatchling
map turtles display delayed emergence, but they are unlikely to experience
long-term anoxia, so development of this overwintering strategy may be more
influenced by spring resource conditions
(Baker et al., 2003
). The
limited anoxia tolerance that is displayed in hatchling map turtles may be
important for survival in a supercooled state. During supercooling, blood flow
may be minimal or nil and the animals may experience a functional hypoxia that
lasts for several days (Birchard and
Packard, 1997
; Hartley et al.,
2000
). Thus an ability to deal with increases in lactate may be
important for surviving these subfreezing episodes in map and painted turtles
(Costanzo et al., 2001a
).
The shorter survival time of hatchling turtles submerged in anoxic water may have some consequences for the life history of a species. Populations of C. serpentina that inhabit aquatic ecosystems with the potential for hypoxic/anoxic winter conditions (e.g. swamps and eutrophic ponds) will probably have large hatchling mortality during winter-kill years unless the hatchlings can find a hibernaculum that contains O2 for most of the winter. Because snapping turtles are a long-lived species, a single year of winter-kill that results in low recruitment may not have a telling effect on the total population; however, several consecutive years of low O2 may produce a population bottleneck. A traditional population bottleneck has been recognized in all species of turtles due to the high mortality associated with nesting and emergence. In this new bottleneck, only those hatchlings that can find microenvironments within the aquatic system that will remain oxygenated for the majority of the winter months will survive to the following spring. It is also possible that there is segregated hibernation among adults and juveniles, with juveniles actively seeking out microenvironments that are less likely to become oxygen deficient, such as streams. New studies are required to elucidate the hibernation habits of hatchlings that spend their first winter submerged, since their hibernacula are currently unknown.
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Acknowledgments |
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