Metabolic and cardiovascular adjustments of juvenile green turtles to seasonal changes in temperature and photoperiod
Department of Zoology, The University of British Columbia, 6270 University Blvd, Vancouver, BC, Canada V6T 1Z4
* Author for correspondence at present address: Joint Institute for Marine and Atmospheric Research, The University of Hawaii - Manoa, 2570 Dole St, Honolulu, HI 96822, USA (e-mail: amanda.southwood{at}noaa.gov)
Accepted 27 August 2003
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Summary |
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Key words: green turtle, Chelonia mydas, metabolism, heart rate, enzyme activity, temperature, season
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Introduction |
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There is evidence that green turtles from some populations may enter a
state of torpor or dormancy during the winter, when environmental conditions
are sub-optimal. Felger et al.
(1976) observed Eastern
Pacific green turtles (Chelonia mydas agassizii) in the Gulf of
California partially buried in the muddy substrate on the seafloor during
winter when TW was less than 15°C; these turtles were
lethargic and unresponsive when handled. Although cold temperatures or changes
in food availability may induce metabolic torpor in green turtles, this does
not seem to be the common response to typical winter conditions. Sonic
tracking studies conducted by Seminoff
(2000
) show that Eastern
Pacific green turtles reside year-round at foraging pastures in the Gulf of
California and can maintain activity during both summer and winter months.
Based on sea surface temperature data and movement patterns of turtles,
Seminoff (2000
) suggested that
the `inactivity threshold' for turtles at this site was 15°C and that
turtles may become torpid when temperatures drop below this threshold. Studies
of the behavior and movements of green turtles from other populations also
indicate that turtles can remain active over a wide range of environmental
temperatures. Mendonca (1983
)
tracked the movements of juvenile green turtles in a subtropical lagoon in
Florida and found that turtles were active and continued to forage at a
TW as high as 34°C in the summer and as low as
18°C in the winter. Likewise, Read et al.
(1996
) observed that green
turtles in subtropical Moreton Bay, Queensland, Australia were active
year-round. Turtles at this study site rapidly fled approaching research boats
during the winter when TW was 15.0-22.7°C and had food
in their buccal cavities upon capture.
Remote monitoring studies and direct observations allow us to monitor the
behavior of green turtles in their natural environment during summer and
winter, but technological and logistical limitations make at-sea measurements
of metabolic and physiological variables quite difficult to accomplish.
Laboratory studies with captive turtles provide an attractive alternative to
field studies because factors such as temperature and photoperiod may be
controlled, and the metabolic and physiological responses to changes in
environmental conditions may be closely monitored. Previous experiments with
captive green turtles have shown that acute decreases in temperature result in
a decrease in metabolic rate over the range of 30-10°C
(Davenport et al., 1982), a
decrease in heart rate over the range of 35-15°C
(Smith et al., 1986
) and
greatly reduced peripheral blood flow over the range of 32-17°C
(Hochscheid et al., 2002
).
However, the metabolic and cardiovascular responses of green turtles to
gradual and prolonged shifts in temperature and photoperiod, such as are
experienced on a seasonal basis, have not previously been investigated.
Green turtles from many populations appear to be capable of maintaining
activity year-round in tropical and subtropical habitats, despite fluctuations
in environmental conditions. The purpose of our study was to investigate the
effects of seasonal changes in temperature and photoperiod on the metabolism
and physiology of green turtles and to assess the ability of green turtles to
acclimate to changing environmental conditions. Maintenance of activity over
the range of temperatures experienced seasonally may be accomplished by either
a low thermal dependence of metabolism and supporting physiological functions
or thermal acclimation of metabolic rate. We exposed captive turtles to an
environmental simulation based on seasonal changes in TW
and photoperiod commonly experienced by green turtles at subtropical
latitudes. Changes in temperature and photoperiod were specifically modeled
after environmental conditions in Moreton Bay, Queensland, Australia
(27°30' S, 153°18' E), as this site harbors a resident
population of green turtles that are known to remain active year-round. Oxygen
consumption
(O2), breathing
frequency (fB) and heart rate (fH) of
turtles were measured during exposure to simulated summer and winter
conditions. We also collected muscle tissue samples from turtles during the
summer and winter simulations so that we could determine the effects of
temperature on the function of important enzymes in aerobic and anaerobic
metabolic pathways. In particular, our goal was to assess the thermal
dependence of these metabolic enzymes and whether metabolic compensation of
reaction rates occurred with prolonged exposure to low TW
during the winter simulation. Blood samples were collected from turtles during
exposure to summer and winter conditions so that hematocrit and plasma levels
of glucose, proteins, ions, creatine phosphokinase and thyroxine (T4) could be
determined.
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Materials and methods |
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All procedures were approved by the University of British Columbia's Animal Care Committee in accordance with guidelines set by the Canadian Committee on Animal Care.
Seasonal simulation
Turtles were exposed to changes in temperature and photoperiod that
simulated seasonal changes in these variables at Moreton Bay, Australia. Mean
TW during summer at Moreton Bay is 26°C, and mean
TW during the winter is 17°C
(Read et al., 1996). In our
study, turtles were initially held at a TW of 26°C for
a period of several months. TW was then decreased by
3°C every 2 weeks until it reached 17°C. Turtles were maintained in
17°C water for 16 weeks and then TW was increased by
3°C every 2 weeks until it was once again 26°C. Photoperiod was
modified based on seasonal sunrise and sunset times at Moreton Bay: 14 h:10 h
light:dark at 26°C; 12.75 h:11.25 h light:dark at 23°C; 11 h:13 h
light:dark at 20°C; and 10.25 h:13.75 h light:dark at 17°C. We defined
`summer conditions' as a TW of 26°C with a 14 h:10
hlight:dark photoperiod. `Winter conditions' were defined as a
TW of 17°C with a 10.25 h:13.75 h light:dark
photoperiod.
Turtle body temperature (TB), specifically the subcarapace temperature, was measured periodically using temperature-sensitive passive induced transponder (PIT) tags (BioMedic Data Systems, Maywood, NJ, USA) implanted beneath left marginal scute #2, just above the left front flipper. The tags had a resolution of ±0.1°C and were calibrated over the range of 20-35°C using a National Institute of Standards and Technology traceable thermometer (0.05°C gradations) before implantation.
Oxygen consumption and breathing frequency
Open-flow respirometry was used to measure oxygen consumption
(O2) of turtles.
Two days before a respirometry trial, the turtle was moved into a smaller
circular fiberglass tank (2 m diameter). The surface of the tank was covered
except for a 30 cm2 area. Turtles were trained to surface in this
area and to breathe under a Plexiglas funnel placed over the water surface. A
one-way respiratory valve (Hans Rudolph, Kansas City, MO, USA) was placed on
the inflow port of the funnel, and a T-junction with one arm leading to a
latex gas collection bag to receive overflow expired gas was placed on the
outflow port of the funnel. Air was pulled through the funnel at a rate of 4.0
l min-1 by an air pump (Shop-vac Canada Ltd., Burlington, ON,
Canada), and air flow was monitored with a mass flowmeter (Omega Engineering
Inc., Stamford, CT, USA). Exhaled air that entered the overflow bag was
eventually pulled back through the T-junction and into the main airstream. A
sub-sample of funnel gas was drawn through Drierite and soda-lime before
passing through an SAE 300 oxygen analyzer at a rate of 150 ml
min-1. The percentage of oxygen (%O2) in the sub-sample
from the funnel was recorded on a computer at a frequency of 1 Hz using
LabTech software. Downward deflections from 20.94% O2
(concentration of O2 in dry air) occurred when the turtle breathed
into the funnel.
O2 was
determined by measuring the area of downward deflections in a data analysis
program (Acqknowledge, BIOPAC Systems, Inc., Santa Barbara, CA, USA). The
respirometry system was calibrated by injecting known quantities of nitrogen
into the funnel. Values for
O2 were
corrected for STPD.
Respirometry trials lasted 3-5 h and were conducted between 10:00 h and 15:00 h. A video camera was set up above the experiment tank and all trials were videotaped so that behavior and breathing frequency could be monitored continuously. The amount of time spent resting or active during the trial was calculated. The turtle was considered to be resting if motionless on the tank bottom. Active periods consisted of turtles slowly paddling around the side of the tank or shuffling along the tank bottom.
O2 and
fB were recorded during exposure to summer conditions
before and 20 weeks after the winter simulation (26-pre and 26-post,
respectively), and at 8 weeks and 16 weeks exposure to winter conditions
(17-8w and 17-16w, respectively). Turtles were fasted for 5-7 days before
respirometry trials in summer conditions and for 10-12 days before trials in
winter conditions to ensure that they were in a post-absorptive V
state (Davenport et al., 1982
;
Hadjichristophorou and Grove,
1983
; Brand et al.,
1999
).
O2 was corrected
for differences in body mass using an exponent of 0.83. This exponent was
derived for green turtles weighing between 0.3 kg and 141.5 kg (ambient
temperature T°=23-27°C;
Prange and Jackson, 1976
) and
is similar to exponents derived for the Aldabra giant tortoise (Geochelone
gigantea; exponent=0.82, mass=0.1-35.5 kg, T°=21-26°C;
Hughes et al., 1971
), 10
species of aquatic turtles (exponent=0.86, mass=0.3-132 kg,
T°=20°C; Bennett and
Dawson, 1976
) and 26 species of lizards (exponent=0.83,
mass=0.001-4.4 kg, T°=30°C;
Bennett and Dawson, 1976
). The
effect of the seasonal simulation on
O2 and
fB was analyzed using repeated measures analysis of
covariance (ANCOVA) with activity level as the co-variate. Activity levels of
turtles during summer and winter trials were compared using repeated measures
analysis of variance (ANOVA). Mean values for
O2 and
fB in summer conditions (26-pre and 26-post combined) and
winter conditions (17-8w and 17-16w combined) were calculated for each turtle,
and Pearson correlation was used to investigate the relationship between mean
O2 and mean
fB during simulated summer and winter conditions.
Metabolic enzyme activity
Muscle tissue samples were obtained so that the effects of temperature on
muscle enzyme activity could be assessed. Muscle tissue samples were obtained
from the flexor tibialis muscle of the rear flipper during exposure to summer
conditions before the winter simulation and after 4 weeksexposure to winter
conditions. The flexor tibialis flexes and retracts the rear flipper and
controls the shape of the flipper for steering
(Wyneken, 2001). Before
excising muscle tissue, the incision area was cleaned thoroughly with 95% EtOH
and Betadine topical antiseptic. A local anaesthetic (2% Lidocaine; Vetoquinol
Inc., Lavaltrie, QC, Canada) was injected into the area from which the biopsy
sample was to be taken. A 1.5 cm incision was made in the skin and
approximately 100-200 mg of muscle tissue was excised using surgical scissors.
Dissolvable sutures were used to sew muscle tissue together and close the
incision wound. The area was treated with topical antibiotic cream (Furacin;
Vetoquinol Inc.) and the turtle was given an intramuscular injection of
antibiotics (5 mg kg-1 Amiglyde-V; Ayerst Veterinarian
Laboratories, Guelph, ON, Canada) to reduce the risk of infection. The tissue
samples were immediately frozen in liquid nitrogen and rapidly transferred to
a -70°C freezer for storage. Tissue samples were stored for 20-22 months
before assays were performed. Activity of citrate synthase (CS), lactate
dehydrogenase (LDH) and pyruvate kinase (PK) were determined for all tissue
samples.
Tissue samples were partially thawed, minced and diluted to 1/10 volume in
ice-cold 75 mmol l-1 Tris-HCl homogenization buffer adjusted for pH
7.5 at room temperature. The dilution was homogenized using a Polytron tissue
homogenizer (model PT10; Brinkman Instruments, Rexdale, ON, Canada) and
sonicated using a MicroUltrasonic Cell Disrupter (model KT5; Kontes, Vineland,
NJ, USA). Whole homogenate was used for all assays and each assay was run in
duplicate. Enzyme activities were measured with a Perkin-Elmer
spectrophotometer [model Lambda 2; Perkin-Elmer (Canada) Ltd, Rexdale, ON,
Canada]. Temperature in the spectrophotometer cells was controlled with a
circulating water bath (MGW Lauda, Lauda, Germany). Enzyme activity was
measured at assay temperatures of 15°C, 20°C, 25°C and 30°C.
The order in which activity was measured at different assay temperatures was
randomized. Stock solutions for assays were prepared using buffers adjusted
for pH at each temperature. All reactions were initiated by the addition of
substrate. The millimolar extinction coefficient () and wavelength at
which the reaction was monitored (
) are indicated for each assay
below.
Enzyme protocols were as follows. LDH: =340 nm,
=6.22, pH
7.5, 50 mmol l-1 imidazole-HCl, 0.15 mmol l-1
ß-nicotinamide adenine dinucleotide (reduced), 4 mmol l-1
pyruvate, 1/1000 tissue dilution.
PK: =340 nm,
=6.22, pH 7.0, 50 mmol l-1
imidazole-HCl, 10 mmol l-1 magnesium chloride, 100 mmol
l-1 potassium chloride, 0.15 mmol l-1
ß-nicotinamide adenine dinucleotide (reduced), 7 mmol l-1
phosphoenol pyruvate, 5 mmol l-1 adenine diphosphate, excess LDH,
1/500 tissue dilution.
CS: =412 nm,
=13.6, pH 8.0, 100 mmol l-1 Tris-HCl,
0.3 mmol l-1 acetyl-CoA, 0.5 mmol l-1 oxaloacetate
(omitted for control), 0.1 mmol l-1
5,5'-dithiobis-(2-nitrobenzoic acid), 1/10 tissue dilution.
All chemicals were obtained from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada).
The effects of the seasonal simulation and assay temperature on enzyme activity were analyzed using repeated measures two-way ANCOVA with turtle body mass as co-variate. Thermal coefficients (Q10 values) were calculated for enzyme activity over the assay temperature range of 15-30°C.
Heart rate
Electrocardiograms (ECGs) were recorded from turtles using custom-built
data loggers (Andrews, 1998).
Electrode assemblies were constructed of 34G polyethylene-coated medical
stainless steel wire (5 cm length; Cooner Wire, Chatsworth, CA, USA) soldered
to waterproof connectors (model MS1F-1; Underwater Systems Inc., Stanton, CA,
USA). The distal tip of the medical wire was stripped of 0.5 cm of the
polyethylene coat. The medical wire portion of the assembly served as the
internal electrode, and the waterproof connectors were externalized so that
electrodes could be interfaced with the data logger. Medical wire was inserted
into the body cavity of the turtle through 2 mm holes drilled through left
marginal scute #9 (LM9) and right marginal scute #5 (RM5) of the carapace 1-2
days before ECG trials. A blunted stainless steel shaft inserted through the
drill hole in scute LM9 was used to guide an electrode to a depth of 5 cm into
the body cavity. The same procedure was used to insert another electrode to a
depth of 5 cm through scute RM5. The positioning and polarity of electrodes
corresponded with ECG Lead II of Einthoven's triangle. The holes in the
carapace were treated with local anaesthetic (2% Lidocaine) as soon as they
were drilled and coated with topical antibacterial gel (Furacin). Electrodes
were secured in the holes with tissue glue (3M; Vet-Bond, St Paul, MN, USA)
and the hole was sealed with fast-curing epoxy (5-Cure; Industrial
Formulators, Burnaby, BC, Canada). The externalized waterproof connector was
glued flat to the surface of the carapace at the site of electrode insertion.
Electrodes remained implanted for a maximum of 5 days,during which time the
turtle was isolated in a 2 m diameter tank (1 m depth) so that other turtles
would not disturb the externalized portion of the electrode assembly.
ECG was recorded during exposure to summer conditions and after 16 weeks exposure to winter conditions. ECG trials were conducted between 10:00 h and 17:00 h in the 2 m-diameter isolation tank, and trials were videotaped so that activity could be monitored. Behavior was categorized as `active' or `resting' in the same manner as for turtles during respirometry trials. When the ECG recordings were completed, the electrodes were removed and the drill holes were treated with topical antibiotics and re-sealed with epoxy. Antibiotics (2.5 mg kg-1 Amiglyde-V) were administered to the turtle intramuscularly every 3 days for 2 weeks following electrode removal to lower the risk of infection. Drill holes healed completely within 4 weeks of electrode removal.
Heart rate was determined from the ECG trace using the Acknowledge data analysis program (BIOPAC systems, Inc). Cardiac intervals (CI) were derived from the ECG trace by measuring the time between consecutive R-R peaks. Values for CI were converted to instantaneous heart rate in beats min-1. Sections of the ECG trace in which the QRS complex could not be distinguished were excluded from data analysis. Mean resting fH was determined for all five turtles but, because of increased background noise due to electrical signals from muscle contraction during swimming, active heart rates could only be distinguished on ECG traces from three turtles. Mean active fH was calculated for these three turtles. Increased EMG activity was associated with breathing episodes for all turtles, so breathing fH could not be determined.
The effects of the seasonal simulation and activity state on fH were analyzed using two-way repeated measures ANOVA.
Blood chemistry
Blood samples (5 ml) were collected from turtles during exposure to summer
conditions and after 16 weeks exposure to winter conditions. All blood samples
were drawn from the venous cervical sinus using heparinized VacutainerTM
tubes with 21Gx1g needles. A subsample of blood was immediately
transferred to duplicate microcapillary tubes and centrifuged so that
hematocrit could be determined. The remainder of the blood was centrifuged at
3000 r.p.m. for 5 min. Plasma was transferred to cryo-safe plastic tubes
(Sarstedt Inc., Montreal, QC, Canada) and stored in a -70°C freezer.
Samples were sent to a pathology laboratory (Central Laboratory for
Veterinarians Ltd, Langley, BC, Canada) for analysis of glucose, proteins, ion
and mineral content and thyroxine (T4). Creatine phosphokinase (CPK) activity
at 37°C was determined using an automated clinical chemistry analyzer
(Dade-Behring Canada, Mississauga, ON, Canada). Paired t-tests were
used to test for significant differences in blood chemistry variables for
turtles during exposure to summer and winter conditions.
Statistics
Statistical analyses described in the previous sections were performed
using JMPIN software (SAS Institute, Inc., Cary, NC, USA). Differences were
considered to be significant at P<0.05. Values are presented as
means ± S.E.M.
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Results |
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There was no significant decrease in fB during exposure
to winter conditions (P=0.288, F=1.444, d.f.=3;
Fig. 1B). Turtles had a mean
fB of 10.8±1.5 breaths h-1 at 26-pre and
12.3±2.3 breaths h-1 at 26-post. Mean fB
decreased to 9.6±3.4 breaths h-1 at 17-8w and 9.1±2.0
breaths h-1 at 17-16w. There was a significant correlation between
mean fB and mean
O2 during
exposure to summer conditions (r=0.982, P=0.018,
t=7.430, d.f.=3; Fig.
3A), but the correlation was not significant in winter conditions
(r=0.514, P=0.375, t=1.040, d.f.=3;
Fig. 3B). Activity level of
turtles had a significant effect on fB
(P<0.001, F=24.638, d.f.=1).
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Turtles were active for an average of 61.0±16.8% of the time during trials at 26-pre and 50.5±12.8% of the time during trials at 26-post (Fig. 1C). The mean time spent active was 38.3±21.3% during trials at 17-8w and 47.8±7.4% during trials at 17-16w. Mean percent time spent active during exposure to winter conditions was not significantly lower than mean percent time spent active in summer conditions (P=0.400, F=1.073, d.f.=1).
Turtle TB was not significantly higher than TW during respirometry trials conducted during exposure to either summer or winter conditions (Student's t-test, P=0.563-0.834, t=0.378-0.612). Growth rates during exposure to winter conditions (0.78±0.12 g day-1 kg-1) were significantly lower compared with summer conditions (1.59±0.10 g day-1 kg-1) (P=0.010, t=4.608).
Metabolic enzyme activity
Activity of the aerobic enzyme citrate synthase at any given assay
temperature was significantly higher in muscle tissue collected during summer
conditions compared with tissue collected during winter conditions
(P<0.001, F=17.313, d.f.=1;
Fig. 4A). CS activity was
significantly affected by mass of the turtle (P=0.002,
F=11.711, d.f.=1), with larger turtles having lower mass-specific CS
activity. The Q10 value for CS activity over the range of
15-30°C was 1.44 for both summer- and winter-acclimated tissue. CS
activity at an assay temperature of 30°C was significantly higher than
activity at all other assay temperatures (P=0.022, F=3.734,
d.f.=3). There was no significant difference in CS activity at assay
temperatures of 15°C, 20°C and 25°C.
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The enzymes LDH and PK showed similar responses to changes in temperature. For both enzymes, activity at any given assay temperature was significantly higher in tissue collected during exposure to winter conditions than in tissue collected during exposure to summer conditions (LDH: P<0.001, F=59.014, d.f.=1; PK: P<0.001, F=25.255, d.f.=1; Fig. 4B,C). Mass did not significantly affect activity of LDH (P=0.230, F=1.499, d.f.=1) or PK (P=0.224, F=1.539, d.f.=1). The Q10 value for LDH was 1.61 for summer-acclimated tissue and 1.48 for winter-acclimated tissue. The Q10 of PK activity was 1.67 for summer-acclimated tissue and 1.69 for winter-acclimated tissue. There was no significant difference in activity at assay temperatures of 20-25°C for either LDH or PK. Activity of LDH and PK at 15°C was significantly lower than at all other assay temperatures, and activity at 30°C was significantly higher than at all other assay temperatures (LDH: P<0.001, F=46.283, d.f.=3; PK: P<0.001, F=33.627, d.f.=3).
Heart rate
Heart rate was significantly lower during exposure to winter conditions
compared with summer conditions (P<0.001, F=89.030,
d.f.=1). Active fH in winter conditions (12.9±0.6
beats min-1) was 46% lower than active fH in
summer conditions (24.0±2.4 beats min-1), and resting
fH in winter conditions (10.2±0.9 beats
min-1) was 48% lower than resting fH in summer
conditions (19.6±1.5 beats min-1)
(Fig. 5). There was a
significant difference between active and resting fH at
both temperatures (P=0.012, F=9.293, d.f.=1).
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Blood chemistry
There was no significant difference in the plasma levels of glucose,
sodium, potassium, calcium, phosphorus, chloride or globulin for turtles
during exposure to summer and winter conditions
(Table 1). Plasma levels of
albumin (P<0.001, t=9.129), total protein
(P<0.047, t=2.845) and T4 (P<0.001,
t=11.918) were significantly lower in turtles during exposure to
winter conditions compared with levels during exposure to summer conditions.
Blood variables that increased significantly during exposure to winter
conditions were plasma CPK (P=0.024, t=3.545) and hematocrit
(P=0.003, t=6.216).
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Discussion |
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Given the pervasive effects of temperature on biochemical reaction rates,
structure and stability of proteins and membranes, and physiological
processes, it was not surprising to see a trend of decreased
O2 with exposure
to a lower temperature (17°C) during the winter simulation
(Fig. 1A).Although
statistically there was no significant difference in
O2 between
simulated summer and winter conditions (P=0.059), the trend warrants
comment. Previous studies with green turtles have provided values for
O2 at various
temperatures, but the thermal dependence of VO2
(i.e. Q10 value) has not been explicitly stated
(Kraus and Jackson, 1980
;
Davenport et al., 1982
),
perhaps because factors other than temperature, such as digestive state or
activity level, were not controlled. Lutz et al.
(1989
) reported a
Q10 of 2.4 for
O2 of loggerhead
sea turtles (4.3-22.7 kg) exposed to acute decreases in temperature over the
range of 30-10°C. A Q10 of 2.4 applied to our green turtle data
results in a predicted
O2 of 0.36 ml
min-1 kg-0.83 at 17°C, a decrease of 54% from the
mean
O2 at
26°C before initiation of the winter simulation. In fact, mean
O2 during
exposure to 17°C (0.58-0.60 ml min-1 kg-0.83) was
24-27% lower than mean
O2 at 26°C
(0.79-0.89 ml min-1 kg-0.83); the magnitude of the
decrease in
O2
of green turtles between summer and winter conditions is much less than that
predicted using a Q10 of 2.4.
One possible explanation for this discrepancy is that turtles undergo some
degree of metabolic compensation during chronic exposure to colder
temperatures. If thermal acclimation of metabolic rate occurs, it should be
reflected at the molecular level by adjustments in metabolic enzyme activity.
One of the most common mechanisms to compensate for decreased kinetic energy
of reactant molecules at low temperature is an increase in enzyme
concentration (Hochachka and Somero,
2002). If a compensatory increase in enzyme concentration occurs
with prolonged cold exposure, maximal enzyme activity in tissue obtained from
cold-acclimated animals is higher than activity in tissue obtained from
warm-acclimated animals when compared at a common temperature. In the current
study, activity of the aerobic enzyme CS in tissue collected during exposure
to 17°C was significantly lower than CS activity in tissue collected
during exposure to 26°C (Fig.
4A), a pattern opposite to what we would expect if a compensatory
adjustment in CS activity had been made during cold exposure (see discussion
below). Therefore, there is no evidence of thermal acclimation of oxygen
consumption at the molecular level.
An alternative explanation for the moderate decrease in
O2 during
exposure to 17°C compared with 26°C is that metabolic rate has a low
thermal dependence over this temperature range. This speculation is supported
by the fact that CS activity had a low thermal dependence
(Q10=1.44) over the 15-30°C range of assay temperatures, and
there was no significant difference in CS activity at assay temperatures of
15°C, 20°C and 25°C. Several species of reptiles display a
metabolic plateau over the temperature range in which they are normally
active, i.e. metabolic rate remains stable and Q10 approaches 1
within a range of several °C (Bennett
and Dawson, 1976
; Waldschmidt
et al., 1987
; Angilletta,
2001
). Penick et al.
(1996
) measured tissue
metabolic rates
(
O2) of isolated
skeletal muscle, heart, liver, intestine and kidneys from freshly killed
juvenile green turtles and found that Q10 values for tissue
O2 were
relatively low within the temperature range of 12.5-27.5°C
(Q10=0.79-1.82). Green turtle tissues generally had a lower thermal
dependence than tissues from cold-climate garter snakes (Thamnopnis
spp.) and eurythermic lizards (Leiolopisma zelandica and Eumeces
obsoletus; Penick et al.,
1996
).
In the current study,
O2 and metabolic
enzyme activity data indicate that green turtles have a low thermal dependence
of aerobic metabolism over the range of temperatures experienced during the
seasonal simulation (17-26°C). If this is the case, then populations of
green turtles that experience seasonal fluctuations in temperature within this
range should be capable of maintaining activity year-round. Field observations
of green turtles at Moreton Bay, the study site that was used to model our
laboratory seasonal simulation, support this speculation
(Read et al., 1996
). It should
be noted, however, that Q10 values typically increase at the lower
and upper limits of an animal's optimal thermal range
(Bennett and Dawson, 1976
;
Hochachka and Somero, 2002
).
Increased thermal dependence of physiological and biochemical processes at
lower temperatures may result in a drastic reduction in activity and metabolic
rate. Laboratory and field studies have shown that green turtles become
quiescent and cease feeding at a TW of 15°C or lower
(Felger et al., 1976
;
Moon et al., 1997
;
Seminoff, 2000
), and green
turtles subjected to rapid decreases in TW within the
range of 8-15°C commonly develop a pathological condition called
`cold-stunning', which results in a loss of respiratory function and buoyancy
control (Sadove et al., 1998
).
Obviously, metabolic and physiological responses to decreases in environmental
temperature depend on both the magnitude of the temperature change and how
quickly the temperature changes.
Plasma T4 levels of vertebrates are commonly associated with metabolic
state, and seasonal variation in plasma T4 has been observed in several
reptile species. Changes in reproductive or developmental state, nutritional
status, general activity levels and temperature may all result in changes in
plasma T4 (Lynn, 1970). Given
that the last of these three factors varied for green turtles exposed to our
seasonal simulation, it is difficult to pinpoint the exact cause of the
decrease in T4 during exposure to winter conditions. The fact that growth
rates, blood protein levels and presumably food consumption were lower during
exposure to winter conditions suggests that nutritional status may play a key
role. This assertion is supported by data from earlier studies, which show
that at a constant temperature increases in food quantity and quality result
in significantly higher plasma T4 levels in juvenile green turtles
(Moon et al., 1999
). Variation
in temperature and photoperiod may also affect T4 production, but it is
difficult to assess the direct effects of these variables in the current
study.
The decrease in plasma T4 during exposure to winter conditions may provide
an explanation for the pattern observed for CS activity during the seasonal
simulation. CS activity during exposure to winter conditions was significantly
lower than during exposure to summer conditions. Several studies have shown
that CS activity of reptiles can be affected by increases or decreases in T4
levels. Lizards injected with T4 or implanted with subcutaneous T4 pellets had
significantly higher CS activity in liver and muscle tissue than control
lizards, whereas thyroidectomy of lizards resulted in significantly reduced
plasma T4 and reduced CS activity (John-Alder,
1983,
1990a
,b
).
Other studies have shown that CS activity, maximal
O2 and endurance
of field-active lizards undergo a pattern of seasonal variation that parallels
changes in plasma T4 levels (Lynn,
1970
; John-Alder,
1984
), which suggests that thyroid hormones may be involved in
regulating aerobic energetic capacities. If green turtles in the field
experience a decrease in T4 levels during the winter months, a concurrent
decrease in aerobic capacity may also occur. Interestingly, and in contrast to
the current study, blood samples collected monthly from juvenile green turtles
at a tropical field site showed no evidence of a seasonal shift in plasma T4
(Moon, 1992
). As noted
previously, there are numerous environmental and physiological factors that
affect plasma T4 levels on a seasonal basis. These factors may be different
between laboratory and field conditions, as well as between different age
classes of turtles and different geographic populations of turtles. The
concurrent decrease in plasma T4 and CS activity during exposure to winter
conditions in our laboratory simulation supports the proposition that aerobic
capacity may fluctuate seasonally, but exercise variables such as maximal
O2 and endurance
capacity would need to be measured to confirm this.
In contrast to the results obtained for CS, activity of enzymes associated
with the glycolytic pathway of energy production (LDH and PK) was
significantly higher during exposure to winter conditions compared with summer
conditions (Fig. 4B,C).
Glycolytic enzymes showed a pattern of thermal acclimation even though they
had a low thermal dependence over the range of 15-30°C
(Q10=1.48-1.69). There was no significant difference in activity of
LDH and PK at 20°C and 25°C, but activity at 15°C was
significantly lower than at all other assay temperatures. One interpretation
of these results is that low thermal dependence of glycolytic enzymes is
sufficient for maintenance of anaerobic function with relatively small shifts
in temperature, but larger shifts in temperature induce a compensatory
response to ensure that enzyme function is preserved. Field observations of
green turtles from sub-tropical populations show that these animals are
capable of quick bursts of activity to escape pursuing predators during both
winter and summer (Read et al.,
1996; Southwood,
2002
). Although green turtles are known to have a high aerobic
capacity (Butler et al., 1984
),
they rely heavily on their large anaerobic reserves for high intensity
activity (Prange and Jackson,
1976
; Jackson and Prange,
1979
; Dial, 1987
;
Baldwin et al., 1989
;
Wyneken, 1997
). Preservation
of anaerobic means of energy production, via low thermal dependence
and/or compensation of biochemical reaction rates, may be critically important
for predator avoidance and survival in a changing thermal environment.
Phosphocreatine (PCr) provides a large anaerobic energy buffer in muscle
cells during burst activity via a reversible reaction catalyzed by
CPK (PCr + ADP + H+ ATP + Cr). This reaction takes place in
the cytosol of cells, and the presence of large amounts of CPK in the
bloodstream is generally used as an indicator of cellular injury or death. In
green turtles, the mean level of plasma CPK during exposure to winter
conditions was 4.6 times higher than during exposure to summer conditions
(Table 1), and the significant
increase in CPK could represent breakdown of muscle tissue during prolonged
exposure to sub-optimal conditions. However, turtles continued to feed and
gain weight and showed no overt signs of malaise during the winter simulation.
Another possibility is that the occurrence of cell death may be the same in
winter and summer, and the increase in plasma CPK is due to increased amounts
of CPK being released from individual cells. A comparison of muscle tissue and
plasma activity of CPK in green iguanas (Iguana iguana) and yellow
rat snakes (Elaphe obsoleta quadrivitatta) showed that low plasma
activity of CPK coincided with low tissue activity, and high plasma activity
of CPK was associated with moderate to large amounts of tissue activity
(Wagner and Wetzel, 1999
;
Ramsay and Dotson, 1995
). It
is possible that elevated plasma CPK during exposure to winter conditions
reflects elevated concentrations of intracellular CPK and therefore
compensation of CPK activity. CPK activity in muscle tissues should be
measured directly to confirm whether or not a compensatory adjustment
occurs.
In addition to the changes we observed in metabolic variables, we also
found that prolonged exposure to winter conditions resulted in alterations in
respiratory and cardiovascular variables. Interestingly, the changes in oxygen
delivery variables that we measured did not match the changes in oxygen
consumption. For example, the relationship between
O2 and
fB was strong and highly significant during exposure to
summer conditions (r2=0.965, P=0.018), but the
relationship between these variables broke down in winter conditions
(r2=0.265, P=0.375)
(Fig. 3). This suggests that
the relative contributions of fB and tidal volume to
ventilation may change with temperature. Kraus and Jackson
(1980
) found that for juvenile
green turtles increases in ventilation between 15°C and 25°C were due
exclusively to changes in tidal volume, whereas increases in ventilation
between 25°C and 35°C were due to changes in fB.
The weak relationship between fB and
O2 during
exposure to winter conditions supports the idea that turtles alter tidal
volume, rather than fB, to match O2 supply to
O2 demand at cooler temperatures.
As with fB, seasonally induced changes in
fH did not correspond well with the changes in
O2. Mean
fH during the winter simulation was 46-48% lower than mean
fH during the summer simulation - approximately double the
decrease observed for
O2 (24-27%). If
cardiac output is primarily controlled by changes in fH,
then the greater decrease in fH compared with
O2 could result
in an imbalance between O2 delivery and O2 demand at
lower temperatures. Hudson and Bertram
(1966
) suggested that
discrepancies in thermal dependence of fH and
O2 could be
explained by alterations in stroke volume, with increased filling time between
systolic contractions at lower temperatures resulting in larger stroke
volumes. The increase in stroke volume could offset the decrease in
fH, so that cardiac output is stabilized and oxygen
delivery is maintained during exposure to cool temperatures. Temperature
effects on stroke volume of sea turtles have not been investigated, but stroke
volume is highly variable in many other species of turtle and is affected by
both activity state and temperature
(White, 1976
). Furthermore,
simulation of winter conditions resulted in a significant increase in
hematocrit. The range of hematocrit observed in our study (32-42%) was within
the range observed for wild loggerhead sea turtles (Caretta caretta;
28-48%), although hematocrit for loggerhead sea turtles either did not change
seasonally or decreased during the winter months
(Lutz and Dunbar-Cooper,
1987
). An increase in hematocrit during the winter, if mirrored by
an increase in hemoglobin concentration, could enhance blood oxygen transport
capabilities, thereby partially offsetting the seasonal bradycardia.
Conclusions
The magnitude of seasonal shifts in temperature and photoperiod, as well as
other site-specific environmental factors such as food availability, may play
a large role in determining seasonal metabolic response. Our study shows that
metabolic enzyme activity and overall metabolic rate of juvenile green turtles
have a relatively low thermal dependence over the range of temperatures
commonly experienced at tropical and subtropical latitudes (17-26°C). Low
thermal dependence of aerobic metabolism and metabolic compensation of
anaerobic means of energy production may allow turtles to remain active
year-round if other environmental conditions are favorable. Results from
previous studies suggest that the pivotal temperature for metabolic
downregulation and torpor in green turtles is close to 15°C
(Moon et al., 1997;
Seminoff, 2000
). Whether or
not other environmental cues contribute to the induction of torpor in this
species remains to be determined.
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References |
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