![Mass conversion efficiency of yolk to tissue at 3 incubation temperatures. A) Regression line fitted to the scatter plot fits the equation: log 10 [hatchling mass] ¼ 0.9151(log 10 egg mass) À 0.0716, r 2 ¼ 0.4952. Closed circles ¼ 26.58C, triangles ¼ 28.58C, and inverted triangles ¼ 30.58C incubation temperatures. Dashed lines ¼ 95% Confidence Interval. B) Least-squares residuals from log-log plot. Error bars ¼ 6 1 Standard Error.](https://www.researchgate.net/profile/Day-Ligon/publication/232683207/figure/fig2/AS:670042769338379@1536762053709/Mass-conversion-efficiency-of-yolk-to-tissue-at-3-incubation-temperatures-A-Regression_Q320.jpg)
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Chelonian Conservation and Biology, 2009, 8(1): 74–83
Ó2009 Chelonian Research Foundation
Temperature Effects During Early Life Stages of the Alligator Snapping Turtle
(Macrochelys temminckii)
DAY B. LIGON
1,2
AND MATTHEW B. LOVERN
1
1
Department of Zoology, Oklahoma State University, Stillwater, Oklahoma 74078 USA [matt.lovern@okstate.edu];
2
Present Address: Department of Biology, Missouri State University, Springfield, Missouri 65897 USA
[dayligon@missouristate.edu] (corresponding author)
ABSTRACT. – Alligator snapping turtle (Macrochelys temminckii) populations have declined across
much of the southeastern United States in recent decades, due at least in part to overcollection.
Recently, however, legal protection from large-scale harvesting has been granted to the species in
all states where it is native, thereby drastically reducing one of the greatest threats to its survival.
There is growing interest in captive propagation of alligator snapping turtles for reintroduction
where populations have been decimated. In conjunction with one such effort, we analyzed the
physiological effects of temperature on embryonic and posthatching development. Results indicate
that extreme high and low incubation temperatures negatively affected embryo survival, and high
incubation temperatures corresponded with shorter incubation time but also produced smaller
hatchlings. The effects of temperature on gonadal differentiation indicated that the upper pivotal
temperature was approximately 27.58C. Posthatching growth was faster at warmer water
temperatures, and there was little to no acclimation of metabolic rate to exposure to either
incubation or water temperature. We conclude that intermediate (27.58–28.58C) incubation
temperatures produce a female-biased mixed sex ratio and maximize hatching success and
hatchling size while increasing incubation duration only slightly over that at the higher
temperatures. In addition, posthatching growth was positively influenced by hatchling body
temperature; therefore, warmer water temperatures (;308C) decreased the time required to rear
turtles to a size suitable for reintroduction.
KEY WORDS. – Reptilia; Testudines; Chelydridae; Macrochelys temminckii; alligator snapping
turtle; incubation; temperature; egg; growth; metabolism; oxygen consumption; embryo;
hatchling turtles; juvenile turtles
Turtles are long-lived organisms characterized by
delayed maturity (resulting in a long generation time), low
fecundity, low nest survival, and high adult survival
(Congdon et al. 1993, 1994). Survivorship in turtles is
generally proportional to body size (Wilbur 1975; Frazer et
al. 1990; Iverson 1991; Haskell et al. 1996; Janzen et al.
2000; but see Congdon et al. 1999) and as a result tends to
increase substantially during the juvenile life stages when
growth rates are highest (Cagle 1946; Dunham and
Gibbons 1990; Bobyn and Brooks 1994).
Presumably, the benefits associated with delayed
maturity yield higher average lifetime fitness than for
individuals that mature earlier. However, the long
generation time exhibited by most turtles makes them
susceptible to chronic environmental disturbances that
impact egg or juvenile survival (Congdon et al. 1993,
1994). Similarly, adverse effects on population demo-
graphics can result from increased adult mortality (Deevey
1947; Congdon et al. 1993, 1994). Commercial collection
of turtles for the pet and food trades represents an
important anthropogenic source of disturbance that
disproportionately impacts larger size classes (review in
van Dijk et al. 2000). Collection for consumption, in
particular, targets larger turtles, which represent the
reproductively mature age classes that otherwise enjoy
high annual survivorship. Although still a serious problem
in many parts of the world, collection of a number of
threatened and endangered turtle species in the United
States has been reduced in recent years by improved state
and federal protection.
Embryonic and juvenile age classes are more likely to
be affected by abiotic environmental disturbances than are
adults. Such events include fluctuating water levels that
flood nests and long-term temperature fluctuations that can
in turn affect a number of variables, ranging from food
availability to the incubation conditions experienced by
embryos (Janzen 1994). For example, whereas most
vertebrates have genetically fixed sex determination, most
turtles exhibit temperature-dependent sex determination
(TSD; Bull 1980; Bull and Vogt 1981). As a result, rising
global temperatures resulting from the emission of
greenhouse gases and other factors present a potentially
serious threat to turtles by skewing population sex ratios
(Fisher 1930; Janzen and Paukstis 1991).
In addition to sex, several other physiological and
morphological characteristics have been identified that
may be influenced by incubation temperature (T
inc
).
Among these are size at hatching (Brooks et al. 1991;
Rhen and Lang 1995), posthatching growth rate (Etch-
berger et al. 1990; Ryan et al. 1990; Brooks et al. 1991;
Bobyn and Brooks 1994; Etchberger 1993; McKnight and
Gutzke 1993; Paez et al. 1995; Roosenburg and Johnson
1995), and temperature acclimation (O’Steen and Janzen
1999; Steyermark and Spotila 2000; Ewert 2008). Because
these variables can reasonably be expected to influence
survivorship among hatchling and juvenile age classes, it
follows that incubation conditions can dramatically affect
average lifetime fitness (Miller 1993; Janzen 1993, 1995;
Bobyn and Brooks 1994).
Although legal protection has been granted to some
turtles, such actions are often taken after a species has
already experienced declines sufficient to compromise
hatchling and juvenile recruitment. In response to severe
population declines, a number of efforts have been made—
with some success—to rehabilitate populations of a few
species through captive rearing of hatchlings to a size that
is expected to substantially reduce mortality following
release (Iverson 1991; Haskell et al. 1996; Caillouet 1998).
Unfortunately, due to a lack of critical data, such efforts
are sometimes made without the benefit of basic
knowledge of the factors affecting reproduction and fitness
(Morreale et al. 1982; Spotila et al. 1987, 1994; Wibbels et
al. 1989; Moll and Moll 2000, 2004). Although such
‘headstart’ programs have the potential for improving
conservation of threatened and endangered turtles, it will
become increasingly important to maximize their efficien-
cy as more turtle species face critical population declines.
Alligator snapping turtles (Macrochelys temminckii)
have experienced population declines in recent decades
and have likely been extirpated in many parts of the
species’ historical range (Pritchard 1989; Ernst et al. 1994;
Wagner et al. 1996; Heck 1998; Riedle et al. 2005). This
species is found in river systems throughout much of the
southeastern United States; it is the largest freshwater
turtle in North America and ranks among the most highly
aquatic species as well (Pritchard 1989; Ernst et al. 1994).
Among the many turtle species throughout the region for
which population declines have been documented, M.
temminckii has been identified by several state and federal
agencies as a particularly promising candidate for
conservation via captive propagation and release.
A recent 3-year survey in eastern Oklahoma, USA,
found M. temminckii in restricted portions of just 4 river
systems within the historical range of the species (Riedle et
al. 2005), and reports of incidental catch by fishermen at 3
additional locations were reported to one of us (DBL) in
2002, 2007, and 2008. Although river impoundments have
likely affected alligator snapping turtle populations in the
state by fragmenting populations and creating barriers to
migration (Pritchard 1989; Moll and Moll 2000), the most
devastating impact has likely stemmed from the harvesting
of adult turtles to meet the demands of the turtle meat
market (Pritchard 1989; Heck 1998).
The US Fish and Wildlife Service was petitioned in
1983 to list the alligator snapping turtle as threatened, but
the petition failed because information available for the
species was deemed insufficient (Heck 1998). In recent
years, however, M. temminckii has been awarded some
level of protection in all states in which it occurs (Roman
and Bowen 2000) and in 2006 was listed in Appendix III
(controlled trade) of the Convention on International Trade
in Endangered Species of Wild Fauna and Flora (CITES
2008). In Oklahoma, M. temminckii is currently listed as a
Species of Special Concern (Ramus 1998), a status that
prohibits possession and export from the state. It is hoped
that this ban will curb further population declines. In
response to apparent extirpation of the species across much
of the state (Riedle et al. 2005), Tishomingo National Fish
Hatchery, in collaboration with Sequoyah National
Wildlife Refuge, initiated a pilot captive-breeding study
in 1999 to assess the merit of using captive-bred stock to
repopulate rivers where alligator snapping turtles were
extirpated.
The objective of this study was to determine
conditions that optimized embryonic development, hatch-
ing success, and postembryonic growth. Here, we report
T
inc
effects on M. temminckii sex, size at hatching, and tail
morphology. Additionally, we measured T
inc
and water
temperature (T
water
) effects on posthatching growth rates,
metabolic compensation, and metabolic thermal sensitiv-
ity. Finally, we evaluated the potential effects of these
variables on survival of captive-reared turtles. These
objectives were designed to be relevant to current captive
propagation/release programs for the species and to
provide insights into the viability of similar programs for
other threatened and endangered turtle species.
METHODS
All procedures for this research were approved by the
Oklahoma State University Institutional Animal Care and
Use Committee (protocol No. AS023), assuring compli-
ance with animal care guidelines (Institute of Laboratory
Animal Research 1996).
Eggs. — Alligator snapping turtle eggs were obtained
in 2002 and 2004 from nests laid by females housed at
Tishomingo National Fish Hatchery. An additional clutch
of eggs was obtained in 2002 from L. Andrews, a private
snapping turtle breeder in north-central Oklahoma. Eggs
were removed from nests and numbered, then transported
to Oklahoma State University within 24 hours of
oviposition. There, they were weighed (60.1 g) and
distributed among plastic shoeboxes containing damp
vermiculite (1:1 water:vermiculite by mass) in a random-
ized block design with clutch serving as the blocking
variable. Eggs were distributed among 6 incubation
temperatures ranging from 238to 318C in 2002. Due to
high egg failure at the upper and lower temperatures used
in 2002 (see Results), incubation was restricted to 3
temperatures (26.58, 28.58, and 30.58C) in 2004. Incubator
temperatures were monitored daily with calibrated ther-
mocouple wires inserted into 1 egg container in each
LIGON AND LOVERN — Temperature Effects on Macrochelys temminckii Embryos and Hatchlings 75
incubator. Each egg box was weighed weekly and, when
necessary, distilled water was added to compensate for
evaporation. Additionally, egg boxes were rotated daily
and eggs were redistributed within each box weekly to
minimize effects of thermal or moisture gradients.
Hatchlings. — Eggs were removed from the plastic
shoeboxes and placed individually in plastic jars lined with
damp paper towel after pipping to retain individual
identification. Hatchlings remained in their individual
containers until residual yolk was internalized (3–8 days),
at which time they were weighed (60.1 g) and midline
carapace length and postvent tail length were measured
(60.1 mm). Additionally, metabolic rate (MR) was
measured in terms of oxygen consumption for each
hatchling in 2004. Following MR measurements, unique
combinations of posterior marginal scutes were marked by
tying loops of dental floss through needle holes to facilitate
long-term identification of individuals (O’Steen 1998), and
each turtle was assigned to 1 of 2 T
water
treatments
maintained at 258and 308C. Turtles were fed a
commercially produced fish-based pellet diet ad libitum
and weighed and measured weekly for 11 weeks.
Metabolic Rate. — Oxygen consumption was mea-
sured via closed-system respirometry (Vleck 1987) and
used to calculate MR (Peterson 1990). Oxygen consump-
tion rate of each turtle was measured at 2 different times at
all 3 T
inc
(26.58, 28.58, and 30.58C): first, 1–2 days after
internalizing yolk and second approximately 6 months
posthatching ( ¯
x¼187 days, range ¼185–198 days).
Metabolic chambers were constructed from 169-,
322-, and 959-mL cylindrical plastic jars with screw-on
lids. A stopcock was inserted through the lid of each jar,
and a thin film of vacuum grease was applied on the inside
of each lid to ensure an airtight seal when the stopcock was
closed.
Prior to the 6-month MR measurements, turtles were
fasted for 4 days to minimize metabolic costs associated
with specific dynamic action and growth. Hatchling
measurements were conducted as soon as residual yolk
was internalized, and therefore digestion and growth likely
still contributed to MR during this early measurement. On
the day of measurement, each turtle was weighed and
placed into a metabolic chamber. Chambers were then
placed inside an environmental chamber for 1.5–2 hours to
allow body temperatures to stabilize and equilibrate to
ambient temperature. With the overhead lights off to
minimize disturbance to the turtles, chambers were
carefully removed from the environmental chamber. After
screwing on lids to create an airtight seal, pretrial air
samples were drawn into 20-mL syringes, also equipped
with stopcocks. The stopcocks on the syringe and chamber
were then closed, the syringes removed, and the time of
sampling recorded. Chambers were placed back into the
dark environmental chamber and removed after approxi-
mately 1 hour, when posttrial samples were drawn from
the stopcock after pumping each syringe several times to
ensure mixing of the air inside. After the final measure-
ment at a given temperature, turtles were moved to an
environmental chamber set at another temperature and the
process was repeated. Measurements at all 3 temperatures
were conducted on the same day.
Oxygen concentrations of all air samples were
analyzed in 10-mL aliquots with a Sable Systems FC-1
oxygen analyzer. Air was drawn from outside the building
at a regulated flow rate of 100 mL/min and through serial
columns of Drierite and Ascarite to remove water and
CO
2
, respectively. Each aliquot was injected into the air
stream, which passed through a small column of Drierite
and Ascarite and then through the oxygen analyzer.
Oxygen consumption by each turtle was calculated as the
difference between the initial and final volumes of oxygen
after correcting for chamber volume (Peterson 1990).
Sex Identification. — Individuals were sexed 267–278
(¯
x¼273) days after hatching via laparoscopic surgery
(Rostal et al. 1994). Food was withheld for 6 days prior to
the procedure to reduce the volume of gut contents. The
turtles were transferred to the veterinary facilities at the
Tulsa Zoo on the mornings that surgeries were performed
by K. Backues, DVM. General anesthesia was achieved
with a mixture of 10 mg/kg Ketamine and 0.1 mg/kg
Medetomidine. After cleaning the site with Chlorhexidine,
a 3–4-mm incision was made posterior to the bridge. A
laparoscope was inserted into the incision, and the gonads
were identified visually. Small tissue biopsies were taken
from 4 individuals and examined histologically to validate
macroscopic determinations. The gonads of M. temminckii
at this age were distinct; ovaries appeared as gray tissue
with varying numbers of primordial follicles lying ventral
to the oviducts; whereas, testes were cream-colored and
highly vascularized. Following gonad identification,
incisions were closed with a suture and surgical adhesive.
Turtles were maintained under moist conditions but out of
water for 48 hours following surgery to ensure recovery
from anesthesia.
Statistics. — Mass and MR values were log
10
-
transformed prior to statistical analyses to normalize the
distribution and homogeneity of variance among treat-
ments. After transformation, assumptions of parametric
statistics were met for all subsequent analyses.
Hatchling mass and tail length were analyzed using
analysis of covariance (ANCOVAs), with T
inc
as a fixed
effect and clutch as a random effect. Initial egg mass was
included as a covariate for mass analyses, and carapace
length was used as a covariate for analyzing tail length.
Growth rate was calculated for each 7-day interval for
77 days after hatching using the equation Growth rate ¼
(mass
n
mass
n1
)3(mass
n1
)
1
3[n(n1)]
1
,
where n¼the last day of each measurement interval. These
growth rate values were then analyzed over 77 days that
growth was monitored using a repeated-measures analysis
in which T
inc
and T
water
were treated as fixed effects, clutch
was treated as a random effect, and hatchling ID was
repeated over the 11 measurement periods. Additionally,
the effects of T
inc
,T
water
(fixed effects) and clutch (random
76 CHELONIAN CONSERVATION AND BIOLOGY,Volume 8, Number 1 – 2009
effect) on the maximum growth rate over any 7-day interval
of each turtle was analyzed using ANCOVA.
Metabolic compensation following acclimation to a
constant temperature (either during incubation or pro-
longed exposure to constant T
water
) was measured shortly
after hatching and 6 months posthatching by measuring
MR at all 3 incubation temperatures: 26.58, 28.58, and
30.58C. Analyses of data from both stages were performed
in a repeated-measures ANCOVA, with T
inc
and ambient
temperature as fixed effects, turtle ID repeated over each
temperature, and mass as a covariate. Six-month measure-
ments were analyzed similarly but with T
water
included as a
third fixed effect.
Metabolic sensitivity to changes in body temperature,
expressed as temperature coefficient (Q
10
) values, were
calculated for each turtle after hatching and at 6 months
posthatching using MR measurements obtained at 26.58
and 30.58C. Separate ANCOVAs were used to analyze
clutch and T
inc
effects on hatchling Q
10
, and clutch, T
inc
and T
water
effects on 6-month-old juveniles.
RESULTS
Eggs. — Eighty-eight alligator snapping turtle eggs
comprising 3 clutches were obtained in 2002. Clutch size
ranged from 15 to 37, but eggs in the smallest clutch
proved infertile (Table 1). One hundred eighty-six alligator
snapping turtle eggs were obtained from 6 nests in 2004.
Clutch size was 17–42 eggs ( ¯
x¼31.2), and hatching
success was varied among clutches, again including 1
infertile clutch (Table 1).
T
inc
strongly influenced hatching success (Fig. 1).
Turtles at 23.08and 24.58C in 2002 appeared fully formed
but failed to pip; whereas, embryos at 31.08C initiated
development but died in the first 3 weeks of incubation.
Hatching success also varied within the narrower T
inc
range in 2004; 26.58and 28.58C exhibited 85% and 73%
hatching success, respectively; whereas, hatch rate fell to
40% at 30.58C.
Incubation duration varied nonlinearly with T
inc
.
There was substantial overlap in incubation duration
among eggs incubated at 28.58and 30.58C but no overlap
between those incubated at 26.58and 28.58C (26.58
range ¼90–99 days; 28.58range ¼80–87 days; 30.58
range ¼75–85 days). In 2004, embryonic development
took an average 11 days longer at 26.58C compared to
28.58C but took an average 3 days longer at 28.58C
compared to 30.58C (Fig. 2).
Variation in egg mass was analyzed separately for
2002 and 2004 due to the likelihood that individual
females contributed clutches in both years. Clutch strongly
influenced egg mass (2002, 2004: p,0.0001); however,
because each clutch was distributed randomly among
Table 1. Clutch size (n), hatching success, and egg and hatchling size for 9 Macrochelys temminckii clutches produced in 2002 and
2004. Mass and length values expressed as mean 61 Standard Error.
Year Clutch nHatching success (%) Egg mass (g) Hatchling mass (g) Carapace length (mm)
2002 1 37 30 26.38 60.19 16.46 60.23 36.51 60.17
2 36 22 24.37 60.18 14.68 60.28 35.11 60.27
3 15 0 24.32 60.49 — —
2004 1 33 79 27.26 60.20 17.17 60.17 35.93 60.19
2 28 75 23.76 60.18 14.72 60.27 33.62 60.29
3 35 0 27.99 60.13 — —
4 31 65 26.59 60.17 16.97 60.14 35.61 60.24
5 42 69 25.99 60.24 17.37 60.14 36.40 60.18
6 17 29 26.83 60.51 16.92 60.55 35.40 60.45
Figure 1. Alligator snapping turtle hatching success by
temperature of incubation in A) 2002 and B) 2004. Bar height
indicates the number of eggs incubated at each temperature, and
the black portion of each bar indicates the percentage that
successfully hatched.
LIGON AND LOVERN — Temperature Effects on Macrochelys temminckii Embryos and Hatchlings 77
treatments, mean egg mass did not differ among T
inc
s
(p.0.05).
Hatchlings. — There was a positive correlation
between log
10
egg mass and log
10
hatchling mass (log
10
hatchling mass ¼0.9151[log
10
egg mass] 0.0716,
r
2
¼0.495; Fig. 3A). Eggs that incubated at 30.58C
produced smaller hatchlings than those incubated at
26.58C or 28.58C (ANOVA: F
2, 94
¼4.84, p¼0.010);
this relationship remained consistent after correcting for
differences in initial egg mass (ANCOVA: F
2, 93
¼3.72,
p¼0.028; Fig. 3B). However, the relationship between
mass and T
inc
became progressively weaker and had
disappeared by the third week posthatching (7 days:
F
2, 86
¼4.60, p¼0.011; 14 days: F
2, 86
¼4.00, p¼
0.022; 21 days: F
2, 86
¼1.35, p¼0.264).
Growth rates during the first 11 weeks after hatching
were affected by T
inc
,T
water
, and age (Repeated Measures
ANOVA—T
inc
3age interaction: F
20, 950
¼2.44, p¼
0.0004; T
water
3age interaction: F
10, 950
¼4.76, p,
0.0001). Tukey’s post hoc tests indicated that turtles
reared in warm water grew consistently faster than those
maintained in cool water in weeks 6–11 (Fig. 4A). In
contrast to this consistent pattern, differences among T
inc
treatments were variable, with no regular pattern emerging
across multiple weeks (Fig. 4A). However, over time,
these modest and variable differences in mass-specific
growth rates translated into consistent differences in mass
among T
inc
treatments (Fig. 4B). At T
water
¼308C, turtles
incubated at 26.58C and 30.58C were larger than those
incubated at 28.58C beginning 8 weeks after hatching;
whereas, at T
water
¼258C turtles from the 2 lower
incubation temperatures were larger than those from
30.58C in weeks 7–11 (Fig. 4B). Averaged across
incubation temperatures, after 11 weeks turtles raised in
308C water had gained more than twice the mass as had
those maintained at 258C (308C: þ31.04 61.47 g; 258C:
þ14.92 60.55 g).
Maximum growth rate (independent of age) was
strongly influenced by T
water
but not affected by T
inc
(T
water
:F
1, 87
¼9.04, p¼0.004; T
inc
:F
2, 87
¼0.89,
p¼0.416; Fig. 5A). The time at which maximum growth
occurred also varied with T
water
but not T
inc
(T
water
:
F
1, 87
¼4.89, p¼0.029; T
inc
:F
2, 87
¼2.68, p¼0.075;
Fig. 5B). Turtles that were maintained at 258C exhibited
average maximum growth rates of 19.23 60.72
mgg
1
d
1
, compared to maximum growth rates of
28.27 60.95 mgg
1
d
1
among turtles raised in 308C
water.
Average postvent tail length was shorter among
hatchlings from T
inc
¼30.58C than from lower T
inc
s, and
was not significantly different between 26.58C and 28.58C
(ANCOVA: F
2, 82
¼12.22, p,0.0001).
Gonadal differentiation followed a pattern consistent
with previously published data (Ewert et al. 1994; Fig. 6).
A high proportion of males (81.4%) was produced at
26.58C; whereas, males constituted 3.3% and 0% of
hatchlings at 28.58C and 30.58C, respectively.
Metabolic Rate. — Hatchling oxygen consumption
rates showed a positive correlation with body temperature
(log
10
[oxygen consumption] ¼0.047 [body temper-
Figure 2. Incubation duration among M. temminckii incubated in
2004 at 3 constant temperatures. Error bars: 61 Standard Error.
Figure 3. Mass conversion efficiency of yolk to tissue at 3
incubation temperatures. A) Regression line fitted to the scatter
plot fits the equation: log
10
[hatchling mass] ¼0.9151(log
10
egg
mass) 0.0716, r
2
¼0.4952. Closed circles ¼26.58C, tri-
angles ¼28.58C, and inverted triangles ¼30.58C incubation
temperatures. Dashed lines ¼95% Confidence Interval. B)
Least-squares residuals from log–log plot. Error bars ¼61
Standard Error.
78 CHELONIAN CONSERVATION AND BIOLOGY,Volume 8, Number 1 – 2009
ature] 1.180; r
2
¼0.549; p,0.0001). Among hatch-
lings, T
inc
induced metabolic compensation (F
2, 84
¼
11.47, p,0.0001; Fig. 7A), though the effect was subtle.
Prolonged exposure to constant T
inc
resulted in slightly
higher MR among turtles incubated at 26.58C compared to
those incubated at 28.58C and 30.58C (Tukey’s post hoc
tests: p,0.01). MR of turtles incubated at 28.58C and
30.58C did not differ (Tukey’s post hoc test: p¼0.185).
Among 6-month-old juveniles, the previously observed
effects of T
inc
on metabolic compensation had disap-
peared, and no effect of recent exposure to constant T
water
was apparent (T
inc
:F
2, 85
¼0.76, p¼0.471; T
water
:
F
1, 85
¼0.02, p¼0.884; Fig. 7A, B).
MR Q
10
values did not differ among hatchlings
incubated at different temperatures (F
2, 17
¼2.94,
p¼0.079; Fig. 8A), and neither T
inc
nor T
water
affected
Q
10
among 6-month-old juveniles (T
inc
:F
2, 86
¼0.15,
p¼0.865; T
water
:F
1, 86
¼0.90, p¼0.346; Fig. 8A, B).
DISCUSSION
Incubation Duration. — The inverse relationship
between the duration of embryonic development and
temperature that was evident in the present study is not
surprising. However, at any given T
inc
, incubation duration
was longer than has been reported for many other
sympatric species (Ewert 1979; 1985). For example,
incubation period at 28.08C varied inversely with latitude
in Chelydra serpentina, ranging from 79 days in Florida to
Figure 4. Changes in A) growth rate and B) mass over time of
alligator snapping turtles incubated at 3 temperatures and
maintained at 2 different water temperatures. Circles ¼26.58C,
triangles ¼28.58C, and inverted triangles ¼30.58C incubation
temperatures. Closed symbols ¼258C and open symbols ¼308C
water temperatures. Error bars ¼61 Standard Error. Figure 5. A) Peak growth rates of alligator snapping turtles from
3 incubation temperatures and reared at 2 water temperatures.
Rates calculated over 7-day intervals. Error bars ¼61 Standard
Error. B) Timing of peak growth during the first 11 weeks post-
hatching. Black bars ¼258C water, gray bars ¼308C water.
Figure 6. Sex ratio of alligator snapping turtles incubated in 2004
(open circles). Closed symbols are data adapted from Ewert et al.
(1994).
LIGON AND LOVERN — Temperature Effects on Macrochelys temminckii Embryos and Hatchlings 79
just 59 days in Michigan (Ewert et al. 2005). In
comparison, we found that M. temminckii originating
from Oklahoma hatched in 82 days at 28.58C. Our results
corroborate the idea that M. temminckii, and other species
that produce relatively large eggs, may be range-limited at
northern latitudes by long incubation periods at relatively
cooler nest temperatures (Ewert 1985). High mortality
among M. temminckii overwintering in nests in captivity
(Grimpe 1987) suggests that this strategy is likely not
utilized to compensate for long incubation at high
latitudes. These limitations imposed on the embryic life
stage may account for the fact that there are records of
adult M. temminckii in Illinois (the northernmost extent of
the species’ range), including 1 gravid female (Phillips et
al. 1999), but no records of nests or hatchlings (Galbreath
1961; Smith 1961; S. Ballard, Illinois Department of
Natural Resources, pers. comm.).
Sex. — In contrast to many turtle species that exhibit
TSD, the pivotal temperature range within which a mixed
sex ratio is produced in M. temminckii spans a wide range
(Ewert et al. 2004). Our results support previously
published data suggesting that no constant temperature
results in 100% male production (Ewert et al. 1994).
However, temperatures that progressively increase during
development can produce higher male ratios than any
single constant temperature (Ewert and Jackson 1994). It
has been hypothesized that the production of females at all
T
inc
s is due to interactions between TSD and genetic sex
determination (GSD) mechanisms (Ewert et al. 1994). This
hypothesis has not been explicitly tested in M. temminckii,
but more recent data from another turtle with TSD suggest
that among-clutch variation in the concentrations of
maternally derived sex steroids deposited in the yolks of
eggs presents a more parsimonious explanation for the
phenomenon (Bowden et al. 2000; Elf 2004).
Hatchling Survival, Size, Growth, and Tail Length. —
Hatchling survival, mass, and tail length were reduced at
the highest incubation temperatures used in this study.
Survival has obvious direct effects on average lifetime
fitness, but even in the absence of embryo mortality, it is
generally assumed that the probability of survival
increases with size among age classes that are at risk of
predation (Janzen 1995). Therefore, high T
inc
s may be
detrimental to the average fitness of M. temminckii, even
when embryo mortality is not affected. However, this
effect could be dampened to some degree by the fact that
turtles from high T
inc
s develop and hatch faster, and
Figure 7. Metabolic response to changes in body temperature
following prolonged exposure to A) constant incubation
temperature and B) constant water temperatures. Symbols in
(A) are as in Fig. 4. B) open squares ¼258C, closed
squares ¼308C water temperatures.
Figure 8. Metabolic rate Q
10
values calculated from O
2
consumption measurements at 26.58C and 30.58C. A) Hatchlings
(black bars) and 6-month-old juveniles (gray bars) from 3
constant incubation temperatures, and B) juveniles after pro-
longed exposure to either 258Cor308C water temperatures. Error
bars ¼61 Standard Error.
80 CHELONIAN CONSERVATION AND BIOLOGY,Volume 8, Number 1 – 2009
therefore, would have greater opportunity to acquire
resources and grow prior to winter torpor.
Severely curled tails among hatchling M. temminckii
have been described, including speculation about the
causes and ecological consequences of such malformations
(McCallum and Trauth 2000). The consequences of
possessing a straight but unusually short tail are not
immediately clear, and its impact on overall fitness may in
fact be minimal. However, possessing a short tail could
affect turtles’ capacity to right themselves when flipped
onto their carapace (McCallum and Trauth 2000), impact
terrestrial locomotion (Finkler and Claussen 1997), or
correlate with other less apparent developmental problems.
Temperature Effects on MR. — The metabolic rates of
hatchling turtles from all 3 T
inc
s scaled positively with
body temperature. T
inc
did not affect sensitivity to
temperature, but the long-term exposure to a single
temperature during development did induce incomplete
metabolic compensation that could dampen the effects of
incubation at extreme temperatures. However, this effect
was short-lived, and M. temminckii exhibited no compen-
satory response following periods of exposure to different
temperatures after hatching. Therefore, it appears likely
that this species has only limited capacity to physiolog-
ically dampen temperature effects. This leaves the species
2 options: it might simply tolerate a wide range of
nonoptimal temperatures, or could restrict activity to areas
and seasons in which suitable temperatures prevail.
Although the first option is certainly a possibility, one
study reported seasonal movements from shallow water in
spring to deeper water during the heat of the summer,
suggesting that at least some seasonal temperature
selection occurs (Riedle et al. 2006).
Conservation. — Much remains to be discovered
about the thermal ecology of M. temminckii; however,
there is sufficient information to make informed recom-
mendations for conservation programs that incorporate
captive hatching and rearing into their protocols. Interme-
diate temperatures (27.58–28.58C) will produce a female-
biased mixed sex ratio. Although bias is not predicted in
natural populations where individual selection dominates
(Fisher 1930), it is reasonable to produce more females
than males when manipulating population demographics
to maximize reproduction. Additionally, female-biased sex
ratios have been observed in several natural populations of
turtles with TSD (Bull and Charnov 1989). The same
temperatures that produce a desirable sex ratio also result
in high embryo survivorship, normal development,
efficient yolk-to-tissue conversion that results in relatively
large hatchlings, and substantially shorter development
times than occurred at lower T
inc
s.
After hatching, turtles grew much more quickly at
308C than at 258C. Because fast growth minimizes the
time that juvenile turtles need to be maintained in
captivity, and/or maximizes the size at which turtles are
released, warmer water temperatures should be utilized
when it is feasible. Other studies have measured effects of
diet on growth and shown that high protein (45%–55%)
will also increase the rate at which turtles grow (Harrell
1998). These results, in combination with studies that have
addressed causes of population decline (Roman and
Bowen 2000), population genetics (Roman et al. 1999;
Hackler 2004), and behavior and population demographics
(Riedle et al. 2005) should be instrumental in maximizing
the success of M. temminckii conservation.
ACKNOWLEDGMENTS
We thank Tishomingo National Fish Hatchery and L.
Andrews for providing turtle eggs for this research. S.
Watkins and E. Ligon assisted with measuring metabolic
rates, and J. Bidwell, E. Ligon, S. Fox, M. Payton, J. Carr,
and an anonymous reviewer read and provided valuable
comments on earlier drafts. We are also indebted to the
Tulsa Zoo and K. Backues, DVM, for assistance with
laparoscopic sex determination. Financial and logistical
support was provided by the Oklahoma State University
Environmental Institute and Department of Zoology,
American Society of Ichthyologists and Herpetologists
Gaige Fund, Chicago Herpetological Society, Chelonian
Research Foundation Linnaeus Fund, Society of Integra-
tive and Comparative Biology Grants-in-Aid of Research,
and Sigma Xi Grants-in-Aid of Research.
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Received: 21 April 2008
Revised and Accepted: 4 March 2009
LIGON AND LOVERN — Temperature Effects on Macrochelys temminckii Embryos and Hatchlings 83
