,. I’ :1’ ..
Comparative Biochemistry and Physiology Part A 120 (1998) 399-403
Thermal independence of muscle tissue metabolism in the leatherback
g turtle, Dermochelys coriacea
David N. Peniclt ‘-"', James R. Spotila ‘, Michael P. O’Connor ", Anthony C. Steyermark “, .
Robert H. George ", Christopher J. Salice “, Frank V. Paladino °
- Department of Bioscience and Biotechnology, Drexcl University, Philadelphia. PA r9104, USA
" Virginia Institute. of Marine Science, Gloucxster Point. VA 23062, USA
‘Department of Biological Sciences, Purdue University, Fort Wayne. IN 46805, USA
Received 29 May 1997; accepted 20 January 1998
Metabolic rates of animal tissues typically increase with increasing temperature andthermoregulatory con-trol in an animal is . U
a regional or whole body process. Here we report that metabolic rates-of isolated leatherback ‘turtle (Dermochelys coriacea)
pectoralis muscle are independent of temperature from 5-38°C (Q0 2 1). Conversely, metabolic rates of green turtle (Chelonia
mydas) pectoralis muscle exhibit a typical vertebrate response and increase with increasing temperature (Q,,,= 1.3-3.0).
Leatherbacks traverse oceanic waters with dramatic temperature differences during their migrations from sub-polar, to equatorial
regions. The metabolic stability of leatherbaclc muscle effectively uncouples resting muscle metabolism from thermal constraints V
typical of other vertebrate tissues. Unique muscle physiology of leatherbaclrs has important implications for understanding
vertebrate muscle function, and is another strong argument for preservation of this endangered species. 0 1998 Elsevier Science
Inc. All rights reserved. ‘
Keywords: Orelania mydas; Dermochelys coriacea; Endangered species; Muscle; Sea turtle; Temperature; Thermal compensation;
Tissue metabolism :
Metabolic rates are among many physiological and
ecological processes. in reptiles that are temperature
dependent [14,15,8,26,37]. The importance of this ther-
mal dependence of physiological function is demon-
strated by the widespread occurrence of behavioral
thermoregulation among reptiles . Regulation of
body temperature may have evolved primarily as a
means of controlling rate functions in animals [7,2,5].
Other adaptations, including brain heater organs in
billﬁsh , regional endothermy in tunas,‘ sharks and
‘ Corresponding author. Present address: Department of Ecology
. and Evolutionary Biology, University of Connecticut, Storrs, CI‘
06259, USA. Tel: +1 860 4860811; fax: +1 860 486020; e-mail:
1095-6433/98/Sl9.00 O 1998 Elsevier Science Inc. All rights reserved.
turtles [3l,32,4], and countercurrent heat exchangers
 and gigantothermy in leatherback turtles (Der-
mochelys coriacea) , increase the ability of aquatic
vertebrates to function in cold water. Knowledge of the
physiological bases that underlie these adjustments is
fundamental to understanding the overall responses of
these organisms to‘ ﬂuctuating thermal environments
Leatherback turtles are among the largest living rep-
tiles, adults ranging in size from 250 to 900 kg [10,29].
They are pelagic, traverse ranges of latitudes greater
than any other reptile, and inhabit waters of 0-30°C
[3,35]. In cold water, they maintain elevated body tem-
peratures (Tb) (25.5°C in 75°C seawater, ), yet in
the tropics have Tb only l~3°C above ocean tempera-
ture [~18]. Metabolic rates of adults at rest (0.387 W
400 ' " " ' D.N. Perrldc e!_aI./Comparative Br'odIanLrtry and Physiology, Part A I20 (1998) 399-403
- kg-.1 an_d.whﬂe as... (1.227 w k,;-*) are intermedi-
ate to thosepredicted by allometric relationships for
reptiles and mammals . These data and mathemati-
cal models indicate that they can use large body size,
circulatory changes, and peripheral insulation to main-
tain warm body temperatures -in frigid waters and to
avoid overheatingin tropical waters. In a preliminary
experiment we lowered the body temperature of two
- adult leatherbacks 5°C with no reduction in resting
metabolic rate (unpublished data). This suggests that
there may be some thermal independence in metabolic
rate of this species. ‘
The objective of this study was to determine the
extent to which the metabolic rates of muscle tissue
were affected by temperature in this animal.
We measured tissue metabolic rates of excised "pec-
toralis muscles from nine leatherback turtles (mass,
280-380 kg) nesting on Plays Langosta, Costa Rica.
We compared these data to metabolic rates of muscle
tissue from ll subadult green turtles (Chelonia mydas)
(mass, 20-30 kg) at the.Cayman Island Turtle Farm,
and to metabolic rates of muscle tissue from three
lizards (Clenosaunzs similis) and two marine toads
(Bufo marinas) tested simultaneously with leatherback
tissues in Costa Rica as controls. We removed
leatherback samples by surgical biopsy and obtained
green turtle samples from ‘farm-reared animals upon
their harvest . We removed muscle tissues from
lizards and toads after sacriﬁce by decapitation. Upon
removal, we placed samples in sterile ice cold, physio-
logic saline (0.9% pH 7.2) with 10 mM glucose added as
an energy substrate. We used sealed sterile saline be-
cause of concerns of bacterial contamination, and for
consistency under uncertain field conditions in the _trop—
ics. We prepared triplicate’ samples on ice and kept
them moist with-saline solution. We quickly trimmed
samples of fascia and vascular tissue, and blotted them
dry before weighing them to ;]:0.5 mg. We teased
samples apart longitudinally, cut them into .1 ~2 mm
long sections, and suspended them in the saline solution
[l1,33,l'7]. We then -placed samples (1 i 0.05 g) into 15
ml respirometry ﬂasks, brought preparation volumes to
3 ml, and immersed ﬂasks in a temperature controlled
water bath (i 0.5°C). We determined metabolic rate by
measuring 02 uptake at 10.20, and 30 min after a 15
min equilibration period at each temperature.
We measured oxygen consumption? in a Gilson Dif-
ferential Respirorneter using standard ‘techniques .
Three subsamples prepared for each tissue sample were
run independently. We used mean metabolic rate for
the three subsamples as the metabolic rate for each
a individual at each temperature (5, 12.5, 20, 27.5, 35, 38
and 40°C). The original temperature protocol‘ was from
S—35°C; the 38 and 40°C temperatures were added
when the '5—35°C trials revealed no signiﬁcant differ-
ence. This shift in temperature protocol for leatherback
samples was reflected in a decreased sample size at
higher temperatures (Fig. 1). Cautionmust be applied
when measuring metabolic rates of excised muscle tis-
‘sue. Ideally we should have measured the metabolic
rate of a whole muscle preparation since teasing apart
the muscle disrupts the cells (, p. 141). However, it
was not practical to measure the metabolism of an
entire pectoralis muscle of a leatherback turtle because
it may weigh as much as 25 kg, limiting diffusion of
nutrients, oxygen and carbon dioxide, and removal of
the entire muscle ‘would be lethal. Since this species is
endangered, this approach was ethically not acceptable.
A similar technique was used by West et al.  to
study in‘ vitro glucose uptake in trout (Oncorhynchus
mykiss) skeletal muscle, by Oikawa and Itazawa [22,23]
to measure the in vitro metabolic rate of muscle tissue
from ﬁsh, and by Penick et al.  to study tissue
metabolism of green turtles. Schmidt-Nielsen  and
Burggren and Roberts  discuss the value of this type
of data on muscle metabolism in their discussions of
scaling and the relationship between metabolism and
body size. While the results reported here may not.
represent the truesresting metabolic rates of muscle in
vivo, they do provide a valid indicator of the relative
metabolic response to temperature of muscles from the
species tested. . -
‘ Given the limitations of this technique, we also com-
pleted several experiments to reduce the error in these
experiments. These controls showed that: (1) There was
no signiﬁcant difference in tissue metabolic rates if
temperature order was randomized (n = 4), or returned
0 10 20 30 40
Fig. 1. Oxygen consumption of pectoralis muscle from green (,1 .~. 11
except at 35°C where 11:4) and leatherback turtles (n=9 from
5—27.5°C. l1==4 at 35°C and n=5 at 38 and 40°C). Error bars
represent 131 SE.
mv. muck er al. /Canlplaratloe Biochernirlry and Physiology, Part A 120 (1993) 399-4433 401
to starting position after -completion of all temperatures
(n=5). This control indicated that while there may
have been some disruption of cellular integrity, the
muscle was still viable;.(2)' There was no difference in
metabolic rate of tissue that were > l0 h old. This
control indicated that tissues remained viable for a time
greater than thatnequired for the experimental proto-
col; (3) There was no difference in metabolic rate when
a turtle Ringer’s solution» (100 mM NaCl, 4 mM KC],
10 mM NaHCO,, 5 mM Nazi-IP04, 2 mM Nal-11014,
0.75 mM CaCl,_ 10 mM ‘glucose and 30 mEq Na
HEPES Buffer) was used instead of the glucose and
saline solution. This indicated that the saline solution
was a sufficient medium for this experiment; (4) There
was no signiﬁcant difference in pH of saline solution
over the course of the run; (-5) There was no difference
in tissue, metabolic rate when lidocaine was used prior
to excising tissues; (6) There was no difference between
expected and observed patterns of metabolic rates of
toad, Bufo marinas, thigh muscle and lizard, Ctenosau-
rus similis, quadriceps musclemeasured simultaneously
with and by identical techniques as the leatherback
We found signiﬁcant differences (P=0.05) in tissue
metabolic rates of green turtle and leatherback muscle
using repeated measures analysis of variance
3. Results A
We found that temperature signiﬁcantly affected
metabolism of subadult green turtle muscle (repeated
measures ANOVA, F = 226, P < 0.0001 for 11 individ-
uals at 5—27.5°C) with values of 30.1 pl 02 g“' h"
(wet mass) at 5°C to 119.5 [1102 g" h" at 35°C (Fig.
1). Metabolic rates were similar at 27.5 and 35°C.
Thermal dependence of tissue metabolism, computed as
Q“, was 2.97 from 5—l2.5°C, 1.56 from l2.5—20°C,
1.31 from 20—27.5’C, and 1.00 from 27.5—35°C. Con-
versely, metabolic rates of leatherback pectoralis muscle
were completely insensitive to temperature from 5-
35°C (57.0—60.S pl 02 g" h"), became variable at
38°C (Qm = 1 from 5—38°C) and decreased signiﬁcantly
at 40°C (33.9 pl 0, .g-'1 11°‘). Metabolic rates of thigh
muscles from the toad, -Bufo marinas, and quadriceps
muscles from the lizard, Ctcnosaurus similis, "used as
controls and measured simultaneously with leatherback
samples, responded as expected‘ for vertebrate tissue
with metabolic rates increasing from‘94 pl 0, g‘ ‘ _h“‘
at 5°C to 145 pl 0, g“ h" at 25°C for the‘ toad and
from 27.5 p10, g”‘ hr? at 5°C to 74.7 pl 0; g"‘ h"
. at 35s@ for the lizard [n¢2 and 3, respectively).
Metabolic rate of leatherback pectoralis muscle’ was
higher than that of green turtle muscle at 5?’C', similar
at 12.5°C,'and lower at higher temperatures,
Untilhnow, temperature dependence of vertebrate
tissue metabolism (0, consumption) over a broadrange
of temperatures was almost universal, and a basic
paradigm of comparative physiology [27,28,7]. Temper-
ature independence of leatherback pectoralis muscle
metabolism is a rare example of perfect metabolic
compensation of tissue metabolic rate over the range of
environmental temperatures experienced by a verte-
brate. Temperature dependence of green turtle pec-
toralis muscle using identical protocols, and Bufo and
Ctenosaurus muscles. using identical test conditions and
run concurrently with leatherback tissues is persuasive
that this phenomenon is real and not a procedural
artefaot. In addition, our extensive controls also sup-
port this conclusion.
Data on other reptiles indicate that their muscle
tissues exhibit temperature dependent metabolic rates in
vitro like those of green turtles, ‘ctenosaurs and marine
toads. I-Ioskins and Alelcsiuk  report the inﬂuence of
temperature (4—34°C), photoperiod and season on oxy-
gen consumption of several different tissues of the
garter snake, Thamnophis sirtalis. There are signiﬁcant
changes in Q“, with temperature and season, with Q“,
values of g:6.5 at low temperatures (4-20°C) and z 2
at_ higher temperatures (20~34°C). This compares with ~
a Q“, of‘2.97 for’5-l2.5°C, 1.56 for 12.5-20°C, 1.31
for 20-27.5°C and l.0 for 27.5—35°C for the green
turtle and a Q", of l.0 for 5—35°C in leatherback turtle
muscle. Morris  reports metabolic-rates for eight
tissues, including muscle of the eurythermic lizard Leia-
plopisma zelandica, from 5—40°C. The Q“, values range
from 2 to 4, indicating a greater thermal dependence
than we measured in green turtle muscle.
Fish tissues generally exhibit temperature dependent
metabolic rates in vitro . However, skipjack tuna
(Katsuwonus pelamis) white muscle in vitro has a con-
stant oxidative metabolism from 5—25°C which in-
creases from 25 to 35°C. Tuna’ red muscle has a very
high metabolic rate which increases dramatically from 5
to 25°C, and is temperature independent from 25 to
35°C. However, this apparent temperature indepen-
dence of tuna muscle is difficult to interpret because of
relatively high variability in metabolic rates and low
sample size [ll].
Standard metabolic rates of some marine inverte-
brates (e.g. sea anemone Actinia, crustacean Nephrops,
snail Littorina, and clam Cardium) are very low, and
relatively independent of temperature over their daily
temperature range, but are thennally sensitive at more
extreme temperatures [l-9-21]. This adaptation allows
muscle operation over changing temperatures without
5 changes in muscle function [l9—2l].
The potential ecological signiﬁcance of this metabolic
phenomenon is illustrated by comparing the natural
402 D.N. Penick er al. Comparative ﬂiodravsértry and Phyﬂobgy, Part A 120 (1998) 399-403
history of leatherbaeks and green turtles. Leatherbacks
are largely .pelagic and frequently occur in extreme,
sub—polar waters. Green turtles inhabit warm, shallow
coastal waters and maintain body temperatures l—3°C
warmer than water temperature . Temperature sen-
sitivity of green turtle muscle tissue may explain their
tropical and sub-tropical distribution. It may also be
responsible for their susceptibility to cold stunning, a
winter phenomenon in which they lose the ability to
swim and dive when exposed to cold water [36,25]. The
metabolic stability of resting leatherback muscle may
facilitate? survival in both cold and warm water, allow-
ing use of a broad thermal niche, not only by adapta-
tions for heat retention [24,4,9], but also by direct
regulation of tissue metabolism, such that pectoralis
muscle functions independently of its thermal
Additional experiments are needed to elucidate the
functional signiﬁcance of this ﬁnding and its molecular
mechanisms. Key rate limiting enzymes in metabolic
pathways need to be assessed with regard to tempera-
ture, K,,,, V,,,_,,, enzyme-substrate affinity, andnumber
of isozymes. Molecular studies are also needed on gene
regulation involved in this system.
We thank the ‘following for technical assistance: M.
Boza, M.T. Koberg, P. Patillo, R. Arauz, R. Byles, J.
and F. Wood, G. Serrano, G. Marin, V. Lance, I.
Naranjo-Arauz, A. List, and our EARTHWATCH and
Drexel volunteers. Protocols were approved by animal
care committees of Drexel and Purdue Universities.
Research supported by NSF (DCB-9019780), National
Geographic Society, EARTHWATCH, and by the Betz
Chair endowment of Drexel University. '
 Avery ‘RA. Field studies of body temperature and thermoregu1a-
tion. In: Gan: C, Pough FH, editors. Biology of the Reptilia,
vol. 12. New York: Atzdemic Press, 198293-166.
 Bennett AF. The evolution of activity capacity. J Exp Biol
 Bleakney IS. Reports of marine‘ turtles from New England and
Eastern Canada. Can Field Nat l965;79:l20—8.
 Block BA, Finnerty JR, Stewart AFR, Kidd 1. Evolution of
endotherrny in fish, mapping physiological traits to a rnolwular
phylogeny. Science 1993;260:210-4.
 Block BA. Evolutionary novelties: How fish have built a heater
out ofmuscle. Am Zool 199l;3l:'Z26-42. ‘
 Burggren W, Roberts J. Rupiration and metabolism. In: Prosser
CL, editor. Environmental and Metabolic Animal Physiology.
New York: Wiley, 199l:353—435. ‘ ‘ _
 Cousins AR, Bowler K. Temperature Biology of Animals. New
‘York: Chapman and Hall, l987:339.
 Dunham AE, Grant raw, Overall KL. interfaces between bio-
’ physical and physiological ecology and the population ecology of
V terrestrial vertebrate ectotherrns. Physiol Zool l989;62:335—55.
 Eclrert SA, Eclzert KL, Ponganis P, Kooyman GL. Diving and
foraging behavior of leatherback sea turtles (Dernioclrely: cari-
acea). Can J Zool 1989;67:2834-40.
 Golf GP,‘Lien J. Atlantic leatherbaclr turtles, Dermachcly: cari-
acea, in cold water off Newfoundland and Labrador. Carr Field
 Gordon MS. Oxygen consumption of red and white muscles
from tuna ﬁshes. Science 1968;l59:87—9.
[121 Greer AB, Lazell ID, Wright RM. Anatomical evidence for a
countercurrent heat exchanger in the leatherbaclt turtle (Der-
mochelys cariacca). Nature 1973;244:181.
 Hoskins MA, Alelrsiuk M. Effects of temperature, photoperiod
and season on in vitro metabolic rates of tissues from
Thamnophts .r-irrali: parietalis, a cold climate reptile. Comp
' Biochcrn Physiol l973;45A:737—56.
 Huey RB. Temperature, physiology, and the ecology of reptiles.
in: Guns C, Pough Fl-1, editors. Biology of the Reptilia. vol. 12.
New York: Academic Press, l982:25-91.
 Huey RB. Physiological consequences of habitat selection. Am
Nat l99l;l37:S9l—Sll5. - '
[161 Huey RB, Bennett AF. Physiological adjustmentsto ﬁuctuating
thermal environments: An ecological and evolutionary perspec-
tive. In: Morimoto RI, Tissieres A, Georgopoles C, editors.
Stress Proteins in Biology and Medicine. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory Press, 199037-59.
 Morris RW. Effects of temperature on metabolic rates of iso-
lated tissues from the eurytherrnic lizard Leiaplopirma zelandica.
Comp Biochem Physiol l980;66A:l27-31. _
 Mrosovsky N, Pritchard PCH. Body temperatures of Der-
" moclrelys cariacea and other sea turtlts. Copeia l97l;197l:624—
 Newell RC,.Pye V1. Seasonal changes in the effect of tempera-
ture on the oxygen consumption of the .win1cle Littorrha litmrea
and the mussel Mytiltu edulir. Comp Biochern Physiol
 Newell RC, Pye V1. Quantitative aspects of the relationship
between metabolism -and temperature in the winlrle Lirtarina
Iiuarza (L). Comp Biochem Physiol 197l;38B:635—50.
 Newell RC, Pye Vl. Temperature-induced variations in the respi-
ration of mitochondria from the winkle, Lrjttorina litlorea (L.).
Comp Biochem Physiol 1971;4013:249.—6l. ~
 Oikawa S, ltanwa Y. Allometric relationship between tissue
respiration and body mass in a marine teleost, porgy Pagrus
major. Comp Biochem Physiol 1993;105A:l29—33.
 Oilcawa S. Itazawa Y. Tissue respiration and relative growth of
parts of body of a marine teleost, porgy Pagru: major, during
early life stages with special reference to the metabolism-size
relationship. Comp Biochem Physiol l993;l05A:741—4.
 Paladino FV, O'Connor MP, Spotila IR. Metabolism of
leatherback turtles, gigantothermy, and thermoregulation of di-
nosaurs. Nature 1990;344:858-60.
 Peniclt DN, Paladino FV, Steyennark AC, Spotila lR. Thermal
dependence of tissue metabolism in the green turtle, Chelonia
mydar. Comp Biochem Physiol 1990;113:293-6.
 Pough FH. Organismal performance and Darwinian ﬁtness:
Approaches and interpretations. Physiol Zool 1939;62:199-236.
 Prosser CL. Comparative Animal Physiology. Philadelphia:
Saunders, 19732966. -
 Prosser CL. Adaptational Biology Molecules to Organisms.
New York: Wiley, l986:784. ’ _
 Rhodin AG, Ogden IA, Conologue GJ. Chondro-osseous mor-
phology of Dermochelyr coriacea, a marine reptile with mam-
malian skeletal features. Nature 1981;290:244-6. ‘
D.N. Penick et al. / Comparative Biocllemiury "and Physiology, Part A 120 (1998) 399-403 403
i  Schmidt-Nielsen Scaling. Why is Animal so Important?
New York: Cambridge University Press, 1984. ,
 Standom EA, Spotiln JR, Foley RE. Regional mdothermy in
. the sea turtle, Chelouia mydas. J Therm Biol l982;7:l59—65.
 Stxmdora EA, Spotila JR, Keinath IA, Shoop CR. Body tem-
peratures, diving cycles, and movement of a mbadult
leatherback turtle, DcmIocheIys- corlacea. Herpetologica
l984;40:l69—76. . '
 Umbreit WW. -Burris RH, Stauffer JF. Mnnometric and Bio-
chemical Techniques. Minneapolis. MN: Burgess. 19724387.
 West TG, Arthur PG, RK, Doll C3, Hochachka P. In
vivo utilization of glucose by heart and locomolory muscles of
exercising rainbow trout (0ncorhynchu.r mykiss). J Exp Biol
 Willgoh: JF. Occurrence of the leathery turtle in the northem
North Sea and oil‘ western Norway. Nature l9S7;179:63—l64.
 Witherington BE, Ehrhnlt LM. Hypothermia stunning and
mortality of marine turtles in the Indian River lagoon system,
Florida. Copeia l989jl989:i989.
 Zimmerman LC, Tracy CR. Interactions between the environ-
‘ ment and ectotherrny and hcrbivory in reptiles. Physiol Zool