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First published online April 17, 2009
Journal of Experimental Biology 212, 1237-1247 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023945
Atmospheric oxygen level affects growth trajectory, cardiopulmonary allometry and metabolic rate in the American alligator (Alligator mississippiensis)
1 Ecology and Evolutionary Biology, University of California, Irvine, CA 92697,
USA
2 Rockefeller Wildlife Refuge, Louisiana Department of Wildlife and Fisheries,
Grand Chenier, LA 70643, USA
* Author for correspondence (e-mail: towerkow{at}uci.edu)
Accepted 3 February 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: hypoxia, hyperoxia, growth, metabolic rate, cardiopulmonary plasticity, ontogenetic allometry
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Graham et al.,
2004; Fan et al.,
2005), environmental physiologists interested in human acclimation
(or lack thereof) to high altitude
(Hochachka, 1998;
Hoppeler and Vogt, 2001;
Erzurum et al., 2007), and
comparative physiologists interested in animal adaptation to oxygen-poor
environments (Frappell et al.,
2002; Bickler and Buck,
2007; Ramirez et al.,
2007). Less attention has been paid to hyperoxia, which is only
encountered by air-breathing vertebrates at hatching or birth
(Mortola, 2001). Effects of
hyperoxic exposure have been studied, for instance, by paediatricians treating
pulmonary insufficiency and airway inflammation in premature mammals
(Denis et al., 2001;
Rehan and Torday, 2003;
Dieperink et al., 2006) and by
exercise physiologists interested in improving human performance
(Nummela et al., 2002;
Romer and Dempsey, 2006;
Stellingwerff et al., 2006;
Perry et al., 2007). Most
studies have focused on relatively acute (from minutes to days) exposure to
hypoxia and hyperoxia, either because short-term exposure better characterised
the physiologic situation (e.g. ischaemic attack, diving response) or because
of the logistic difficulties of maintaining vertebrate animals under constant
non-normoxic atmospheres. Acute and chronic exposure, however, can have
drastically different effects on physiologic function–for instance,
metabolic depression is a frequent response of many organisms to acute, but
not chronic, hypoxia (Jackson,
2004; Ramirez et al.,
2007).
The influence of hypoxia on physiologic function may also have been a
critical environmental factor during the course of vertebrate evolution.
Recent models of Earth's atmospheric composition during the Phanerozoic Eon
show that oxygen levels might have risen as high as 30–40% in the
Permian, and dropped as low as 12% in the Late Triassic and Early Jurassic
(Bergman et al., 2004;
Berner, 2006). Considering that
the oxygen supply to respiring tissues is in large part determined by the
atmospheric oxygen level, the physiology of extinct vertebrates is likely to
have been affected by contemporary hypoxia and hyperoxia, and may have been
quite different from the physiology of extant vertebrates living in normoxia
(with oxygen comprising 20.94% of air). Hence, changing levels of atmospheric
oxygen over the past 550 million years must have influenced the course of
vertebrate evolution (McAlester,
1970; Erwin,
1993). With a few recent exceptions
(Graham et al., 1995;
Dudley, 1998;
Berner et al., 2003;
Falkowski et al., 2005;
Huey and Ward, 2005;
Ward et al., 2006),
atmospheric oxygen levels and other aspects of the abiotic environment are
rarely considered by palaeobiologists attempting to reconstruct the
physiological function of extinct animals (e.g.
Chinsamy and Hillenius, 2004;
Padian and Horner, 2004;
Fastovsky and Weishampel,
2005). In fact, some studies dismiss episodes of hypoxia or
hyperoxia as insufficient to explain patterns in vertebrate diversity,
focusing on global CO2 levels instead
(Knoll et al., 2007). To date,
most attempts to estimate the growth rate and metabolism of extinct species
have been based upon modern functional analogues breathing normoxic air, with
the atmospheric oxygen content of air at approximately 21%. Findings of these
studies are appropriate for our understanding of the physiological responses
of extant organisms adapted to today's atmosphere. Yet, it is clear that rates
of any sustainable activity, such as growth, locomotion and digestion, will be
dependent on sufficient oxygen being available to support aerobic
respiration.
In order to illustrate and understand the potential effects of chronic
hypoxia and hyperoxia on physiologic function of vertebrates, we incubated
eggs and subsequently raised hatchlings of the American alligator
(Alligator mississippiensis, Daudin 1801). We measured whole-body
growth and metabolic rates of alligators raised under three oxygen levels:
12%, 21% and 30%. These levels were chosen so as to provide boundary
conditions (using the most recent GEOCARBSULF model) for the likely minimum
and maximum oxygen levels that vertebrates experienced in the past
(Berner, 2006).
The alligator was chosen as a model experimental species because it is an
ectothermic representative of the large clade Archosauria, which encompasses
all crocodilians, phytosaurs, pterosaurs and dinosaurs (including birds).
Archosaur origins can be traced back to Euparkeria
(Ewer, 1965;
Carroll, 1988) of the Late
Permian; the fossil record of the earliest crocodilian Protosuchus
(Colbert and Mook, 1951;
Crompton and Smith, 1980) can
be traced back to the Late Triassic. Given their phylogenetic position and
highly conserved morphology throughout their evolutionary history,
crocodilians are often thought to retain many characteristics of basal
archosaurs. Although crocodilian metabolic and cardiopulmonary physiology
might have changed over the course of their evolutionary history
(Seymour et al., 2004),
crocodilians have obviously survived and thrived despite large-scale
fluctuations in atmospheric oxygen. In addition, the oxygen transport system
of crocodilians (unlike that of lizards) is known to exhibit plasticity in
response to long-term metabolic stress
(Owerkowicz and Baudinette,
2008). This suggests their high phenotypic adaptability to novel
environments and metabolic demands.
At this point, it is important to acknowledge a distinction between
evolutionary adaptation and acclimation-induced phenotypic plasticity. The
former describes how a species genetically responds to environmental change
(such as % O2), whereas the latter is a measure of an organism's
(specific genotype) capacity to change its phenotype in response to an
environmental stimulus. Although selection studies and the paradigms of
experimental evolution have now gained popularity among comparative
physiologists (see `Focused Issues' in Physiol. Biochem. Zool. vol.
80, nos 4–6, July–August 2007), these approaches are logistically
impossible in experiments on slow-growing and slow-reproducing ectothermic
vertebrates (with an inter-generation time of 5 years or more). Hence, studies
of acclimation and phenotypic plasticity (such as this study) may provide the
next best alternative to understanding of the palaeophysiology of extinct
vertebrates in non-normoxic atmospheres
(Berner et al., 2007).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) marked with
pencil, and eggs from each clutch randomly assigned to one of three
incubators.
Egg incubators were constructed out of shallow plastic containers, filled
with moist vermiculite [initial vermiculite:water ratio 2:1
(Crossley and Altimiras,
2005)] and maintained at 30.0±0.5°C inside a walk-in
environmental chamber. Full humidity was maintained in the incubator by
passing the air supply through a water-bubbler and lightly misting the eggs
every other day. The water content of the vermiculite was not monitored,
because eggs were not buried in vermiculite but laid on top of it in a shallow
dimple. Each incubator was sealed in a large plastic bag and supplied with
hypoxic, normoxic or hyperoxic air. Hypoxia (12% O2) was achieved
by mixing compressed air with nitrogen, and hyperoxia (30% O2) by
mixing compressed air with oxygen.
Airflow to each incubator was approximately 750 ml min–1, controlled and monitored by a rotameter (Cole Parmer, Vernon Hills, IL, USA). Inflation of the plastic bags was checked several times a day and used as confirmation that each incubator was under slight positive pressure from the mixed air/gas supply, i.e. incubator atmosphere was unlikely to be affected by inward leakage of room air. Air from each incubator was regularly sampled and checked with O2 and CO2 sensors (P-61B and N-22, respectively; Applied Electrochemistry Instruments, Pittsburgh, PA, USA), connected to O2 and CO2 analysers (S-3A and CD-3A, respectively; Applied Electrochemistry Instruments). Mixed gas flow rates were adjusted to maintain the O2 level within 0.5% of 12%, 21% or 30% (appropriate for each treatment group), and to make sure that the CO2 level remained <0.3%.
After setting the eggs in incubators, every other week we confirmed that
eggs remained viable. Initially, we did so by checking whether the white
opaque band widened beyond its original markings, as its width corresponds to
the extent of the underlying chorioallantoic membrane
(Ferguson, 1985). If it had
not, the embryo was assumed to be dead and was discarded. Once the
chorioallantois had spread under the entire egg surface, we resorted to
briefly candling each egg to view embryonic activity every other week. If no
embryonic movement in response to light tapping of the eggshell could be
detected, the egg was opened (to check for embryonic viability) and
discarded.
Misting and viability checks required opening of incubators and exposure of eggs to room air, i.e. hypoxic and hyperoxic conditions were occasionally interrupted by acute normoxia. These disturbances, however, were short (each misting was accomplished in less than a minute, and each viability check in under 15 min) and occurred infrequently (misting every other day and viability checks every fortnight). Thus, for the most part, embryos remained under the desired atmospheric conditions for the majority of the incubation time.
Hatching times of individual alligators were recorded and pooled for each group in 8 h bins. We used a Wilcoxon/Kruskal–Wallis rank sums test to test for significant differences in hatching times.
Post-hatching maintenance
Alligator hatchlings (N=6 in hypoxia, N=27 in normoxia,
N=20 in hyperoxia) were maintained under the same oxygen levels as
during incubation. For 3 months after hatching, animals were raised at
30°C with a relative humidity >95% and a 12 h:12 h light:dark cycle.
Animals were kept in plastic tubs (0.6 mx0.4 mx0.3 m) enclosed in
a large transparent plastic bag, with 6–7 animals per tub. Each
container was supplied with air (at airflow >4 l min–1) of
appropriate % O2 for each treatment group. Air in the containers
was monitored daily with O2 and CO2 analysers, and flow
rates were adjusted to maintain O2 within 0.5% of the desired level
and the CO2 level below 0.3%. Animals had daily access to water and
were fed every other day ad libitum (lean ground beef, generously
sprinkled with powdered mineral/vitamin mix). From week 6 onward, alligators
also received live crickets ad libitum twice a week. For the last
week, food was withheld, so standard metabolic rates could be measured on
fasted animals. Holding tanks were cleaned daily.
Every other week, starting at hatching, alligators were weighed (±1 g) and measured for total and snout-to-vent length (±1 mm), as well as head length and post-orbital head width (±0.1 mm). A repeated-measures analysis of variance (RM-ANOVA) was used to test whether treatment (% O2, nested within clutch) had a significant effect on the post-hatching growth trajectories of the three groups.
Animal husbandry necessitated opening the holding tanks daily and exposure of the hatchlings to room air. As with egg incubation, animals were out of their specific oxygen environments as briefly as practically possible. Each feeding (placement of food in tank) took less than a minute, cleaning took under 10 min, and measurements under 15 min.
Metabolic and breathing rate measurements
Rates of oxygen consumption
(
O2) and carbon
dioxide production
(CO2) were
measured in 3 month old alligators (N=6 for hypoxia, N=14
for normoxia and N=10 for hyperoxia; each treatment group comprised
two clutches) under absorptive (within a day of feeding) and post-absorptive
(fasted for 5–6 days) conditions. All metabolic measurements were made
at 30°C, under full humidity and in complete darkness.
For each metabolic measurement, animals were placed in individual respirometry chambers, fashioned from PVC pipes and tightly capped at both ends. Water (200 ml) was placed in each chamber to prevent dehydration stress of the animals. A constant, unidirectional flow (300–500 ml min–1) of humidified oxygen/nitrogen gas (with % O2 appropriate for each treatment) was maintained across each chamber with a Cameron GF-3 gas mixing flowmeter (Cameron Instrument Co., Port Aransas, TX, USA). Excurrent flow was subsampled at 120 ml min–1 and passed through a column of Drierite, a LI-6251 carbon dioxide analyser (LI-COR, Lincoln, NE, USA) and an Oxzilla oxygen analyser (Sable Systems International, Las Vegas, NV, USA). Data were collected at 50 Hz using the MP100 A/D board and AcqKnowledge 8.3.1 software (Biopac, Goleta, CA, USA) on a Dell Inspiron 1100 personal computer.
Metabolic measurements were initiated at least 8 h after the animal was
last handled, the minimum time necessary for the alligator respiratory
parameters to return to resting values
(Hartzler et al., 2006). For
each animal in each state (absorptive and post-absorptive), three measurements
(each of 25–30 min duration) were taken over the course of 12 h.
Individual metabolic rates were calculated as
O2 from the
difference in fractional O2 content in incurrent
(FIO2) and excurrent
(FEO2) air, using equation 3a from
Withers (Withers, 1977). The
average of O2
measurements under absorptive conditions was taken as the absorptive metabolic
rate (AMR), and the lowest
O2 measurement
under post-absorptive conditions was taken as the standard metabolic rate
(SMR).
In order to compare metabolic rates of animals from different treatment
groups, we ran an analysis of covariance (ANCOVA) of
O2 measurements,
with body mass (Mb) as covariate. We found AMR to scale
proportional to Mb0.69, and SMR to scale
proportional to Mb0.65, with no significant
interaction effects between body mass and treatment. These allometric
exponents are close to those reported for resting alligators at 25°C
[
Mb0.69
(Smith, 1975)], and for
estuarine crocodiles across a range of ecologically relevant temperatures
[20–33°C, Mb0.65–79
(Wright, 1986)]. Hence, we
used an average exponent (Mb0.67) to
mass-correct individual metabolic rates (i.e.
VO2/Mb0.67) and
applied an ANOVA (with post hoc Tukey–Kramer test) to compare
the three O2 treatment groups.
We estimated breathing rates for each animal under absorptive or
post-absorptive conditions by counting distinct dips in the
FEO2 trace and peaks in the
FECO2 trace, based on the assumption
that these corresponded with individual exhalations. In each case, the number
of exhalations obtained from the raw O2 trace corresponded with the
number obtained from the raw CO2 trace. We calculated breathing
rate by dividing the number of exhalations by the time period over which they
occurred. ANCOVA did not detect a significant scaling relationship between
breathing rate and body mass. Unlike mammals and birds where breathing rate
scales approximately proportional to
Mb–0.25
(Frappell et al., 1992a;
Frappell et al., 2001), among
ectothermic reptiles breathing rate is mass independent in lizards (e.g.
Frappell and Daniels, 1991)
and, apparently, in crocodilians (this study). Hence, we did not correct
breathing rate estimates for body mass, and used an ANOVA (with post
hoc Tukey–Kramer test) to compare breathing rate between the three
O2 treatment groups.
Tissue harvest
Blood samples were taken from the supraspinal vein of the 3 month old
alligators, within 2 days of the last (standard) metabolic measurement, just
prior to killing. Haematocrit fraction was determined in triplicate using
heparinised micro-capillary tubes spun in a capillary centrifuge (Hermle
Z231M, Gosheim, Germany) at 6000 g for 3 min. Haematocrit
values were arcsin square root transformed, and compared between groups using
a Wilcoxon/Kruskal–Wallis rank sums test (with post hoc
Tukey–Kramer test).
Prior to tissue harvest, animals were anaesthetised with isoflurane, killed
by exsanguination and dissected. We excised major visceral organs (liver,
lungs, heart) and select skeletal muscles (rectus femoris and diaphragmaticus)
from hatchling alligators within a day of hatching, and from juvenile
alligators (13 weeks old) within 2 days of collecting SMRs (see below). In
addition, we divided the heart ventricle into the right ventricular free wall
(RV) and the left ventricle (left ventricular free wall + interventricular
septum; LVS), and calculated the RV/LVS ratio
(Fulton et al., 1952). Organs
were excised, cleaned of blood and connective tissue, lightly blotted with
gauze and weighed (±0.01 mg) on an analytical balance (Mettler AB104,
Toledo, OH, USA). For hatchlings, the remnant yolk sac and yolk-free body mass
(±0.1 g) were also recorded.
We used a one-way ANOVA on log-transformed whole-body variables of
hatchlings: total mass, yolk-free mass, yolk sac mass, total length,
snout-to-vent length and head length. We also used a one-way ANOVA to compare
wet masses of individual organs (liver, lungs and heart) in 1 day old
hatchlings and 3 month old juveniles. We compiled measurements from hatchlings
and juveniles to generate logarithmic linear regressions against body mass and
establish allometric scaling relationships for individual viscera and select
skeletal muscles. For each regression, we used an ANCOVA (with body mass or
organ mass as covariate) to test for significant differences between
treatments and for body mass–treatment interaction, with 
set at
0.05.
All experiments were carried out with the approval and in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of California, Irvine.
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RESULTS |
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Compared with their normoxic siblings, accretion in body mass was slower in hypoxic alligators and faster in hyperoxic ones (clutch-nested RM-ANOVA, F11,41=14.147, P<0.001; Fig. 2A). Similarly, the increase in total, snout-to-vent and head lengths was slower under hypoxia, and faster under hyperoxia (RM-ANOVA, F11,41>11.646, P<0.001; Fig. 2B).
Ontogenetic allometry of visceral organs and skeletal muscles
At hatching, wet masses of major visceral organs–liver (ANOVA,
F2,53=33.136, P<0.0001), lungs (ANOVA,
F2,30=9.628, P<0.001) and heart (ANOVA,
F2,53=4.362, P<0.05)–were significantly
smaller in hypoxic hatchlings in comparison to their non-hypoxic siblings
(Fig. 3A–C). Three months
later, a similar pattern was evident for wet masses of liver (ANOVA,
F2,34=101.801, P<0.0001), lungs (ANOVA,
F2,34=5.070, P<0.05) and heart (ANOVA,
F2,34=29.465, P<0.001;
Fig. 3A–C).
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Enlargement of both ventricular chambers contributed to cardiac hypertrophy
in hypoxic alligators, as evidenced by individual regressions of RV and LVS on
body mass (Table 1). RV showed
a slightly negative allometry
(Mb0.92±0.02) and LVS showed slightly
positive allometry (Mb1.11±0.02), but
both were significantly enlarged in hypoxic alligators
(Table 1). The RV/LVS ratio
decreased slightly with age (from hatchling to juvenile) but remained
significantly higher in hypoxic alligators in both age groups. In hatchlings,
the RV/LVS ratio was 0.77±0.03 in hypoxic alligators, 0.58±0.04
in their normoxic siblings and 0.58±0.03 in their hyperoxic siblings
(Wilcoxon/Kruskal–Wallis rank sums test, 
2=13.047,
P<0.01; Fig. 4). In
juveniles, the RV/LVS ratio was 0.54±0.03 in hypoxic alligators,
0.46±0.01 in their normoxic siblings and 0.45±0.02 in their
hyperoxic siblings (Wilcoxon/Kruskal–Wallis rank sums test,
2=8.047, P<0.02;
Fig. 4).
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When wet masses of representative skeletal muscles (rectus femoris, diaphragmaticus) were compared, both scaled with positive allometry (Mb1.19) with animal body mass and showed no significant differences between treatments (Table 1).
Haematocrit (%) was significantly higher in hatchlings of the hypoxic group
(28%) compared with their normoxic (22%) and hyperoxic (21%) siblings
(Wilcoxon/Kruskal–Wallis rank sums test, 2=7.686,
P<0.05). This difference became more pronounced in juvenile
alligators (Fig. 5), as
haematocrit decreased with age in both normoxic (16%) and hyperoxic (13%)
groups, but increased in the hypoxic group (36%; Wilcoxon/Kruskal–Wallis
rank sums test, 2=15.407, P<0.001).
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Metabolic and breathing rates
AMR was significantly higher (ANOVA, F2,27=3.729,
P<0.05) in hyperoxic animals than in normoxic animals
(Fig. 6). SMR was approximately
twice as high in hypoxic and hyperoxic alligators as in their normoxic
siblings (ANOVA, F2,27=6.368, P<0.01). The
difference in metabolic rate due to digestion and absorption (i.e.
AMR–SMR) was significantly higher in hyperoxic animals than in their
hypoxic siblings (ANOVA, F2,27=4.200, P<0.05).
The ratio of AMR:SMR was significantly higher in the normoxic group than in
the hypoxic group (ANOVA, F2,27=3.704,
P>0.05).
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O2 per breath
than did normoxia (ANOVA, F2,27=10.852,
P<0.001).
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DISCUSSION |
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), amphibians
(Bradford and Seymour, 1988;
Mills and Barnhardt, 1999),
mammals (Mortola et al., 2000;
Zamudio et al., 2005;
Julian et al., 2007); as well
as reptiles: squamates (Herman and
Ingermann, 1996; Andrews,
2001), turtles (Kam,
1993), crocodilians
(Warburton et al., 1995;
Crossley and Altimiras, 2005)
and birds (Wangensteen et al.,
1974; Black and Snyder,
1980; Xu and Mortola,
1988; Dzialowski et al.,
2002; Crossley et al.,
2003b) (reviewed by Chan and
Burggren, 2005). Most recently, domestic chicken eggs have become
a popular model to study the effects of chronic hypoxia on embryonic growth at
altitude (Giussani et al.,
2007). Some of the earlier studies measured significantly lower
blood oxygen saturation and depressed metabolic rates in embryos exposed to
chronic hypoxia, and used these findings to explain why hypoxic embryos show
reduced utilisation of egg yolk and reduced somatic growth
(Kam, 1993;
Crossley and Altimiras, 2005;
Giussani et al., 2007;
Tintu et al., 2007). Although
we did not measure embryonic metabolic rates, we did observe smaller yolk-free
body mass and greater remnant yolk sac mass in hypoxic hatchlings. This
supports the hypothesis that, while yolk supply is definitely not limiting, a
constraint in oxygen supply is responsible for lower rates of yolk catabolism
and tissue anabolism in vertebrate embryos incubated in hypoxia.
Post-hatching growth of alligators is also stunted under chronic hypoxia
(Fig. 2). Other studies have
shown hypoxia to depress the post-hatching growth trajectory in fish
(Dabrowski et al., 2004;
Foss et al., 2003) and mammals
[humans (Frisancho and Baker,
1970); rats (Cunningham et
al., 1974; Mortola et al.,
1990; Sekhon and Thurlbeck,
1995); mice (Fan et al.,
2005)]. Growth retardation in mammals, however, appears to be
species dependent. Species adapted to fossorial hypoxia [e.g. hamsters
(Frappell and Mortola, 1994)]
and those with highly precocial neonates [e.g. guinea pigs
(Hsia et al., 2005)] show a
normal growth trajectory under chronic hypoxia, possibly because they do not
exhibit hypoxia-induced hypometabolism. In those mammalian species whose
growth is sensitive to atmospheric hypoxia, growth retardation may be the
result of two factors: metabolic depression
(Mortola, 2004) and reduced
food intake (Sekhon and Thurlbeck,
1995; Daneshrad et al.,
2001). Among reptiles, post-hatching growth has not been
investigated under chronic hypoxia, and ours is the first report to document
hypoxia-induced growth retardation in this diverse vertebrate group.
The mechanism of hypoxia-induced growth retardation in vertebrates is not
well understood, but different mechanisms probably account for growth
retardation in mammals and alligators. Alligator hatchlings are highly
precocial and do not show metabolic depression under chronic hypoxia. Instead,
growth retardation in alligators is probably caused by reduced food intake,
elevated maintenance metabolism, or a combination of the two. Evidence
regarding the effect of chronic hypoxia on food conversion efficiency is
lacking, but in farmed fish hypoxia depresses food consumption, not food
conversion efficiency (Foss et al.,
2002). Similarly, we have observed, but not quantified, a lower
food intake in hypoxic alligators, compared with normoxic hatchlings. Resting
metabolism (see below) was slightly higher in hypoxic alligators than in their
normoxic siblings. Thus, it may well be that a lower net energy balance (=
energy in–energy out) was available to the hypoxic alligators for
conversion into tissue and for growth.
Growth under hyperoxia
Growth of alligator embryos is not enhanced by incubation under
hyperoxia–normoxic and hyperoxic hatchlings had similar masses of the
yolk-free body and the remnant yolk sac. As such, our data agree with two of
three other investigations into embryonic growth under chronic hyperoxia in
air-breathing vertebrates: the Eastern fence lizards incubated at 32%
O2 (Andrews, 2001),
and the Northern bobwhite quail incubated at 60% O2
(Williams and Swift, 1988).
This is not surprising, considering that haemoglobin saturation of arterial
blood in alligators is complete at normoxic conditions
(Busk et al., 2000), and the
small amount of extra oxygen dissolved in blood plasma under hyperoxia is
unlikely to make a difference to relatively slow-growing species. In contrast,
domestic chicken embryos showed enhanced growth in air containing 40–60%
O2, but late-term growth retardation in 70% O2
(Metcalfe et al., 1981;
McCutcheon et al., 1982;
Stock et al., 1983). Domestic
chickens, which have been selectively bred for fast somatic growth, are
perhaps more likely to exhibit growth acceleration in response to elevated
atmospheric oxygen, at least until yolk energy intake becomes limiting [an
explanation which has been proposed for the growth reversal observed at 70%
O2 (Stock et al.,
1983)].
Unlike embryonic alligators, hatchling alligators grow faster under
hyperoxia than under normoxia. We propose that the enhancement of
post-hatching growth elicited by hyperoxia may stem from energetic savings due
to a lower air convection requirement in hyperoxic alligators. Cost of
breathing constitutes a significant fraction of resting metabolism of
reptiles, reported at approximately 13% for alligators
(Wang and Warburton, 1995).
Given that PO2 of inspired air does not appear
to affect tidal volume in resting alligators
(Skovgaard and Wang, 2007),
and assuming that wet lung mass corrrelates with tidal volume in alligators as
it does in mammals (Stahl,
1967), each breath of hyperoxic air supplies more oxygen than a
breath of normoxic air. Energy thus saved may be channelled toward body growth
of the hatchling. In addition, exposure to hyperoxia may prevent hatchlings
from reducing their metabolic rate (see below). The resulting sustained
hypermetabolic state may allow hyperoxic hatchlings to maintain a higher rate
of food digestion, absorption and assimilation, and hence growth rate, when
compared with their normoxic and hypoxic siblings.
Ontogenetic allometry of select visceral organs and skeletal muscles
Chronic exposure to hypoxia, but not hyperoxia, results in significant
differences in scaling characteristics of the visceral organs and skeletal
muscles in alligators; hence we focus our discussion in this section on the
hypoxia-induced plasticity.
Hypoxia-reared alligators show a distinctive ontogenetic allometry of major
visceral organs (Fig. 3).
Although liver mass scales to the same exponent of body mass
(Mb0.80) for all three treatment groups, liver
mass is consistently smaller for hypoxic alligators
(Fig. 3A,D). A developing trend
in liver hypotrophy was observed in embryonic alligators at different times of
egg incubation and at different levels of chronic hypoxia [15% and 10%
O2 (Crossley and Altimiras,
2005)]. Similarly, chicken embryos in chronic hypoxia (15%
O2) exhibit a reduction in liver growth in late development
(Tintu et al., 2007). Because
the liver is the major metabolic centre of the vertebrate body, its small size
may be a reflection of the reduced growth rate (or a functional constraint on
the growth rate) of hypoxic hatchlings. We do not know whether liver
hypotrophy is a general response of immature vertebrates to chronic hypoxic
exposure, because most studies of vertebrate acclimation to hypoxia do not
report liver mass, concentrating instead on discussing the pulmonary and
cardiovascular systems.
Lungs show a different growth trajectory before and after hatching. Lungs
of hypoxic hatchlings are smaller than those of normoxic and hyperoxic
alligators, which suggests their relative under-development. This is somewhat
surprising, as one might expect lung development to be accelerated at the same
time as the heart undergoes hypertrophy (see below), so that cardiopulmonary
function can be matched at hatching time. This suggests that growth of
non-functional lungs in hypoxic embryonic alligators is reduced similar to
that of the liver. Three months later, however, although absolute lung mass is
still lower in hypoxic animals (Fig.
3B), the regression shows accelerated growth of functional lungs
in hypoxic hatchlings (Fig.
3E). Unfortunately, we did not measure lung morphologic parameters
other than their wet mass. Although we cannot exclude the possibility of
pulmonary oedema accounting for the relatively high wet mass of hypoxic
alligator lungs, it is unlikely that pulmonary oedema would have persisted for
3 months. In rat pups, the initial pulmonary oedema is resolved within a week
of chronic exposure to hypoxia, and followed by accelerated alveolar
proliferation (Bartlett and Remmers,
1971). Multiple other studies of pulmonary acclimation to hypoxia
in young rodents have also reported greater dry mass of lungs, larger alveolar
and capillary volume, and increased surface area of respiratory exchange
surfaces (Burri and Weibel,
1971; Cunningham et al.,
1974; Mortola et al.,
1990; Sekhon and Thurlbeck,
1996; Hsia et al.,
2005). If similar remodelling of lung tissue can be substantiated
for hypoxic alligators, it would suggest compensatory lung growth occurs in
both postnatal mammals and hatchling reptiles, once functional lungs are
exposed to the reduced oxygen content in breathed air.
In neonate mammals, lung wet mass is an accurate indicator of lung vital
capacity (Mortola, 2001).
Assuming this mass–volume relationship also holds true for the
non-homogeneous lungs of reptiles, it suggests that the positively allometric
growth of lungs in hypoxic alligator hatchlings
(Mb1.44) affords them greater lung volume
per unit body mass compared with their non-hypoxic siblings
(Mb0.73). An increase in the surface area
of respiratory parenchyma, epithelial thickness or capillary volume may also
account for the observed positive allometry of wet mass of lungs in hypoxic
animals. Pneumotachographic measurements of lung volume (tidal volume, vital
capacity) and a histomorphometric analysis of lung structure will be necessary
to establish differences in pulmonary parameters between hypoxic and normoxic
alligators.
During post-hatching ontogeny, wet heart mass scales with slightly positive
allometry (Mb1.07) in all groups but is
significantly greater in hypoxic alligators
(Fig. 3F). Despite its smaller
absolute wet mass, the heart is significantly larger relative to body mass in
hypoxic alligators, both at hatching and 3 months later
(Fig. 3C,F). This suggests that
cardiac hypertrophy in response to hypoxia begins during embryonic
development, as previously observed in late-term embryos
(Crossley and Altimiras,
2005). This is not surprising. Unlike the non-functional lungs,
the heart of embryonic alligators is responsible for blood convection and
supplying developing tissues with sufficient oxygen, which is taken up at the
chorioallantoic membrane. Faced with a reduced oxygen supply in the
atmosphere, the heart responds with hypertrophy in order to increase cardiac
output and maintain oxygen delivery to the tissues.
The relatively high RV/LVS ratio in hypoxic animals, especially in
hatchlings (Fig. 4), is similar
to what has been observed in rodents acclimated to hypoxia
(Nouette-Gaulain et al., 2005;
Macarlupú et al.,
2006), and appears to be a reflection of sustained pulmonary
hypertension (Rabinovitch et al.,
1979). The decrease in the RV/LVS ratio from hatchlings to
juveniles, as reflected in the slightly divergent scaling of RV (negative
allometry) and LV (positive allometry;
Table 1), may be a result of
decreasing pulmonary vascular resistance post-hatching, but this remains to be
verified experimentally.
Cardiac hypertrophy in hypoxic alligators may also be driven by their high
haematocrit. Haematocrit in hypoxic alligators increases significantly
post-hatching, while it decreases in alligators under normoxia or hyperoxia
(Fig. 5). High haematocrit
(polycythaemia) results in greater blood viscosity, which in turn increases
vascular resistance and cardiac work. High haematocrit has been shown to be
necessary for mice to develop cardiac hypertrophy in response to hypoxia
(Macarlupú et al.,
2006).
Altogether, hypoxia appears to be responsible for inducing cardiopulmonary plasticity in alligator hatchlings: positive allometry of lung growth, right ventricular hypertrophy and higher haematocrit. In combination, this suite of morphologic changes (and probably many others not investigated in this study) may allow hypoxic alligators to support an elevated demand for oxygen by their tissues despite a reduced atmospheric oxygen supply.
At this point, it would be appropriate to compare scaling coefficients from
this study with those obtained by others. Alas, there are very few published
studies on intraspecific ontogenetic allometry. Although numerous accounts of
interspecific scaling in neonates
(Mortola, 2001) and adults of
different sizes (e.g. Tenney and Tenney,
1970; Schmidt-Nielsen,
1984; Calder,
1996; Seymour and Blaylock,
2000; Lindstedt and Schaeffer,
2001; Withers and Hillman,
2001) have been published, interspecific scaling is a combination
of physiologic and phylogenetic signals, and often shows a different
allometric relationship with animal size from intraspecific (ontogenetic)
scaling. This makes any attempted comparisons of intraspecific and
interspecific scaling difficult to interpret. Our literature search uncovered
only two studies, one on the Australian agamid lizard, Ctenophorus
(Amphibolurus) nuchalis
(Garland and Else, 1987), and
one on the laboratory rat, Rattus norvegicus
(Stewart and German, 1999).
Liver scaled with more negative allometry in the alligator
(Mb0.80) than in either the agamid lizard
(Mb0.91) or the lab rat
(Mb0.98). Data on agamid lung mass are not
provided, but lungs of normoxic (and hyperoxic) alligators appear to scale
quite similar (Mb0.73) to lungs of female rats
(Mb0.66). On the other hand, the slight
positive allometry of the alligator heart
(Mb1.07) is closer to that of the agamid lizard
(Mb1.11), and quite different from the strong
negative allometry of the rat heart (Mb0.74),
despite the fact that crocodilian heart ventricle is structurally more similar
to the mammalian heart than the agamid one. Finally, the alligator rectus
femoris muscle scales similar (Mb1.20) to the
thigh musculature of the agamid lizard (Mb1.16)
and the gastrocnemius of the lab rat (Mb1.16 in
males and Mb1.29 in females). It is hard to
draw any conclusions about the similarity of ontogenetic allometry in
alligators with other vertebrates, given the paucity of data.
Metabolic rates
Compared with normoxia, hypoxia does not significantly change the AMR of
recently fed alligators, but does increase the SMR of fasted alligators
(Fig. 6). This is in contrast
to most studies of acute or short-term hypoxic exposure, which generally
results in temporary metabolic depression
(Hicks and Wang, 1999;
Hicks and Wang, 2004;
Platzack and Hicks, 2001;
Jackson, 2004;
Ramirez et al., 2007). In most
newborn mammals (e.g. rats), chronic exposure to hypoxia also results in
hypometabolism (Mortola,
2001). In contrast, the metabolic pattern of our hypoxic alligator
hatchlings resembles that of hypoxia-adapted young mammals (e.g. hamsters),
which maintain similar metabolic rates under normoxia and hypoxia
(Frappell and Mortola, 1994).
Further experiments are obviously needed to explain why the SMR of
hypoxia-acclimated alligators is not depressed, but higher energetic costs of
oxygen uptake and distribution (in terms of lung ventilation and
cardiovascular convection) are a distinct possibility.
Hyperoxia induces a chronic elevation of metabolic rate in juvenile
alligators, under both absorptive and post-absorptive conditions compared with
the normoxic group (Fig. 6).
This does not appear to be a general vertebrate response to hyperoxia.
Hypermetabolism has been reported in hyperoxia-reared fish
(Foss et al., 2003), but not
in pre-metamorphic amphibians (Territo
and Altimiras, 1998). Acute hyperoxic exposure in neonate mammals
(Mortola and Tenney, 1986;
Frappell et al., 1992b) also
induces a hypermetabolic response, but whether it is sustained over long-term
hyperoxic exposure is not known. We suggest a plausible mechanism that may
account for our observation–hyperoxic alligators may be unable to reduce
their SMR because hyperoxic exposure inhibits cardiac shunting. This effect
has been demonstrated in internally pipped chicken embryos
(Sbong and Dzialowski, 2007),
which possess a cardiac shunt via the ductus arteriosus, analogous to
the crocodilian cardiac shunt via the left aorta
(Ewer, 1950). The similarity
of cardiovascular control in embryonic birds and alligators
(Crossley et al., 2003a;
Crossley et al., 2003b;
Crossley et al., 2003c)
suggests that hyperoxia may inhibit shunting in alligators. As cardiac
shunting can induce hypometabolism in reptiles
(Hicks and Wang, 1999;
Hicks, 2002), chronic
inhibition of shunting due to atmospheric hyperoxia is likely to cause a
hypermetabolic state in alligators. Direct measurements of blood flow pattern
in the heart outflow tract of normoxic and hyperoxic alligators are required
to confirm this hypothesis.
The absolute postprandial metabolic elevation (i.e. AMR–SMR)
increases with atmospheric oxygen level and is significantly higher in
hyperoxic alligators than in hypoxic ones. We use AMR–SMR, instead of
the specific dynamic action (SDA), as a measure of the average aerobic cost of
food digestion and absorption (McCue,
2006). Accurate SDA determination in crocodilians usually requires
fasting the animal before and after the feeding bout for at least 5–6
days (Coulson and Hernandez,
1979; Busk et al.,
2000; McCue, 2006;
Starck et al., 2007). We did
not measure SDA because the necessity of withholding food prior to SDA
measurements would have interfered with the concurrent growth study. Given the
regular feeding regimen, however, we are confident that the AMR–SMR
difference is probably an accurate, if rough, approximation of chronically
sustained SDA. We suggest two mutually inclusive explanations for the trend of
increasing AMR–SMR with atmospheric oxygen level.
;
Starck and Beese, 2001;
Starck et al., 2007). A low
AMR–SMR of hypoxic alligators may be a reflection of their relatively
small liver (Fig. 3A and D) and
digestive tract (this study, data not shown), hence a limited capacity for
food digestion, absorption and assimilation in hypoxic animals. ;
Hicks et al., 2000), the
AMR–SMR may indicate differences in food intake between treatment groups
(assuming similar food conversion efficiency). The ad libitum feeding
regimen did not quantify the food intake for individual animals, but we have
observed hypoxic alligators to ingest less food than normoxic and hyperoxic
animals. Altogether, hypoxic animals may have less metabolised energy available for growth because of a volitionally restricted diet. In order to elucidate differences in postprandial metabolic elevation under hypoxia and hyperoxia, additional experiments are required which will control for meal size and measure food conversion efficiency at different oxygen levels.
Breathing rates and O2 per breath
Acute exposure to hypoxia is known to stimulate higher breathing rates in
vertebrates, but as animals acclimate to chronic hypoxia they undergo hypoxic
desensitisation (Powell et al.,
1998) and their breathing rate slowly returns to normal (while
their tidal volume increases). Hypoxic alligators in our experiment showed a
similar breathing rate to their normoxic siblings during both absorptive and
post-absorptive conditions (Fig.
7). As we did not measure breathing rate in young hatchlings, we
cannot ascertain whether the similarity between hypoxic and normoxic animals
stems from the former not being affected by 12% O2 or having
acclimated to it during the three months post-hatching. An atmospheric
composition of 12% O2 may not have been sufficient to cause an
observable increase in breathing rate, as much lower oxygen levels (<5%)
are usually needed to elicit a hypoxic ventilatory response in alligators
(Wang and Warburton, 1995;
Skovgaard and Wang,
2007).
Acute hyperoxia has been shown to reduce breathing rate in mammals
(Mortola and Tenney, 1986;
Hertzberg et al., 1990;
Bavis, 2005). Hyperoxic
exposure is also known to reduce the sensitivity of neonate rats to acute
hypercapnia (Hertzberg et al.,
1990) and acute hypoxia
(Donnelly et al., 2005).
Hyperoxic alligators in our experiment consistently showed a lower breathing
rate than normoxic animals, despite higher metabolic rates and (presumably)
greater CO2 accumulation in their lungs and blood during apnoeic
periods between breaths. This suggests that the respiratory rhythm generator
in alligators is suppressed as long as sufficient oxygen is present in the
lungs, which accounts for the lower breathing rate in hyperoxic animals.
A combination of high metabolic rate and low breathing rate of hyperoxic
alligators means that each breath supports a significantly greater rate of
oxygen consumption than it does in normoxic animals
(Fig. 7). Breathing in reptiles
can be costly–the cost of breathing in alligators has been estimated at
13% of standard metabolism (Wang and
Warburton, 1995) [but see a more recent contrary opinion
(Skovgaard and Wang, 2007)],
and even higher in lizards (Milsom and
Vitalis, 1984; Andrade and Abe,
1999) and turtles (Kinney and
White, 1977; Vitalis and
Milsom, 1986). Assuming the cost of breathing is similar in
hyperoxic and normoxic animals, hyperoxic alligators are saving a significant
amount of energy by taking fewer breaths. Energy saved by bradypnoea can be
channelled towards tissue growth in juvenile alligators.
Experimental palaeophysiology
This study reports on the acclimation, not adaptation, of alligator growth
and metabolism to non-normoxic atmospheres. Nevertheless, changes in
physiologic function due to acclimation (phenotypic plasticity) often drive
directional selection (evolutionary adaptation), even if the magnitude of
acclimatory and adaptive responses to the same stimulus is different
(Garland and Kelly, 2006). For
instance, the hypoxic ventilatory response to acute exposure is exaggerated in
recent migrants to high altitude and blunted in native residents of high
altitude, but in both groups the directionality of the physiologic response
(increase in minute ventilation) is the same
(Hochachka, 1998). Admittedly,
acclimation studies cannot elucidate exactly which genes underlying the
morphologic and physiologic plasticity are adaptive, and might be selected and
retained over generations. Despite this caveat, we posit that our results on
normoxia-adapted alligators reared under chronic hypoxia and hyperoxia may
provide insight (albeit indirect and limited) into the physiology of extinct
vertebrates contemporary to non-normoxic atmospheres. As stated by Knoll and
colleagues (Knoll et al.
2007): "Physiology provides the proximal interface between
organisms and their environment. Thus, physiological inferences gleaned
directly from fossils or from their living relatives can illuminate the causes
and consequences of major extinctions and other events in the history of
life". Viewed from the palaeontologic perspective, our results of
increased growth rate of alligators under chronic hyperoxia are in agreement
with the observation of a general trend of increasing body size (and
presumably growth rate) among placental mammals during the Late Cretaceous and
the Tertiary, when atmospheric oxygen levels were rising
(Falkowski et al., 2005). It
would be interesting to see whether similar changes in gross body size can be
detected in other vertebrate lineages surviving large fluctuations in
atmospheric oxygen.
Many studies have used extant vertebrates in order to understand the
physiologic function of their extinct relatives, but most have not paid
attention to the prevalent environmental conditions to which extinct
vertebrates were subjected. As argued by Berner and colleagues
(Berner et al., 2007), a new
discipline of `experimental palaeophysiology' is needed, which would consider
animal physiology in its appropriate palaeo-environment. As our study
demonstrates, basic physiologic functions (such as resting metabolism) of
extinct vertebrates were probably significantly altered at times of global
hypoxia and hyperoxia. Though beyond the scope of this study, it is easy to
envisage hypoxia and hyperoxia also altering patterns of aerobic and anaerobic
activity. These would probably have translated into differences in energy
budgets, predator–prey interactions and ecologic community structure of
vertebrates contemporary to global hypoxia or hyperoxia. Further experiments
are required to address these aspects of physiology of extinct vertebrates.
More elaborate experiments, approximating global conditions prevalent at
different times in the vertebrate evolutionary history, will permit the
effects of multifactorial interaction (e.g. O2, CO2 and
temperature) on vertebrate physiology to be addressed. Eventually, a more
complete picture of extinct vertebrate life can emerge from the
laboratory.
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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