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First published online October 31, 2008
Journal of Experimental Biology 211, 3627-3635 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020958
External gills and adaptive embryo behavior facilitate synchronous development and hatching plasticity under respiratory constraint
Department of Biology, Boston University, Boston, MA 02215, USA
* Author for correspondence (e-mail: kwarken{at}bu.edu)
Accepted 22 September 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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50% longer than development to hatching
competence, and development is synchronous across perivitelline oxygen levels
(PO2) ranging from 0.5–16.5 kPa. Embryos
maintain large external gills until hatching, then gills regress rapidly. We
assessed the respiratory value of external gills using gill manipulations and
closed-system respirometry. Embryos without external gills were oxygen limited
in air and hatched at an external PO2 of 17
kPa, whereas embryos with gills regulated their metabolism and remained in the
egg at substantially lower PO2. By contrast,
tadpoles gained no respiratory benefit from external gills. We videotaped
behavior and manipulated embryos to test if they position gills near the
air-exposed portion of the egg surface, where
PO2 is highest. Active embryos remained
stationary for minutes in gills-at-surface positions. After manipulations and
spontaneous movements that positioned gills in the O2-poor region
of the egg, however, they returned their gills to the air-exposed surface
within seconds. Even neural tube stage embryos, capable only of ciliary
rotation, positioned their developing head in the region of highest
PO2. Such behavior may be critical both to
delay hatching after hatching competence and to obtain sufficient oxygen for
normal, synchronous development at earlier stages.
Key words: embryo behavior, gills, hatching, hypoxia, phenotypic plasticity, respiration
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Booth, 1995;
Cohen and Strathmann, 1996;
Seymour et al., 2000). Both
parental traits and egg mass features that improve egg oxygenation are well
documented [e.g. egg ventilation
(Fernández et al.,
2000), clutch size and shape
(Strathmann and Strathmann,
1995), convection channels
(Pinder and Friet, 1994)].
Embryo traits could also improve oxygen uptake ability. Here, we assess the
contribution to respiration of embryo behavior and the external gills of
embryos and hatchlings of red-eyed treefrogs.
Hatching frees animals from the diffusion barrier of the egg and can be
induced prematurely by hypoxia in many taxa
(Petranka et al., 1982;
Warkentin, 2007). In some
species, hatching is both advanced by hypoxia and delayed by hyperoxia,
suggesting that it occurs at a respiratory threshold
(Latham and Just, 1989). While
solving a physiological problem, hatching earlier may, however, create an
ecological one. More mature hatchlings typically have greater sensory
development and/or locomotor abilities
(Fuiman, 2002), both of which
can reduce mortality from predation (Sih
and Moore, 1993; Warkentin,
1999a; Gomez-Mestre et al.,
2008). If predators or other factors in the post-hatching
environment impose selection for later hatching, then mechanisms to extend
embryonic development will be favored
(Warkentin, 2007). For embryos
under respiratory constraint these include traits that improve oxygen supply
or oxygen uptake capacity.
Anurans have four respiratory surfaces: skin, lungs, external gills and
internal gills. Their external gills are transient embryonic structures that
are, in many species, little more than short externalized capillary loops of
equivocal functional value (Burggren and
Just, 1992). Moreover, some amphibian and fish embryos develop
normally without convective oxygen transport, suggesting that diffusion is
sufficient to supply their oxygen needs
(Flores and Frieden, 1969;
Pelster and Burggren, 1996;
Territo and Burggren, 1998).
In other anurans, both morphological elaboration of the external gills and
environmentally regulated gill regression suggest that these structures serve
an important function (del Pino and
Escobar, 1981; Channing,
1993).
The behavior of embryos and fetuses has been viewed primarily from a
developmental perspective (Smotherman and
Robinson, 1996). For instance, embryo movements play a role in
neuromuscular and skeletal development
(Robinson et al., 2000;
Pitsillides, 2006). Embryo
movements have seldom been examined for immediate utility, and functional
roles for embryo behavior are documented in few contexts, mostly late in
embryonic development [e.g. environmentally cued hatching
(Warkentin and Caldwell, in
press); care solicitation vocalizations
(Brua, 2002)]. Behavior may,
however, have immediate utility at earlier stages
(Goldberg et al., 2008).
Study organism and hypotheses
Red-eyed treefrogs, Agalychnis callidryas (Cope), attach their
eggs to vegetation over ponds and swamps, and tadpoles fall into the water
upon hatching. The embryos hatch rapidly, up to 30% before their modal
spontaneous hatching age, in response to egg-stage threats, including
egg-eating snakes and wasps, pathogenic fungus and submergence underwater,
which drowns eggs too young to hatch
(Warkentin, 2007). Early
hatched tadpoles are developmentally premature
(Warkentin, 1999b), and more
vulnerable to aquatic predators than are full-term hatchlings
(Warkentin, 1995;
Warkentin, 1999a). Embryos
hatch by performing movements that rupture the egg capsule and propel them
from it. This behavior is cued by vibrations in snake attacks
(Warkentin, 2005) and by
hypoxia in flooding (Warkentin,
2002).
Red-eyed treefrog eggs are large (5 mm diameter when
hatching-competent) and closely packed in gelatinous clutches, so each egg is
only partly exposed to air. The perivitelline oxygen level (partial pressure;
PO2) at the center of eggs varies
substantially, and embryos develop synchronously at
PO2 from 0.5–16.5 kPa. Moreover,
hatching-competent embryos tolerate PO2 as low
as 0.5 kPa without hatching; such hypoxia can slow development, induce
hatching and kill embryos of other anurans with terrestrial eggs
(Warkentin et al., 2005).
We hypothesize that the ability of A. callidryas to maintain rapid embryonic development and delay hatching despite very low PO2 depends on four things: (1) retention of external gills until hatching; (2) spatial variation in PO2 within eggs, with at least a small well-oxygenated region; (3) functional contribution of external gills to oxygen uptake; and (4) behavioral positioning of external gills in the well-oxygenated region of the egg. The first two have already been demonstrated; we briefly review the evidence. We then measure the value of A. callidryas' external gills for oxygen uptake and assess how embryos behaviorally position their gills.
The timing of gill regression in A. callidryas is plastic. It
normally occurs immediately after hatching, regardless of hatching age; young
hatchlings regress the gills rapidly but even the oldest embryos retain them
(Warkentin, 2000b). Gill
regression depends more on oxygen availability than on hatching per
se. Embryos under hyperoxia or in eggs removed from the clutch to
increase surface exposure regress the gills, whereas tadpoles in hypoxic water
without access to air retain them
(Warkentin, 2000b;
Warkentin, 2002). Moreover,
embryos induced to regress their external gills, under temporary hyperoxia or
prostaglandin treatment, hatch rapidly from egg clutches in air
(Warkentin, 2002).
Measurements of PO2 at two locations within
4-day-old A. callidryas eggs showed strong gradients, despite
constant ciliary circulation of the perivitelline fluid.
PO2 was 12.4±0.8 kPa just underneath the
air-exposed surface, compared with 3.4±0.3 kPa deep inside the eggs
(mean ± s.e.m. here and throughout, N=30)
(Warkentin et al., 2005). Here
we repeat those measurements with younger eggs (2 days old) to assess
developmental change in the gradient.
The external gills of A. callidryas are long (25% of total
length), branched structures with great positional flexibility
(Warkentin, 1999b).
Hatching-competent embryos curl within the egg so most of their skin faces the
hypoxic egg interior or is pressed against parts of the egg capsule adjacent
to other eggs or their leaf substrate. By contrast, the gills offer a
spatially flexible gas exchange surface that could be positioned adjacent to
the air-exposed egg surface. We assess the contribution of gills to oxygen
uptake by comparing metabolic rates of embryos and tadpoles with and without
external gills across different oxygen levels. We quantify natural patterns of
embryo position and movement, and embryo responses to experimental
repositioning, to assess if embryos actively position their gills near the
air-exposed egg surface.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Warkentin, 1999b), we use
embryonic age as a proxy for developmental stage; plasticity in gill
regression makes standard staging tables misleading for hatching-age animals.
For detailed discussions of development, age and staging of A.
callidryas see (Warkentin,
1999b; Warkentin,
2002; Warkentin,
2007). This research was conducted between June and August 2006 and 2007 under permits from the Smithsonian Tropical Research Institute and the Panamanian National Authority for the Environment (Autoridad Nacional del Ambiente), and approved by the Animal Care and Use Committee of Boston University.
Respirometry and manipulations of gill regression
We used closed system respirometry to measure metabolic rates of individual
A. callidryas embryos and hatchlings with and without external gills
across a range of oxygen levels. Measurements were conducted in airtight glass
respirometry chambers (Unisense, Aartius, Denmark). We used a 0.41 ml chamber
for measurements of embryos in air, and a larger chamber for measurements of
tadpoles in aged tap water (2.09 ml water + tadpole volume). A glass-covered
magnetic stirrer in the larger chamber, separated from the tadpole by
stainless steel mesh supported on a glass ring, continuously mixed the water.
The combination of stirring speed (240 r.p.m.) and mesh size (0.4 mm) was
chosen to ensure complete mixing within the chamber based on dye
visualization, and to not disturb tadpoles in pilot tests. For all
measurements, we sealed the capillary tube probe port and the lid to the
chamber with water. Seal effectiveness was verified by a pilot experiment in
which the PO2 of degassed, hypoxic water in the
chamber remained constant for 2 h.
We used an optical oxygen sensor (Microx TX2, Precision Sensing; Regensburg, Germany) with a fiberoptic microprobe (optode) to measure the partial pressure of oxygen (PO2) within the chamber. The optode does not consume oxygen. It was mounted in a needle with the tip slightly extruded. PO2 was sampled once per second and recorded on a laptop computer. The probe was calibrated daily before measurements in water-saturated air and anoxic water formulated with NaSO3. The high point calibration was checked between animals and both calibration points were rechecked at the end of each day. Optode drift was never over 3%, thus we did not correct for it. Oxygen measurements were automatically temperature-compensated by the sensor using a thermal probe touching the respirometry chamber. Temperatures ranged from 25 to 28°C.
To assess bacterial oxygen consumption we conducted five blank trials of 2 h each, under each of the two measurement conditions (0.41 ml respirometry chamber filled with air, 2.09 ml respirometry chamber filled with aged tap water). Bacterial O2 consumption was undetectable in air. We used the average bacterial O2 consumption in aged tap water (0.044±0.019 µmol O2 h–1) to correct measurements of tadpole oxygen consumption.
We measured PO2 over time as animals consumed oxygen in four treatments: embryos with and without external gills and newly hatched tadpoles (hatchlings) with and without external gills (N=11 per treatment). All animals were 5 days old and of similar size. Their opercula had formed, yolk sacs were streamlined but large and still undivided into gut coils, and beaks had begun to keratinize. We only used clutches that were healthy, with no evidence of predator contact or pathogen infection, and only eggs that were developing in synchrony with their siblings and other eggs of the same age. No siblings were included in the same treatment, and each individual was measured only once.
We ran pilot experiments to determine pre-test rearing conditions to induce
or prevent gill regression. Gill condition was assessed at x30
magnification under a dissecting microscope. To measure respiration of a
gilled embryo, we separated a single egg from its clutch and placed it in the
center of the floor of the respirometry chamber. Eggs thus had 75%
air-exposed surface, near the high end of exposures recorded in clutches
(Warkentin et al., 2005). The
chamber was sealed and PO2 of the air around
the embryo recorded until the embryo hatched. Embryos were observed
continuously throughout the experiment (9.17±0.66 h per embryo) and
retained easily visible, well perfused external gills that extended past the
end of their yolk sac (
1.9 mm). When the embryo hatched, the
PO2 at hatching was recorded, and the mass of
the egg and embryo were determined.
To induce gill regression we reared embryos in an oxygen-enriched
environment. Individual 3-day-old eggs were separated from their clutches and
suspended on 3 mm diameter hexagonal nylon netting, fully exposing the egg
surface except for a narrow strip occluded by the nylon filament
(Warkentin, 2000b). The
suspended embryos were placed in a container with a constant flow of a
humidified 40% oxygen, 60% nitrogen mixture. Measurements of
PO2 at the center of such eggs at 4 days ranged
from 17.4 to 17.9 kPa (17.7±0.001 kPa, N=7), substantially
higher than the 0.5–9.0 kPa (4.0±0.3 kPa, N=41) recorded
from 4-day eggs in clutches (Warkentin et
al., 2005). On the morning when embryos were five days old,
individual eggs were placed in the respirometry chamber. The chamber was
partially flushed with 40% O2 gas (starting
PO2: 32.6±1.1 kPa) and then sealed.
PO2 was recorded until the embryo hatched, then
hatching PO2 was recorded, the mass of the egg
and embryo determined, and external gills examined. Final gill length was
0.08±0.05 mm; eight of 11 animals had no visible gills. Experiment
durations were 9.64±0.75 h, similar to those with gilled embryos
(Wilcoxon rank-sum test: z=0.296, P=0.77).
For measurements of gilled tadpoles we induced individual embryos to hatch directly into the larger chamber, which was filled with air-saturated aged tap water. We sealed the chamber immediately, excluding air before hatchlings could fill their lungs. For measurements of tadpoles without gills, we induced gill loss in embryos as above, then induced these animals to hatch into the water-filled chamber. Trials ran until the PO2 in the water surrounding the tadpole reached approximately 0.7 kPa (1.78±0.05 h), at which point the mass of the tadpole was determined and the external gills examined. All gilled tadpoles retained external gills (1.9±0.1 mm), and no animals induced to regress gills had visible gill remains.
Analysis of respirometry data
We calculated the rate of oxygen consumption
(
O2; as a
measure of metabolic rate) at different PO2
over 5 min and 30 min periods in the tadpole and embryo trials, respectively
(1 kPa decrease in both cases). To smooth noise in optode readings, we
calculated O2
from the difference between PO2 averaged over
30 s at the start and end of each period. We used average
PO2 over the entire period as a measure of the
oxygen level corresponding with each
O2 value. To
determine the volume of medium (air or water) in the chamber from which
O2 was consumed, we subtracted individually estimated egg or
tadpole volume from the chamber capacity. For eggs, we measured the diameter
of five eggs near the test individual in its clutch, and used the average to
estimate volume based on spherical geometry. For tadpoles, we used the wet
mass of the experimental individual and the average density of tadpoles, based
on the wet mass and volume of a sample of 5-day hatchlings with unfilled lungs
(N=40 tadpoles from five clutches).
We calculated the critical oxygen level (Pcrit), below
which animals could no longer regulate their metabolic rate, for each
individual following the method of Yeager and Ultsch
(Yeager and Ultsch, 1989), as
implemented in a MatLab script written by E. Dzialowski.
Pcrit was determined as the break point between two linear
regressions of
O2 on
PO2, minimizing r2 for the
regression above Pcrit. We calculated unconstrained
O2 for each
animal by averaging
O2 across all
data points above Pcrit. For animals with at least four
data points below Pcrit, we also calculated the slope of
that regression. Data were analyzed in STATA v.9.0. We tested for effects of
hatching, gill regression and their interaction on Pcrit
and the slope of the regression below Pcrit with ANOVAs,
and on unconstrained
O2 with ANCOVA,
with wet mass as a covariate. Hatching PO2 was
heteroscedastic and therefore tested nonparametrically.
Measurement of perivitelline PO2 gradient
We measured perivitelline PO2 at two
locations within each of 23 2-day-old eggs, from five clutches (three to five
eggs each): just under the air-exposed egg surface and deep within the egg,
between the center of the egg and the far wall, touching neither embryo nor
egg capsule. Embryos were in early Gosner stage 18 (muscular response)
(Gosner, 1960). Measurements
were made using the Microx TX2 and a needle-mounted probe following the method
of Warkentin et al. (Warkentin et al.,
2005).
Manipulation of embryo position
To test the hypothesis that embryos actively maintain their gills near the
air-exposed egg surface we manipulated embryo position and monitored
subsequent behaviors. We selected 10 eggs of intermediate surface exposure in
each of five clutches at three ages: 3 days (Gosner stage 22, tail fin
circulation), 2 days (stage 18, muscular response) and 1 day (stage 16, neural
tube). All eggs were fully surrounded by other eggs in the vertical plane of
the clutch and adhered at the back to a leaf, thus exposed to air only at the
front. All embryos used started with their external gills (3 days) or
developing head (1 and 2 days) facing the air-exposed portion of the egg. We
haphazardly assigned five eggs per clutch to each treatment. Using a blunt
probe we carefully turned each embryo within its egg capsule. Test embryos
(N=25/age) were positioned with their external gills or developing
head in the oxygen-poor region at the back of the egg. Controls
(N=25/age) were manipulated in a similar manner, but positioned with
their external gills or developing head at the air-exposed surface. We
recorded all behaviors of each manipulated embryo. We watched each 3-day
embryo until it returned its gills to the surface, stopping after 60 s
post-manipulation for those that did not move. We watched each 2-day embryo
for 120 s. For 1-day embryos, whose only movement is slow ciliary rotation in
the horizontal plane, we drew their position in dorsal view at 60 s intervals
for 5 min then measured their angular rotation with reference to the
exposed–deep axis of the egg and the covered and exposed egg surfaces.
Temperatures during experiments were 28.6±2.1°C.
Analyses were conducted in STATA v.9.0. For 2- and 3-day eggs, we tested the effect of position on `time-to-move' with ANOVA. We conservatively assigned time watched as time-to-move for control embryos that did not move in tests, and time to return gills to the surface as time-to-move for experimental embryos that took multiple movements to accomplish this. Neither clutch nor a clutch-by-position interaction were significant in either case, nor did they improve model fit as indicated by the Akaike Information Criterion (AIC). At 1 day, angular rotation of embryos was intractably heteroscedastic. Based on the lack of clutch effects for older eggs, we report Wilcoxon rank-sum test results, pooling eggs across clutches. Paired sign tests on clutch average values are also significant. For time-to-move we assigned 6 min for 1-day embryos that did not move in the 5 min observation period.
Videotape analysis of embryo behavior
To quantify embryo behavior in relation to oxygen gradients within eggs we
videotaped ten 3-day and ten 5-day A. callidryas clutches for 1 h
each. After positioning each clutch vertically in front of the camera we left
embryos undisturbed for 5 min before recording. Temperatures during recordings
were 31.7±0.2°C. We estimated the proportional surface exposure of
each egg in the field of view based on egg geometry
(Warkentin et al., 2005).
Rogge and Warkentin initially independently estimated exposure, and results
were fully consistent. We analyzed the behavior of four embryos per clutch:
the two with the least and the two with the most air-exposed surface in the
field of view (27±2% and 67±2% exposed, respectively). We only
used eggs for which we could see the entire air-exposed surface. We counted
all gross muscular movements during the 1 h recording for each embryo,
classifying each behavior as either a twitch (muscular contraction with no
change in position) or a move (muscular contraction with position change). The
final position after each move was scored on the basis of gill location:
either `surface' if at least some part of one gill was visible at the
air-exposed surface or `deep' if no part of either gill was at the exposed
surface. To determine if the time embryos spend in a position depends on their
gill location, we used the time trace on the videotape to record the time each
embryo spent in up to six positions (following different moves): three surface
and three deep positions. Individual positions sampled were at least 10 min
apart except in a few cases where the embryo made three or fewer moves to a
position type.
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RESULTS |
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O2=0.261±0.006
µmol O2 h–1, N=44; mass-specific
O2=
18.46±0.39 µmol O2 h–1
g–1, N=43; all F1,38
2.7, all
P0.11; mass data are lacking for one embryo).
There was a significant hatching-by-gill regression interaction effect on
Pcrit (F1,40=19.5, P=0.0001).
For embryos, Pcrit was lower with external gills than
without them, whereas for tadpoles gill regression did not change
Pcrit (Fig.
2A; Tukey's post-hoc tests, embryo P=0.0002,
tadpole P=0.89). Hatching also decreased Pcrit
for animals both with and without external gills
(Fig. 2A; Tukey's
post-hoc tests, both P=0.0002). There was no effect of gill
regression, or its interaction with hatching, on the slope of the regression
of O2 on
PO2 below Pcrit (both
F1,360.9, both P0.35), but the slope was
steeper for tadpoles than for embryos (tadpoles: 0.038±0.003 µmol
h–1 kPa–1, N=22; embryos:
0.015±0.003, N=18; F1,36=36.9,
P<0.0001; Fig. 1).
Embryos without external gills hatched at a higher
PO2 than did embryos with gills
(Fig. 2B; Wilcoxon rank-sum
test, z=3.98, P=0.0001). Embryos without external gills also
tolerated less of a drop in PO2 below
Pcrit, before hatching, than did embryos with gills
(Fig. 2; 4.6±0.7
vs 7.5±0.7 kPa; Wilcoxon rank-sum test,
z=–2.40, P=0.017).
|
), there was an oxygen gradient in 2-day-old eggs (Sign
test: P<0.0001). In all 2-day eggs measured, the surface
PO2 was higher than the
PO2 deeper within the egg. The difference was
7.0±0.7 kPa (range 1.1–16.2 kPa); interior
PO2 averaged 6.9±0.6 kPa, whereas
surface PO2 was 14.0±0.7 kPa.
Embryo response to position manipulation
All embryos capable of muscular response returned their external gills or
head to the well oxygenated part of the egg, near the air-exposed surface,
soon after we positioned their gills deep inside the egg
(Fig. 3). Experimental animals
moved sooner than did controls with gills positioned at the air-exposed egg
surface (3-day embryos, F1,48=163.9, P<0.0001;
2-day embryos F1,48=18.9, P=0.0001;
Fig. 4). All 3-day experimental
embryos rapidly returned their gills to the surface, on average in 15±2
s, while only 24% of controls moved within 60 s. All 2-day experimental
embryos, newly capable of muscular response, returned their gills to the
surface in 47±7 s, while only 44% of controls moved within 120 s.
All experimental embryos at the neural tube stage, before developing gills and capable only of ciliary rotation, rotated their developing heads toward the air-exposed surface (Fig. 5). They were more likely to move, moved sooner, and moved farther than control embryos (Wilcoxon rank-sum tests: moved at all, z=–4.802, P<0.0001; time-to-move, z=5.386, P<0.0001; angle moved, z=–6.1, P<0.0001; Fig. 6). On average they moved 83±7 deg. in 5 min; eight embryos by then had their heads fully exposed at the egg-air interface, six were partially exposed, and 11 still next to the egg-covered surface. By contrast only 36% of controls moved; average rotation was 6±2 deg.
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DISCUSSION |
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50% longer, developing substantially and, thereby, increasing their
chances of surviving as tadpoles with aquatic predators
(Warkentin, 1995;
Warkentin, 1999b). Among
amphibians, this species has relatively large eggs and develops at warm
temperatures; consistent with this, A. callidryas embryos have high
metabolic rates [for a review, see Seymour and Bradford
(Seymour and Bradford, 1995)].
These embryos maintain rapid development and refrain from hatching at
perivitelline PO2 as low as 0.5 kPa, and
develop synchronously across a broad range of
PO2
(Warkentin et al., 2005), even
though hypoxia in natural flooding or experimentally manipulated gas mixtures
causes hatching or death (Pyburn,
1970; Warkentin,
2000a; Warkentin,
2002). How can we reconcile these apparently conflicting facts? We
propose that the concentrated, spatially flexible gas exchange surface of the
external gills, strong oxygen gradients within individual eggs, and
oxygen-sensitive embryo behavior allow terrestrial A. callidryas
embryos to exploit a spatial refuge from hypoxia, and that these traits may be
critical for both normal development and hatching plasticity.
External gills in embryonic oxygen uptake and delayed hatching
The external gills of A. callidryas embryos appear to confer a
substantial oxygen uptake benefit. Embryos with external gills were able to
regulate their metabolism at external PO2 well
below the Pcrit of gilless embryos
(Fig. 2), and indeed below the
Pcrit of some smaller, cooler anuran eggs [e.g.
Pseudophryne bibroni, 2.8 mm diameter eggs, hatching stage
O2 1.05
µlh–1 (Seymour and
Bradford, 1987; Seymour and
Bradford, 1995) and Pcrit 19 kPa
(Seymour et al., 1991) at
12°C, vs A. callidryas 4.9 mm, 5.85µlh–1 and
13.4 kPa at 25–28°C].
Age-matched externally gilled and gilless A. callidryas were
indistinguishable in size, non-branchial aspects of gross morphology, and
oxygen uptake under conditions of ample supply. Thus there is no evidence that
rearing conditions affected their oxygen requirements. It is possible that the
oxygen-enriched rearing conditions designed to induce gill regression also
affected other respiratory traits, such as hematocrit or blood chemistry. We
did not assess these variables, thus cannot be sure gill regression was the
only factor affecting oxygen uptake ability. Nonetheless, the dramatic
difference in Pcrit between gilled and gilless embryos is
consistent with a major role of these structures in embryonic respiration. The
rapid hatching of A. callidryas following rapid,
prostaglandin-induced external gill regression
(Warkentin, 2002) is also
consistent with a respiratory role of the gills. Indeed, the fact that induced
hatching begins under 20 min after prostaglandin treatment limits the
possibility that other respiratory traits that change more slowly also
contribute to the hatching induction.
The metabolic rate of embryos without external gills in eggs with 75%
of their surface exposed for gas exchange was oxygen limited even in air
(Fig. 2). This surface exposure
is near the highest we have observed in natural clutches (80%), and most eggs
are substantially less exposed (Warkentin
et al., 2005). Were such gilless embryos to occur in nature, they
would be oxygen limited, and we would expect variation in surface exposure to
generate variation in development rates. By contrast, externally gilled
embryos in eggs with as little as 15% air-exposed surface develop in synchrony
with their highly exposed clutchmates
(Warkentin et al., 2005).
Their rapid, synchronous development appears to depend on the external
gills.
For both gilled and gilless embryos, the PO2 that induced hatching was well below Pcrit (Fig. 2), indicating that embryos tolerated some metabolic constraint within the egg. Consistent with this tolerance, in natural clutches, embryos with very low air exposure can develop – albeit more slowly than their clutchmates – and hatch late but not necessarily developmentally premature (K.M.W., personal observation). Tolerance of metabolic constraint by hatching-competent embryos suggests that, at least in the absence of other cues indicating egg-stage risk, a more advanced developmental stage at hatching is prioritized over maximizing development rate per se.
More extreme hypoxia can, however, kill embryos, for instance when clutches
are submerged in pond water (Pyburn,
1970). Under such conditions, premature hatching is clearly a
better option, and embryos hatch. The level of hypoxia that embryos tolerate
without hatching differs more between gilled and gilless animals than does
their Pcrit (Fig.
2). Gilless, hatching-competent embryos remain in only a small
subset of eggs in natural clutches
(Warkentin, 2002), suggesting
that most eggs are sufficiently hypoxic to put gilless embryos beyond their
tolerance for metabolic constraint. Although we do not know the safety margin
that embryos allow between lethal hypoxia and hatching, eggs lacking any
air-exposed surface typically fail to develop and die (K.M.W., personal
observation). Poorly exposed eggs that support normal development of gilled
embryos may also be sufficiently hypoxic to kill gilless embryos. Thus the
presence and function of embryonic gills will affect the range of clutch
structures that support normal development, as well as the balance between
water loss and oxygen exchange in a terrestrial environment
(Strathmann and Strathmann,
1989; Strathmann and Hess,
1999).
Tadpole respiration and rapid gill regression upon hatching
We detected no effect of external gills on the oxygen uptake of tadpoles in
water without access to air. Moreover, the unconstrained metabolic rate of
tadpoles did not differ from that of developmentally matched embryos.
Consistent with results from other anamniotes
(Barrionuevo and Burggren,
1999), Pcrit decreased upon hatching. The
indistinguishable Pcrit of gilled and gilless tadpoles
also suggests that non-branchial respiratory traits are unlikely to contribute
substantially to the higher O2 uptake capacity of gilled embryos,
compared with gilless embryos. If such were the case, we would expect this
superior capacity to also be evident in tadpoles.
Because neither embryos nor tadpoles in our experiment had access to air,
the key difference in their respiratory environment was the spatial
distribution of oxygen. Tadpoles were in well-mixed water, in which all
respiratory surfaces – skin, internal gills and external gills –
would be exposed to similar PO2. Embryos were
exposed to oxygen gradients in the egg. Although the single eggs in our
experiment were highly exposed, at most half the embryo's skin faced the
air-exposed surface, with the rest facing the hypoxic egg interior or the
portion of egg capsule against the impermeable glass bottom of the container.
We tested A. callidryas early in the period of hatching competence,
when their internal gills are poorly developed
(Warkentin, 1999b). Thus most
gas exchange presumably occurred via skin or external gills.
Cutaneous respiration, although insufficient for A. callidryas within
terrestrial eggs, therefore appears adequate to support unconstrained
metabolism of hatchling tadpoles in even moderately hypoxic water
(Fig. 2). This sufficiency of
cutaneous gas exchange for tadpoles is consistent with the rapid regression of
external gills that normally occurs upon hatching
(Warkentin, 2000b).
Tadpoles hatched into very hypoxic water retain their external gills
(Warkentin, 2000b). Since the
tadpole gills alter neither Pcrit nor the slope of the
O2/PO2
relationship below Pcrit, under experimental conditions
gill retention would confer no metabolic benefit. It might simply reflect
mechanisms of gill retention more relevant within the egg. Alternatively,
facultative retention of external gills might benefit hatchlings in hypoxic
ponds, which often hang with gills near the air-water interface (K.M.W.,
personal observation). Similar behavior and facultative external gill
retention has been reported for other anuran tadpoles [Hypsiboas
(Hyla) rosenbergi
(Kluge, 1981),
Stephopaedes anotis (Channing,
1993)].
Adaptive embryo behavior
The positions of A. callidryas embryos are highly non-random. Most
of the time embryos have their external gills positioned near the air-exposed
egg surface, where oxygen is highest. They may not specifically shape
movements to position their gills near air; they often end up with gills in
other places, particularly in eggs with low air-exposure. However, embryos
that come to rest with their gills in a high-oxygen region remain in that
position for a relatively long period (Fig.
7). By contrast after experimental repositioning or spontaneous
movements that place their gills in a low-oxygen region embryos move again
within seconds. Indeed, the time that active embryos remained in such
positions was remarkably consistent across ages and contexts (10±1 s
for 3-day embryos after spontaneous movements, 15±2 s for 3-day embryos
after experimental repositioning, and 15±1 s for 5-day embryos after
spontaneous movements). This may reflect the time necessary for oxygen
gradients, based on flow patterns of perivitelline fluid, to re-establish
after the turbulence caused by embryo movements, and for embryos to either
assess oxygen levels at their gills or begin to feel a constraint on
O2 uptake.
Considering the efficacy of cutaneous respiration even in moderately
hypoxic water and its inadequacy in the egg, and the fact that external gills
do not improve O2 uptake in well-mixed water, it is unlikely that
these gills would contribute much to embryonic respiration if they were not
positioned in high-oxygen regions of the egg. Thus appropriate embryo behavior
is necessary for the gills to confer a respiratory benefit, and appears
critical both for rapid, synchronous development and for delayed hatching,
after hatching competence. Indeed, the initial response of A.
callidryas embryos to oxygen stress is position change; under hypoxia
embryos change position within the egg many times at short intervals before
hatching (J.R.R. and K.M.W., personal observations). These movements are like
the gill repositioning movements and distinct from hatching movements
(Warkentin et al., 2007).
Even before the development of gills that allow spatially focused oxygen
uptake, and a circulatory system that distributes oxygen to tissues, the
oxygen demand of different parts of the embryo varies. For instance, yolk is
relatively inert and developing nervous tissue is more metabolically active.
Embryos newly capable of muscular response (Gosner stage 18) took longer to
turn 180 deg. than did more developed, active embryos, but nonetheless rapidly
returned their developing head to the air-exposed region of their egg after
displacement. The earliest we could rotate embryos within the egg was stage 16
(neural tube), shortly after the onset of ciliary rotation at stage 15. After
displacement these early embryos, without heart, brain, blood or gills,
rotated within the egg to bring their developing head back to the
best-oxygenated region. We measured substantial oxygen gradients within eggs
at early stage 18 (the onset of muscular response), and they probably exist
before this. Local oxygen levels affect tissue metabolism, thus we hypothesize
that developmental benefits accrue from positioning the developing head in a
region of higher oxygen. If this is correct, the ciliary rotation of embryos
to face the air-exposed surface represents an adaptive behavioral response to
environmental variation. Pond snail embryos also respond behaviorally to
hypoxia, increasing ciliary rotation and repositioning themselves within the
oxygen gradient inside their eggs (Kuang
et al., 2002; Goldberg et al.,
2008). This behavior is mediated by the first neurons to develop
in the embryo (Kuang et al.,
2002).
The behavior of embryos is known to play a role in neuromuscular and
skeletal development in preparation for functions that occur later in life,
after hatching or birth (Colman and
Lichtman, 1993; Pitsillides,
2006). Particularly at early developmental stages, this is the
predominant functional framework for investigations of embryo behavior.
Research on adaptive plasticity in hatching has demonstrated that embryo
behavior, and appropriate responses to environmental cues, can be also of
immediate importance for survival
(Warkentin, 2007;
Gomez-Mestre et al., 2008;
Warkentin and Caldwell, in
press). Similarly, behavioral interactions of bird embryos,
shortly before hatching, with parents and siblings appear to have immediate
functions in care solicitation and hatching synchronization
(Brua, 2002). The behavioral
responses of A. callidryas embryos to oxygen gradients within the
egg, like the ciliary spinning of pond snail embryos under hypoxia
(Goldberg et al., 2008),
represent apparently adaptive behavioral responses to environmental variation
in ovo at much earlier developmental stages.
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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