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First published online November 28, 2008
Journal of Experimental Biology 211, 3800-3807 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017913
Phenotypic differences in terrestrial frog embryos: effect of water potential and phase
Adaptational and Evolutionary Respiratory Physiology Laboratory, Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia
Author for correspondence (e-mail:
p.frappell{at}latrobe.edu.au)
Accepted 25 September 2008
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Summary |
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=0 kPa) were better hydrated than embryos incubated with
a vapour water source (v=0 kPa), and grew to a larger size.
Eggs incubated in atmospheres with lower v values showed
significant declines in mass and in the thickness of the jelly capsule, while
embryos primarily showed reductions in dry mass, total length, tail length and
fin height. The most significant deviations from control (v=0
kPa) values were observed when the v of the incubation media
was less than the osmotic water potential () of the
embryonic interstitial fluid (approximately –425 kPa). Despite the
caveat that a v of 0 kPa is probably difficult to achieve
under our experimental conditions, the findings indicate the importance for
eggs under natural conditions of contacting liquid water in the nesting
substrate to allow swelling of the capsule.
Key words: water balance, water potential, osmotic pressure, vapour pressure, relative humidity, isopiestic, embryo, Geocrinia victoriana, Myobatrachidae, Anura, amphibian
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXReferences |
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). In terrestrial breeding species without parental
care, selection of nest sites with adequate water availability is critical, as
eggs lose water freely from their jelly capsule and are at risk of desiccation
(Bradford and Seymour, 1988;
Mitchell, 2002). The jelly
capsule consists of a matrix of acid mucopolysaccharides, which swells in the
presence of water to form a liquid water reservoir
(Salthe, 1963;
Beattie, 1980) and buffers
embryonic water loss in the short term (days). However, high mortality can
result from prolonged incubation in terrestrial environments with less than
optimal water availability (Martin and
Cooper, 1972; Bradford and
Seymour, 1988). Further, the growth rate of embryos that develop
terrestrially is reduced in dry conditions and embryos have a lower tissue
water content, metabolise less yolk and may have lower mass-specific rates of
oxygen consumption (Taigen et al.,
1984; Bradford and Seymour,
1988; Mitchell,
2002).
When eggs are first oviposited, the egg (or perivitelline) membrane tightly
binds the ovum. After fertilisation, the perivitelline membrane separates from
the ovum, creating a fluid-filled space that allows the embryo to rotate
freely (Salthe and Duellman,
1973; Elinson,
1987). Under conditions of water stress, the volume of the
perivitelline space does not increase and the jelly capsule swells only
slightly. Embryos developing within smaller capsules have a reduced surface
area for gas exchange and may risk becoming hypoxic, especially during the
later stages of development when
O2 is relatively
high. Other damaging effects, including body asymmetry and head depression,
may result from development in a dehydrated egg if the embryo is unable to
rotate freely within the perivitelline space, and tail lesions can occur if
the embryo adheres to the perivitelline membrane
(Bradford and Seymour, 1988;
Mitchell, 2002). In anuran
species in which embryos hatch into a water body to become free-swimming,
feeding tadpoles, such developmental anomalies will potentially be detrimental
to swimming performance and may enhance predation risk.
The driving force for water movement is decided by the free energy
(G) of a mass of water, such that water will move spontaneously from
high water potential to low water potential (see Appendix). For aquatic frog
eggs, osmotic water potential () is the driving force for
exchange of water between the embryo and its environment, with the
perivitelline membrane acting as a semi-permeable membrane. However, in
terrestrial eggs, the matric (m), vapour (v)
and osmotic components of water potential are all important drivers of water
flux, and both liquid and vapour sources of water can be incorporated into the
egg.
Embryos of the Victorian smooth froglet, Geocrinia victoriana
(Anura: Myobatrachidae; Boulenger 1888) from southeastern Australia develop in
moist depressions under leaf litter or grass on the periphery of water bodies
that flood following winter rains
(Littlejohn and Martin, 1964).
Water availability for the embryos will be variable during development. At
times the eggs may be submerged or with no liquid water available in the nest.
Hatching is stimulated by full submersion of the egg during the winter rains
(Martin and Cooper, 1972).
Other species of Geocrinia (G. leai; G. laevis) deposit
their eggs in elevated situations, usually attached to grass stalks
(Main, 1965;
Martin and Cooper, 1972);
hence, this genus is an excellent model for examining the relative importance
of the matric, vapour and osmotic components of water flux in terrestrial
anuran eggs. The relative humidity (RH) above G. victoriana egg
masses developing in natural nests ranges between 85 and 100%, depending on
nest exposure (Martin and Cooper,
1972). Depending on soil matric and osmotic properties (which were
not measured), these humidities could potentially result in relatively low
water potentials and consequently low water availability to the embryos. The
survival limit of terrestrial anuran embryos incubated on filter paper
substrates ranges between –25 kPa (Bryobatrachus nimbus) and
–400 kPa (Pseudophryne bibronii)
(Bradford and Seymour, 1988;
Mitchell, 2002).
In this study we compared the morphology and metabolic traits of G. victoriana embryos incubated at a range of low vapour water potentials with those of embryos incubated in contact with liquid water.
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MATERIALS AND METHODS |
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Individual eggs were cleaned of debris by gentle rolling on damp tissue,
and total clutch size, number of live embryos and developmental stage
(Gosner, 1960) were recorded.
Diameters of the yolk, perivitelline membrane and capsule were measured while
embryos were briefly submerged (1–2 min) in distilled water (to prevent
refractive errors), using an ocular micrometer under a stereomicroscope. This
process did not appear to change the initial egg mass. Developmental stage of
the eggs ranged between Gosner stages 1 and 9, and embryos were estimated to
be no more than 2 days old when entering the experiment. Eighty-four eggs
selected randomly from each egg mass were used in the experiments.
Establishment of water potential treatments
Previous studies of the effects of water potential on the embryonic
development of frogs and reptiles have used wetted substrates (e.g. filter
paper or vermiculite) for which a relationship between substrate water content
and m had previously been determined (e.g.
Miller and Packard, 1992). In
contrast, v can be controlled by allowing vapour pressure
(PH2O) to come into equilibrium with a reference
solution of known molality; often referred to as the isopiestic technique
(Solomon, 1951;
Winston and Bates, 1960;
Muth, 1977).
We established six v treatments between approximately
–20 and –550 kPa (Table
1) using appropriate molalities of NaCl solutions calculated from
van 't Hoff's equation for an incubation temperature of 12°C (see
Appendix). Deionised water was used as the control treatment
(v=0 kPa). Approximately 500 ml of each NaCl solution (or
deionised water) was poured into a 1-l airtight container, and a 5 mmx5
mm stainless steel mesh shelf covered with two layers of Kimwipe®
(WypAll® X50, Kimberly-Clark, NSW, Australia) was suspended approximately
20 mm above the liquid. Two eggs from each egg mass were placed in each
container on the Kimwipe® into a labelled, 13 mm diameter nylon plumbing
olive, and Vaseline® was smeared around the lids of all incubation
chambers to ensure they were airtight. A second 0 kPa treatment in which eggs
were able to contact liquid water (=0 kPa) was also
created. In this case the mesh shelf was set at the bottom of the container
and was flooded with deionised water to a depth of 
1 mm. Six replicates
of each of the eight treatments were randomly arranged on a shelving unit in a
controlled-temperature (CT) room at 12±1°C (L:D, 11 h:13 h), close
to the field-measured temperature (see Results), with one container from each
treatment per shelf. Mean temperature ±0.2°C measured inside the
containers ranged from 11.7°C on the bottom to 12.2°C on the top
shelf. While this temperature variation for a given v may
result in changes in RH of 1–2%, the experimental design ensured that
there was a container from each treatment on each shelf and, thus, any changes
in RH were consistent across all treatments.
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The incubation chambers were prepared >3 weeks before any egg clutches
were collected. The Kimwipe® was assumed to have reached the
m of the NaCl solution (or H2O) in less than 24 h;
preliminary testing by following the mass changes in the Kimwipe® revealed
that the Kimwipe® reached the maximum achievable water content for a given
v in less than 1 day.
Egg clutches were collected over a 10 day period and thereafter containers
were continuously sealed ensuring v was undisturbed. After 47
days of incubation the oldest embryos reached hatching stage 26
(Gollmann and Gollmann, 1991)
and were removed for measurement of morphological traits and rate of oxygen
consumption
(O2). Removal of
embryos from the remaining clutches occurred over the next 10 days when each
clutch was 47 days old.
Morphological measurements
The diameters of the capsule and perivitelline membrane of embryos at
Gosner stage 26 were measured using a stereomicroscope with an ocular
micrometer while eggs were briefly submerged in distilled water. The eggs were
then blotted on damp tissue, weighed on a Mettler AE240 balance (Columbus, OH,
USA), and the embryos hatched by gentle rolling on damp tissue to remove the
capsule layers. Embryos were immediately preserved in Tyler's fixative
(Tyler, 1962). Dorsal and
lateral photographs of the preserved embryos were taken using a digital camera
attached to the stereomicroscope and measurements of total length,
snout–vent length and tail lengths, heights and widths were made from
the images using tpsDig 1.31 image analysis software (F. J. Rohlf, State
University of New York, Stony Brook, NY, USA). Measurements of 10 live embryos
were compared with those made of the same embryos preserved for 4 weeks
(approximately the time experimental tadpoles were fixed) to test for any
effect of the fixative on tadpole dimensions. The gut was dissected from the
body and carcasses were oven dried for 1 h at 50°C, then stored over
silica gel for 3–4 days before dry masses were recorded
(Mitchell and Seymour,
2000).
Rate of oxygen consumption
O2 was
determined for unhatched stage 26 embryos from selected treatments
(=0 kPa, v=0, –22, –105 and
–493 kPa) prior to morphological measurements by measuring the decay in
the partial pressure of oxygen (PO2) in a
sealed chamber with a Clark electrode (OXY040A, Rank Brothers Ltd, Cambridge,
UK). The electrode was positioned at the bottom of the chamber and polarised
with a current of 60 mV, and a rotating magnet caused a magnetic stirrer to
rotate continuously, eliminating any boundary layer formed by electrode oxygen
consumption. The embryos were placed in 5% Holtfreter's solution
(approximately isotonic with pond water) on a stainless steel mesh stage above
the stirrer, and the chamber was maintained at 12±0.2°C using water
pumped from a water bath though a surrounding jacket.
Electrodes were calibrated at the beginning and end of each run with
air-bubbled 5% Holtfreter's solution (PO2
20.81 kPa) and electrical zero was used to indicate
PO2=0 kPa. Any drift that occurred was assumed
to be linear. PO2 was recorded at 1 Hz (Chart,
Powerlab AD Instrument, Bella Vista, NSW, Australia) and linear regression
used to determine the change in PO2 with time,
after correcting for baseline drift. Embryonic
O2 (in
µlh–1) was calculated from
Eqn 1:
 | (1) |
O2 was then
calculated on a mass-specific basis using gut-free dry body masses (see
above).
Osmolality of perivitelline and interstitial fluid
Samples of perivitelline fluid were collected from spare stage 26 embryos
that were incubated at 12±0.5°C on tissue flooded with distilled
water. The outer capsule layers were removed by gentle rolling on damp tissue,
and the embryo was immersed in paraffin oil and perivitelline fluid collected
in 10µl glass microcapillary tubes after rupturing the perivitelline
membrane with a needle.
Embryonic interstitial fluid was collected from 15 stage 26 embryos killed
by immersion for 5 min in MS222 (150 mg l–1). Immersion time
was short to prevent any disturbance of osmotic balance. Groups of four to
five embryos (to obtain sufficient volumes) were blotted on damp tissue, their
tails were removed, and heads and tails were separately homogenised in 1.5 ml
Eppendorf tubes using a Teflon pestle. The Eppendorfs were centrifuged for 3
min and the supernatant collected in microcapillary tubes. The osmolality of
10 µl samples of the perivitelline fluid and interstitial fluids was
measured with a vapour pressure osmometer (Vapro 5520, Wescor, Logan, UT,
USA), and calculated using
Eqn A1 (see Appendix).
Analysis
Data are presented as means ± 1 s.e.m., and percentage data were
arcsine transformed for statistical analysis. Weighted linear regressions and
statistical comparisons were performed using Minitab (v14), but the liquid
water treatment (=0 kPa) was not included in the
regressions as water was exchanged with the embryo in a liquid phase rather
than a vapour phase. For other analyses the saturated water vapour treatment
(v=0 kPa) was treated as the control and all comparisons were
made to this treatment (ANOVA with Dunnett's comparisons). Statistical
significance was assumed at P<0.05.
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RESULTS |
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v
(Table 2;
Fig. 1). The diameters of the
perivitelline membrane and capsule, and consequently capsule thickness, also
decreased with decreasing v
(Table 2; Figs
1 and
2). Eggs from atmospheres below
–493 kPa had significantly smaller perivitelline diameters and thinner
capsules than control eggs, while eggs in atmospheres below –206 kPa had
significantly smaller capsule diameters (Figs
1 and
2).
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There were no differences in whole (embryo+gut) wet or whole (embryo+gut)
dry mass between the various v treatments
(Table 2;
Fig. 3), but dry gut-free
embryo mass decreased with decreasing v. The relative
proportion of dry gut to dry body tissue was influenced by v,
with embryos reared in the driest conditions assimilating less yolk than
better hydrated embryos. Total embryo length decreased with decreasing
v (Tables 2 and
3;
Fig. 2). Stage 26 embryos
raised at v=0 kPa were longer than those embryos raised in
atmospheres below v=–493 kPa. Tail length was shorter
with decreasing v, but snout–vent length was only
significantly different between embryos raised at v=0 and
embryos raised at –105 kPa and –493 kPa (Tables
2 and
3;
Fig. 2). Body height only
differed between embryos from v=0 kPa and at
v=–533 kPa (Table
3). Fin height decreased with decreasing v. There
was no effect of incubation v on tail muscle height or tail
width (Tables 2 and
3;
Fig. 2). Fixed embryo
dimensions were 95% of live embryo dimensions (P=0.001, Student's
t-test).
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Comparison of liquid and vapour phase
There were significant differences between G. victoriana eggs
incubated on a wet substrate (=0 kPa) and the control
(v=0 kPa) eggs. Eggs incubated on a wet substrate were 4.8
times heavier than control eggs, the perivitelline membrane and capsule
diameters were significantly greater and the jelly capsule was thicker
(Fig. 1). Stage 26 embryos
raised at =0 kPa were 18% heavier and 14% longer than
embryos reared at v=0 kPa
(Fig. 3). The greater length of
=0 kPa embryos was due to both greater snout–vent
length and greater tail length. Fin height of embryos raised at
=0 kPa was 23% higher than for control
(v=0 kPa) embryos (Table
3; Fig. 2).
Osmolality of perivitelline and interstitial fluid
The osmolality of perivitelline fluid measured in unhatched stage 26
embryos was 10±2 mosmol kg–1, which was equivalent to
a of –24±2 kPa (N=16). The
osmolality of interstitial fluid from the tails of unhatched stage 26 embryos
was 179±2 mosmol kg–1 (N=3), equivalent to a
of –399±15 kPa. The osmolality of the heads
and residual yolk material was 194±1 mosmol kg–1
(N=3), equivalent to a of –441±2
kPa. On average, the osmolality of the entire embryo (tail+head) was
186±2 mosmol kg–1, equivalent to a
of –424±5 kPa.
Rate of oxygen consumption
There were no differences in the dry mass-specific rate of oxygen
consumption (O2,
µlh–1 mg–1) for eggs at stage 26 that had
been raised at different v values or between the control and 0
kPa embryos (Fig.
4). The pre-hatching stage 26
O2 across all
treatments averaged 0.92±0.09 µlh–1
mg–1.
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v on the percentage of embryos
surviving to hatching stage 26 (P=0.302) with survival averaging
37.12±7.10% (N=42 containers) across the v
treatments. There was also no effect of v on the number of
embryos that were able to hatch when flooded (P=0.067), which
averaged 74.01±5.23% (N=42 containers). The percentage of
v=0 kPa versus =0 kPa embryos
surviving to hatching was not different (P=0.17), averaging
39.17±8.21% and 40.00±2.24%, respectively. Similarly, the
hatching rates of embryos raised at v=0 kPa versus
=0 kPa were also not different (P=0.994),
averaging 74.81±9.96% and 85.40±2.30%, respectively.
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DISCUSSION |
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=0 kPa) were larger and heavier than eggs in saturated
vapour (v=0 kPa; Fig.
1), and the resulting embryos were more hydrated
(Fig. 3) and had longer tails
with higher fins (Table 3). The
two numerically identical water potentials produced two different phenotypes.
Why does the egg swell more when placed in shallow pure water (hence hydraulic
pressure gradient is negligible) than in 100% RH above the water surface (i.e.
when the two conditions have the same water potential)? A likely explanation
is that small spatial or temporal gradients in ambient temperature across the
incubation chambers prevent equilibrium between the liquid reservoir and the
atmosphere around the eggs. The containers were unlikely to be absolutely
isothermal, a condition that is further complicated by temperature cycling of
the CT room. Small changes in temperature can substantially alter
v above a solution. For example, at a v of
–22 kPa the RH is 99.98% (see Table
1). An equivalent RH can be achieved above distilled water if the
air temperature rises approximately 0.003°C, though this would be
transient because saturation vapour pressure above the water would eventually
be reached. The effect of any temperature differentials across the CT room as
a whole will overall have an equal effect on each treatment as the containers
were randomised within the CT room. Presumably, the small temperature
differentials within the incubation containers add to the differences seen in
eggs incubated above liquid water compared with those incubated with access to
liquid water, as changes in ambient temperature alter v only
and not in pure water. An increase in ambient
temperature, or opening of the chambers, will cause a decrease in
v, a situation that will not be reversed when ambient
temperature is decreased as RH cannot exceed 100%. The equilibrium was
presumably re-established well within 24 h as preliminary testing revealed
that a Kimwipe® under similar conditions reached maximal water content
within 1 day. Another possibility is that the metabolic heat of the embryo
elevates the temperature of the egg a very small amount above the ambient air
temperature, hence establishing a water potential gradient where water may be
lost as vapour. Fourier's law of heat conduction for a spherical shape using
the diameter of the egg and metabolic rate of the embryo reported in this
study, and assuming the egg is primarily water (thermal conductivity of water
0.6 W m–1 K–1), reveals that the
temperature would be raised by 0.000004°C. This increase in egg
temperature would result in a water potential between the egg and the
saturated atmosphere at 12°C of –0.0347 kPa; a very small driving
force. Debate concerning the effects of temperature on liquid and vapour
exchange of water in reptilian eggs has shown the importance of temperature
differences on vapour pressure (see
Ackerman et al., 1985;
Thompson, 1987).
Alternatively, the swelling of the egg in liquid water is related to the
gel-like properties of the capsule. Cameron and colleagues
(Cameron et al., 1990) showed
that about 80% of a frog egg's water is osmotically inactive, and further
suggested that this water is bound to cell proteins; an idea that supports the
notion of the cytoplasm being a gel. The swelling of gels does not simply
depend on the difference in ionic concentration between the gel and the medium
in which they are placed but on the elasticity of the polymer matrix, with the
swollen gel exerting lower restraining forces on the diffusing water molecules
(Yasuda et al., 1971;
Gunt et al., 2007). Water
diffusivity for hydrogels has been shown to be greater when the gels are fully
hydrated (liquid phase) compared with the diffusivity of the hydrogel in a
vapour phase of equivalent water potential (e.g. 4-fold increase for
well-hydrated keratin) (Gunt et al.,
2007). Hence, at the same water potential, the egg in liquid will
have a higher flux due to a higher diffusivity of water and reach equilibrium
earlier. In over-swelling, flux is halted (it has been postulated) because of
the increased cross-linking of randomly distributed polymers in the gel, which
also act to decrease gel compliance (Kelly
et al., 1995). Alternatively, stretch-activated ion channels,
which have been found in Xenopus oocytes
(Yang and Sachs, 1989), may
help to limit swelling of the perivitelline space.
Despite potential experimental issues with achieving a v of
0 kPa, our experiments show the importance for eggs of contacting liquid water
in the substrate to allow for swelling of the capsule.
Fitness consequences of development in dehydrating atmospheres
Major components of offspring fitness such as embryonic survival or size
can be used as an indication of the effect of the incubation environment on
lifetime fitness (Arnold,
1983). In our study, embryos incubated in atmospheres of
decreasing v showed significant declines in whole-egg mass,
perivitelline membrane diameter, capsule thickness, dry mass and total length.
In particular, tail lengths were significantly shorter for embryos reared in
the driest conditions, similar to B. nimbus hatching-stage embryos
where a decline in total length at lower was due to
proportionally shorter tails (Mitchell,
2002). Smaller larvae have lower foraging efficiency and may
develop more slowly, but in the case of terrestrial embryos, higher rates of
yolk assimilation associated with high during incubation may also allow
hatchlings to metamorphose earlier and, hence, spend less time in a vulnerable
stage of their life cycle (Arnold,
1983). Post-hatching monitoring would be required to determine the
optimum v range for terrestrial development in G.
victoriana, but our results imply that incubation at v
values below –400 kPa reduces embryonic fitness and is therefore likely
to have a detrimental effect on adult fitness. For example, in anurans, larval
size is usually positively correlated with adult size, and larger female
G. victoriana are more successful breeders
(Scroggie, 2001).
Another specific morphological trait likely to influence larval fitness is
fin area. This study, like another on B. nimbus
(Mitchell, 2002), found that
fin area was reduced with decreasing water availability
(Fig. 2). As tail fins are
highly vascularised and hence must be an important respiratory surface,
reduced tail fin area could potentially affect rates of metabolism, but we
found no effect of incubation v on the rate of oxygen
consumption
(O2). At least
one study has shown that possession of larger tail fins does not enhance
tadpole swimming speed (Van Buskirk and
McCollum, 2000a), but larger tail fins may give tadpoles some
advantage in escaping predation attempts, as predators are more likely to
damage fins rather than tail muscle. For example, larval Hyla
versicolor are able to lose up to 30% of their tail fin though predatory
attempts without affecting swimming performance
(Van Buskirk and McCollum,
2000b).
Egg mass decreased as water potential (v) decreased
(Fig. 1), but there was no
associated decline in the hydration state of embryos with decreasing
v (Fig. 3),
demonstrating the buffering capacity of the jelly capsule in preventing
embryonic water loss. The only significant deviation in embryonic water
content from the control was for embryos incubated on the wet substrate
(=0 kPa), whose wet body mass was 18% greater than that of
the v=0 kPa embryos. Dry body mass did not vary between any of
the treatments, therefore embryos from the =0 kPa
treatment simply had a higher water content per unit of dry body mass.
However, the proportions of gut and body (gut-free embryo) that constituted
the total dry mass varied linearly amongst the treatments
(Fig. 3), similar to other
studies of anurans and reptiles where `wetter' embryos converted more yolk
into tissue (Packard et al.,
1987; Bradford and Seymour,
1988; Miller and Packard,
1992).
We detected no differences in metabolic rate across our treatment groups, a
result that differs from studies of B. nimbus where
O2 of embryos
reared at –25 kPa was 72–81% of the
O2 of embryos
incubated at 0 kPa (Mitchell,
2002). The average
O2 across all
treatments of 0.92±0.09 µlh–1 mg–1
was very similar to the 0.92 µlh–1 mg–1
we predicted using a previously published equation
(Seymour and Bradford, 1995),
where O2 is
related to ovum volume, and average
O2 is corrected
to 12°C using a Q10 of 2.5 for Geocrinia
vitellina (Mitchell,
2001). As
O2 is directly
related to aerobic metabolism (Mortola and
Gautier, 1995), we assumed that embryos in our different
treatments were metabolising at the same rate per unit of tissue. However, to
have assimilated a greater proportion of yolk into tissue
(Fig. 3), the more hydrated
embryos must have had a higher mass-specific
O2 at some stage
in embryonic development.
Survival to hatching stage and hatching success were not influenced by
v across the 0 to –533 kPa range studied, which is
comparable to a study of E. coqui that found no effect of on
embryonic survival between –50 and –550 kPa
(Taigen et al., 1984). Studies
of other Australian Myobatrachid frogs with terrestrial eggs show different
effects of low water potentials; P. bibronii have high survival to
hatching stage at high (>–50 kPa) but very low survival at
less than –400 kPa (Bradford
and Seymour, 1988), whereas the survival of B. nimbus
embryos is severely impaired at –25 kPa
(Mitchell, 2002), suggesting a
reliance on wet conditions.
Conclusions
Embryonic development of the terrestrial-breeding frog G.
victoriana was affected by the vapour water potential of the humid
atmosphere in which embryos were raised. The difference in morphology was
marked, with embryos incubated in atmospheres of decreasing v
showing declines in egg mass, perivitelline membrane diameter, capsule
thickness, dry mass, and tail and total length. In the driest conditions
(v=–533 kPa, corresponding to a RH of 99.5% at
12°C), embryos were significantly smaller. Eggs and embryos were also
smaller when raised in a saturated atmosphere compared with those raised in
contact with pure liquid water, despite these treatments having the same water
potential (v==0 kPa). Presumably, it would
be most advantageous for G. victoriana to lay its eggs in saturated
atmospheres with access to liquid water, a possibility that was observed in
some nest site choices in the field. This would ensure larger hatchlings,
which would potentially confer an advantage in terms of foraging
efficiency.
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APPENDIX |
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) is decided by the free
energy (G) of a mass of water, such that water will move
spontaneously from high potential to low potential. The total water potential
is the sum of a number of component water potentials, including gravitational
(g), pressure (p), matric (m),
osmotic () and vapour (v) water
potentials. g is related to the gravitational pull and
p is due to pressure differences from atmospheric pressure,
and both are considered negligible in the present study, though turgid eggs
would have a positive p. The potential due to matrix effects
(e.g. fluid cohesion and surface tension) is due to adsorption of water onto
surfaces. The addition of solutes to water will affect the osmotic potential
of the solution. For ideal dilute solutions (in kPa) can
be calculated from the van 't Hoff equation in terms of solute concentration:
 | (A1) |
=0.
The vapour water potential (v, in kPa), the water potential
of vapour in air, is also determined from the van 't Hoff relation such that:
 | (A2) |
 | (A3) |
 | (A4) |
Rearranging Eqn A4, it is seen
that Nw is equivalent to the relative humidity
(RH=PH2O/PS=Nw)
above the solution. Hence:
 | (A5) |
If
nw
 | (A6) |
 | (A7) |
in an aqueous solution is the same as that for vapour
phase transport established by the v above the solution. LIST OF ABBREVIATIONS
O2
g
m
p
v
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXReferences |
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