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First published online January 31, 2007
Journal of Experimental Biology 210, 722-731 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02702
Oxygen in egg masses: interactive effects of temperature, age, and egg-mass morphology on oxygen supply to embryos
1 Department of Biological Sciences, Clemson University, Clemson, SC 29634
USA
2 Division of Biological Sciences, University of Montana, Missoula, MT 59812
USA
* Author for correspondence (e-mail: moran{at}clemson.edu)
Accepted 20 December 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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A simple mathematical model is developed to provide a quantitative means of estimating primary and interactive effects of the different factors. We also show that in T. diomedea the gel itself is the main barrier to oxygen transport into egg masses, and that the metabolic activity of embryos increases substantially when embryos are artificially released from the capsules that contain them within the gel mass.
Key words: oxygen, egg mass, nudibranch, marine, temperature, size
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Lambert et
al., 2003; Kojima et al.,
2004; Levin,
2006). For individuals, benthic egg masses reduce the window of
vulnerability to predation on free-living larvae in the plankton, and egg-mass
gel may also provide protection from predators and parasites
(Benkendorff et al., 2005),
buffer embryos from adverse physical conditions
(Biermann et al., 1992;
Woods and DeSilets, 1997;
Przeslawski et al., 2004), and
alter metabolic gas levels around embryos
(Cohen and Strathmann, 1996;
Lee and Strathmann, 1998;
Strathmann and Hess, 1999;
Strathmann, 2000). Here we
focus on the last factor; in particular, we examine the relationship between
the structure of gelatinous egg masses and their internal oxygen profiles.
Oxygen levels inside egg masses can be extremely low and can retard
development or kill embryos. Low oxygen levels have been described from masses
of many marine invertebrates (Booth,
1995; Cohen and Strathmann,
1996) and aquatic vertebrates
(Seymour and Roberts, 1991;
Pinder and Friet, 1994) and
stem from straightforward principles of mass balance; respiration by embryos
(or other commensal organisms, such as bacteria) within an egg mass draws down
oxygen levels within the mass, while oxygen is replenished by transport from
the environment via diffusion down a partial pressure gradient.
Because diffusion of oxygen in water is so slow and egg masses can be large,
the equilibrium oxygen level within masses (at which rates of consumption and
supply are equivalent) can be quite low, and these low oxygen levels are
correlated with delayed development of internal embryos
(Strathmann and Strathmann,
1989; Strathmann and
Strathmann, 1995). Slow development and embryo death are
overwhelmingly caused by low oxygen levels rather than buildup of toxic
metabolic byproducts in the egg mass, because developmental delays can be
reversed or prevented entirely with supplemental oxygen
(Strathmann and Strathmann,
1995).
However, beyond the basic observations that oxygen is often depleted inside
masses more rapidly than it can be replenished via diffusion, and
that hypoxia can delay development or kill embryos
(Strathmann and Strathmann,
1995; Seymour et al.,
1995), little is known about the complex relationships among
egg-mass structure, embryo age, and the opportunities and constraints imposed
by environmental effects on embryonic metabolism. Here we examine the
quantitative effects of four factors that, a priori, we expected
would influence oxygen levels in egg masses: temperature, embryonic age,
density of embryos, and size of egg masses. The first three factors should all
be positively associated with volume-specific oxygen demand (= metabolic
density), because increases in temperature, embryonic age, and density of
embryos within an egg mass should all result in increased oxygen consumption.
The last factor, egg mass size, should affect both total metabolic demand for
oxygen (all else being equal, larger egg masses will hold more embryos) and
the geometry of the supply–demand relationship. In particular, larger
egg masses will have smaller ratios of surface area for oxygen acquisition per
volume of oxygen demand. This study is unique in that it builds on previous
studies by including temperature effects, examining the interactions of
temperature with previously studied factors (density of embryos, egg mass
size, and age), and directly measuring rates of oxygen consumption in addition
to oxygen distribution.
A second goal of this work was to examine how packaging of embryos within
the egg mass affects embryonic metabolism. In most taxa embryos are not
embedded directly in the gel matrix of the egg mass; rather, they are packaged
singly or multiply into liquid-filled egg capsules that are contained within
and are in direct contact with the gel, and these capsules can have direct
consequences for oxygen diffusion (e.g.
Cronin and Seymour, 2000). In
opisthobranch gastropods, embryos develop in these capsules within the egg
mass from the fertilized embryo until immediately prior to hatching from the
mass as free-swimming veligers or metamorphosed juveniles. Embryos develop
cilia and begin rotating early in development and, by the late veliger stage,
are swimming vigorously inside the capsule.
The presence of an encapsulated stage within the egg mass leads to two
important questions about the effects of encapsulation on oxygen budgets that
are necessary to understand oxygen distribution within the mass as a whole.
First, does encapsulation itself affect embryo metabolic rates? In principle,
the total metabolic rate of an egg capsule should be predictable from the
summed metabolic rates of the embryos in it; this rate can be calculated by
measuring the per-embryo metabolic rate of free embryos using a variety of
established methods (e.g. Moran and
Manahan, 2004). However, the total metabolism of a group of
embryos confined in a capsule may not be predictable from free-embryo rates.
For example, encapsulation may suppress larval metabolism by physically
restricting swimming activity, or through tranquilizing substances in the
capsule fluid (Marthy et al.,
1976).
A second, related question concerns oxygen transport. When embryos are
packaged in capsules, oxygen transport occurs through (at least) three
distinct materials: the outer gelatinous matrix, the egg-capsule wall, and the
liquid inside the egg capsule. Most work on oxygen profiles has considered
just oxygen in the gel itself [for exceptions in frogs, see Seymour and
Bradford (Seymour and Bradford,
1987) and Seymour et al.
(Seymour et al., 1991)]. How
important a barrier to oxygen movement is the egg capsule wall and interior
liquid?
We examined these questions in both natural egg masses of the nudibranch Tritonia diomedea Bergh 1894 and in artificial egg masses made using low-melting-temperature agarose and embryos of the sand dollar Dendraster excentricus Eschscholtz 1831. Using T. diomedea, we measured the effects of temperature and embryo age on metabolic rates of embryos that were either (1) removed from the mass, but left in the capsules in which multiple embryos are embedded together in the mass; or (2) removed from both the mass and capsules. To examine the effects of embryo density and egg mass size on oxygen profiles, we constructed artificial egg masses by embedding fertilized eggs of D. excentricus into low-melting point agarose and varying embryonic density and egg mass size. Together, our results show that the four factors we tested, temperature, embryonic age, embryo density, and egg-mass size, each affect oxygen profiles by themselves. More notably, however, the strongest effects were seen when two or more factors interacted, and these interactions have substantial bearing on egg mass design.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Egg masses and larval development of Tritonia diomedea have been
described (Hurst, 1967;
Kempf and Willows, 1977;
Strathmann, 1987). Within the
gelatinous sleeve, capsules are either loosely attached to each other in a
coiled chain or are unattached (Fig.
1). Embryos develop in egg capsules within the gelatinous mass and
hatch as fully formed veliger larvae, which are released into surrounding
water when the aging egg mass begins to disintegrate (10–14 days at
10.5–12°C) (Kempf and Willows,
1977).
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Respiration
Respiration rates of embryos were measured using end-point determination
methods (Marsh and Manahan,
1999). In short, liberated embryos or capsules containing embryos
were suspended in 0.2-µm filtered seawater in small (
700 µl)
respiration chambers. To test for concentration-dependent effects on
respiration, a range of numbers of capsules (from 1 to 6) or liberated embryos
(from 10 to 1500) were added to each chamber; no such effects were seen
(Fig. 2). Each set of
measurements was replicated by using 6–7 chambers per treatment. Animals
were incubated in the respiration chambers at experimental temperatures for
4–6 h, after which 300 µl subsamples were taken from each chamber
with a temperature-equilibrated gas-tight syringe. Oxygen tension was measured
in each sample using a polarographic oxygen sensor (Model 1302, Strathkelvin,
Glasgow, UK). The number of animals in each chamber was then counted in one of
two ways. For liberated embryos, we directly counted all embryos in each vial.
For egg capsules, we either (1) directly counted embryos in all capsules and
summed them for each vial, or (2) counted embryos in 20 haphazardly chosen
capsules from the same mass, calculated the mean number of embryos per
capsule, and multiplied the number of capsules in a given vial by this
average. The latter method was used for temperature experiments because time
constraints precluded immediate counts of embryos in each egg capsule.
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Oxygen consumption per embryo or per capsule was calculated as the slope of the regression line of oxygen consumed per hour against number of embryos or capsules in each chamber (Fig. 2).
Effects of encapsulation on embryonic respiration
Respiration rates of individual embryos may change depending on whether
embryos are free or encapsulated. We therefore measured respiration of both
whole capsules containing embryos and of liberated embryos. These paired
measurements were made at three developmental stages: embryos classified as
`early' (1 day old, between early cleavage and gastrulation), `mid'
(early veligers, unshelled but ciliated) or `late' (mature veligers near
hatching, 2 weeks old). Paired comparisons were made at 12°C on
encapsulated vs freed embryos from one egg mass at each developmental
stage.
Temperature and age effects
To determine how respiration rates changed with temperature, we measured
oxygen consumption of embryos from `early', `mid' and `late' (see above) egg
masses at two temperatures, 12°C and 21°C. Because free embryos
exhibited higher metabolic rates than encapsulated ones, we used encapsulated
embryos in these experiments to more closely estimate natural oxygen demand in
egg masses. At each stage of development we estimated the Q10 of
metabolic rate using the standard equation:
 | (1) |
Oxygen in egg masses
PO2 inside egg masses or in individual egg capsules was
obtained using a Clark-style O2 microelectrode (model 737GC, 15
µm tip, Diamond General, Ann Arbor, MI, USA) connected to a picoammeter
(Chemical Microsensor I, Diamond General). The electrode was calibrated in
bag-filtered seawater that was bubbled with air or pure N2.
Calibration water was held at constant temperature with a water-jacketed
calibration cell connected to a recirculating water bath. The electrode was
always calibrated at the experimental temperature (12 or 21°C). Signals
from the picoammeter were logged once per second onto a computer running
ExpeData software (v 0.2.48, Sable Systems, Las Vegas, NV, USA). Water
temperature was also logged continuously using a T-type thermocouple connected
to a thermocouple meter (TC-1000, Sable Systems).
Two main structural features of egg masses could impede oxygen movement: the gel-filled egg mass sleeve itself, or the capsule walls surrounding individual batches of embryos. To distinguish between these possibilities, we measured levels of oxygen inside egg capsules that were residing in situ in short sections of otherwise intact egg mass, or inside egg capsules that had been freed by gently squeezing them from the cut end of an egg mass section (see Fig. 1).
For in situ measurements, short sections of egg mass were
submerged and pinned onto Nitex mesh that had been hot-glued across the
opening of a short length of PVC tubing (1'' i.d.). For measurement of
PO2 inside freed capsules, the mesh was replaced with a
black PlexiglasTM disc (black to improve capsule visibility) into which
fine grooves had been cut. Individual capsules (each containing 100
embryos) were positioned into the grooves using a plastic pipette.
In all cases, the platform was mounted onto a post fixed to the bottom of a
small glass jar (70 ml total volume) filled with bag-filtered seawater.
Air was bubbled gently into the water to maintain ambient oxygen levels near
air saturation. Temperature was controlled by submerging most of the jar into
the recirculating water bath (set to 12°C or 21°C). The electrode tip
was viewed under a stereo microscope and positioned using a micromanipulator.
The tip could be placed reliably into egg capsules within intact sections of
egg mass. Placing the tip into free capsules was more difficult; unless the
capsule was caught in a groove, it would slip away from the tip as pressure
was applied. However, once the tip had penetrated into a capsule, the tip and
capsule together could be lifted off the PlexiglasTM support and
suspended free in the bulk seawater, thereby avoiding local oxygen depletion
within the grooves.
We examined the effects of both age and temperature on oxygen levels.
Sections from two different, large Tritonia egg masses were used: one
was less than 12 h old and the other was approximately 2 weeks old. The older
mass contained vigorously swimming veliger larvae (close to hatching). For
both masses, small sections of egg mass (each 6 cm long) were sampled
from six different locations. Oxygen levels inside egg capsules in three each
of young and old sections were obtained first at 12°C. Subsequently, a few
egg capsules were gently squeezed from the cut ends of each section, and
measures of oxygen inside free, suspended capsules were obtained. The three
other masses from each mass were ramped from 12°C to 21°C over 15 min,
always in air-bubbled seawater (during this time, the electrode was
recalibrated at the higher temperature). After we had allowed egg mass
sections to equilibrate to the new temperature for 1 h, oxygen levels were
again measured inside egg mass sections and in free capsules.
Artificial egg masses
Artificial agarose masses containing pre-hatching embryos of Dendraster
excentricus were created from low-melting point agarose gel after the
methods of Strathmann and coworkers
(Strathmann and Strathmann,
1989; Strathmann and
Strathmann, 1995; Lee and
Strathmann, 1998), with some modifications. Adult D.
excentricus were collected in Puget Sound, Washington and maintained in
running seawater. Adults were spawned and gametes fertilized following
published procedures (Strathmann,
1987). Newly fertilized embryos were allowed to settle in two
changes of freshly filtered seawater and poured gently back and forth between
two 1 l beakers to loosen jelly coats. Embryos were then gently washed on a 40
µm screen, which retained most embryos but removed the loosened jelly
coats. De-coated embryos were then allowed to settle for a third time and
concentrated embryos were drawn up from the bottom of the beaker with a glass
pipet. Five replicate 10 µl aliquots were counted to determine total
concentration and number of embryos.
To make artificial egg masses, a 2% solution of agarose and seawater was
cooled with stirring to 30°C. 1 ml of a known concentration of embryos at
the 2–4-cell stage of development was added to 2 ml of this solution,
stirred briefly, and immediately drawn up by mouth suction into a soft-sided
cylindrical mold (plastic straws). The mold was placed into 12°C seawater
and allowed to set for 5 min. Set masses were liberated by gently
squeezing molds underwater and were maintained in running 12°C seawater
until used for measurements.
To assess the effect of size and embryo density on oxygen gradients, six artificial masses were created in three sizes and at two densities for a total of six different treatments: high-density small, medium and large masses, and low-density small, medium and large masses. The three low-density masses contained embryos at a density of 1.33 µl–1 and high-density masses contained 14.6 embryos µl–1. Small, medium and large masses were 2.5, 4 and 10.5 mm in diameter, respectively. To investigate the effect of temperature, O2 gradients were measured in all mass types as above (for Tritonia) at two temperatures, first 13°C and then 22°C, with several hours of equilibration at the test temperature prior to making electrode measurements.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Effects of age and encapsulation on embryonic respiration
Respiration rate of embryos increased with embryonic stage of development.
Early embryos had the lowest metabolic rates, and pre-hatching mature stages
(late) had the highest (Fig.
3). There was no significant difference between the metabolic
rates of encapsulated vs liberated embryos at the earliest stage of
development (Fig. 3;
respiration rates=1.36 pmol embryo–1 h–1 for
each group, comparison of slopes; t=0.027, d.f.=9, P=0.98).
At mid and late stages, liberated embryos had substantially higher metabolic
rates than did encapsulated embryos (Fig.
3; liberated embryos have metabolic rates 1.7-fold and 2.2-fold
higher than encapsulated embryos at the mid and late embryonic stages,
respectively). This effect was significant at both stages (mid,
t=2.45, d.f.=9, P=0.037; late, t=5.23, d.f.=9,
P=0.0005).
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Temperature and age effects
In masses at all three stages of development, metabolic rate was
substantially higher at 21°C than at 12°C
(Fig. 4). This held true both
for direct measurements on capsules (Fig.
4A) and for estimated per-embryo metabolic rates
(Fig. 4B). Q10
values for metabolic rate of embryos in capsules were 2.1, 2.5 and 2.6 for
embryos from the early, mid and late-stage masses, respectively.
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Artificial egg masses
Embryos of D. excentricus developed normally in artificial egg
masses up to the point of hatching from the fertilization envelope, and all
O2 electrode measurements were made prior to this point. All three
manipulated variables (egg mass size, embryo density and temperature) had
large and interactive effects on radial oxygen profiles
(Fig. 6B). Under most
combinations of density and size, for example, small-diameter (2 mm) masses
had oxygen levels that were close to air saturation throughout. The main
exception (for the smallest diameter masses) was the high-density,
high-temperature treatment, which showed distinctly low
PO2 in the center. At the two larger egg-mass sizes (4 and
10 mm diameter), however, density and temperature effects were pronounced,
with the largest egg masses exhibiting anoxia throughout most of the egg mass
in the high-density treatment.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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We approached this problem by measuring the effects of four factors, identified a priori, on embryo metabolism and oxygen distributions in egg masses. The effects of the first two factors, temperature and embryo age, were measured using natural egg masses produced by the nudibranch T. diomedea. The effects of two other factors, embryo density and egg-mass size, were not feasible to manipulate experimentally in natural masses and so were evaluated in conjunction with temperature in artificial egg masses. Our results suggest that each of the four factors can, independently, affect oxygen distributions in egg masses; however, the strongest effects on oxygen distribution were seen when two or more factors interacted. In other words, in our experiments, and likely in nature as well, any particular factor will have the greatest biological effects when combined with changes in other factors. Below we discuss each factor individually and in combination.
Temperature
Temperature has pervasive effects on oxygen consumption and transport. In
many ectotherms at normal operating temperature, metabolic demand for oxygen
increases substantially with temperature (Q10=2–3) whereas
oxygen transport by diffusion, a physical process, increases slowly
(Q10<2). The consequence of such differential sensitivity is
that higher temperatures lead to relative oxygen shortage
(Woods, 1999;
Pörtner, 2001;
Woods and Hill, 2004).
We measured metabolic rates of embryonic T. diomedea at two
temperatures, 12°C and 21°C, and metabolic rates were always higher at
the warmer temperature (Q10 values = 2.1–2.6). These data
indicate that indeed oxygen demand increases rapidly with temperature. In both
intact egg masses of T. diomedea and artificial egg masses
(Dendraster embryos), higher incubation temperatures led to lower
oxygen levels within masses. However, while the effects of temperature were
strong in artificial masses, they were more modest in natural masses of T.
diomedea. This pattern suggests that, for T. diomedea, rising
metabolic activity caused by warmer temperatures may be partially offset by
compensatory changes in the conductance of egg-mass material (though not in
the egg-capsule wall, as this had a negligible effect on intracapsular oxygen
levels in our experiments). Changes in conductance could occur through
chemical alteration or physical thinning of the mass jelly; similar phenomena
have been seen in egg-containing structures other molluscs (e.g.
Cronin and Seymour, 2000;
Brante, 2006). Alternatively,
this pattern could be explained by differential sensitivity of metabolic rate
to hypoxia in the two species. Embryos of T. diomedea occur naturally
in egg masses while the free-living embryos of D. excentricus do not,
suggesting that metabolic rates of embryos of the two species might respond
differently to hypoxia.
In either case, this observation suggests that simple mass-balance models
(e.g. Woods, 1999) may be
misleading; i.e. that incorporating diffusion as a purely physical process,
with a physical Q10<2, does not necessarily capture
temperature's true effects on oxygen availability to internal tissues. Rather,
a model incorporating temperature- and hypoxia-driven changes in both
metabolism and material conductance may give more realistic results. Indeed,
temperature-driven changes in conductance would seem to be advantageous for
aerobic ectotherms, as a means of avoiding internal hypoxia at high
temperatures.
Was 21°C a reasonable high temperature for the species we used? We
think so, for several reasons. First, embryos of both species (T.
diomedea and D. excentricus) have been successfully reared at
21°C (Strathmann, 1987),
and we observed no ill effects of these warmer temperatures on embryos in our
experiments. Second, our measured Q10 values for embryo metabolism
were a biologically reasonable 2.1–2.6, suggesting that the warm
temperature (21°C) did not impair metabolism (as would be expected if the
high temperature were at or above the critical thermal maximum). Finally, both
D. excentricus and T. diomedea have dispersive larvae and a
broad geographic distribution [D. excentricus, Alaska to Baja
California (Mooi, 1997);
T. diomedea, Alaska to Panama
(McDonald, 1983)], so it is
realistic to assume that embryos of both species are eurythermal.
Age of embryos
Metabolic rates of encapsulated embryos increased more than fourfold from
early cleavage to late veliger stages (Fig.
3), which is consistent with the increase in activity we observed
over development. All else being equal, such an increase would lead to a
fourfold higher metabolic density within egg-mass gel as embryos age from the
newly fertilized embryo to a pre-hatching veliger, leading to higher oxygen
deficits within older egg masses. This deficit could be partially offset if
internal embryos were developmentally delayed relative to outside embryos,
which has been observed in gelatinous egg masses of several species
(Strathmann and Strathmann,
1995). In this case, because of reduced metabolic demand at the
center of masses compared to the outside, gradients in older egg masses would
be smaller than predictions made based on chronological age alone.
Egg-mass size and density of embryos
In general, at a fixed embryo density, larger mass size should depress
central oxygen levels. Likewise, for a given mass size, oxygen levels should
be depressed by greater densities of embryos. We tested these two predictions
directly by measuring radial oxygen profiles of artificial egg masses in which
both parameters were independently altered. We found that even modest
increases in the diameter of cylindrical egg masses led to large declines in
central PO2 (for the same density and temperature)
(Fig. 6B). This result likely
explains why very few species package embryos into thick egg masses [e.g. like
Polinices (Booth,
1995)]; rather, like T. diomedea, they minimize diffusion
distances by producing very long coil- or ribbon-shaped masses
(Hurst, 1967). In addition,
higher densities of embryos within masses depressed internal oxygen
concentrations, as would be expected from the increased metabolic density at
higher concentrations of embryos. The resulting internal hypoxia would, in
natural masses, likely be disadvantageous because it would lead to delayed
development of internal embryos (Lee and
Strathmann, 1998).
Interactions between factors
The most general conclusions to emerge from our study are that, first,
temperature plays an important role in oxygen availability in gelatinous egg
masses; and, second, that interactions among factors are the rule, not the
exception. To take two examples from the present work, (i) high temperatures
depressed oxygen levels in egg masses when embryos were at later stages of
development, but not at early cleavage stages; and (ii) the effects of high
embryo density on oxygen profiles were more pronounced in larger masses than
in smaller ones.
This point about interactions should perhaps be obvious, because we superimposed effects of multiple factors onto a simple underlying mass balance involving just oxygen consumption and transport. Factors affecting either of these two basic components will undoubtedly interact with one another in determining internal oxygen concentrations. However, our data direct attention to the multiple pathways by which egg mass design could vary within and between species and how environmental temperatures might affect the range of possible egg mass structures. For example, the smallest artificial egg masses that we created never had low internal PO2 regardless of temperature or embryo density, but in larger masses both factors became important. Thus, at warm temperatures a large egg mass may experience strong constraints on the density of embryos while a small mass may not. Under very cold environmental conditions, in contrast, constraints on egg mass size and embryo density may be reduced by strongly depressed metabolic demand; therefore, egg masses in cold regions may evolve morphologies that would be nonfunctional in warmer regions.
Such interacting systems are good candidates for analysis by mathematical
modeling. Here we demonstrate how to integrate our data into a model framework
used by other workers (Harvey,
1928; Gerard, 1931;
Lee and Strathmann, 1998;
Woods, 1999), and others. The
model specifies relationships among egg mass size, shape, O2
diffusion coefficient, embryo density, and ambient and central O2
concentrations. One form of the equation
(Lee and Strathmann, 1998)
calculates the maximal size of an egg mass at which the central O2
concentration just goes to zero:
 | (2) |
), which is reasonable in gelatinous egg masses where
D is similar in gel and water and O2 capacitance or
solubility does not vary spatially within the egg mass (see
Lee and Strathmann, 1998).
For egg masses of T. diomedea we assumed a shape factor
(F) of 4, corresponding to an infinite cylinder. The actual value may
be somewhat lower given that the egg mass cord is sinuous and loops back on
itself. We have no direct measures of D, but use Lee and Strathmann's
estimate that D (at 20°C) in egg mass gel is 75% of D in
water (1.58x10–5 cm2 s–1)
(Lee and Strathmann, 1998).
Here we use this value for T. diomedea at 21°C and
1.24x10–5 cm2 s–1 for
D at 12°C (Denny,
1993). The surface concentration of oxygen,
CR, depends on flow regime
(Lee and Strathmann, 1998); in
low flows, boundary layers of water around the egg mass depress oxygen
concentrations to below ambient, whereas higher flows will give increasingly
negligible boundary layers. In nature, egg masses of T. diomedea are
subtidal and therefore likely experience moderate to high water flows.
Therefore here we ignore boundary layer effects and CR is
assumed to be the same as the ambient oxygen concentration, which itself is
assumed to be air saturated at the particular temperature under
consideration.
We have direct measures of both embryo metabolic rates
(Fig. 4) and central oxygen
levels (Fig. 5). Lee and
Strathmann (Lee and Strathmann,
1998) provide estimates of embryo density at the zygote stage for
T. diomedea (0.19 embryos µg–1 wet mass gel).
Generally in opisthobranch egg masses embryo density falls with age, because
capsules expand gradually. Although Lee and Strathmann did not measure
age-related changes in density in T. diomedea, several other species
studied showed declines of approximately 40% by the veliger stage and here we
assume a similar decline for T. diomedea. All parameter values are
summarized in Table 2. Eqn 2
calculates maximum egg mass radius (or thickness) (Rmax)
at which the central oxygen concentration just falls to zero. Calculated
values for Rmax (0.15–0.41 cm; see
Table 2) were slightly larger
than actual radii measured from calibrated digital photographs of the egg
masses (see Fig. 1)
(0.09–0.13 cm), but estimated and measured values were remarkably
similar overall. Two subtle patterns are worth mentioning. First, estimated
values of Rmax were 20–200% larger than measured egg
mass radii, suggesting a built-in safety margin. Such a margin may reflect
simply that egg masses function optimally (for embryos) when central oxygen
levels do not drop to zero (see Fig.
5). However, the apparent safety margin may also reflect factors
not incorporated directly into the model. For example, because the egg mass
cord loops back on itself, the effective diameter of an egg mass may be larger
than its single-cord diameter would indicate; or water flow velocities may be
low enough under some circumstances that boundary layers substantially reduce
CR. Second, calculated Rmax decreased
substantially during development because the greater metabolic rates of older
embryos more than offset falling embryo densities. Indeed, values of
Rmax late in development were most similar to measured egg
mass radii, suggesting that egg mass physiology and morphology are designed to
ensure adequate oxygen flux primarily at the veliger stage, near hatching.
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Effects of encapsulation
Two unexpected results of our study were that (1) encapsulation had a
strong effect on metabolic rate of embryos, and (2) the capsule wall did not
provide a barrier to oxygen diffusion; oxygen gradients across the capsule
wall were negligible at both cold and warm temperatures. From a practical
perspective, the latter finding simplifies the modeling of oxygen flux into
Tritonia egg masses because the walls of the egg capsules can be
effectively ignored. From a physiological perspective, our data suggest that
egg capsule membranes are surprisingly permeable to oxygen considering that
they are strong and durable enough to retain swimming embryos for >10 days.
How these capsules achieve such high gas permeability is not known and merits
closer ultrastructural examination.
The first finding, that encapsulation had a strong effect on metabolic rate of embryos, has implications for understanding the physiology and evolution of egg mass structure in general. Early in development, free embryos of T. diomedea had metabolic rates that were indistinguishable from encapsulated embryos; in advanced developmental stages, in contrast, free embryos had approximately double the metabolic rate of their encapsulated siblings. There are several possible explanations. First, embryos released from their capsules might be spurred to greater activity simply by stress. We feel this is unlikely, however, for a number of reasons: (i) embryos that had been removed from the mass were normal in appearance and behavior for at least 24 h after release, (ii) removal of embryos from their capsule required considerably less manipulation and disturbance than removal of capsules from the egg mass, and (iii) the increase in metabolic activity of late-stage embryos was measured close to the natural hatching time at a point when many larvae had already hatched from the older parts of the same mass.
A second, more likely possibility is that encapsulation depresses embryonic
metabolism through some mechanism other than oxygen deprivation (because
capsules did not retard oxygen flux). We envision three potential mechanisms.
First, the physical constraints of living within the egg capsule may restrict
embryo movement such that encapsulated embryos expend less energy on swimming,
velar retraction, foot movements, and other activities, than free larvae.
Second, while oxygen diffuses freely across the capsule membrane, other
substances such as metabolic wastes produced by embryos may not; embryonic
metabolism may be reduced by the concentration of these metabolites within the
capsule. We feel this second possibility is unlikely because oxygen
availability, rather than buildup of metabolic wastes, was the primary
limiting factor in embryonic development of a related opisthobranch
(Strathmann and Strathmann,
1995). Third, other compounds in the egg capsule fluid introduced
by the adult or by embryos themselves may act directly to reduce embryonic
metabolism; this phenomenon is found in egg capsules of some cephalopods, in
which the capsular fluid exhibits a `tranquillising' effect on embryonic
activity (Marthy et al.,
1976).
Low metabolic rates of encapsulated embryos likely have biological
significance if, at a given stage of development, encapsulated embryos utilize
less oxygen and less energy than would free-living larvae. Lowered per-embryo
oxygen consumption would result in lower oxygen demand throughout an egg mass,
thus reducing the need for energetically expensive gel to provide spacing
between embryos. Likewise, free-living larvae fuel metabolism and growth by
feeding on exogenous sources in the plankton; much of their energy expenditure
may be related to their need to swim, feed and grow. In contrast, encapsulated
embryos likely rely largely on endogenous reserves or intracapsular nutrition
provided at oviposition by the parent (e.g.
Goddard, 1991;
Moran, 1999;
Wilson, 2002). By reducing
stage-specific metabolic rates, species may produce hatched larvae or
juveniles of higher energetic content than if embryos were metabolizing at
high free-larval rates throughout encapsulated development. Indeed, if
metabolic depression is a common feature of encapsulated embryos, this
phenomenon may be an important physiological correlate of evolutionary changes
in developmental mode.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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