Fertilization ecology of egg coats: physical versus chemical contributions to fertilization success of free-spawned eggs
University of North Carolina, Department of Biology, CB 3280, Chapel Hill, NC 27599, USA
E-mail: podolsky{at}unc.edu
Accepted 19 March 2002
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Summary |
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Key words: jelly coat, target size, egg size, sperm swimming, fertilization success, energy investment, broadcast-spawning, sand dollar, Dendraster excentricus
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Introduction |
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In addition to mediating chemical interactions, however, accessory
structures can also alter the egg as a physical target for sperm. Jelly coats,
follicle cells and egg hulls can double or triple the effective diameter of a
free-spawned egg (Strathmann,
1987), substantially increasing cross-sectional (`target') area
while also altering egg buoyancy. The potential importance of egg size for
fertilization success was highlighted by Levitan
(1993
), who reasoned that
under sperm limitation, larger eggs would be fertilized with greater
probability because they present a larger target for sperm. As an alternative
to the conventional view of accessory structures as primarily chemical in
their action, here I test the hypothesis that accessory structures around
free-spawned eggs can enhance fertilization success through a change in
physical target size. This hypothesis has received speculation in previous
studies (Rothschild and Swann,
1951
; Vogel et al.,
1982
; Epel, 1991
;
Podolsky and Strathmann,
1996
), and elsewhere I showed that overall target size is a good
absolute predictor of the probability of fertilization
(Podolsky, 2001
). Previous
studies have not, however, evaluated the importance of physical relative to
other (e.g. chemical) effects of accessory structures on fertilization
success.
Given several possible roles in fertilization, accessory structures could evolve in response to more than one factor. Using a combination of approaches, however, one can estimate the current importance to fertilization of a physical change in target size relative to other effects. I first use removal experiments to measure the effect of jelly coat presence on fertilization. Because this treatment potentially confounds the effects of physical target size with other effects, I then use a model of fertilization kinetics to predict the effect on fertilization of a size change equal to jelly coat removal. With the experimental data and the model predictions, I address the following questions. (1) What is the effect of jelly coat removal on fertilization rate? (2) How much of this effect can be accounted for by a simple change in target size? (3) Which parameters that could influence collision (e.g. sperm swimming speed) are influenced by the presence of jelly? (4) How does the effect of jelly on egg buoyancy influence fertilization? (5) What is the relative energetic cost of jelly as a means of enlarging target size? (6) What accounts for the remarkably large size of some accessory structures?
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Materials and methods |
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The echinoid jelly coat is a glycoprotein-polysaccharide complex
(Suzuki, 1989). In D.
excentricus, the jelly coat is a firm, transparent, smoothly spherical
shell with embedded pigment cells (Burke
and Bouland, 1989
; Podolsky,
2001
). For size measurements, eggs were viewed in a suspension of
Sumi ink, which is visibly excluded by the clear jelly coat
(Schroeder, 1980
). The coat is
compressed inside the gonad but expands to full size within moments of contact
with sea water (Podolsky,
2001
). Fertilization is clearly evidenced by inflation of the
vitelline envelope below the jelly coat and by cleavage. Embryos hatch within
1 day at ambient sea water temperatures, but jelly can disperse earlier
(Strathmann, 1987
).
Following convention and to avoid confusion, I use the term `ovum' to refer strictly to the reproductive cell not including the jelly coat, and `egg' to refer to an ovum with or without a jelly coat.
Removal of jelly coats
To maintain distinct treatments, these experiments require a species with
jelly coats that persist on eggs unless actively removed. Attempts to remove
jelly coats just after spawning by sieving, centrifugation and shaking were
not completely successful and resulted in some egg damage. I therefore used a
standard brief exposure to mildly acidified sea water to hydrolyze the coat
(e.g. Harvey, 1956;
Schroeder, 1986
). For each
fertilization trial, I removed jelly coats from half a cohort of freshly
spawned eggs. The sea water was adjusted to approximately pH 5-5.5 by adding
drops of a weak HCl solution to the well-stirred egg suspension. Presence of
the jelly coat on a sample of eggs was checked each minute using Sumi ink.
After jelly coats had dispersed, I thoroughly washed both batches twice in
filtered sea water, and standardized egg concentrations. Normal pH was
restored by washing because jelly-free ova became unpredictably sticky when
readjusting pH chemically. Ten trials were conducted using this removal
method.
To test whether experimental results were an artifact of the acidification
method, for five additional trials I used a second method of jelly coat
removal. Each half-cohort was poured gently through Nitex mesh (153 µm
diameter) after brief washing in a Ca2+-free, neutral pH isotonic
buffer (500 mmoll-1 NaCl, 27 mmoll-1 KCl, 2
mmoll-1 EDTA, pH 7.8 at 23°C) that weakens the coat and
facilitates its mechanical removal
(Salmon, 1982;
Suprenant, 1986
). Eggs were
otherwise washed and handled as above.
Fertilization assays
Standard assays (Vogel et al.,
1982; Mita et al.,
1984
; Levitan et al.,
1991
; Styan, 1998
)
were used to measure fertilization kinetics (proportion fertilized as a
function of sperm concentration) for eggs with and without jelly coats. Assays
were done in 20 ml glass tubes held at 13°C. Each trial included 32
experimental tubes (8 sperm concentrations x2 treatments x2
replicates). Final sperm concentrations ranged from 10-2 to
104 µl-1 in powers of 10. Eggs from the two
treatments (`intact' and `coat-free') were added to the top of tubes
immediately after sperm, to produce final egg concentrations of 0.05
µl-1 in a total volume of 10ml. Fertilization was blocked after
15 min by addition of an equal volume of 0.5 moll-1 KCl
(Schuel, 1984
). 15 min was
more than adequate, based on time-course studies
(Hagström, 1956b
;
Hagström and Markman,
1957
), for sperm that had contacted the jelly coat to penetrate to
the egg surface. I counted under a compound microscope the proportion of eggs
fertilized in a sample of 150-200 eggs from each vial; replicates were
averaged.
Fertilization kinetics model
I used a standard fertilization kinetics model
(Vogel et al., 1982) to
predict the effect of a change in target size on fertilization that would
result from changes in spermegg collision. I did not include
modifications to the model by Styan
(1998
) because (1) at sperm
concentrations used in these experiments, there is little evidence of
polyspermy for intact eggs; the vast majority undergo normal cleavage and
development (consistent with observations by
Schuel and Schuel, 1981
;
Nuccitelli and Grey, 1984
;
Dale, 1985
), and (2) since
jelly removal could influence properties of the egg that control polyspermy
(Hagström, 1956a
;
Schuel and Schuel, 1981
),
evaluation of the importance of target size per se required a
performance measure that was equivalent between treatments (e.g. lifting of
the fertilization envelope, diagnostic of fertilization) rather than one that
could confound fertilization frequency and polyspermy rate (e.g. normal
development).
The Vogel et al. (1982)
model is based on two assumptions, that sperm swimming direction is random and
that sperm attach permanently to the first egg encountered. Vogel et al.
(1982
) and Levitan et al.
(1991
) tested the model for
echinoid species and found good correspondence between predictions and data
given variation in several parameters. The model was subsequently used to
predict effects on fertilization of variation in gamete characters (Levitan,
1993
,
1998
;
Podolsky and Strathmann, 1996
;
Styan, 1998
). Here I use the
model to predict the relative change in fertilization success given a size
change equivalent to removing (or adding) a jelly coat.
The model predicts the proportion of eggs fertilized () given
a set of initial conditions:
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For each trial and treatment, I used the fitted ß to estimate ß/ß0 and computed the log-transformed sperm concentration at which 50% fertilization would be achieved (logFC50). Within each trial I then determined logFC50 using the ratio ß/ß0 characteristic of intact eggs, but adjusting target size to that of coat-free eggs. This calculation represents the expected effect of reducing target size while holding constant all size-independent parameters, including ß/ß0. I used logFC50 to estimate the proportion of the difference in fertilization between intact and coat-free eggs that is predicted by the change in target size alone. Given the exponential structure of the model, log-transformed values are on an appropriate scale for comparison. To demonstrate this, an equivalent analysis can be carried out using ß/ß0 for coat-free eggs and adjusting the target size to match that of intact eggs. These reciprocal procedures produce identical results, as they should, only when FC50 values are compared on the logarithmic scale.
Parameter estimates
The model includes three measured parameters (egg size with and without the
jelly coat, sperm swimming speed and sperm half-life), and three that were
controlled during experiments (sperm concentration, egg concentration and
sperm-egg contact time).
(1) Egg size
Using an ocular micrometer on a compound microscope, I measured 10 ova and
fully expanded jelly coats for each of six females. I used the grand mean for
this parameter in the model (average CV for egg size was approximately 3.5%
both within and among females; Podolsky,
1995).
(2) Sperm speed
Sperm diluted in filtered sea water (104µl-1) were
placed in a covered chamber of approximately 1.2 mm depth on a
temperature-controlled (13°C) stage. Sperm were videotaped at an optical
plane half way through the chamber (0.6 mm, or approximately 67 sperm body
lengths, from the chamber walls) to minimize wall effects
(Winet, 1973;
Gee and Zimmer-Faust, 1997
).
Recordings were done in the time interval 8-10 min after dilution. A time-date
generator recorded time to the nearest 0.01 s (at 30 frames s-1),
and sperm paths were later digitized. I measured 20 sperm per treatment,
scoring only those that had covered, in a typical helical path, at least half
of the field (>300 µm) while in focus.
In addition to measuring sperm speed at a standardized concentration in
filtered sea water, I tested for effects of two factors that varied in
experiments and could have influenced sperm speed. (a) For echinoid sperm,
respiration is inversely related to concentration (the `respiratory dilution
effect' or RDE; Chia and Bickell,
1983), which could alter swimming speed. To test for this effect,
I measured speeds at four concentrations (103.5, 104,
104.5 and 105 sperm µl-1). Although these
concentrations are at the high end of the range used in fertilization assays,
the RDE should be apparent in this range
(Chia and Bickell, 1983
) and
lower concentrations did not provide enough sperm for analysis. (b) Although
the sperm of D. excentricus and most echinoids do not show chemotaxis
(Miller, 1985
), exposure to
sea water that previously held eggs (`egg-water') is known in other species to
increase sperm activity and longevity
(Suzuki, 1989
;
Bolton and Havenhand, 1996
).
For two males at two temperatures (10 and 20.5°C) I compared swimming
speeds in filtered sea water and in `egg-water', which was prepared by
occasionally stirring a suspension of fresh eggs held at 4°C in filtered
sea water for 3 h before the eggs were removed.
(3) Sperm longevity/contact time
Sperm `half-life' is the time after dilution when fertilization drops to
50% of its initial value. To estimate half-life, for seven males I measured
fertilization at 5-8 time points after sperm dilution at the upper six
concentrations used in fertilization assays. Diluted sperm were held at
ambient water temperatures (11-13°C). Time points used depended on sperm
concentration, ranging from 0 to 6 h for the most dilute and from 0 to 24 h
for the most concentrated. Sperm were diluted in egg-water, because
preliminary measurements suggested that exposure could reduce longevity
(Bolton and Havenhand, 1996)
and thereby provide a conservative estimate of half-life. In all trials, sperm
were allowed contact with eggs for 15 min before KCl addition. For each sperm
concentration and replicate, I performed a linear regression of log(% eggs
fertilized + 1) on time after dilution, and used the regression equation to
calculate a half-life for each concentration
(Levitan, 1993
).
Effect of the jelly coat on egg sinking
The fertilization kinetics model of Vogel et al.
(1982) treats eggs as if they
were stationary, using sperm speed alone to estimate rates of sperm-egg
collision. In reality, eggs of most species are not neutrally buoyant, and
their motion could contribute to the collision coefficient ß0.
In addition, an egg sinking more rapidly through a sperm cloud would encounter
more sperm per unit time, but would be in the presence of sperm for a shorter
time (i.e. contact time,
).
In species that lack chemotaxis, a sinking egg can be considered to move
through an isotropic field of non-directional swimming sperm, as previously
modeled for planktonic predators and prey, respectively
(Gerritsen, 1980). In the
original model, the rate coefficient of encounter (Ec,
here relabelled
) depended on three
variables: the swimming velocities of predator and prey
(uf and usl, denoting the faster and
slower) and the radius of detection (Rd):
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For eggs sinking through a finite cloud of sperm, sperm-egg contact time
(*) will be related directly to cloud diameter
(dc) and inversely to egg sinking speed
(ue), or
*=dc/ue. In laboratory
assays sperm are initially distributed uniformly, eggs are released at the top
of the cloud, and dc is fixed by container depth.
Typically, eggs are allowed to settle and `contact time' is assumed to be the
length of time before fertilization is artificially stopped. In practice, for
part of this time eggs will be concentrated at the container bottom, where
fertilization rate is predicted to be strongly depressed by a high effective
egg concentration (Vogel et al.,
1982
). Based on this prediction, I assume that fertilization at
the bottom of the container is negligible relative to during the sinking
period. The original model (no effect of egg sinking) and the model with this
assumption (maximum effect) represent the two extremes of the potential
contribution of egg sinking to collision rate under these conditions. The
parameters used are summarized in Table
1.
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To estimate the sinking speed of eggs with and without jelly coats, I
assumed that eggs were spherical and used Stokes' equation
(Vogel, 1981):
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Organic cost of ovum and jelly
To estimate organic investment I measured ash-free dry mass (AFDM) of eggs
with and without jelly coats (Crisp,
1984; Omori and Ikeda,
1984
; Jaeckle,
1995
). AFDM of jelly was calculated by subtraction. Using AFDM to
estimate total organic cost assumes that material costs reflect costs of
synthesis, and ignores the differing energetic values of lipids, carbohydrates
and proteins. These simplifications produce a conservative estimate of the
ratio of ovum to jelly AFDM, because costs of synthesis and energetic values
are greater for the major constituents of ova (protein and lipid;
Turner and Lawrence, 1979
)
than of jelly (carbohydrate and protein;
Paine, 1971
;
Crisp, 1984
;
Suzuki, 1989
).
For each of eight females, I divided a cohort of fresh eggs and desalted
both halves in isotonic (3.5%) ammonium formate (NH4COOH). After
exposure, jelly coats could be removed from one sample by pouring the eggs
through a mesh screen (153 µm). Eggs were allowed to settle and then
resuspended in fresh buffer. I counted ten well-mixed subsamples to estimate
the remaining number of eggs. I then collected eggs on a pre-ashed and
pre-weighed GF-C filter under low vacuum
(Omori and Ikeda, 1984). The
filtered sample was dried to constant mass at 60°C for 24h, weighed, ashed
at 500°C for 12h, and reweighed. The difference in filter masses before
and after ashing was divided by egg number to calculate AFDM per egg. Control
filters that had been treated with ammonium formate were handled and measured
in the same way.
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Results |
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Model parameters
Among six females, egg diameters (mean ± S.E.M.) with and without
the jelly coat were 311.9±4.3 µm and 128.8±1.7 µm,
respectively. Considering that eggs are nearly spherical, average volumes of
the jelly coat and ovum were approximately 1.48x10-1 and
1.12x10-2 µl, respectively, a ratio of 13.2.
Among seven males, average sperm swimming speed at 13°C was
195.0±9.8 µm s-1. Swimming speed did not vary as a
function of sperm concentration over the 1.5 orders of magnitude tested
(F3,159=0.28, P=0.84; see also
Rothschild and Swann, 1950),
nor as a function of whether sperm had been diluted in egg-water or filtered
sea water (F1,8=0.438, P=0.53;
Podolsky, 1995
). Swimming
speed varied significantly among males (F4,159=3.42,
P=0.01; see also Gee and
Zimmer-Faust, 1997
).
As expected, sperm half-life increased as a function of sperm
concentration, but was high at all concentrations
(Fig. 3). Because average
half-life even at the lowest sperm concentration was more than 10 times longer
than the imposed contact time of 15 min, the parameter was set to 15 min
(Vogel et al., 1982
).
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Model fit and prediction of the effects of size change
For each trial and treatment, I fitted the kinetics model (Equation 1) to
fertilization data using the parameter values summarized in
Table 1. The model accounted
for 92-99% of the variation in fertilization percentage. The fitted parameter
ß (Table 2) was then used
for each trial and treatment to plot fertilization kinetics as a function of
sperm concentration. Using these curves, I calculated the log-transformed
sperm concentration that would result in 50% fertilization
(Table 2,
logFC50).
I then used the model to predict the effect on fertilization of a change in target size equal to removal of the jelly coat. For each trial, I generated a prediction curve by holding constant all size-independent parameters (egg concentration, contact time, sperm speed and ß/ß0 for intact eggs) and setting target size equal to the value for coat-free eggs. For each prediction curve I calculated an expected logFC50 (Table 3), and the proportion of the difference in observed logFC50 between intact and coat-free eggs that was predicted by the change in target size. On average, 54% of the difference between treatments could be accounted for by a simple change in target size alone [95 % CI=(44.4,63.7); Table 3].
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Organic content and specific gravity of ovum and jelly
Measurements of AFDM for eight females gave values of 0.260±0.025
and 0.217±0.021 µg (mean±S.E.M.) for eggs with and without
jelly coats, respectively. The ovum thus constituted 83.6±9.1 % of the
AFDM per egg; given differences in volume, the ovum has 67.3 times the organic
density of jelly. As a result, the addition of an average jelly coat enlarges
volume approximately 14-fold and target area almost sixfold, but increases
organic cost per egg only 1.19-fold.
As described earlier, the latter value is a maximum estimate, because the
major components of the ovum (protein and lipid) have higher energy content
than those of jelly (protein and carbohydrate). To derive a minimum estimate,
I assumed that jelly was all carbohydrate (lowest in energetic density) and
used available data on the composition of ova. For nine species of echinoderms
with eggs of a size similar to those of D. excentricus, the average
percentage compositions of protein, lipid and carbohydrate were approximately
63 %, 31 % and 6 % (Turner and Lawrence,
1979), which had average energetic contents of 23.7, 39.6 and 17.2
kJ g-1, respectively (Crisp,
1984
). According to these figures, the jelly coat may increase
organic investment per propagule by as little as 1.12-fold. Jelly therefore
comprises from 10 to 16 % of the organic material cost of an egg.
Intact eggs and coat-free eggs sank through Percollsea water mixtures below specific gravities of 1.025 and 1.054, respectively. By subtraction, taking into account relative volumes, the specific gravity of jelly is around 1.023, close to that of sea water (1.0225 at 13 °C).
Incorporation of egg sinking into the model
To incorporate egg sinking, I considered the speed at which eggs moved
through the sperm cloud as well as the total time they remained suspended in
the cloud (Vogel et al.,
1982). The addition of a jelly coat increases egg size, but
reduces egg density; these changes have opposing effects on sinking speed
(Equation 3). The calculated net effect of adding a coat is a decrease in
relative sinking speed, from 223 to 104 µm s-1. In theory this
decrease would continue up to more than twice the observed size of jelly
coats, at which point the effect of size would overcome the effect of density
and sinking would begin again to increase
(Podolsky, 1995
).
The collision coefficient increases
only slightly when egg sinking is incorporated into the model
(Fig. 4A). However, the total
volume of water `cleared' of sperm by the passing egg (the product
) increases steadily
with an increase in jelly coat size (Fig.
4B). Depending on the water conditions, an intact egg may
therefore have an additional benefit to spermegg collision of remaining
suspended longer in a sperm cloud. To examine the potential contribution of
egg sinking to assay results, I reanalyzed the fertilization data to produce
new predicted logFC50 values, taking into account
treatment differences in
and
(Table
3). With egg sinking included, 73.3 % of the difference between
intact and coat-free treatments was predicted by changes in size and density
combined [95 % CI=(60.0,86.6); Table
3].
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Discussion |
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Which aspects of jelly coat chemistry are likely to account for effects of
jelly removal that were not related to size or density? The potential roles of
chemical interaction before and during spermegg contact are
well-established (SeGall and Lennarz,
1981; Suzuki,
1989
; Vacquier and Moy,
1997
), but the importance of any particular role can vary among
species. In some, specific jelly components induce the sperm acrosome reaction
(Vacquier and Moy, 1997
) and
may therefore be lost in the process of jelly removal, while in others, the
role of jelly is less directly causal
(Aketa and Ohta, 1977
).
Similarly, although sperm-activating peptides (SAPs) associated with jelly can
stimulate sperm activity under certain conditions, their specific action and
overall benefits to fertilization remain uncertain. According to one leading
hypothesis, SAPs may stimulate sperm primarily to counteract the negative
effects on sperm of jelly coat acidity, which is presumably maintained for a
different function (Suzuki,
1989
). In experiments presented here, dilution in egg-water had no
significant effect on sperm swimming, indicating that SAP-enhanced swimming
speed was probably not a factor in fertilization differences between intact
and coat-free eggs.
Given the evident physical and chemical benefits of a jelly coat for
spermegg encounter, it is worth noting some apparently conflicting
reports in the literature. Some studies have similarly reported a `cost' to
jelly removal (Lillie, 1915;
Tyler, 1941
;
Rothschild and Swann, 1951
;
McLaughlin and Humphries,
1978
), some report little effect
(Loeb, 1915
;
Farley and Levitan, 2001
), and
at least one investigator promoted a consistent `benefit' (Hagström,
1956b
,
1959
;
Hagström and Markman,
1957
). In a brief review of this history, Vacquier et al.
(1979
) concluded that variable
results were due to differences in methods (e.g. for jelly coat removal) and
species biology (e.g. portion of jelly removed, role of jelly coat chemistry).
However, it is equally important to recognize that variable conclusions have
been based on measurements of different phenomena under different conditions.
Specifically, studies that reported benefits (e.g.
Hagström, 1956b
;
Vacquier et al., 1979
) were
concerned with the short-term (i.e. seconds) kinetics of sperm attachment or
fertilization at sperm-saturation. Under these conditions, the time course of
spermegg collision may be short relative to penetration and attachment,
and removing a jelly barrier could increase such rates. In contrast, studies
that found costs to jelly removal measured the total proportion fertilized
under sperm-limited conditions conditions where the time course of
collision regulates fertilization success
(Vogel et al., 1982
), and a
smaller target size therefore reduces the proportion fertilized. Awareness of
this distinction is critical to interpreting results. For example, if sperm
are killed within a time period shorter than needed to penetrate the jelly
coat as typically done in `rate' measurements (for estimates of the
time course of jelly penetration, see
Rothschild and Swann, 1951
;
Hagström, 1956b
,
1959
;
Hagström and Markman,
1957
; Dale, 1985
)
then studies could confound time-dependent `rates' with ultimate
`proportions' and conclude that jelly is insignificant or even detrimental to
fertilization success (Hagström,
1956b
; Farley and Levitan,
2001
).
Size and cost of accessory structures
What accounts for the strikingly large size of many accessory structures
(Strathmann, 1987)? Given
substantial differences in organic density between ovum and jelly, an
extracellular coat provides an efficient means of extending target size far
beyond the ovum size that may be optimal for larval development
(Podolsky, 2001
). Upper limits
on coat size could be set by diminishing returns of extra-embryonic investment
(Lee and Strathmann, 1998
) or
by costs associated with increased polyspermy at large target size
(Styan, 1998
; but see below).
Beyond its efficiency, jelly may also be more effective for target
enhancement, because an ovum the size of an intact egg would also sink 12
times faster. Calculations showed that eggs sinking faster through a sperm
cloud contact more sperm per unit time but a smaller total number of sperm.
Given the benefits of prolonged suspension, it is probably not a coincidence
that large accessory structures can bring eggs close to neutral buoyancy.
Buckland-Nicks (1993
) suggested
a similar `parachuting' function for the hulls of chiton eggs.
While this study documents one consequence of the presence of a jelly coat,
optimal coat size could be influenced by other functional requirements.
Although chemical effects on sperm could select for large coat size, the
relationship between jelly volume and chemical sperm activation is unknown,
and residual jelly remaining after coat removal can be sufficient for the
acrosome reaction (Vacquier et al.,
1979). On the other hand, accessory structures have been
implicated in several processes where size could be important, including the
prevention of both polyspermy (Lambert and
Lambert, 1981
; Patricolo and
Villa, 1992
) and hybridization
(De Santis and Pinto, 1991
;
Vilela-Silva et al., 2002
).
Coat thickness, for example, could help to regulate the arrival of sperm at
the egg surface, through chemically mediated agglutination or physical delays
in penetration (Schuel, 1984
).
Jelly coats might thereby benefit fertilization at both low and high sperm
concentrations, by increasing sperm collision and regulating sperm passage,
respectively (R. D. Podolsky, in preparation). Finally, jelly coats have also
been cited for a role in egg or embryo protection
(Szollosi, 1964
;
Chia and Atwood, 1982
),
including the stabilization of eggs under extreme shear forces as spawn is
released through the echinoderm gonoduct
(Thomas and Bolton, 1999
;
Thomas et al., 1999
). However,
because this latter benefit accrues before the coat has expanded
(Podolsky, 2001
) it is
unlikely to explain large variation in coat size.
Among the structural materials that could enlarge target size (e.g. jelly
coats, hulls, follicle cells), jelly may have an additional advantage: because
most size expansion occurs after release
(Podolsky, 2001), jelly is
especially suitable for packing inside the female test. In D.
excentricus, a jelly coat compressed to just 50% of its full thickness
triples the space available for egg storage relative to a rigid structure of
the same final size. In similar fashion, adults of chiton species that brood
young tend to be smaller than broadcast-spawning relatives, and their eggs
tend to have more reduced and flattened hulls
(Eernisse, 1988
). This pattern
is consistent with the hypothesis that limitations on storage space
(Buckland-Nicks, 1993
), and
modes of reproduction where target size and buoyancy have a weaker effect on
sperm egg-encounter, will favor the evolution of smaller egg accessory
structures.
Generalizations of this analysis
The strength of conclusions about the magnitude of the physical role of
accessory structures depends on at least three assumptions. First, I assume
that the fertilization kinetics model (Equation 1) predicts the effect of a
physical change in target size, as summarized in the collision parameter
ß0. Information for D. excentricus and other
echinoids (Vogel et al., 1982)
supports both basic assumptions of the model (random swimming and permanent
attachment): sperm of D. excentricus and most echinoids do not show
chemotaxis (Miller, 1985
), and
the acrosome reaction following spermegg contact incapacitates sperm,
regardless of whether they attach permanently
(Vacquier, 1979
). In species
without chemotaxis, physical target size is a good absolute predictor of both
spermegg collision (Farley and
Levitan, 2001
) and the probability of fertilization
(Podolsky, 2001
) under
sperm-limited conditions. Clearly, the definition of `target size' would be
complicated by chemotaxis, because attractants can change the effective
distance at which a sperm `contacts' the target
(Jantzen et al., 2001
). An
additional contribution of chemotaxis would support the general conclusion
that accessory features, whether physical or chemical, provide an economical
alternative to investment in the ovum for increasing target size.
Second, I assume that jelly coat removal did not damage ova. Fertilization
reached 100% under sperm saturation in all trials, indicating that all eggs
could potentially be fertilized. The pH used for coat removal in trials was
higher than those reported to cause damage (pH=3.5-4.5;
Hagström, 1956b;
Vacquier et al., 1979
) and is
in a range reported to avoid damage (Loeb,
1915
) or actually to enhance fertilization rate
(Hagström, 1959
;
Vacquier et al., 1979
).
Indistinguishable results using an independent removal method indicate that
mild acidification was not the cause of reduced fertilization at intermediate
sperm concentrations, a confirmation paralleled in earlier work
(Hagström and Markman,
1957
). Given the need for a resilient coat during experiments, in
practice any removal method runs a risk of damaging some ova. Jelly
coats have been removed without apparent ill effect by mechanical shear forces
associated with several thousand revolutions in a beaker
(Farley and Levitan, 2001
);
the alternative method used here was judged to be the gentlest available. In
any case, if some damage to ova occurred with any method, then the proportion
of the total effect that the model attributed to physical target size would be
reduced. That is, my estimates of the importance of egg size and density
relative to jelly coat chemistry would then be conservative.
Third, I assume that results from laboratory experiments are relevant to
natural conditions. Application of these results depends on understanding
particular patterns of flow in the field and how they affect gamete movement
(Young et al., 1992;
Levitan, 1995
). Dendraster
excentricus inhabits a wide range of habitats, from intertidal to shallow
subtidal and from calm to high current or surge-influenced
(Merril and Hobson, 1970
;
Telford, 1983
;
Emlet, 1986
). Particular
depth, current, and wave conditions will determine the distribution of eggs
after spawning and, therefore, the importance of factors like egg sinking. As
demonstrated here, differences in egg suspension time even over short vertical
distances can have a big influence on the probability of spermegg
collision. Even for organisms that occur and potentially spawn in higher
energy habitats (Denny and Shibata,
1989
; Mead and Denny,
1995
), target size is likely to play a role in spermegg
collision regardless of whether gametes move through self-propulsion, gravity
or water motion. The primary goal of this analysis has been to estimate the
relative importance of physical versus chemical contributions of the
jelly coat. Given that water motion could interfere more with chemical cues
used by sperm to encounter eggs, the relative importance of physical
attributes could be greater under turbulent conditions.
Responses to environmental variation at the gamete stage
Renewed interest in the fertilization ecology of marine invertebrates has
focused mainly on the importance of adult traits: body size, aggregation,
synchrony, habitat location and population size and density
(Pennington, 1985;
Yund, 1990
;
Levitan, 1991
;
Denny et al., 1992
;
Levitan et al., 1992
;
Oliver and Babcock, 1992
;
Babcock et al., 1994
;
Levitan and Young, 1995
;
Atkinson and Yund, 1996
). This
study adds to growing evidence for gamete traits as responses to variation in
fertilization conditions. In addition to exploring the ecological role of
sperm chemotaxis (Jantzen et al.,
2001
), recent work has highlighted several physical traits,
including: sperm size, morphology and energy storage in low-energy deep sea
environments (Eckelbarger,
1994
); egg size (Levitan,
1993
) and shape (Podolsky,
1995
) and their effects on sperm egg encounter; egg accessory
structures and their role in protecting eggs from shear during and after
spawning (Mead and Denny,
1995
; Thomas et al.,
1999
) and in guiding sperm to the egg surface
(Buckland-Nicks, 1993
);
positive egg buoyancy and the compression of gamete interactions into two
dimensions at the airwater interface
(Oliver and Willis, 1987
); and
spawn viscosity and its adjustment in response to flow conditions (Thomas,
1994a
,b
;
Meidel and Yund, 2001
). These
examples, involving properties of sperm, eggs and aggregate spawned material,
illustrate the important relationship between gamete structural traits and
physical processes in understanding the fertilization ecology of
broadcast-spawners.
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Acknowledgments |
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References |
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