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Marcie Levine
ABSTRAC
Eggs from solitary and colonial tunicates were fed to adult Ascidia
ceratodes and Ciona intestinalis. Those eggs which are normally spawned,
A. ceratodes and C. intestinalis, were recovered unharmed from fecal
pellets and embryonic development observed to continue. Dechorionated
eggs and viable tadpoles, however, were digested. Furthermore, eggs which
are invariably brooded, those of Clavelina huntsmani and Botryllus sp.,
were not recovered intact. Fertilized Stronglyocentrotus purpuratus eggs
were also able to survive ingestion by the adult A. ceratodes. A correlation
was noted between protease-insensitivity of the chorion and fertilization
membrane and the resistance to digestion. It is suggested that the proteo¬
lytic insensitivity of the extra-embryonic investments serves to protect
eggs normally spawned from digestion by filter-feeding adult tunicates.
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INTRODUCTION
Many marine organisms produce eggs covered by tough membranes and
coats. Not only does this packaging make the eggs difficult to study, but
removal of these layers is accomplished only by strenuous means (Berg,
1967). This has long been a point of interest among invertebrate embryol¬
ogists, generating some rather creative solutions, such as digestion by
crab stomach juice (Berrill, 1932) and popping eggs out of a pipette and
lancing their investments with a tungsten needle (Muneoka, 1979).
This study centers on ascidian eggs, which are unique in the
intricacies of their extra-embryonic membranes. Two species of echinoderm
eggs were also briefly examined. The tunicate egg (Fig. 1) is covered by
vacuolated cells, known as test cells, which float more or less freely in
the perivitelline space. They are surrounded by a non-cellular envelope,
the chorion, upon which is attached another set of highly vacuolated cells,
the follicle cells (Reverberi, 1971). Many functions have been suggested
for these extra-cellular packagings. The chorion, the focus of this study,
is often noted to be the site of specific sperm recognition and is thought
to be responsible for preventing polyspermy, self-fertility, and inter¬
specific fertilization (Rosati and DeSantis, 1978). The chorion is also
considered to be the binding site of the spermatozoa (DeSantis et al., 1979).
although the presence of an acrosome in the ascidian spermatozoon has been
a matter of discussion (Kubo et al., 1978; Woollacott, 1977).
This paper demonstrates a further ecological function of this
extra-embryonic investment: that of protection of the egg against digestion
by filter-feeders. Eggs of both solitarytunicates, which are normally
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spawned, and colonial tunicates, whose eggs are brooded, are examined.
along with sea urchin and starfish eggs. Given the limited number of
eggs studied, it appears that those eggs which are spawned correlate with
a much greater degree of proteolytic insensitivity. It is suggested that
this protease insensitivity of the extra-embryonic chorion confers the
ability to survive ingestion by the adult.
MATERIALS AND METHODS
Handling of gametes:
Adult tunicates were obtained during April and May from the inter¬
tidal region at Mussel Point and from the fouling communities of the
Monterey Marina in Pacific Grove, Ca., and kept in running sea water at
100
12 . Gametes were removed with a pasteur pipette from the gonaducts of
the solitary ascidians, Ascidia ceratodes and Ciona intestinalis, and from
the atrial chamber of the colonial ones, Clavelina huntsmani and Botryllus,
sp. Eggs were rinsed three times in paper filtered sea water and kept in
sea water at 12
; undiluted sperm of the solitary tunicates were used
fresh or stored for several hours at 40.
The shedding of Stronglyocentrotus purpuratus gametes was induced
by an intracoelomic injection of 0.5M KCl. The eggs were stored in
filtered sea water at 12 and the undiluted sperm kept at 40
Immature oocytes of Pisaster ochraceus were obtained by dissecting
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out the ovary, which was then minced and filtered through a Nitex screen.
This preparation was washed three times by centrifugation in Ca-free
artificial sea water, and the oocytes were resuspended in filtered sea
0
water and stored at 12
Removal of follicle cells:
The follicle cells of A. ceratodes and C. intestinalis were
removed mechanically by drawing the eggs up into a syringe and forcing
them through a tip of small enough aperture to approximate the diameter
of the chorion. A standard 31-gauge needle was successful.
Removal of the chorion:
Eggs were dechorionated enzymatically, according to a procedure
developed by Dr. Charles Lambert of California State University at
Fullerton, using crude porcine pancreatic lipase (Sigma) (lmg/ml) and
10 mM dithiothreitol (Sigma) at 16 and pH 9.1. Due to the fragility
of the dechorionated eggs, fresh eggs were placed in a supporting cylin¬
drical tube, whose bottom had been removed and lined with a 50 u Nitex
mesh. The enzyme-egg suspension was stirred constantly with a plastic
paddle until observation under a light microscope demonstrated that
753 of the eggs were free of their extracellular investments (about 3
hours). The dechorionated eggs were then washed three times in filtered
sea water, using the Nitex mesh support.
Feeding experiments:
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6.
Eggs from the ascidians, A. ceratodes and C. intestinalis, were
fertilized with fresh sperm in pH 9 sea water (C. Lambert, personal
communication), and fertilization was assessed 2-4 hours later by evidence
of cleavage. A series of mechanically defolliculated and enzymatically
dechorionated eggs and tadpoles were also prepared, as well as P. ochrageus
and fertilized and unfertilized S. purpuratus eggs.
In order to assess digestion of eggs by the filter-feeding adult.
mature A. ceratodes and C. intestinalis were placed in a glass bowl, so
that uningested eggs could be easily recovered. A relatively dense sus¬
pension of eggs (300-350/ml sea water) and carmine, a biological marker.
was prepared and the number of eggs in 1 ml aliquots were counted under a
light microscope. A pasteur pipette was carefully placed just outside
the oral siphon of the adult and known aliquots were added dropwise to
the incoming feeding current. Animals were then put back into running
sea water to insure a constant food supply and the uningested eggs were
immediately recovered from the bottom of the bowl and the quantity noted.
The carmine labelled fecal pellets were recovered 6-26 hours later, washed
well in filtered sea water, and observed under a light microscope. The
number of intact and damaged eggs found in the fecal pellets was assessed.
and embryos were further incubated in fresh sea water at 12 to assay
the continuation of development.
Enzymatic digestion:
Digestion of the extra-embyonic investments was tested with;
Crude porcine pancreatic lipase (Sigma) (lmg/ml) and 10 mM dithiothreitol.
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pH 9.1; Pronase, a nonspecific protease isolated from Streptomyces
griseus (Sigma) (5 mg/ml), pH 7.8; and Trypsin,,2X crystallized from
bovine pancreas, dialyzed and lyophilized (Sigma) (5mg/ml), pH 7.6.
All experiments were carried out at 16, using a dilute egg concentra¬
tion (no greater than a monolayer) and effectiveness of digestion scored
as noted above.
Scanning electron microscopy:
Eggs of A. ceratodes, C. intestinalis, C. huntsmani, and Botryllus
sp. were fixed in 18 gluteraldehyde, 18 formaldehyde, 708 sea water, and
1.28 HEPES, pH 7.3. When preparing the mounts for gold spattering, the
eggs were gently squashed so as to cause some fragmentation of the extra¬
cellular layers.
RESULTS
Table I summarizes the fate of ingested eggs by adult A. ceratodes.
Parallel experiments were also carried out with C. intestinalis, with
similar results, although a strict numerical analysis was not done.
The eggs of the solitary tunicates, which had acomplete set of extra¬
cellular layers when fed to the adults, are found intact in fecal pellets.
except for the loss of follicle cells. These eggs do not fertilizé after
passing through the digestive tract; however control eggs, which were not
fed to the animals, also don't fertilize (most likely due to storage time
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after removal from the animal). Eggs fertilized prior to ingestion do
continue to develop and 448 hatch into normal viable tadpoles. Similar
results were seen in the mechanically defolliculated eggs.
A different type of result was seen in those eggs of solitary
tunicates which had been enzymatically dechorionated. Only 1-38 were
recovered intact in the fecal pellets. Furthermore, ingested live tadpoles
were only found in fragments after ingestion by the adult.
The eggs and embryos of the colonial ascidians do not pass through
the digestive tract of A. ceratodes intact. Less than 38 of the whole eggs
and embryos ingested were recovered unharmed.
Unfertilized eggs of S. purpuratus appeared to be digested, whereas
fertilized eggs (2-cell stage) with a fertilization membrane were recovered
intact. It appeared that cleavage had progressed, but further develop¬
ment was not assessed.
Unfertilized P. ochraceus eggs caused a most violent rejection
response, consisting of immediate closure of both siphons, body contrac¬
tion, and then a forceful ejection of water from the oral siphon. However,
those eggs which were ingested by means of forceful and persistent feeding.
were rarely recovered intact in the fecal pellets.
In vitro digestion experiments were also carried out on the
various eggs and the results are summarized in Table II. Eggs of the
solitary tunicates, A. ceratodes and C. intestinalis, appear to be
pronase and trypsin-insensitive and digestable by lipase. However, when
placed in lipase for ten minutes and then put into a protease solution.
dechorionation occurs quickly. Digestion of the chorion of eggs of the
colonial tunicates, C. huntsmani and Botryllus sp., on the other hand,
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does not occur in lipase and takes place quickly in pronase and trypsin,
No difference was noted in the sensitivity to digestion of fertilized and
unfertilized eggs.
The fertilization membrane of the urchin S. purpuratus was found
to be sensitive to both lipase and pronase, although the latter digestion
occurs more slowly. P. ochraceus eggs are lipase-insensitive and pronase
and trypsin-sensitive, analogous to colonial tunicates.
Comparison of the investments of colonial and solitary tunicate
eggs was done with the scanning electron microscope (Fig. 2a-d). It is
interesting to note the relative size of the eggs: colonial tunicate eggs.
with a mean diameter of about 250u, being 3-4 times as large as solitary
ones. Follicle cells are much more pronounced in the latter, appearing
as a single layer of elongated conical-shaped cells attached to the
outer surface of the chorion. Pitted surfaces can be seen, most likely
corresponding to vacuoles. Conversely, in the colonial tunicate eggs.
follicle cells appear as thin, symmetrical cells pressed flat onto the
chorion. The structure of the chorion also seems to be a thicker, more
complex and intricate matrix in the eggs of the solitary tunicates.
DISCUSSION
Most research on eggs has been done from a developmental perspective.
It is interesting however to look from an ecological viewpoint and focus
on the problem of survival of eggs and developing embryos. This study
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10.
centered on spawned and brooded tunicate eggs, resulting in the discovery
of the ability of those spawned to resist digestion by filter-feeders.
presumably by means of a protease-insensitive extra-embryonic membrane.
This study shows that when a series of eggs of solitary tunicates
are fed to adult A. ceratodes, only those with a chorion are recovered
intact and continue to develop. The chorion therefore seems to function
as a protective device against digestion by filter-feeders. Two modes
of action are proposed: the chorion is impermeable to normal digestive
enzymes and the egg itself is very delicate and the chorion serves as
a supporting envelope, protecting the egg from mechanical fragmentation.
The latter idea is supported by the observed fragility of the enzymatically
dechorionated egg (Reverberi, 1971); however, this fragility may have re¬
sulted from incubation in the enzymes. On the other hand, tadpoles.
which have much more formal integrity, yet no chorion, are also digested.
suggesting that enzyme action may be more important,
Fully-packaged eggs of colonial tunicates are rarely recovered
intact. These eggs are normally brooded and would never be presented
with the problem of digestion by filter-feeders in the wild. The chorions
of these eggs are easily digested by pronase and trypsin,
Unfertilized sea urchin eggs appear to be digested by mature A.
geratodes, whereas fertilized eggs possessing a fertilization membrane
are not. The membrane appears to be sensitive to 0.58 pronase, however
proteolytic digestion occurs much more slowly than in the colonial ascidian
eggs. The change in protease sensitivity of the Stronglyocentrotus egg
noted by Berg (1967) during the first few minutes after insemination is
thought to be caused by crosslinking of tyrosyl residues producing an
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11.
elevated, hard, DTT-insoluble fertilization membrane (Foerder and
Shapiro, 1977). The increased intricacies of the membrane is comparable
to the more organized chorion in solitary tunicate eggs than in colonials,
The correlation between the protease-insensitivity of the membranes
of solitary tunicates and urchins and their resistance to digestion by
adult A. ceratodes and C. intestinalis is fascinating. The observations
that eggs without these investments are not recovered intact from fecal
pellets seems to put the barrier to digestion at the chorion and fertiliza¬
tion membrane.
Solitary ascidian eggs only respond to protease after a short treat¬
ment with lipase. Lipid content in the chorion has been demonstrated by
various staining procedures (M. Poenie, personal communication). This seems
to suggest that lipids somehow mask the proteins in the chorion membrane.
as a protective device against proteolytic digestion. The hypothesis
of the hatching enzyme in A. ceratodes as a trypsin-like compound (Berrill,
1932) would suggest that this masking effect is found only on the external
surface of the chorion. It is noteworthy that lipase also dissolves the
fertilization membrane of S. purpuratus; further investigation seems to
be warranted concerning the lipid content of this investment,
Unfertilized starfish eggs seem to attack the problem of digestion
from another angle: prevention of ingestion. Ingested P. ochraceus eggs
cause a violent ejection reaction, although eggs comparable in size, such
as those of A. ceratodes, are ingested easily. Perhaps saponin compounds
found in Pisaster eggs (Ikegami et al., 1967) function to provide a chem¬
ical defense against potential predators. The fertilization membrane could
12.
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also function as a further barrier against digestion.
Organisms have developed two basic strategies of reproduction
(Thorson, 1949): producing few eggs and taking good care of them, or
producing many eggs and releasing them immediately. Many marine inverte¬
brates, including urchins and starfish, favor the latter strategy.
Tunicates, however, present both patterns, which seem to roughly correlate
with the social organization of the individual species: colonial ascidians
tend to brood few eggs (Fig. 3), while solitary ascidians spawn many
thousands (Fig. 4).
Solitary tunicates deal with the problem of survival of spawned
eggs by the brevity of the pelagic embryonic stage (18-20 hours), post¬
poning the more involved processes of development and growth until the
benthic post-metamorphic stage. Pelagic eggs, however, are still presented
with the problem of being eaten. Spawned ascidian eggs especially are
in danger of digestion by the parent tunicate - out the atrial siphon
and into the oral one (Fig. 4).
In the scope of this study, it was seen that spawned eggs and
embryos have a protease-insensitive investment and not only are able to
pass through the digestive tract of the adult A. ceratodes undisturbed,
but embryonic development continues to the larval stage. It is therefore
suggested that there is a comparable survival strategy among some marine
invertebrates; namely, a protease-insensitive extra-embryonic membrane,
such as a chorion or a ferilization membrane, serves to protect the egg
and embryo from digestion by filter-feeders.
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13.
ACKNOWLEDGEMENTS
I would like to thank Dr. David Epel for his suggestions and
encouragement during my work. The unlimited enthusiasm and energy of Dr.
Donald P. Abbott proved an invaluable inspiration. And a special note of
thanks goes to Carl Johnson for his patience, insight, and enthusiastic
support.
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14.
LITERATURE CITE
BERG, W.E. 1967. Some experimental techniques for eggs and embryos of
marine invertebrates. pp. 767-776. in: F.H. Wilt and N.K. Wessells
(eds.), Methods in Developmental Biology. Thomas Crowell Co., New
York.
BERRILL, N.J. 1932. Mosaic Development of the Ascidian Egg. Biol. Bull.
73:37-78.
DESANTIS, R., J. GENNARO, AND F. ROSATI. 1979. A study of the chorion and
the follicle cells in relation to the sperm-egg interaction in the
ascidian, Ciona intestinalis. Develop. Biol. 74:490-499.
FOERDER, C.A. AND SHAPIRO, B.M. 1977. Release of ovoperoxidase from sea
urchin eggs hardens the fertilization membrane with tyrosine
crosslinks. Proc. Nat. Acad. Sci. USA 74:4214-4218.
IKEGAMI, S., S. TAMURA, AND H. KANATANI. 1967. Starfish gonad: action
and chemical identification of spawning inhibition. Science.
158:1052-1053.
KUBO, M., ISHIKAWA, M., AND NUMAKUNAI, T. 1978. Differentiation of
apical structures during spermiogenesis and fine structures of
the spermatozoon in the ascidian Halocynthia roretzi. Acta,
Embryol. Exp. 3:283-295.
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15.
MUNEOKA, K. 1979. A new method for mechanical dechorionation of ascidian
eggs. Develop. Biol. 74:486-489.
REVERBERI, G. 1971. Ascidians. pp. 507-550. in: G. Reverberi (ed.).
Experimental Embryology of Marine and Fresh-Water Invertebrates.
North Holland Publishing Co.
ROSATI, F. AND R. DESANTIS. 1978. Studies of fertilization in the ascidians;
1. Self-sterility and specific recognition between gametes of Ciona
intestinalis. Exp. Cell Res. 112:111-119.
THORSON, G. 1950. Reproductive and larval ecology of marine bottom inverte¬
brates. Biol. Rev. 25:1-45.
WOOLLACOTT, R.M. 1977. Spermatozoa of Ciona intestinalis and analysis of
ascidian fertilization. J. Morphol. 142:77-88.
e
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TABLE CAPTIONS
TABLE I: Results of ingestion of eggs by adult Ascidia ceratodes. (+-
intact, development continued. +/no FC-intact egg without follicle
cells, development continued. O-fragments.)
TABLE II: Sensitivity of chorion to enzymatic digestion. (+=enzyme¬
sensitive. O-enzyme-insensitive. NA-data not available.)
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TABLE I
Eggs from;
Ascidia ceratodes
-whole
-without follicle cells
-without chorions
-tadpoles
Ciona intestinalis
-whole
-without follicle cells
-without chorions
-tadpoles
Clavelina huntsmani
Botryllus sp.
Stronglyocentrotus purpuratus
-unfertilized
-fertilized
saster ochraceus
Recovery of ingested
eggs in fecal pellets
888 (N=978)
918 (N=1056)
28 (N=1124)
08 (N=589)
868 (N=1138)
898 (N=995)
18 (N=946)
08 (N=358)
38 (N=1037)
28 (N=586)
28 (N=1274)
928 (N=1189)
28 (N=1184)
17.
Assessment
/no FC
/no FC
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TABLE II
5 5

+

—
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19.
FIGURE CAPTIONS
FIGURE 1: Extra-embryonic layers of ascidian egg.
FIGURE 2: Scanning electron micrograph of ascidian eggs. (FC-follicle cells.
CH-chorion. TC-test cells.)
(a) Ascidia ceratodes
(b) Ciona intestinalis
(c) Clavelina huntsmani
(d) Botryllus sp.
FIGURE 3:Reproductive strategies of colonial ascidians. Few large eggs.
broods eggs, embryonic development 4-8 days, larval development
6-8 hours.
FIGURE 4: Reproductive strategies of solitary ascidians. Many small eggs.
eggs spawned, enbryonic development 18-20 hours, larval development
24 hours.
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FIGUDF 1
dg
T
follicle cells
20.
chorion
test cells
egg
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FIGURE 2a
21.
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FIGURE 2b
22.
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FIGURE 20
23.
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FIGURE 2d
24.
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FIGURE 3



Sa





Botryllus colony
25.
brooding eggs
Ventral view of Botryllus zooid
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FIGURE 4
Oral
Siphon
26.
eggs spawned
......
...
Atrial
Siphon
Lateral view of Ascidia ceratodes