NEIL ALLEN
NTRODUCTTON
This study exanines metamorphosis and postmetamorphic
development of the solitary tunicate Ascidia ceratodes
(Huntsman, 1912). This account follows morphogenic
and behavioral events from larval settlement to the
fifth week of its life cycle. Previous works such as
those of Berrill (1947), Millar (1954), Scott (1952),
and Trason (1957), give accountsof larval and oozooid
development in a number of solitary and compound species.
however, a description of the growth and behavior of
Ascidia ceratodes through the first month has not
previously been done.
The abundanceof Ascidia ceratodes along the western
coast of North America (Abbott and Newberry,1980) and
its ease of growth and culture in the laboratory make
this species an interesting subject of study as has
been demonstrated by Lambert (1970) and King (1975)
who have investigated other aspects of the life cycle
of this species.
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MATERIALS AND METHODS
Adult specimens of Ascidia ceratodes were col¬
lected from styrofoam floats in the Monterey Marina,
Monterey, California, during the months of April and
May 1981. Gametes were removed in the laboratory and
fertilization was carried out in five inch finger bowls
which were kept in running seawater at 12-1470.
About 24 hours after fertilization, hatching occured and
the free swimming larvae were transfered to shallow
pyrex baking dishes lined on the bottom with glass
slides. The slides were covered with about 2 cm of
seawater. The larvae would settle on the glass slides
in 1-2 days. The slides were then placed in open
slide racks with the backs removed, and maintained on a
sea table with enough fresh running seawater to
cover the slides. The process of metamorphosis and
development which followed was monitered with a
compound light microscope. Prolonged observations
of the oozooids on the slides was made possible
by placing the slides in a petri dish filled with
seawater, using a U-shaped glass rod to hold the slides
off of the bottom of the dish. The slides could
then be turned over for eithera dorsal or ventral
view. Drawings were made with a camera lucida.
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RESULT
LTAMORPHOSI
Metamorphosis takes place 1-2 days after hatching.
The free swimming larvae orient on the substrate
allowing the three anterior adhesive papillae to adhere
to the surface. This is accompanied by rapid wiggling
of the tail, which may last up to 10 minutes. When this
movement ceases and the larva is fixed to the substrate
the axial complex of the tail begins to contract
anteriorly towards the body in a process which lasts
from 20-40 minutes, (fig. 1). At the end of this
process, the axial
complex, which has folded
over on itself, appears as a ball of brown tissue at
the posterior end of the oozooid (fig. 2-A). This tis¬
sue remains for 1-2 weeks before being completely
removed by phagocytic cells. The transparent cuticle
layer of the tail remains on the oozooid for up to
several hours before being washed away. At this
time, rotation of the body through about 90 degrees
occurs, after which the ventral side of the body
comes to lie parallel with the substrate. No
apparent adult organs can be seen for a period of
about the first 24 hours. During this time, the
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darkly pigmented sensory organs, the resorbed tail.
three ampullae and the relatively undfferentiated
tissue of the rudimentary organs can be seen (fig. 2-B).
The endostyle forms at 24-36 hours after settling.
and appears as a blunt elongated tissue mass parallel
with the anterior-posterior axis of the body. About
six hours later the heart, gut and two pairs of
stigmata can be seen (fig. 2-0). Tail remanantts and
sensory organs disappear by the end of the second
week.
AMPULLAE
Three ampullae form shortly after settling as
outpockets of ectodermal tissue, radiating out along
the substrate from the ventral side of the oozooid
(fig. 2-B). These elongate and remain for about one
week, after which 6-8 longer bifurcating finger like
ampullae develop by five weeks (fig. 4). At this
stage, the ampullae have heocoelic lumina in which
bloos cells can be seen circulating.
HEAE
The heart, which develops at the posterior end
of the endostyle, is almost transparent, and beats
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arythmically for the first several hours (fig. 2-C).
After this, the heart in a biphasic manner, pumping
anteriorly, the posteriorly for 20-50 beats in each
direction. After about 72 hours a visable circulation
of bloos cells can be seen in the subendostylar
vessel adjacent to the heart. At this time the
number of heart beats in each direction ranges from
50-100 with an average of about 80. The heart
elongates with time, and may be up to one fourth the
length of the oozooid at five weeks (fig. 5).
Circulation through discrete channels in the pharynx,
tunic and ampullae is easily observed at two weeks
and becomes more complex with the growth of the oozooid.
PHARYNX ANI
TIGMATA
The outline of the pharynx is visible at 24-48
hours after the beginning of metamorphosis (fig. 2-C).
At this time, two pairs of stigmata form, one on
the left side of the oozooid and one pair on the
right. Several hours later ciliary action begins
in the stigmata. The stigmata move in counter¬
clockwise metachronal waves. Two atrial siphons are
present at this time, along with two searate atrial
cavities, one above each pair of stigmata. The oral
siphon becomes better defined at the anterior end of
the oozooid (fig. 2-C).
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The peripharygeal groove appears within the first week
extending from the anterior of the endostyle on the
ventral side, posteriorly to the area of the brain(fig.3-A0.
tigmata multiplication proceeds as in figure 7.
During the second week, three more stigmata are
added to the pharyngeal wall on each side of the
oozooid (fig. 3-B), two between the original proto-
stigmata and one posteriorly. By the third week
the four anterior stigmata on each side pinch off to
form a second row, and one more is added posteriorly
to make ten on each side. At this time, papillae
are forming on the interior of the pharyngeal wall,
and will eventually give rise to the internal
and
begin
longitudinal vessels.A the atrial siphons
to move closer together towards the dorsal centerline
of the oozooid(fig. 4). During the fourth week, the
stigmata pinch off again to form four rows on each
side of the pharynx. At this point, the left and right
atrial siphons,and atrial cavities
begin to merge (fig. 5). Internal longitudinal
vessels now are visible.
TRACT
DIGES
The digestive tract is apparently functional
at 60-72 hours coinciding with the formation of four
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oral tentacles. Feeding can be observed with the aid
of carmine suspensinn added to the seawater surrounding
the oozooid. Mucus sheets formed at the endostyle and
moving dorsally of each side of the interior of the
pharynx to the dorsal lamina can be seen trapping the
fine particles of carmine. The dorsal lamina is out-
lined by the carmine as the mucus sheets are formed
into a rope and passed posteriorly to the esophagus.
Feeding is thus demonstrated at about 72 hours after
settling. Particulate matter entering the oral siphon
reaches the esophagus in 1-2 minutes. Resulting
fecal pellets are released into the atrial cavity in
1-2 hours. The esophagus, located at the posterior
of the pharynx, is U-shaped and curves dorsally and
anteriorly to the stomach, which appears as a dark
sphere when filled with food particles. The intestine
passes anteriorly below the pharynx before emptying
into the right atrial cavity of oozooids less than
five weeks old. When the atrial cavities have fused,
the intestine empties into the right side of the atrial
cavity (fig. 3-5). At two weeks the pyloric gland is
visible arising as a single bifurcating duct, from
the wall of the i stomach at the junction of
the intestine (fig. 3-5). Just anterior to the pyloric
gland is a diverticulum of the intestinal wall. This
caecum is of unknown function (fig. 3-5).
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SPONTANEOUS CONTRACTIONS
Spontaneous contractions can be
observed
from the onset of ciliary action in the stigmata at
about 48 hours after settling. These contractions
occur at intervals of 30 seconds to 10 minutes, and
involve a movement in which the body of the oozooid
shortens and the oral siphon closes. Similar ennt¬
ractions in response to physical diturbanceof the
oozooid are seen later in development at about 72
hours. During these contractions the cilia in the
stigmata stop beating and seem to project into the
pharynx. Beating resumes when the animal relaxes.
BOD
Y ORIENTATION
Though the oozooid rests on its ventral side
a marked tendancy to lean to thesleft is observed from
the first week after metamorphosis begins. Thus
the endostyle on the ventral side appears skewed to
the right and the dorsal lamina appears somewhat
to the left when the oozooid is viewed dorsally.
10
NEIL ALLEN
DISCUSSION
The larvae of solitary ascidians are somewhat
less developed when metamorphosis begins, than are
the larvae of most compound ascidians. As an ex¬
ample, the oozooid of the compound ascidian Distaplia
occidentalis begins feeding within 30 minutes after
settlement (Cloney,1978) while three days aré required
for Ascidia ceratodes to begin feeding. This pro-
longed period offers a good opportunity for obser¬
vations of organogenesis.
An outline of early developmental changes can
be seen on fig.6. Adhesive ampullae are the first
structures to develop, securing the oozooid to the
substrate. Further organogenesis lasts 1-2 days
before functional heart, gut and stigmata are formed.
The organs involved in filter feeding must develop
and become functional beforethe oozooid yolk store
runs out. The endostyle, peripharyngeal band, oral
and atrial siphons, active stigmata and digestive
tract all Pecome visible within hours of each other.
Actual feeding takes place 12-24 hours later. It
is interesting to note that the onset of feeding also
occurs at the time of the development of oral tent¬
acles. Feeding may coincide with the actual ppening
of the siphons which have beervisible for almost
24 hours before feeding but may be covered by a thin
layer of epidermis, or it may coincide with the development of
which
tentacles"are necessary as regulatory structures
to control the type of particulate matter takenin.
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11
The development of stigmata in Ascidia ceratodes
generally follows the outline given by Garstang (1892).
in which the stigmata are arranged in a series of
rows, placed transversly to the longitudinal axis
of the oozooid. However, the exact sequence of addition
of stigmata is somewhat different than Garstang
described. Instead of a row of six protostigmata
forming before a second row forms by transverse
division of the protostigmata, the sixth protostigma
forms after the second row appears (fig. 7).
The purpose of spontaneous contractions is not
known, but it is interesting to note that they occur
before filter feeding begins, and also before
contractions in response to physical disturbance
takes place. The latter, which may be a defense
mechanism, is more easily explained.
At the termination of this study, no gonadal
development was seen, nor was the full compliment
of stigmata formed.
NEIL ALLEN
SUMMARY
Fertilized eggs of Ascidia ceratodes hatch in
about 24 hours. Resulting free swimming larvae
settle and begin metamorphosis 1-2 days later.
Development of the major organs involved in filter
feeding occurs 36-48 hours after settlement. Feeding
begins at 60-72 hours. Spontaneous contraction
begin at about 48 hours and contractions in response
to physical disturbance at 72 hours.
C
NEIL ALLEN
ACKNOWLEDGMENT
I would like to thank Dr. Bonald P. Abbott,and
Dr. David Epel for their help in this project.
os
Mo
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13-+
LITERATURE CITED
Abbott, Donald P. and Andrew Todd Newberry, 1980.
Urochordata: The Tunicates. pp. 177-226 in: Intertidal
Invertebrates of California. Morris, Robert H.,
Donald P. Abbott and Eugene C. Haderlie. Stanford
University Press.
Berrill, N.J. 1947. The development and growth of
Ciona. J. Mar. Biol. Assoc., 26:6, pp. 616-625.
Cloney, Richard, 1978. Ascidian metamorphosis:
Review and analysis, pp. 255-282. in: Settlement
and metamorphosis of marine invertebrate larvae.
Elsevier/ North-Holland Biomedical Press. pp. 255-282.
Garstang, Walter, 1892. On the development of
stigmata in ascidians. Proceedings of the Royal
Society. 51: 505-513.
King, Robert Emil, 1975. The population biology of
Ascidia ceratodes in Monterey Harbor California.
Masters thesis, San Francisco State University,
Lambert, C.C., 1970. Genetic transcription during the
development and metamorphosis of the tunicate Ascidia
ceratodes. Diss. Abst. Int. 31B 1970: 1731.
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1+
—5
Millar, R.H., 1954. The development of the ascidian
Pyura microcosmus (Savingy). J. Mar. Biol. Assoc. U.K.
33: 403-107.
Scott, Sister Florence Marie, 1952. The developmental
history of Amaroecium constellatum. III. Metamorphosis.
Biological Bulletin. 103(2) : 226-241.
Trason, Winona Bethune, 1957. Larval structure and
development of the oozooid in the ascidian Euhermania
claviformis. J. Morph. 100(3): 509-546.
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LEGEND FOR
AM
ampullae
atrial siphon
ATS
brain
CU
tail cuticle
endostyle
END
esophagus
heart
IN
intestine
oral siphon
PAF
adhesive papillae
peripharyngeal band
PB
ynx
pha
stigmata
stomach
STM
sensory pigments
2
tail
tentacles
TU
tunic
pyloric gland
gastric caecum
dorsal lamina
DL
FIGURES 1-
16
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Figure 1 : Process of tail resorption in Ascidia
ceratodes larvae, beginning at point of
attatchment to substrate. Time O through
20 minutes.
Figure 2 : A. Dorsal view of 1 day old oozooid.
B. Dorsal view of 2 day old oozooid.
C. Dorsal view of 3 day old oozooid.
Figure 3 : A. Dorsal and ventral view of 1 week
old oozooid.
b. Dorsal and ventral views of 2 week
old oozooid.
Figure 4 : Dorsal and ventral views of 4 week old
oozooid.
Figure 5 : Dorsal and ventral views of 5 week old
oozooid.
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t:.O. min
t: 5 min
t: 1O min
t: 1 5 min
t: 20 min

EIG1

ASCIDIA CERATODES LARVAE
D

7






L

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6

V



M
CU


N



Imm
18

1
—
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DAY
2DAYS
3 DAYS
A
o

ENO
kkkkv
FIG 2

O
10

ES
mm
A
DORSAL VIEW
STM

19
WEEK
2: WEEKS


e
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DORSAL VIEW
0



EN


1

6
FIG 3

E
ENO
B
mm
STM
0
EVD
20
VENTRAL VIEW


G
A0

GC
5
——
4 WEEKS
END

O5




511
mm

RVT



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DORSAL VIEW
VENTRAL VIEW
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90
FIG 4



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END
— P
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5 WEEKS
VENTRAL VIEW
FIG 5

EN
A
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ood
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00

HM
ES

mm

22
DORSAL VIEW
END

—

8c

J

IMT
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23
FIGURE 6
A- tail resorption
B- formation of ampullae
C- endostyle
D- heart
E- digestive tract
F- primary protostigmata
G- atrial siphons
H-oral siphon
I- peripharyngeal band
J- oiliary action
K- spontaneous contractions
L- oral tentacles
M- feeding
N- contractions due to physical disturbance
O- circulation
P- fecal pellets
Time schedual of important early developmental events
beginning at larval settlement.
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24

L

—

oouo-zod
NEIL ALLEN
FIGURE 7
A schematic representation of the development of
stigmata on the right side of the pharynx, to the
time at which the atrial cavities fuse.
- represents new stigmata
- represents newly diveded stigmata
aoisod
3
8 8

8

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88 80
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