ABSTRACT:
Specimens of Ciona intestinalis were observed in
the lab and in their natural habitat for evidence of
spontaneous contractions. In the laboratory oxygen
tension and temperature were varied to induce spontaneous
contraction modulation. It was found that Ciona intestinalis
contracts at an average rate of 6.3 times h' at 15-15.50
in nature. Intermediate levels of oxygen tension have
no effect on contraction rate but increasing temperature
increases contraction rate. 3 contraction types can
be isolated in Ciona intestinalis (cross-oral, cross-atrial,
and both siphons). At higher contraction rates more
cross-oral and fewer "both" contractions occur. Observations
at high contraction rates indicate that contraction
durations occur between 10-15 sec with little variation.
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INTRODUCTION:
The behavioral repertoire of solitary ascidians
is rather limited. They are benthic filter feeders;
clearing pariticles of organic matter from water pumped
through their brachial basket by beating cilia. This
feeding current is occaisonally interrupted by behavioral
events known as spontaneous contractions. Spontaneous
contractions differ from induced contractions in that
they occur without a visible external trigger. Hecht
(1918) presented the first detailed description of
spontaneous contractions in Ascidia nigra. However,
after many years of investigation the purpose of spon¬
-taneous squirting in ascidians is still a matter of
controversy.
Hoyle (1953) did extensive studies on spontaneous
contractions in which he calculated high rates of water
intake due to muscular contraction. He proposed that
spontaneous contractions represent a supplementary feeding
mechanism. However, later measures of ciliary water
flow (Jorgensen 1966 and Carlisle 1966) showed that water
flow due to ciliary activity is actually 30 times greater
then muscular water renewal. These new current calculations,
coupled with the observation that contractions eject
good unfiltered water and suck in equally good water,
reopened the spontaneous contraction controversy.
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Speculation on the significance of spontaneous
squirting has ranged widely. Suggestions have included,
mixing of water adjacent to oral siphon (Jorgensen 1966),
cleaning of brachial wall of accumulated sediment, gamete
release, fecal pellet ejection (Goodbody 1974), food
particle sorting (MacGinitie 1939), and a suggestion
that there is an ancestral tie to the pulsations of the
salps (Hecht 1916).
The rythmic contractions of ascidians has been
reported in many species but variation and regulation
of spontaneous contractions has been sparsly studied.
Yamaguchi (1931) and Hoyle (1953) are in agreement that
starvation causes the spontaneous contraction rattee to
increase. Shumway (1978) observed that Ciona intestinalis
has a rate of 14 contractions h at 10°0 and that contraction
rate decreased as external salinity decreased. Many
aspects of environmental regulation of spontaneous
contraction rate have never been studied.
The characteristics of contractions in ascidians
have only been briefly touched upon by previous investi¬
-gators. Yamaguchi (1931) has the only detailed descrip¬
-tion of more then one distinct spontaneous contraction:
type. In Styela clava he observed and described several
varieties of spontaneous contractions but he made no
effort to suggest the functional purpose of contraction
varieties.
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Gibb
My investigation is aimed at pinpointing more environmental
factors which modulate spontaneous contraction behavior.
Few measurements of naturally contracting ascidians have
been made. Little is known about contraction characteristics.
By recording natural contraction rates then observing
contractions under varying environmental conditions it
may become clearer what the purpose of spontaneous behavior
is in tunicates.
Oo o.
a
Gibb
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METHODS AND MATERIALS:
The ascidian Ciona intestinalis was chosen as the
subject for all contraction experiments because of its
size and easily observable contraction behavior. The
animals were collected from the fouling community that
hangs down from the underside of the floating docks in
the Monterey marina.
Spontaneous contractions were observed in four
different ways.
1. Direct observations were made of the contraction
frequencies of Ciona intestinalis hanging from the dock.
24 clearly observable animals were watched for 1 h. each.
2. Animals were collected from the marina and placed
in laboratory aquariums supplied with circulating seawater
at 12-140. The animals are normally attatched upside
down to the bottom of the docks. To provide support and
natural orientation the animals were clipped at the viseral
end and hung upside down in an aquarium. Strings of
contracting Ciona intestinalis were then observed using
a color videotape set up. (fig. 1) This allowed easy
observation of large numbers of animals at once and the
ability to replay contractions the observer missed. 12
animals were observed for 4 h and 6 more animals were
observed for 1 h.
3. For longer term observation the animals were attatched
to strain gauges. Ciona intestinalis was connected to
E
O
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Gibb
the strain gauge via a small alligator clip 1 cm below
the oral siphon. Contraction of an animal was converted
into an analog signal which was recorded on a polygraph
with adjustable time base. (fig. 2) After the animals
were allowed to adjust to the clips they appeared to
contract normally.
4. Direct observations of contracting rows of Ciona
intestinalis allowed more careful observation of contraction
types.
The first experiment done involved measuring contraction
rates for animals in stagnated and circulating water.
The
animals were hooked to the strain gauge and allowed to
sit for 8 h. in a.3.8.1 tank of filtered seawater at
18°0. After 8 h. contraction measurements were started.
Stagnation conditions involved higher temperature, lack
of food, increase in metabolic wastes, and small changes in
oxygen tension. After 5 h. circulation indicates the
addition of 14 C water with food present. After 1 h. the
temperature of the tank changed from 18 C to 14 C.
Temperature variation represents one of the factors
of stagnation so experiments involving temperature change
were done. An array of 12 animals were hung in a 75.7 1
aquarium and observed for 6 h. The water in the tank was
aerated and stirred but unrenewed. Between each one hour
observation an aquarium heater brought the temperature up
2°0 over a 1 h. period. Aeration of the water was provided
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by an aquarium pump. At the end of the experiment the
water temperature was allowed to drop to 19 C and one
additional 1 h observation was obtained.
Bubbling nitrogen through seawater took the oxygen
out of the water for dissolved oxygen study.
Ciona intestinalis embryos were raised in the lab
for contraction observations. Sperm and eggs were extracted
from healthy Ciona intestinalis and placed together in
glass dishes. Slides were placed in to bottom of the
glass dishes for tadpoles to settle on. After 4 days
slides were removed from dishes and maintained in wooden
racks in circulating seawater.
o
Contractions / Hour
NGSOJOOO
+
5
Contractions / Hour
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Gibb
RESULTS:
Contraction rates vary widely between individuals
and in the same individual over time. In marina observations
some animals contracted 1 time h" and others contracted
20 times. The temperature of the marina ranged between
15-15.5 0. The dissolved oxygen content of the water
was 9.5ppm. The average Ciona intestinalis contraction
rate in the marina is 6.3 contractions h with a standard
deviation of 6.0. Observations at the marina revealled the
sensitivity of Ciona intestinalis to passing pedestrians.
The animals responded to vibration by quick closure of
both siphons with no body contractions. Subsequent
observations were made at off hours and laboratory
observations were conducted in an undisturbed basement
aquarium room.
The first environmental factor analysed was the
amount of dissolved oxygen in the seawater surrounding
the ascidian. Temperature ranged from 15-15.5°0. When
oxygen tension dropped from ambient (9.5ppm) down to 5. 5ppm.
fig. 3 shows that contraction rate varies only slightly.
Strain gauge measurements done at temperatures of
13 0  10 produced contraction rates that were very
close to those obtained at the Monterey marina and on
video observation. (fig. 4)
The first modulation of contraction rate was observed
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Contractions / Hour
8


8
G
8-
8-
D

Contractions / Hour
8

8
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in animals in stagnant (filtered water at room temp.)
and fresh seawater. (fig. 5) The animal maintained a
contraction rate of 30 contractions h'+ 3 until the
circulated ambient seawater brought the contraction rate
down to 11 contraction h. One or more factors involved in
stagnation conditions increased contrastion rate.
Temperature turned out to be a contraction modulating
factor. (fig. 6) As ambient temperature rises Ciona
intestinalis contracts more rapidly and vigorously.
Confirmation of the reversibility of this effect is
demonstrated by decrease in the contraction rate as
temperature drops back to 19'0. Marina, video, and strain
gauge data fits into the bottom of the temperature corre¬
-lation.
During observations of contraction rates it became
clear that 3 isolated contraction types occur in Ciona
intestinalis. By carefully hooking up a strain gauge
to an especially active animal it is possible to get a
record of the different kinds of contractions which can
be read like an electrocardiogram. Spike shape is
characteristic of contracton type. (fig 7)
Contraction types included:
1. Both - siphons narrow to small openings and the
circular muscles in the upper third of the body tighten
to force water out both siphons.
6 7
a Contraction Types
atrial siphon
oral siphon
Both Siphons
Relaxed


fecal pellet
Time (min)
water ejection
Cross Atrial
Cross Oral

—
6
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2. Cross Atrial - oral siphon closes and contraction
of circular muscles causes ejection of water out the atrial
siphon.
3. Cross Oral (opposite of cross atrial ) - atrial
siphon closes and contraction of circular muscles cause
ejection of water out the atrial siphon.
Often the cross-atrial of cross-oral contraction
is followed by closure of both siphons for a short time.
The cross-atrial and cross-oral reaction causes a stronger jet
of water then the constriction of both siphons.
Some Ciona intestinalis contract more vigorously
then others. However, the duration of a spontaneous
contraction often stays constant over long periods. On
strain gauge measurements over 2 h time periods animals
were observed to contract for an average of 11.5 sec.
with a standard deviation of 1.7 sec regardless of
contraction variety. Circular muscle constriction is
a major part of speontaneous contraction behavior but
seldom is notable shortening of longitudinal muscle fibers
involved. The maximum contraction rate for Ciona intestinalis
under temperatures of 25 C is 90-95 contractions h*!.
This contraction rate was obtained as the upper range
in the temperature modulation experiment. At high
temperatures Ciona intestinalis will often contract
rapidly for a period of time then contract without subsequent
relaxation.
a
88
O
D

O
5
O
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Gibb
Observation of contraction types show that cross-atrial
contractions are associated with gamete and fecal removal
from the atrial cavity. Often a series of several cross¬
-atrial contractions preceeds ejection of waste or sexual
products. Manual insertion of large particles into the
oral-siphon of Ciona intestinalis will evoke cross- oral
behavior but in regular spontaneous contraction sequences
particles were not observed to be discharged from cross-
-oral contractions.
Measurements of contraction types in stagnant and
circulating water were done on 2 animals under stagnation
conditions and 14 animals in circulating ambient water.
Calculations indicate that at high contraction rates
more cross-oral contractions occur and fewer contractions
of both siphons. The frequency of cross-atrial contractions
stays about the same.
Observations of Ciona intestinalis embryos were
made on freghly settled animals. In the observation of
7 embryos the start of spontaneous contractions was
correlated with the start of ciliary action on the developing
igmata. At 5 days Ciona embryos contract every-2 min.
At this early stage embryos are insensitive to vibration.
As embryos grow older (10 days) they become more sensitive
to vibrations and their contraction rate decreases to
every 5 min.
Gibb
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Discussion;
No thorough studies have been done on the environmental
modulation of spontaneous contractions in solitary ascidians.
Regulation of contraction rate has been observed with
starvation (Hoyle 1953 and Yamaguchi 1931) and salinity
fluctuations (Shumway 1978) but no mention of temperature
modulation has been made. The only mention of the temperature
effect is (Polimanti 1911) who noticed high rates of
contraction at 30°0 but made no observations under normal
conditions. This study has shown that temperature increases
contraction rate with a 0 of about 6 over the temperature
range of 15 to 25°0. This is a dramatic temperature
response and certainly warrants further investigation,
especially seasonal and acclimation aspects.
My values for average normal contraction rate clustered
around 3-8 contractions h" at 13P0.+1. "This is much
sower then previously published values of 14 contractions
h" at the lower temperature of 10°0 (Shumway 1978). The
close correlation between video, marina, and strain gauge
observations provides confidence that animals in the
laboratory were contracting in a way similar to their
natural behavior.
Suggestions that particle sorting may be a result
of spontaneous contractions (MacGintie 1939) are unlikely
considering the marina observations. The Monterey marina
has water containing high amounts of plankton and suspended
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organic particles yet this doesn't effect contraction
rate.
Yamaguchi (1931), like this author described cross¬
oral, cross-atrial, and simultaneous closure of both
siphons as 3 isolated contraction types. He was able
to determine that in Styela clava each siphon has its
own pacemaker activity. Isolated siphons contract at
rates which are different from intact animals. Further
experiments may show that Ciona intestinalis has a similar
intrinsic siphonic rate. The different contraction types
could be postulated to be an integration of different
pacemaker regions.
Based on my observations it appears that regular
spontaneous contractions are an integration of 2 rhythms.
First, the cross-atrial contractions are tied to waste
and sexual release. The other contractions are a reaction
to environmental changes. Ciona intestinalis is sampling
it's environment continuously. As a benthic organism
it has only one escape from unpleasant enveronmental change.
Ciona intestinalis contracts away from or blows away any of
the environment it finds noxious. Environments with high
salinity, high temperature or no food produce a high
maintained rate of contraction as long as the irritation
persists. Spontaneous contractions are not strictly
spontaneous but result from environmental stimulation
rather than the more obvious mechanical stimulation.
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Gibb
Further work needs to be done on the modulation of contraction
types as a part of spontaneous contractions. It may
be that in ideal conditions contractions would be entirely
of the cross-atrial variety.
SUMMARY:
1. Ciona intestinalis contracts rhythmically in nature
at an average rate of 6.3 contraction h'.
2. Between animals contraction rates are highly
variable.
3. Intermediate values of oxygen tension have no effect
on contraction rate.
4. Raising temperature causes the average contraction
rate of Ciona intestinalis to increase
5. The duration of contractions stays constant over
long periods of time.
6. 3 seperate contraction types can be observed in Ciona
intestinalis.
7. The ratio of these 3 contraction types changes with
contraction rate.
CAPTIONS FOR FIGURES:
fig. 1. Apparatus used for video observation of contracting
Ciona intestinalis
fig. 2. Strain gauge and polygraph set up for long term
measurement of contraction rate in Ciona intestinalis.
fig. 3. Average contraction rates, and standard deviations
(sd) of contraction rates for Ciona intestinalis.
fig. 4.
train gauge observations of average contraction
rate for normally contracting Ciona intestinalis
in a small temperature range (13°0 +1 °0).
Stagnation observation. Changes in average
fig.
contraction rate with exposure to stagnation
(filtered water, 18 C, lack of food, and build up
of metabolic waste) and circulation (14 0, moving
water, food, no build up of wastes).
Temperature observations. Changes in average
fig.6.
contraction rate with changes in temperature.
(Dotted line) strain gauge measurements. (Solid
line) direct observation. (Square) reversal of
temperature back to 19°0 for last direct observation.
(Rectangle) marina observations. (Triangles)
train gauge measurements at 3 temperatures.
fig. 7. Contraction types. (a) Illustrations of 4 contraction
types with emphasis on the role of the siphons.
(b) Sample of strain gauge data. A - cross-atrial
contraction which is followed by fecal pellet
ejection. B - Both siphons contracting. O - cross¬
-oral contraction. R - relaxed resting state.
table 1.
Variation in contraction type with contraction
rate. Stagnation measurements made on 2 animals.
Circulation measurements made on 14 animals.
ACKNOWLEDGEMENT
l'd like to express my thanks to Hopkins Marine
Station for offering me an opportunity and a place to
work; Professor Don P. Abbott for his instruction;
Professor William Gilly for his technical help; Faylla
Chapman for scrounging materials; and Professor Charles
H. Baxter for the thoughtful patience that inspired my
work.
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