Rice and Cotsirilos
-la
Abstract
Fecal pellets of seven species of tunicates (Ascidiacea)
were collected from animals inhabiting the same intertidal
habitat near Pacific Grove, California in order to assess the kinds
material ingested. Two additional species were studied but
of
their fecal pellets were not examined. Dry weight per pellet
was established for all species and 5.0 x 10 5g of fecal matter
from each were compared microscopically. Of a variety of ma¬
terials tabulated, diatoms and dinoflagellates were the most
abundant. Species that had very high numbers of diatom frustules
also had a large number of digested diatoms. Tunicates pro¬
ducing pellets with large numbers of dinoflagellates were of a
different species and morphology than those showing diatoms to
dominate.
Five species of tunicates were selected for laboratory feed¬
ing studies and involved solitary, social, and colonial animals.
Graded sizes of two Macrocystis species ( brown algae) and sand
grains were offered. Larvae of the barnacle Pollicipes polymerus
(Sowerby,1833) were fed to the three largest animals (Styela mon-
terevensis Dall, 1872; Ascidia ceratodes Huntsman, 1912; and
Perophora annectens Ritter, 1893) and Ascidia tadpoles were
introduced to the two solitary species. The acceptance and re¬
jection of these food items was observed in connection with mouth
size and tentacular screens. Items with diameters too large to pass
the screen without striking a tentacle were found in fecal pellets
while smaller ones were occasionally rejected. Our observations
indicate that the tentacles show important activity when encountered
with potential food particles.
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Rice and Cotsirilos
Introduction
In their surveys of size and shape of marine invertebrate
fecal pellets, Edge (1934) and Arakawa (1971) demonstrated that
pellets from the few species of tunicates they examined differ¬
ed in morphology. Whether these differences were specific to
the species, as shown by Arakawa (1971) for the three species
of Styela, or different because of food choice was not consider¬
ed by these authors.
While mechanisms associated with feeding such as pumping
rates, pumping rhythm, filtration rates and filtration efficiency
have been studied in ascidians (Fiala-Médioni 1978 a ,b,
and e), the fate of particles taken in during these active pro¬
cesses has not been looked into. That many of these particles
serve as food is a reasonable surmise, but some such particles
may be ingested by accident.
A study of size, type and number of particles ingested in
field populations of tunicates was undertaken. Laboratory feed¬
ing studies were subsequently performed to determine both poten¬
tial food kind and size and also how a tunicate might actively
exclude particular items from entering its digestive tract.
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Rice and Cotsirilos
Methods and Materials
The following ascidians: Styela monterevensis (Dall, 1872),
Ascidia ceratodes (Huntsman, 1912), Clavelina huntsmani (Van Name,
1931), Perophora annectens (Ritter, 1893), Synoicum parfustis
(Ritter & Forsyth, 1917), Polyclinum planum (Ritter & Forsyth, 1917),
Archidistoma molle (Ritter, 1900), Archidistoma psammion (Ritter &
Forsyth, 1917) and Aplidium solidum (Ritter & Forsyth, 1917) are
available in the intertidal off Mussel Point at Hopkins Marine Station,
and were chosen for this study. A channel subject to heavy surf
action was selected in order to obtain Styela which appeared to
prefer the well-exposed, low intertidal zones. Species were collect¬
ed within a 10 m radius. Barnacles, clams, and other encrusting or¬
ganisms were trimmed off the colonial species to prevent mixing of
waste materials. Tunic and substratum surfaces were washed free of
debris. Specimens were placed in cut polyvinylchloride tubing (15 cm
diameter) with Nitex 0.15 mm weave floors which allowed for water
exchange but prevented fecal pellet loss. One hundred pellets were
collected from each species, except from Styela which produces con¬
siderably larger pellets. Eighteen millimeters of Styela fecal
matter was obtained. Pellet weights were obtained in order to con¬
duct a cross species comparison of food content between identical
masses of fecal matter. All samples were rinsed twice in fresh
distilled water to remove salt residue. Twelve glass vials were baked
for 1 h at 70° C. and then vacuum desiccated for 48 h. Crystalline
phosphoric acid was the desiccating agent. Dry vial weights were
obtained with a Mettler balance accurate to 1.0x 10 ' g. Pellet
samples were
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Rice and Cotsirilos
gathered in 2 ml of fresh deionized water and placed in separate
vials, leaving five controls. The same baking and drying pro¬
cedures were repeated and dry weight per pellet was established
for each species. An average weight reduction of 1.1 x 10
(R = 1.0 x 10  g to 1.2 x 10"4 g) was observed for the five
control vials and was added to dry pellet weights in order to
correct for them. Water weight as a source of error was elimin¬
ated by obtaining dry weight data and vials were handled with
forceps to avoid fingerprint weight.
In late May these species were collected within a 10 m
radius. In the laboratory, tunicates were rid of extraneous
external material, and 1.0 x 10" g of fecal matter was collect¬
two glass slides
ed from each species. Pellets were evenly spread between,and a
grid was etched to determine area. Half of the total fecal area
was examined at 450 X from different sections of the grid in
order to observe random parts of the fecal matter.
Laboratory feeding studies were performed on Styela,
Ascidia, Perophora, Synoicum and A. psammion. Digestive times
for each species were obtained in order to predict elimination
time of a given ingested particle. Collected animals were
placed in the Nitex containers for a minimum of 1 h. A carmine¬
seawater suspension was gradually introduced after verifying
that zooids were relaxed and expanded. Due to its larger size,
Styela was placed on a non-toxic clay base in a beaker receiving
running water. After 10 min of active carmine ingestion, sub¬
sequently voided pellets were rinsed in carmine-free water and
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Rice and Cotsirilos
examined microscopically for remnant carmine. The first appear¬
ance of red particles enveloped within the mucous rope was scored
as a laboratory digestive time (table 1).
Food material included Macrocystis spp., Pollicipes larvae,
Ascidia tadpoles and sand grains. Detrital Macrocystis spp.
blades were collected and allowed to decompose for 3 days in
stagnant water before grinding in a blender. Algal pieces of
2.20-1.35 mm, 1.35-1.00 mm, 1.00-0.56 mm, 0.56-0.25 mm, 0.25-
0.15 mm, 0.15-0.10 mm, and less than 0.10 mm were obtained by
straining through Nitex and geological sieves. Animals were
initially fed the smallest algal sizes mixed with carmine to
habituate them to the food item. Running water assured continual
plant fragment circulation. At 5 min intervals small amounts of
the next largest size category were added. Pellets were chron¬
ologically examined and size of largest algal fragments were
noted (see figure 3).
Pollicipes larvae studies were carried out in still water
under the dissecting microscope to allow for behavioral response
observations. Species were simultaneously introduced to carmine,
larvae and algal fragments of size 0.25-0.15 mm after habituation
to the maximum algal fragment proven to be acceptable in the feeding
studies. Larvae were present during active feeding on carmine
and algae. Fecal pellets were examined for larval remains.
Sand grains and Ascidia larvae were fed to Styela and
Ascidia. The inert particles proved too heavy for Styela to suck
into its curved siphon so the animal was inverted. Styela was
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Rice and Cotsirilos
fed algae of known maximum size acceptance to demonstrate its
ability to feed while inverted. Sand grains were then intro¬
duced and responses were noted.
Mouth sizes and tentacular screens were measured with an
ocular micrometer and sketched to scale (figure 4). Mouth dimen¬
sions and tentacular shape, organization and orientation were
measured for Perophora, Synoicum and A. psammion during carmine
intake and diagrams are replicas of a feeding zooid. Styela and
Ascidia tentacular networks were taken from preserved and anasthe-
tized animals respectively and measured from the inside. Tentac¬
ular clearance, a measure of the largest sphere and rectangle
that could slip through the tentacles without contact, was estab¬
lished for each species (figure 4).
Rice and Cotsirilos
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Results—Fecal pellet contents
Figure 1 shows in descending order, the abundance of dia¬
toms within a 50 ug fecal sample from each of seven species of
tunicates. Mean weight per pellet was calculated for each species
and is shown in table 1. A large number of empty diatom frustules
occurred in identical fecal weights of all the species. A
portion of these could have been present in the water column
and a portion entering as food. Dinoflagellates, the second
most numerous item found in the pellets were within the size
ranges of the diatoms, but of different shapes.
Table 2 divides the seven species into two groups accord¬
ing to individual or colony shape. Polyclinum and Synoicum
are lobed colonies attached to the substratum by stalks.
These two were placed in Group L along with Clavelina and Styela
which are also lobed with their oral siphons elevated above the
substratum. Aplidium solidum, A. psammion and A. molle comprise
Group M, which are encrusting mounds growing in close proximity
to the substratum. Fecal pellets from species in Group L con¬
tain more dinoflagellates per ug than species in Group M.
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Rice and Cotsirilos
Discussion—Fecal pellet contents
It is assumed that all species were exposed to uniform
environmental conditions since they were collected from the same
site within a 10 m radius. Some of the species, however, had
several times as many diatom remains in their fecal pellets than
did others. Species which take in the most diatoms leave the
highest proportion of diatoms digested. (Digested diatoms are
defined as those frustules partially or completely emptied of
contents.) This correlation indicates that they are efficiently
digesting the food source which is most often seen in their
fecal pellets.
Fecal pellet architecture was compared between species in
light of the data presented in figure 1. No correlation was
observed between fecal pellet contents and pellet structure.
Any settling of diatoms upon or close to the substratum
could potentially create higher densities there and facilitate
ingestion by encrusting species such as A. molle and Ap. solidum.
If diatoms come to rest upon the tunics of these colonies then
they are in an excellent position to be taken into the oral
siphons.
The greater number of dinoflagellates in pellets from
Group L indicate that species with oral siphons located some
distance above the substratum are more likely to ingest dino¬
flagellates. Whether this is due to a lower abundance of dino¬
flagellates close to the substratum or to some selective action
of the intake mechanism is not clear. Since most of the dino¬
flagellates counted ranged in size from 15-20 um, and tentacular
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Rice and Cotsirilos
clearances ranged from 80-230 um, it is hard to visualize :
mechanism by which tunicates could actively select dinoflagellat
Rice and Cotsirilos
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Results--Feeding
The two Macrocystis species were ingested by all five tun¬
icates. Animals with larger mouth sizes ingested greater sized
algal fragments (figure 3). Large plant material tended to be
rejected unless the individual was introduced and habituated to
smaller food items first. When algal clumps entered oral siphons,
a violent contraction of the pharyngeal cavity and subsequent
expulsion of the plant matter occurred. Algal fragments were
larger or comparable to the largest inorganic rocks found during
pellet examinations.
Dimensions of the largest sand grains found in waste prod¬
ucts of each species exceeded the tentacular clearance dimensions.
Largest dimensions of algal patches also exceeded tentacular
clearance restrictions in all species except Synoicum.
When individually introduced, Pollicipes larvae were ingested
and digested by the largest sized animals—Styela, Ascidia, and
Perophora. Larvae fed in large quantities, however, were
actively rejected by all three tunicates. Larvae dimensions ex¬
ceed or match tentacular clearance estimates but are smaller than
all mouth sizes (figure 4). Ascidia larvae averaging 0.85 mmx 0.12
mm were introduced to and digested by Styela and Ascidia.
Sand grains of 0.6 mm cross-section were fed to Styela
and Ascidia. It was impossible to experimentally introduce
smaller grains. Twenty-five grains were individually placed into
their oral siphons at one minute intervals and all were rejected.
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Rice and Cotsirilos
Discussion—Feeding
Data suggest that algal pieces are the largest particles
acceptable to the five tunicates. However, tentacular clear¬
ance measurements indicate that many particles found in fecal
pellets must have contacted the screen without eliciting
pharyngeal contractions. Among such particles were sand grains
larger than the largest algal fragment ingested by Ascidia. On
the other hand, animals fed sand grains of 0.6 mm cross-section
immediately after ingestion of larger sized algae consistently
rejected these grains. The parameter most likely to prevent in¬
gestion of a grain particleappears to be its mass rather than its
size. This conclusion contradicts a study made by Hecht on Ascidia
atra ( - Ascidia nigra) who claimed "The selection of its food-
if mere exclusion may be called selection-- is made on the basis
of size, and rejection depends on the mechanical stimulation by
the larger particles" (Hecht, 1918). Sand grains dropped
through the water column into the oral apertures were immediately
expelled; particles placed into the siphon would occasionally sit
within the tentacular mesh for several minutes before rejection.
Gravity's effect on a sand grain is considerably more pronounced
than on an algal fragment of comparable size. The disparity in
size acceptance is clearly depicted for Styela in figure 3, which
accepted plant fragments larger but less massive than the largest
rocky material. Algal fragments descended slowly and often were
accelerated by the animal's own intake suction. In the sand
particle study which produced 100% rejection, a grain's velocity
greatly exceeded the oral intake velocity. Impact force of grains
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Rice and Cotsirilos
against the tentacular screen, especially when dropped through
the water column, exceeded that of algal fragments and provoked
rejection.
Observations on oral tentacular sensitivity were also made
in Hecht's study of A. nigra. He performed similar sand grain
experiments. Pharyngeal contractions following ingested sand
grains led him to discount earlier hypotheses by Roule and
Lecaze Duthiers et Delage who both suggested non-innervation of
tentacular screens. Our study supports Hecht's theory that oral
tentacles are innervated and capable of excluding certain items
from entering the digestive tract.
Pollicipes larvae were used to determine if live animals
were acceptable to tunicates. Their mass was less than the
rejected rocks and they were not limited to free-falling motion
due to their own mobility. Observations on Synoicum indicated
that larvae could actually swim against an incurrent of carmine
and were often seen swimming in the siphon opening but not
actually striking the tentacles. An errant leg of the larva
against the tentacles would elicit a brief contraction, ejecting
the barnacle larva from the oral siphon. Entanglement within
the tentacular screen and ensuing frenzy of beating appendages
by the larva initiated violent contractions by the tunicates,
preventing ingestion of barnacle larvae. Styela and Ascidia
accepted larvae readily. It is plausible that these largest
ascidians, due to their powerful water intake, frequently ingest
larvae parts (or particles of equal mass) and are therefore
insensitive to them. Larvae may also be moving so rapidly
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Rice and Cotsirilos
that they span the tentacle mesh with little chance of eliciting
a rejection response. Barnacles force-fed in large numbers to
both species were readily rejected; presumably their additive
mass and appendage movements are enough to stimulate an expul¬
sive contraction by the larger ascidians. Perophora repeatedly
repulsed larvae but fecal analysis indicated that they were indeed
ingested and digested by this tunicate. Careful procedures in
larvae selection ensured that only live animals were fed, thus it
can be assumed that larvae cleared the tentacular screens,
regardless of appendage movements. Perophora only allowed the
smallest of larvae to pass into its gut, suggesting that they
stimulated the tentacles the least.
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Rice and Cotsirilos
Summary-fecal pellet contents
1. Diatoms and dinoflagellates account for a large percentage
of a tunicate's diet.
2. An increase in diatom content in identical fecal weights
correlates well with increasing numbers of digested diatoms.
3. No correlation is seen between the gross structure of fecal
pellets and their contents.
4. In our sample, species morphology appeared related to
dinoflagellate abundance in their fecal pellets. Perhaps
the taller individuals (Styela) or stalked colonials are
feeding upon a water column containing more dinoflagellates
than are those species growing closer to the substratum.
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Rice and Cotsirilos
Summary—Feeding
1. All five species involved in the feeding study consistently
accepted algal pieces larger than tentacular clearance dim¬
ensions, indicating that while tentacular contact occurred,
ingestion was still allowed.
Consistent rejection of sand grains smaller than readily
2.
accepted algal patches suggests that a screening mechanism
is indeed available to tunicates but does not operate strictly
on size of the particle encountered. This rejects Hecht's
(1918) appraisal that ascidians select their diet on the
basis of size alone which would predict that larger particles
are preferentially excluded.
The force of contact applied by an ingested particle on the
tentacle network is closely correlated to its probability
of being rejected. Dropped sand grains, accelerated by
gravity, are more likely to be ejected than a grain of com¬
parable size gently rolled into the oral siphon.
Hecht (1918) proposed that detection of ingested matter
occurred at the tentacle screen, suggesting innervation of
these tentacles. We support this conjecture.
4.
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Rice and Cotsirilos
Suggestions for further study
1. An interesting follow-up to this project would be to elucidate
the maximum sized particle a species might be able to digest.
Perhaps tentacular irritation can be bypassed by anastheti¬
zation, in which case a large organic particle could be pass¬
ed through the tentacles into the branchial basket. Ob¬
servations on the fate of this artificially fed particle
could be made after the animal recovers.
2. Mechanism used to construct the species specific fecal
architecture and location of these mechanisms within the
digestive tract could be studied by examining the consis¬
tency and shape of fecal material at different sites of the
tract.
c
Rice and Cotsirilos
Acknowledgement
We thank Dr. Isabella A. Abbott and Dr. Donald P. Abbott
for their encouraging suggestions and willingness to spend
long hours with us.
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Rice and Cotsirilos
Literature cited
Arakawa, Kohman Yi 1971. Studies on the faecal pellets of marine
invertebrates (excluding molluscs) I. Publ. Seto Mar. Bio. Lab.,
19: 231, 239-241.
Edge, Elton R. 1934. Faecal pellets of some marine invertebrates,
Am. Midl. Nat.. 15 (1): 82-84.
Fiala Medioni, Aline 1978a. Filter feeding ethology of benthic
invertebrates (ascidians).III. Recording of water current
in situ—rate and rhythm of pumping, Mar. Biol. 45: 185-190.
Fiala Medioni, Aline 1978b. Filter-feeding ethology of benthic
invertebrates (ascidians).IV. Pumping rate, filtration rate,
filtration efficiency. Mar. Biol. 48: 243-249.
Fiala Medioni, Aline 1978c. Filter-feeding ethology of benthic
invertebrates (ascidians).V. Influence of temperature on
pumping, filtration and digestion rates and rhythms in
Phallusia mamillata, Mar. Biol., 48: 251-259.
Hecht,Selig 1918. The physiology of Ascidia atra Lesueur.I. General
physiology.II. Sensory physiology. J. Exp. Zool. 25: (1) 266-270.
0
Rice and Cotsirilos
Table 1
Although captions for fecal matter are given in
terms of micrograms per pellet, Styela data are
represented as micrograms per length. Amount of
fecal matter is represented as number of individual
fecal pellets.
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Rice and Cotsirilos
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Rice and Cotsirilos
Table 2 Group L contains lobe shaped species and
Group M contains mounded colonies.
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Rice and Cotsirilos
-22-
Table 2

Number of dinoflagellates in the
Group L
50 microgram sample of fecal pellets
Polyclinum planum
148

Clavelina huntsmani
118




Synoicum parfustis
115



Styela montereyensis
114

Group M


Aplidium solidum
88

45
Archidistoma psammion




37
Archidistoma molle


—
e
Rice and Cotsirilos
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The column of numbers on the left represents the
Figure 1
percentage of all diatoms found in the fecal
pellets of each species which were either partially
digested or completely empty frustules. A number
has been placed at the tip of the diatom bars to
facilitate exact comparisons.
Rice and Cotsirilos
-24-
Percent of all diatoms
Figure
which are digested
k
Clavelina huntsmani
388
2

H

90
30
H
Archidistoma molle
34

—
+
57
94

23
—
Aplidium solidum
—23
88 S
20
H
Polyclnum planum
199

88



Styela montereyensis
—17
II
I

80


Archidistoma psammion
—80
II
Ldiatoms
76
10
22
I dinoflagellates
Synoicum parfustis
178
animals / misc.
s
74

Hother algae


L
200
50
100
50
Number of items in 5 x IO gramsof fecal pellets

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Rice and Cotsirilos
Figure 2
In order to show details better, most of the pelle
are shown enlarged by the given factors. For in¬
stance, the sketch for Aplidium solidum must be
reduced by a factor of six in order to make a scaled
comparison with Styela. Arrow indicates separation
point between two Styela pellets. The Styela is 8.5
times longer than life size.
Rice and Cotsirilos
Figure 2
Styela montereyensis
1

Clavelina huntsmani

2X
Bol

Synoicum parfustis





Polyclinum planum
5X

Archidistoma molle
side view
3X

Aplidium solidum
6X
3)

cross section
2

-26

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Rice and Cotsirilos
Figure 3 Horizontal line within a bar indicates the smaller
of two dimensions. Vertical line superimposed on
mouth size data indicates range of mouth sizes that
were measured. Pollicipes larvae were measured along
their greatest dimension which involved distances
between laterally projecting appendages.
Rice and Cotsirilos

N
E
5

wr . .
1020
azo00
9



11
0



EHDE
vaao
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r 1 1 0 l


—


2092•0
H
5
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*
t10 18h0
O


O2br

L

0



*.
t
L
w4ro 1780
o
jj
so se talleel

0
o

kk


T
——



aa-
2OX60
l

—

S
-28-
e
Rice and Cotsirilos
Figure 4 Diagram of mouth is depicted in the top row.
Circles and rectangles in rows 2 and 3 repre¬
sent the largest area that can fit through the
tentacular screen without touching a tentacle.
Largest sand grain found in fecal matter of
each animal is represented schematically in the
bottom row. All diagrams for Styela, Ascidia,
and Perophora are enlarged by a factor of 4.3.
Diagrams for Synoicum and A. psammion are
drawn at 2X relative to the other three species.
Rice and Cotsirilos
——

I

J
O
O
*-
L
C
C

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