805
INTRODUCTION
Cirriformia spirabrancha (Moore, 1904) is a small polychaete
worm found in large numbers along the California coast (Ricketts
and Calvin, 1952). The worm, which bears a number of tentacles on
the anterior end of the body, lives buried in the sand or mud with
its tentacles usually out of the substrate and waving in the water.
The worm is found both intertidally and subtidally. Being basically
a sedentary animal and living in large clumps, it, or its tentacles,
should offer a food source to a number of predators. This, however.
has not been observed and no previously published literature men-
tions any predation on C. spirabrancha.
This paper is an attempt to determine whether, in fact, there
is significant predation on adult C. spirabrancha and to report an
amine present in the animal that may lessen predation.
MATERIALS & METHODS
Predation observations
Observations were carried out in the field in Pacific Grove
and Montery, California. These were made at all times of the day
and night and throughout the tidal cycle. Observations were made
both by wading in hip boots and also by scuba diving up to a depth
of 15 feet. All types of predation were looked for including pred-
ation by other invertebrates, by fish and also by shore birds.
After several weeks of field work, suspected predators were taken
into the laboratory for closer scrutiny.
The animals were maintained in 5 gallon aquaria filled with
running sea water. Two aquaria were set up for each species of
predator. One contained 4 inches of sand and mud and the other
had no substrate. Suspected predators were left in a tank for +
days with 10 specimens of C. spirabrancha and then specimens of
Notomastus tenuis Moore, 1909 and Lumbrineris erecta (Moore, 1904),
two other marine polychaetes, were placed in the tank and left for
another 24 hours.
Predators observed in the laboratory were: the polychaetes
Nereis grubei (Kinberg, 1866) and Glycera convoluta Keferstein,
1862; the crab Cancer gracilis Dana, 1852; the nudibranch
Hermissenda crassicornis (Eschscholtz, 1831); two types of small
intertidal fish, Oligocottus snyderi Greeley and Clinocottus
analis (Girard) (As determined by keys in Light, et. al., 1954).
Attempts were also made to directly feed the fish different
sections of C. spirabrancha, tentacles, pieces of body wall only,
and also pieces of gut alone.
Extraction and isolation
1. Assay. The assay used to detect the presence or absence
of the noxious substance in C. spirabrancha was soaking N. tenuis
or L. erecta in C. spirabrancha extract for 5 minutes and then
offering these to the fish. The same thing was also tried with the
steam distillate of extract of C. spirabrancha. If the fish rejected
the food it was assumed the noxious substance was present; if the
fish ate the food immediately it was considered absent.
2. Extraction. Extract was made of the entire body, of
tentacles alone, of body wall alone and also of the anterior and
posterior parts of the body alone. Extract was made by grinding
C
07
the tissue with mortar and pestle and then homongenizing it with
a teflon homongenizer. The extract was centrifuged for 10 minutes
at 2000 rpm and the sediment disgarded.
3. Alkaline steam distillation. Extract (not centrifuged) was
raised to a pH 9.5 by the addition of 1ON NaoH and placed in a 25 ml
test tube connected by plastic tubing to a second test tube placed
in an ice bath. The tube in the ice bath was connected by a second
piece of tubing to an asperator. The tube containing the extract
was heated in a water bath and the distillate collected under
vacume.
4. Purification. Extract was mixed with an equal volume (50
ml) of IN HCl. This was centrifuged for 10 minutes at 2000 rpm
and the supernate saved. The pellet was resuspended in 30 ml of
IN HCl and again centrifuged as before. The two supernates were
pooled and then made strongly basic by the addition of lON NaoH.
This solution was extracted twice with 150 ml of anhydrous ether.
The aqueous phase was disgarded and the ether washed twice with
50 ml. of water. The ether was extracted five times with 10 ml
of 1N HCl which was pooled, washed once with ether (25 ml), and
kept for analysis.
5. Identification. The first attempt at identification was
the Hinsberg procedure for distinguishing between primary, secondary,
and tertiary amines. Further attemps at identification were those
given for amines by McElvain (1946).
RESULTS
1. Predation observations. Table 1 summerizes the reactions
208
of the suspected predators to C. spirabrancha. All of the predators
would eat N. tenuis or L. erecta before C. spirabrancha. The
immediate reaction by the fish tested was to spit out all parts
of C. spirabrancha taken into the mouth. All parts of the worm
were repulsive to the fish. This reaction still continued after
14 days of starvation. The only change was an increase in the number
of attempts to eat the worm. One piece was taken in the mouth 33
times between 7 different fish before being abandoned as unpalatable.
2. Presence of noxious substance. N. tenuis and L. erecta,
which normally are immediately devoured by the fish, were like-
wise spit out after being soaked in C. spirabrancha extract.
This reaction lasted, on the average, about 1 minute--the time
it would take for the water to wash the extract from the soaked
worms. After this, the fish would eat the worm. Extracts of
various parts of the body worked equally well with no localization
of the adverse compound apparent.
N. tenuis and L. erecta soaked in distillate of C. spirabrancha
extract were again unpalatable to the fish until the distillate
washed off. Worms soaked in the residue left after steam distillation
and then introduced to the fish were immediately eaten by the fish.
Injecting 1 cc of extract down the throats of several fish
produced no ill effects although the fish tried to cough it up
after it was placed in their throats.
The steam distillate had the extremely pungent odor of C.
spirabrancha extract and the ether extraction product in alkali
solution had the same characteristic odor.
209
3. Identification of noxious substance. Upon addition of the
benzenesulfonyl chloride in basic solution a white precipitate
appeared. This, however, disappeared upon dilution with water.
When made acidic a second white precipitate appeared. Although
a primary amine by the reaction in acid, the appearent positive
reaction in alkali suggests that there is a second functional
group on the molecule.
DISCUSSION
The obvious lack of predation on C. spirabrancha points to
some means of protection for the worm. On the basis of the results
it is evident that this protection is two-fold.
The first means of protection is the substrate itself. C.
spirabrancha lives in the black sand and mud found in the inter-
tidal region. This mud would probably discourage predation by
those animals which normally don't burrow as deeply as C. spira¬
brancha (i.e. H. crassicornis).
Secondly, and more important, is the bad taste of the worm
itself. Although not poisonous, the worm is noxious enough that
predators choose other prey before C. spirabrancha.
The bad taste seems to be from a compound found throughout
the body tissue. This compound can be seperated from the body
extract and the remainder of the extract is completely palatable.
The compound appears to be a primary amine-its further structure
being unknown at present.
SUMMARY
The lack of predation on C. spirabrancha seems to be due mainly
R
TABLE 1
Predation on C. spirabrancha
REACTION IN
REACTION TO
PREFER OTHER
ANIMAL
SUBSTRAT
WORMS *
EXPOSED WORM
Pos.* Neg.*
Pos.
Neg.
Neries grubei
2. Glycera convoluta
X
3. Cancer gracilis
X
4. Hermissenda
X
X
crassicornis
Oligocottus snyderi
X
X
6. Clinocottus analis
X
X
* Positive reaction means at least 1 worm eaten in 4 day period
Negative reaction means all 10 worms present after 4 days
** N. tenuis and L. erecta eaten before C. spirabrancha
C
to a compound, found within the body tissue, which has thus far
been identified only as a primary amine. This compound seems to
make C. spirabrancha unpalatable to other animals.
ACKNOWLEDGEMENTS
I'd like to thank, among others, my advisor, Dr. Pearse, for
his help and also Dr. Phillips without whose help I couldn't have
completed the chemical end of this paper.
This work supported in part by the Undergraduate Research
Participation Program of the National Science Foundation Grant
GY-4369.
BIBLIOGRAPHY
Light, S.F., Smith R.I., Pitelka F.A., Abbott D.P. & Weesner F.M.
(1954) Intertidal Invertebrates of the Central California Coast,
University of California Press, Berkeley, California.
McElvain, Samuel M. (1946) The Characterization of Organic Compounds,
The MacMillian Company, New York.
Ricketts and Calvin (1952) Between Pacific Tides, Stanford University
Press, Stanford, California
80
C