The ability to regenerate has been found to vary greatly among poly-
chaetes. At one extreme are Ctenodrilus (Korscheldt) and Dodecaceria
(Dehorne 1933; Martin 1933), which can regenerate completely from single
REGENERATE
segments. At the other extreme is Aphrodite, which cannotany lost seg-
ments (Clark and Clark 1962). Extensive work on the caudal regeneration
of a Nereid, Nereis diversicolor, has shown that the pygidium regenerates
within eight days of caudal amputation, and as many as twelve segments
regenerate within thirty days. However, it has been noted that there is
much individual variation in the number of regenerated segments (Clark
and Evans 1961).
There has been little study of Cirratulid regeneration. One species,
Dodecaceria, is able, as stated above, to regenerate entire worms from
individual segments. Another Cirratulid, Cirrineris, has been found to
regenerate a prostomiumand six to seven segments within ten days, and a
pygidium and twenty segments within sixteen days (Stone 1935).
In this investigation, the regenerative ability of the Cirratulid
Cirriformia spirabrancha (Moore, 1904) was tested with respect to various
parameters such as size of the animal and location of the wound. The
results of these experiments show that, under the laboratory conditions
used, C. spirabrancha does regenerate certain body parts, but at a slow
rate, and possibly to a limited extent, in comparison to Cirrineris and
certain other polychaetes, Nereis diversicolor (Clark and Bonney 1960)
and Nephtys (Clark and Clark 1962).
Materials and Methods
The C. spirabrancha used in these experiments were collected in
Monterey yacht harbor in spring 1968. The animals were kept in the lab-
oratory no longer than three days before being used.
It was found that unlike N. diversicolor, which autotomizes immediately
anterior to the point at which pressure is applied with tweezers (Clark
and Bonney 1960), C. spirabrancha remains intact after being squeezed.
This necessitated making a more unnatural wound in order to amputate
caudal segments. The appropriate cuts were made with a razor blade while
the animals were anaesthetized in isotonic magnesium chloride solution.
Within a few minutes after the animal is cut, the wound is closed by con-
striction of the circular muscles of the two or three segments nearest
3/
the wound, and by swelling of the gut. It was reasoned that the most
critical period for the occurance of infection was before this process
of wound closure was complete. Therefore, following the operations, the
animals were kept for fifteen or thirty minutes in a container of sea
water to which had been added 100,000 i.u./1. of penicillin and 100 mg./1.
of streptomycin. The animals were then moved to aquaria containing 13-15°0.
sea water, in which groups of twenty-five to thirty animals were maintained
without feeding for the duration of the experiments.
Results
Wound closure, appearance of regeneration
As stated above, wound closure is accomplished by constriction of
muscles and expansion of the gut of the segments nearest the wound. This
process is similar to findings with other polychaetes. If a wound has
become well healed, the last segment is rolled inward posteriorly, providing
a small, smooth opening for the gut.
The first signs of regeneration appear in ten to twenty days in smaller
(40-60 mm) animals, and within sixteen days in larger (70-100 mm.) animals.
At this time a small bulb of tissue can be seen on the posterior surface
of the last segment, ventral to the anal opening. Within six more days,
in both large and small animals, the pygidium has acquired the size and
shape of a normal pygidium. In no case was concrete evidence of regeneration
of segments found in these experiments.
Sex
An experiment was carried out to determine if ability to regenerate were
affected by the sex of the animal. However, the only identifiable external
feature distinguishing sexes in C. spirabrancha is the pale yellow coloring
of males filled with sperm. Using this feature as a criterion for separ-
ating large animals into two groups, one of "obvious males" and another of
animals of indeterminate sez, the caudal twenty per cent of the animals
was removed. Although all of the "obvious males" had died within eight
days, more than half of the animals of indeterminate sex were still healthy
after eighteen days. One member of this group was still alive and healthy
at the end of the thirty-three day observation period, although there was
no evidence of any regeneration.
Size
To determine if size affects regenerative ability, animals of indeter-
minate sex were divided into three groups: thick animals seventy to one
hundred millimeters long, thin animals seventy to one hundred millimeters
long, and animals forty to sixty millimeters in length. Within sixteen
days, all of the longer animals were dead or decaying. Over half of the
shorter worms, however, were still alive and healthy after twenty days,
and were beginning to regenerate pygidia.
Region of cut
It has been found in work on Nereids that there is an axial gradient
of regenerative potential (Golding 1967). To determine if such a gradient
exists in C. spirabrancha, three experiments were conducted. In the first,
large animals of indeterminate sex were cut into six sections. Although
no regeneration was found, there were differences in length of survival
times for different areas of the animal (Text fig.la). In the second exper-
iment, the posterior 1/10, 1/5, and 2/3 respectively of three groups of
larger worms was removed. Cuts were made in the same three locations on
3
small animals in the third experiment. Results of these experiments can
be compared in Text-figs. 1 and 2.
Brain extirpation
Brain extirpation experiments were performed by slicing off the anterior
tip of the prostomium, resulting in a small wound and little loss of tissue.
Control groups consisted of worms from which a wedge of tissue, approxi-
mately equal in volume to the tip of the prostomium, was removed from the
dorsal surface of the prostomium, just posterior to the brain. When the
supraesophogeal ganglion was removed from a group of small worms, the wounds
healed within six days, and noticeable replacement of tissue began after
thirteen days. A light band of tissue, possibly a regenerating supraesoph-
ogeal ganglion, was observed in most of the animals after nineteen days.
In another series of experiments, the brain and either the posterior
1/5 or the posterior 2/3 of caudal tissue were removed from large and small
worms. Control groups from which similar lengths of caudal tissue were
removed, were used as well. The only group surviving these operations was
the control group consisting of small worms from which the caudal ten per
cent had been removed. Approximately half of this group was alive thirteen
days after the operation, at which time all of the worms in the other groups
had died.
When resuilts of sinilarly treated groupe from different erperinents
are compared, the data is sometimes contradictory. For example, half of the
animals of indeterminate sex used in the experiment on effect of sex on
regenerative ability survived eighteen days. However, similar worms, used in
the experiment on effect of size, were all dead within sixteen days, even
though the percentage of material removed was the same as above, Similar
variation was seen in the brain extirpation experiment. The members of
two control groups died within eleven days, whereas over half of the animals
in a third control group were healthy fourteen days after amputation.
IDENTIFICALION  REEENERADON
Regeneration in the polychaete Nephtys is identifiable by the lighter
color of the regenerated segments (Clark and Clark 1962). C. spirabrancha
were found in the field having posterior segments distinctly lighter than
the rest of the worm, suggesting that regeneration had taken place. However,
animals kept in the laboratory became bleached from their original dark
green color to light brown. Regenerating pygidia were the same light brown
color. It is therefore possible that segments were regenerated in some
instances, and went unnoticed because they were the same color as the older
segments. This possibility seems unlikely however, since no difference
in size or parapodial development of caudal segments was observed.
Tentacles
The tentacles of C. spirabrancha arise from two general areas: a band
running laterally across the dorsal side of the animal between the fourth
and fifth segments, and singly, from the dorsal side of the notopodia, at
apparently random intervals down the length of the animal.
C. spirabrancha were found in the field having no tentacles between
the fourth and fifth segments. These were kept as experimental animals,
and died within ten days. In a similar laboratory experiment, all tentacles
between the fourth and fifth segments were removed from thirty worms, with a
small scissors. These animals were dead after seven days.
In another experiment, one tentacle, the anterior-most blood filled
tentacle on the right side of the prostomium, was removed from twenty-five
worms. The amputation resulted in the blood being squeezed from the
tentacle stub remaining after amputation. Within two days, many of these
382
stubs had filled with blood. After nine days, those tentacles which had
regenerated had reached the length (15-20 mm.) of many of the intact
tentacles, and as do the older intact tentacles, had developed white and
green portions. After twenty-seven days, five of the ten surviving animals
had regenerated tentacles, which were twenty to twenty-two millimeters in
length.
Discussion
A wide range of caudal regeneration rates has been found in this
study of C. spirabrancha. However, even the fastest regeneration time is
much slower than those reported for Cirrineris, even though the Cirrineris
were kept in bowls of sea water and presumably starved (Stoned 1935), as
were the C. spirabrancha in these experiments. The fantastic regenerative
ability of Dodecaceria, a member of the same family as Cirriformia, provides
an even greater contrast to the observed slow regeneration rates.
It is not known if number of segments or size is a good indication of
the age of the animal but as can be seen in Text-figs. 1 and 2, there does
appear to be correlations between the size of the animal, and ability to
survive wounding, rate of regeneration, and ability of different regions to
regenerate.
As also noted (Text-fig. 2b), an axial gradient of regenerative ability
may be present in small animals, since regenerative rate increases with
the amount of tissue removed, which is in agreement with the results of
Golding (1967). However, it must be noted that my data measures the length
of time required to regenerate a pygidium, whereas Golding measured the
number of segments regenerated in a given time.
The results of the two experiments on larger animals show better
survival in the posterior and anterior regions. Also, ability to regener-
ate a pygidium was found only in the anterior and posterior regions of
large worms. The reasons for the poorer survival and longer time before
commencement of pygidial regeneration of large animals may be related to
growth rate and age. Large animals have done most of the growing that they
will do. The proliferating area of the pygidium is perhaps slowing prod-
uction of new segments, and segment production may have stopped entirely.
If this is the case, it seems logical that adults would have less ability
to regenerate lost or injured body parts. But if some areas are to retain
more regenerative potential than others, it stands to reason that the ends
of the animal, the regions which are probably exposed the most to injury
and predation, would have the higher potential.
The data collected on regeneration in larger animals appears to support
the conclusions drawn from studies on wound survival. Larger worms did not
regenerate if the wound was made in the middle of the animal. Regeneration
did occur when wounds were made in posterior or anterior regions. The
first signs of regeneration in these areas appeared sixteen days after
amputation. This is intermediate to the range of regeneration rates found
in smaller animals.
Although experiments with "obvious males" and worms of indeterminate
sex did not subdivide the animals into the two sexes, it did indicate that
males with sperm have less ability to survive caudal amputation. This poor
survival ability could be characteristic of males, or of males with sperm,
or of animals of either ser having gametes. In any case, these results
indicated that sexually mature males are poorly suited for studies of
regeneration.
36
Experiments concerning the effect of the brain on caudal regeneration
have been done by several workers on N. diversicolor. In these studies
the worms were able to survive simultaneous removal of the supraesophogeal
ganglion as well as some caudal tissue, although they were not able to
regenerate any caudal segments (Clark 1960, 1961,1962), (Herlant-Meewis
1964), (Golding 1967). This contrasts with the results with Cirriformia,
from which removal of both the brain and caudal segments resulted in death.
Loss of the brain was probably not the sole cause of death however, because
animals from which only the brain was removed survived quite well. The
shock of the two simultaneous wounds may be the cause, since two of the
three control groups died within thirteen days.
According to the results of experiments in which tentacles were removed,
Cirriformia cannot survive a large loss of tentacles from one location.
Since animals lacking tentacles were found in tle field and since the tent-c
acles are exposed to injury more than any other part of the worm, the
failure of the worm to survive the loss of many tentacles is surprising.
However, the loss of a single tentacle at one time may be more likely
under natural conditions, and the ability of the animals to survive and
replace single tentacles is therefore not surprising. It is of interest,
however, that so few of the animals (1/5-1/4) were capable of replacing
the lost tentacle.
That the conditions under which the animals were handled and kept were
not the best for Cirriformia can be seen by the high mortality rate of the
animals. The fact that the animals were starved during the experiments
may be the major factor in their poor survival, although results of starving
other polychaetes show much lower mortality rates in most cases.
38.
Another possible factor contributing to the high mortality rate might
be the practice of keeping the animals clumped in groups of up to thirty
worms. This close contact probably causes the spread of infection from
decaying animals to healthy animals. As a check on this possibility, intact
worms were added to three groups of animals from which caudal tissue had
been removed. With only one exception, all the intact animals had died of
infection ty the time the amputated animals had died.
All these factors, and probably several others, add up to great
ambiguity regarding the regenerative ability of Cirriformia. Where experi-
mental groups were duplicated, results are sometimes contradictory. However,
it can only be hoped that the groups compared to each other to yield the
conclusions, having been carried out at the same time, and using the same
techniques, have fewer variable differences and therefore more validity
than those contradictory results of experimental groups operated on at
different times.
Summary
1. Regeneration of the tip of the prostomium, the tentacles, and the
pygidium occurs in C. spirabrancha.
2. Regeneration of caudal segments may occur, but at a rate too slow
to be observed during the available time period. Regeneration of the
supraesophogeal ganglion may also occur.
3. There appears to be, in general, greater capacity for wound healing
and faster pygidial regeneration in smaller animals than in larger animals.
4. The presence of an axial gradient of regenerative ability in
small animals, and certain areas of increased wound healing ability and
386
regenerative ability in larger worms is indicated by results of several
experiments.
5. The conditions under which these experiments were conducted were
probably not best suited for survival and regeneration of C. spirabrancha.
Contradictory results were sometimes obtained from duplicate experiments.
Therefore the results of these experiments cannot be regarded as conclusive
proof of the conclusions drawn from them, although further experimentation
may reveal the causes of the discrepancies.
Acknowledgements
I wish to thank Mr. Roger Szal for his advice and technical assistance
throughout this research, and Dr. David Epel, for his advice and criticism
of this paper. This work was supported in part by the Undergraduate
Research Participation Program of the National Science Foundation Grant
GY-4369.
3
leferences
Clark, M. E. & Clark, R.B. (1962). Growth and Regeneration in Nephtys.
Zool. Jb. Physiol. Bd. 10, 24.
Clark, R.B. & Bonney, D.G. (1960). Influence of the Supra-oesophogeal
Ganglion on Posterior Regeneration in Nereis diversicolor. J. Emb.
101.
exp. Morph. 8, 112.
Clark, R.B. & Evans, S.M. (1961). The Effect of Delayed Brain Extirpation
and Replacement en Caudal Regeneration in Nereis diversicolor. J.

Embryol. exp. Morph. 9, 97.
Golding, D.W. (1967). Neurosecretion and Regeneration in Nereis diversicolor.
Gen. Comp. Endocrin. 8, 348.
Herlant-Meewis, H. (1964). Regeneration in Annelids. Advances in Morpho-
genesis, 4, 155.
Stone, R.G. (1935). Regeneration in the Cirratulid Cirrineris. Tortugas
Lab., Carnegie Inst. 29, 1.
20

2

30
5
6
2
3
OF CuTS
LOCATIONS


A
L

TExr-FIG. 1a.
LARGE ANINAL CuT
INTO SIX SECTIONS,
4-5

A

LA
TEXT-FIG. 1 b.  GROUPS OF LARGE
AMMALS CuT IN DIFFERENT REGIONS.
у оу оие оу ооооу ооoe
IEXT-FIG.
AMPUTATION. (1a) POSITION OE BAR ON X-AXIS CORRESPONDS
ONE OE SIX SECTIONS INTO WAICA ANIAAL WAS cur:
h) Posоуоу ое оооуео оае оу у-оуs ооввEsрos
JO REGION OF ANIMAL IN wUICN CuT WAS MADE.
TIME (IN DAYS) FOS LAST MEMBER DIE
TIAE (M DAYS) FOR DERTH OF 50% OF GROUI
20
o
LOCATIONS OF CuTS
O
8
TEXT-FIG. Ra. LARGE AMMAES.
a
TEXT-FIG. A b, SMALL ANIMALS.
TEXT-FIG. A. TE BEFORE FIRST EVIDENCE OF
REGENERATION IS OBSERVED IN (Ra) ANMMALS 70-100mm.
IN LENGTA AND (Rb) IN ANIMALS 40-60 mm. IN
LENETH. POSITIDN OF BAR ON X-AXIS CORRESPONDS
TO LOCATION OF WOUND FOR THAT
GRDUP.
100