ABSTRACT
The cirratulid polychaete, Cirriformia spirabrancha,
was found to tolerate anaerobiosis up to forty-eight hours.
However, the biological advantage for the organism of anae-
robiosis is approximately twenty-four hours. Longer regimes
leave the animal moribund and subject to environmental ac-
tivities such as wave action which would disintegrate the
worm before recovery from anoxia. The length of the tentacles
and body increases as oxygen tension is lowered, but once
anaerobic conditions are reached the tentacles contract and
coil tightly, while the body remains elongate. Cyanide, for
reasons still unknown, is fatal within twenty hours. Whether
carbohydrates, which comprise 0.97% of the dry body weight,
are utilized during anaerobiosis is still unknown.
INTRODUCTION
The cirratulid polychaete, Cirriformia spirabrancha is
found in black mud of low oxygen content in intertidal and
subtidal areas along the central California coast. Other
polychaetes found in similar area, such as Arenicola (von
Brand, 1946), are capable of surviving anaerobiosis for per-
iods of several days. Little is known, however, about the
anaerobic potentialities of cirratulid worms. The present
investigation shows that Cirriformia can tolerate up to
forty-eight hours of anaerobiosis.
MATERIALS and METHODS
Anaerobic conditions were established in two ways. In
the first, worms were plaeed in soft glass Pasteur pipettes,
and the wide end of the pipette was heat sealed in a Meker
burner. The pipette was then filled, using a syringe, with
sea water deoxygenated by one hour of bubbling with nitrogen,
and the fine end was then heat sealed as above. Calculations
based on their known respiratory rate (Gladfelter, 1968) in-
dicate anaerobic conditions were established within one hour
under these conditions.
In a second method, sixteen ounce jars were half-filled
with either mud or fine glass beads, fifteen to twenty worms
placed on this substrate, and the jar filled with sea water.
An oxygen electrode (Yellow Springs Instrument Company,
Model 51 0, Meter) was inserted through the lid, and the lid
sealed with lubriseal (Arthur H. Thomas Company, Philadelphia).
Anaerobic conditions were then established by allowing the
worms to utilize the available oxygen as determined by the
oxygen electrode reading.
Survival was assayed by removing the worms from the anaerobic
vessels and placing them on fine glass beads in finger bowls
in running sea water. If no movement was immediately evident
they were prodded with a glass stirring rod.
Carbohydrate determinations were made colorimetrically
by the phenol-sulfuric acid method of Dubois (1956) at 490 m
on a Beckman DU spectrophotometer with Gilford attachment.
Whole animals were homogenized in 2 ml of 70% ethanol, and
then brought to a final volume of 10 ml with 70% ethanol.
O.l ml of a saturated sodium sulfate solution was added to
facilitate precipitation (Van Handel, 1965), and the suspen-
sion centrifuged for twenty minutes on a clinical centrifuge.
Two ml of this supernatant was then analyzed for carbohydrate
as described above. The pellet was then resuspended in 3 ml
of cold 10% trichloroacetic acid (TCA), boiled for one min-
ute, and centrifuged. Two ml of the supernatant, diluted
ten to one with 10% TCA, was then tested for total carbohy-
drate. Concentrations of sugar were determined from a stan-
dard curve of absorbance against micrograms of sugar.
RESULTS
Although worms could tolerate anaerobic conditions up
to sixty hours, forty-eight to fifty hours was the extreme
for most individuals. Recovery periods of two to three days
were required for animals after forty-eight hours of anaer-
obiosis. In many cases, however, after "living" for three
days, the animals would then die.
A survival period of up to forty-eight hours was found
for anaerobiosis, but worms could only survive a maximum of
twenty hours in 0.001 M cyanide. The reasons for this are
unclear, but may be related to nonspecific effects of cya-
nide on other enzymatic systems (Bellamy, 1968).
Behavioral observations showed that as oxygen tension
decreased, the length of the tentacles increased, but once
anaerobic conditions were established the tentacles coiled
tightly. After twenty-four hours in the sixteen ounce jars
in an anaerobic regime, animals moved out of the substrate
and lay in an elongate form on the surface. As the body can
respire at normal rates without tentacles (Gladfelter, 1968)
this may be a behavioral mechanism for utilizing any trace
oxygen.
Upon removal from anaerobic regimes of twenty-four hours
duration or less, burrowing began within a few hours. After
thirty-six hours of anaerobiosis recovery to a burrowing be-
havior requires one to two days. Worms under anaerobiosis
for forty-eight hours were moribund, with a few individuals
burrowing after a recovery period of three days. Many worms
from forty-eight hour anaerobic regimes, however, died after
exhibiting movement for three days.
The biological advantages arising from the ability to
tolerate anaerobic conditions would appear to be limited to
approximately twenty-four hours or less. Periods of longer
duration leave the animal in such a moribund state that en-
vironmental activity such as wave or current action would
disintegrate the animal before recovery from the effects of
anoxia.
Tests for total carbohydrate changes during anaerobiosis
over a forty-eight hour period were inconclusive. Variance
within the populations tested was extreme, and ranged from
697 micrograms of carbohydrate per gram dry wieght to 22,456
Agm per gram dry weight. Total carbohydrate appears to be
o.97% of the dry weight. Difficulty in establishing con-
sistent standard curves casts some doubts on the validity of
the method of preparation. The possibility that carbohydrate
metabolism is not utilized as a msjor pathway must be con-
sidered until metabolic end products are determined. Both
control and experimental snimals existed in a starved con-
dition, but aerobic worms showed much more activity than
anaerobic worms, and must have used some stored energy source,
which could decrease differences in levels, and increase
varibility.
SUMMARY
The cirratulid polychaete, Cirriformia spirabrancha,
was found to tolerate anaerobiosis up to forty-eight hours.
However, the biological advantage for the organism of anae-
robiosis is approximately twenty-four hours. Longer regimes
leave the animal moribund and subject to environmental ac-
tivities such as wave action which would disintegrate the
worm before recovery from anoxia. The length of the tentacles
and body increases as oxygen tension is lowered, but once
anaerobic conditions are reached the tentacles contract and
coil tightly, while the body remains elongate. Cyanide, for
reasons still unknown, is fatal within twenty hours. Whether
carbohydrates, which comprise 0.97% of the dry body weight,
are utilized during anaerobiosis is still unknown.
TABLE 1
Number surviving after
Number of hours of
Number of worms
3 days
anaerobjosis
10
12
10
10
48
10
Tolerance of Cirriformia spirabrancha to anaerobic
Table 1.
conditions.
TABLE 2
Number surving after
Number of worms
Number of hours in
48 hours
cyanide
20
Table 2. Tolerance of Cirriformia spirabrancha to 0.001 M
cyanide
ACKNOWLEDGMEN!
I would like to express my sincere thanks to Drs. D.
Epel and J. Pearce, and to the faculty and staff of Hopkins
Marine Station for their help and advice. This work was
supported in part by the Undergraduate Research Participation
Program of the National Science Foundation Grant GY-4369.
BIBLIOGRAPHY
Bellamy D. & Petersen J.A. (1968) Anaerobiosis and the
toxicity of cyanide in turtles. Comp. Biochem. Physiol.
24, 543-548.
Dubois M. et all (1956) Colorimetric method for determin-
ation of sugars and related substances. Anal. Chem.
28, 350-356.
Gladfelter, E. (1968) Certain aspects of the respiration
iformia spirabrancha.
rate of the cirratulid polychaete, Cir
Reasearch report, Hopkins Marine Station.
4. Van Handel E. (1965) Estimation of glycogen in small
amounts of tissue. Anal. Biochem. 11, 256-265.
5. von Brand T. (1946) Anaerobiosis in Invertebrates.