4

INTRODUCTION
Little is known about digestive enzymes in polychaetes.
Where general classes of enzymes have been identified, their
origins and sites of action have often remained undetermined.
have attempted to describe the carbohydrases in the gut of
Cirriformia spirabrancha, a polychaete which inhabits sulfide-
rich mud and sand and apparently eats a widely varied diet of
microscopic debris. Laminarin, starch, and cellulose have
been found to be degraded by extracts of the gut of the annelid,
and enzyme action tentatively located in the stomach and fore-
intestine. The roles of the worm and its intestinal bacteria
in this digestion were examined without definitive results.
MATERTALS AND METHOLS
Large worms were collected fresh daily in the intertidal
area during low tide at Fisherman's Wharf, Monterey, California.
The specimens were anesthesized for one hour in magnesium chlor-
ide-sea-water solution, pH 7.7, and the alimentary tube then
dissected out. About .3ml. of tissue and contents was yielded
by the intact gut of a worm; .lml. by a gut opened and washed
free of contents. The gut was homogenized in the cold with a
Teflon pestle in a glass tube using an artificial sea-water (ln-
stant Ocean, Aquarium Systems Inc., Wickliffe, Ohio) as the momo-
genization medium. The supernatant after light centrifugation
in the cold was used as the enzyme extract.
Seven sugar substrates were tested: laminarin (Pierce
Chemical Co.), agar (Difco Chemical Co.), and chitin (Eastman
Chemical Co.) as 0.5% suspensions; fucoidin (Pierce Chemical
Co.), and alginic acid (Pierce Chemical Co.) as 0.5% solutions;
soluble starch (Malinkrodt Chemical Works) as a 0.14 and
carboxymethylcellulose (Hercules Powd. Co.) a 1.0% solution.
A one ml. aliquot of extract, one ml. of sugar substrate,
and three ml. of Tris-maleate buffer (GOMORRI, 1955) com-
prised the incubation medium.
Sugar breakdown was assayed by the Somogyi method as
adapted by Nelson (1944) for reducing sugar. AKlett-
Summerson photoelectric colorimeter was used to determine
per cent absorption at 540mu. and this value read against
a standard glucose curve to find amount of reducing sugar
liberated. Appropriate enzyme and sugar controls were con-
ducted. Correction for dilution was made to obtain a fig-
ure for enzyme potency in one ml. of gut tissue or tissue
and contents.
Two methods were used to determine the ability of the
intestinal bacteria of C. spirabrancha to secrete carbohy-
drases. First, the particulate fraction after centrifuga-
tion, which contained the bacteria present in te homogenate,
was washed ten thousand-fold in sterilized artificial sea-
water by alternate resuspension and centrifugation to di-
lute enzyme concentration. Resuspended it was used as a
"bacteria extract." One-tenth ml. aliquots of this solution
were spread on a sterile growth medium consisting of 0.1%.
yeast extract (Difco), 1% starch, 2% agar, and sea-water.
After seventy-eight hours the plates were washed with po-
tassium iodide-iodine solution and breakdown of starch de-
termined by the presence of clear areas. Aliquots of the
supernatant upon the initial centrifugation, which was free
3.
of bacteria, were also plated to provide a comparison of
activity.
Secondly, one ml. aliquots of the enzyme-free extract
were incubated in the usual manner and assayed for degrada-
tion of sugar by freshly secreted enzyme at twelve-minute
intervals over a seventy-two minute period.
RESULTS
Three sugars were broken down by extracts of the gut:
laminarin, starch, and cellulose. No positive evidence was
found for hydrolysis of the other substrates. Opening and
washing the gut resulted in extracts with very greatly de-
creased activities. Figure 1 shows the amounts of break-
down by one ml. of gut in one hour expressed in glucose¬
equivalent units. Activity was greatest using a buffer at
pH 5.5. Reduced degradation took place at pH 6.2, and there
was practically none at ph 7.5.
Enzyme action was found in extracts from three portions
of the gut: stomach, fore-intestine, and rear-intestine. The
buccal cavity, pharynx, and esophagus homogenized together
showed no activity. Values for starch and laminarin break-
down by region are found in Figure 2. Similar assays for
degradation of cellulose were not made.
My attempts to determine the role of bacteria in enzyme
secretion have not yielded positive results. Colonies were-
established on the nutrient plates, but even after three days
they broke down little starch relative to the amout degraded
by bacteria-free enzyme extract. No satisfactory time course
for hydrolysis of sugars was derived. That for starch is the
most intelligible and reproducible; it is shown in Figure
III. Evidently, most of the activity of the enzyme occurs
within twelve minutes. Time courses with enzyme-free bac-
teria extract showed no breakdown and consequently no secre-
tion of enzyme during incubation.
DISCUSSION
It is somewhat surprising that only three of the seven
algal polysaccharides tested were degraded in the gut of
C. spirabrancha. In view of the worm's non-specific eating
habits a wide digestive capability would seem most useful.
Other common inhabitants of the intertidal such as the gas-
tropods Tegula funebralis and Helix pomatia (Reviewed by
WILBUR and YONGE, 1966) possess large complements of carbo¬
hydrases. Before conclusions can be drawn, however, about
which enzymes might be expected to occur in the gut of C.
spirabrancha, more information about sugar digestion in
other polychaetes is needed.
Although they are suggestive, the places of enzyme ac-
tivity found do not strictly delineate the sites of enzyme
origin or physiological function. Surely digestion can be
expected in the stomach and fore-intestine; it is breakdown
in the rear-intestine which should be questioned. This area
is characterized by a thin, transparent gut wall, scant blood
supply, and gut contents which are in a pellet- or string-
form. These factors indicate that profitable digestion prob-
ably does not take place in the rear-intestine.
Digestion in polychaetes is at least predominantly ex-
tracellular. (Meglitsch, 1967), and this fact complicates
identification of the source of enzymes. The very low ac-
195
tivities of extracts from opened and washed gut tissue nei-
ther proves nor disproves enzyme synthesis in the gut walls.
No histochemical examination of the gut has been made and it
is not known if secretory-type cells are present.
It was thought that possibly magnesium ions in the anes-
thesizing and dissecting solution caused the release of en-
zymes from tissue before the gut was transfered for homogen-
ization. Preliminary studies with dissection made in sea-
water show a continued absence of laminarinase or amylase in
gut tissue. A cellulase, may be present in significant amounts;
difficulty persists, however, with a turbidity in the colored
cellulose solutions.
Microscopic examination of the gut contents of C. spira-
brancha reveals bacteria in large numbers in all regions.
Strains of bacteria have been shown capable of breaking down
starch, cellulose, agar, chitin, and a carrageenin. In many
animals the role of bacteria in digestion is important; in
others they are present in the gut, but not thought signifi-
cant in the secretion of enzymes. The cultures obtained showed
that the bacteria of C. spirabrancha can secrete an amylase,
but its potency may be limited. The time courses made indi-
cate the bacteria do not secrete an enzyme under the exper-
imental conditions. Further study of this problem should be
carried out. For example, large quantities of the bacteria
could be cultured and the degree to which they are able to
digest and utilize the various sugar substrates tested. (cf.
GALLI and GIESE, 1959) Until the presence of enzymes in the
gut wall of C. spirabrancha is demonstrated, the bacteria of
the annelid will remain a possible source of its carbohydrases.
99
SUMMARY
Laminarin, starch, and cellulose are found to be de-
graded by extracts of the gut of Cirriformia spira-
brancha.
The stomach and fore-intestine of the annelid are iden-
tified as the probable sites of enzyme action.
Differentiation of the abilities of C. spirabrancha
to synthesize carbohydrases was attempted without
definitive results.
ACKNOWLEDGEMENTS
I would like to thank Dr. David Epel for his continued
advice and assistance in my work.
This study supported in part by the Undergraduate Research
Participation Program of the National Science Foundation, Grant
GY-4369.
O
REFERENCES
GALLI, D.R. and GIESE, A.C. (1959) Carbohydrate digestion in
a herbivorous snail, Tegula funebralis. J. Exptl. Zool.
140, 415-440.
GOMORRI, G. (1955) Preparation of buffers used in enzymatic
studies. In Methods in Enzymology (Edited by COLOWICK, S.
and KAPLAN, N.) Vol. I, p.143. Academic Press, New York.
MEGLITSCH, P.A. (1967) Invertebrate Zoology. p.600. Oxford
University Press, London.
NELSON, N. (1944) A photometric adaption of the Somogyi method
for the determination of glucose. J. Biol. Chem. 160, 69-73.
WILBUR, K.M. and YONGE, C.M. (1966) Physiology of Mollusca.
Vol. II, pp.70-71. Academic Press, New York.
Figure I: Sugar breakdown. Mgm. glu-
cose-equivalent sugar degraded in one
hour by the extract of one ml. of gut.
pH 5.5.
Gut opened
Intact
and washed
gut
0.5
34
Starch 0.1% solution
32
Laminarin O.5% suspension
Cellulose 1.0% solution
*Preliminary
figure II: Sugar breakdown by gut re-
gion. Mgm. glucose-equivalent sugar
degraded in one hour by the extract of
one ml. gut tissue and contents. ph 5.5.
Laminarin
Starch
0.5% suspension
0.1% solution
Buccal cavity
Pharynx
Esophagus
56
Stomach
42
Fore-intestine
Rear-intestine
20
Figure III: Time course of starch break-
down. Abscissa: minutes of incubation.
Ordinate: mgm. glucose-equivalent sugar
degraded by the extract of one ml. gut
tissue and contents. Sugar solution is
0.1%; pH 5.5.