CARBOHYDRASE ACTIVITY IN THE
INTESTINAL TRACT OF PAGURUS SAMUELES
(Arthropoda: Malacostraca)
Benjamin Hourani
Höpkins Marine Station
Stanford University
Pacific Grove, Calif.
June 1, 1965
An examination of the gut contents of Pagurus samuelis
(Stimpson, 1859) reveals red, green, and brown algae, as well
as various diatoms, bacteria and portions of tissue from other
marine animals. With such a variety of food sources, it seem-
ed of interest to determine the digestive carbohydrase activi-
ties of this animal.
The substrates used in this study are all present in
the organism's environment and thus present a potential source
of energy to P. samuelis. The survey to follow is not intended
to be a characterization of the carbohydrases of this decapod
crustacean, but rather an examination of the digestive capabil
ities necessary for splitting linkages in various structural
and reserve carbohydrates in the marine envirorment.
Materials and Methods
The following substrates were used as one percent solu-
tions without further purification: maltose (Difco Laboratories,
C.P.), cellobiose (Calif. Corp. for Biochemical Research),
and sucrose (C & H Sugar Co.). Whatman 41 filter paper served
as the substrate in testing for cellulase activity. Laminarin
and fucoidin were prepared from Fucus by the method of Black,
Dewar, and Woodward (1951-1952) and also used as one percent
solutions. One percent gels and 0.1 percent solutions of agar
(Consolidated Laboratories), and alginate (Kelco Co., commer-
cial grade) were also prepared. These gelled substrates were
solidified within glass tubing, covered with tissue extract,
and layered with toluene. Substrate, to determine chitinase
activity, was prepared by dissolving large pieces of chitin
(Eastman Kodak, practical grade) in 967 sulfuric acid. The
solution was then poured into a large volume of water, preci-
pitating the carbohydrate as a fine powder. After settling
for three days, the mixture was centrifuged, washed, and pre-
pared in a one percent suspension.
The alimentary canal was divided into three anatomical
sections: foregut (stomach and esophagus), midgut (excluding
caeca), and hepatopancreas.
To determine enzymatic activity, freshly collected
crabs from China Point in Monterey, California, were dissect-
ed, and the appropriate gut section excised from a number of
animals until sufficient tissue for study was obtained. The
tissue was washed several times in sea water (all visible gut
contents removed), weighed, and homogenized in a tissue grinder
with a Teflon pestle. Homogenization was performed in 0.2M
phosphate buffer at either a pll of 5.7 or 7.5. The above pro¬
cedure was carried out at 00C so as to retard any loss of ac¬
tivity due to denaturation or autolysis. After centrifuging
for fifteen minutes, an appropriate aliquot of the supernatant
was added to substrate, layered with toluene to prevent grouth
of microorganisms, and incubated at 15-160c in sea water.
Enzyme-buffer and substrate-buffer controls were run simul-
taneously with the reaction mixture. The solutions were tested
for activity at 1, 5, and 24 hours. If activity was indicated,
an identical test was run with the number of samplings great-
ly increased.
Enzymatic activity was quantitatively determined by
testing an aliquot of the mixtures after deproteinization
with barium hydroxide and zinc sulfate (Somogyi, 1945). The
amount of reducing sugar was determined by the Somogyi method
(Somogyi, 1945 and 1952) using Nelson's colorimetric modifica-
tion (Nelson, 1944). Measurements were made on a Klett-Summerson
Photoelectric Colorimeter, using a green filter (654).
An increase in reducing sugar over the sum of the two
controls was considered as an indication of carbohydrase ac-
grerert ut
tivity. Estimations of monosaccharides perdeprewene based
on a standard curve of 10-200 micrograms of reducing sugar
specific to the carbohydrate being tested. In addition, when-
ever activity was indicated, a glucose-enzyme control was in-
serted in an identical test to determine reducing sugar con-
sumption by the crude tissue extract.
After testing foregut, midgut, and hepatopancreas for
the nine substrates mentioned above, an attempt was made to
determine if the anterior midgut diverticula (caeca) possessed
any maltase activity.
Experimental Results
Digestion of carbohydrate reserves.
Laminarin is the reserve carbohydrate of the sublitoral
brown algae and is especially abundant in Laminaria. All
tests using this polysaccharide as a substrate gave no indication
of the presence of an enzyme capable of splitting the 1,3
beta linked glucose units of this carbohydrate. Tests were
performed with the three sections of the gut and at both ph
5.7 and 7.5.
2. Digestion of structural components of algae.
Hydrolytic activity capable of splitting the 1,3 beta
galactose linkage of agar and the 1,4 beta mannuronic acid
linkage of alginate was not detected in extracts from any
section of the intestinal tract. These results were support-
ed by the persistence of the marked border at its initial
level between tissue extract and carbohydrate gel within a
small piece of glass tubing. In addition, no carbohydrase
activity was detected when dilute solutions of these struc
tural polysaccharides were tested.
In testing for cellulose activity, Whatman l filter
paper was incubated in tissue extracts of appropriate sections
of gut and observed periodically for a total of 72 hours.
The intact, seemingly undamaged filter paper indicated that
in each tissue studied, no hydrolysis of the substrate was
occurring. Thus no activity capable of splitting the 1,4
beta glucose linkage was observed at pll 5.7 or 7.5. Reducing
sugar tests, performed on the above tissues, supported these
qualitative findings.
The hydrolysis of fucoidin, a sulfate ester of 1,2
alpha fucose, also did not occur in foregut, midgut, or
hepatopancreas extracts at either ph studied.
3. Hydrolysis of chitin.
Chitin, a major structural component in the hard body
parts of insects and crustaceans, was prepared in a one per
cent suspension. Under the conditions of the experiment no
indication of hydrolysis was observed.with material obtained
from sections of the intestinal tract buffered at both ph's.
4. Digestion of oligosaccharides.
Tests with midgut extracts from P. samuelis indicated
absence of an enzyme capable of splitting the 1,4 beta-glucose
linkage of cellobiose or the 1,2 alpha glucose-beta fructose
linkage of sucrose. In testing for maltase activity, a slight
amount of hydrolysis of the 1,4 alpha glucose linkage was
indicated at pll 5.7. However, the amount of reducing sugar
increase over controls was below the minimum sensitivity of
the experimental procedure. The midgut was therefore considered
inactive in digesting the above three disaccharides at both ph
5.7 and 7.5 under experimental conditions. The foregut and
hepatopancreas were also found incapable of hydrolyzing su¬
crose and cellobiose at the above ph's.
Table 1 and Figure 1 indicate a high degree of hydro-
lysis of the 1,4 alpha-glucose linkage of maltose by hepato
pancreas extracts. A very rapid hydrolysis of the substrate
can be noted at pll 5.7. A total of 139 micrograms of glucose
produced per milligram of tissue was found at the end of 24
hours. At pH 7.5 hydrolysis was much slower, although a
value of 186 micrograms reducing sugar per milligram of tissue
was attained after an equivalent period of incubation.
J4
R

I


2

L
S

d
8
8
1
8
O
Figure 1.
Hydrolysis of maltose by extracts
prepared from hepatopancreas tissue.
O pH 7.5,
pH 5.7,
key:
A
— glucose-enzyme control at pH 5.7.
20.
r4
18
17
10
N 450
4
130
12
110
10
90
7o

40
20
20
10


2
4
A—A-

—

8
10
12
HOURS
14
O
Figure 2.
Hydrolysis of maltose by extracts
prepared from foregut tissue.
key:
O pH 7.5,
• pH 5.7,0
—glucose-enzyme control.at pH 5.7.
4
20

19
170
10
1
140
1
2 12
10
1o
90
70
10
50
40
30
20
20
—A
lao


NOURS
Figure 2 and Table 1 indicate less maltase activity
in extracts of foregut tissue. Again, hydrolysis occurs ra-
pidly at a ph of 5.7, reaching a value of 138 micrograms per
milligram of tissues after 24 hours. At ph 7.5 hydrolysis
was delayed as in the hepatopancreas until after 6 to 7 hours
of incubation and reached a total of 119 micrograms per milli-
gram tissue for a 24 hour period of incubation. It is signi-
ficant that in both tissues buffered at ph 7.5, activity
was demonstrable only when the ph of the solutions had dropped
to between 5.0 and 6.0 The increase in hydrogen ion concentra-
tion is probably due to the production of acidic produets of
carbohydrate metabolism.
Under the experimental conditions involved, the an-
terior midgut diverticula showed no maltase activity at either
pll.
Discussion
It is interesting that under the conditions of the ex
periment, no laminarase, alginase, fucoidase, cellulase, cello-
biase, agarase, or sucrase activity was observed in enzyme
extracts of foregut, midgut, and hepatopancreas. As stated
before, sources of the above carbohydrates were revealed upon
examination of gut contents. Diatoms and other microorganisms,
epiphytic on algae may constitute another source of food
obtained when materials containing the above sugars are eaten.
Moreover, the author observed P. samuelis eating partially
decayed algae, both in the field and in the laboratory. Thus,
the possibility of partial digestion of carbohydrates, prior
to intake by the crab is suggested. This could allow the animal
to use otherwise indigestible food sources.
The possibility of digestion by intestinal microflora
must not be eliminated, although no attempt was made to cul-
ture microorganisms from the gut segments in this study.
Nevertheless, it seems improbable that any significant diges-
tive complement is provided by microflora (Vaterman, 1960).
Chitinase activity was not found in the three sections
of the gut. This finding is in good agreement with observa-
melt
tions made by Norman Richardson (1965) studyingmmkt induction
in this crab. His study took place simultaneously with this
survey at Hopkins Marine Station. After two weeks of observa-
tions, he reported that he had never witnessed a crab eat its
discarded carapace after molting.
Waterman (1960) has described amylase activity in other
crustaceans. Sullivan (1965) has more specifically observed
this enzyme in the hepatopancreas and foregut of P. samuelis.
Therefore, it is not surprising that an active maltase has
been demonstrated in this animal. In the studies of Sullivan
(1965), paper chromatograms of starch degradation products
showed glucose and only traces of maltose. Therefore, it ap
pears likely that preparations having amylase activity contain
maltase as well.
It is significant that the alpha glucosidase activity
in the hepatopancreas is much greater than in the foregut.
This finding is in good agreement with Waterman (1960), who
lists the organ as the major source of digestive enzymes in
the crustaceans. Moreover, Waterman lists the ph optima for
crustacean maltase as 5.0-6.0. A more rapid hydrolysis at
pHi 5.7 than 7.5 for both foregut and hepatopancreas extracts
was observed. It was noted that the first indication of ac-
tivity by extracts buffered at pll 7.5 occurred only after the
reaction mixtures had dropped in ph to between 5.0-6.0. It
can therefore be concluded under the conditions of the exper-
iment, that the ph optimum for maltase activity in both tissues
is in the slightly acidic range.
The loss of reducing sugar demonstrable in the glucose-
enzyme controls indicates an important property of the crude
enzyme extracts. An initial consumption of glucose is indicated
by the drop in reducing sugar. Enzymes of the glycolytic
sequence or those of the pentose shunt might well explain the
initial decrease. The subsequent increase in reducing sugar
can possiblg be attributed to the hydrolysis of glycogen. present
in the crude extract. The above indicates one basic objection
to the work reported in this paper, ince. no attemptrwas made to
purify the enzymes of the gut by protein fractionation. It
is difficult to control the enzymatic reaction occurring in a
brei as crude as that obtained by the method used, since the
enzyme extract contains a wide variety of cellular enzymes
in addition to other hydrolytic enzymes. As shown by the glu-
cose enzyme control some of the oxidative enzymes present in
such a mash could conceivably degrade or alter the monosac-
charides liberated by a digestive enzyme, masking its chemical
detection. As a result, the activity of a carbohydrase capable
of handling a specific carbohydrate, even if present in the
gut extract,might not be demonstrable.
Turning to the anterior midgut diverticula, an attempt
was made to determine if the organ possessed maltase activity.
The results were negative, in good agreement with Sullivan
(1965), who found the tissue lacking amylase activity.
Summary
Under the conditions of the experiment, enzyme extracts,
prepared from the hepatopancreas, foregut, and midgut of
P. samuelis were found to show no activity capable of hy¬
drolyzing the following carbohydrates: laminarin, fucoidin,
cellulose, cellobiose, sucrose, agar, and alginate.
Alpha glucosidase activity was detected using maltose
in extracts of hepatopancreas and foregut, the former
producing a higher reaction rate. Both extracts reacted
more rapidly at a pll of 5.7 than at pll of 7.5. Midgut
extracts indicated no hydrolysis of the 1,4 alpha glucose
linkage.
No chitinase activity was observed in any of the sec
tions of the gut studied.
Extracts of the anterior midgut diverticula showed
no increase in reducing sugar over controls when tested
for maltase activity.
76.
LITERATURE CITED
Black, W. A. P., W. J. Cornhell, E. T. Dewar and F. N. Wood-
ward 1951. Manufacture of algal chemicals: Laboratory-
scale isolation of Laminarin from brown marine algae.
Journ. Appl. Chem. 1: 505-517.
Black, W. A. P., E. T. Dewar, and F. N. Woodward 1952. Manu¬
facture of algal chemicals: Laboratory-scale isolation of
Fucoidin from brown marine algae. Journ. Sci. of Food
and Agricult. 3: 122-129.
Nelson, N. 1944. A photometric adaptation of the Somogyi
method for determination of glucose. Journ. Biol. Chem.
153: 375-380.
Somogyi, M. 1945. Determination of blood sugar. Journ. Biol.
Chem., 160: 69-73.
1952. Notes on sugar determination. Ibid., 195:
19-23.
Richardson, Norm. 1965. Personal communication.
Sullivan, Phillip 1965. Unpublished paper on amylase activity
in P. samuelis done at same time and location as this paper.
Waterman, Talbot H. 1960. The Physiology of Crustacea Vol.
1. Academic Press, New York. 670 pp.