0

Abstract
1. Four digestive carbohydrases, laminarinase, maltase,
chitinase, and amylase have been demonstrated in Balanus
nubilis (Darwin, 1854).
2. The tissues showing highest enzyme activity were
as follows: laminarinase, last half of the midgut: maltase,
foregut: amylase, glandulae pancreaticae: and chitinase,
foremost part of the midgut.
Introduction
Preliminary investigation of the gut of Balanus nubilis
(Darwin, 1854) revealed a, myriad of kinds of particulate
matter including diatoms, other algae, nauplii, and molted
exoskeletons. Such a diverse particulate diet would require
a variety of carbohydrases for efficient digestion. The
literature contains a comprehensive anatomical analysis of
the digestive tract of Balanus improvisus (Darwin, 1854) by
S. R. Tornava (1948), but there are no references to digestive
arbohydrase activity in(
rripedae.
The first part of this study consisted of a survey of
the entire digestive tract for the presence of enzymes capable
of hydrolysing a variety of glycosidic linkages. The second
part of the study examined anatomical regions of the gut
for their enzyme content.
S
O
Materials and Methods
All animals used in this study were collected at Old
Fisherman's Wharf, Montery, California. Only animals fresh
from the field were used in these studies because animals
maintained in running sea water in the laboratory were found
to have considerably reduced enzyme activity.
In this study the alimentary canal was divided into five
anatomical parts: the foregut, the fatty tissue very similar
to the coeca hepatica as described by S. R. Tornava (1948),
the glands on the coeca hepatica which are quite similar
to the glandulae pancreaticae as described by S. R. Tornava
(1948), (see figure 5) the first half of the midgut, and
the last half of the midgut plus the hindgut.
The entire digestive tract or gut segments were evcised
from the barnacles and homogenized immediately in chilled
0.2 M acetate buffer (Gomori, 1955) at pH 5.5. The use of
tris (hydroxymethyl) aminomethane buffer (Gomori, 1955) at
pH 7.5 yeilded extracts with reduced activity. Generally a
ten per cent homogenate was prepared using a glass homogen¬
izer equipped with a Teflon pestle. The extract was centri¬
fuged in a refrigerated centrifuge at 4000 rpm. Enzymatic
activity was determined on a one ml. aliquot of the resulting
preparation without further purification.
The following substrates were tested: fucoidin (Pierce
Chem. Co.), maltose hydrate (Calbiochem), lactose (Calbiochem).
melibiose (Calbiochem), sucrose (Calbiochem), raffinose
pentahydrate (Calbiochem), stachyose tetrahydrate (Sigma
O
Chem. Co.), inulin (Calbiochem), amygdalin (Sigma Chem. Co.).
salicin (Sigma Chem. Co.), agar (Difco Chem. Co.), gentiobiose
(Calbiochem), amylose (Nutritional Biochemical Corp.).
dextran (Pharmacia, Uppsala, Sweden), pectin (Calbiochem).
arbutin (Sigma Chem. Co.), alginic acid (Pierce Chem. Co.).
gum arabic (Baker and Adams), glycogen (Calbiochem), starch
(Baker and Adams), quercitrin (K & K Laboratories), laminarin
(Pierce Chem. Co.), melezitose dihydrate (Calbiochem).
turanose (Calbiochem), cellobiose (Calbiochem), a,a-trehalose
(Calbiochem), and Rutin (Nutritional Biochemical Corp.).
A sample of purified kappa carrageanin was provided by
Dr. John H. Phillips of Hopkins Marine Station.
Chitin (Eastman Chem. Co. Technical Grade) was purified
by dissolving it in fifty per cent sulfuric acid and pre¬
cipitating it in distilled water.
A polyribose was extracted form Endocladia by dissolving
the alga in cold water with a Waring Blender, precipitation
vith ethanol, deproteinization with chloroform and amyl
alcohol, and drying with acetone.
All substrates were used as 0.3 per cent solutions
except amylose and chitin which were added as a suspension.
The test mixture was composed of one ml. of substrate, one
ml. of homogenate, and three mls. of buffer. The results
of all enzyme assays were corrected by subtracting the reducing
sugar values for enzyme and substrate blanks.
Reducing sugar was determined by the method of Somogvi
as modified by Nelson (1944). This method was found to be
0

reproducible to 21.5 micrograms in the range from 25-200
micrograms. Dilution of the reaction mixture due to de¬
proteinization brought the theoretical amount of reducing
sugar that could be released by enzymatic action within the
range.
The potenoy of the extracts was evaluated in terms
of their protein content as measured by the method of Lowry
(1951).
Results
Extracts prepared by homogenizing the entire digestive
tract were tested for their ability to hydrolyse a variety
of substrates. Figure 1 shows the time course of release
of reducing sugar from glycogen, starch, and amylose. The
amount of substrate added would have yeilded approximately
200 micrograms of reducing sugar as glucose or half this
amount with respect to maltose. This theoretical limit was
not reached suggesting a progressive degration of the enzyme
in these crude homogenates, perhaps by proteolyses. The
lower activity observed with amylose is probably a reflection
of the low solubility of this substance.
Following the detection of amylase activity the gut
was subdivided as described above and extracts of the various
regions were tested as well as extracts of the whole digestive
tract.
Figure 2 shows the hydrolysis of maltose by various
extracts. The theoretical limit of reducing sugar as glucose
S.
O
e
would have been 100 micrograms.
Figure 3 shows the hydrolysis of laminarin by various
extracts. The theoretical limit of reducing sugar as glucose
would have been 200 micrograms. When the sample was allowed
to incubate for two hours, a peak of 160 micrograms was
reached at one hour.
Figure 4 shows the hydrolysis of chitin by various extracts.
The theoretical limit of reducing sugar as glucoseamine
would have been 200 micrograms. When a whole gut extract
was permitted to incubate for five hours there was a linear
increase in reducing sugar to a final value of 71 micrograms.
No hydrolysis of the following substrates was observed:
fucoidin, lactose, melibiose, sucrose, raffinose pentahydrate,
stachyose tetrahydrate, inulin, amygdalin, salicin, agar.
gentiobiose, dextran, pectin, arbutin, alginic acid, gum
rabic, quercitrin, kappa carrageanin, melezitose dihydrate.
turanose, cellobiose, a,a-trehalose, rutin, and the Endocladia
polyribose.
Table I shows enzymatic activity expressed in terms
of micrograms of reducing sugar released per millegram of
protein for various regions of the digestive tract. The
glandulae pancreaticae showed the highest amylase activity
and relatively high maltase and laminarinase activity.
However, activity for these substrates was not limited to
this gland. The foregut has substantial maltase and laminarin-
ase activity which may be due to the presence in this gut
C
5
segment of the glandulae salivales described by S. R. Tornava
(1948). Highest laminarinase activity was found in the
second half of the midgut. High enzyme activity was also
observed in the first half of the midgut where most of the
chitinase activity was demonstrated.
The sum of the activities of the individual segments
of gut exceeded the activity of the whole gut extract. This
discrepancy would suggest the presence of enzyme inhibitors
in some of the tissues or degradation of the carbohydrases
due to proteolytic enzymes.
Since the glandulae pancreaticae are located on the
coeca hepatica and do not border on the gut itself, it was
desireable to determine if there is a duct leading from
these glands to the lumen of the gut. Figure 5 shows several
duct openings in the anterior part of the midgut. While it
was not possible to follow the ducts through the mass of the
coeca hepatica, injection of methylene blue into the glandulae
pancreaticae revealed the presence of ducts terminating at
the duct openings described above.
It should be noted that when the coeca hepatica was
dissected, it was impossible to separate it from part of the
midgut. It is therefore possible that there was no enzymatic
activity here at all.
Disoussion
Digestive enzyme activity in Crustacea has been reviewed
" Most
of th
H. J.
tudi
been
in deca
da,
ly Aste
nd
mado. Con-
siderable maltase and
ase ac
strated
n de
and their
have
ns
5.O
6.0. Marked activity of extracts fi
was
noted, as desribed
above.
ttle
ativity
DH 7.
inase
ati
great extant in ot
ity have been demonstrated to ai
ea.
most all
stacea have
ne or more
of glandula
appe
nth
thought
ake part in
In Malaco
raca digestive juices are produced
by
almos
ese tissues
However, in othe
Crustace
duc
n presumably takes place in
the
aend
and the midgut, altho
lac
In Balanus nubilis it was sho
t digestive carbohydrases are located th
he fo:
ratio
and mi
the fe
ula
itho
gut,
pancreaticae, and first part of the midgut.
rement-
to Dr. Johr
The autho
muchin
owle
Phill
his assistance in the preparation of this stu
ps for
le
O
O
References
Darwin, C. H. (1854), A Monograph on the sub-class Ci
ipedia
(Balanidae, Verrucidae, etc.). Ray society, London,
p. 30+.
Gomori, G. (1955), Preparation of buffers used in enzymatic
studies. cited in Methods of Enzymology Vol. I, ed.
by Colowick, S. and Kaplan, N., Academic Press, Inc.
New York, pp.138-146.
Rosebrough, Farr, and Randall (1951), Protein meas-
Low
urement with the Folin Phenol Reagent. J. Biochem
193:265-275.
Nelson (1944), A Photometric Adaptation of the Somogyi
method of determination of Glucose. J. Biochem 153:
375-381.
Tornava, S. R. (1948), Alimentary canal of Balanus improvisus.
Acta. Zool. fenn. 52:4-52.
Somogyi, M. (1937), A reagent for the Copper-Iodometric Determ¬
ination of Very Small Amounts of Sugar. J. Biochem.
117:771-779.
Vonk, H. J. (1960), Digestion and Metabolism, cited in The
Physiology of Crustacea Vol. I, ed. by Waterman, Talbot H.,
Academic Press, New York and London, pp.295-297.
6.
C
C
Figures
Figure 1. Time course for release of reducing sugar
from  starch, □ glycogen, and A amylose by extract of
whole digestive tract. Substrates were added as one ml. of
0.3 per cent solution or suspension. All reducing sugar
values are corrected by subtraction of substrate and enzyme
blanks.
Figure 2. Time course for release of reducing sugar
from maltose by extracts of E whole gut, □ foregut,
O glandulae pancreaticae, A coeca hepatica, O first half
of midgut, and A last half of midgut plus hindgut. Substrate
was added as one ml. of 0.3 per cent solution. All reducing
sugar values are corrected by subtraction of substrate and
enzyme blanks.
Figure 3. Time course for release of reducing sugar
from laminarin by extracts of E whole gut, □ foregut,
Oglandulae pancreaticae, A coeca hepatica, O first half
of midgut, and A last half of midgut plus hindgut. Substrate
was added as one ml. of 0.3 per cent solution. All reducing
sugar values are corrected by subtraction of substrate and
enzyme blanks.
Figure 4. Time course for release of reducing sugar
from chitin by extraots of A whole gut, O coeca hepatica,
and □ first half of midgut. Substrate was added as an
approximately 0.3 per cent suspension. All reducing sugar
values are corrected by subtraction of substrate and enzyme
blanks.
6.
C
10
Figure 5. Diagram of first part of digestive tract of
Balanus nubilis. a - glandulae pancreaticae, b - coeca
hepatica, c - foregut, d = first part of midgut, e = ducts
leading to glandulae pancreaticae, f = opening to foregut.
Table 1. Micrograms of reducing sugar released per
millegram of protein in one hour of incubation.

64
C
ve
(
O
30.




—


100
5
20
Minole
65
o

5.
re we


Minstas
— O


—


30


C
6
O

5
——
0


0
20
O
(0.
30
5.
Anctes
(thy
67
O
C
345
2
re
(





7
77
Pute
C
tse
minsis
ka-
—
27.

3
12
—


e
0

20
—
—.—
Cua
5
25
-


21.


——
—



1.0





C

—

—
5

2