Summary
B-D-glucosidase activity increased during development
of the barnacle, Pollicipes polymerus. The activity increased
18.7-fold from early blastula through the nauplius stage and
at all stages exhibited a 3:l ratio of soluble to particulate
form and was activated by the detergent Triton X-100. A
similar activity was also found in the adult digestive tract.
The possible roles of the exhibited activity are discussed.
1

Introduction
Hydrolytic enzymes of Crustacea have been studied exten-
sively in the adult stages but almost nothing is known
about the embryonic synthesis of these proteins. Since the
midgut cells become specified around the 33 cell stage, it
seems probable that digestive enzymes might also be forming
simultaneously(1). An outgrowth of cells in the posterior
midgut of free-swimming larvae, or nauplii, of the barnacle
Balanus balanoides, were found to be very similar to those
of the adult digestive gland (2,3).
Lack of knowledge concerning specific enzymes, either
for the digestion of yolk proteins or for later use in the
adult barnacle, prompted this study of a B-D-glucosidase
during development in the barnacle Pollicipes polymerus.
Activity of the enzyme was assessed for five developmental
stages and, for comparison, in the adult barnacle.
Materials and Methods
Obtaining Embryos
Adult Pollicipes were collected at Mussel Point near
Monterey, California in April and May, 1982. The embryos
are brooded in the mantle cavity in a pair of oval disks or
ovigerous lamelli. The embryos were inspected and classified
into one of the following five developmental stages: early
blastula, just after the anterior and posterior midgut
rudiment cells first become specified; limb rudiments,
development just begun of the naupliar limb rudiments; naupliar
appendages elongated; nonmotile nauplius; motile nauplius.
Assay of Enzyme Activity
The lamelli were gently homogenized on ice with a Dounce
homogenizer in a chilled buffer solution of 0.05M Tris, O.05M
Citrate, pH 4.0, unless otherwise specified. In the assays
for the determination of intracellular compartmentalization,
O.5M KCl was added to the buffer to insure an isotonic assay
medium. Determination of B-D-glucosidase activity, using
the synthetic substrate, p-nitrophenyl B-D-glucoside (Sigma
Chemical Co.) was modified after that of Fedecka-Bruner et al (4).
The assay mixture contained 0.2 ml of 0.02M substrate, 0.2 ml
1.0% Triton X-100, 0.4 ml homogenate, and 1.2 ml buffer.
After incubation at 30°C (60 minutes unless specified other¬
wise), the reaction was terminated by addition of 1.0 ml of
O.5M Naoh. The mixture was centrifuged at 27,000 g at 0°0
for 10 minutes and the supernatant then read in a Gilford
spectrophotometer along with its corresponding blank, against
distilled water at 420 mu. The blank was prepared by adding
the homogenate together with the Naoh. The B-D-glucosidase
activity is expressed in nanomoles of p-nitrophenol liberated
per milligram protein per 60 minutes (4). Protein was
measured by the method of Bradford using bovine serum albumin
as a protein standard (5).
Results
ph Optimum and Linearity with Time
Fig, 1 shows that the maximum activity of the enzyme
occured at a pH 3.0 however, at this pH, the time course was
not linear (data not shown). The activity at pH 4.0 is
within 80% of the activity at ph 3.0 and the time course is
linear for up to 60 minutes at this pH (Fig. 2). For this
reason, the assays were done at pH 4.0 for 60 minutes.
Linearity of Rate with Protein Concentration
Fig. 3 shows that the enzyme activity is directly
proportional to the protein concentration for up to 1800 ug
of added protein. For this reason, the protein concentration
was adjusted so that the final concentration was always less
than 1800 ug with most assays containing between 500 and 1000
ug protein.
Enzyme Activity in Development
Fig. 4 shows that the enzyme is present in the early
blastula stage of development and increased 18.7-fold through
the nauplius stage. Even though the embryos were classified
into five stages, there was very little variation except in
the motile nauplius stage. This stage exhibited 337 +103
nanomoles per mg protein per 60 minutes. For this reason.
five additional collections and assays of naupliar stages
were made to insure that the results were indicative of that
stage of development. The large deviation observed in the
naupliar stage can easily be explained by the fact that the
embryos are brooded for 30 days and as such, the stage
classified as the naupliar stage could represent a longer
time span than the other stages of development (6). This argument
is strengthened by the fact that, whereas the ovigerous
lamelli of the other stages have a characteristic texture and
color, the color of the motile nauplius stages were seen to
range from an orange-brown to a gray color. It was observed
that this progression of color was associated with an increase
in the accumulation of B-D-glucosidase activity.
Enzyme Compartmentalization
To determine if the enzyme is particulate at some point
in development, the homogenate of the early blastula and
motile nauplius were centrifuged (27,000 g for 30 minutes)
in the presence and absence of 0.27 Triton X-100. The
supernatants were removed by pipet and saved for later assay
of activity; the pellets were resuspended in a volume of
buffer equivalent to that of the supernatant. The assays were
then run with 1% detergent present in each mixture. Ihe
absence of Triton X-100 during the centrifugation resulted
in 75% activity in the supernatant and 25% in the pellet.
In the presence of Triton X-100, the activity shifted to 947
in the supernatant and 6% in the pellet. This behavior was
true in both stages.
A pH optimum curve, similar to Fig. 1, was run on the
supernatant and pellet (27,000 g for 30 minutes) in the
absence of Triton X-100 to determine if the soluble and
particulate activity were due to different isozymes. In the
assay mixtures buffered to pH 3.0, 4.0, 5.5, and 6.5, the
activity, in each case, was 75% supernatant and 25% pellet.
The total activity at each ph was found to be similar in ratio to the
activity found in the earlier ph optimum curve (Fig. 1).
Perhaps a 27,000 g spin was inadequate to pellet a
particulate activity. Centrifugation was therefore carried
out at 100,000 g for 60 minutes to ascertain if any additional
activity could be sedimented. The same 3:1 ratio of activity
was found, as in the two fractions of a 27,000 g spin. The
assay was also run with both fractions in the presence or
absence of 1% Triton X-100; an incréase in enzyme activity
was seen in both insoluble and the soluble supernatant
fraction when Triton X-100 was present (1.6-fold increase).
This result indicates that the soluble enzyme is directly
activated by the detergent.
These results suggest that one enzyme is responsible for
the activity. Twenty-five percent of the enzyme appears to
be particulate and the soluble form is activated by the
detergent, Triton X-100; the effect of Triton X-100 on the
particulate fraction is related to its effect on the
solubilization of the enzyme.
A final enzyme assay was run on the intestinal tract of
the adult barnacle. The juices of the intestine were obtained
by inserting a fine-pointed pipet in the tract and extracting
the juices contained in the lumen. The tract was then removed
and homogenized. Assays were run on the juices and the
supernatant and pellet (27,000 g for 30 minutes) of the homog.
enate. The juices of the lumen contained a majority of the
activity. The homogenized gut tract showed activity in the
supernatant and pellet with a ratio of 3:1, supernatant:pellet,
as was found in the embryonic stage. All fractions (juices
and homogenized gut) were also activated by the detergent.
. . .
Discussion
The results of this study show the presence of a B-D-

glucosidase in Pollicipes embryos and nauplii. The activity,

h increased 18.7-fold from the early blastula through the
nauplius stage, exhibited a 3:l ratio of soluble to particulate
form in every stage. The enzyme was activated by the detergent
Triton X-100. A B-D-glucosidase activity was also present in
the adult digestive tract and this enzyme was activated
1.5-fold by Triton X-100 and similarly exhibited a 3:1
soluble:particulate nature. No further comparisons were made.
but these results suggest that the adult and embryonic forms
are isozymes. There are two major possibilities for the
presence of a B-D-glucosidase in embryonic stages. One
hypothesis is that the enzyme is used during embryogenesis,
as for digestion of yolk constituents. A second hypothesis
is that the enzyme activity is accumulating in the midgut cells
for later use in the swimming nauplius and following larval
stages.
If the first proposal is correct, the enzyme could be
functioning intracellularly, as in a lysosome, or extracellularly.
as a component of a developing but functioning embryonic gut.
A lysosomal role would seem to be excluded by the largely
soluble nature of the enzyme (the increased activity induced
by the detergent did not result from release of the enzyme
from a particle; as shown, the soluble enzyme itself was
detergent activated). An alternate possibility however, is
that the yolk cells themselves are acidic and functioning as
a " cellular lysosome." If so, these large cells would be
broken during the homogenization procedure and release the
soluble enzyme. An extracellular function of the enzyme might
also be possible. The yolk cells are present in the midgut
during development and the midgut might be functional at this
time in utilizing yolk constituents. A problem with the above
concept of embryonic utilization is that there is no evidence
for a B-linked carbohydrate in the embryo; the major known
store is glycogen, which would be degraded by glycogen
phosphorylase or an a4-D-glucosidase (7,8).
The alternative explanation is that the enzyme is not.
used during embryogenesis but represents accumulation of the
enzyme for later use in the feeding larval stages. The role
for a B-D-glucosidase activity then could be for digesting
laminarin or cellulase, which would be major constituents of
a planktonic or detrital diet. A laminarinase has been found in the
midgut of adult barnacles, Balanus nubilus and a cellulase
is present in the " sterile midgut of adult lobster and
crawfish with a ph optimum of 4.0 to 4.5 which appears to be
much lower than the pH optimum of 5.0 to 6.0 found for other
carbohydrases (9,10,11).
Studies on synthesis of adult digestive enzymes could
explain the 3:1 soluble:particulate nature of this enzyme
and activation by detergents. If it is indeed digestive, the
enzyme is probably being accumulated in the hepatopancreatic
gland.
In adults, digestive enzymes are stored in enzymatic
granules which are distended with fluid, forming a large
vacuole, or mature secretory cell (11,12). The contents of
these cells are extruded into the lumen for extracellular
digestion. It has been shown that a refractory period is
necessary for mature cells and enzyme granules to form (11).
The soluble activity could represent the enzyme present in
mature or almost mature secretory cells, while the particulate
activity could represent enzyme granules or newly forming cells.
Homogenization could release the enzymes from secretory cells
prematurely since it was found that even slight pressure on
the cover slip of a slide of hepatopancreatic gland cells
caused the breakage of the distended mature cells (12). As
regards activation by detergent, it is interesting that the
hepatopancreatic gland also stores and secretes bile salts
into the lumen; the activating effect of Triton X-100 might
mimic the activity of a natural detergent in the intestine(Il).
There is insufficient data to decide between these various
alternatives. Clearly, further studies on the digestive
capabilities of barnacle embryos would be of interest.
O
Special thanks to Dr. David Epel for his help in this
study and in the preparation of this manuscript. His
insights and constructive criticisms were indispensable in
the production of this text.
References
1 Anderson, D. T., On the embryology of the cirripede
crustaceans, Tetraclita rosea (Krauss), T. purpurascens
(Wood), Chthamalus antennatus (Darwin) and Chamaesipho
columna (Spengler) and some considerations of crustacean
phylogenetic relationships, Phil. Trans. R. Soc. B. 256(1969)
183-235.
2 Walley, L. Jane, Studies on the larval structure and
metamorphosis of Balanus balanoides (L.), Phil. Trans. R.
Soc. B, 256 (1969)237-280.
3 Anderson, D. T., Embryology and Phylogeny in Annelids and
Arthropods, Pergamon Press, Oxford, 1973, pp. 322-329.
4 Fedecka-Bruner, B., Anderson, M. and Epel, D., Control of
enzyme synthesis in early sea urchin development: aryl
sulfatase activity in normal and hybrid embryos, Develop.
Biol., 25(1971)655-670.
5 Bradford, Marion M., A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding, Analyt. Biochem.,
72 (1976) 248-254.
6 Morris, R. H., Abbott, D. P. and Haderlie, E. C.,
Intertidal Invertebrates of California, Stanford University
Press, Stanford, 1980, pp. 514-515.
7 Green, J., Crustaceans. In G. Reverberi (ed.), Experimental
embryology of marine and fresh-waster invertebrates, North-
Holland Publishing Company, Amsterdam, 1971. pp. 312-362.
8 Barnes, H., Studies in the biochemistry of cirripede eggs,
J. mar. biol. Ass. U. K., 45(1965)321-339.
9 Harnden, D. G., Digestive carbohydrases of Balanus nubilis
(Darwin, 1854), Comp. Biochem. Physiol., 25(1968)303-309.
10 Kooiman, P., The occurence of carbohydrases in digestive
juice and in hepatopancreas of Astacus fluviatilis Fabr.
and of Homarus vulgaris M.-E., J. Cell. Comp. Physiol.
63(1964)197-201.
11 Vonk, H. J., Digestion and Metabolism. In T. H. Waterman
(ed.), The Physiology of the Crustacea, Vol. 1, Academic
Press, New York, 1960, pp.291-316.
12 Van Weel, P. B., Processes of secretion, restitution, and
resorption in gland of mid-gut (glandula media intestini)
of Alya spinipes Newport (Decapoda- Brachyura), Physiol.
Zool., 28 (1955)40-54.
Legends
Fig. 1. pH optimum of B-D-glucosidase activity in the
motile nauplius stage. Activity is expressed as 10 2 mole
substrate hydrolyzed/mg protein/60 minutes.
Fig. 2. Linearity of B-D-glucosidase activity as a function
of time. Activity is at ph 4.0 and is expressed as 10-2
mole substrate hydrolyzed/mg protein. Source of enzyme was
the motile nauplius stage.
Fig, 3. Linearity of rate as a function of protein concentration.
Results are expressed as change in optical density/60 minutes
of assay mixture. Source of enzyme was the motile nauplius
stage.
Fig, 4. B-D-glucosidase activity during developmental stages
of Pollicipes polymerus. For the motile nauplius, n-8; for all
other stages, n-3. Vertical lines indicate range, bars indicate
mean, and colored bars indicate standard deviation. Activity
is expressed as 10-9 moles substrate hydrolyzed/mg protein/60
minutes.
500
400
300-
200

10
m
600
500
L 400
300
200
100
D
ph
60
TIME (minutes)
30
90
120
:
-.-
6
L

1000
2000
PROTEIN CONCENTRATION (ug)
3000
500

400:
300
20
100

(n=3)
motile nauplius-(n=8)


L
limb
naupliar nonmotile
early
blastula rudiments appendages nauplius
motile
naupsius