ADDITIONAL INFORMATION, IF ANY, CONCERNING AUTHORS, ADDRESS, TITLE, OR CITATION DATA
PLEASE TYPE ABSTRACT DOUBLE SPACED BELOW
Beppu, William J. (Hopkins Marine Station of Stanford University.
Pacific Grove, California). A Comparison of Carbohydrate
Capabilities in Four Species of Acmaea (Mollusca: Gastropoda;
Prosobranchia). The Veliger
The carbohydrate digestion of four limpets, Acmaea scabra,
A. digitalis, A. limatula, and A. scutum was studied. The presence
Kagt
of a -carrageeninase, andagarase, a laminarinase, a fucoidinase.
an alginase, and an amylase was found in all species. Someohydrase
correlation of available sources of food with enzyme activity
was found. Carbohydrase levels in starved animals were compared
with levels in non-starved animals. A decrease in enzyme
activity during starvation was found, and the amount of this
decrease could be correlated with height in the intertidal, higher
species (A. scabra and A. digitalis) showing less decrease than
lower species (A. limatula and A. scabra). This correlation
might be related to food retention time and to feeding
behavior.-Author.
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62
CALBOIRDRAT
CAPABILTTIES
COMPARIS
IN FOUR SPECIES OF
ACMAEA
Gastropoda:
Prosobranchia
llusca:
william J. Beppu
Hopkins Marine Station of Stanford Univers
Pacific Grove, California
William J. Beppu
An examination of the distribution of the genus
Acmaea in the intertidal revealed that the various species
have their highest population densities in different tidal
zones. One of the characteristics of these zones is a dif-
ference in their algal flora. These various intertidal
algae, which synthesize a variety of polysaccharide mate-
rials (see Peat and Turvey, 1966) constitute a potential
source of food to herbivorous scrapers such as Acmaea.
ess at
The assimilation of these materials would require a varie
carbohydrases and led to the present comparative study
of the carbohydrases present in the digestive tracts of
four species of Acmaea.
MATERIALS AND METHODS
The species chosen for this study were: Acmaea
abra (Could, 1846); A.
igitalis Eschscholtz, 1833; A.
limatula Carpenter, 1864; and A. scutum Eschscholtz, 1833.
Large representatives of these species were collected from
areas of their maximum population density at Pescadero
Point, Monterey County, California.
To remove the digestive tract, the animals were
anaesthetized in isotonic magnesium chloride isotonic with
seawater, and the entire digestive tract along with associ-
ated glands (digestive gland, buccal salivary gland, and
William J. Beppi
esophageal salivary gland) excised and placed in 37 sodium
chloride in an ice bath. An enzyme extract was prepared,
containing 1 part tissue, 8 parts 36 sodium chloride, and 1
part of a saturated solution of ovomucoid (ovomucoid dis-
olved in either 0.1 Macetate buffer, ph 5.5, or 0.1 M Tris
puffer, ph 7.2). This mixture was homogenized in a tissue
grinder and then centrifuged for 30 seconds at half maximum
speed in an International Clinical Centrifuge. Ovomucoid,
prepared by the method of Fredericq and Deutch (1949), was
used as an inhibitor of proteolytic enzymes since initial
experiments indicated low carbohydrase activity, possibly
resulting from degradation of the enzymes by proteolysis.
of ovomucoid permitted detection of higher levels of
anzyme activity, and was therefore used in all reported
periments.
One ul of ensyme extract was incubated with l ml of
polysaccharide solution in 3 ml of buffer. Two buffers
were used, either 0.1 M acetate at pli 5.5,or 0.1 M Tris at
p 7.2. The polysaccharides included in this study were
arch (Baker and Adamson, Reagent grade), agar (Difco),
arrageenin Kappafraction) isolated from Rhodoglossum spp.
and laminarin, fucoidin, and alginic acid isolated from
. The mixtures were incubated for 1 hour at
vetia
200C. Enayme and substrate controls were similarly incubated
Liam J. Bepu
All tubes, including enzyme and substrate controls, were
layered with toluene as a bactericide, after Galli (1956
At the end of the incubation period enzyme activity
was stopped by sodium hydroxide-zinc sulfate precipitation,
and aliquots were assayed for reducing sugar by a modifica-
tion of Nelson's procedure (1944). This modification was
he substitution of sodium hydroxide for barium hydroxide
prevent precipitation of sulfated oligosacharides
formed during enzymatic hydrolysis of K-carrageenin and
fucoidin. The optical density of the final solution was
measured in a Klett-Summerson photoelectric colorimeten
using a green filter. Nwo standard glucose solutions,
1509 and 509, were run with each set of assays, and all
readings were evaluated by comparison with a glucose
standard curve. Values represent an average of two deter
minations of total reducing sugar released in the incubation
mixture, and correspond to the activity of 0.1 ml of tissue
xtract. The assay was found to be reproducible within 10
percent. The minimum value considered to be significant in
the tables was taken to be 50 ug.
REs
Algal associations:
To correlate diet with digestive activity, a rough
survey was made of the collection area. Heights for maximum
Lliam J. 5
concentration of larger specimens were estimated as 5 ft
for Acmaea scabra, 4 ft. for A. digitalis, 3 ft. for A.
limatula, and 2.5 ft. for A. scutum. Algae growing in the
spective collection areas were: A. scabra, microscopic
lgae only; A. digitalis, microscopic algae, Ralfsia spp.,
and some Peysonnelia spp.; A. limatula, microscopic algae,
Sigartina spp., Endocladia spp., and Pelveti-
Balfsia s
. andA. scutum, microscopic algae, Ralfsia spp.,
Sigartina spp., and Porphyra spp. The microscopic algae ir
this area were varied green and red algae (Haven, 1966).
his is not meant to be a definitive list of the dietary
onstituents of these animals, but rather an observation
of the algae available for food in the limited areas from
which the organisms were collected.
Enzyme activit
In the first experiment, the animals were "starved"
in an aquarium scraped free of algae, and kept in the dark.
After 4-6 days of starving, extracts of the gut were made,
and ensyme activity determined.
The results are recorded
in Table 1.
The results show the presence of a powerful amylase
with higher activity at ph 5.5 in all'species. An alginase
was also demonstrated in all species. All species examined
William J. B.
xcept Acmaea limatula exhibited a fucoidinase with highes
activity at pH 5.5. Small amounts of agarase and laminari-
nase were found in A. scutum, A. scabra, and A. digitalis
a K-carrageeninase also being present in the two latter
digitalis and A. scabra show the greatest
species.
overall activity, with sionificant amounts of enzyme activi
demonstrated for all substrates tested.
na second experimental series, animals were col
lected fresh from the field and extracts prepared within
Enzyme activities determined are shown in
three hours.
Table 2
powerful amylase was again evident, but wit
these animals small amounts of activity for all substrate
digitalis showed the
were found in all species. Acmae.
greatest overall activity.
comparison of the starved animals with the non-
tarved animals shows, in general, greater activity in non
ved animals. However, some species differences are
ident Although there are some deviations for particular
ubstrates, there is a general grouping in the effect of
tarvation, starvation causing a much greater decrease in
activity in Acmaea
limatula and A. scutum than in A. scabra
and A. digitalis.
Amylase activity was particularly affected.
William J. Beppu 6
DISCUSSION
Green and red algae, which contain starches, K-carrageenin,
and agar (Peat and Turvey, 1965), are available to all species
of Acmaea studied, and the high recorded amylase activity
correlated well with this. Brown algae, containing alginic acid,
fucoidin, and laminarin (Peat and Turvey, 1965), are available
to all species except A. scabra. Although this correlates well
with the low alginase activity found in A. scabra, it is not
reflected in the laminarinase or fucoidinase activity.
Eaton (1966) has made a careful study of the diet of A. limatula
and A. pelta. A similar study of the other species would be
desireable.
The data on changes in carbohydrase activity due to star¬
vation suggest that the degree of these changes is a function
of height in the intertidal zone, higher species (Acmaea scabra
and A. digitalis) showing loss drop in activity than lower species
(4. limatula and 4. scutum). Two possible reasons for this
difference are food retention time and feeding behavior. In fresh
animals, the lower species oliminated food that was only partly
digested at a much greater rate than the higher species. In
the starved animals, the remains of well-digested food were still
found in the gut of the two higher species, whereas nothing was
found in the gut of the two lower species. This difference in
food retention time correlates well with the changes in enzyme
activity during starvation.
The feeding behavior of tho higher species has also been
found to be more sporadic than that of the lower species. The
lower species, being splashed or under water a greater amount of
the tine than the higher species, are thus able to move and feed
for longer periods of time. Trus the higher species must
68
William J. Beppu
retain and digest food more efficiently, as was found, and are
therefore, not as susceptible to the effects of deprivation of
food.
SUMARY
The carbohydrate digestion of four limpets, Acmaea scabra,
A. digitalis, A. limatula, and A. scutum was studied. The
presence of a K-carrageeninase, an agarase, a laminarinase, a
Tucoidinase, an alginase, and an amylase was demonstrated for
fods avaae
all species. Some correlation of with enzyme activity was
found. Carbohydrase levels in starved animals were compared with
levels in non-starved animals. A decrease in enzyme activity
during starvation was found, and the amount of this decrease
could be correlated with height in the intertidal, higher species
(A. scabra and A. digitalis) showing less decrease than lower
species (A. limatula and A. scutum). This correlation might be
and
related to food rotention time feeding behavior.
ACKOLEDG
My sincere thanks to Dr. John H. Phillipps for purified
samples of algal polysaccharides and for his guidance during
the entire study. Many thanks to Dr. David Epel for his great
assistance in preparing this paper. This work was made possible
by Grant GY806 from the Undergraduate Research Participation
Program of the National Wcience Foundation.
7
64
O
William J. Beppu
8
LITERATURE CITED
Eaton, Charles
1966. The Activity and Food of the File Limpet, Acmaea
limatula. The Veligen, this volume.
Frederice, Eugene & H. F. Deutch-
1949. Studies on ovomucoid. Journ. Biol. Chem 181: 499-509
Galli, Donald Richard
1956. Carbohydrate digestion in an herbivorous marine snail,
Terula funebralis. Haster of Arts Thes., Stanford Univ.; 153 pp.
Haven, Stoner
1966. Personal communication
Nelson, Norton
1944. A photometric adaptation of the Somogyi method for the
determination of glucose. Journ. Biol. Chem. 153: 375-380
Peat, Stanley & J. R. Turvey
1965. Polysaocharides of marine algae. Pp. 1-45, Progress in
the chemistry of organic natural products, v. 23. New York
Wien & Springer-Verlag viii-397 pp., 58 fig.
70
page 1:
Footnotes
Permanent Address:
William J. Beppu
William J. Beppu
72
Aelt 1
Carbohydrase Activity of Starved Animals
kkak


Substrates
Opecies
pH
Fucoidin Alginic Acid
Agar
Laminarin
K-
rageenin
Starch
Total Reducing Sugar Released in 1 Hour

-
e
5.5
65.8 ug
32 ug
197A
1324g
19748
3520 ug
A. scabra
43.9
7.2
109
1530
168
298
298
224
3420
5.5
112
A. digitalis
55.9
1440
7.2
2760
20.8
A. limatula
5.5
811
239
7.2
82.0
82.0
61.5
1660
103
5.5
41.0
A. scutum

681
7.2
Opecies
A. scabra
A. digitali
A. limatula
A. scutum
l
William J. Beppu
78
19812 2
Carbohydrase Activity in Non-Starved Animals
—

Substrates
pI
K-carrageenin
Laminarin
Fucoidin Alginic Acid Starch
Total Roducing Sugar Released in 1 Hour





128 ug
5.5
51.4g
77.0Ag
51.4Ag
Opg
3440 gg
7.2
77.0
1980
100
250
275
225
3850
25.0
150
100
7.2
1950
51.4
103
103
103
51.4
5.5
3680
O
7.2
206
1870
5.5
300
198
175
150
150
3320
125
50.0
100
7.2
50.0
1775