CARBOHIDRASES IN TWO SPECIES OF LITTORIVES
Cornelia Farnum
May 30, 1964
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In the field both Littorina planaxis and Littorina scutulata are
observed feeding by radular action on substrates containing a variety
of microscopic blue green and green algae. Macroscopic algae are
known to be a natural component of the diet of L. scutulata; L. planaxis
only feeds on the large marine algal types when starved under laboratory
conditions. In addition, large quantities of bacteria and detritus
are revealed on examination of stomach contents. Feeding does not
appear to be highly selective. Extracts of the gut of both species
could, therefore, be expected to contain a variety of carbohydrases
capable of splitting linkages in the structural and reserve polysaccarides
of the marine enviornment. The bacterial contribution to snail digestion
must also be considered.
The following survey is not a characterization of the carbohydrases
of the littorines, but rather an examination of the digestive potential-
ities of these snails. Although the pH of the littorine gut was not
determined experimentally, a value of 5-6 or 6.5-7.5 is reported for the
digestive tract of many marine molluscs. (Freter and Graham, 1962).
Initial tests on L. planaxis showed activity to be considerably greater
at 5.5 than 7.5; therefore, subsequent tests were run at the lower
value. It cannot be assumed that the pH optimum of a particular enzyme
is the level at which it is functioning in the intact animal. By test-
ing at a pH calculated to be that of the alimentary tract one can
qualitatively analyze the enzymes of ecological significance to the
test organism.
Procedure and Metho
Snails were collected daily and starved for a period of twelve
hours. This procedure allowed time for a reduction of both the algal
1. Arthur Lyon Dahl, personal communication.
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703
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content and the bacterial infestation of the gut. Both esophagus and
midgut (stomach and digestive gland) were tested. Midgut extracts also
included gonadal tissue which probably contributed a large amount of
inert protein. Thus, enzyme activity on the stomach and digestive
gland is probably higher per milligram of tissue than experimental results
indicate.
For L. planaxis siail weight ranged from one to two grams, average
1.4; esophagus wet weight range .0046-.0028, average .0037; midgut
wet weight range .032-.059, average .045 grams. Snail weight in L. scutulata
averaged .4018 grams; esophagus wet weight range .0018-.0021, average
.0020 grams; midgut wet weight range .015-.021, average .018 grams.
Substrates of the polysaccarides starch, glycogen, agar and inulin were
prepared in one per cent solutions. For cellobiose, turanose and maltose
60 micrograms were present in the test solution; with melibiose and
sucrose this amount was increased to 100 micrograms.
The enzyme extracts were prepared by homogenization in five
milliliters of buffer with subsequent centrifugation for fifteen minutes.
One ml. of supernatant was added to one ml. of substrate and eight ml.
of buffer. This reaction mixture, layered with toluene, was incubated
at room temperature. Deproteinization with sodium tungstate in dilute
Høso (Haden, 1923) was used to stop the reaction. The mixture was
sampled at 0, 4. 12, 24, 48, and 72 hours and reducing sugar determined
by the Somogyi method (Somogyi, 1952; Velson, 1944). Measurements
were made with a Klett-Summerson Photoelectric Colorimeter using a
standard curve from 10 to 100 micrograms specific to the reducing sugar
being tested. Controls of buffer and substrate and buffer and enzyme
were run simultaneously with the reaction mixture.
Since various sugars give differing amounts of color by the Somogvi
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method, quantitative interpretation of experimental results is limited.
Although sugar increase by hydrolysis was always considered in respect
to the structural components of the polysaccarides and disaccarides
being tested, the exact proportions of different reducing sugars
present in the extractcontrols could not be determined. It is probable
that in this case hydrolysis of snail glycogen is occuring with glucose
the principle sugar.
Bacteria were isolated from the snail gut on a medium composed
of 500 ml. sea water, 500 ml. distilled water, 15 grams agar, 1 gram
peptone and 10 grams starch. A medium without starch but containing
filter paper was used to detect bacteria with cellulase activity.
Results
Active amylases capable of breaking down both starch and glycogen
were found in the midgut of both L. planaxis and L. scutulata, with
lesser activity per milligram of tissue in the esophagus. See figures
2 and 3. However, extracts of both tissues show an equal ability to
hydrolyze maltose, a disaccaride with the same alpha 1,4 linkage
present in starch and glycogen. This maltase was present in both
species. See figures 4 and 5. Alpha glucosidase activity for sucrose,
though positive for both tissues in both littorines, was weak compared
to the rate of hydrolysis of maltose. The alpha 1,3 fructose linkage
in turanose was split by the midgut of both species.
Cellulase activity was determined qualitatively by mixing enzyme
extracts with buffer and strips of Whatman #l filter paper, layering
with toluene and checking for increase in amount of reducing sugar
over the controls. Filter paper was dissolved overnight by mdgut
extracts from both species. Differences in ability to digest cell
walls of Porphyra and Ulva though not of Enteromorpha, Cladophora,
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Carbohydrases of Littorina planaxis and littorina scutulata
Substrate Linkage L. planaxis L.planaxis L. scutulata L. seutulata
midgut
esophagus
midgut
esophagus
STARCH
alpha 1,4
alpha 1,6
(pH 7.5)
glucose
GLYCOGEN
alpha 1,4
alpha 1,6
(pH 7.5
glucose
AGAR
beta 1,3
galactose
(pH 7.5)
INULIN
beta 1,2
fructose
CELLULOSE
beta 1,4
glucose
no test
CELLOBIOSE beta 1,4
gluose
MALTOSE
alpha 1,
luose
MELIBIOSE alpha 1,6
glucose
galactose
TURANOSE
alpha 1,
glucose
fructose
SUCROSE
alpha 1,2
glucose
beta
fructose
All run at pH 5.5. 4 means activity found under experimental conditions.
- means no activity found.
Figure 1
Pelvetia or Endocladia may reflect differences in the physical natures
of the substrates.
Galactosidase activity was weak in the extracts of esophagus of
both species when tested on the alpha 1,6 galactose linkages of melibiose.
Midgut extracts failed to show any activity. See figures 6 and 7.
However, the midgut of both L. planaxis and L. scutulata showed a limited
capability of hydrolyzing the beta 1,3 galactose linkages in agar while
esophagus extracts showed no increase in reducing sugar over controls.
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The only tissue containing activity for the 1,2 fructose linkages
of inulin was the hindgut of L. scutulata.
These results are summarized in figure 1.
Bacteria were cultured from the esophagus, stomach and digestive
gland of both L. planaxis and L. scutulata. Groups of both starved
and actively feeding snails were studied. Plates innoculated from these
different sources showed no significant differences after one week
of incubation.
Although a variety of bacteria grew, only the most common type,
one forming large white colonies, was positive for starch consumption
when tested with iodine. Vo bacteria capable of digesting either
cellulose or agar were detected after five weeks of incubation.
Discussion
Although at least one strain of bacteria capable of digesting
starch was detected, the presence of an active amylase in both eso-
phagus and midgut essentially discounts the possibility that intestinal
microflora are necessary for starch digestion. The lack of cellulose
digesting bacteria is also significant since coupled with the presence
of a very active cellulase in the midgut of both species and active
cellobiases in both tissues of both species, the cellulose digestion
of these herbivorous snails can be considered independent of the enzyme
contribution of bacterial simbionts.
Amylase activity on starch and glycogen appeared more active per
milligram of tissue in the midgut than in the esophagus in both L. planaxis
and L. scutulata. To the contrary, activity on the same alpha 1,4
linkage in maltose was comprable in both tissues. This would suggest
that the amylase and maltase are separate enzymes.
Although esophageal extracts of both species split starch and
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glycogen to a comprable degree, midgut extracts were found to release
greater amounts of reducing sugar from glycogen than starch over a
given time interval. This observation suggests a difference in the
specificity of the enzymes in these two tissues. The release of greater
amounts of reducing sugar from glycogen could be due to the action of
an alpha amylase in conjunction with a beta amylase. Combined activity
could degrade up to 80-90% of the substrate (Baldwin, 1957). Although
there may be inhibition of the enzyme near the branch points in glycogen,
an amylo 1,6 glucosidase may be breaking these branch linkages. This
ability would appear specific to alpha 1,6 glucose glucose linkages
since midgut extracts were found incapable of hydrolyzing the alpha
1,6 glucose galactose linkages in melibiose.
Amylase activity at pH 5.5 on starch and glycogen in the esophagus
of L. scutulata wasweak compared to that in L. planaxis. See figures 2
and 3. At plI 7.5 the esophagus of L. scutulata showed greatly increased
hydrolysis, while esophagus extracts of L. planaxis at this pl failed
to break down either polysaccaride.
Both the disaccarides maltose and sucrose contain alpha glucosidic
linkages. Snail enzymes hydrolyzed maltose quickly, but sucrose only
after an extended time. This indicates that probably two enzymes are
present. It is possible that the sucrase was studied not at its pl
optimum. Since beta fructosidase activity on inulin was limited to
the hindgut of L. scutulata, the sucrase is probably not a beta
fructosidase but rather an alpha glucosidase.
It is concluded that the carbohydrases in the littorines are
produced by the snails themselves; little reliance is placed upon in¬
testinal bacteria during digestion. Vo attempt was made to characterize
O
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enzymes found in the gut of the littorines for pH and temperature
optima, specificity, or change in activity with change in diet. Yet
it is clear that whether by many specific carbohydrases or by a number
of general enzymes, both L. planaxis and L. scutulata are capable
of breaking down structural and reaerve polysaccarides and disaccar-
ides of the marine enviornment to monosaccarides which can in turn be
absorbed to provide energy to these snails
Summary
1. Enzyme extrots of the esophagus and midgut of Littorina
planaxis and Littorina scutulata were found to contain amylases
capable of splitting starch and glycogen. Only one strain of bacteria
isolated from these organs could digest starch.
2. Alpha glucosidase activity was detected on maltose and
sucrose. The alpha 1,3 glucose galactose linkage in turanose was
split by the esophagus of both species.
3. Esophagus extracts showed galactosidase activity on agar
and melibiose.
4. No cellulose digesting bacteria were detected. However,
cellulase activity was found in the midgut extracts and cellobiase
activity in both tissues of both species.
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//2
BIBLIOGRAPHY OF WORKS CITED
BALDWIN, ERVEST.
namic Aspects of Bioschemistry.
Cambridge: University Press, 1957.
FRETER AVD GRAHAM: British Prosobranch Molluscs: Their
Functional Anatomy and Ecology. Dorking England:
Bartholomew Press, 1962.
HADEN, R. L:"A Modification of the Folin-Wu Method for
making protein free blood filtrates."
Journal
of Biological Chemistry 56: 469-471. (June) 1923.
NELSON. "A Photometric Adaptation of the Somogyi Method
for the Determination of Glucose." Journal of
Biological Chemistry. +153 (1944) p. 375.
SOMOGYI, MICHAEL: "Notes on Sugar Determination." Journal
of Biological Chemistry. +195. (1952) p. 23.