TRODUCTION
Several workers have reported the presence of high concentrations
of amino acids in the excretory fluid of Crustaceans (reviewed by Prosser
and Brown, 1961; see also Delaunay, 1927; Dresel and Moyle, 1950). For
example, as much as 18 to 27% of the nitrogen in Maja urine is reportedly
present in the form of free amino acids (Delaunay, 1927). However, there
seems to be little information available on the identity of amino acids
excreted by Crustacea nor is it clear how widespread this phenomena is
in the class.
In the present report the excretory fluid of an intertidal acorn
barnacle was analyzed for free amino acids. Eight amino acids were
separated and identified by techniques of paper chromatography.
MATERIALS AND METHODS
Excretory fluid was collected from the mantle cavity of the barnacle
3alanus glandula Darwin, 1854. The organism was removed from the rock
in such a way that the calcareous bottom plate was left on the rock but
the tissue layer forming the floor of the mantle cavity was left intact.
Through this semi-transparent membrane the mantle cavity fluid was
collected with a syringe. Approximately 75 organisms were needed to
collect one milliliter of fluid.
One milliliter of the fluid was deproteinized by addition of one
volume of 10% trichloroacetic acid. The TCA was removed with four to five
ether extractions. The pH was adjusted to pH 7 with O.1 N NH),OH and the
solution desalted by passage through Dowex 50 (H* form). The amino acids
were eluted with 1 N NHOH, evaporated to dryness in a flash-evaporator
at 50° C., and the residue redissolved in 1/2 milliliter of distilled
water. For analysis of blood amino acids, 1/4 milliliter of Balanus
glandula blood was collected from the main ventral blood sinus, added
mediately to an equal volume of TCA, and processed in the same manner.
Tive to ten microliters of solution were applied in 0.5 microliter
aliquots to Whatman l paper. The initial chromatograms, which first
indicated the presence of amino acids, were prepared by two-dimensional
ascending chromatography using n-butanol-acetic acid-water (l:1:5) as
the first solvent, followed by n-butanol-methyl ethyl ketone-water (2:2:1).
nal identifications were made on amino acids separated by one-dimensional
descending chromatography run simultaneously with 1 per cent amino acid
standards in systems of n-butanol-acetic acid-water (4:1:5) and n-butanol-
methyl ethyl ketone-water (2:2:1) in a chamber saturated with cyclohexyl-
amine fumes (Mizell and Simpson, 1960). The chromatograms were dried in
a 75° C. oven for 30 minutes, sprayed with.25% ninhydrin in acetone with
78 v/v acetic acid, and heated at 85° C. for 15 minutes.
Identification of amino acids was made on the basis of the character-
istic colors produced by ninhydrin in the presence of cyclohexylamine
(Mizell and Simpson, 1960), on the basis of their co-chromatography in
the above two solvents with amino acid standards, and on the basis of
results with specific colorimetric location reagents. The location reagents
used were platinic iodide reagent for sulfur-bearing amino acids (Block, 1958),
sulphanilamide reagent for histidine (Block, 1958), Sakaguchi reagent for
arginine (Block, 1958), 0.28 isatin in acetone followed by Erlich reagent
for hydroxyproline and proline (Smith, 1965), the Sullivan procedure for
cystine and cysteine (Stekol, 1959), 1-nitroso 2-naphthol test for tyrosine
(Feigl, 1954), and the Elson-Morgan test for the presence of N-acetyl-
glucosamine in the blood (Kabat, 1961).
na
RESUL!
The results showed the presence of eight amino acids in the mantle
cavity fluid. Table I lists those present and indicates the relative
intensity of the ninhydrin-positive spots. Amino acid composition of the
blood was also determined (Table I). A major difference between blood
and mantle cavity fluid is that the blood had hydroxyproline and traces
of proline, while the mantle cavity fluid had large amounts of proline
and no hydroxyproline. Serine and leucine, which were present in the
mantle cavity fluid, were not present in the blood. The concentration
of lysine seemed to be far greater in the mantle cavity fluid than in
the blood. These differences can also be seen in Figure I, which depicts
a chromatogram in which the amino acids of blood and mantle cavity fluid
were simultaneously separated. The figure also shows the position of
an unidentified yellow, ninhydrin-positive substance with an approximate
Rf of 10 in n-butanol-acetic acid-water.
SSION
DIS
The above results show that eight amino acids are present in barnacle
mantle cavity fluid, and that these are not completely identical to those
present in barnacle blood. Of particular interest is the inverse
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relationship between mantle cavity fluid and blood in the content of
proline and hydroxyproline. These differences suggest that the amino
acids found in the mantle cavity fluid are not contaminants from the
blood.
Why amino acids are excreted by Crustacea is not known. One
explanation suggested by Parry (1960) is that the Crustacea cannot
ficiently metabolize all amino acids. The present findings, showing
that eight amino acids are excreted, would require modification of
this hypothesis, since it seems doubtful that the organism would have
lost almost one-half of the genetic machinery involved in amino acid
catabolism. It is conceivable, however, that the excretion of these
amino acids represents an overload of amino acids resulting from an
excessive amount of protein in the diet.
Amino acid excretion could also represent an alternate mechanism
for avoiding toxic concentrations of ammonia, since ammonia could accumulate
when the animal is exposed at low tide and injure the adult or its brooded
larvae. If this is the case, the organisms have chosen the extremely
expensive route of simply not oxidizing a large number of amino acids.
his would seem extremely wasteful, especially since several of the amino
acids found, such as lysine, have very low nitrogen/carbon ratios.
Norman (1967) has found that embryos in the ovigerous lamellae can
utilize lysine and leucine for protein synthesis. Since these amino acids
were found in the mantle cavity, there is possibly some utilization of
these amino acids by the larvae. However, amino acid excretion does not
C
depend on the presence of the larvae, since the amino acid content
was identical during and after the reproductive season.
Further study of the mantle cavity fluid, blood, and diet are
required to clarify the significance of this aminchacid excretion.
SUMMARY
Separated by paper chromatography, eight amino acids were identified
lam
in tho mantio cavity fluid of
glandula. They are glutamic acid,

lysine, proline, alanine, valine, serine, leucine, and arginine. These
amino acids do not compare identically with those found in the blood of
the same organism, one main difference being the presence of hydroxy-
proline and traces of proline in the blood, and the absence of hydroxy-
proline and large concentrations of proline in the mantle cavity fluid.
Reasons for the presence of amino acids in the mantle cavity are discussed.
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TABLE I

AMINO ACIDS IN MANTLE CAVITY FLUID AND BLOOD
NTNEV
TY OF MIRIYDRIN SPOT
AMINO ACID
PRESENCE IN BLOOD
MANTLE CAV.
Y FLUID
—-
Arginine
+ ++
+
Lysine
—
—
Aspartic Acid
—
Cysteine
Glutamine
Asparagine
—
Cystine
—
—
Glycine
Hydroxy
- + —
roline
+ +
— —
Alanine
+ +
Proline
—
Cysteic Acid
—
—
Histidine
Valine
+ +
—
Tyrosine
Leucine
Tryptophan
Phenylalanine
Glutamic Acid
+ -
— -
Serine
—
Taurine
O
C
key
———
TABLE I CONT


not present
+
trace present
+ —
resent in moderate amounts
+ + ++ present in large amounts

2
-—
FIGURE I
D PHOTOGRAPH OF ACTUAL CHROMATOGRAM
r09
Chromatogram I (on left) - in n-butanol-acetic acid-water
left - mantle cavity fluid
right - 19 standards of aspartic acid and hydrox
proline
Chromatogram II (on right) - in n-butanol-acetic acid-water
left - mantle cavity fluid
right - blood (notice presence unidentified spot)
28
REFERENCES
Block R. J., Durrum E. L. and Zweig G. (1958) A Manual of Paper Chroma¬
tography and Paper Electrophoresis. 2nd edition, Academic Press,
New York.
Delaunay H. (1927) Bull. Sta. Biol. Arcachon 24: 95-214, 1927.
Dresel E. B. and Moyle V. (1950) Nitrogen Excretion by Amphipods and
Isopods, J. Exp. Biol., 27, 210-225.
Feigl F. (1954) Spot Tests. Elsevier Pub. Co., Houston.
Kabat E. A. and Mayer M. M. (1961) Experimental Immunochemistry, 2nd ed.,
Charles C. Thomas Publisher, Springfield, Illinois.
Mizell and Simpson (1960) Paper Chromatography of Amino Acids, J. Chroma-
tography, 5, 157-160.
Norman D.
Parry G. (1960)Excretion. in The Physiology of Crustacea by Waterman,
Academic Press, New York.
Prosser C. L. and Brown F. A. (1961), Comparative Animal Physiology,
2nd ed., W. B. Saunders Company, Philadelphia.
Smith I. (1965) Chromatographic and Electrophoretic Techniques, 2nd ed.,
Interscience Pub., New York.
Stekol J. S. (Preparation and Determination of Sulfur Amino Acids and
+
Related Compounds. in Methods in Enzymology, V. Ill, Academic Press,
New York.
20
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0
Acknowledgements
The author wishes to thank Dr. David Epel, Dr. J. H. Phillips,
and Dr. D. P. Abbott for their advice and helpful assistance during the
study.
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