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BARIBAULT, WILLIAM H. (Hopkins Marine Station of Stanford
University, Pacific Grove, California). Nitrogen
Excretory Products in the Limpet Acmaea (Mollusca:
Gastropoda: Prosobranchia). The Veliger
Nitrogen excretory products were examined in the genus
Acmaea by a microdiffusion technique for urea and ammonia and
a colorimetric technique for uric acid. Five species were
examined, varying in vertical distribution. Similar concen¬
trations of ammonia, urea, and uric acid were found in all five
species. A large amount of intraspecific varition in the
distribution of these compounds was noted, especially in the
high intertidal forms Acmaea scabra and Acmaea digitalis. These
variations might reflect individual differences in enzyme
activity, individual differences in diet, or adaptive responses
to the environment. E
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4
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Nitrogen Excretory Products In The Limpet Acmaea
(Mollusca: Gastropoda: Prosobranchia)
by
William H. Baribault
Hopkins Marine Station
Stanford University.
Pacific Grove, California
X Footnott
William H. Baribault
NTRODUCTION
Examination of excretory products in the molluscs
indicates that more terrestial forms tend to be urico¬
telic and aquatic forms tend to be ammonotelic. This is
especially evident in Needham's classic study of the
littorinid snails where an ascending order of uric acid
production was found to be directly correlated with
increasing height in the intertidal zone (Needham, 1935).
als
In this regard, members of the genus Acmaea present an
interesting contiuum. On the Monterey Peninsula,
populations of the five common species of this genus
are found varying from +0.0 feet to +5.0 feet. To see if
any correlations could be found between intertidal zona¬
tion and nitrogen excretory patterns, a comparative
analysis of excretory products in this genus was made.
METHODS AND MATERIALS
The five species of Acmaea used in this investi¬
gation were Acmaea digitalis Eschscholtz, 1833; A, lima¬
tula Carpenter, 1844; A: scutum Eschscholtz, 1833; ap shs
823 and A. scabra (Gould, 1846). All species were collected
from the intertidal area at periods of low tide on
Pescadero Point in Monterey County, California.
The same collection procedure was followed for all
animals studied. Each animal was removed from the rock
William H. Baribault
substratum, with care to avoid loss of fluid from the
mantle cavity, and placed into a capped glass vial
containing 5 ml of artificial sea water (Harvey, 1954).
The animals were kept in the vials at sea water tempera¬
ture for two to three hours. The artificial sea water
(ASW) was decanted into a graduated centrifuge tube, the
limpet removed, and each vial rinsed with 2 ml of ASW.
Each animal was then gently squeezed, anterior end down
over the centrifuge tube, to empty the mantle cavity of
any remaining fluid. The pooled fluid was brought to
10 ml with ASW, centrifuged to remove any particulate
matter, and the supernatant decanted and frozen until
used for analyses.
Total nitrogen, ammonia nitrogen, and urea nitrogen
were assayed by the microdiffusion method of Ternberg (1965).
The total nitrogen was determined on a 1 ml sample after
wet ashing with 1 ml of sulfuric acid saturated with cop¬
per sulfate. Ashed samples were diluted with 5 ml of
distilled water, and 4 ml of 10 N sodium hydroxide were
substituted for the sodium carbonate solution used in
Ternberg's original method.
Urea was determined as ammonia after a thirty minute
incubation with 0.1 ml of a 50 mg/ml solution of urease
(obtained from Matheson, Colman, and Bell) in distilled
water. Uric acid was determined colorimetrically by the
method of Sobrinho-Simões (1965). Prior removal of
proteins was found to be unnecessary.
William H. Baribault
Gaseous excretion of ammonia was determined by pla¬
cing the intact animal into a dry diffusion apparatus
for 24 hours. Gaseous ammonia was collected by absorp¬
tion on alkaline filter paper, and analyzed for ammonia
by Ternberg's procedure.
The microdiffusion assay procedure was calibrated
with ammonium sulfate solutions of known concentrations
and found to be extremely reproducible (Figure 1) with
triplicate and duplicate determinations on the same sample
revealing small standard deviations (eg., Acmaea scutum
NH, - N, 7.0 + 0.25 ug).
RESULTS
Ammonia, urea, and uric acid were found to be ex¬
cretion products in all species of Acmaea investigated.
Some species showed wider individual varations in
amounts of certain excretory products than other species.
For example, the values for ammonia nitrogen in Acmaea
digitalis ranged from 0.4 to 25 ug N, whereas for Acmaea
scutum the range was much less, varying from 6 to 10 ug
N (Table 1). In terms of individual varation, the
greatest variability in ammonia nitrogen was found in A.
digitalis, followed by A. scabra, A. pelta, A. limatula,
and A. scutum. Variation in uric acid was greatest in
A. pelta, followed by A. scabra, A. limatula, A. scutum,
and A. digitalis.
William H. Baribault
The percentage distribution of the various nitro¬
genous compounds, based upon mean values, is shown in
Table 2. The comparison between the five species indicates
that relatively more ammonia is excreted by Acmaea dig¬
italis and A. scabra, that less uric acid is excreted
by A. digitalis, and that A. scabra and A. scutum excrete
less urea. It can also be seen that greater than 80%
of the total nonprotein nitrogen can be accounted for
by these methods.
Gaseous ammonia was found to be excreted in the five
species of Acmaea examined with the dry diffusion tech¬
nique described above. Those forms found higher in the
intertidal zone, A. scabra and A digitalis, produced
more of the gaseous product than the lower intertidal
forms, A. scutum, A. limatula, and A pelta. The mean
value after 24 hours was 2.9 ug NH,-N for the higher
species and 2.0 ug NH-N for the lower ones.
DISCUSSION
The microdiffusion and colorimetric techniques,
coupled with the collection procedure, haveprovided a
means of analysis of individual specimens of the genus
Acmaea. The extreme standard deviations obtained (eg.,
in A. digitalis, NH,-N 8.9 + 8.3 ug) indicate very large
individual differences. The deviations did not result
from the assay procedure, which, as previously indicated,
6
William H. Baribault
TABLE CAPTION
TABLE I
Relative ug nitrogen excretory products with
standard deviations for eight animals for each
species. Rest nitrogen is the difference between
the total nitrogen and the sum of the three pro¬
ducts.
TABLE II
Percentage excretory products calculated
rom mean values of table I.
50
O
1.0
0.9
O.8
).7
0.6
O.4
O.3
0.2 -


O.1

kakaaka-
1 2 3 4 5 6
U9 NH3 N
William H. Baribault
Figne
NH3 N
Urea N
Uric Acid N
Rest N
Total N
William H. Baribault
NITROGENOUS EXCRETORY PRODUCTS IN ACMAEA
Acmaea
Acmaea
Acmaea
Acmaea
Acmaea
scabra
limatula
digitalis
scutum
pelta
10.6 + 6.3
8.9 + 8.3
8.5 + 3.9
7.5 + 1.8
9.25 + 4.6
3.75 + 3.2
5.9 + 4.2
7.3 + 5.2
6 f1.5
11.1 + 4.2
14.6 + 4.9
14.7 + 4.3
7.9 + 1.7
13.3 + 2.9
17,8 + 6.7
5.0 + 3.5
5.0 + 2.9 2.8 + 2.7
.7 1 4.2
5.75 + 4.7
33.0 + 7.9
27.8 + 7.1 83.3 1 8.2 82.0 + 7.3 43.9 + 9.1
able
S.
NH3 N
Urea N
Uric Acid N
Rest N
Total N
William H. Baribault
RIBUTION OF NITROGEN PRODUCTS
PERCENTAGE DI
Acmaea
Acmaea
Acmaea
Acmaea
Acmaea
Pelta
digitalis
limatula
scutum
scabra
21
32
25.5
23.5
32
25.5
22
14.5
11.4
21
40.5
44.5
44.5
42.5
28.5
13
12
19.5
18.5
100
100
100
100
100
ae
illiam H. Baribault
CAPTIONS
FIGURE I
Calibration of diffusion apparatus with ammo-
nium sulfate solutions using optical density as a
measure of ug concentration.
TABLE I
Relative ug nitrogen excretory products with
standard deviations for eight animals for each
species. Rest nitrogen is the difference between
the total nitrogen and the sum of the three pro¬
ducts.
TABLE II
Percentage excretory products calculated
from mean values of table I.
William H. Baribault
highl
was reproducible. These maximal and minimal
components of variability, only seen in comparing indi¬
viduals, would be masked by methods that measure pooled
mara
. In particular, these results demonstrate
the value of individual analysis. Although average
values can be obtained, as in Table 2, the interspecific
differences observed might therefore be misleading.
Several hypothses can be advanced to explain the
large individual variation in the relative amounts of
excretory nitrogen. One is that enzyme content varies
within the population, resulting in different distri¬
butions of excretory nitrogen products when comparing
individuals. This is especially suggested by William's
work on biochemical variation within individuals of
the same species (Williams, 1966). Another explanation
is that individual variations in the diet would result
in the observed distribution of nitrogen products. A
third possibility is that the limpet changes its major
products of nitrogen excretion as a result of exposure
to air during the tidal cycle. This is suggested by
Needham's (1935) proposal that more terrestial animals
tend to excrete uric acid, whereas more aquatic forms
tend to excrete ammonia. This hypothesis is also sugges¬
ted by the larger variations in the higher forms, which
would have been exposed to longer, and more variable,
periods of dryness. If correct, this could suggest a
S.
William H. Baribault
unique adaptation of the higher forms to secrete more
uric acid when dry than when wet. Such a transition
would be of adaptive value, since ammonia and urea at
high concentrations are toxic.
Although urea was found to be excreted by all species
examined, the metabolic mechanism of its formation is
not clear. Campbell (1966) found no arginase in the
spP
digestive gland of an Acmaea, suggesting the absence of
the ornithine cycle in this species. However, Campbell
only examined the digestive gland, leaving open the pos¬
sibility that the enzyme might be present in other tissues.
While his results possibly eliminate the ornithine cycle,
other pathways based upon purine degradation could be
alternative mechanisms for urea production. For example,
in fishes, products of purine breakdown from uric acid
give rise to urea via the enzyme allantoicase (Laskowski,
1951). This pathway is also suggested by hi
preliminary experiments indicating formation of urea by
minced digestive gland in an ASW- uric acid solution.
The finding that all three products are present,
and the reported absence of arginase, suggests the pos¬
sibility that the source of ammonia nitrogen is by
protein catabolism, whereas the urea and uric acid pro¬
ducts might result from purine catabolism.
The nature of the  nitrogen is not
5
William H. Baribault
clear. Qualitative tests with 0.25% ninhydrin solution
show the presence of peptides and amino acids in the
samples obtained from the limpets. This may account
for some of the 13-18% unidentified nitrogen.
SUMMARY
Nitrogen excretory products were examined in the
genus Acmaea by a microdiffusion technique for urea and'
ammonia and a colorimetric technique for uric acid.
Five species were examinéd, varying in vertical distribution.
Similar concentrations of ammonia, urea, and uric acid
were found in all five species. A large amount of
intraspecific variation in the distribution of these
compounds was noted, especially in the high intertidal
forms Acmaea scabra and Acmaea digitalis. These variations
might reflect individual differences in enzyme activity,
individual differences in diet, or adaptive responses
to the environment.
William H. Baribault
ACKNOWLEDGEMENTS
I am indebted to Dr. John Phillips for his
advice and encouragement during this investigation,
Appreciation is extended to Dr. David Epel for his
helpful recommendations in the preparation of the
manuscript. The work was made possible by Grant
GY 806 from the Undergraduate Research Participa¬
tion Program of the National Science Foundation.
William H. Baribault
LITERATURE CITED
CAMPBELL, J. W.
Distribution of arginase activity in molluscs.
1966.
Comparative Biochemistry and Physiology. 17: 259-270
HARVEY, H. W.
1954.
Chemistry and fertility of sea waters. Cambridge
University Press
LASKOWSKI,
The Enzymes, Chapter 27, ed. Sumner and Myrbäck,
1951.
Academic Press, New York
NEEDHAM, J.
1935.
Problems of nitrogen catabolism in invertebrates.
Correlations between uricotelic metabolism and habitat
in the phylum Mollusca. Biochemical Journal 29:
238-251
HO-SIMOES, M.
SOBRIN
A sensitive method for the measurement of serum
1965.
uric acid using hydroxylamine. Journal of Laboratory
and Clinical Medicine 65: 665-668
IRNBERG, J. L.
1965. Colorimetric determination of blood ammonia. Jour¬
nal of Laboratory and Clinical Medicine 56: 766-776
WILLIAMS, R. J.
Individuality in nutrition: Effects of Vitamin A¬
1966.
Deficient and other deficient diets on experimental
animals. Proceedings of the National Academy of Science
55: 126-134
59
William H. Baribault
FOOTNOTES
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60
William H. Baribault
FIGURE CAPTIONS
FIGURE I
Calibration of diffusion apparatus with ammo-
nium sulfate solutions using optical density as a
measure of ug concentration.
c