Michael J. Miille
Abstract
Uptake of Amino Acids by Metridium senile
ABSTRACT
1. Metridium senile incorporated between 30% and 60% of
the 7C-labeled glysine, valine, glutamic acid, lysine,
and an algal mixture when they were present at natural
concentrations.
33% of the glysine and 66% of the glutamic acid incorporated
by the anemone was metabolized to C0.. This could account
for 12% to 24% of the animals total CO, pbduction.
The rates of release for glysine and glutamic acid are
calculated. There is a net loss of glysine and a net
uptake of glutamic acid by the anemone.
In starved versus fed anemones, the starved animals
take up glysine and produce 1700, in significantly
higher amounts. The advantage of this to Metridium
senile is discussed.
Michael J. Miille
Page 2
Uptake of Amino Acids by Metridium senile
INTRODUCTION
Since the early 1960's many marine organisms have been
shown to take up dissolved organics such as amino acids and
glucose. Stephens gives a good summary of this work (Stephens
1968) and concludes that it can be energetically significant
to the animal. The major work on coelenterates has been done
over the past 4 years by Schlichter on Anemonia sulcata. He
has found that there is active uptake of the amino acids from
sea water, that this uptake is through the ectoderm, and
suggests that the animal could satisfy much of their energy
requirements in this way (Schlichter, 1974). However, it still
remains to be demonstrated that there is a net uptake of amino
acids, and the amounts which are removed from sea water are
nutritionally significant.
The animal used in the study reported here was Metridium
senile, (Linnaeas, 1767) collected in Monterey Bay from an
area of high dissolved organic content, little water movement
necessary for particle feeding, and therefore, from a situation
in which direct uptake of dissolved organics would be a benefit
Michael J. Miille
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Uptake in Amino Acids by Metridium senile
to the animal. These anemones were used to measure uptake,
and release of amino acids, and the contribution of such
materials to C02 production by fed and starved animals.
MATERIALS AND METHODS
Metridium senile were maintained in the laboratory in
running water. They were fed squid every three days prior
to use. Animals weighing approximately 2 grams were placed
in a 250 ml. Erlenmeyer flask with 100 ml. of filtered sea
water and allowed to settle 24 hours prior to the start of
an experiment. Filtered instant ocean containing 170-labeled
amino acids or glucose at concentrations found in coastal waters
were used, i.e. 60/m/1 for amino acids (Clark, Jackson, North,
1972) and 804g/1 for glucose (Riley, Chau, 1970). These
preparations were maintained at 11 to 12 C. The decrease in
dissolved organic material was measured by removing iml. samples
from the flask after thorough mixing by transfer to a scintillation
vial. 1002 was removed by the addition of IN H»SO4. One hour
was allowed for the loss of volatile 0 before restoring the
Page
Michael J. Miille
Uptake of Amino Acids by Metridium senile
origidnal pH by addition of IN NaoH prior to the addition of
New England Nuclear Aquasol and counting.
To measure the amounts of 1700, given off by the animal,
a 50ml. Erlenmeyer flask stoppered with Top stoppers, K-882310,
fitted with plastic center wells, K-882320, both from Kontes
Glass Co. Viveland, N.J. was used as a trap. The well contained
a filter paper wick wet with .15 ml. of ß-phenalethylamine. Two
1002 was
ml. samples were used in these determinations.
released by lowering the pH with the addition of IN H,SO). This
system was allowed to stand for 24 hours before removal of the
wick for counting.
Gaseous 100, released by the animal during incubation
was trapped and measured in a similar way. Samples were counted
using a Nuclear-Chicago Unilux II scintillation counter.
RESULTS
Uptake
The following materials at sea water concentrations were
studied: glucose, imCi/12mg; lysine, 312 mCi/mmole; valine,
Michael J. Miille
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Uptake of Amino Acids by Metridium senile
260 mCi/m mole; glutamic acid.1 mCi/27mg; glysine, .imCi/7.2 mg;
and New England Nuclears Amino Acid -70 (u.1.) Mixture, which
contains amino acids present in the same relative proportions
as found in a typical algal protein hydrolysate..Each ofthese
materials was tested for rates of uptake on two separate animals.
At the end of 12 hours exposure to the 70-labeled materials, the
animal was dried, weighed, and ground in a mortar before extraction
with 5 ml. of 70% ethanol for 24 hours to obtain free amino acids
and other extractable substances. An additional 5 ml. of ethanol
was added and the mixture centrifuged at 2,000 r.p.m.'s. A
sample of both the supernatent and insoluble residue wascounted.
The results are shown in Table #1. C0,, alcohol soluble,
and insoluble portions are expressed as percent of the total
'C taken up. Figure l shows the rates of uptake over the
entire 12 hours. Uptake for the first 2 hours is linear. Also,
20%-50% of the amino acids taken up were metabolized to C0..
were selected
Glysine and glutamic acidfor further study because of
the difference in their conversion to C0, and incorporation
into the amino acid pools.
Michael J. Miille
Page 6
Uptake of Amino Acid by Metridium senile
Bacterial Affects on Uptake
The contribution to catabolism by bacteria associated
with the anemone was assessed by observing uptake and conversion
to CO, in the presence and absence of Streptomyocin at a concentratio
of 50/g/ml. Uptake by 5 different animals in the presence of
this antibiotic was compared with the uptake by 5 animals in the
absence of the bacteristatic substance. Incubation was for only
Whours. The results are presented in Table f2.
Streptomyocin had no significant affect on the amount of
C-labeled amino acids taken up or catabolized. In this experiment
which utilized a larger number of animals, a correlation between
body weight and percent uptake, r-.85, p.01, was observed and
permitted percent uptake to be expressed in terms of the gram
dry weight of the anemone. Note that almost twice as much
glutamic acid was converted to labeled CO, as glysine.
To further substantiate the minimal involement in the effects
observed, 4canemones were shaken in 50ml. of sea water to release
bacteria attached to their outer surfaces.. The anemone was then
removed, labeled glysine and glutamic acid added, and the decrease
in specific activity measuredover 24 hours. Results are shown in
Table f3. During the first 4 hours bacteria had little affect
Michael J. Miille
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Uptake in Amino Acids by Metridium senile
on the amount of labeled material removed from solution. After
reult
24 hours in the presence of streptomyocin a similiar/was found.
In the absence of the antibiotic a significant affect apparently
due to bacteria, was observed.
Release of Amino Acids from Metridium senile
In this experiment six animals, described in Table #2, were
incubated in the labeled substrate solutions for 4 days to permit
equilibration of labeled and unlabeled amino acids in the animals
tissue. At the end of this period the animal was washed four
times in sea water and then placed in 100ml. of filtered sea
water. The release rates of labeled material were then measured
as a function of the increase in non-CO, carbon-14 in the sea
water. At the end of the experiment the anemones were extracted i
in 70% ethanol, and the levels of 170 -labeled amino acids
determined. The results are shown in Table #4. The anemone exhibits
a net release of glysine over the 4 hour period, and a net uptake
of glutamic acid.
Uptake in Fed versus Starved Anemones
Ten Metridium senile, varying in weight by no more than
.2 grams wet weight, were collected from the same clone. They
were placed in beakers and maintained in running filtered sea water
for two weeks. During the following two weeks, only 5 of the 10
Michael J. Miille
Page 8
Uptake of Amino Acids by Metridium senile
were fed squid three times a week. At the end of this period,
all ten were put in 10ml. of 170-labeled gylsine, and uptake
and CO, release were measured as before.
The results are shown in Table #5. The difference in results
was assessed significant. In both cases the starved animals
removed amino acids and catabolized them at a significantly
higher rate, p(.05, p(.Ol respectively.
DISCUSSION
The results of the experiments performed indicate that
Metridium senile is capable of absorbing amino acids and glucose
from sea water at natural concentrations. The rates of uptake
are similar to those found by Schlichter an Anemonia sculcata
(Schlichter, 1974). The figures show that uptake is linear for
the first two hours, followed by a leveling off to a final
concentration. One possible explanation for the results is that
a minimum concentration was reached, below which the animal
could no longer absorb the amino acids. These minimal values
range from 1.2 x 10"' moles/liter for valine to 6.9 x 10 moles/li
Page 9
Michael J. Miille
Uptake of Amino Acids by Metridium senile
for glysine. These results can be explained by the reports
that uptake rates exhibit enzyme kinetics. (Taylor, 1969; Shick,
1975; Stephens 1963). The observed uptake of amino acids and
conversion to CO, cannot be attributed to bacteria because
of the high uptake rates in the presence of streptomyocin. Twice
as much of the incorporated glutamic acid was converted to C02 as
compared to glysine. To compare the 10,/ 1200, ratio in the
animal, and determine the nutritional contributions of dissolved
glysine and glutamic acid, the CO, excretion values for Metridium
as determined by Parker were used. (Parker, 1923). This shows
glysine could account for 12.5% of the animals CO, and glutamic
acid 24.4%. These values suggest that dissolved organics may
account for substantial amounts of the anemone's nutrition in
some situations.
The release rates from the anemone were calculated for
glysine and glutamic acid following the guidelines established
by Johonnes, Coward, and Webb, (1969). A net release of glysine was
found, while there was a net uptake of glutamic acid. This is
energetically resonable if one compares the amounts which are
used in metabolism, and it demonstrates the same results which
Ferguson found in starfish (1971). Because of the high concentratio
gradient, 102:1, the uptake of glutamic acid may be explained
Michael J. Miille
Page 10
Uptake of Amino Acids by Metridium senile
by active transport, as found by Schlichter (1974).
The increased utilization of dissolved amino acids by
starved Metridium was demonstrated, showing the possible selective
advantage to uptake of dissolved organics during food deprivation.
A similar increase in 00, production by starved Aurelia aurita
was shown by Shick (1975), who reached the same conclusion.
One possible explanation for this result would be a difference
in the freedamino acid pools of fed and starved animals. However,
in the same experiment, Shick (1975) showed that the total
free amino acid poncentration in the pools is little affected
by starvation, supporting the theory that increased uptake is a
response to starvation.
The possible importance of dissolved organics to Metridium
senile has been demonstrated by this study. However, no conclusive
statement as to their quantitative contribution to the animals
nutrition can be made until the 100,/1200, ratio for an
animal over a long period of time has been measured. Further
studies of this aspect are anticipated.
Page 11
Michael J. Miille
Uptake of Amino Acids by Metridium senile
ACKNOWLEDGMENTS
Iwould like to express my sincere thanks to the faculty
and staff of Hopkins Marine Station, especially to Dr. John
Phillips and Dr. Robin Burnett for their advice during this
study and assistance in preparing this paper. My thanks
also to Susan Ball for typing this paper and to Kim Suelzle
and Carol Rodgers for their assistance in running two of
the experiments.
Michael J. Miille
Page 12
Uptake of Amino Acids by Metridium senile
LITERATURE CITED
Chau, Y. K., J. P. Riley
1970. Amino acid concentration found in sea water.
Deep Sea Res. 13:1115-1121
Clark, M. E.; G. A. Jackson, and W. J. North
Dissolved Free Amino acids in Southern California Coastal
Waters. Limnol. & Ocean. 17(5):749-756
Ferguson, J. C.
117
Uptake and release of free amino acids by starfish.
Bio. Bull. 141:122-129
Johannes, R. E., S. J. Coward, K. L. Webb
1969
Are dissolved amino acids an energy sourde for marine
invertebrates?
Comp. Biochem. Physiol. 29:283-288
Schlichter, D.
1274
The influence of physical and chemical factors on the uptake
by Actinians of amino acids dissolved in sea water.
Mar. Biol. 25:279-290
Shick, M. J.
1475
Uptake and utilization of dissolved organics by Aurelia
aurita scyphistomae: temperature effects on the uptake
process; nutritional role of dissolved Amino acids.
Bio. Bull. 148:117-140
Parker, G. H.
723
he excretion ot cnbon dioxide sy velaxes
contracted sen anemones.
J. 6en, Physiol. S: 45-64.
Page 13
Michael J. Miille
Uptake of Amino Acids by Metridium senile
Stephens, G.
1963
Uptake of Organic Molecules by Aquatic Inverts.- II. Accumulation
of amino acids by the bamboo worm, Clymenella Torquata.
Comp. Biochem. Physiol. 10:191-202
Stephens, G.
1968
Dissolved organic matter as a source of nutrition for
marine organisms.
Am. Zoo. 8:95-106
Taylor, A. G.
1969
The direct uptake of amino acids and other smallmolecules
from sea water by Neres virens sars.
Comp. Biochem. Physiol. 29:243-250
Michael J. Miille
Page /4
Uptake of Amino Acids by Metridium senile
Figure Caption
Decrease in the counts-per-minute of a given substrate
Fig. 1.
during the experimental period. Each point represents
the counts in 1 ml. of solution. Circled numbers
correspond to the animals number in Table f1.
Michael J. Miille
Page 15
Uptake of Amino Acids by Metridium senile
Table Captions
Percent uptake (calculated as nitial - final g.p.M.)
Table 1.
initial
and distribution of 10-labeled materials in Metridium
senile. Distribution of the 170 is given as percent
of total incorporated 170.
Table 2.
Percent uptake and 1002 production by Metridium
senile in solutions with C-labeled glysine and
glutamic acid, with and without an antibiotic
present.
Table 3.
Percent uptake of 1C-labeled glysine and glutamic acid
by bacteria, with and without an antibiote present.
Table 4.
Net movement of glysine and glutamic acid is calculated
by determing the total uptake to total release ratio.
All values for uptake, release, 170. Amino acids, and
total amino acids are given in mMol/gm. dry wt.
Percent uptake of glysine and 100, production
Table 5.
is shown for fed and starved Metridium senile.
Michael J. Miille
Uptake of Amino Acids by Metridium senile
TABLE f1
% Alcohol % Alcohol
Substrate
Soluble
% Uptake
% C02
Insoluble
41.5
27.3
8.3
Glucose 1
3.1
44.3
1.6
Glucose 2
25.9
2.5
44.4
20.8
26.0
Lysine 1
1.3
2.6
27.0
22.7
Lysine 2
31.4
29.4
Valine 1
34.4
38.0
2.3
46.0
Valine 2
42.0
7.1
29.7
3.4
Glutamic acid 1
75.0
22.0
38.6
53.0
Glutamic acid 2
39.2
25.0
9.3
49.0
Glysine 1
67.0
6.5
12.5
Glysine 2
49.5
7.0
67.0
37.0
33.4
Algal mix 1
57.0
22.6
5.1
2.5
Algal mix 2
20.6
39.1
30.0
Michael J. Miille
Uptake of Amino Acids by Metridium senile
TABLE #2
Substrate
Weight
% Uptake
(grams)
Glysine 1
33.8
.72
38.4
.90
10
3
32.8
.71
29.3
.85
29.7
.47
X =32.8
Glysine + Strept. 1
37.0
52.9
13.2
1.07
27.9
.60
-31.9
X=38.4
Glutamic Acid 1
44.1
1.78
39.0
1:65
39.0
1.60
44.7
1.80
31.8
1.20
X=39./
Glutamic acid+
46.3
Streptomyocin
41.6
29.1
36.5
1.36
49.3
1.12
6-40.5
Kused for mensure
ofrat of relense.
men
7002 Percent of
Total Uptake
33
41
34
30
67
68
62
Michael J. Miille
Uptake of Amino Acids by Metridium senile
TABLE #3
% Uptake
Substrate
L hours
0.6
Glysine
Glysine + Streptomyocin
1.1
Glutamic acid
1.8
Glutamic acid + Streptomyocin
0.6
% Uptake
24 hours
24.0
1.3
37.0
2.6
0
Qo-
+
+ 0
O
8
5
5
0 3
5

ON

—
ON
N 8
3


oo

21
a

S
O
5
8
O

5
N
S


—5
o
80
— 0
X
9
10

5
55

2.

o la
5
Michael J. Miille
Uptake of Amino Acids by Metridium senile
TABLE 15
% Uptake
Anemone
of Glysine
24.6
Non-fed 1
35.4
32.8
3
36.3
33.6
30.5
Mean
27.2
Fed
24.7
25.6
3
24.8
21.2
24.7
Mean
1002 Percent of
Total Uptake
56.9
58.2
49.6
55.8
60.4
56.2
44.4
40.9
43.4
15.5
44.1
43.7
e
Michael S. mitte
Uptnke of Amino Heid by Meteidium sente
100
22o,00
looc
2og
Glucose
000
oo
Tizood
0000

10.000
140,000
6000
120,000
Tme Goas)
loc
oo
10,000
000
oo,o00
400
Valine.
Eo000
500
Oo,000
700
00
O
70,000
1000
4
2
6
Tme (houzs)
2o
3400
300
270
Glysine
2000
99000
330000
E22oo
500
210,000
60
1400
140000
1000
o
2
—
Fioage
21
LySINE
—
Time (hoans)
Glufmic Heid
O
àp
Choues)
Time
Algal Mixtuge
10
72.