TRODUCTION
Lug
That adult marine invertebrates can concentrate amino acids

irom a dilute solution has now been demonstrated for several dif-
ferent organisms (Stephens and Schinske, 1961; Stephens, 1963, 1961:
Ferguson, 1967). Recent studies have shown that sea urchin embryos
can likewise transport exogenous amino acid and incorporate it in-
to their protein (Mitchison and Cummins, 1966; Epel, 1967) and that
there is an increased rate of both uptake and incorporation follow-
ing fertilization (Hultin, 1953; Gross, 1961; Epel, 1967). In

these eggs the incorporation(peaks) at the mid-gastrula stage and
e
subsequently decreases (Guidice, Vittorelli and Monroy, 1962).
Little data is available, however, on rates of protein synthesis
in the later larval stages.
The present report concerns experiments on uptake and incorp-
oration of C-Il lysine and C-14 leucine by embryos of the barn-
acle,
anus glandula (Darwin). Six distinct stages of develop-
ment have been used in this study, ranging from early cleavage
stages through the hatching nauplius.
The results of this research indicate that there are one
herk

and possibly two peaks in the rate of incorporation of amino acids
between cleavage of the first blastomere and hatching.
As was
had chee ef eune
found with sea urchins, one of these is (around the mid-gastrula
tage. This study also suggests that these embryos possess a
transport system for concentration of amino acid.
1 Th 7e e
16.
-2-
MATERIALS AND METHODS
taining and Handling the Eg
388
Adults of Balanus glandula were collected from rocks on China
Point near the Hopkins Marine Station and transferred immediately
a
to the lab where they were maintained in a circulating water bath
at 16° C. The embryos (found in sheet-like plates known as ovig-
erous lamellae) were then taken from the barnacles, observed under
a compound microscope to determine the stage of development,
weighed, and immersed in millipore-filtered sea water at 16° C.
All tests were made within two hours (of removing the barnacles ote vwee
from the rocks and no longer than one hour after the ovigerous lam¬
ellae were taken from the mantle cavity. All incubations with the
C-1l amino acids were performed at pH 7.7, which corresponds to
the pH of the mantle cavity fluid.
Classification of the Embryonic Stages
The classification of the developmental stages is adapted from
that described by Barnes (1965) for Balanus balanoides and Balanus
balanus. The stages used herein have been characterized in the
following manner.
B. the formation and growth of the blastoderm over the yolk,
C. the formation of the mesoblast and hypoblast (segmentation
of the blastula),
E. demarcation of the nauplius appendages (Barnes' stages D
and E),
F. the development of the appendages and the origin of the
16
-3-
labrum and gut; further development of the appendages and the origin
of the body cavity (Barnes' stages F and G),
H. final stages of development; andincrease in transparency
and the formation of the nauplius eye, and
1. the hatching nauplius (some nauplii still in the egg sack
but in the process of hatching).
Volume Determination
Cirriped eggs are especially difficult to work with since dur-
ing the earlier stages they are held tightly together in the ovig-
erous lamellae and cannot be washed free of this tissue. Since
fferent sets of eggs had to be compared on a volume basis in order

to calculate the results of the experiments this proved to be a
ss tupeete o
complication. As a result, a method for determining the volume of
the eggs was devised.
Pieces of ovigerous lamellae were weighed and the number of
eggs in the pieces counted. From this a curve was prepared establish¬
tanut
mall
Seaza
ing a wet weight:number-of-eggs relationship (only an approximation
since weight density changes during development, described by Barnes,
1965, were disregarded). The volume of a single egg was then cal¬
culated from the egg dimensions, assuming that the egg has the shape
den dd gen sa a
of an oblate spheroid of volume 1/3 Nabe (a=60,1 b-984). The volume
of a single egg was calculated to be 1.16 X 10-° ml. With this a
Eugd.
wet weight: egg volume relationship was made.
Preparation of the Ee
A number of micro-organisms was sometimes found on the surface
of the tissue binding the eggs together and on the eggs themselves.
16.
0
In order to be reasonably certain that these and any traces of bact¬
eria present would not affect the results of the experiments ad¬
versely the ovigerous lamellae were washed h-6 times with millipore-
filtered sea water amd incubated in sea water containing .25 mg/ml
streptomycin (Sigma Chemical Co.) and 150 units/ml penicillin (Sigma
Chemical Co.).
Isotope Incorporation
In all of the experiments the pair of lamellae from one barnacle
was separated and one lamella was incubated with C-11 lysine while
the other was incubated with C-14 leucine. Three different con¬
centrations of amino acid was used:.1c/ml sea water, .33/c/ml
20
we u
and 1.7c/ml.The duration of the incubations was twenty minutes.
After incubation the samples were washed with 0.001 M C-12
mino acid and then 2ml of 5% trichloroacetic acid was added to the
washed eggs in a Dounce homogenizer. The eggs were then homogen-
ized and the TCA-homogenate centrifuged at 1130g for 15 minutes in
a Servall RC-2B centrifuge. Part of the TCA-soluble supernatant
was then collected, extracted twice with either, dried on a
e
planchet and (its activity counted in a Nuclear Chicago gas flow
counter with mica window.
wep
The TCA-insoluble precipitate was resuspended in the TCA and
mg of bovine serum albumin was added. This was then heated for
twenty minutes at 90° C, filtered through 24 Schleicher & Schuell
glass fiber filters and the residue then washed four times with 5%
TCA-O.O5 M C-12 amino acid and twice with ether:ethanol:chloroform
uhe
(2:2:1). The filters were glued to planchets and counted as above.
16
0
-5.
Isotopes
The specific activities of the uniformly labeled C-11 amino
acids (International Chemical and Nuclear Corporation) were:
C-14 lysine, 200 mc/m; C-1h leucine, 198 mc/mM.
LS
rs detee
For all developmental stages determinations were made of the
amount of free isotope in the TCA-soluble phase and the amount of
isotope incorporated into protein (TCA-insoluble phase). The degree
of active uptake was estimated by comparing the total uptake into
the eggs with the a mount of isotope originally present in an equal
erca
volume of external medium.
As the different lamellae varied in size, the results are
expressed in counts per minute/gram wet weight. This mode of ex-
pression allows a comparison of thé different stages with other.
55
However, absolue comparison between stages is extremely difficult
since a long developmental sequence has been here arbitrarily div¬
letaen
ided into six phases. The actual time from fertilization to fully¬
formed nauplius is not known for B. glandula, but estima tes from
other species suggest that the duration is at least twenty-four
days. Thus, stages placed in the same category (e.g., Stage E)
might actually be four to six days apart in time since fertilizat¬
ion.
CA-soluble Phase
In all experiments C-1 amino acid was found in the TCA-soluble
fraction of the embryos, and the amount of labeled amino acid in this
a med
Hos
neded
Eg
Eug
od o
er u de
)stated
16.
O
-6
lerwen
O Rag
fraction differed yith the different developmental stages.There
was usually variation between experiments but the radioactivity
of the TCA-soluble fraction was generally the highest apt one of
dee
the three early stages (see figure 1). Figure 1 also shows dif¬
10
ferences between leucine and lysine uptake in different experiments.
This differenco is apparently real, and does not result from the
difficulties in staging the embryos, since the lamellae used to
compare leucine and lysine uptake were taken from the same animal.
insoluble Phase
TCA¬
Incorporation of labeled leucine and lysine was always lowest
at stage B (blastoderm migration over the yolk) and generally in-
creased to a peak at stages C-E (segmentation of the blastoderm
to demarcation of the limb buds). The rate always decreased to
Then uha
slightly lower levels at the later stages.
Total Incorporation
The total amount of C-11 lysine and C-1 leucine found within
the egg was always greater than the concentration of the C-1h amino acid
external to the eggs. The ratio of these concentrations (inside/
outside) varied from.8 to 8.0, suggesting a type of active trans-
port for these eggs. Although total uptake increased with increas-
ing amounts of added C-14 amino acid, the ratio inside/outside
decreased, suggesting a saturation of the "active transport" sys¬
tem.
Table ! is a listing of the calculated numerical results of
the experiment illustrated in figure 1-1. All of the data used
to plot the graphs were compiled in this way for each experiment.
Eug
16
-7-

ESCUSSIO
Much variation between different experiments was found which
can probably be attributed to the aforementioned difficulties of
classifying embryos with respect to age. Irrespective of these
variations, it was found that the early stages always incorporated
Aal
less amino acid into protein than the later stages, and that
Tyu
peak incorporation occurred at stages C-E.
Interpretation of the above data is complicated by an apparent
bypassing of the endogenous amino acid pool by the added C-Il amino
acids. The data show that at low external amino acid concentrations
———
(Fig. 1-111) greater than 503 of the amino acid taken into the cell
(ct. Bres, 1063)
was incorporated into protein. As Bala
glandula probably con-

tains a large amino acid "pool", the high percentage
o
of incorporation observed can be explained by a bypassing of the
Adt en
gut an ?
"pool" by exogenous amino acid when transported across the cell
Lee?
merbrane into the cell, or by isotope discrimination. A similar
type of compartmentation has been suggested to exist in the sea
te Ge
ax
urchin embryo (Berg, 1965). This compartmentation, if it is this,
is much larger in barnacle embryos, and may seriously hinder the
interpretation of results obtained with C-11 amino acids. If this
compartmentation is of similar magnitude throughout all stages,
however, the general pattern found for incorporation is probably
correct.
The above results also indicated that barnacle embryos can
utilize exogenous amino acids. It is interesting that two of the
amino acids used, leucine and lysine, are also found in the fluid
167
C
O
-8-
of the mantle cavity of B. glandula (Bergman, 1967). As the embryos
are bathed in this fluid it is possible that these compounds are
utilized by the embryos during their development.
O

ANOWE
Special thanks are extended to Dr. David Epel for providing
the inspiration, guidance and friendship which made this project
a most enjoyable and instructive one. I am equally grateful for
the assistance afforded me by all members of the staff of the
Hopkins Marine Station.
—p-
BIDLTOGRAPHY
Barnes, H., Studies in the biochemistry of Cirripede eggs, (1963),
J. Mar. Biol. Ass., U.K.,
15; 321-339
Berg, W., (1965), Exptl. Cell Res.,
10; 169
Epel, D., Protein synthesis in sea urchin eggs: A "late" response
to fertilization, (1967), Proc. Nat. Acad. Aci.,
57,
899-906
Ferguson, J., Utilization of dissolved exogenous nutrients by the
fishes, Asterias Forbesi and Henrica S.
anguinolenta,
(1967), The Biol. Bull.
132; 161-172
Gross, P.R., and G.H. Cousineau, Macromolecule synthesis and the
niluence of actinomycin on early develöbment, Exptl. Cell
Res., (1961),
368-395
33;
Guidice, G.M., L. Vittorelli and A. Monroy, Investigationon the
protein metabolism during the early development of the sea
urchin eggs and embryos, (1958), Acta Embryol. Morph. Exper.
5; 113
Hultin, T., Incorporation of N-15 labeled glycine and alanine into
the proteins of developing sea urchin eggs, (1951), Exptl.
Cell Res.,
191-196
3;
Mitchison, J.M. and J.E. Cummins, The uptake of valine and cytidine
by sea urchin embryos and its relation to the cell surface,
(1966), J. Cell Sci., 1; .35-17
Stephens, G.C., and R. Schinske, Uptake of amino acids by marine
invertebrates, (1961), Liminol. and oceanog.,
6;
175-181
Uptake of organic material by aquatic invertebrates.
II Accumulation of amino acids by the bamboo worm, Clymenella
torquata, (1963), Biochom., Phisiol.,
10;.191-209
Uptake of organic material by aquatic invertebrates.
—2
lil Uptake of glycine byybrackish water annelids, (1964),
Biol. Bull, 126; 150-162
170
O

LEGED
Figure 1: Three representative experiments with diffeeent con¬
centrations of C-11
mino acid. 1-I).lc/ml
1-II) .33c/ml
—
-11I) 1.7c/ml
Table 1: A list of the calculated data for the experiment illust¬
rated in Fig. 1-I
O
17
0
2.
2-
4.
2-


C-la leucine

11
CE
i

1
E H
C14 lysine

1

CE
B
H
ETCA-insoluble x10-
TCA-soluble 10•9
II


C E
H





III

EH
Fig.
17
O
O
TABLE 1
amno
cpm
cpm
com
wet y calculated
weight volume
external TCA-sol TCA-insol
age acid
leu 0.0092 2.8x10-3
1590 1760 11
lys 0.00h9 1.5x10-3

950 1980
leu
lys 0.0085 2.6x10-3 1930
3210 619
leu 0.00hh 1.lx102
865
2680 277
lys 0.0061 1.9x10-3
1382
1920
Ml
leu 0.0085 2.6x10
1760
1300
50
lys 0.0206 6.3x10-3
5100
3860 118
leu 0.0123 3.8210"2
2000
1320 757
lys 0.0122 3.7x10-3
2780
1580 120
leu 0.0233 7.1x10-3
3510
1120 110
lys 0.0196 6.0x10-3
1150
3680 391
—
7 in grams
external exogenous cpm/equal volume of eggs
170
O
APV
SUPTIAR
Emt
ryos of Bala
nus
andula will concentrate amino acid from
an external source and incorporate it into protein. Moreover
the pattern of incorporation varies between different embryonic
stages. It has been found that the lowest incorporation occur
at the early stages and the highest during the middle stages of
development.
C
9804 N. E. 14th Street
Bellevue, Washington
July 18, 1967
Dear Dr. Epel,
I am wondering if you have had time to read my
paper again and pass any kind of judgement on it. I understand
that you are probably pretty busy with your summer course so
I don't mean to push you into anything. What I really want is
an opinion. I have been asked by a medical school to which
I am applying to write a short essay about any subject which I
have studied and feel confident with. My studies last quarter
would be ideal for this I think but I'm still not sure if you
think my findings are valid enough. I suppose this is related
to whether or not my paper is publishable. Any opinions of
yours at this time would be appreciated.
I certainly hope that the sun is shining a little
brighter now than it did last qurter and that "Big Bertha" is
functioning for Raffin!
Sincerely,
Ao
175