STRACT
7. Six crotenoid pigments, -carotene, isozeaxanthin (?), lutein.
zeaxanthin, an astaxanthin ester, and astaxanthin were isolated
from the bodies and stalk fluid of Pollicipes polymerus, althoughi
the proportions differ in each case.
2. Similar extracts of the different developmental stages of
ovigerous lamellae showed the presence of the same six carotenoids
with the exception of isozeaxanthin.
3. A decline of the amount of astaxanthin ester and lutein and the
apparent simultaneous increase of astaxanthin with maturation of
the nauplii was observed.
4. Astaxanthin was found to be bound to a lipo-protein in the stalk
fluid and ovigerous lamellae but not in the bodies.
5. The presence of protein bound astaxanthin in the lamellae
appears to decline with maturation.
0. The involvement of the astaxanthin lipo-protein complex which
is stored in the stalk fluid is discussed.
78
INTRODUCTION
Much of the investigation concerning the pigmentation of the
Crustacea has been centered on the decapods although some recert
investigations have been made on species of both copepods (Herring,
1967) and isopods (Lee, 1966a & b). In the majority of these studies
astaxanthin (4-4'-di-keto, 3-3'-di-hydroxy/-carotene) has been
found to be very abundant and more or less characteristic of the
class. Furthermore, the presence of astaxanthin in the form of
protein complexes has been shown to be frequently linked to rø¬
productive and developmental processes (Cheesman, et al, 1966).
Some research on the pigmentation of stalked barnacles (Class
Cirripedia) has been carried out by Ball (1911) and Fox and
Crozier (1967). Ball's study revealed that the blue pigment in
the ovaries and developing embryos of Lepas fascicularis is a
carotenoprotein with astaxanthin attached to euglobuin. Fox and
Crozier discovered that a similar blue color found in the body of
L. fascicularis is likewise the result of an astaxanthin protein
complex. These two complexes are typical carotenoproteins, both
showing absorption maxima as a broad band at about 600mu. [Cheesman,
et al, 1966). Furthermore, in Ball's study the developing nauplii
were found to change color from blue to pink, reprèsenting the
liberation of astaxanthin from the protein complex. Green (1965)
has suggested that this breakdown of the carotenoprotein during
development, a process commonly seen to occur in crustaceans, may
coincide with the peak of proteinase activity and thus represent
a mechanism during development by which protein stores can be re¬
leased to the embryos at specific stages of development.
The present investigation of Pollicipes polymerus was prompted
by the similarity of this barnacle's bright orange stalk fluid to
78
-2-
ovarian tissue suggesting the presence of a carotenoprotein which
might be involved in the devolopment of the nauplii, perhaps in a
manner similar to that described above. The ovigerous lamellae,
moreover, were observed to change color during development from a
bright orange, similar to that of the stalk fluid, to yellow
and then brown, further suggesting such a relationship.
The present study is an an invetigation of the carotenoids and
carotenoproteins present in both adult and immature barnacles.
their stalk fluid, and various stages of the ovigerous lamellae.
MATERTALS AND METHODS
Animals
Barnacles were collected both at The Hopkins Marine Station.
Pacific Grove, California and approximately one quarter mile south
of the mouth of Malpaso Creek near Yankee Point along California
Highway Wl. They were either brought back to the laboratory and
stored in a deep freezer at -7°0 until needed or the ovigerous la¬
mellae were dissected in the field and frozen as soon as'possible.
The stalk fluid was collected by cutting whole clumps of barnacles
from the rocks and allowing the stalks to drain. The material
was then centrifuged before being used.
xtraction of Carotenoids
The carotenoids were extracted from the bodies, ovigerous
lamellae, and stalk fluid with acetone. Petroleum ether (b.p.
20-10°0) was added and the carotenoids transferred to the petroleum
ether phase by the addition of water. The petroleum ether solu-
tion was repeatedly washed with water until free of acetone, dried
with anhydrous sodium sulfate and concentrated under a stream of
nitrogen.
The carotenoids were initially seperated chromatographically
on collunns of aluminum oxide (Woelm neutral - activity grade 1
for chromatography, Eschwege, Deutschland). The aluminum ogide
was activated by exposure to 11000 for one hour in an oven prior
to packing the columns. The columns, which were of two sizes.
measured approrimately l om x 15 cm and 1.5 om x 17 cm, respectively.
Figments were placed on the columns in petroleum ether and developed
with acetone-petroleum ether mixtures of different proportions.
The resulting bands were eluted and transfered again to petroleum
ether. Further purification was carried out on columms containing
75
a mixture of magnesium oxide and "celite" (1:1 w/w). These columns
were likewise developed with acetone-petroleum ether mixtures.
Pigment Identification
In all cases it was necessary to confirm results by comparing
samples of known carotenoids (kindly supplied by Professor B.C.L.
Weedon, University of London) with the extracted pigments by means
of thin layer chromatography. Shandon equipment was used through-
out and the plates were spread with aluminum oxide (Aluminiumoxid
G-nach Stahl, E. Merck AG., Darmstadt, Deutschland) to a depth of
200mu. The plates were developed in one of four acetone-petroleum
ether mixtures: 5% for carcenes, and 253, 302, and 50% for the di¬
keto and di-hydroxy derivatives. It shoud be noted that the
recorded Re values can be considered as valid for individual
plates only, since identical conditions from plate to plate were
not possible even under the most careful conditions.
Saponification was carried out by dissolving the pigments in
a 12% solution of KOH in methanol, followed by incubation in the
dark at room temperature overnight. After saponification the
pigments were re-extracted with petroleum ether.
The chromatographic and partition characteristics were recorded
and the absorption maxima of the extracted pigments determined in
petroleum ether, diethyl ether and carbon disulfide. A Beckman
DK2A Ratio Recording Spectophotometer was utilized for all such
recordings.
Quantitative determinations were based on the extinction at
the wavelength of maximum absorbance in solutions of a given volume.
The relative amount of each pigment is given as a percentage of
the total. It should be noted that these values are only approxi-
76
-5-
mations as no allowances were made for differences in molar extinction.
Protein Complex
Protein complexes were extracted from the body, stalk fluid.
and ovigerous lamellae with O.2M phosphate buffer pH7 (KH.PO. 4
NaghPo). The extracted protein complex was then precipitated
with saturated ammonium sulfate, centrifuged, redissolved in dis-
illed water, and placed on columns of DEAE-cellulose (Whatman
DE 50). The proteins were then eluted by stepwise increase in
the ionic concentration of the perfusing buffer. The DEAE-cellulose
was prepared by first washing with normal NaoH, then distilled
water, normal Hol and finally again with distilled water. Columns
were prepared by packing a slurry of the purified exchange medium
in distilled water. The columns measured approximately 1.5 x 20 cm
and were thoroughly washed with 0.2M phosphate buffer, pH/, followed
by distilled water before the protein solutions were added.
Lipid Identification
Tentative identification of the class of lipids present in
the stalk fluid was carried out according to the method of Bloor
(cf. Mangold, 1962). 10 parts of stalk fluid was extracted with 20
parts of ethanol-diethyl ether (3 to 1) for 24 hours, concentrated
under nitrogen and plated on thin layer plates of Silica Gel G
(E. Merck AG, Darmstadt, Deutschland) spread to a depth of 200u.
The plates were developed with a mixture of petroleum ether, diethyl
ether, acetic acid (90:10:1 v/v). The lipids were detected with
a 0.05% solution of Rhodamine B in 96% ethanol under U.V. light as
dark violet spots on a pink background.
o
RESUI
Bodies of Mature Adults
The bodies were dissected from the calcarious plates and ex¬
tracted and the pigments separated as outlined above. A total of
six carotenoid pigments were isolated (Fig 1). These were O-caro-
tene, isozeaxanthin, lutein, zeaxanthin, an, astaxanthin ester.
and astaxanthin. The specific characteristics of these pigments
are given below (Table 1).
Fraction 7,/-carotene. Fraction 1 elutes from alumina with 1-23
acetone in petroleum ether as a yellow band. It is epiphasic when
partitioned between both 90% and 95% methanol and petroleum ether.
The absorption spectrum in petroleum ether (119-176mu) is in close
agreement with those reported for /-carotene. (Goodwin, 1951)
Fraction ! is inseparable from known samples of/-carotene when
co-chromatographed on thin layer plates of alumina. On such
chromatographs (using 5% acetone in petroleum ether as the solvent
nixture) both show Re values of 0.98.
Fraction 2, Isozeaxanthin (l-l'-dihydroxy-B-carotene) ? ? Fraction
2 eluted from alumina as a yellow band with 30% acetone in petroleum
ether, slightly ahead of Fraction 3. However, it was seperable from
Fraction 3 on columns of MgO-celite and on such columns could be
eluted with 15% acetone-petroleum ether. It was mostly hypophasic
to 90% methanol and completely hypophasic to 958 methanol both
before and after saponification. When this fraction was plated
gainst a known standard of isozeaxanthin in a 258 acetone-petro-
leum ether mixture, the isozeaxanthin (R.-O.82) plated slightly
ahead of Fraction 2 (R.-O.79). However, large amounts of lipid
present in the sample made identification by thin layer chromato-
graphy difficult. The sample exhibited absorption peaks at.llõmu
78
-7-
and 472mu in petroleum ether which is close to those recorded
for isozeaxanthin (Goodwin, 1954). Iodine catalyzed isomeri¬
zation was carried out according to the method of Zechmeister
(cf. Davies, 1965) Under these conditions isozeaxanthin formed
a series of five isomers while excess lipid interfered with the
results obtained with a similar treatment of the sample solution.
The identification of isozeaxanthin, therefore, must remain con-
jecture at this time since lackof material prevented further
investigation.
Fraction 3, Astaxanthin ester. This fraction elutes with isozeaxan-
thin from alumina with 30% acetone in petroleum ether as a purple
band. It is separable from isozeaxanthin, however, on columns of
Ngo-celite by elution with 100% acetone. The ester reveals a
single absorption maximum at Lóõmü in petroleum ether. The fraction
is 65% hypophasic before saponification and completely hypophasic
afterwards. The saponified pigment is inseparable from astaxanthin
(Re-O.18) in a solvent mixture of 30% acetone in petroleum ether
when co-chromatographed.
Fraction 1-Lutein. This pigment together with Fraction 5 eluted
slowly from alumina with 70% acetone-petroleum ether as a bright
yellow band. However, it was separable from Fraction 5 on Mgo¬
celite and could be eluted from the latter with 153 acetone in
petroleum ether. This fraction was hypophasic to 90% and 958
methanol both before and after saponification. The absorption
maxima in petroleum ether were ll5mu and 173mu. When co-chro-
matographed with a known sample of lutein in 25% acetone-pet-
roleum ether as a solvent mixture, the fractions were inseperable
(Rg-O.16)
75
-8-
Fraction 5 - Zeaxanthin. This pigment elutes with lutein from col-
umns of alumina with 70% acetone in petroleum ether. However, when
rechromatographed on Mgô-celite it is clearly separable as an
orange-yellow band above lutein and can be eluted with 60% acetone¬
petroleum ether. Its absorption maxima in petroleum ether are
5Omu and 176mu. It is completely hypophasic to both 903 and 958
methanol. The pigmenttis inseparable from authentic zeaxanthin
but separable from lutein and isozeaxanthin when co-chromatographed
in a 25% acetone-petroleum ether solvent mixture.
Fraction 6 - Astaxanthin. This fraction elutes from columns of
alumina as a purple streak with a 20% mixture of glacial acetic
acid in methanol. The pigment was completely hypophasic in both
908 and 958 methanol both before and after saponification. It was
insepgrable when co-chromatographed with known astacene. Like-
wise, it gave a single absorption maximum in petroleum ether at
170mu which was identical to the spectrum of known aståcene. It
should be noted that up to 30 of the pigment was estimated to be
lost on the column.
Bodies of Immature barnacles
Hilgard (1960) in her study of reproduction in the barnacle
P. polymerus reported that individuals with a breadth of less than
17.2 mmwere never found to produce ovigerous lamellae. Barnacles
smaller than this were collected and the pigments extracted and
seperated. When compared to extracts of mature animals, no dif¬
ference could be found in the kinds of carotenoids isolated.
Stalk Fluid
The dark orange fluid which bathes the ovarian tissue in the
stalk was collected and the carotenoids extracted and separated
3
-9-
chromatographically. The same six fractions were again present
although in different proportions.
Ovigerous Lamellae
The ovigerous lamellae were collected and divided into three
groups representing progressively advanced stages of development.
These could be easily denoted by their overt color: bright orange,
yellow, or brown. Bright orange lamellae were found to correspond
to Barnes' (1965) stages A, B, and C. The yellow lamellae paralleled
his D and E stages while the brown lamellae were the same as his
stages G and H.
Carotenoids were extracted from the three groups and chro¬
matographed as outlined earlier. The pigments isolated were -caro-
tene, lutein, zeaxanthin, an astaxanthin ester, and astaxanthin.
No astaxanthin was found, possibly due to the small amount of mater-
ial available.
Quantative Determinations
Table 2 lists the relative amounts of the individual carotenoids
obtained from the bodies of mature and immature barnacles, stalk
fluid, and the three stage of ovigerous lamellae studied. These
are represented as a percentage of the total carotenoid content.
All the carotenoids originally isolated are present in approx-
imately equal amounts in both mature and immature animals. The
carotenoids of the stalk fluid are similar to those of the body ex-
tracts with the exception of almost negligable amounts of the ester
and about 203 more astaxanthin. The early lamellae extracts were
similar to those of the mature bodies except for a slightly higher
amount of -carotene and the absence of isozeaxanthin. When compared
to the earlier lamellar stages the middle and late lamellae exhibit
a 10% increase in astaxanthin and a corresponding decrease of about
16%-20% in lutein and the ester.
Protein Comple
Phosphate buffer extracts of the orange stalk fluid yield an
orange water soluble pigment with a single absorption maximum in
the visible range at about 170mu (Fig 2). This orange pigment was
precipitated with saturated ammonium sulfate and after centrifuga-
tion the resulting pellet was redissolved in distilled water and
run through a DEAE-cellulose column as outlined earlier. The
solution eluted from the column with distilled water but left
behind some orange material believed to be lipid in character,
which could not be eluted even with the addition of methanol.
The
material which had eluted still retained some of its original
orange color, but upon standing overnight turned yellow. An
acetone extract was made which freed the carotenoid with the con-
sequent formation of a white flocculent protein precipitate. The
carotenoid was transferred to petroleum ether in which it exhibited
a single absorption peak at 170 mu. When plated against a known
mixture of the carotenoids found in the bodies of adult barnacles
(solvent mixture of 25% acetone-petroleum ether), astaxanthin
was found to be the only pigment present.
The presence of the same astaxanthin protein complex was ver¬
ified in all three stages of ovigerous lamellae by its unique
spectral characteristics and by thin layer chromatography.
Although no quantitative determinations were carried out the abun-
dance of this protein complex appeared to decrease as the lamellae
matured. No carotenoprotein was found in the bodies of adults.
Lipid Determina!
tion
When a portion of the phosphate buffer extract of the stalk
fluid was mixed with diethyl ether, a yellow pellet formed at the
C
0
-11-
interface. This and the unusual behavior of this extract suggested
the presence of a lipo-protein complex. An initial determination
of the class of lipids present in this fluid was carried out as
itlined earlier (cf. Mangold, 1962). The thin layer chromatograph
of the lipid extract suggested the presence of a single lipid group.
saturated hydrocarbons.
DISC
TON
Body
This study has shown the presence of six carotenoids in the
bodies of mature P. polymerus. Astaxanthin and a single astaxan-
thin ester represent about 55% of the total carotenoid component
of this species. The only other pigment in substantial quantity
is lutein which represents about 31% of the total. This is in
general agreement with other studies of crustacean pigments in
which astaxanthin and lutein have both been found to be character-
istic, the former often amounting to well over one-half the total.
For example, in a recent study of the decapod Carcinus maenas
Gilchrist and Lee (1967) found that in the epidermis astaxanthin
accounted for 56% and lutein represented 212 of the total carotenoid
present.
It is interesting to compare the results of this study to those
obtained by Fox and Crozier (1967) from their work on the body
pigmentation of another stalked barnacle, L. fascicularis. They
found that carotenes accounted for 24.18 of the total carotenoid
while the remaining 75% was astaxanthin. This astaxanthin was,
however, in the form of a blue carotenoprotein whereas no carotenoid
is protein bound in the bodies of Pollicipes.
When the immature bodies of Pollicipes were extracted, no dif-
ference in the kind or relative abundance of carotenoids was found.
This data suggests that Pollicipes does not metabolize its large
amount of astaxanthin from carotene precursors since the presence
of possible intermediates was never established as it has been in
the case of other crustacea (cf. Lee 1966a; Gilchrist & Lee 1967).
Perhaps, though, metabolism in Pollicipes could take place very
-13-
rapidly in one of the larval stages or at some other specific time
so that its occurance would not have been detected in this study.
Fluid
The characteristics of the stalk fluid and its contained
protein-complex in particular are extremely unusual. When freshly
collected the fluid is a dark orange with absorption character-
istics similar to those expected for the unbound carotenoid, but
upon standing overnight it turns yellow with a concomitant loss
of absorption in much of the visible range (Fig 2). The same
carotenoids present in the body are also found here except that
the astaxanthin is bound in some way to what appears to be a lipo-
protein. Although all six carotenoids were precipitated from the
fluid by the addition of ammonium sulfate only astaxanthin was
eluted from a DEAE-cellulose column suggesting that it alone is
truly bound to protein.
Numerous theories concerning the possible roles of caroteno-
protein in invertebrates have been suggested (Cheesman, et al, 1966).
Among these are, stabilization of the protein configuration, pro-
tective coloration against solar radiation, and protective color-
ation as a method of concealment. Protein stabilization seems
unlikely in polymerus because the fluid appears to be highly un-
stable. Since the fluid is completely bounded by a thick black
epidermis, it could not possibly provide protection either against
solar radiation or predation.
Lipo-protein complexes which have been noted in the blood of
many invertebrates may be involved in the transport of carotenoids.
since linkage to a protein makes fat soluble carotenoids water soluble
(Cheesman, et al 1966). A carotenoid attached to a lipo-protein
-14-
could possibly mediate the transport of large water soluble
molecules across lipid bound membranes. Such properties could
be important at the onset of reproduction when the bransfer of
critical supplies for egg production is essential. That a sim-
ilar astaxanthin lipo-protein was found in the developing lamellae
suggests that the protein bound astaxanthin in the stalk fluid
is used in some manner by the nauplii during maturation.
Polymerus' stalk fluid thus appears to act as a store of this as-
taxanthin lipo-protein complex for use during reproduction.
A change in the relative amounts of carotenoid pigments during
development was observed. The percentages of %-carotene and zea-
xanthin seemed to remain roughly the same throughout development.
No isozeax
ithin was detected. Both lutein and the astaxanthin
ester, however, showed a relative decrease in abundance in the late
stages of development. It is not known, though, whether this de¬
cline and the increase in free astaxanthin which was also observed
are actually related. Thus, although the percentage of non-ester-
ilied astaxanthin in the late stages is almost identical with the
percentage of astaxanthin bound to the lipo-protein in the stalk
fluid, it is uncertain whether or not this represents the direct
removal of protein-bound astaxanthin. The possibility of the up-
take of astaxanthin derived from the carotenoid lipo-protein
complex of the stalk fluid exists, but the most likely source of
this free astaxanthin is the breakdown of the lipo-protein complex
found in the early stages or de-esterification.
That carotenoproteins are broken during some stage of devel-
opment is a well known fact. Lwoff (1927) observed the breakage
-15-
of a blue carotonoprotein in the eggs of the copepod Idya furcata
and the resulting transfer of the freed carotenoid to the nauplier
eye. Green (1965) also observed a similar process in Cladocera
where the carotenoprotein becomes restricted to the embryonic fat
cells as soon as they are formed, and once the link with the pro¬
tein is broken the free carotenoid passes into fat droplets.
Green speculates that this linkage of protein and carotenoid and
its consequent stabilizing effect removes the protein from the
possibility of attack by certain enzymes. Furthermore, he suggests
that the breakdown of a carotenoprotein may coincide with the peak
of proteinase activity.
Ball (1941) found that the blue astaxanthin protein complex
in Lepas broke at a definite point in the development of the
nauplii. A similar series of events apparently occurs in Pollicipes.
The percentage of lipo-protein bound astaxanthin likewise appears
to decline sharply with advancing development, although not all
of the complex is broken when the late stages are reached. The
protein complex in polymerus is, furthermore, so unstable that to
postulate any stabilization of the protein configuration due to
the presence of astaxanthin seems highly improbable. Rather, the
enhancement of the movement of water soluble molecules across
lipid-bound membranes, the transport of fat soluble carotenoids
in an aqueous medium or the stabilization of lipid membrane seem
to be the most probable functions of this carotenoid lipo-protein
complex.
P. polymerus, thus, parallels other invertebrates by the abun-
dance of astaxanthin found throughout its body. This astaxanthin,
moreover, appears to be taken directly from plant food sources
C
-16
rather than metabolized from carotene precursors. In the stalk
fluid and ovigerous lamellae the astaxanthin is bound to a highly
instable lipo-protein which possibly results in some form of en-
hanced nutrient transport during development of the ovigerous
lamellae.
IGURE
Carotenoids from mature bodies as they appear on alumina
when developed with petroleum ether - acetone mixtures.
e percentage of acetone needed to elute each of the pigments
is included.
Fareron No.
C
C
.

.

PAMENT
ASTAXANTHN
ZEAXANTHIN
LOTEIN
ASTA ZANTMN
F51R
LSOEFANANTNIN 7)
G-eROTENE
*
ELUTE
2 eton
INCOH
of nree
70% ACETENE
f perevt
30J NCETONE
Retrove
C
90
TABLE 1
Absorption maxima in petroleum ether, diethyl ether, and carbon
disulfide of each of the carotenoids isolated from the mature bodies
of P. polymerus.
Absorption maxima (mu)
Fraction Pigmer
Pet. Ether Diethyl Ether Carbon Disulfide
180-510
B-carotene
Ll9-176
150-176
177-506
Isozeaxanthin (?)
lh6-172
119-177
Astaxanthin
500
Ester
167
168
Lutein
169-501
112-170
Ll5-171
119-176
175-506
Zeaxanthin
150-176
Astaxanthin
172
500
172
p
TADLE 2
The relative percentages of the pigments isolated from the mature
body,
ature body, stalk fluid, early ovigerous lamellae,
intermediate lamellae, and late lamellae.
mature immature stalk
early
middle
late
body
pigmer
body
lamellae
lamellae
lamellae
Ö-carotene
119
68
112
88
10%
Isozeaxanthin(?)
19
Astaxanthin
Ester
268
258
39
288
208
8%
Lutein
3118
32%
20%
338
368
Zeaxanthin
59
112
39
23
38
Astaxanthin
298
319
579
60%
238
339
O
IURE 2
Absorption spectrum of (A) the orange stalk fluid of P.
polymerus and (B) the redissolved p
recipi
ated protein
complex from this fluid in 0.2M phosphate buffer, pH7.
C


od
21
ACKNOLELGEME
This research was supported in part by a Gail H. Calmerton
Scholarship for undergraduates from Stanford University. I am
deeply indebted to Professor Welton L. Lee for his invaluable
advice and assistance and for reading this paper. My special thanks
to my roomates and fellow students George Kelso, Robert MacDonald,
and Douglas Norman for their encouragement and camaraderie.
—---
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pr
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