Pigmentation in N. grubei
K. Carlson
Abstract
Pigmentation in the polychaete worm Nereis grubei has
two components. The dark green bile pigment biliverdin
occurs in granules in the body wall and in the coelom.
An unidentified blue-green pigment was found in the body wall
and in oocytes of spawning females. Protective coloration
is enhanced by both biliverdin and the unknown pigment.
Structural differences appear to be responsible for the color
change during oocyte maturation in Nereis grubei.
igmentation in N. grube
K. Carlson
Introduction
Theevivid green color of the polychaete worm Nereis grubei
during its spawning season has been noted by many authors (Reish
1954, Schroeder 1968). Gould and Schroeder (1968) observed an
oocyte color change of blue to yellow green during matura¬
tion. During this time the color of oocytes tends to dom¬
inate the normal year-round darker green color of the body wall.
The purpose of this work is to determine the nature of the
colored pigments in spawning females of Nereis grubei.
Materials and Methods
Nereis grubei containing eggs were collected in gastro¬
clonium holdfasts at Hopkins Marine Station and Stillwater
Cove, Monterey County, California. Eggs were removed from
forty worms by pipette and their bodies cleaned by washing
with Instant Ocean. Both body wall and eggs were frozen un-
til used.
The body walls were extracted with cold acetone (5°C)
while grinding, yielding an orange solution. A green color
remained. The procedure of Bingham (1975) was followed in
attempting to purify the orange body wall extract. Spectra
were taken in acetone with a Beckman DK-2A spectrophotometer,
Tests of H,SO, addition (Fox 1953), SbCl3 addition (Fox,Crane,
and McConnaughney 1948), and staining by Sudan Black B (Work
and Work 1969) on filter paper were used. The unknown pigment
Pigmentation in N. grubei
K. Carlson
was chromatographed on 500 preparative silica plates with
fluorescent-indicator and developed with 25% acetone in hex¬
ane. Relevant tests and spectra were obtained from the de-
veloped bands.
The green pigment remaining was extracted with 5% HOl
in methanol to give in solution a bright green color. This
green solution was extracted into chloroform by addition of
water. The Gmelin reaction (Fox 1953), zinc acetate-iodine
test (With 1968), and conc. HoSOp destruction were carried out.
Spectra were obtained in acidified methanol.
Only blue colored eggs were used for extraction. They
were rinsed over a glass filter and ground with distilled
water. When rinsed the eggs immediately turned to yellow-green.
Removed to a phosphate buffer (KHoPOL and Na»HPOL) pH 7 ac¬
cording to the procedure of Lee (1966) followed by centri¬
fugation (Gilchrist 1968) failed to extract the pigment in-
to the aqueous solution. Acetone was added, yielding a clear
yellow solution; repeated extraction removed all color from
residue. A preliminary drying was carried out with sodium
sulfate. Purification and characterization were done as
with the body wall orange pigment.
Results
The green color of N. grubei has two components: a
darker color found in the body wall and in particles within
the coelom, and a blue-green color found in both the body
Pigmentation in N. grubei
K. Carlson
wall and eggs of spawning females. These colors were extrac¬
ted and analyzed separately. The darker color was found to
be biliverdin, as Dales (1954) found in Nereis diversicolor,
and the blue-green color was due to unidentified pigments ex¬
hibiting many properties of the chromolipoids.
Dark green pigment of body wall: The spectrum in 5%HCl/
methanol gave the following peaks:
369 sharp
690-700 broad
This absorption closely resembles the spectrum of known
biliverdin (Sigma Chemical Company, St. Louis, Mo.). Both
spectra are shown in Fig. 1. The Gmelin test for bile pig-
ments was positive and a red fluorescence indicating a bila¬
triene was obtained after addition of zinc acetate and iodine.
Destruction by conc. HoSOp distinguished this pigment from
mesobiliverdin.
The biliverdin pigment in N. grubei occurs as granules
of varying sizes, often 9-10u, throughout the body wall. Con-
centrations of biliverdin are found anteriorally, the dorsal
side. This is in contrast to Dales (1954) who found less
biliverdin in this area and instead a darker color due to
carotenoids. Also, large (usually 2504) particles of very
concentrated pigment were found free in the coelom. When the
green color was dissolved away from these particles mucous
like fragments remained. Most probably these are coelomic
Pigmentation in N. grubei
K. Carlson
cells (Dales 1954). Granule sizes found in N. grubei are an
an order of magnitude greater than those found by Dales in
N. diversicolor.
Extracted orange pigment of body wall: This pigment
gave a negative Gmelin reaction and was soluble in acetone,
hexane, chloroform, slightly soluble in ethanol, and insol¬
üble in water. The spectrum obtained in acetone is shown in
Fig. 2. In hexane under UV (Black Ray UVL 21, Ultraviolet
Products Inc.) the pigment gave a bluish fluorescence on
the top layer and a green fluorescence in a lower layer.
Addition of conc. H,SO gave a color change from yellow to
cloudy orange, not the blue or green characteristic of car¬
otenoids. Addition of SbCla also failed to give a blue re¬
action and instead cloudy orange. The pigment was stained by
Sudan Black B.
Chromatography (25% acetone in hexane) revealed four
colored bands with the Re values shown in Fig. 3. In hexane
all solutions were pale yellow, band two (Re 0.46) was the
most highly colored.
Viewed in ultraviolet light the chromatograph revealed
extensive fluorescent impurities throughout the eluted zone.
Fluorescence varied from green and blue to a bright purple
band between bands two (Rg 0.46) and one (Rg 0.34).
The data and tests obtained from the chromatograph after
elution with methanol are given in Table 1. The control is
Pigmentation in N. grubei
K. Carlson
from material eluted from an undeveloped silica gel plate.
Pigment of Oocytes: Yellow-green eggs collected from a
small number of N. grubei when frozen turned reversibly to the
blue color of the less mature oocytes.
After extraction of the blue colored eggs the yellow
solution in acetone gave the absorption spectra shown in Fig.
2. Under ultraviolet light the hexane solution fluoresced
whitish purple. Addition of conc. HoSO,, gave a change of
yellow to cloudy orange. Addition of SbCl, yielded an orange
brown solution. The pigment was stained by Sudan Black B.
Co-chromatography with the unknown body pigment revealed
identical bands both in visible and ultraviolet light.
Discussion
The biliverdin in N. diversicolor was found by Dales
(1954) to be derived from hemoglobin. Considering the sim¬
ilarities of occurence it seems reasonable to assume the same
derivation for the biliverdin found in N. grubei. It is not
known whether the amount of biliverdin in N. grubei increases
in sexual epitoky during the spawning season as in N.
diversicolor. The large particles found in the coelom seem
to indicate that large amounts of biliverdin are in the pro-
cess of being removed to the gut (Dales 1954).
In N. diversicolor Dales (1954) found a darker area
dorsally and anteriorally, due to carotenoids, and speculated
its role was protective coloration. It is interesting to note
Pigmentation in N. grubei
K. Carlson
that the same area is darkened by N. grubei, although in
this case colored by biliverdin. Protective coloration seems
likely. Shorebirds were seen feeding in the algal fields from
which N. grubei were collected. The biliverdin matches well
with the light to dark green color of gastroclonium holdfasts.
Though gastroclonium was found to vary in color from yellow-
green to dark green to brown, its holdfasts retained the same
green color. During the spawning season (February to June
when females become a noticable bright blue-green, Schroeder
(1968) found an N. grubei migration to gastroclonium from
the brown colored kelp Egregia menziesii. Migration to a
protectively colored environment is well correlated with color
development by spawning females.
The oocyte color change during maturation seems to be
the result of structural differences in the tissues. Along
with this color change Gould and Schroeder (1969) found in¬
tensive stratification of the cytoplasm in mature oocytes.
Evidence presented here, based on co-chromatography and
spectral similarities, indicates that the blue-green color
in body wall and oocytes of N. grubei results from the same
pigments.
Because of impurities, I was unable to satisfactorally
characterize the blue-green color of body wall and eggs of
N. grubei. After chemical tests, insufficient color remained
to purify by re-chromatographing. Experiments with controls
Pigmentation in N. grubei
K. Carlson
of known carotenoids gave negative tests for carotenoids when
combined with the unknown pigment. Carotenoids may be pre¬
sent and not detectable because of inadequate purification.
Yet the green color (a possible carotenoprotein) would
neither extract with water nor buffer solutions, both used
to extract green carotenoproteins. (Cheesman, Lee, Zagalsky
1967).
The same argument of insufficient purification holds in
the attempt to characterize the unknown as chromolipoids. In
this case, however, many tests (solvent solubilities, conc.
HSO. SbCl3, and Sudan Black B staining) indicate their pre-
sence. It is not known what effects impurities would have
on chromolipoid detection. Yellow chromolipoids in the poly¬
chaete Thoracophelia mucronata were found by Fox, Crane and
MoConnaughney (1948). A green chromolipoid was found by
Tessier (1929) in a number of hydroids. It is not known
whether a chromolipoid could be responsible for a green color
and subsequent change to yellow with acetone extraction.
Sterols are known to absorb in ultraviolet light and
though these should have been removed by saponification.
their presence may be responsible for the high spectral
absorption in the ultraviolet region.
.
Pigmentation in N. grubei
K. Carlson
Summary
Pigmentation in the polychaete worm Nereis grubei has
two components. The dark green bile pigment biliverdin
occurs in granules in the body wall and in the coelom.
An unidentified blue-green pigment was found in the body wall
and in oocytes of spawning females. Protective coloration
is enhanced by both biliverdin and the unknown pigment.
Structural differences appear to be responsible for the color
change during oocyte maturation in Nereis grubei.
HCHME
LINE


COTTONF
0
Sigmentation in N. grubei
K. Carlson



Acknowledgements
To Dr. Frederick A. Fuhrman for his time, advice, and
constant help.
10
Pigmentation in N. grubei
K. Carlson
References
Bingham, Alpheus, 1975. Amphibian carotenoids: Occurence of
Lutein in Agalychnis dacnicolor. Unpublished re-
search report No. 2. Department of Chemistry
Stanford University.
Cheesman, D. F., W. L. Lee and P. F. Zagalsky
1967
Carotenoproteins in Invertebrates.
Biol. Rev.
42: 132-160.
Dales, R. P. and G. Y. Kennedy.
On the diverse colors of
sicolor. J. Mar. Biol. Ass. U.K.
Nereis diver
33: 699-708.
Fox, D. L.,
1953. Animals Biochromes and Structural Colors,
Cambridge University Press, pp. 191, 273.
Fox, D. L., S. C. Crane and B. H. McConnaughey, 1948. A
Biochemical Study of the Marine Annelid Worm,
Thoracophelia mucronata. J. Mar.Res. 7:567-85.
Gilchrist,
B. M., 1968. Distribution and relative abundance
of carotenoid pigments in Anostraca (Crustacea:
Branchiopoda). Comp. Biochem. Physiol. 24: 123-147.
Gould, M. C., and P. C. Schroeder, 1969. Studies on oogenesis
in the polychaete annelid Nereis grubei. Bio. Bull.
136: 216-225.
Lee, W. L., 1966. Pigmentation of the Marine Isopod Idothea
granulosa (Rathke). Comp. Biochem. Physiol. 19:
13-27.
Reish, Donald J., 1954. The Life History and Ecology of the
Polychaetous Annelid Nereis grubei. Allan Hancock
Found. Publ. Occas. Pap. No. 14: 1-74.
Schroeder
P. C., 1968. On the life history of N. grubei,
a polychaete annelid from California. Pac. Sci.
22: 476-481.
Teissier,
G. and M. Volkonsky, 1929. Bull. Soc. zool. Fr.
54: 599.
With, Torben K., 1968. Bile Pigments, Academic Press, p. 13.
Work, T. S. and E. Work, 1969. Laboratory Techniques in Bio-
chemistry and Molecular Biology, Vol. I, John Wiley
and Sons Inc., p. 531.
11
12
Pigmentation in N. grubei
K. Carlson
Captions
Fig. 1
Absorption Spectra of Unknown and Known Biliverdin.
Solvent: 5% HCl/methanol.
Absorption Spectra of Unknown Oocyte and Body
Fig. 2
Wall Pigments in N. grubei. Solvent: Acetone.
Fig.
Chromatograph of Extracted Orange Pigment in
N. grubei.
Table 1
Eluted Bands from Chromatograph of the Orange Pig-
ment in N. grubei.
Fig. 4
Absorption Spectra of Eluted Bands from Chromatograph
of the Orange Pigment in N. grubei. Solvent: Hexane.
E
S
0TTO
—
e
—
—
8
Re.9
Rf.-46
Rf.34
origin
fig.3
—

I


solvent front
pale
yellow
ellow
pale
orange
pale
orange
N



—
E

N
N
N
e






Ge

N


N

08



2 7
0

+
s




—

Der


5