Published January 1, 1978
Microspectrophotometric Evidence for
Two Photointerconvertible States of Visual
Pigment in the Barnacle Lateral Eye
BARUCH
MINKE and KUNO
KIRSCHFELD
From the Department of Physiology, The Hebrew University-Hadassah Medical School,
Jerusalem, Israel, and the Max-Planck-Institut f/Jr Biologische Kybernetik, 7400 T/ibingen,
West Germany
INTRODUCTION
T h e b a r n a c l e visual p i g m e n t has b e c o m e a n i m p o r t a n t m o d e l b i s t a b l e visualp i g m e n t s y s t e m , in w h i c h b o t h s t a b l e states, r h o d o p s i n a n d m e t a r h o d o p s i n , a r e
p h y s i o l o g i c a l l y active ( H i l l m a n et al., 1972; H o c h s t e i n et al., 1973; M i n k e et al.,
1973a,b; M i n k e et al., 1974). T h e c o r r e l a t i o n b e t w e e n t h e a c t i v a t i o n o f t h e
s t a b l e states o f t h e b a r n a c l e visual p i g m e n t a n d a p h y s i o l o g i c a l e f f e c t h a s b e e n
e s t a b l i s h e d o n t h e basis o f p i g m e n t s p e c t r a d e d u c e d f r o m m e a s u r e m e n t s o f t h e
e a r l y r e c e p t o r p o t e n t i a l (ERP) ( H o c h s t e i n et a l . , 1973; M i n k e e t a l . , 1973a;
M i n k e et al., 1974). T h e E R P a c t i o n s p e c t r a m e a s u r e d in Balanus amphitrite
i n d i c a t e t h a t t h e r h o d o p s i n a b s o r p t i o n has a p e a k at 532 n m a n d c a n b e
p h o t o i n t e r c o n v e r t e d to a d a r k - s t a b l e m e t a r h o d o p s i n with a p e a k at 495 n m .
S h i f t i n g t h e p i g m e n t f r o m r h o d o p s i n to m e t a r h o d o p s i n with a r e d l i g h t a f t e r
b l u e a d a p t a t i o n i n d u c e s a n e x c i t a t o r y p r o c e s s t h a t m a n i f e s t s itself in a p r o l o n g e d
depolarizing afterpotential (PDA) which far outlasts the stimulus. PhotoconvertT H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 71, 1978 " p a g e s 3 7 - 4 5
37
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A B S T R A C T Microspectrophotometrically derived difference spectra from the
barnacles Balanus amphitrite a n d B. eburneus show that a blue illumination after an
orange illumination causes a decrease in absorption in the blue region and an
increase in absorption in the green-yellow region, with an isosbestic point a r o u n d
535 nm. Orange-following-blue illumination causes the reverse changes. T h e dark
time between the adapting and measuring lights has no influence on the data.
T h e results confirm previously r e p o r t e d ERP measurements which indicate that
the barnacle visual pigment has two photointerconvertible dark-stable states. If
one assumes a Dartnall n o m o g r a m shape for the two absorption spectra, a best fit
to the observed difference spectra is obtained with nomograms peaking at 492 nm
and 532 nm, with a peak absorbance ratio a r o u n d 1.6:1. These two n o m o g r a m s fit
very well the ERP action spectra o f metarhodopsin and rhodopsin, respectively,
thus indicating that the ERP is a reliable measure o f visual-pigment changes in the
barnacle. T h e existence of a photostable blue pigment is demonstrated in B.
eburneus and in some of B. amphitrite receptors, and the possible influence of this
photostable pigment on the various action spectra measured in the barnacle is
discussed.
Published January 1, 1978
38
T H E J O U R N A L OF GENERAL PHYSIOLOGY • VOLUME 7 1 - ! . 9 7 8
MATERIALS
AND
METHODS
The measurements were carried out on excised ocelli of B. amphztrite and B. eburneus
which were obtained from Eilat, Israel, and Woods Hole, Mass., respectively. The
reflecting tapetum was removed under white illumination and the eye was mounted in a
closed quartz chamber containing artificial seawater on a Peltier element for controlling
temperature. All measurements were carried out at a temperature of approximately 6°C
which was necessary to get stable recordings. The single beam microspectrophotometer
was composed of a Leitz (D-6330 Wetzlar) UV-microscope photometer UVMP, equipped
with Zeiss (D-7082 Oberkochen) ultrafluar condensor and ultrafluar x32 objective and
UV projective, selected for minimal chromatic aberration. The photomultiptier was EM
9558 Q (EM1 Electronics, Hays, Middlesex, England) selected for low dark current. For
the difference spectra (Fig. 1), transmission of the photoreceptor was measured directly,
at a fixed wavelength, after an adaptive illumination, which was switched off during the
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ing the p i g m e n t back from m e t a r h o d o p s i n to r h o d o p s i n with blue light induces
an inhibitory process and results in depression or prevention o f the PDA
(Hochstein et al., 1973; Minke et al., 1974). Hochstein et al. (1973) also f o u n d :
(a) that there is a good fit between the ERP action spectrum of r h o d o p s i n and
the action spectra o f the late receptor potential (LRP) and PDA induction; and
(b) that the ERP action spectrum o f m e t a r h o d o p s i n fits the action spectrum of
PDA depression.
Brown and Cornwall (1975) recently presented a study of the barnacle B.
eburneus lateral ocelli in which they failed to d e m o n s t r a t e by direct p h o t o m e t r i c
m e a s u r e m e n t s the photointerconvertibility o f the visual pigment responsible
for PDA induction and depression. T h e y also f o u n d by m i c r o s p e c t r o p h o t o m e t tic m e a s u r e m e n t s in an intact p h o t o r e c e p t o r p r e p a r a t i o n that illumination with
wavelengths longer than 540 nm i n d u c e d an absorbance decrease between 550
and 420 nm with m a x i m u m change between 480 and 510 nm, and an absorbance
increase at wavelengths shorter than 420 n m with an isosbestic point at 420 nm.
T h e y obtained similar results by p h o t o m e t r i c measurements o f ocelli extracts.
In contrast to Hochstein et al. (1973), they did not find a correlation between
pigment changes and the various physiological effects: (a) their light-induced
c u r r e n t action spectrum has a m a x i m u m at 540 nm, but they could not find a
pigment with equivalent absorption; (b) their PDA depression action spectrum
has a m a x i m u m at 510-520 nm, also without a g o o d correlation to their
m i c r o s p e c t r o p h o t o m e t r i c results. T h e r e f o r e , they c o n c l u d e d that the correlation between a photointerconvertible p i g m e n t system and the p h e n o m e n a of
PDA induction a n d depression has not been established.
We present here m i c r o s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s indicating (a) that
there exist two photointerconvertible dark stable states o f p i g m e n t in the
barnacle lateral ocelli; (b) that the microspectrophotometrically derived difference spectrum agrees very well with a difference spectrum calculated f r o m two
Darmall n o m o g r a m s peaking at 492 nm and 532 nm, which fit very well the
ERP action spectra o f m e t a r h o d o p s i n and r h o d o p s i n , respectively, m e a s u r e d
by Minke et al. (1973a).
This r e p o r t should t h e r e f o r e help to clarify the conflicting findings of
Hochstein et al. (1973) and Minke et al. (1973a) on the one h a n d , and those o f
Brown and Cornwall (1975) on the other.
Published January 1, 1978
MINKE AND KIRSCHFELD
Two Photointerconvertible States of Visual Pigment
39
RESULTS
Fig. 1 shows the microspectrophotometrically derived difference spectra f r o m a
single p h o t o r e c e p t o r o f the barnacle lateral eye m e a s u r e d in the species B.
eburneus. Fig. 1 inset gives an example o f the absorption m e a s u r e m e n t s : the
absorption at 495 n m is smaller after saturating (more light did not affect the
results) blue than after saturating o r a n g e adaptation, and the reverse is true
for absorption at 570 nm.
T h e points o f the difference s p e c t r u m were calculated f r o m the absorption
differences m e a s u r e d at various wavelengths after blue and o r a n g e saturating
adaptation. In all the m e a s u r e m e n t s it was possible to shift the p i g m e n t
"forward" to a g r e e n - a b s o r b i n g p i g m e n t with a difference s p e c t r u m peak
located a r o u n d 570 n m and "backward" to a blue-absorbing p i g m e n t with
difference s p e c t r u m peak located a r o u n d 485 rim. Between m e a s u r e m e n t s (in
the spectral r a n g e f r o m 380 to 650 nm), the p r e p a r a t i o n was illuminated
alternately with saturating blue (495 or 442 nm) and o r a n g e (596 nm) lights,
which shift the p i g m e n t to either the green-absorbing or the blue-absorbing
states o f the p i g m e n t , respectively. We c o n f i r m e d that the d i f f e r e n c e - s p e c t r u m
changes only in amplitude a n d not in shape when o t h e r wavelengths are used
for shifting the p i g m e n t f r o m one state to the other. T h e m a g n i t u d e o f the
difference-spectrum peaks varied a m o n g individual animals, as indicated by
the two sets o f points (O, O) which were m e a s u r e d in two different animals. It
was possible to shift the p i g m e n t forward a n d backward many times over a
period o f h o u r s without any accumulation o f a UV p i g m e n t absorbing maximally at 380 n m , as r e p o r t e d by Brown and Cornwall (1975). T h e isosbestic
point o f the various d i f f e r e n c e - s p e c t r u m m e a s u r e m e n t s was always in the
r a n g e o f 530-540 nm. Occasionally, with the same positions o f the m a x i m u m ,
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measurement. The diameter of the measuring beam was 0.2-0.1 of the diameter of the
photoreceptor cell. Fig. 1 inset shows that the measuring intensity was weak enough to
prevent measurable shifting of the pigment, and also that the measuring light was stable
over the time of measurement. This was checked at regular intervals at specific
wavelengths. The intensity of the monochromatic adapting light (Fig. 1) was strong
enough to shift all the shiftable visual pigment molecules in approximately 5 s. For the
absolute extinction spectrum (Fig. 3), first a run through the spectrum without a
photoreceptor in the beam-path 0to [k]) was measured. Then the photoreceptor was
inserted (by means of a Servosystem) into the beam path, and a second run through the
spectrum was recorded (Jt [•]). Finally, the receptor was withdrawn and J0 [?~] was
measured once more. The extinction spectrum (E [k]) was calculated according to E D~]
= log (Jo/Jt) + K, Jo and Jo control did not differ by more than ~8%, so no corrections
for intensity drifts were necessary. Since there is always other (photostable) tissue in the
path of the measuring beam besides the photoreceptor itself, K (the zero line in Fig. 3)
is not determined.
For the derivation of extinction spectra from difference spectra a standard computer
program of K. Hamdorf and P. Schlecht was used. The program calculated the
difference of two Dartnall nomograms (Fig. 2 A) to give the best least-squares fit to a
given microspectrophotometrically measured difference spectrum. The wavelengths of
maximum extinction of the two nomograms and their peak-absorption ratio were free
parameters in the calculations.
Published January 1, 1978
40
THE J O U R N A L OF GENERAL PHYSIOLOGY " VOLUME 71 "
1978
m i n i m u m , a n d isosbestic point, the relative size o f the m i n i m u m at 570 n m was
smaller c o m p a r e d to the m a x i m u m at 485 n m . We tested the stability o f the
green- and blue-absorbing states o f p i g m e n t by waiting in the d a r k u p to 30
min between the o r a n g e a n d blue a d a p t i n g lights a n d the m e a s u r i n g light, a n d
f o u n d no influence o f the d a r k time on the results. We also m e a s u r e d the
difference spectra in B. amphitrite. T h e dissection o f this p r e p a r a t i o n was m o r e
difficult because the animals were smaller a n d the t a p e t u m was m o r e a d h e r e n t
to the cells. Accordingly, we did not have the c o m p l e t e spectra, but did succeed
in showing that the peaks a n d isosbestic point fall at the same wavelengths as
for B. eburneus. T h e results show directly that the p h o t o r e c e p t o r s of b o t h
species have a p i g m e n t with two dark-stable photointerconvertible states.
Fig. 2 A shows two Dartnall n o m o g r a m s p e a k i n g at 492 n m a n d 532 n m with
T~'~islion rm~a~rod at
JOSnm
570nm
i
~
Ios
~ O,,..,,,.O,..,.----:
-
ool
I
I
I
I
45O
50O
55O
6OO
65O
WcM~mgth (nml
IqG~RE 1. Difference spectra from saturating monochromatic orange adaptation
(596 nm interference filter, Schott, Depal) vs. saturating monochromatic blue
adaptation (485 nm [O] and 442 nm [©] interference filters, Schott, Depal) in two
different preparations in B. eburneus. ]met, Transmission meaurements at 570 nm
and at 495 nm after saturating monochromatic blue (442 nm) and orange (596 nm)
adaptation. These measurements together with othcrs were used in the main
figure (O).
400
a ratio o f the peaks o f 1.6:1, respectively. A calculated difference s p e c t r u m
f r o m these curves gives the best fit to the results o f Fig. 1 (O). We used the data
o f Fig. 1 (O) for the c u r v e fitting (Fig. 2B) since they are f r o m the p r e p a r a t i o n
that gave the o p t i m u m signal-to-noise ratio a n d the best long-time stability (see
m e a s u r e m e n t s at 495 a n d 570 n m in Fig. 1). T h e calculated difference s p e c t r u m
is d e m o n s t r a t e d in Fig. 2 B (smooth curve), t o g e t h e r with a replot of o n e set o f
m e a s u r e m e n t s p r e s e n t e d in Fig. 1. T h e curves o f Fig. 2 A also fit very well the
action spectra o f m e t a r h o d o p s i n a n d r h o d o p s i n that were d e d u c e d f r o m ERP
data (Minke et al., 1973a).
T h e second difference s p e c t r u m (Fig. 1, @) by m e a n s o f the same calculation,
yields two Dartnall n o m o g r a m s p e a k i n g at 494 n m a n d 533 n m with a ratio o f
the peaks o f 2.1 : 1, respectively.
T h e r e are differences a m o n g the p e a k wavelengths a n d the s h a p e of the
various action spectra o f the barnacle p h o t o r e c e p t o r s r e p o r t e d in the literature
(Stratten a n d O g d e n , 1971; Shaw, 1972; Hochstein et al., 1973; Brown a n d
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~ (~X) O~
i
Published January 1, 1978
Two Photointerconvertible States of Visual Pigraent
MINI~E AND KIRSCHFELD
41
Cornwall, 1975). T h e s e differences m a y arise f r o m photostable p i g m e n t s which,
in addition to the visual p i g m e n t s , have b e e n r e p o r t e d to exist in several
i n v e r t e b r a t e p h o t o r e c e p t o r s (Kirschfeld a n d Franceschini, 1977; Kirschfeld et
al., 1977), a n d can also be d e m o n s t r a t e d in the barnacle.
T h e p h o t o r e c e p t o r s o f B. eburneus show a strong yellow color, while those o f
B. amphitrite are usually pale, without color, but sometimes yellow. Fig. 3
presents extinction spectra as m e a s u r e d in B. amphitrite yellow ([-1) a n d B.
amphitrite pale (11). T h e extinction s p e c t r u m o f the yellow r e c e p t o r has a p e a k
0.03
uu
1
I
400
450
I
I
I
i
500
550
600
1
I
I
002
.~_
ool
002
~
0.01
• ~-
o.oo
=/.,o
O0 °
o
o
'NJ
2
0.01
I
400
[
450
I
SO0
Wavelength
I
550
Inm)
I
600
FmVRE 2. A, Two Darmall nomograms with peak wavelengths at 492 nm and
532 nm, with a ratio of the peak absorption of 1.63:1, respectively. The difference
between these curves gives the best fit to the difference spectrum of Fig. 1 (O). B,
Computer-calculated difference spectrum from Fig. 2 A (
) that give the best
fit to the photometrically derived difference spectrum of Fig. 1 (O). The points
(O) and the dashed curve are a replot of the corresponding points of Fig. 1 (O)
for comparison.
close to 450 n m with a s h a p e typical o f c a r o t e n e ? A very similar a b s o r p t i o n
s p e c t r u m was m e a s u r e d by us in B. eburneus ( u n p u b l i s h e d data) a n d was seen in
the r h a b d o m e r e o f the central r e c e p t o r (no. 7) o f the fly (Kirschfeld a n d
Franceschini, 1977).
DISCUSSION
Two Photointerconvertible States of Visual Pigment
T h e a g r e e m e n t between the photometrically derived difference spectra and
that calculated f r o m two Dartnall n o m o g r a m s , p e a k i n g at 492 a n d 532 n m , is
Kirschfeid, K., R. Feiler, and N. Franceschini. Manuscript in preparation.
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0.00
Published January 1, 1978
4~
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
71
- 1978
very good. T h e 492 a n d 532 n m Dartnall n o m o g r a m s of Fig. 2 A also fit the
ERP action spectra o f m e t a r h o d o p s i n and r h o d o p s i n , respectively (Minke et
al., 1973a). T o see how g o o d the fit is between the actual ERP m e a s u r e m e n t s
and the m i c r o s p e c t r o p h o t o m e t r i c results, we calculated a difference s p e c t r u m
f r o m curves that give the best fit to the ERP data o f Minke et al. (1973a, Fig.
5), using a m e t a r h o d o p s i n - t o - r h o d o p s i n p e a k - a b s o r p t i o n ratio a r o u n d 1.6:1,
respectively, a n d f o u n d a very g o o d fit to the data o f Fig. 1. T h e ratio o f the
peak a b s o r p t i o n o f m e t a r h o d o p s i n to r h o d o p s i n o f a r o u n d 1.6:1 agrees with
similar data derived f r o m o t h e r invertebrates ( H a m d o r f a n d S c h w e m e r , 1975).
This ratio was m e a s u r e d in the barnacle by Minke et al. (1974) in two
i n d e p e n d e n t ways with the ERP. T h e ratio f o u n d in one way was 4:1 a n d in the
other, 1.6:1. T h e reason for this difference might possibly arise f r o m an effect
o f a photostable p i g m e n t (see below).
2
B P ~ B ~ I H } i ~ |
•
I
i
I
~I ~
400
450
500
550
•
•
~
•
7
600
•
~
•
~I ~
650
•
•
~
•
r =='="
700
Wovelength(nm)
FIGURE 3. Absolute absorption spectra measured from photoreceptors of the
lateral eyes of B. amphitrite yellow ([]) and pale (U) receptors. The spectrum with a
peak close to 450 nm is very similar to that of a carotene.
T h e good fit between the photometrically derived difference spectra and the
s p e c t r u m calculated f r o m the d i f f e r e n c e o f the two Dartnall n o m o g r a m s a n d
f r o m the ERP action spectra strongly suggests that the same visual-pigment
system is m e a s u r e d by both techniques a n d that the ERP is a reliable m e a s u r e
o f the visual-pigment changes in the barnacle.
W h e n we c o m p a r e o u r m i c r o s p e c t r o p h o t o m e t r i c results with those o f B r o w n
and Cornwall (1975), we note that the m a j o r difference arises f r o m their
inability to convert the blue-absorbing state o f p i g m e n t ( m e t a r h o d o p s i n ) to the
g r e e n - a b s o r b i n g state (rhodopsin). Instead, they f o u n d an irreversible accumulation o f p i g m e n t in the UV. This finding might be e x p e c t e d in an extract o f
p h o t o r e c e p t o r s , since in o t h e r crustacea t h e r e is evidence for a r e d u c t i o n in the
stability o f m e t a r h o d o p s i n in extracts, which causes an irreversible increase in a
U V - a b s o r b i n g p h o t o p r o d u c t , probably retinaldehyde (Goldsmith, 1972). T h e
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g
Published January 1, 1978
MINKE AND KIl~SCHFELD TWOPhotointerconvertibleStates of Visual Pigment
43
fact that they f o u n d similar d i f f e r e n c e spectra in both extracts and intact
p h o t o r e c e p t o r s - p r e p a r a t i o n s suggests that their intact p r e p a r a t i o n was d a m a g e d
d u r i n g their e x p e r i m e n t a l procedures, possibly due to long strong-adapting
lights. In o u r e x p e r i m e n t s we used much shorter adapting lights (seconds
instead o f minutes) and, in addition, we lowered the t e m p e r a t u r e to 6°C in
o r d e r to get stable recordings. Since the previously r e p o r t e d ERP action spectra
(Hillman et al., 1972) did not show consistent differences between measurements
at low (8°C) and at r o o m t e m p e r a t u r e , we assume that the low t e m p e r a t u r e
d u r i n g o u r m i c r o s p e c t r o p h o t o m e t r i c experiments did not affect the m e a s u r e d
spectra.
Possible Effects of the Photostable Blue Pigment
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T h e microspectrophotometrically derived absorption spectrum o f r h o d o p s i n
fits very well the action spectra o f the LRP and PDA induction, that o f
m e t a r h o d o p s i n fits the action spectrum o f the PDA depression, as r e p o r t e d by
Hochstein et al. (1973).
T h e PDA depression action spectrum and the action spectrum o f lightinduced c u r r e n t , r e p o r t e d by Brown and Cornwall (1975), have maxima at 510520 n m and 540 nm, respectively. A difference spectrum o f two Dartnall
n o m o g r a m s with the same peaks does not fit the data o f Fig. 1 as well as does
the spectrum p r e s e n t e d in Fig. 2 B.
T h e question arises o f why there is a difference between the peak o f the
action spectrum o f PDA depression (510-520 nm) as r e p o r t e d by Brown and
Cornwall (1975) in B. eburneus and the absorption peak o f m e t a r h o d o p s i n (492
nm), since we expect that the transition f r o m m e t a r h o d o p s i n to r h o d o p s i n
induces PDA depression in B. amphitrite (Hochstein et al., 1973) as well as in B.
eburneus.
T h e existence o f a photostable blue pigment in the p h o t o r e c e p t o r s o f B.
eburneus, and its usual absence in B. amphitrite might possibly explain the
discrepancy between the action spectra o f PDA depression r e p o r t e d by Brown
and Cornwall (1975) in B. eburneus and by Hochstein et al. (1973) in B. amphitrite.
T h e function o f such photostable pigments can be manifold (Kirschfeld and
Franceschini, 1977): a) T h e y may act as a screening pigment which in the case
of the barnacle could induce a red shift in the action spectrum derived f r o m
activation o f m e t a r h o d o p s i n , so that it will better fit the action spectrum o f
PDA depression r e p o r t e d by Brown and Cornwall (1975). b) T h e y may also act
as "sensitizing-pigments", that is as photostable pigments that absorb light and
transfer the energy to a photochemically active pigment. Such an "antenna"function o f photostable pigments, (e.g. of carotenes) is well known in photosynthesis (Govindjee, 1975), but has recently also been demonstrated in photoreceptors o f the fly (Kirschfeld et al., 1977). However, assuming a resonance F6rstertype e n e r g y transfer f r o m d o n o r to acceptor (F6rster, 1966), we expect a blue
shift in the action spectrum o f m e t a r h o d o p s i n , if the blue pigment in the
barnacle acts as an a n t e n n a pigment. In o u r present state of knowledge it is
difficult to know which mechanism is more effective in the yellow receptors of
the barnacle.
In contrast to the action spectrum which can be influenced by a photostable
Published January 1, 1978
44
THE
JOURNAL
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PHYSIOLOGY
• VOLUME
71 • 1 9 7 8
pigment, the shape of the photometrically derived difference spectrum should
b e u n a f f e c t e d by a c t i v a t i o n o f a p h o t o s t a b l e (i.e. u n b l e a c h a b l e ) p i g m e n t . T h i s
m a y e x p l a i n w h y we h a v e a g r e e m e n t b e t w e e n t h e p h o t o m e t r i c a l l y d e r i v e d
d i f f e r e n c e s p e c t r u m (Fig. 1) f r o m t h e yellow r e c e p t o r s o f B. eburneus a n d t h e
c a l c u l a t e d d i f f e r e n c e s p e c t r u m f r o m E R P d a t a m e a s u r e d in r e c e p t o r s o f B.
amphitrite, w h i c h u s u a l l y d o n o t h a v e t h e p h o t o s t a b l e p i g m e n t .
A d e t a i l e d i n v e s t i g a t i o n o f t h e r o l e o f t h e p h o t o s t a b l e p i g m e n t in t h e yellow
r e c e p t o r s o f B. eburneus s e e m s h i g h l y d e s i r a b l e .
We are very grateful to Prof. P. Hillman and Dr. S. Hochstein for their very helpful comments and
discussions. We thank Mr. R. Feiler for his help in the microspectrophotometric measurements,
Prof. K. Hamdorf and Dr. P. Schlecht for the use of their standard computer program to calculate
pigment spectra from difference spectra, Prof. H. Stieve and P. Hillman for the barnacles, and Dr.
P. McIntyre for reading the English manuscript.
This research was supported in part by a grant from the United States-Israel Binational Science
Foundation (B.S.F.)Jerusalem, Israel.
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Published January 1, 1978
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