Developmental changes in the cone visual pigments of black bream Acanthopagrus butcheri
1 Department of Zoology, University of Western Australia, WA 6009,
Australia
2 School of Biological Sciences, University of Bristol, Woodland Road,
Bristol, BS8 1UG, UK
Present address: Vision Touch and Hearing Research Centre, University of
Queensland, Brisbane, QLD 4072, Australia
* Author for correspondence (e-mail: jshand{at}cyllene.uwa.edu.au)
Accepted 11 September 2002
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Summary |
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Key words: vision, retina, cones, opsin, microspectrophotometry, fish, black bream, Acanthopagrus butcheri
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Introduction |
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The life history of many fish involves changes in spectral environment
during migration from one body of water to another. In association with such
migrations, the spectral sensitivity of the fish may also change. The changes
can be the result of the loss of a cone class from the retina; alternatively,
there may be physiological changes in the visual pigments within a cell type
(for a review, see Beaudet and Hawryshyn,
1999). Visual pigment absorption characteristics are governed in
two main ways: by the amino acid sequence of the transmembrane protein (the
opsin), and/or whether the chromophore that binds with the opsin is retinal
(vitamin A1-based) or 3,4-didehydroretinal (vitamin
A2-based), and changes in either moiety affect visual pigment
spectral sensitivity (for a review, see
Loew, 1995
). For example, in
the eel Anguilla anguilla, shorter wavelength sensitivity is
facilitated by the rod chromophore changing from to 3,4-didehydroretinal to
retinal as the adults begin their breeding migration from rivers to the deep
ocean (Carlisle and Denton,
1959
; Wood et al.,
1992
), whereas a switch in opsin expression has been implied in
the short wavelength-sensitive single cones of pollack Pollachius
pollachius (Shand et al.,
1988
), and the long wavelength-sensitive double cones of the
goatfish Upeneus tragula, as the habitat and feeding behaviour of the
fish change during development (Shand,
1993
).
In contrast to the retinae of adults, the pre-flexion larval stages of many
species of teleost possess only one morphological cell type, single cones, and
it is not until a later stage of development, often the time of metamorphosis
from larval to juvenile stages (the time of fin ray formation and body
pigmentation), that double cones and rods are observed
(Blaxter and Jones, 1967;
Blaxter and Staines, 1970
;
Ahlbert, 1973
;
Evans and Fernald, 1993
;
Pankhurst and Eagar, 1996; Shand et al.,
1999
). The possession of only one morphological cell type in the
larval retina raises the question of whether only one visual pigment is
expressed at this stage, and what the spectral absorption characteristics
might be. In addition, it is not known whether there are visual pigment
changes at the time that single cones begin to associate with neighbours and
form double cones.
We performed a microspectrophotometric study of the photoreceptors of black bream, Acanthopagrus butcheri, during their early life history to determine whether changes in visual pigments in juvenile fish are initiated during structural changes in the retina associated with metamorphosis, or correlated with the timing of ontogenetic changes in habitat and behaviour. In addition, we analysed the visual pigment measurements to determine the possible mechanisms by which the spectral characteristics are being altered in black bream.
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Materials and methods |
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Larvae were reared according to procedures outlined by Jenkins et al.
(1999). From hatching to 25
dph they were held in a semi-intensive green water system, so called because
the microalgae, Nannochloropsis oculata, which are present to provide
a food source for rotifers upon which the larval bream feed, colour the water
green. Between 22 and 25 dph the larvae were transferred to clear water and
fed a diet of cultured branchiopod brine shrimps (Artemia spp.). From
55 dph the juveniles were gradually shifted onto a diet of dried pellets
(0.2-0.4 mm diameter, Nippai ML®, Fuki and Co. Ltd). The fish were reared
under fluorescent room light (Philips Coolwhite, 36W tubes) on a 14 h:10 h
light:dark regime.
Preparation for microspectrophotometry
Experimental procedures were approved by the University of Western
Australia Ethics Committee and followed the guidelines of the National Health
and Medical Research Council of Australia. Fish were dark-adapted for at least
2 h prior to immersion in a lethal dose of methanesulphonate (MS 222;
Sigma-Aldrich Pty, 1:2000 w/v in seawater). For examination by
microspectrophotometry, preparations of unfixed retinal tissue were teased
apart in teleost phosphate-buffered saline, containing 10% dextran (Sigma 250k
RMM), on a rectangular 50x22 mm No. 1 coverslip (Marienfeld, Germany).
During the early larval stages (SL 2-4 mm), whole fish were placed on
the coverslip and teased apart with forceps. With larger fish it was possible
to first remove the eyes and dissect the retina on the coverslip. The retinae
of juveniles (SL <20 mm) could be dissected prior to transferring
small pieces (1-2 mm2) to the coverslip. In all cases the
preparation was covered with a smaller (19 mm2) No. 1 coverslip and
sealed with nail varnish to prevent dehydration of the sample. All
preparations were carried out in infrared illumination provided by a bank of
28 infrared emitting diodes and visualised using an infrared image converter
(FJW Industries, USA).
Measurement of visual pigment absorbance spectra
A single-beam wavelength-scanning microspectrophotometer (MSP) was used to
measure the absorption characteristics of the photoreceptor outer segments.
The MSP described previously (Partridge et
al., 1992), has been modified recently to improve the optics and
hence transmission of short wavelengths to the specimen. Briefly, light from a
quartz-halogen bulb was focused onto a holographic grating monochromator
(Jobin Yvon). The output from the monochromator was linearly polarised, using
a calcite crystal, and used to illuminate a variable rectangular aperture that
controlled the dimensions of the measuring beam (typically 1-2x3-5
µm, depending on photoreceptor dimensions). The aperture plane was then
focused by a series of fused-silica biconvex lenses (Oriel) and a Zeiss
Ultrafluar (x32, NA 0.4) objective into the plane of the specimen on a
micrometer-manipulated microscope stage. Above the stage, a Zeiss Neofluar
objective (x100, NA 1.3) imaged the measuring beam onto the photocathode
of a photomultiplier (R3896, Hamamatsu, Japan). The signal from the
photomultiplier was digitised and recorded by a CTM05 counter timer board in a
personal computer, which also controlled the scanning process. To view the
sample and align the measuring beam, the specimen was illuminated with
infrared wavelengths and the image was directed to an infrared-sensitive video
camera, the image being viewed on a video monitor. Spectral absorbance
measurements were made by placing the outer segment in the path of the
measuring beam and scanning over the wavelength range 350-750 nm. Data were
recorded at each odd wavelength on the `downward' (long-wavelength to
short-wavelength) spectral pass and at each interleaved even wavelength on the
`upward' (short-wavelength to long-wavelength) spectral pass. Only one sample
scan was made of each outer segment, but this was combined with two separate
baseline scans from an area adjacent to the outer segment being scanned. The
two absorbance spectra thus obtained were averaged to improve the
signal-to-noise ratio of the absorbance spectra used to determine the
max values. Following these `pre-bleach' scans, outer
segments were bleached with white light from the monochromator for 2-4 min and
an identical number of sample and baseline scans made subsequently. The
post-bleach average spectrum thus obtained was deducted from the pre-bleach
average to produce a difference spectrum for each outer segment and confirm
the presence of visual pigment. Photoreceptor dimensions were measured for
each cell scanned.
Analysis
Baseline and sample data were converted to absorbance values at 1 nm
intervals and the upward and downward scans were averaged together by fitting
a weighted three-point running average to the absorbance data
(Hart et al., 2000).
Absorbance spectra were normalized to the peak and long-wavelength offset
absorbances, obtained by fitting a variable-point unweighted running average.
Following the method of MacNichol
(1986
) a regression line was
fitted to the normalized absorbance data between 30% and 70% of the normalized
maximum absorbance at wavelengths longer than that of the absorbance peak. The
regression equation was used to predict the wavelength of maximum sensitivity
(
max) and fit the visual pigment template following the
methods of Govardovskii et al.
(2000
). Acceptable pre-bleach
spectra (Levine and MacNichol,
1985
; see Partridge et al.,
1992
) had a characteristic `bell-shaped' curve with a clear alpha
peak, low noise and flat long wavelength tail above the wavelength at which
the absorbance had fallen to less than 0.5% normalized maximum absorbance, and
those of similar
max were averaged and reanalysed. For
display, averaged spectra were overlain with an A1 visual pigment
template of the same
max, generated using the equations of
Govardovskii et al.
(2000
).
To investigate how the shift in long wavelength-sensitive cone
max occurs, and specifically to determine whether
chromophore and/or opsin exchanges take place during development and underlie
the observed sensitivity changes, we examined the relationship between the
running average
max, referred to as the over-the-top (OTT)
max, and the full width at half maximum (FWHM) bandwidth of
the visual pigment curves at the 50% normalized absorbance point. OTT
max was used in preference to values derived from fitting
visual pigment templates so that
max values were not biased
by the goodness-of-fit of the template. Although absorbance spectra were all
best-fitted by an A1 rather than A2 visual pigment
template, some deviations (notably increased FWHM bandwidth) from the
mathematical template were noted in measured spectra that did not appear to be
due to classical MSP artefacts such as photoproduct build up, bypassing light
or scatter at short wavelengths. In view of the possibility of visual pigment
co-expression, selection of absorbance spectra that are good fits to
mathematical template spectra might lead to a bias for the selection of
spectra that represent only one opsin rather than a mixture. Using the OTT
max is a more objective way of classifying spectra,
although the chance of including spectra that contain artefacts not due to
spectral absorbance by one or more visual pigments is necessarily increased.
MSP absorbance spectra from both aquarium-reared and wild-caught fish were
included in this analysis. Computer models were constructed using the
rhodopsin (A1) and porphyropsin (A2) visual pigment
templates of Govardovskii et al.
(2000
) to calculate the
bandwidth of mixtures of visual pigment, simulating an exchange of chromophore
and/or a change in the underlying opsin. In all cases the ratio of
bandwidth:OTT
max was calculated for the range of observed
OTT
max values (approx. 520-575 nm), and plotted with the
empirical data. Similar modelling methods were not attempted for the short
wavelength-sensitive cones, as there were too few data.
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Results |
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To show the changes in visual pigments with age, the mean
max for each cone class in each individual aquarium-reared
fish is shown is shown in Fig.
2. The variance in the rod data is typical for MSP measurements of
cells with the dimensions measured in this study
(Shand, 1993
) and no change in
the
max of rod pigments was observed. In the cones, there
is a change in the
max, in both the short- and
long-wavelength absorbing cones, from approximately 100 days, with a wide
spread in the
max of the cone records, up to 160 days.
Cones of the size we measured would not give the variance in
max values observed unless there were differences in the
opsin mixtures between cells, indicating the possibility that a number of
different pigments are expressed in both cone classes during this time.
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Between 5 and 20 dph, when the fish are in their larval stage and all
records were from single cones, two classes of visual pigment were recorded
with max at approximately 425 nm or 534 nm. Between 21 and
40 dph, as the fish undergo metamorphosis, double cones were first observed.
At this time all records close to 425 nm were recorded from single cones;
records close to 534 nm were from both double (62.5%) and single (37.5%)
cones. From 41 dph onwards, all long wavelength records were from double
cones. Between 108 and 154 dph, all single cones were found to have a
max displaced to between 450 nm and 482 nm and the double
cones had a mean
max at 539 nm. Fish aged between 160-172
dph and adult were found to have single cones with a mean
max at 476 nm and long-wavelength-sensitive double cones
with
max between 545 and 575 nm.
From the time of their formation, long-wavelength-sensitive double cones
were observed with the visual pigments in the two outer segments differing by
up to 5 nm. The difference in the max of the double cones
was variable during the time of change in visual pigments but showed a
significant increase as the fish grew (regression analysis;
y=0.038x+2.7404, P=0.017, d.f.=33,
r2=0.166). The maximum difference in
max observed between visual pigments in the two outer
segments of the double cones was 19 nm.
A frequency histogram showing the max of all long
wavelength-sensitive cone absorbance spectra from all fish, calculated using
the running average
max (OTT), is shown in
Fig. 3A. The range in observed
OTT
max values runs from approximately 520 to 575 nm. These
records were used to investigate the relationship between bandwidth and
max shown in Fig.
3B. Also plotted in Fig.
3B is the modelled relationship between bandwidth:OTT
max and the OTT
max for changes in
chromophore and opsins. The data are expressed as bandwidth:OTT
max because this ratio is invariant with
max for any pure visual pigment. Thus all A1
pigments have a ratio close to 0.20 (A1-boundary on the graph), and
all porphyropsins have a ratio close to 0.24 (A2-boundary on the
graph). The data fall within these boundaries, indicative of visual pigment
mixtures. As shown in Fig. 3B,
for
max values greater than approx. 525 nm the bandwidth of
the empirical measurements is generally less than that calculated for mixtures
of an A1 pigment with a
max of 520 nm
(P5201) and an A2 pigment with a
max
of 575 nm (P5752). Fig.
3B also shows the corresponding relationship between bandwidth and
max for mixtures of two A1 pigments,
P5201 and P5751, and three A1 pigments
expressed sequentially, in mixtures of P5201 with P5501,
and P5501 with P5751. In order to compare how well these
various simple models fit the data, the sums-of-squares differences between
the data and the five models were calculated. Division by the degrees of
freedom (229) provided estimates of the variances, the error between the
models and the data. In order of goodness of fit these models, with variances,
are: P5201-P5501-P5751
(2.67x10-4); A1-boundary
(3.79x10-4); P5201-P5751
(1.01x10-3); A2-boundary
(1.04x0-3); P5201-P5752
(2.88x10-3). The best-fitting model is therefore that
involving the expression of three opsins, and an Fmax test
(Sokal and Rohlf, 1995
) was
conducted to compare the variance of this model with that of the
next-best-fitting model [A1-boundary; which implies an
(implausible) multiplicity of rhodopsin pigments of varying
max]. This insensitive test shows that the three-opsin
model provides a significantly better fit (Fmax=1.417;
d.f.=2, 228; P<0.01) than the A1 boundary, and
therefore than all other models.
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Discussion |
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At the time of double cone formation in black bream the green-sensitive
cones are those that become associated with one another to form double cones,
the violet-sensitive cells remaining single cones. The ability to identify
neighbouring cells with similar opsin expression is likely to take place by a
cell signalling mechanism, possibly the same mechanisms involved in formation
of the photoreceptor mosaic during retinal growth
(Cameron and Easter, 1995;
Raymond et al., 1995
;
Stenkamp et al., 1996
).
Behavioural significance of visual pigment changes
During the larval and early juvenile stages, black bream are found in
shallow estuarine waters feeding on plankton
(Sarre, 1999). The presence of
short wavelength-absorbing cones during the early stages may aid the detection
of plankton by increasing the contrast of short wavelength-absorbing or
reflecting zooplankton, as has been suggested for other shallow-water
planktivorous teleosts with similar visual pigments
(Bowmaker and Kunz, 1987
;
Browman et al., 1994
;
McFarland and Loew, 1994
;
Britt et al., 2001
). In black
bream, the shift in the absorption of both cone types to longer wavelengths
begins when they move to deeper, lower light intensity, tannin-stained water,
with reduced proportions of short wavelength light. Combined with the move to
deeper water, black bream also begin to change their feeding strategy from
planktivory to feeding from the substrate, a change in behaviour that is
correlated with a shift in the main visual axis and relocation of the ganglion
cell area centralis to more dorsal regions of the retina
(Shand et al., 2000
). Thus, a
combination of changes in both feeding strategy and spectral qualities of the
water need to be considered when rationalising the visual pigment changes.
The factors that initiate the changes in the visual pigments and control
wavelength specificity are unknown. The variance in the max
of the long-wavelength-sensitive cones is unusual and will have consequences
for wavelength discrimination and hence the behaviour of the fish. We have
also noted a degree of variability in the timing of the changes between
different individuals. In addition, animals obtained from the wild appear to
initiate the changes at a smaller size, and possess longer-wavelength
sensitivity following the changes, than fish reared in clear-water aquarium
conditions under the artificial lights used in this study (J. Shand and N.
Thomas, personal observations). Experiments to determine whether it is colour
or intensity of the environmental light that initiates changes in the visual
pigments are underway. Nevertheless, the ability to respond to environmental
factors by regulation of wavelength specificity during development would be an
appropriate adaptation for fish living in a variable light environment, such
as the temperate estuaries in which black bream are found.
Mechanism of visual pigment changes
From our data and the modelling, we propose that the shifts in spectral
sensitivity in black bream are due to changes in opsin gene expression, rather
than to a switch in chromophore from retinal (A1) to
3,4-didehydroretinal (A2). The observed range of change cannot be
due to a single opsin in association with A1 shifting by
chromophore exchange to A2 via intermediates with mixed
chromophores, because the max range is too great. For
instance, the A2 analogue of a 520 nm
max
A1 would have a
max of approximately 545 nm
(Parry and Bowmaker, 2000
).
Given the fact that neither the rod pigments, nor any cone data, are
better-fitted by A2 templates, it is likely that there is little or
no A2 chromophore in the retina of black bream. Although our
modelling suggests that the shift can best be explained by the expression of
at least three opsin genes, forming visual pigment mixtures in the double cone
outer segments, this conclusion is necessarily tentative. Indeed, the exact
mechanism underlying the
max shift must remain a matter of
conjecture until molecular biological studies are completed.
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Acknowledgments |
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