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First published online April 18, 2008
Journal of Experimental Biology 211, 1495-1503 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012047
The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri
1 School of Animal Biology, University of Western Australia, Crawley, WA 6009,
Australia
2 UCL Institute of Ophthalmology, 11–43 Bath Street, London EC1V 9EL,
UK
3 Australian Equine Genetics Research Centre, School of Biomedical Sciences,
University of Queensland, Brisbane, Qld 4072, Australia
4 Sensory Neurobiology Group, School of Biomedical Sciences, University of
Queensland, Brisbane, Qld 4072, Australia
* Author for correspondence (e-mail: jshand{at}cyllene.uwa.edu.au)
Accepted 21 February 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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and β, and
LWS) opsins were obtained and their expression levels in larval and
adult stages examined using quantitative RT-PCR. The changes in spectral
sensitivity of the cones were related to the differing levels of opsin
expression during ontogeny. During the larval stage the predominantly
expressed opsin classes were SWS1, SWS2B and
Rh2A, contrasting with SWS2A, Rh2Aβ and
LWS in the adult. An increased proportion of long
wavelength-sensitive double cones was found in fishes reared in the short
wavelength-reduced conditions and in wild-caught animals, indicating that the
expression of cone opsin genes is also regulated by environmental light.
Key words: photoreceptor, cone, spectral tuning, fish, microspectrophotometry, environmental light
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Bowmaker and Hunt,
2006).
Teleost fishes, with their wide range of natural habitats, have become the
model for examining the relationship between ambient light and the spectral
sensitivity of visual pigments; it has been shown that species from bodies of
water with differing spectral irradiance tend to possess visual pigments that
are related to the most abundant wavelengths
(Bowmaker et al., 1994;
Bowmaker and Hunt, 2006;
Douglas et al., 2003;
Loew and Lythgoe, 1978;
Lythgoe et al., 1994;
Partridge et al., 1989;
Partridge et al., 1992a).
Furthermore, the ontogenetic migrations of fishes from one body of water to
another or to greater depths is accompanied by changes in the spectral
sensitivity of the retina in many species (for reviews, see
Beaudet and Hawryshyn, 1999;
Collin and Shand, 2003). For
example, those that migrate to deeper water during ontogeny exhibit a
narrowing in the spectral range of their pigments as the spectral range of the
light environment also narrows (Bowmaker
and Kunz, 1987; Shand et al.,
1988; Shand, 1993;
Hope et al., 1998).
The spectral sensitivity of a visual pigment is dependent on the amino acid
sequence of the opsin protein component of the molecule. There are four opsin
classes in vertebrate cone photoreceptors: short wavelength-sensitive 1 (SWS1)
pigments with peak sensitivities (
max) in the
UV–violet region of the spectrum, short wavelength-sensitive 2 (SWS2)
pigments with max in the blue region, middle
wavelength-sensitive rod-like (Rh2) pigments with max in
the green region, and long wavelength-sensitive (LWS) pigments with
max in the yellow–red region (for reviews, see
Bowmaker and Hunt, 2006;
Yokoyama, 2000). The
max of a visual pigment also depends on whether the
chromophore present in the pigment molecule is derived from vitamin
A1 (retinal) or A2 (3,4-didehydroretinal) giving a
rhodopsin or porphyropsin pigment, respectively. The latter pigments are long
wavelength shifted compared with the former, with a greater effect at longer
wavelengths (Whitmore and Bowmaker,
1989).
It was originally assumed that chromophore changes or the loss of a cone
type from the photoreceptor mosaic would be the main way in which changes in
spectral sensitivity are facilitated (for reviews, see
Bowmaker, 1995;
Beaudet and Hawryshyn, 1999).
However, changes in opsin expression were subsequently implicated in a number
of species such as the pollack (Shand et
al., 1988), goatfish (Shand,
1993) and flounder (Evans et
al., 1993), and were shown to account for the blue–green
sensitivity shift in the rods of the eel
(Archer et al., 1995;
Hope et al., 1998). More
recently, changes in cone opsin expression have been demonstrated in zebrafish
(Chinen et al., 2003;
Takechi and Kawamura, 2005),
Pacific pink salmon (Cheng and Novales
Flamarique, 2004), rainbow trout
(Veldhoen et al., 2006;
Cheng and Novales Flamarique,
2007) and cichlids (Spady et
al., 2006).
A recent microspectrophotometric study of the black bream Acanthopagrus
butcheri revealed a changing pattern of cone photoreceptors with
different max values in fish at different developmental
ages (Shand et al., 2002). In
general terms, larval fish have cones that absorb maximally at shorter
wavelengths, while juveniles and adults have longer wavelength sensitivity.
However, a degree of variability was noted and animals from the wild were
found to have longer wavelength-absorbing pigments than their aquarium-reared
counterparts. It was predicted from modelling changes in bandwidth of the
visual pigment that a change in opsin expression was taking place rather than
a simple switch from an A1 to an A2 chromophore. In
order, therefore, to establish the basis for the visual pigment changes, we
have identified the different rod and cone opsins expressed in the black bream
retina and used quantitative RT-PCR (qPCR) to determine their expression
levels in larval and adult fishes. In addition, we have sought to establish
whether the cone class/visual pigment changes are pre-programmed developmental
events or are influenced by environmental light by using
microspectrophotometry (MSP) to compare fishes reared in two different light
regimens with wild-caught fishes at equivalent ages.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Experimental procedures were approved by the University of Western Australia
Ethics Committee and followed the guidelines of the Australian Code of
Practice for the Care and Use of Animals for Scientific Purposes.
For MSP, 100 larval fish were obtained at day 4 and transferred to rearing
tanks. The cohort was divided between two experimental lighting conditions:
(1) broad spectrum fluorescent lighting (Philips Coolwhite, 36 W) and (2)
short wavelength-reduced lighting as is typical in estuarine conditions in
which chlorophyll and tannins absorb short wavelength light
(Lythgoe, 1979). To achieve
the tannin-like conditions, yellow acetate filters (no. 764, Lee Filters,
Andover, UK) surrounded the tanks and this treatment is referred to as the
yellow filter group hereafter. The broad spectrum tanks were enclosed within
neutral density acetate filters (optical density 0.2) to equate the intensity
(quanta) of the treatments at approximately 70 µW cm–2
(400–700 nm). The spectral characteristics of the two rearing conditions
and the transmission characteristics of the yellow filter are shown in
Fig. 1. For the standard
conditions, fishes were examined by MSP during the larval (0–40 days;
N=6), post-settlement (41–100 days; N=8), juvenile
(101–210 days; N=8) and adult stages (>1 year and 10 cm
standard length; N=6). Fishes from the yellow filter conditions were
sampled during the larval (N=4), post-settlement (N=6) and
juvenile (N=6) stages. It was not possible to rear adult fish in the
yellow filter group within the time frame of the investigation. In addition,
post-settlement (N=4), juvenile (N=6) and adult fish
(N=4) were caught from their natural environment, the estuarine
section of the Swan River, Western Australia, and the max
of their photoreceptors determined by MSP within 1 week of capture. All
attempts to capture live larval fish from the wild were unsuccessful. For
molecular investigation, fishes reared in broad spectrum lighting were sampled
at two stages: larval aged 20 days (<5 mm standard length) and adults.
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Microspectrophotometry
Fishes were dark adapted for at least 2 h before being killed by immersion
in a lethal dose of methanesulphonate (MS222, Sigma-Aldrich Pty, Castle Hill,
NSW, Australia; 1:2000 w/v in seawater). For MSP examination, preparations of
unfixed retinal tissue were teased apart in teleost Ringer solution (0.1 mol
l–1 of the following: NaCl, KCl,
Na2HPO4, CaCl2) containing 10% dextran
(Sigma, 250 000 Mr), on a rectangular 50 mmx22 mm
no. 1 coverslip. 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 under infrared
illumination and visualised using an infrared image converter (FJW Industries
Inc., Chicago, IL, USA).
A single-beam wavelength-scanning microspectrophotometer was used to
measure the absorption characteristics of the photoreceptor outer segments.
The microspectrophotometer has been described previously
(Partridge et al., 1992b),
although recent modifications have been made to improve the optics and hence
transmission of short wavelengths to the specimen
(Shand et al., 2002). Spectral
absorbance measurements were made by placing the outer segment of the
photoreceptor in the path of the measuring beam and scanning over the
wavelength range 350–750 nm. A single bidirectional scan (750 nm to 350
nm to 750 nm) was made for 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 subsequently made.
The post-bleach average spectrum thus obtained was subtracted from the
pre-bleach average to produce a difference spectrum for each outer segment and
thereby confirm the presence of visual pigment.
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 normalised to the peak and long wavelength-offset
absorbances, obtained by fitting a variable-point unweighted running average.
Following the method of MacNichol
(MacNichol, 1986), a
regression line was fitted to the normalised absorbance data between 30% and
70% of the normalised maximum absorbance at wavelengths longer than that of
the absorbance peak. The regression equation was used to predict the
max and fit the visual pigment template following the
methods of Govardovskii et al.
(Govardovskii et al., 2000).
Acceptable pre-bleach spectra (Levine and
MacNichol, 1985; Partridge et
al., 1992b) all had a characteristic `bell-shaped' curve with a
clear alpha peak, low noise and a flat long wavelength tail above the
wavelength at which the absorbance had fallen to less than 0.5% normalised
maximum absorbance. To establish the cone classes present at different
developmental stages, the max values of the spectra of
individual photoreceptors were presented as frequency histograms. Changes in
the frequency of records within each cone class at each stage of development
in fishes either reared in the different lighting regimens or wild-caught were
compared using contingency chi-square.
Amplification of gene sequences
Fish were anaesthetised by immersion in MS222 (1:2000 w/v in seawater). Due
to the small size of the larval fish, whole heads were pooled and used for
extraction of mRNA. Three pools of 20 larval heads and four separate adult
retinae were used. Prior to the isolation of RNA, eyes were placed in RNAlater
(Ambion, Austin, TX, USA) to minimise RNA degradation. All tissue samples were
initially stored at 4°C overnight to maximise the diffusion of the RNA
preservation solution throughout the whole tissue and then stored at
–80°C for further analysis.
RNA samples were treated with DNase to remove any contaminating genomic DNA. Following isolation of total RNA using Epicenter Technologies MasterPureTM complete RNA purification kit (Madison, WI, USA), retinal mRNA was prepared using either the Qiagen Oligotex mRNA purification kit (Doncaster, Vic, Australia) or the Quickprep micro mRNA purification kit (Amersham Biosciences, Little Chalfont, UK). Genomic DNA was isolated from liver tissue using a standard phenol/chloroform method.
cDNA was prepared using Expand reverse transcriptase and oligo dT primers,
and rod and cone opsin gene fragments were PCR amplified using the primers
listed in Table 1. A
full-length copy of the Rh2A sequence was obtained using
5'- and 3'-RACE with a primer pair specific for
Rh2A. For Rh2Aβ, the sequence was obtained as
two overlapping fragments, using primer pairs for
Rh2Aβ1 and 2, which were subcloned into pMT4
to give a full-length sequence.
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qPCR analysis of opsin expression
First strand cDNA, prepared from total RNA (1 µg) extracted from whole
heads of larvae and the retinae of adult fish as described above, was used for
qPCR. Individual visual pigment transcripts were quantified using
gene-specific forward and reverse primers designed specifically to amplify
SWS1, SWS2, Rh1, Rh2 and LWS opsin transcripts
(Table 2). In addition, forward
and reverse primers were designed to amplify transcripts of the house-keeping
gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to use as an
internal control to correct for sample-to-sample variation.
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In all cases, regions of divergence between the different opsins were
identified and 30-mer oligonucleotides were designed manually with
non-conserved nucleotides present at the 3'-end of all forward and
reverse primers to facilitate amplification of specific transcripts. The
specificity of each primer was assessed and the production and validation of
qPCR protocols was performed as previously described
(Davies et al., 2007). All
standard curves (semi-log plot of PCR cycle value for above background
threshold value against log of input DNA concentration) gave line gradients
very close to –3.32. The efficiency of the reaction is calculated from
the line gradient value; in this study, all amplification efficiencies were
close to 100% and the standard curve plots showed a very high coefficient of
determination (R2>0.99, P<0.01). In all
cases, the triplicate reactions gave very small error bars showing a high
reproducibility of amplification at each point. All primer combinations
traversed at least one exon–exon boundary and yielded amplicon lengths
of 336±8 bp. Assays were performed in triplicate with three independent
cDNA templates (40 ng), using 1x Platinum SYBR Green qPCR
SuperMix-UDG-Master mix kit (Invitrogen, Paisley, UK), and forward and reverse
primers at 200 nmol l–1. A Corbett Life Science Rotor-Gene
qPCR detector was used to detect SYBR Green reporter dye fluorescence and data
were analysed offline. A typical protocol took 2 h to complete and included an
initial denaturation step at 95°C for 10 min, followed by 40 cycles of
95°C for 20 s (denaturation), 56°C for 20 s (annealing) and 72°C
for 30 s (extension), with a plate read at the end of the extension phase for
each cycle. Melting and standard curves were generated for each amplicon using
a 10-fold serial dilution (1 pg to 10 ng) of input template. Fluorescence was
quantified as previously described (Davies
et al., 2007).
Phylogenetic analysis
Neighbour joining (Saitou and Nei,
1987) was used to construct phylogenetic trees from opsin amino
acid sequences after alignment with ClustalW
(Higgins et al., 1996). The
degree of support for internal branching was assessed by bootstrapping with
1000 replicates using the MEGA2 computer package
(Kumar et al., 2001).
In vitro expression and analysis of recombinant pigments
The Rh2A and Rh2Aβ sequences were cloned into
the pMT4 expression vector using forward and reverse primer pairs for
Rh2A and Rh2Aβ1
(Table 1). Human embryonic
kidney (HEK-293T) cells were transiently transfected with 7 µg per plate of
opsin-pMT4 recombinant expression vector by GeneJuice (Merck, Hoddesdon, UK),
using thirty 90 mm plates per experiment. After 48 h, transfected cells were
harvested and washed four times with phosphate-buffered saline (PBS, pH 7.0)
and stored at –80°C until required. The recombinant visual pigments
were generated by suspending the cells in PBS, followed by incubation with 40
µmol l–1 11-cis-retinal in the dark. The
membrane-bound pigments were solubilised and purified by immunoaffinity
chromatography using the anti-Rho1D4 antibody coupled to a CNBr-activated
Sepharose column as previously described
(Molday and MacKenzie, 1983),
eluted, and stored on ice.
Chilled reconstituted visual pigment samples were subjected to
spectrophotometric analysis and absorbance spectra were recorded in the dark
using a Spectronic Unicam UV500 dual-beam spectrophotometer. Subsequently, the
samples were bleached by exposure to fluorescent light for 10 min.
Spectrophotometric recordings were repeated three times per sample and the
bleached spectra were subtracted from the dark absorbance spectra to produce
difference spectra for the calculation of max for each
expressed black bream visual pigment. The resultant visual spectra were
overlaid with visual pigment templates
(Govardovskii et al., 2000)
and best-fit spectral curves were obtained using the Solver add-in function in
Microsoft Excel to vary the max. As absorbance spectra are
distorted by the underlying absorbance and scatter of the protein, difference
spectra were used as the more accurate estimation of max
values.
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RESULTS |
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max values ranging from 418 to 430 nm (425 nm class) and
from 520 to 545 nm (535 nm class). A single cone with max
at 457 nm was also identified. By the post-settlement stage, the number of
cones with max values between 430 and 475 nm had increased,
indicating a transition to longer wavelength sensitivity. The majority of the
long wavelength-sensitive cones were still centred on 535 nm, but a few cones
with longer max values were present. By the juvenile stage,
the 425 nm class was absent and the short wavelength class had
max values centred on 475 nm. In the long wavelength cones,
there was a range of max values between 520 and 576 nm but
with classes centred on 535, 555 and 570 nm. In the adult, the majority of
longer wavelength-sensitive cones had max values between
540 and 575 nm. Overall, therefore, there was a clear temporal shift in
sensitivity of the cone photoreceptors to longer wavelengths.
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The two Rh2 genes were found to be closely related, with 96%
identity of the predicted amino acid sequence. Phylogenetic analysis indicated
that they belonged to the Rh2A group
(Parry et al., 2005) and their
proximity in the tree indicates that they had arisen from a recent gene
duplication within the black bream lineage. The finding is similar therefore
to the Rh2A and Rh2Aβ genes in cichlids, which
also derive from a duplication within their own lineage
(Parry et al., 2005). The
black bream genes have accordingly been designated Rh2A and
Rh2Aβ.
The two SWS2 genes were found to be more divergent with only 84%
identity of the predicted amino acid sequence and fell into the separate
SWS2A and SWS2B clades. Similar variants were also found in
cichlids, killifish and medaka (Spady et
al., 2006). The gene duplication is more ancient therefore than
the Rh2A gene duplication and probably occurred near the base of the
Paracanthoptergian/Acantopterygian radiation. The black bream sequences have
been designated SWS2A and SWS2B based on their phylogenetic
placement.
A SWS1 opsin was also identified even though no UV-sensitive cones
were found by MSP in this or our previous study
(Shand et al., 2002). The
discrepancy is, however, not unexpected as sampling by MSP of UV-absorbing
cones presents technical difficulties. From the coding sequence, the opsin
would be expected to generate a UV-sensitive pigment (see below).
The opsin sequences from black bream have been deposited in GenBank under
the following accession numbers: Rh1, DQ354577; LWS,
DQ354578; Rh2A, EU090913; Rh2Aβ, EU090914;
SWS2A, DQ354580; SWS2B, DQ354581; SWS1,
DQ354579.
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. Only very low levels of SWS2A, Rh2Aβ and
LWS transcripts were detected. In adult samples, however, the
predominantly expressed opsins switched to SWS2A, Rh2Aβ and
LWS, consistent therefore with the overall shift shown in
photoreceptor sensitivity to longer wavelengths evident in
Fig. 2. Note also the 25-fold
rise in the relative level of Rh1 expression from the larval stage to
adult. In the larval stage, the combined expression of cone opsins represented
40% of the total (rod + cone) expressed opsin but the value fell to only 10%
in the adult, as the result of a substantial increase in the expression of
Rh1 opsin.
Since whole heads were used to obtain RNA at the larval stage, opsins
expressed in the pineal gland will be included in the sample. Relevant to this
is the report that SWS1 opsin is expressed in the embryonic pineal gland of
the halibut (Forsell et al.,
2001; Forsell et al.,
2002), although retinal expression was also seen. It is possible
therefore that the absence of UV tracings amongst the MSP records reflects a
true absence of UV cones and that the SWS1 opsin is expressed only in the
pineal gland. This is, however, unlikely, especially since the relative level
of SWS1 transcript is similar to that for SWS2B, with the latter correlating
with one of the two cone classes found at relatively high frequency by MSP in
the larval retina. However, the possibility that a small amount of SWS1
expression may be attributable to the pineal gland cannot be excluded.
Correlation of cone opsins and visual pigment classes
Based on the spectral ranges already known for different opsin classes, it
was possible to relate cone class with visual pigment gene. The expression of
the different cone opsin genes was compared with the frequency of the
different cone classes in larval and adult fish. As shown in
Fig. 5, the predominant
transcripts in the larval retina were from the SWS2B and
Rh2A genes and these correlate with the 425 and 535 nm cone
classes, whereas the transcripts in the adult retina arose largely from the
SWS2A, Rh2Aβ and LWS genes, which correlate with the
475, 555 and 570 nm cone classes. On this basis, the pigment expressed in each
cone class can be identified as follows: 425 nm class, SWS2B; 475 nm class,
SWS2A; 535 nm class, Rh2A; 555 nm class, Rh2Aβ; 570 nm class,
LWS.
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In vitro expression and regeneration of the Rh2 pigments
Since the max value of the Rh2Aβ pigment at 555 nm
is very long wavelength shifted compared with orthologous pigments from other
species, the spectral characteristics of both Rh2A pigments were examined by
in vitro expression of a recombinant opsin, followed by pigment
regeneration with 11-cis-retinal and spectral analysis. As shown in
Fig. 6, the fitted Govardovskii
opsin template gave a max of 527 nm for Rh2A,
consistent therefore with the average max of the 535 nm
cone class. Surprisingly, however, the Rh2Aβ pigment gave a similar
max at 534 nm, which is significantly shorter than the
average value of 555 nm obtained by MSP. A possible explanation for the
discrepancy is that the adult pigment has a mixed A1/A2
chromophore.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), that changes in opsin expression underlie the changes in
spectral sensitivity of the cone photoreceptors in black bream.
The MSP data obtained at four developmental stages spanning the period from
the larval stage to adult identified five cone classes with
max values around 425, 475, 535, 555 and 570 nm. In the
larval retina, the two most abundant cone classes were the 535 nm and the 425
nm classes. The 535 nm class progressively falls in frequency through the
post-settlement and juvenile stages and the 425 nm class disappears by the
adult stage. Coincidental with these reductions, the 475, 555 and 570 nm
classes increase in frequency through these stages. The max
changes of the visual pigments are closely mirrored by changes in the
expression of the six cone opsin genes and these data have enabled the
different visual pigments to be assigned to a particular opsin gene.
Phylogenetic analysis identified the cone opsins as members of the
SWS1, SWS2, Rh2 and LWS gene classes. Two copies of
SWS2 (A and B) and two copies of Rh2, both
belonging to the Rh2A clade
(Parry et al., 2005), were
also found. The 425 nm cone class expresses the SWS2B gene and the
535 nm cone class expresses the Rh2A class. These two genes
represent the `larval' variants as expression in the adult switches to the
SWS2A gene, which encodes the 475 nm pigment, and the
Rh2Aβ gene, which encodes the 555 nm pigment. In addition, the
adult retina expresses the LWS gene, which encodes the 570 nm pigment
at a substantially higher level than in the larval retina. The SWS1
gene that was amplified from retinal cDNA is not expressed in the adult retina
but represents approximately 14% of total cone opsin gene expression in the
larval stage. The SWS1 opsin gene most probably specifies a
UV-sensitive pigment since it possesses Phe86. This residue is generally
sufficient to specify a UV-sensitive pigment
(Hunt et al., 2007). MSP,
however, failed to find a corresponding class of cones, most probably due to
technical reasons.
Amino acid substitution at nine different sites has been linked to spectral
shifts in the SWS2 pigments of Teleostei
(Cowing et al., 2002) and
Amphibia (Takahashi and Ebrey,
2003). In the black bream, however, substitution is found at only
one of these sites, with non-polar Cys at site 94 in `larval' SWS2B replaced
by non-polar Ala in `adult' SWS2A. Substitution at this site is responsible
for the spectral difference between SWS2 pigments of the newt and bullfrog
(Takahashi and Ebrey, 2003),
but in contrast to the black bream pigments, polar Ser in the newt is replaced
by non-polar Ala in the bullfrog to give a 14 nm short wavelength shift. It is
unlikely therefore that a Cys to Ala change would produce a similar spectral
shift. The molecular basis for the 50 nm difference between the SWS2A and
SWS2B pigments remains to be established.
The max values obtained for in vitro expressed
Rh2A and Rh2Aβ pigments are very similar at 527 and 534 nm,
respectively. The Rh2A gene, with its strong bias towards
larval expression, links the 527 nm pigment with the 535 nm cone class, and
expression of the Rh2Aβ gene is coincident with the appearance
of the 555 nm cone class in the adult. The 534 nm value obtained for the
in vitro expressed pigment from the Rh2Aβ gene is
therefore short wavelength shifted by 21 nm compared with the MSP value. Such
a discrepancy could arise either from an undefined post-translational change
that occurs only in the intact photoreceptor or from the presence of some
A2 chromophore that long wavelength shifts the
max in situ. The spectral analysis carried out
previously (Shand et al.,
2002) does not rule out the presence of some A2-based
pigment.
The amino acid sequences of the Rh2A and Rh2Aβ opsins in the
black bream differ at only 13 sites. Of these, only five are in transmembrane
regions, with one of these latter sites involving a change from a non-polar
(Val in Rh2A) to a polar (Thr in Rh2Aβ) residue. However, none of
the 13 residue differences are at sites previously shown to be involved in
spectral tuning, so it is perhaps not surprising that the in vitro
max values of the A1 pigments are very similar
at 527 and 534 nm, respectively.
Spectral shifts between different LWS pigments are generally due to the
residues at five sites, 164, 181, 261, 269 and 292
(Yokoyama and Radlwimmer,
2001). At each site, the bream LWS opsin sequence had the amino
acid (Ser164, His181, Tyr261, Thr269 and Ala292) associated with long
wavelength-shifted pigments. The combination of residues at these key tuning
sites would be expected therefore to generate a pigment with a
max around 565 nm, which is consistent with the average
max value of 570 nm for the black bream LWS pigment as
determined by MSP.
The developmental changes in opsin gene expression in black bream show some
similarities to those described in the cichlid Oreochromis niloticus
[(Nile tilapia (Spady et al.,
2006)]. In this species, expression of the SWS1 gene is
also confined to larval and juvenile stages and the LWS gene also
shows a very substantial rise from the larval stage to adult. However, three
Rh2 genes are present in the Nile tilapia, a pair of recently
diverged genes, Rh2A and Rh2Aβ as found in black
bream, and an Rh2B gene, which arose from a more ancient duplication.
The expression of the Rh2 genes in Nile tilapia differs from that in
the black bream with Rh2B expressed in the larvae, whereas
Rh2A and β are expressed throughout development. The
SWS2A and B genes in the Nile tilapia also show
developmental changes, with both forms showing an increase in expression from
larva to juvenile but only SWS2A continuing at a high level into the
adult. In black bream, the SWS2B variant is clearly the `larval' form
and SWS2A is the `adult' form.
At the larval stage, Rh1 opsin gene expression represents about
60% of total opsin gene expression in black bream. The percentage rises,
however, to reach about 90% in the adult, which reflects the substantial
increase in the frequency of rod versus cone photoreceptors during
development (Shand et al.,
1999). MSP data show that the Rh1 pigment has a
max at 508 nm (Shand et
al., 2002). The major tuning sites for teleost Rh1 pigments
identified previously (Hunt et al.,
2001) are at residues 83, 122, 261 and 292 with Asp, Glu, Phe and
Ala, respectively, at these sites in the black bream pigment. A similar
combination of residues was also found in the goldfish with a
max of 492 nm (Johnson
et al., 1993) and in a deep-sea fish, Phycis blennoides,
with a max of 494 nm
(Hunt et al., 2001). It
remains uncertain therefore which other sites are responsible for the
16–18 nm long wavelength shift present in the black bream pigment.
Effects of environmental light
The effect of rearing black bream under short wavelength-reduced conditions
is to increase the frequency of longer wavelength-sensitive cone classes; such
changes would be expected to generate a substantial increase in the
sensitivity of the fish to long wavelength light. In contrast, studies on the
blue acara, Aequiodens pulcher, reared under monochromatic blue light
revealed a reduction in the frequency of the single cone class maximally
sensitive to blue light (Kroger et al.,
1999; Kroger et al.,
2003; Wagner and Kroger,
2000; Wagner and Kroger,
2005). In adult black bream, the frequency ratios between 550 and
570 nm cone classes rose from 1:0.8 in the standard condition fish to 1:10.5
in the wild-caught fish. This result suggests that the double cones in adult
fish switch from an approximately equal frequency of outer segments with the
550 and 570 nm pigments in the standard condition fish to a majority with only
the 570 nm pigment in wild-caught fish. The effect of exposure to light
reduced in short wavelengths would appear therefore not to be on the
production of double cones but on the type of pigment produced in their outer
segments.
There are two possible mechanisms by which the opsin changes could be
taking place. In the Pacific salmon and rainbow trout
(Cheng and Novales Flamarique,
2004; Cheng and Novales
Flamarique, 2007), individual single cones have been shown to
switch from expressing SWS1 to SWS2 during development, whereas in the
zebrafish new opsins are expressed in newly differentiated photoreceptors as
the retina progressively grows (Takechi
and Kawamura, 2005). Our results imply both mechanisms may be in
operation. In black bream, the visual pigment changes are not abrupt and the
MSP results give intermediate max values during the shift
to longer wavelengths, indicating that both the original and new visual
pigment are present within the individual outer segments during the transition
phase. Such intermediate max values would be expected
because new opsins expressed in cone outer segments become mixed with those
already present within an outer segment
(Bok and Young, 1972) and are
consistent with the findings in salmonids. It should also be noted, however,
that during the larval and juvenile stages the black bream retina undergoes
rapid growth by addition of cells at the retinal margins
(Shand et al., 1999) and the
observations of the differing proportions of double cone
max values between the post-settlement and juvenile fish in
the different light environments (Fig.
7 this study) indicate that the environmental light is also
fine-tuning the expression of opsins in the newly developed cells. In
situ hybridisation studies are needed to confirm how the two mechanisms
may be interacting to bring about changes in opsin expression across the
retina of black bream.
Intraspecific variation in spectral sensitivity has been observed using
compound action potentials from ganglion cells in the three-spine stickleback
from lakes with differing spectral light qualities
(McDonald and Hawryshyn,
1995). In addition, differences in the levels of cone opsin
expression have been related to variation in the frequency of cone classes in
bluefin killifish from different habitats
(Fuller et al., 2004).
Seasonal changes in spectral sensitivity, attributed to changes in
chromophore, have also been reported in a number of freshwater species (for
review, see Bowmaker, 1995).
The black bream investigated here were reared under a regimen that shifts the
ambient light to longer wavelengths, which, while designed to be similar to
the natural environment, did not reproduce the seasonal changes in light
quality in which black bream are found. The ability to respond to changes in
environmental light by altering opsin expression in the long
wavelength-sensitive double cones, both during growth and in established
populations of cells, is likely to be an advantage for fishes in a variable
estuarine environment. Our study also has implications for fisheries'
restocking programmes in which hatchery-reared fish are released as juveniles
to replenish wild stocks. Survival of juveniles, released into a light
environment that differs from that of the rearing conditions, may be affected
by inappropriate spectral sensitivity for visual tasks such as feeding and
predator avoidance.
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Acknowledgments |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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