Adaptations to an extreme environment: retinal organisation and spectral properties of photoreceptors in Antarctic notothenioid fish
1 Institute of Ophthalmology, University College London, 11-43 Bath Street,
London, EC1V 9EL, UK
2 Department of Animal Biology, University of Illinois at Urbana-Champaign,
515 Morrill Hall, 505 S. Goodwin Avenue, Urbana, Illinois 61801,
USA
* Author for correspondence (e-mail: d.hunt{at}ucl.ac.uk)
Accepted 18 April 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: icefish, visual pigment, retina, photoreceptor
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both rod and cone photoreceptors are present in the retinae of notothenioid
fishes (Meyer-Rochow and Klyne,
1982). Single and double cones have been reported in a number of
species (Eastman and Lannoo,
2003
; Meyer-Rochow and Klyne,
1982
; Miyazaki et al.,
2002
), and Miyazaki et al.
(2001
) identified a square
mosaic that is formed with double cones at the sides and single cones in the
centre and at each of the four corners. Cones in corner positions have been
shown to contain a UV-sensitive visual pigment in the retinae of a number of
teleost species (Avery et al.,
1983
; Bowmaker et al.,
1991b
; Hárosi and
Hashimoto, 1983
; Hisatomi et al.,
1996
,
1997
;
Whitmore and Bowmaker, 1989
),
so it is possible that UV sensitivity is also present in notothenioid fishes.
Meyer-Rochow and Klyne (1982
)
reported an increase in the overall proportion of rods and a decrease in cones
in three species of notothenioid fishes as depth of habitat increased; the
deeper dwelling species, Dissostichus mawsoni, has a higher
concentration of rods with longer outer segments as compared to the two
shallower living species, Trematomus borchgrevinki and T.
bernacchii.
In the present study, we have examined the organisation of the retina in a
number of notothenioid species that live in different depth habitats. We have
focused on the classes of photoreceptors present, and the spectral
characteristics of the associated visual pigments have been determined by
microspectrophotometry (MSP). The corresponding visual pigment genes have been
sequenced and the peak absorbance (max) of encoded cone
pigments confirmed by in vitro expression of the opsin protein and
regeneration with 11-cis-retinal. Finally, the organisation of the
retina has been examined by conventional histology and in situ
hybridisation with opsin cRNA probes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Liver and spleen were flash frozen in liquid nitrogen for subsequent DNA extraction. Whole eyes were dissected from the fish, the lens was removed, and the eye cup was flash frozen in liquid nitrogen for subsequent RNA isolation. For microspectrophotometry, fish were dark adapted for several hours in aquarium tanks. Eye capsules were then dissected in dim red light at 4°C, and lightly fixed for 1530 s in 2%glutaraldehyde in notothenioid fish PBS (0.1 mol l1 phosphate buffer, 0.1 mol l1 NaCl, pH 7.4), and stored in light-tight bottles with fresh notothenioid PBS containing penicillin (100 U ml1), streptomycin (0.1 mg ml1), and amphotericin B (0.25 µg ml1) at 4°C. For retinal histology, either the lens and vitreous humour was removed from the eye prior to fixation or fixative was injected into the vitreous humour of the intact eye with a 30-gauge needle. In both cases, the eye capsules were then fully immersed in 4% (w/v) paraformaldehyde in notothenioid PBS.
Microspectrophotometry
Absorption spectra of individual photoreceptors were determined using a
computer-controlled modified Liebman dual beam microspectrophotometer. By
means of an infrared converter, the measuring beam (normally about 2
µm2 cross section) was aligned to pass transversely through a
given photoreceptor outer segment, while the reference beam passed through a
clear space adjacent to the photoreceptor. Spectra were scanned from 750 nm to
350 nm in 2 nm steps and back from 351 nm to 749 nm. To estimate the peak
absorbance (max) of each outer segment, a standardised
computer program was employed. A detailed description of the experimental
procedures and methods of analysis have been published previously
(Bowmaker et al., 1991a
).
Genomic DNA extraction
High molecular mass genomic DNA was prepared from frozen liver or spleen.
Tissue was ground with a pre-chilled pestle and mortar, and the pulverized
tissue was digested (1:10 w/v) in lysis buffer (10 mmol l1
Tris-HCl, pH 8.0, 100 mmol l1 NaCl, 250 mmol
l1 EDTA, 0.5% SDS and 100 µg ml1
proteinase K) at 60°C. The digest was then extracted twice with an equal
volume of Tris-HCl-buffer-saturated phenol (pH 8.0), and then once each with
phenolchloroform and chloroform. The genomic DNA in the supernatant was
dialyzed against 0.5x TE (5 mmol l1 Tris-HCl, 0.5 mmol
l1 EDTA, pH 8.0), treated with RNaseA (50 µg
ml1), re-dialyzed and stored at 4°C until use.
RNA isolation
Frozen eye cups were ground to a fine powder in a pre-chilled pestle and
mortar, and total RNA was extracted with the Ultraspec RNA isolation reagent
(Biotecx, Houston, TX, USA), which is based on the single-step acidic phenol
extraction of Chomczynski and Sacchi
(1987).
Southern blot and hybridisation
Genomic DNA was digested with the restriction enzyme EcoRI,
separated by gel electrophoresis, transferred to a nitrocellulose membrane by
capillary action in 20x SSC and fixed by baking at 80°C for 3 h. The
blot was incubated with radiolabelled probes for the four vertebrate cone
opsins, SWS1, SWS2 (short-wave sensitive 1 and 2), Rh2 (middle-wave sensitive)
and LWS (long-wave sensitive). The SWS1 probe was cloned from D.
mawsoni retinal cDNA and encompassed the entire coding region. The SWS2,
Rh2 and LWS clones were obtained from retinal cDNA isolated from black bream,
Acanthopagrus butcheri (J.A.C. and D.M.H., unpublished data). In all
cases, the probes were cloned into the pGEM-T-Easy plasmid (Promega,
Southampton, UK). The opsin inserts were excised by restriction enzyme
digestion and radioactive probes were synthesised using the Ready-To-GoTM
DNA Labelling Beads (-dCTP) (Amersham Biosciences, Little Chalfont,
Bucks, UK), following the manufacturer's instructions. The membranes were
prehybridised for 3 h at5065°C (depending on probe specificity) in
a buffer containing 0.05 mol l1 PO4, 4x
SSC, 5x Denhardt's reagent, 5 mg ml1 denatured,
fragmented salmon sperm DNA, 0.3% (w/v) SDS and 0.15% sodium pyrophosphate
(NaPPi). The membrane was then hybridised against the SWS1, SWS2, Rh2 or LWS
opsin probes overnight at 5060°C. The blot was washed twice in
6x SSC, 0.5% (w/v) SDS and twice in 3x SSC, 0.5% SDS at
5065°C before exposure to X-ray film.
Northern blot and hybridisation
Approximately 6 µg of total retinal RNA of each species were
electrophoresed on a 1.2% agarose/2.2 mol l1 formaldehyde
gel, vacuum blotted onto Hybond N (Amersham) nylon membrane, and
UV-crosslinked (Stratalinker, Stratagene, La Jolla, CA, USA). The membrane was
prehybridised at 55°C in QuickHyb solution (Stratagene), and hybridised at
55°C to opsin probes generated from random-primed, 32P-labelled
sea bream opsin cDNA (SWS2, Rh2 and LWS) and P. borchgrevinki opsin
(SWS1) cDNA. Hybridised blots were washed exhaustively in 2x SSC/0.1%
SDS, followed by 0.1x SSC/0.5% SDS up to 50°C, and autoradiographed
on X-ray film.
PCR cloning and sequencing of SWS1, SWS2 and Rh2 opsins
Standard polymerase chain reaction (PCR) conditions were used with either
30 ng cDNA or 100 ng of genomic DNA in a 50 µl reaction volume. A cDNA
fragment of about 800 bp of SWS1 opsin was first obtained from P.
borchgrevinki retinal RNA by reverse transcription-PCR amplification. The
first strand cDNA was synthesised using Superscript II reverse transcriptase
(Invitrogen), and used for the PCR amplification of a cDNA fragment with
degenerate primers VSWS1F and VSWS1R (Table
1) designed from an alignment of vertebrate SWS1 opsins.
Sequencing of the RT-PCR product and BLASTP of translated sequence verified
that the fragment was from an SWS1 mRNA. SWS1-specific primers were designed
based on this partial sequence to generate overlapping 5' and 3'
fragments inclusive of UTRs by 5'RACE (rapid amplification of cDNA ends)
and 3'RACE. For 5'RACE, the first strand cDNA was dC-tailed with
terminal deoxynucleotide transferase and amplified using the SWS1-specific
reverse primer paired with the 5' RACE adaptor primer (5'RACE kit,
Invitrogen). The 3'RACE product was amplified with the SWS1-specific
forward primer paired with lock-docking oligo(dT) (NV(T)20).
Primers to the 5'UTR and 3'UTR (P.borchSWS1F and P.borchSWS1R)
were used to amplify full-length SWS1 cDNA from P. borchgrevinki, D.
mawsoni and T. loennbergii first strand cDNA.
|
Degenerate PCR was also used to amplify the Rh2 opsins from four species,
D. mawsoni, P. borchgrevinki, N. angustata and G. acuticeps.
Degenerate primers Green1F and Green596R were used to amplify an approximately
600 bp product from D. mawsoni cDNA, which showed homology to teleost
Rh2 opsins. The 3' cDNA sequence was obtained by 3' RACE using the
FirstChoiceTM RLM-RACE kit (Ambion, Austin, TX, USA) and the D.
mawsoni-specific primers Green3'O and Green3'I. To extend the
5' sequence and to obtain upstream sequence information, a genomic
walking method with genomic DNA was used as described by Dominguez and
Lopez-Larrea (1994) with the
initial PCR performed with the universal primer UNI33 and the D.
mawsoni-specific GreenWkO primer. The inner nested PCR was performed with
the universal primer UNI17 and the gene-specific GreenWkI primer. The complete
Rh2 sequences for P. borchgrevinki, N. angustata and G.
acuticeps were amplified with the primers D.mawsGF and D.mawsGR.
The SWS2 opsin DNA was PCR amplified from T. loennbergii genomic DNA with the degenerate primers blue400F and blue818R using a low annealing temperature of 4854°C. This fragment encompassed exons 3 to 5. To extend the 3' sequence of the gene, a PCR with the primers T.loen3'I and MZbluestop was performed on T. loennbergii genomic DNA.
All PCR-amplified products were analysed by gel electrophoresis and extracted using WizardTM columns (Promega). The eluted DNA was cloned into the pGEM-T-EasyTM vector (Promega) and sequenced on an ABI 3100 using the BigDye® Terminator v3.1 Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA, USA) V.2 or V.3 and vector-specific primers.
Expression of recombinant opsins
The entire coding sequences for D. mawsoni SWS1 and Rh2 opsins
were amplified from retinal cDNA with Pfu DNA polymerase, using
primer pairs D.mawsUVF/D.mawsUVR and D.mawsGF/D.mawsGR containing
EcoRI and SalI restriction sites (shown underlined in
Table 1). The resulting
products were then cloned via these restriction sites into the
expression vector pMT4, which contains the sequence for the 1D4 epitope from
bovine rod opsin downstream of and in-frame with the SalI site
(Franke et al., 1988).
The pMT vector containing either the SWS1 or Rh2 coding sequence was
transfected into HEK-293T cells with Genejuice (Invitrogen) according to the
manufacturer's instructions. Thirty 90 mm plates were used per transfection
and the cells were harvested 48 h post-transfection, and washed with 1x
PBS. The visual pigments were regenerated in 1x PBS with 40 µmol
l1 11-cis retinal in the dark. The pigments were
then incubated with 1% (w/v) dodecyl-maltoside and 20 mg ml1
phenyl methyl sulphonyl fluoride (PMSF) and isolated by passage over a
CNBr-activated Sepharose binding column coupled to an anti-1D4 monoclonal
antibody (Molday and MacKenzie,
1983).
Absorption spectra were recorded in the dark using a dual-path
spectrophotometer (Spectronic Unicam, Cambridge, UK). Pigments were either
bleached by exposure to light for 15 min or acid denatured by incubation with
10.8 µl 1 N HCl for 10 min. The max value for each
pigment was determined by subtracting the bleached or acid denatured spectrum
from the dark spectrum to produce a difference spectrum. This was then fitted
to a standard Govardovskii rhodopsin A1 template
(Govardovskii et al., 2000
)
and the
max determined.
In situ hybridisation
Enucleated eyes were fixed in 4% (w/v) paraformaldehyde in 1x PBS
overnight and then washed briefly in fresh 1x PBS, and stored in
1x PBS until use. Whole mounts were prepared by removing the retinal
pigment epithelium (RPE) and placing 1 cm wide strips, photoreceptor layer
side up, on glass slides. Coverslips were placed on top and weighted down so
as to keep the sample flat. The slides were left in 25% (w/v) sucrose in
1x PBS overnight, and then stored at 80°C (with coverslips
removed). The retinal tissue to be sectioned was left with RPE attached and
cut into 0.5 cm squares, placed in 25% (w/v) sucrose overnight and then
embedded in OCT medium. Sections 2 µm thick were cut on a cryostat.
Digoxigenin (DIG)-labelled antisense and sense RNA probes were synthesised from the D. mawsoni SWS1 and Rh2 opsin clones and from the sea bream SWS2 opsin clone in pGEM-T-Easy using a DIG RNA labelling kit (Roche Diagnostics Ltd., Lewes, UK). The level of probe cross-hybridisation, as assessed by dot blot analysis, was found to be very low (data not shown). For hybridisation, retinal whole mounts and sections were rinsed in 1x PBS and left to dry for 2 h. Whole mounts were washed in PBS with 0.1% (w/v) Tween (PBST), treated with proteinase K (10 mg ml1) for 15 min, fixed with 4% (w/v) paraformaldehyde in 1x PBS for 20 min and then washed in PBST before being incubated in hybridisation buffer for 1 h at 60°C. Retinal sections were treated as described above but without proteinase K treatment. DIG-labelled RNA probe was then added at an approximate concentration of 0.5 µg ml1 and left to hybridise overnight at 60°C. After multiple washes in SSC and PBST, hybridisation was detected with anti-DIG alkaline phosphatase Fab fragment and labelled for visualisation with a solution of Nitrobluetetrazoleum (NBT) (18.75 mg ml1) and BCIP (9.4 mg ml1). Slides were viewed under a light microscope and images taken with a Nikon digital camera.
Histology
Enucleated eyes were transferred into 2% (w/v) paraformaldehyde, 2% (w/v)
glutaraldehyde in 1x PBS overnight. The large size of the D.
mawsoni eyes (a diameter of approximately 5 cm) made manipulation of the
whole retina very difficult, so fragments were dissected from the eyecup, with
the RPE intact, and dehydrated before storage in historesin for 24120 h
at 4°C. The fragments were then embedded in Technovit embedding hardener
(Heraeus, Hanau, Germany) and flat mounted on to slides. The eyes of T.
hansoni are considerably smaller (1.5 cm diameter) so the whole retina
with RPE attached was dissected and flat mounted. 0.52.5 µm thick
sections were cut with a glass blade and dried on to slides. The cells were
stained with 1% (w/v) Toluidine Blue before being coverslipped. Images were
captured using a Nikon digital camera attached to a light microscope.
Phylogenetic analysis
Neighbor-joining (Saitou and Nei,
1987) was used to construct phylogenetic trees from opsin
nucleotide coding 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
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Southern and northern hybridisation
In order to determine which cone opsin genes are present in the
notothenioid fish genome, Southern analysis of genomic DNA from D.
mawsoni was carried out using cone opsin gene probes. As shown in
Fig. 2, the SWS1, SWS2 and Rh2
probes identified single bands in EcoRI digests, confirming the
presence of the SWS1, SWS2 and Rh2 genes. In contrast, the LWS probe did not
generate a signal, indicating that this gene is not present in the genome of
this species.
|
|
Amplification and sequencing of opsin genes
SWS1 opsins
The SWS1 coding sequence was amplified from retinal cDNA using a
combination of RT-PCR and 5' and 3' RACE. Primer details are in
Table 1. Full coding sequences
were obtained for five species, D. mawsoni, N. angustata, P.
borchgrevinki, T. loennbergii and P. macropterus
(Fig. 4A). These opsins showed
an average of 90.7% amino acid identity, with no consistent differences
between the two notothenioid families that these species represent.
|
|
|
The deduced amino acid sequence is shown in Fig. 5,aligned with the goldfish SWS2 sequence. This sequence was then used to design a primer pair, T.loenF and T.loenR (Table 1), that successfully amplified a short 250 bp fragment of the SWS2 coding sequence (data not shown) from retinal cDNA of two species, P. borchgrevinki and G. acuticeps, thereby confirming expression of the SWS2 gene in the notothenioid fish retina.
Rh2 opsins
The coding sequence for the Rh2 opsin gene was initially amplified from
D. mawsoni retinal cDNA using degenerate primers Green1F and
Green596R (Table 1). From this
600 bp fragment, 3' RACE and genomic walking primers
(Dominguez and Lopez-Larrea,
1994) were designed to complete the coding region. Primers
designed to the 5' and 3' ends of the coding region were then used
to amplify full coding sequences from P. borchgrevinki, G. acuticeps
and N. angustata cDNA. The translated sequences are shown aligned in
Fig. 6A. Phylogenetic analysis
(Fig. 6B) shows that the
notothenioid sequences form a clade with goldfish Rh2 with a bootstrap value
of 100. The sequences are approximately 93% identical to each other at the
amino acid level and show an 84% identity with cichlid (Metriaclima
zebra, GenBank acc. no. AF247122), and a 74% identity with cyprinid
(goldfish, GenBank acc. no. L11866) Rh2 opsins. Their identity as Rh2 cone
pigments was further confirmed by in vitro expression of the D.
mawsoni opsin in HEK 293T cells followed by regeneration of the pigment
with 11-cis-retinal. The difference spectrum for the pigment
(Fig. 6C) fits a Govardovskii
template at 488 nm, which closely matches the value of 490 nm obtained from
single and double cone photoreceptors by MSP, and differs from the rod pigment
which has a
max of around 500 nm as determined by MSP
(Table 3).
Although the amino acid sequences of the Rh1 and Rh2 opsins are similar,
Rh2 pigments show higher rates of regeneration and meta II decay than Rh1
pigments, and this difference has been shown, for chicken pigments, to be
largely dependent on the residue present at site 122
(Imai et al., 1997).
Interestingly, the notothenioid Rh2 pigments differ at this site, with three
species following chicken Rh2, with Gln, but one, N. angustata,
following chicken Rh1, with Glu (Fig.
6A).
|
En face cryosections probed with the antisense Rh2 showed hybridisation in both peripheral and central retina (Fig. 8) whereas neither the SWS1 nor SWS2 probes showed positive labelling (data not shown).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As down-welling light passes through a body of water, it becomes
progressively attenuated with a maximum penetration in the clearest oceanic
water to around 1000 m. This attenuation is greater, however, at longer and
shorter wavelengths, such that after a few hundred metres, down-welling light
is reduced to a narrow band of radiation between 470 and 480 nm in the
blue-green region of the spectrum (Jerlov,
1976). In Antarctic waters, light penetration is further reduced
by snow and thick sea-ice cover with a peak transmission around 500 nm
(Pankhurst and Montgomery,
1989
; Perovich et al.,
1998
). These reduced light levels under the ice will therefore
limit photopic vision to the upper 150 m and scotopic vision to a maximum
depth of around 300400 m (Morita et
al., 1997
; Pankhurst and
Montgomery, 1989
) in summer months. In the winter, these
limitations will be substantially greater.
The longwave spectral attenuation of light as it passes through ice and
water is the most probable evolutionary basis for the loss of the LWS pigment
and red-sensitive photoreceptors in notothenioid fish. In fact, the loss of
long-wave sensitivity is not unusual in fish, particularly in species from
low-light environments such as the deep ocean
(Douglas et al., 2003;
Levine and MacNichol, 1979
;
Loew and Lythgoe, 1978
;
Yokoyama and Tada, 2000
;
Yokoyama et al., 1999
) and
deep lakes. The species flock of cottoid fish in Lake Baikal is an example of
the latter situation; amongst these species, only the very surface dwelling
have retained red cones (Bowmaker et al.,
1994
). What remains unclear is why the UVS pigment has been
retained in notothenioid fish where the penetration of UV light though snow
and sea ice would also be extremely limited.
Only P. borchgrevinki retinal RNA gave a positive signal by
northern analysis with an SWS1 opsin probe, although expression of an SWS1
opsin in the retinae of other species is indicated by the successful
amplification of the full SWS1 coding sequences from retinal cDNAs.
Presumably, only a small number of SWS1 receptors are present in these latter
species such that the SWS1 mRNA levels are below detection level by northern
analysis. No UV cones were identified by MSP, although this may be a sampling
problem arising from the small number of UV cones that may be present in the
retina. UV sensitivity of the SWS1 pigment, if present, is a consistent
finding in teleost fish and the UV sensitivity of the pigment in D.
mawsoni was confirmed directly by in vitro expression and
regeneration. The key amino acid for this UV sensitivity is Phe at site 86
(Hunt et al., 2004); all five
notothenioid fish studied possessed this residue so it is probable that the
other four species also possess a UV pigment. Many fish possess UV sensitivity
while immature to aid plankton foraging, but it is then lost upon maturity
(Bowmaker and Kunz, 1987
;
Loew et al., 1993
).
Notothenioid fish may also follow this pattern, with a low number of UV cones
retained into the adult, although the positive signal by northern analysis
seen with P. borchgrevinki retinal RNA may be associated with a
higher frequency of UV cones in this cryopelagic species that lives in the
platelet ice layer on the underside of surface sea ice; the higher levels of
UV light just below the ice that this species will encounter may have led to
the retention of more UV cones than in other species.
SWS2 opsin expression in retinal RNA was confirmed by northern analysis in six species, by PCR in two species, P. borchgrevinki and G. acuticeps, and by the amplification, cloning and sequencing of 800 bp of the SWS2 gene from the genomic DNA of a third species, T. loennbergii. Surprisingly, although northern analysis gave a positive, albeit faint, signal with P. borchgrevinki retinal RNA, no blue cones were identified by MSP. This may again be due to sampling limitation although a population of single cones was identified by MSP that appear to have outer segments devoid of pigment. It may be that these cones originally contained the blue-sensitive SWS2 pigment but that fixation had denatured the pigment.
All five species of notothenioid fish examined by MSP gave a
max for the MWS cones of around 490 nm, a common value for
Rh2 pigments in teleost fish, and this value was also obtained for the D.
mawsoni Rh2 pigment by in vitro expression and regeneration with
11-cis retinal. This also agrees exactly with the photopic spectral
sensitivity as determined by electrophysiology in P. borchgrevinki
(Morita et al., 1997
) and in
T. bernacchii (Pankhurst and
Montgomery, 1989
). Although the
max values for
the pigment in the different notothenioid species are essentially identical,
they do differ at site 122, which has been implicated in determining the rate
of pigment regeneration and meta II decay in chicken rod and Rh2 cone pigments
(Imai et al., 1997
). In
chicken rod, charged Glu122 is present with uncharged Gln in chicken Rh2. The
pigment in N. angustata departs from this rule however, with Glu
present rather than Gln, although it is difficult to see why the Rh2 pigment
in N. angustata should be more rod-like than the pigments in the
other three species. Moreover, a comparison of other fish rod and Rh2 cone
opsin sequences shows that the residue present at site 122 is not tightly
conserved, with Glu present in the Rh2 pigments of goldfish and
Metriaclima zebra, and either Glu, Gln or Val present in the rod
pigments of different species of deep-sea fish
(Hunt et al., 2001
). In view
of this, it would seem unlikely that this site plays the same key role in the
regulation of the rate of regeneration in fish as it does in chicken pigments.
However, it would appear to have an impact on spectral tuning, with the
substitution of Gln by Glu causing a 1415 nm shortwave shift in
zebrafish Rh2 pigments (Chinen et al.,
2005
).
MSP identified both partners of all the double cones as green sensitive, and single cones as either blue or green sensitive. In situ hybridisation with an Rh2 probe also demonstrated that double cones express the Rh2 pigment, both in peripheral and central retina. Although SWS single cones were routinely identified by MSP, the SWS2 probe showed positive staining only in transverse sections of peripheral retina, indicating either that blue cones are only present in the peripheral retina or that the expression level in the central retina is below detection by in situ hybridisation. No signal for UV cones was obtained by in situ hybridisation with the SWS1 probe, and no UV cones were identified by MSP, although PCR experiments did confirm that SWS1 mRNA is present in the retina of D. mawsoni.
This absence of identifiable UV cones raises a major question regarding the
organisation of the different spectral cone classes within the retinal cone
mosaic. In a number of species of Trematomus, there is a typical
square cone mosaic with both central and corner single cones
(Meyer-Rochow and Klyne, 1982;
Miyazaki et al., 2002
;
Miyazaki et al., 2001
).
Miyazaki et al. have assumed that because the corner cones in many teleosts
are UV sensitive, this will also be the case in the notothenioids. Our data,
both molecular and MSP, indicate that this cannot be the case, at least not
throughout the retina. During MSP experiments, we often encountered
preparations where two small single cones were clearly aligned on either side
of a double cone in the position of corner cones. These single cones were,
however, green sensitive and spectrally identical to the double cones. We
infer from this that at least in some regions of the retina, the mosaic
squares are composed of double cones and corner single cones that are all
green sensitive, surrounding a blue-sensitive central single cone.
The organisation of the cone receptor mosaics is different in the retinae
of two related nototheniid species, D. mawsoni and T.
hansoni. The deeper dwelling D. mawsoni (mainly around 1600 m,
but sometimes up at 300500 m to feed) has double and single cones
positioned in a row mosaic. All double cones in a single row lie in the same
orientation, with single cones forming rows in between the doubles.
Meyer-Rochow and Klyne (1982)
described this as a hexagonal arrangement of double cones surrounding a pair
of single cones, but this would appear to be a consequence of the slightly
disordered row array (Fig.
10). Cone row arrays are commonly found in fish that have a
reduced demand on photopic vision, e.g. in deeper dwelling species where
vision is largely dependent on the scotopic system, and in species with a
non-predatory lifestyle. The lack of a retinomotor response in the D.
mawsoni retina (Meyer-Rochow and
Klyne, 1982
) is also consistent with a light environment that
lacks significant fluctuations in intensity as found at depth in the ocean.
The second species examined, T. hansoni, is demersal but lives in
shallower water (down to 550 m). The retina of this species is organised into
a typical square mosaic in which the double cones form the sides of a square,
each orientated 90° to its closest neighbours. In contrast to the previous
study by Miyazaki et al.
(2001
), there appears to be
only a single morphologically distinct class of central single cones, with
corner cones absent, which may explain the failure to identify UV cones by
MSP. Two distinct retinal arrangements are therefore present in two related
species within the same family. However, the visual pigments of the deeper
dwelling species do not differ from the more shallow living fish: indeed, the
max values for rod, Rh2, and SWS2 pigments match almost
exactly across all notothenioid species studied.
In general there is a trend in deep-water fish for the maximum
sensitivities of the visual pigments of both rods and cones to be displaced to
shorter wavelengths with increasing depth of habitat, and for the cone
population to be reduced and eventually lost. This is most evident in deep-sea
fish that have pure rod retinae with max around
470480 nm (Bowmaker,
1995
; Douglas and Partridge,
1997
; Partridge et al.,
1989
), tuned to the maximum transmission of oceanic water and/or
to the maximum emission of bioluminescence, and in the cottoid species flock
of Lake Baikal (Bowmaker et al.,
1994
; Hunt et al.,
1996
). Why is similar spectral tuning not apparent in the
different species of notothenioid fish and how is optimal sensitivity achieved
further down the water column? Light penetrating through bare Arctic sea ice
has a peak transmittance at around 500 nm and substantially reduced intensity,
and the effect of a snow covering will be to reduce intensity further but not
to alter the spectral characteristics
(Perovich et al., 1998
).
Transmission on through the clear waters of the Antarctic (algal bloom is
limited to the austral summer) would be expected to lead to a further
attenuation of intensity and shift in the wavelength of maximal penetration
towards 480 nm. Under these restricted photic conditions, the notothenioid
rods with
max at about 500 nm and the dominant double cones
with
max at 490 nm would appear to be tuned to the
available down-welling light (Morita et
al., 1997
). Also, most bioluminescence peaks in the same spectral
region (Nicol, 1969
;
Herring, 1983
). Increased
sensitivity is achieved in the deeper-dwelling species such as D.
mawsoni by an increase in the proportion of rod photoreceptors and an
increase in the length of the outer segments
(Meyer-Rochow and Klyne,
1982
). This is a common mechanism in deep-sea fish for photon
capture in a dim light environment. Furthermore, the more shallow water
species organise the cone receptors in a square mosaic to ensure optimal
packing of cones in the retina, since these fish presumably rely more on
photopic vision. Behavioural studies on both shallow and deep living species
may help to identify how the different retinal arrangements affect chromatic
and overall light sensitivity.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avery, J. A., Bowmaker, J. K., Djamgoz, M. B. A. and Downing, J. E. G. (1983). Ultraviolet receptors in a freshwater fish. J. Physiol. 334,23P .
Bowmaker, J. K. (1995). The visual pigments of fish. Prog. Retinal Eye Res. 15, 1-31.
Bowmaker, J. K. and Kunz, Y. W. (1987). Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the brown trout (Salmo trutta): age-dependent changes. Vision Res. 27,2101 -2108.[CrossRef][Medline]
Bowmaker, J. K., Astell, S., Hunt, D. M. and Mollon, J. D.
(1991a). Photosensitive and photostable pigments in the retinae
of Old World monkeys. J. Exp. Biol.
156, 1-19.
Bowmaker, J. K., Thorpe, A. and Douglas, R. H. (1991b). Ultraviolet-sensitive cones in the goldfish. Vision Res. 31,349 -352.[CrossRef][Medline]
Bowmaker, J. K., Govardovskii, V. I., Shukolyukov, S. A., Zueva, L. V., Hunt, D. M., Sideleva, V. G. and Smirnova, O. G. (1994). Visual pigments and the photic environment: the cottoid fish of Lake Baikal. Vision Res. 34,591 -605.[CrossRef][Medline]
Cheng, C.-H. C. (1998). Evolution of the diverse antifreeze proteins. Curr. Opin. Genet. Dev. 8, 715-720.[CrossRef][Medline]
Chinen, A., Matsumoto, Y. and Kawamura, S.
(2005). Reconstitution of Ancestral Green Visual Pigments of
Zebrafish and Molecular Mechanism of their Spectral Differentiation.
Mol. Biol. Evol. 22,1001
-1010.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156 -159.[CrossRef][Medline]
Cowing, J. A., Poopalasundaram, S., Wilkie, S. E., Robinson, P. R., Bowmaker, J. K. and Hunt, D. M. (2002). The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments. Biochem. J. 367,129 -135.[CrossRef][Medline]
DeVries, A. L., Komatsu, S. K. and Feeney, R. E.
(1970). Chemical and physical properties of freezing
point-depressing glycoproteins from Antarctic fishes. J. Biol.
Chem. 245,2901
-2908.
DeVries, A. L., Vandenheede, J. and Feeney, R. E.
(1971). Primary structure of freezing point-depressing
glycoproteins. J. Biol. Chem.
246,305
-308.
Dominguez, O. and Lopez-Larrea, C. (1994). Gene walking by unpredictably primed PCR. Nucl. Acids Res. 22,3247 -3248.[Medline]
Douglas, R. H. and Partridge, J. C. (1997). On the visual pigments of deep-sea fish. J. Fish Biol. 50, 68-85.[CrossRef]
Douglas, R. H., Hunt, D. M. and Bowmaker, J. K. (2003). Spectral Sensitivity Tuning in the Deep-Sea. In Sensory Processing in Aquatic Environments (ed. S. P. Collin and N. J. Marshall). New York: Springer-Verlag.
Eastman, J. T. (2005). Nature of the diversity of Antarctic fishes. Polar Biol. 28, 93-107.[CrossRef]
Eastman, J. T. and Lannoo, M. J. (2003). Diversification of brain and sense organ morphology in Antarctic dragonfishes (Perciformes: Notothenioidei: Bathydraconidae). J. Morphol. 258,130 -150.[CrossRef][Medline]
Fasick, J. I., Applebury, M. L. and Oprian, D. D. (2002). Spectral tuning in the mammalian short-wavelength sensitive cone pigments. Biochemistry 41,6860 -6865.[CrossRef][Medline]
Fletcher, G. L., Hew, C. L. and Davies, P. L. (2001). Antifreeze proteins of teleost fishes. Ann. Rev. Physiol. 63,359 -390.[CrossRef][Medline]
Franke, R. R., Sakmar, T. P., Oprian, D. D. and Khorana, H.
G. (1988). A single amino acid substitution in rhodopsin
(lysine 248leucine) prevents activation of transducin.
J. Biol. Chem. 263,2119
-2122.
Gon, O. and Heemestra, P. C. (1990). Fishes of Southern Ocean (ed. O. Gon and P. C. Heemestra). Grahamstown, South Africa: JLB Smith Institute of Icthyology.
Govardovskii, V. I., Fyhrquist, N., Reuter, T., Kuzmin, D. G. and Donner, K. (2000). In search of the visual pigment template. Vis. Neurosci. 17,509 -528.[CrossRef][Medline]
Hárosi, F. I. and Hashimoto, Y. (1983). Ultraviolet visual pigment in a vertebrate: a tetrachromatic cone system in the dace. Science 222,1021 -1023.[Medline]
Herring, P. J. (1983). The spectral characteristics of luminous marine organisms. Proc. R. Soc. Lond. B 220,183 -217.
Higgins, D. G., Thompson, J. D. and Gibson, T. J. (1996). Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266,383 -402.[Medline]
Hisatomi, O., Satoh, T., Barthel, L. K., Stenkamp, D. L., Raymond, P. A. and Tokunaga, F. (1996). Molecular cloning and characterization of the putative ultraviolet-sensitive visual pigment of goldfish. Vision Res. 36,933 -939.[CrossRef][Medline]
Hisatomi, O., Satoh, T. and Tokunaga, F. (1997). The primary structure and distribution of killifish visual pigments. Vision Res. 37,3089 -3096.[CrossRef][Medline]
Hunt, D. M., Fitzgibbon, J., Slobodyanyuk, S. J. and Bowmaker, J. K. (1996). Spectral tuning and molecular evolution of rod visual pigments in the species flock of cottoid fish in Lake Baikal. Vision Res. 36,1217 -1224.[CrossRef][Medline]
Hunt, D. M., Dulai, K. S., Partridge, J. C., Cottrill, P. and
Bowmaker, J. K. (2001). The molecular basis for spectral
tuning of rod visual pigments in deep-sea fish. J. Exp.
Biol. 204,3333
-3344.
Hunt, D. M., Cowing, J. A., Wilkie, S. E., Parry, J., Poopalasundaram, S. and Bowmaker, J. K. (2004). Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates. Photochem. Photobiol. Sci. 3, 713-720.[CrossRef][Medline]
Imai, H., Kojima, D., Oura, T., Tachibanaki, S., Terakita, A.
and Shichida, Y. (1997). Single amino acid residue as a
functional determinant of rod and cone visual pigments. Proc. Natl.
Acad. Sci. USA 94,2322
-2326.
Jerlov, N. G. (1976). Marine Optics. Amsterdam: Elsevier Scientific.
Kiss, A. J., Mirarefi, A. Y., Ramakrishnan, S., Zukoski, C. F.,
Devries, A. L. and Cheng, C.-H. C. (2004). Cold-stable eye
lens crystallins of the Antarctic nototheniid toothfish Dissostichus
mawsoni Norman. J. Exp. Biol.
207,4633
-4649.
Kumar, S., Tamura, K., Jakobsen, I. B. and Nei, M.
(2001). MEGA2: molecular evolutionary genetics analysis software.
Bioinformatics 17,1244
-1245.
Levine, J. S. and MacNichol, E. F., Jr (1979). Visual pigments in teleost fishes: effects of habitat, microhabitat, and behavior on visual system evolution. Sens. Processes 3, 95-131.[Medline]
Littlepage, J. C. (1965). Oceanographic investigations in the McMurdo Sound, Antactica. Biology of Antactic seas 2. Antarctic Res. 5,1 -37.
Loew, E. R. and Lythgoe, J. N. (1978). The ecology of cone pigments in teleost fishes. Vision Res. 18,715 -722.[CrossRef][Medline]
Loew, E. R., Macfarland, W. N., Mills, E. and Hunter, D. (1993). A chromatic action spectrum for planktonic predation by juvenile yellow perch, Perca flavescens. Can. J. Zool. 71,384 -386.
Meyer-Rochow, V. B. and Klyne, M. A. (1982). Retinal organization of the eyes of three nototheniid fishes from the Ross Sea (Antarctica). Gegenbaurs Morphol. Jahrb. 128,762 -777.[Medline]
Miyazaki, T., Iwami, T., Yamauchi, M. and Somiya, H. (2001). `Accessory corner cones' as putative UV-sensitive photoreceptors in the retinas of seven adult nototheniid fishes. Polar Biol. 24,628 -632.[CrossRef]
Miyazaki, T., Iwami, T., Somiya, H. and Meyer-Rochow, V. B. (2002). Retinal topography of ganglion cells and putative UV-sensitive cones in two Antarctic fishes: Pagothenia borchgrevinki and Trematomus bernacchii (Nototheniidae). Zoolog. Sci. 19,1223 -1229.[CrossRef][Medline]
Molday, R. S. and MacKenzie, D. (1983). Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. Biochemistry 22,653 -660.[CrossRef][Medline]
Morita, Y., Meyer-Rochow, V. B. and Uchida, K. (1997). Absolute and spectral sensitivities in dark- and light-adapted Pagothenia borchgrevinki, an Antarctic nototheniid fish. Physiol. Behav. 61,159 -163.[CrossRef][Medline]
Nicol, J. A. C. (1969) Bioluminescence. In Fish Physiology (ed. W. S. Hoar and D. J. Randall), pp. 355-400, Vol. III. New York: Academic Press.
Pankhurst, N. W. and Montgomery, J. C. (1989). Visual function in four Antarctic nototheniid fishes. J. Exp. Biol. 142,311 -324.
Parry, J. W., Poopalasundaram, S., Bowmaker, J. K. and Hunt, D. M. (2004). A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment. Biochemistry 43,8014 -8020.[CrossRef][Medline]
Partridge, J. C., Shand, J., Archer, S. N., Lythgoe, J. N. and van Groningen-Luyben, W. A. (1989). Interspecific variation in the visual pigments of deep-sea fishes. J. Comp. Physiol. A 164,513 -529.[Medline]
Perovich, D. K., Longacre, J., Barber, D. G., Maffione, R. A., Cota, G. F., Mobley, C. D., Gow, A. J., Onstott, R. G., Grenfell, T. C., Pegau, W. S. et al. (1998). Field observations of the electromagnetic properties of first-year sea ice. IEEE Trans. Geosci. Remote Sens. 36,1705 -1715.[CrossRef]
Raymond, J. A. and DeVries, A. L. (1977).
Adsorption inhibition as a mechanism of freezing resistance in polar fishes.
Proc. Natl. Acad. Sci. USA
74,2589
-2593.
Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,406 -425.[Abstract]
Whitmore, A. V. and Bowmaker, J. K. (1989). Seasonal variation in cone sensitivity and short-wave absorbing visual pigments in the rudd, Scardinius erythrophthalmus. J. Comp. Physiol. A 166,103 -115.
Yokoyama, S. and Tada, T. (2000). Adaptive evolution of the African and Indonesian coelacanths to deep-sea environments. Gene 261,35 -42.[CrossRef][Medline]
Yokoyama, S., Zhang, H., Radlwimmer, F. B. and Blow, N. S.
(1999). Adaptive evolution of color vision of the Comoran
coelacanth (Latimeria chalumnae). Proc. Natl. Acad. Sci.
USA 96,6279
-6284.