Multiple photopigments from the Mexican blind cavefish, Astyanax fasciatus: a microspectrophotometric study

Vision Research 43 (2003) 31–41 www.elsevier.com/locate/visres

Multiple photopigments from the Mexican blind cavefish, Astyanax fasciatus: a microspectrophotometric study Juliet W.L. Parry b

a,*

, Stuart N. Peirson b, Horst Wilkens c, James K. Bowmaker

a

a Division of Visual Science, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK Division of Neuroscience and Psychological Medicine, Department of Integrative and Molecular Neuroscience, Imperial College School of Medicine, London W6 8RF, UK c Zoologisches Institut und Zoologisches Museum, der Universit€at Hamburg, Hamburg 20146, Germany

Received 4 July 2002; received in revised form 9 September 2002

Abstract The cave-dwelling (hypogean) form of the teleost Astyanax fasciatus is blind, having only subdermal eye rudiments, but nevertheless maintains intact opsin genes. Second generation offspring of a cross between these and the normally sighted surface (epigean) form inherit opsin genes from both ancestries. A study of the expressed hypogean opsins of the hybrids, in comparison to the epigean forms, was undertaken by microspectrophotometry. The hybrid population showed considerable variation in the visual pigments of double cones, with evidence for two groups of cells with kmax intermediate to those of the epigean pigments. Possible explanations for these intermediate pigments are discussed, including the hypothesis that they may represent hybrid genes similar to the genes for anomalous cone pigments in humans. Evidence was also found for ultraviolet-sensitive single cones and for an additional MWS pigment. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Astyanax; Visual pigment; Opsin; Hybrid gene; Double cone; Cave fish

1. Introduction The neotropical characid fish, Astyanax fasciatus, is distributed widely across Mexico and southern Texas. It is found in a variety of forms, including surface living or epigean varieties and cave dwelling or hypogean forms. The epigean fish is the most widely spread, and is ÔnormalÕ in appearance. Hypogean forms have developed through populations becoming trapped in subterranean caves approximately 1 million years ago (Wilkens, 1988) and these varieties differ most notably from the epigean fish by reduced eyes and skin pigmentation. Indeed, the majority of hypogean populations are blind, having only rudimentary subdermal eyes, but may compensate for their lack of eyesight with improved senses such as taste, olfaction and lateral line (Peters, Schacht, Schmidt, & Wilkens, 1993; Wilkens, 1988).

*

Corresponding author. Tel.: +44-20-7608-6996; fax: +44-20-76086850. E-mail address: [email protected] (J.W.L. Parry).

Isolated blind population of Astyanax have been used in the study of eye development (Behrens, Langecker, Wilkens, & Schmale, 1997; Culver & Wilkens, 2000; Langecker, Schmale, & Wilkens, 1993; Wilkens, 1988). In hypogean forms the eyes are subjected to a process of ontogenetic regression: they initially parallel those in epigean forms, but normal development does not proceed beyond the third day, after which the eye degenerates into the adult rudimentary form (Langecker et al., 1993). It has been suggested that regulatory, rather than structural genes are responsible for this regression. The effects of the Ôeye genesÕ are additively polygenic so that, depending on the number of affected genes, both gradual changes and large jumps in phenotype can arise. However, members of individual isolated populations share the same phenotype (Culver & Wilkens, 2000; Langecker et al., 1993; Wilkens, 1988). The rod opsin gene is briefly expressed during the early morphogenesis of the degenerate eye, as photoreceptors start to be formed on the third day, although outer segments never develop. Sporadic cell death occurs in all layers of the developing retina,

0042-6989/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 2 - 6 9 8 9 ( 0 2 ) 0 0 4 0 4 - 2

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beginning on the second day and rod opsin is only found in the outer nuclear layer of the retina for a very limited time (Langecker et al., 1993). The final eye rudiment is about 1% of the size of that of the epigean, with the lens and number of visual cells completely reduced. The degree of reduction of other structures correlates with the eye size, but the retinal rudiment, when present, consists only of inner nuclear, inner plexiform and ganglion cell layers, even in the largest and most developed rudimentary eyes (Langecker et al., 1993; Wilkens, 1988). The retinae of epigean fish contain single and unequal double cones in addition to rods. According to Kleinschmidt and H arosi (1992), the double cones are long-wave-sensitive and contain visual pigments with kmax close to 596 and 554 nm, whereas the single cones are short-wave-sensitive with kmax around 453 nm. The rods absorb maximally at 520 nm. The visual pigments appear to be about a 50/50 mixture of A1 (retinal) and A2 (3, 4-didehydroretinal) chromophores. A striking feature of the two longer-wave pigments is that they are both ionochromic, exhibiting spectral shifts to shorter wavelengths in the absence of chloride ions. This is a recognised feature of visual pigments that belong to the long-wave-sensitive (LWS) family of opsins (Bowmaker, 1998; Kleinschmidt & H arosi, 1992; Wang, Asenjo, & Oprian, 1993; Zak, Ostrovsky, & Bowmaker, 2001) and suggests that not only the 596nm pigment, but also the 554-nm pigment should be classed as LWS. The presence of two differently tuned cone pigments from the same opsin family has been confirmed from studies of the hypogean genome from which rod and cone opsins have been sequenced (Yokoyama & Yokoyama, 1990a,b, 1993; Yokoyama, Starmer, & Yokoyama, 1993). This is unusual in lower vertebrates and parallels the situation in primates where the L (red) and M (green) cones also contain pigments from the LWS class of opsin (Nathans, Thomas, & Hogness, 1986). To avoid confusion these will be referred to as LR and LG genes. The sequences of three genes (one LR and two variants of LG ) in Astyanax show the same spectral tuning sites as the primate pigments (Asenjo, Rim, & Oprian, 1994; Merbs & Nathans, 1992; Nathans, Thomas et al., 1986; Neitz, Neitz, & Jacobs, 1991; Yokoyama & Yokoyama, 1990a; Yokoyama et al., 1993) and it can be inferred that they (or at least the LR and one of LG genes) are expressed as the two longer-wave pigments identified by microspectrophotometry (Kleinschmidt & H arosi, 1992). However, lower vertebrates normally possess a middle-wavesensitive cone, based on a separate class of opsins (MWS or RH2 or rod-like) (Bowmaker, 1998; Heath, Wilkie, Bowmaker, & Hunt, 1997; Johnson et al., 1993; Yokoyama, 2000), but it would appear that in Astyanax the LG gene has supplanted the normal MWS

pigment gene. Nevertheless, such a gene has been identified in the Astyanax genome (Register, Yokoyama, & Yokoyama, 1994). It has been suggested that an LG gene could be expressed in the pineal organ (Yokoyama & Yokoyama, 1990b), since electrophysiological studies of the pineal of the epigean form indicated the presence of two middle-wave-sensitive pigments with kmax at about 494 and 525 nm (Tabata, 1982). Although the hypogean genes appear to be intact open reading frames, and the kmax predictions from standard L class tuning sites agree with the microspectrophotometric data from epigean forms, it has not been established that the epigean and hypogean opsins are identical. A comparison of a phylogenetically old and strongly eye-reduced population of hypogean fish with that of a phylogenetically young, less eye-reduced population, showed numerous changes between the sequences (Yokoyama, Meany, Wilkens, & Yokoyama, 1995). These consisted mainly of cytosine to thymidine changes, but also a deletion in the LG sequence of the old population with respect to that of the new (Yokoyama et al., 1995). The phylogenetically young hypogean population shows a much greater variability in phenotype than the older populations possibly because of being in status nascendi as a cave fish. These natural populations have provided much data on the genetics of eye regression, but phenotypically similar specimens can be created under more controlled conditions by laboratory crossing of epigean and hypogean forms. The first generation of such a cross gives individuals with intermediate eye size, but the F2 generation gives a much broader, but continuous range of phenotypes, incorporating both parental forms (Wilkens, 1970; Wilkens, 1988). These hybrids allow the study of the effects of hypogean genes within a less restricted background. Within an F2 hybrid crossing, individuals will be found expressing hypogean opsin genes within intact functional photoreceptors. We have conducted a study of such pigments by microspectrophotometry, comparing ÔcontrolÕ epigean fish with two F2 hybrid phenotypes: those showing an epigean phenotype with respect to eye size, and those with smaller, but still functional eyes. The visual pigments of the epigean population were found to be essentially identical to those previously reported (Kleinschmidt & H arosi, 1992), though the amounts of A2 chromophore relative to the A1 form varied quite widely. However, the hybrid populations showed considerable variation in the visual pigments of the double cones with evidence for two groups of cells with kmax intermediate to those of the epigean LR and LG pigments. Evidence was also found for an ultraviolet-sensitive single cone and the possibility of an additional MWS pigment.

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2. Methods 2.1. Materials Hybrid fish were bred from a cross between hypogean fish from the Molino cave (Tamaulipas, Mexico) (Mitchell, Russel, & Elliott, 1977) and epigean fish from Rio Teapao (Tabasco, Mexico). The Molino population belongs to the phylogenetically old and strongly eyereduced cave fish, having only subdermal eye rudiments containing no sensory cells, no outer nuclear layer nor outer plexiform layer, and no lens rudiments. Fish were kept at 27 °C and in darkness or dim light to prevent fighting of the epigean fish. Adult specimens were used in all experiments. 2.2. Microspectrophotometry Fish were dark-adapted overnight, then sacrificed by cervical dislocation. Eyes were enucleated, hemisected and the anterior portion discarded. The retina was then separated from the pigment epithelium and small samples prepared for measurement. All preparation was carried out under a dim red safelight. The method of tissue preparation, recording spectra, and the design of the microspectrophotometer, a modified ÔLiebmanÕ dual beam instrument, are described elsewhere (Bowmaker, Astell, Hunt, & Mollon, 1991; Liebman & Entine, 1964; Mollon, Bowmaker, & Jacobs, 1984). Spectra were recorded at 2-nm intervals from 750–350 nm, and from 351–749 nm on the return scan. The outward and return scans were averaged. A baseline spectrum was measured for each cell, with both beams in an unoccupied area close to the cell, and subtracted to form the final spectrum. Two (or more) baseline spectra were recorded for each cell and averaged. Cells were routinely bleached by exposure to a beam of white light from the monochromator and the post bleach spectrum recorded from which difference spectra were calculated. 2.3. Analysis of absorbance spectra Spectra were analysed using a computer program that fits the appropriate A1 (Knowles & Dartnall, 1977), or A2 (Schwanzara, 1967) (or mixture) visual pigment template to the data. The templates are expressed on an abscissal scale of log frequency, since absorbance curves of visual pigments have almost the same shape when expressed on such an abscissa (Bowmaker et al., 1991; Mansfield, 1985). The relationship beaks down at short wavelengths and in the case of UVS cones a slightly narrower template was employed (H arosi, 1994). kmax were determined in two ways. First, using points on the long-wave limb of the absorbance spectrum, and secondly, from equal numbers of points on either side of

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the peak. Spectra were selected for further analysis by the application of criteria (for the rationale underlying these criteria, see Bowmaker et al. (1991)). For each individual fish, spectra were initially somewhat arbitrarily subdivided into groups by spectral location and bandwidth. The average of spectra from each kmax group was then best fitted to a mixed template as a tool to estimate kmax and chromophore content. Since data from any one fish were limited, a mean chromophore ratio was arrived at from all the cone pigment groups, on the simple assumption that all spectral classes of cone had similar chromophore ratios. It was clear that rods often exhibited a different chromophore ratio from cones, so that these were treated separately. In the case of LR and LG pigments, the estimations from individuals with significantly different chromophore ratios, suggested visual pigment pairs of about 566/612 and 536/568 nm respectively (see Section 3). A number of hypogean fish appeared to have pure A1 pigments with kmax at about 536 and 566 nm, strongly supporting the values arrived at from the analysis of bandwidths from individuals with mixed chromophores. Having established a chromophore ratio for each fish, individual spectra were then reanalysed using the mixed template and reassigned to spectral classes. The kmax for each class of pigment, as given in the results, was determined from the right-hand limb of the average spectrum of all the selected spectra. The mean kmax (and standard deviation) for each class of pigment was also calculated from the kmax for each individual spectrum. All calculations were performed on absorbance spectra, but estimates of kmax from the difference spectra differed by only a few nanometres. Conversions of kmax between A1 , A2 and mixed forms were performed using A1 kmax ¼ 400  ðLnðA2 kmax Þ  5:0Þ (Parry & Bowmaker, 2000). 2.4. Sectioning of retinae After dark-adaptation overnight, enucleation was performed as above, but eyes were fixed in 2% glutaraldehyde/2% formaldehyde prior to hemisection. Eyecups were dehydrated flat and embedded in epon. Sections were cut en face at 1 lm intervals and stained with toluidene blue.

3. Results 3.1. Photoreceptor organisation The retinal mosaics of the epigean population were typical of teleosts in general, with central single cones and double cones forming the sides of the squares (Fig. 1A), though the degree of mosaic organisation varied across the retina. In contrast, the F2 hybrids showed a high level of variability, ranging from extremely ordered

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Fig. 1. Semi-thin sections through retinae of dark-adapted Astyanax cut perpendicular to the axis of the photoreceptors to show the arrangement of the cone mosaic. Panel (A) shows a section from an epigean fish: a square mosaic, each unit consisting of four double cones around a central single cone. Example squares are denoted by white boxes. Panels (B)–(D) show sections from different large-eye phenotype hybrid fish. (B) Row mosaic with many triple cones (white arrow heads). (C) Highly disorganised and sparse cone distribution. (D) Intermediate cone organisation. The oblique section shows a regular square array of single cones (left), but a degenerate row array of double cones (right). The black arrow head indicates a triple cone. Scale bar 20 lm.

to very disorganised cone mosaics. Some individuals appeared very much like the epigean population with clearly organised row or square mosaic patterns, but with a higher incidence of triple cones (Fig. 1B). These replaced double cones within the mosaic and caused a disruption of the regularity. Other individuals showed an extreme reduction in the number of photoreceptors with a loss of any mosaic organisation (Fig. 1C). This reduction was not limited to the smaller-eyed phenotypes, but also occurred in those with full-sized eyes. The disruption of the mosaic in some individuals also differed between cone classes (Fig. 1D). In the individual shown, the single cones maintained a relatively regular square pattern, whereas the double cones formed a very degraded row pattern. 3.2. The complement of cone photopigments in the epigean population Double cones and both large and small single cones were observed. The double cones were LWS with the majority containing two different pigments, having kmax between about 540 and 560 nm or 565 and 590 nm (Fig. 2). Large single cones also contained pigments identical to the double cones. Small single cones were short-wave

sensitive with kmax between about 445 and 450 nm (Fig. 2). Variability in the kmax of a given class of cone, most notable in longer-wave pigments, is usually the result of variable mixtures of A1 and A2 chromophores and this appears to be the case in Astyanax. Individual fish from the epigean population tended to fall into two groups possessing cones with either 20% or 70% A1 (Fig. 2B and C), though other percentages were also observed. The percentage mixtures were based on the bandwidths of the mean absorbance spectra of the two long-wave pigments. From these ratios it is possible to estimate the kmax of the two A1 =A2 pigment pairs (Parry & Bowmaker, 2000) which are close to 536/567 nm for the shorter-wave and 563/606 nm for the longerwave. The distribution of the kmax of individual cones containing the two longer-wave pigments is shown in Fig. 3 from fish exhibiting a 70% A1 =30% A2 mixture (the most common form). The spread of the kmax of individual cones in the longer-wave group was around 14 nm, whereas the distribution of the shorter-wave group was considerably more extensive with ÔoutliersÕ extending the spread to some 27 nm (Fig. 3). A minority of double cones contained the shorter-wave pigment in both members, though double cones with the longer-wave

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Wavelength, nm Fig. 2. Normalised mean absorbance spectra for cone pigments in hybrid and epigean fish. (A) spectra from hybrid fish containing pure A1 , full lines represent pure rhodopsin templates with kmax at 560, 533, 448 and 401 nm respectively. (B) Spectra from epigean fish, full lines represent mixed rhodopsin/porphyropsin templates with 70% A1 and kmax at 572, 547 and 449 nm respectively. (C) Spectra from epigean fish, full lines represent mixed rhodopsin/porphyropsin templates with 20% A1 and kmax at 602, 560 and 451 nm respectively. Triangles, LR ; circles, LG ; squares, SWS; diamonds, VS. For clarity, LR and LG have been truncated at short wavelengths and VS truncated at long wavelengths.

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Wavelength, nm Fig. 3. Histogram showing the spectral distribution of the kmax of individual cones from epigean fish with a 70% A1 =30% A2 pigment mixture in the cones. The two main populations of LG and LR cones have mean kmax at 547 and 572 nm respectively. The A1 equivalent kmax are about 537 and 559 nm respectively. Only cells with kmax greater than 500 nm are plotted. Bin size is 1.5 nm.

single cones were identified that contained a pigment with a much shorter kmax , in the region of 400 nm (Fig. 2A). Large single, double and triple cones contained longer-wave pigments, again similar to the epigean population. However, in contrast to the epigean fish, many individuals from the F2 hybrids showed pure A1 cone pigments (Fig. 2A), though a smaller number did possess mixtures with A2 in variable amounts. The tendency to pure A1 cones was observed more frequently in the hybrid fish with full-sized eyes. The distribution of the kmax of the individual cones, however, was markedly different from that of the epigean population (Fig. 4). In addition to the two clear populations with kmax centred around 536 and 566 nm, there were many cones that contained a pigment with kmax falling between these populations, a feature not seen in the epigean fish. These intermediate pigments cluster around 545 and 552 nm (Fig. 4). The mean absorbance spectra of the four pigment groups (Fig. 5) clearly demonstrate that all the spectra are similar in shape, but spectrally displaced from each other.

pigment in both members were not observed. A small number of triple cones were identified, containing similar pigments to the double cones. 3.3. The complement of cone photopigments in the F2 hybrid population The cone classes found in hybrids were basically similar to those seen in the epigean fish. Small and large single cones were observed, as were double and triple cones with the latter often having an increased incidence relative to the epigean population. A population of small single cones contained a SWS pigment with kmax around 447 nm, similar to the epigean form, and small

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Wavelength, nm Fig. 4. Histogram showing the spectral distribution of the kmax of individual cones from F2 hybrid fish with pure A1 pigments in the cones. Note, in comparison with Fig. 3, the additional cones with kmax falling between the LG and LR populations. Only cells with kmax greater than 500 nm are shown. Bin size is 1.5 nm.

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Relative absorbance

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Wavelength, nm Fig. 5. Normalised mean absorbance spectra of long-wave cone pigments from hybrid fish containing pure A1 pigments. Triangles, LR (kmax longer than 557 nm); diamonds, longer intermediate population (kmax between 550 and 556 nm); squares, shorter intermediate population (kmax between 541 and 549 nm) and circles, LG (kmax less than 540 nm) (cf. Fig. 4). Full lines are pure A1 templates with kmax at 566, 553, 544 and 533 nm respectively.

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The majority of double cones contained a 536/566-nm pairing, but the intermediate pigments were found paired with any other pigment. Double cones containing the shorter wave pigments in both members were again observed, but some double cones containing only longer-wave pigment were also identified. This resulted in a slightly higher incidence of double cones identical in pigment content compared to the epigean population. Triple cones, where all members were measurable, appeared to contain the same pigment in at least two members.

Fig. 7. Normalised mean absorbance spectra of pineal (closed symbols) and rod (open symbols) photopigments from two hybrid fish, fitted with appropriate visual pigment templates (solid and dashed curves, respectively). A: individual possessing pure A1 pigments with kmax of the pineal pigment at 503 nm and of the rod pigment at 511 nm. B: individual possessing pure A2 pigments with kmax of the pineal pigment at 518 nm and of the rod pigment at 535 nm.

with almost pure A1 , whereas the other contained about 50% A2 (Fig. 6). In the epigean population, most fish possessed rods with about 70% A1 , although some individuals were found to have rods with pure A1 pigment. 3.5. Pineal organ

Rods had kmax between about 510 and 530 nm with an estimated pigment pair of about 511/533 nm (Fig. 6). It was notable that the chromophore ratio in the rods was found to vary independently of that in the cones. For example, two individual hybrid fish that had pure A1 in their cones had different mixtures in their rods, one

Only a single spectral class of photopigment was found in the pineal organ in both epigean and hypogean populations, although the A1 :A2 ratio varied between individuals. The pineal photopigment had a kmax that was always somewhat shorter than the kmax of the rods (Fig. 7) and the chromophore ratio appeared similar between rods and pineal photoreceptors. In individuals with pure A1 in the rods, the kmax of the pinealocytes was close to 500 nm, but in individuals with A2 -dominated rods, the kmax of the pinealocytes was closer to 520 nm (Fig. 7).

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Wavelength, nm Fig. 6. Normalised mean absorbance spectra of rods from two different F2 hybrid fish, each possessing cones containing pure A1 pigments. Filled squares from an individual with rods with a pure A1 pigment and best fitted with a rhodopsin template with kmax at 508 nm (full line). Open circles from another fish with rods containing a 50% A1 =50% A2 mixture and best fitted with an appropriate template with kmax at 521 nm (full line).

4.1. Triple cones In epigean fish the retinal photoreceptors are arranged in a typical teleost pattern consisting of a square mosaic, in which oriented double cones form a square around short single cones, although the regularity of the mosaic is variable. In the majority of the F2 hybrids, the underlying arrangement tends more toward a row for-

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mation, but the mosaic can become markedly disturbed and disorganised. This disruption to the retinal organisation is assumed to be dependent on the degree of hypogean inheritance of any individual hybrid. A striking feature in some hybrids with relatively organised cone mosaics was the prevalence of triple cones that occur rarely in the epigean form. The triple cones occupied the position of the double cones in the cone array and so tended to disrupt the regularity of the mosaic. Triple cones occur in a number of different species of fish (e.g. Collin, Collin, & Ali, 1996; Collins & MacNichol, 1979; Collins & MacNichol, 1984; Narayanan & Khan, 1995; Reckel, Melzer, & Smola, 1999; Reckel, Melzer, & Smola, 2001) and, as in Astyanax, tend to occupy the same position as double cones within the receptor mosaic, although often in limited areas of the retina. However, in the Black Sea anchovy, Engraulis encrasiocholus, triple cones occupy the majority of the retina (Zueva & Govardovskii, 1991). Strings of multiple cones sometimes occur during development (Shand, Archer, & Collin, 1999), but are subsequently lost during the reorganisation of the retina into the adult form where double cones, with the occasional triple cone, are retained. Triple cones may also occur after repair of retinal damage (Cameron & Easter, 1995). Clearly, the morphology of cone types in teleosts is relatively plastic and the presence of significant numbers of triple cones in the hybrid Astyanax may simply be the accidental result of the disruption of retinal development genes in the parental hypogean variety. The function of triple cones where they occur normally is difficult to determine and may not be significantly different from that of double cones. 4.2. Visual pigments and chromophore variability The epigean fish were found to vary widely with respect to the ratio of the A1 :A2 chromophores. Generally, the fish could be divided into two groups, with either 70% A1 or 20% A1 retinal in their cones, although there were some exceptions. Variations in the ratio of A1 to A2 will change the spectral sensitivities of the cones, and a number of temperate species of teleosts take advantage of this to change their spectral sensitivity either seasonally, to match changes in water colour or turbidity (Bowmaker, 1995; Bridges, 1972; Dartnall, Lander, & Munz, 1961) or, in migratory species, to adapt between bluer oceanic waters and greener fresh waters (Beatty, 1966; Bridges, 1972). Astyanax, as a non-migratory neotropical species, is not exposed to such changes in environment, which may be reflected in the apparent lack of rigid control over its chromophore ratio. The three cone pigments identified in the epigean form comprise Ôpigment pairsÕ with the kmax for those containing pure A1 at about 447, 536 and 566 nm and the respective pure A2 variety kmax around 454, 567 and

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611 nm. These data are in good agreement with previously published values for Astyanax (Kleinschmidt & Harosi, 1992). The same pigments were observed in the hybrid population, which also showed a lack of fixed chromophore ratio. However, in marked contrast to the epigean fish, over half of the hybrid fish studied possessed almost pure A1 pigments in their cones. Rods from both populations were also found to vary in their chromophore content, although the range of chromophore ratios was more restricted than in cones. The majority of rods measured contained either 70% of the A1 chromophore or possessed a pure A1 pigment. Both the epigean and the hypogean populations contained the same pigment pair, with the pure A1 version having a kmax at about 511 nm and the pure A2 equivalent at about 533 nm, which agrees with previously published data (Kleinschmidt & Harosi, 1992). The chromophore ratio in the rods was often found to be different from that in the cones. This was most noticeable in some hybrid fish that showed the pure A1 chromophore in cones, but had a percentage of the A2 chromophore incorporated in the rods (Fig. 6). The chromophore ratio can vary not only between rods and cones in the same retina, but also between two spectrally distinct classes of rod within the same retina, as in some species of deep-sea fish (Bowmaker, Dartnall, & Herring, 1988; Partridge, Shand, Archer, Lythgoe, & van Groningen-Luyben, 1989).

4.3. Intermediate long-wave photopigments The hybrid fish possessed the expected A1 cone pigments with kmax at about 536 and 566 nm, but it was notable that cones containing pigments intermediate to these were also present. The intermediate pigments fell into two groups with kmax close to 545 and 552 nm (A1 ) (Fig. 4). Although this is a striking difference between the epigean and the hybrid fish, we cannot exclude the possibility that the broadening in the longer-wave region of the middle-wave cone histogram derived from the epigean fish (Fig. 3) is due to small number of cones containing an intermediate pigment. Nevertheless, the substantial number of these ÔintermediateÕ cones in the hybrid fish suggests that their occurrence can be attributed to the parental hypogean genome. A number of possibilities can be suggested to account for these intermediate cones. First, they could represent chromophore ratios different from those in the ÔnormalÕ LR and LG cones. However, this would seem highly unlikely, since the intermediate pigments are located within the double cones along with the ÔnormalÕ pigments. Further, the intermediate cones in any given individual, including hybrid fish with pure A1 pigments, can also be fitted with the same template as used for their other visual pigment classes (Fig. 5).

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A second explanation for these ÔintermediateÕ cones is that they contain a mixture of the two ÔnormalÕ pigments, a consequence of the coexpression of their two opsins. A somewhat similar situation of cone polymorphism has been proposed for the guppy, Poecilia reticulata (Archer & Lythgoe, 1990) in which it was suggested that the polymorphism was based on only two visual pigments which occurred either on their own in cones with kmax at 533 and 572 nm or as a mixture in a 543-nm intermediate cone class. However, if coexpression were the explanation in Astyanax, it is difficult to account for the two groups of intermediate cones observed. Coexpression might be expected to be either random, giving an even distribution of intermediate pigments, or at a set ratio where both pigments rare fully expressed. The presence of two different, yet controlled ratios, though possible, would be surprising. Another explanation relates to the identification of two LG gene sequences in the hypogean genome (Yokoyama & Yokoyama, 1990a). If the two LG genes were expressed as two spectrally distinct pigments, at least three cone spectral distributions could be expected, though the four distributions observed would require either a second LR gene sequence or coexpression. However, both LG gene sequences contain identical residues at known tuning sites for this class of pigment, so that the additional LG sequence would require novel tuning sites to be expressed as a spectrally distinct pigment. Alternatively, opsin polymorphism could be responsible for the intermediate wavelength pigments. Since the cave-dwelling ancestors are blind, they might be expected to experience genetic drift in their opsin genes. If this occurred at one or more of the tuning sites, a spectral distinct pigment could be produced. However, this is unlikely, since there are thought to be only three major tuning sites in this class of opsin that produce significant shifts in kmax (see below). The chance of random mutation affecting any of the three sites, while not producing other changes that cause dysfunctional pigments is very low, especially if there is no selection pressure to maintain the functionality of the opsin. Additionally, the opsin genes sequenced by Yokoyama and Yokoyama (1990a) were from the genome of a blind fish and showed no genetic drift at the identified tuning sites. A further, more likely possibility, is that the ÔintermediateÕ pigments are derived from hybrid genes. Humans (and other old world primates) have L and M cone pigments encoded by homologous LR and LG genes, caused by a duplication of the ancestral L-class gene. These genes are found in tandem array on the X chromosome and the proximity of two such highly similar sequences can lead to unequal crossing-over during meiosis. This process leads to the creation of new, hy-

brid pigments. In most humans the array consists of an LR gene followed by two or more LG genes, but more complex arrays can occur which include hybrid genes. Normally, only two genes in the array are expressed (Deeb & Motulsky, 1996; Hayashi, Motulsky, & Deeb, 1999; Macke & Nathans, 1997; Nathans, Merbs, Sung, Weitz, & Wang, 1992; Nathans, Piantanida, Eddy, Shows, & Hogness, 1986; Nathans, Thomas et al., 1986; Neitz, Neitz, & Kainz, 1996; Sjoberg, Neitz, Balding, & Neitz, 1998). The three major tuning sites in the human opsins, position 180 in helix IV and positions 277 and 285 in helix VI, are occupied by alanine, phenylalanine and alanine respectively in the LG pigment, but by serine, tyrosine and threonine respectively in the LR pigment. Site 180 is coded for in exon 3, but sites 277 and 285 are coded together in exon 5, so that in hybrid genes, composed of mixtures of exons from the two genes, sites 277 and 285 are rarely, if ever, separated. Simplistically, this results in two intermediate hybrid pigments (or families of pigments), a shorter-wave LR 180  LG 277/ 285 and a longer-wave LG 180  LR 277/285 (Asenjo et al., 1994; Merbs & Nathans, 1992). Expression of such hybrid genes can lead to anomalous trichromacy, since the kmax of an anomalous pigment will be displaced to a spectral location intermediate between the normal LR and LG pigments. In Astyanax, the LR and LG genes are of the same evolutionary class as the human opsin genes and have the same characteristic residues at the equivalent tuning sites in their opsins. If the duplication event that presumably formed the second L-class gene in Astyanax caused it to be inserted into the genome close to the original copy, the arrangement would be similar to that in humans. In these circumstances, the genes would be susceptible to unequal crossing over during cell division, with the formation of hybrid genes. Such hybrid genes, similar to those in humans, would be expressed as two ÔanomalousÕ cone pigments, spectrally intermediate to the normal LR and LG pigments, as observed in the F2 hybrid fish. The high incidence of these ÔanomalousÕ pigments in the F2 hybrids presumably reflects the hypogean genome. Although, unlike humans, the hybrid pigments are expressed in addition to the LR and LG pigments (and in the hybrid fish almost any pairing of the normal and/or intermediate pigments occurred in the double cones), they will nevertheless cause colour vision anomalies. This impairment of colour vision would cause selection against hybrid pigments, which would therefore, although easily formed, be kept to a low level within the epigean population. In contrast, there would be no selection pressure in the hypogean genome, and hybrid genes, once created, would remain and deteriorate no faster than the parental LR and LG sequences.

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4.4. Other additional photopigments 4.4.1. Ultraviolet-sensitive cones A rare population of single cones with kmax around 400 nm was identified in the F2 hybrid fish. This cone type has not been reported previously and was not observed in the epigean fish. Nevertheless, this cone class must have been present in the epigean ancestors and presumably occurs in the epigean fish in the present study. The apparent absence of this cone class in the epigean fish is probably a consequence of their rarity and the random sampling inherent in microspectrophotometry. Since ultraviolet-sensitive cones are relatively common in shallow living teleosts (Bowmaker, 1995; Losey et al., 1999; Partridge & Cummings, 1999), their presence in Astyanax is not unexpected. Ultravioletsensitive cones often correlate with the presence of corner cones in a square mosaic (Bowmaker, 1995; Bowmaker & Kunz, 1987; Lyall, 1957). The regular square mosaic from an epigean fish (Fig. 1A) shows few, if any, corner cones, possibly accounting for the scarcity of measurable ultraviolet-sensitive cones within this study. 4.4.2. Middle-wave sensitive cones The genome of Astyanax not only includes the LG and LR genes, but also a gene belonging to the middle-wavesensitive class or rod-like cone opsins (Register et al., 1994). Is this gene expressed in the retina? In both the epigean and the F2 hybrid fish there are indications of a small number of cones with kmax some 10 nm shorter than the majority LG cones (Figs. 3 and 4). It is not possible from the microspectrophotometric data to determine whether these cones simply represent a shortwave ÔtailÕ of the LG population or are evidence of the expression of the MWS gene. In many teleosts the MWS gene is expressed in double cones with kmax somewhere between about 480 and 530 nm and the possibility of its expression in Astyanax cannot be excluded. If it is expressed in the retina, then the functional significance of the presence of both an LG and an MWS pigment remains obscure. 4.5. Pineal organ Only one photopigment was found in the photoreceptors of the pineal organ of Astyanax, whether epigean or hybrid. This pigment, unique to the pineal, exhibited a kmax 10–15 nm shorter than that of the retinal rod, depending on chromophore content, and is characteristic of the pineal exorhodopsin (Mano, Kojima, & Fukada, 1999; Peirson & Bowmaker, 1999; Philp, Bellingham, GarciaFernandez, & Foster, 2000). Since only a single pigment could be identified, it appears unlikely that any retinal cone opsin genes are expressed in the pineal, as proposed by Yokoyama (Yokoyama & Yokoyama, 1990b). The presence of a

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single pigment also appears to contradict the identification electrophysiologically of two pineal photopigments by Tabata (1982). However, these two pigments were identified from different individual fish and it may be that they represent a single pigment but with different chromophore composition. The kmax presented here range from about 500 to 520 nm and a Ôpigment pairÕ in the region of 500 nm will have a separation in the order of 20 nm. This is somewhat smaller than the difference observed by Tabata (495–525 nm), but the discrepancy may reflect the very different nature of the experimental procedures. 4.6. Maintenance of opsin genes The hypogean genome appears to have maintained the opsin genes as intact open reading frames. Although it has not been established that these sequences are identical in epigean and hypogean forms, our data suggest that both forms encode pigments that are functionally the same. For each pigment measured in the epigean fish, one of equivalent kmax has been found in the F2 hybrid fish. The intermediate pigments were much more common in the hybrid fish, suggesting a hypogean ancestry and if these pigments are the products of hybrid genes, the parental LR and LG sequences must also be intact. The maintenance of the opsin genes in the hypogean population may reflect the possibility that they are required during development (e.g. Langecker et al., 1993). 4.7. Colour vision in Astyanax fasciatus Astyanax potentially possesses eight functional retinal photopigments––a rod pigment and seven cone pigments consisting of UV/VS, SWS, MWS and up to four LWS (LR , LG and two intermediate pigments). However, the basic spectral distribution of the cone pigments in the epigean form, with LG =LR double cones and SWS and possibly VS single cones, is typical of many shallow water teleosts. This complement of cone pigments probably supports tetrachromatic colour vision, as has been demonstrated in the goldfish (Neumeyer, 1992). The complication of the possible additional cone pigments in the red/green spectral region of the epigean variety and their higher incidence in hybrid fish, most likely does not increase the dimensions of colour space for Astyanax, but simply leads to mild forms of anomaly.

Acknowledgements This work is supported by the Leverhulme Trust. We thank Peter Munro and Glen Jeffery for assistance with histology.

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