Enhanced Retinal Longwave Sensitivity Using a Chlorophyll-Derived Photosensitiser in Malacosteus Niger, a Deep-Sea Dragon ﬁsh with Far Red Bioluminescence

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Vision Research 39 (1999) 2817–2832

Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon ﬁsh with far red bioluminescence

R.H. Douglas a,*, J.C. Partridge b, K.S. Dulai c, D.M. Hunt c, C.W. Mullineaux d, P.H. Hynninen e

a Department of Optometry and Visual Science, Applied Vision Research Centre, City Uni6ersity, 311–321 Goswell Road, London EC1V 7DD, UK b School of Biological Sciences, Uni6ersity of Bristol, Woodland Road, Bristol BS81UG, UK c Institute of Ophthalmology, Uni6ersity College London, Bath Street, London EC1V 9EL, UK d Department of Biology, Uni6ersity College London, Darwin Building, Gower Street, London WC1E 6BT, UK e Department of Chemistry, Uni6ersity of Helsinki, PO Box 55 (A.I. Virtasen aukio 1), Fin-00014 Helsinki, Finland Received 15 July 1998; received in revised form 2 December 1998

Abstract

Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin u u \ pigment pair with max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments ( max 550 nm) observed in two other red light producing stomiids. The presence of only a single opsin gene in the M. niger genome was conﬁrmed by molecular and cladistic analysis. To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M. niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Fish; Retina; Photosensitiser; Bioluminescence; Visual pigment; Opsin

1. Introduction peak emission of most bioluminescence, produced by over 80% of all animals inhabiting the deep-ocean, is While most shallow water fish have a large range of also around the same wavelengths as the remaining wavelengths potentially available for vision (ca. 320– sunlight (Herring, 1983; Widder, Latz & Case, 1983; 850 nm), deep-sea fish are exposed to a spectrally more Latz, Frank & Case, 1988). Not surprisingly therefore, restricted environment. Two sources of radiation are the vast majority of deep-sea fish visual pigments have available in the deep-sea: downwelling sunlight and u absorption maxima ( max) within the range 470–492 nm bioluminescence. In ideal conditions enough solar radi- (Partridge, Archer & Lythgoe, 1988; Partridge, Shand, ation penetrates to depths of around 1000 m to allow Archer, Lythgoe & van Groningen-Luyben, 1989; Par- vision in the most sensitive fish (Denton, 1990). Due to tridge, Archer & van Oostrum, 1992; Douglas, Par- spectral filtering by the water column, this residual tridge & Hope, 1995; Douglas & Partridge, 1997), spacelight is composed primarily of a narrow band of coinciding with both the most common bioluminescent radiation between 460 and 490 nm (Kampa, 1970; emission maxima and the wavelengths that most easily Jerlov, 1976; Kirk, 1983; Frank & Widder, 1996). The penetrate water. Although the wavelength of maximum emission of * Corresponding author. Tel.: +44-171-4778334; fax: +44-171- most deep-sea bioluminescence matches the remaining 4778355. E-mail address: [email protected] (R.H. Douglas) downwelling sunlight, there are exceptions, perhaps the

0042-6989/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0042-6989(98)00332-0 2818 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 most striking of which are three genera of deep-sea observed in A. tittmanni and P. microdon. However, dragon fish (Malacosteus, Aristostomias and Pachysto- microspectrophotometry has shown that the outer seg- mias; order Stomiiformes, family Stomiidae), which, in ments of M. niger also contain one or more additional addition to blue bioluminescence emanating from pos- pigments with absorption maxima around 670 nm that torbital light organs, also have suborbital photophores are not bleached significantly by light (Fig. 6B; Bow- producing far-red bioluminescence with spectral emis- maker et al., 1988; Partridge et al., 1989). We present sions peaking sharply at wavelengths beyond 700 nm evidence that these pigments, which we identify as (Denton, Gilpin-Brown & Wright, 1970; Widder, Latz, bacteriochlorophyll derivatives, are used as a photosen- Herring & Case, 1984; Denton, Herring, Widder, Latz sitiser by M. niger to enhance its sensitivity to longwave & Case, 1985). To facilitate perception of their own radiation such as its own bioluminescence. A short longwave bioluminescence, all three genera have been shown, using conventional retinal extracts and microspectrophotometry (MSP), to have two visual pigments, that are longwave shifted compared to those of u other deep-sea animals ( max approximately 515 and 550 nm) (O’Day & Fernandez, 1974; Knowles & Dart- nall, 1977; Somiya, 1982; Bowmaker, Dartnall & Her- ring, 1988; Partridge et al., 1989; Crescitelli, 1989, 1991). The sensitivity of Malacosteus niger to long-wave radiation is further enhanced by the possession of a bright red, long-wave reflecting, astaxanthin-based, tapetum (Denton & Herring, 1971; Somiya, 1982; Bow- maker et al., 1988). However, although such visual pigments will enhance the sensitivity of these animals to their own longwave bioluminescence, the match between these pigments and a bioluminescent signal with emission maximum above 700 nm is still far from perfect. Consequently, using spectrophotometry of retinal wholemounts, which iso- lates visual pigments that are not readily measurable in retinal extracts (Douglas et al., 1995), we have been able to demonstrate the existence of a third, even longer u wave absorbing, visual pigment ( max approximately 588 nm) in the retina of Aristostomias tittmanni (Par- tridge & Douglas, 1995) and, in confirmation of limited previous work (Denton et al., 1970, 1985), we have isolated a similar visual pigment in the retinae of u Pachystomias microdon ( max approximately 595 nm) (Douglas, Partridge & Marshall, 1998a). Since these extremely longwave sensitive visual pigments most likely have retinal as their chromophore, and as the retinae of these animals are known also to contain 3,4-dehydroretinal, we have speculated that both Aris- tostomias and Pachystomias may also have a fourth, even longer wave absorbing, porphyropsin within their Fig. 1. Malacosteus niger visual pigments in detergent extracts. (A) u retina ( max approximately 650 nm) (Douglas et al., Absorbance spectra of a retinal extract of M. niger following various 1988a; Partridge & Douglas, 1995). Such extreme far durations of exposure to monochromatic light. In order of decreasing red sensitivity will allow these stomiid dragon fishes to absorbance at 500 nm the absorbance spectra are: (a) initial measurement of the unexposed extract 10 min after the addition of 20 ml1M use their own longwave bioluminescence, which will be hydroxylamine to 150 ml of extract; (b) after 5 min exposure to invisible to all other deep-sea animals, for covert illumi- monochromatic light of 671 nm; (c) another 10 min 671 nm; (d) nation of prey and intraspecific communication im- another 15 min 671 nm; (e) another 20 min 671 nm; (f) another 30 mune from detection by potential predators (Partridge min 671 nm; (g) another 40 min 671 nm; (h) another 60 min 671 nm; & Douglas, 1995). (i) 2 min 501 nm; (j) another 10 min 501 nm. (B) Normalised difference spectra (dashed lines) constructed using the curves shown Here we report that M. niger has visual pigments in Fig. 1A; (k) a–b; (l) i–j. These were best fit to templates (solid with u values close to 520 and 540 nm, but lacks the u max lines) revealing a porphyropsin with max 540 nm, and a rhodopsin u \ u very longwave sensitive visual pigments ( max 550 nm) with max 515 nm. R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2819 report of some of these data has appeared previously of solution to move the spectral absorbance of the (Douglas, Partridge, Dulai, Hunt, Mullineaux, Tauber photoproducts formed during the bleaching process to et al., 1998b). shorter wavelengths. After a 10 min reaction period, a second spectral scan of the unbleached preparation was performed. 2. Materials and methods Subsequently, each sample was exposed to a series of bleaches at one wavelength. Quartz cuvettes holding 2.1. Initial treatment of material the samples were removed from the spectrophotometer and positioned in front of a bleaching light comprising Four individual M. niger were caught using a 50 m2 a 75 W stabilised Xenon arc lamp and Balzer B40 rectangular midwater trawl during cruise 122 of the interference filters (full bandwidth at half maximum R.R.S. Challenger in the waters south of Madeira in the transmission 10 nm). Following a bleach of a given North-eastern Atlantic. All animals, which were dead duration (1–120 min) the sample was returned to the on removal from the net, were transferred to light-tight spectrophotometer and rescanned before being ex- containers as soon as they came aboard and kept in posed to another bleach. Each preparation was sub- darkness floating in iced sea water. In a darkroom, jected to six to nine consecutive monochromatic retinae were removed from hemisected eyes and either exposures until most of the visual pigment had been used immediately or frozen in PIPES buffered saline (20 bleached. This was followed by an exhaustive exposure mM PIPES, 180 mM NaCl, 19 mM KCl, 1 mM MgCl2, at 501 nm to ensure complete bleaching of all visual 1 mM CaCl2) and stored at −70°C in light-tight pigments (e.g. Fig. 1A and Fig. 2A). All absorption containers. This, and all subsequent analyses of visual spectra were zeroed at 700 nm. Such bleaches were pigments, were performed in dim red light. performed on four extracts derived from one M. niger (bleaching wavelengths 630, 637, 654 and 671 nm), 2.2. Visual pigment extraction three extracts from another (654, 671 and 702 nm) and three outer segment suspensions from a third fish (654, The retinae of two fish were homogenised separately 671 and 702 nm). For comparison two extracts from in PIPES buffered saline with 100 ml 200 mM n-dode- the retinae of a related stomiid, Chauliodus sloani, cyl-b-D-maltoside per 1.0 ml of saline, mixed by slow were exposed to bleaches of 654 and 671 nm. rotation for1hatroom temperature and centrifuged at 23 000×g for 10 min at 4°C. The supernatants, which 2.4.1. Calculation of photosensiti6ity contained the visual pigment, were frozen for later From the bleaches described above it is possible to analysis. derive the relationship between the cumulative photon 2.3. Visual pigments in cell suspension exposure of the preparation and the total amount of pigment bleached for the wavelengths tested. The 2 To obtain pure outer segment suspensions for trans- overall integrated intensity (W/m ) of each monochro- mission spectrophotometry at sea, the retinae of one matic bleaching light at the cuvette within the bleach- individual were homogenised in PIPES buffered saline ing chamber was measured using either a Spex with 20% sucrose followed by centrifugation (1 h at Industries 1681 Minimate-2 spectrometer system with 23 000×g at 4°C) above PIPES buffered saline con- SCADAS software (for M. niger) or a Macam Q101 taining 40% sucrose. Rod outer segments, which accu- radiometer (for C. sloani ). These measurements were −1 −2 mulated at the density interface, were removed and converted to photons s m , and multiplied by the resuspended in 20% sucrose. This suspension was di- total number of seconds that the preparation had been vided into four aliquots which were used immediately. exposed to the light, to give a measure of the total cumulative photon exposure. The total amount of 2.4. Spectrophotometry of pigment extracts and outer photopigment in the preparation at the start of the segment suspensions experiment was estimated by calculating the area un- der the difference spectrum (450–650 nm for M. niger; Absorption spectra of both extracts and suspensions 410–600 nm for C. sloani ) resulting from the subtrac- were determined using a Shimadzu UV2101-PC spec- tion of the absorption spectrum of the completely trophotometer. To counteract the inevitable large bleached pigment from that of the unbleached pigment amounts of scatter induced by suspensions, the spec- following the addition of hydroxylamine. Similar cal- trophotometer was fitted with an integrating sphere culations were performed for each bleach and the re- (Shimadzu ISR-260) when scanning such preparations. sulting area expressed relative to the total amount of After initial scans of the unbleached preparations, 20 pigment present at the start of the experiment (Figs. 4 m m lof1MNH2OH (pH 6.5) was added per 150–200 l and 5). 2820 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832

suspensions during the course of photosensitivity experiments (see above) nonetheless allow an accurate determination of visual pigment content. The difference spectrum between the unbleached preparation following the addition of hydroxylamine and the ﬁrst longwave bleach, describes the absorption spectrum of the most longwave sensitive visual pigment, while the difference spectrum between the fully bleached preparation and the previous bleach specify the spectrum of the most shortwave sensitive visual pigment. Any other visual pigments would become apparent in difference spectra u involving intermediate bleaches. The max values for the visual pigments described by these difference spectra were determined by ﬁtting either the rhodopsin template of Partridge and De Grip (1991) or the porphyropsin template of Stavenga, Smits and Hoenders (1993), using methods described by Douglas et al. (1995).

2.5. Analysis of rod opsin gene

Genomic DNA was isolated from liver samples that had been stored at −70°C. DNA extraction was carried out using a standard phenol-chloroform method.

2.5.1. Amplification and sequencing An opsin gene was PCR amplified as two partially overlapping fragments from genomic DNA using degenerate primer pairs (forward 5%-WWWWWWATGAAYGGYACAGAGG-3%; Fig. 2. Malacosteus niger visual pigments in outer segment suspen- reverse sions. (A) Absorbance spectra of a retinal rod outer segment suspen- 5% AGAGGTYRGMHAYNGCVAGGTTWAG-3%; sion of M. niger following various durations of exposure to and forward monochromatic light. In order of decreasing absorbance at 500 nm 5%-NBBCTGGSYGCBTACATGTTC-3%; the scans are: (a) initial measurement of the unexposed extract 8.5 m m reverse min after the addition of 20 l 1 M hydroxylamine to 200 lof % % suspension; (b) after 15 min exposure to monochromatic light of 671 5 -TCTTBCCRCAGMAWARWGTGG-3 ). nm; (c) another 15 min 671 nm; (d) another 30 min 671 nm; (e) These were direct sequenced using the FS cycle se- another 45 min 671 nm; (f) another 60 min 671 nm; (g) another 60 quencing kit (Perkin Elmer) and an ABI 373a se- min 671 nm; (h) another 60 min 671 nm; (i) 5 min 501 nm. (B) quencer. The amplified region of the gene excluded 17 Normalised difference spectra (dashed lines) constructed using the bps at the 5% end and 93 bps at the 3% end of the gene. curves shown in Fig. 2A; (j) a–b; (k) h–i. These are best fit by a u u porphyropsin template with max 533 nm, and a rhodopsin with max 522 nm respectively (solid lines). 2.5.2. Southern blot analysis Genomic DNA was digested with the restriction en- donucleases AluI, HinfI and HindIII and the products 6 u 6 2.4.2. Determination of isual pigment max alues electrophoresed in a 1% agarose gel with 1×TAE A retinal extract, as well as an outer segment suspen- buffer. The gel was prepared for transfer using standard sion, represents a mixture of all the accessible visual methods and blotted using 20×SSC. The nylon mem- pigments within a retina, as well as any other pigments brane (Magna Charge) was pre-hybridised at 55°C for 1 and/or impurities. In order to isolate the visual pig- hin1×Denhart’s solution containing 100 mg/ml ments within such mixtures spectrophotometrically, salmon sperm DNA. A probe consisted of a 259 bp they must be subjected to partial bleaching (Knowles & fragment amplified from the 5% end of the M. niger Dartnall, 1977; Douglas et al., 1995). Although only gene, radiolabelled by the random priming method with one standard partial bleach was performed on an outer [a-32P]-dCTP. Hybridisation was carried out at 52.5°C segment suspension in this study, the bleaching proto- for 48 h, followed by 20 min washes at room tempera- col used on both retinal extracts and outer segment ture in 2×SSC and 0.5% SDS, at 52.5°C in 1×SSC R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2821

Fig. 3. The opsin gene in Malacosteus niger. (A) Southern blot of restriction enzyme-digested M. niger genomic DNA hybridised at low stringency with a 259 bp M. niger opsin gene probe. Lane 1: HindIII, Lane 2: AluI, Lane 3: HinfI. Arrows indicate the positions of molecular weight markers (1 kb ladder and X174/HaeIII digest). (B) Amino acid sequence of M. niger opsin aligned with the rod opsin of goldfish (Johnson, Grant, Zankel, Boehm, Merbs, Nathans et al., 1993). The horizontal lines indicate the positions of the a-helical transmembrane regions. Amino acid identity is indicated by a dot, missing data by a dash. and 0.5% SDS, and at 52.5°C in 1×SSC and 0.1% slitwidths of 2.5 nm. The fluorescence emission spec- SDS. The membrane was autoradiographed at −80°C trum was generated with excitation at 418 nm, while the with Kodak XAR film using intensifying screens. excitation spectrum (roughly equivalent to the absorption spectrum of the pigment) was obtained by measur- 2.5.3. Phylogenetic analysis ing the emission at the peak wavelength of 678 nm. The MEGA computer package (Kumar, Tamura & Nei, 1993) was used to generate an opsin phylogenetic 2.6.2. Isolation, purification and absorption tree by the method of neighbour-joining (Saitou & Nei, spectroscopy 1987) from the frequency of nucleotide substitution in A total of 2 ml of thawed retinal suspension was pairwise comparisons of opsin sequences. The tree was placed in a partition system consisting of 10 ml rooted using the Drosophila Rh3 gene as an outgroup. methanol and 10 ml n-hexane, cooled to −20°C prior The degree of support for internal branching was as- to use and homogenised for 10 min using a sonicator sessed by bootstrapping with 500 replicates. (Branson 200), before being transferred into a separation funnel. After equilibration, the phases were sepa- 2.6. Analysis of the photosensitising pigment rated and the upper phase was re-extracted with 10 ml pure lower phase and the lower phase with 10 ml pure Cell suspensions were prepared at sea from one indi- upper phase of the equilibrated n-hexane-methanol-wa- vidual by placing both retinae in PIPES buffered saline ter (10:10:2) solvent system. The two hexane phase and agitating the mixture for a few seconds using a extracts were combined, as were the two methanol vortex mixer, followed by centrifugation (15 300×g, phase extracts. Absorption spectroscopy showed the 4°C, 10 min) and resuspension of the pellet in PIPES photosensitising pigment to be confined to the buffered saline containing 20% sucrose. This was frozen methanol phase. The combined methanol phase ex- for later analysis. In such preparations, the material is tracts were therefore diluted with distilled water and as close as possible to its native state. Some was utilised extracted with chloroform (2×6 ml). The combined directly on thawing for fluorescence spectroscopy, while chloroform extracts were then filtered through What- the photosensitiser was isolated from the remainder and man no. 1 filter paper in a small glass funnel and identified using absorption spectroscopy and electron concentrated to a volume of 0.5 ml. This solution, impact mass spectrometry (EIMS). which was faintly green in colour, was evaporated to dryness in an argon stream and the residual pigments 2.6.1. Fluorescence spectroscopy were dissolved in pure diethyl ether. After methylation

Fluorescent excitation and emission spectra of the with ethereal CH2N2 (diazomethane) for 0.5 h at room unpurified M. niger retinal cell suspension were temperature, the pigments were further purified by recorded at room temperature with a Perkin-Elmer preparative thin-layer chromatography (TLC) on silica- LS50 spectrofluorimeter using excitation and emission gel plates (Merck’s Si 60, particle size 5–40 mm, on a 2822 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 glass support, 3×20 cm; thickness of the silica layer 0.2 mm; eluent chloroform–acetone, 20:1, v/v). The resulting single spot of the chromatogram was scraped off and, following extraction with diethyl ether, the absorption spectrum of the photosensitising pigment it contained determined using a Cary 5E UV-Vis-NIR spectrophotometer.

2.6.3. Electron impact mass spectrometry The methyl esters of the puriﬁed photosensitising pigment, which from their absorption and ﬂuorescence spectra appeared to be magnesium-free chlorophyll derivatives (approximately 3 mg), were analysed using a Micromass Quatro II triple quadrupole mass spectrometer and methyl pheophorbide a as a standard. Each m sample was dissolved in 40 lofCH2Cl2 (methylene Fig. 5. Effectiveness of two different wavelengths at bleaching visual chloride). Using a syringe, a 2 ml aliquot was loaded pigment extracts of Chauliodus sloani. The abscissa is represented as onto the electrically heatable coil of the probe and the relative exposure as the photometer employed to measure light inten- solvent was allowed to evaporate before inserting the sity was not calibrated in absolute units. probe into the mass spectrometer. The sample was desorbed into the ion source by passing a programmed heating current through the coil. The most important tuning parameters for positive ion EI were: electron energy 70 eV, emission current 200 mA and source temperature 180°C.

3. Results

3.1. Visual pigments of M. niger

Bleaching of both retinal extracts (Fig. 1) and outer segment suspensions (Fig. 2) revealed the presence of only two visual pigments in the retinae of M. niger. Partial bleaching of seven pigment extracts indicated a u porphyropsin with, on average, a max at 542 nm and a u rhodopsin with max 517 nm. An average of four outer segment suspensions suggested similar pigments with u max values of 534 and 522 nm. Given the known u relationship between the max of rhodopsin and porphyropsin pigments (Dartnall & Lythgoe, 1965; Whitmore & Bowmaker, 1990), these measurements indicate a rhodopsin/porphyropsin pigment pair based on a single opsin. In none of the preparations was there any sign of a visual pigment at longer wavelengths. The unusual multipeaked appearance of the extracts and suspensions (Figs. 1A and 2A) is due to the presence of a chlorophyll-derived photosensitising pigment within the outer segments (see below).

3.2. The rod opsin gene

Spectrophotometric partial bleaching suggested the existence of only a single opsin within the M. niger Fig. 4. Effectiveness of different wavelengths at bleaching the visual pigments of Malacosteus niger. (A) Retinal extract; and (B) outer retina (see above). To verify this, degenerate primers segment suspension. Qualitatively similar results were obtained for a based on the sequence of ﬁsh rod opsin genes were used second retinal extract (data not shown). to amplify an opsin gene from M. niger genomic DNA R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2823

(gene bank accession no. AJ224691). The identifica- M. niger (Fig. 3A). However, it is not possible, by tion of this gene as encoding a rod opsin was confi- this method, to be entirely certain that other opsin rmed by cladistic analysis (data not shown) that genes are not present, since their identification de- grouped the gene with other rod opsins in a phyloge- pends on an unknown extent of sequence homology netic tree and by the absence of introns, a feature to the probe. The deduced amino acid sequence of that appears unique to rod opsin genes of teleost the M. niger opsin, aligned with that of a typical fishes (Fitzgibbon, Hope, Slobodyanyuk, Bellingham, shallow water teleost, the goldfish Carassius auratus, Bowmaker & Hunt, 1995; Hope, Partridge, Dulai & is shown in Fig. 3B. A number of amino acid Hunt, 1997). A 259 bp 5% fragment of this gene was residues which are known to be important for the used to probe a Southern blot of restriction enzyme- function of visual pigments are conserved in the M. digested M. niger genomic DNA. The stringency con- niger opsin. These include Lys-296, the site of the ditions of this hybridisation were adjusted such that Schiff base linkage to retinal (Bownds, 1967; Wang, cross-hybridisation between the probe and members McDowell & Hargrave, 1980), Glu-113, the Schiff of other opsin gene classes, if present, would be ex- base counterion (Sakmar, Franke & Khorana, 1989; pected. In all cases, only a single band was seen, Zhukovsky & Oprian, 1989; Nathans, 1990a; Sakmar, consistent with the presence of a single opsin gene in Franke & Khorana, 1991), two Cys residues at positions 110 and 187 which form a disulphide bond (Karnik & Khorana, 1990), and Glu-134 and Arg-135 which are thought to interact directly with the G- protein transducin (Franke, Konig, Sakmar, Khorana & Hofman, 1990). As expected for a rod opsin, the chloride-binding pocket found in longwave cone pigments (Wang, Asenjo & Oprian, 1993) is absent from this protein. Compared to the rhodopsin pigment in rod photoreceptors of the goldfish, the rhodopsin pigment of M. niger is longwave shifted by around 18 nm. Previ- ous work on mammalian rod and cone pigments has shown that spectral tuning residues are generally located in the a-helical transmembrane regions of the protein (Merbs & Nathans, 1993; Asenjo, Rim & Oprian, 1994). Two-dimensional crystallographic analysis of bovine rod opsin has demonstrated that the seven helices of the opsin protein form a central hy- drophilic pocket that contains the chromophore (Schertler, Villa & Henderson, 1993) and a model based on conserved residues across 206 G protein linked receptor proteins (Baldwin, 1993) can be used as a framework for orientating each helix with respect to the exterior lipid membrane and the central hy- drophilic pocket. Only non-conservative or polar to non-polar changes in residues that are located either adjacent to this pocket or face another helix appear to affect the spectral tuning of the resulting pigment (Nakayama & Khorana, 1990; Nathans, 1990b; Merbs & Nathans, 1993; Asenjo et al., 1994; Hunt, Fitzgibbon, Slobodyanyuk & Bowmaker, 1996). Ap- plying these rules, there are only six potential tuning substitutions between the goldfish and M. niger opsins (Table 1). Of these, only substitutions at sites Fig. 6. (A) Fluorescent excitation (dotted line) and emission (solid 83 (Nathans, 1990b; DeCaluwe´, Bovee-Geurts, Rath, line) spectra of an unpurified M. niger retinal cell suspension in 20% Rothschild & de Grip, 1995) and 261 (Merbs & sucrose in PIPES buffered saline. (B) Absorbance spectrum of the purified M. niger photosensitising pigment (dashed line), along with Nathans, 1993; Asenjo et al., 1994; Yokoyama, Knox spectra of standard samples of pheophorbide a (solid line) and & Yokoyama, 1995) are known from site directed pyropheophorbide a (dotted line) prepared from pure chlorophyll a. mutagenesis to cause spectral shifts. 2824 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832

Table 1 Substitutions at potential amino acid tuning sites

u b Species max (nm) TMI TMII TMIII TMV TMVI

51a 82a 83a 168a 208a 261a

M. niger 517 Ser Ala Asn Ser Tyr Tyr Carassius auratus 499 Gly Ser Asp Ala Phe Phe

a Numbers refer to amino acid sites at which the identiﬁed substitutions are present. b TM, transmembrane domains.

3.3. Photosensiti6ity ment, which migrated with the same speed as the reference compounds, pheophorbide a methyl ester

As would be expected if light were being absorbed (Rf =0.54) and pyropheophorbide a methyl ester (Rf = directly by the visual pigments, 630 and 637 nm illumi- 0.53), prepared from pure chlorophyll a (Hynninen, nation is the most effective at bleaching M. niger visual 1991). When this spot was removed from the plate and pigments, while 702 nm light is the least effective (Fig. extracted with diethyl ether, the UV-visible absorbance 4). However, inconsistent with a direct action of light spectrum of the resulting purified compound (Fig. 6B) on the visual pigments, exposure to 671 nm is always was similar to that of standard samples of pheophor- more effective at bleaching them than a 654 stimulus. bide a and pyropheophorbide a prepared from pure The data can, however, be explained if the unusual chlorophyll a, and also virtually identical to the spec- outer segment pigment with an absorption maximum trum of the methyl ester of chlorobium (Cbm) around 667 nm (Fig. 6B) is acting as a photosensitiser. pheophorbide 650, fraction 2 (Holt, 1966). The absorp- In C. sloani, whose retinae do not contain a photosensi- tion spectrum of the purified pigment is similar to the tising pigment, however, 654 nm illumination is more fluorescent excitation spectrum of the simple retinal effective than 671 nm light (Fig. 5), consistent with homogenate (Fig. 6A), indicating that no major chemi- visual pigment bleaching being the result of direct cal changes took place during the isolation process. The action of light on the visual pigment of this species. peaks corresponding to the Qx and Qy transitions are slightly red-shifted in the fluorescence excitation spec- 3.4. Identity of photosensitising pigment trum. This is probably due to pigment-pigment or pigment–protein interactions when the pigment is in its 3.4.1. Fluorescence spectroscopy native state. Although the retinal cell suspensions contained relatively high concentrations of visual pigments and 3.4.3. Electron impact mass spectrometry carotenoids (the latter derived from the astaxanthin- The electron impact mass spectrum obtained from based tapetum of M. niger; (Denton & Herring, 1971; the reference compound, methyl pheophorbide a, (Fig. Locket, 1977; Somiya, 1982; Bowmaker et al., 1988)), 7a) is identical with that described in the literature only the photosensitising pigment fluoresced apprecia- (Budzikiewicz, 1978). The spectrum shows the M+ ion bly. Both the excitation and emission spectra (Fig. 6A) at Da/e 606 and the main fragment ions at Da/e 547, + suggest a magnesium-free chlorophyll derivative such as 519 and 459, representing [M– CO2CH3] ,[M– + pheophorbide ( Pheide) a or pyropheophorbide CH2CH2CO2CH3] and [M– CO2CH3 – CH2CH2CO2- + ( pyroPheide) a. Note in particular the excitation peaks H– CH3] , respectively. The spectrum derived from at 514 and 544 nm, corresponding to the Qx (1, 0) and the sample containing the methyl esters of the chloro- Qx (0, 0) transitions, respectively. These peaks are of phyll derivatives isolated from M. niger retinae, how- lower intensity when a central chelated magnesium ion ever, is very different, showing a more complex series of is present (Weiss, 1978). signal batteries (Fig. 7b). Many of the highest signals show a regular separation of 14 mass units, suggesting 3.4.2. Isolation, purification and absorption spectroscopy that the sample contained a series of homologues. When homogenised retinal suspensions were parti- Because the UV-visible spectrum of the purified M. tioned in the hexane/methanol system, the photosensi- niger photosensitising pigment closely matched the tising pigment was found entirely in the methanol spectrum for a Chlorobium pheophorbide 650 methyl phase, indicating that it possessed a free propionic acid ester, fraction 2 (Holt, 1966), the mass numbers shown residue, not esterified with a long-chain alcohol such as in Fig. 7b were compared with those calculated for the phytol or farnesol. The TLC analysis showed only one methyl esters of 132-demethoxycarbonyl-3-devinyl-3-(1- spot for the methyl esters of the photosensitising pig- hydroxyethyl) pheophorbide a homologues (methyl R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2825

Fig. 7. Electron impact mass spectra of (a) methyl pheophorbide a and (b) the puriﬁed Malacosteus niger photosensitising pigment. esters of 31(R,S)-hydroxy-phytochlorin homologues) These methylated Chlorobium pheophorbides are (Fig. 8, Table 2, 1a–n). The methylation is necessary to derived from the known (Smith & Goff, 1985; Smith, make the derivatives sufﬁciently volatile for EIMS. Goff & Simpson, 1985) Chlorobium chlorophyll 660 2826 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832

4. Discussion

4.1. Visual pigments of M. niger

Retinal detergent extracts, as in Aristostomias sp. (O’Day & Fernandez, 1974; Knowles & Dartnall, 1977; Bowmaker et al., 1988; Crescitelli, 1991; Partridge & Douglas, 1995) and P. microdon (Bowmaker et al., 1988), reveal two visual pigments in the M. niger retina u ( max values approximately 517 and 542 nm present study; see also Bowmaker et al., 1988) confirming mi- crospectrophotometric studies (Bowmaker et al., 1988; Partridge et al., 1989). These pigments are considerably Fig. 8. Structures of known Chlorobium pheophorbides (Bpheides longwave shifted compared to those of other deep-sea c/d) and Chlorobium chlorophylls (Bchls c/d). The structures of the fish. Unfortunately, the high optical density of the various homologues are indicated in Table 2; BPheides c, 2a–f; longwave reflecting tapetum of M. niger masks any Bpheides d, 2g–n (1a–n are the corresponding methyl esters); and BChls c, 3a–f; BChls d, 3g–n. visual pigment in a retinal wholemount preparation, which in A. tittmanni and P. microdon indicated the presence of a third, even longer wave absorbing, pig- u ment ( max values approximately 588 and 595 nm, respectively: Douglas et al., 1998a; Partridge & Dou- (renamed bacteriochlorophyll c) and Chlorobium glas, 1995: see also Denton et al., 1970, 1985). We feel chlorophyll 650 (renamed bacteriochlorophyll d) homo- the retinal wholemount technique owes its success to logues (Fig. 8; structures 3a–f and 3g–n, respectively). the fact that the visual pigments are as close as possible Also all mass numbers of the expected main fragment to their natural state, since the photoreceptor outer ions (Table 2) can be found in the mass spectrum of segments remain intact and the retinae are not sub- Fig. 7b. The highest intensities of the signals corre- jected to any chemical reagents. As an alternative we sponding to the expected main fragments from deriva- therefore isolated M. niger photoreceptor outer seg- tives 1g–n (Table 2) suggest the predominance of the ments by suspending them in a 20% sucrose solution, Chlorobium pheophorbide 650 homologues in M. niger which, like retinal wholemounts, involves minimal dis- outer segments. The mass spectrum also exhibits molec- ruption of photoreceptor membranes. However, such ular ions above 622 Da/e with increments of 14 mass suspensions, in contrast to the wholemounts of A. units, suggesting the presence of unusually high molecu- tittmanni and P. microdon, still revealed only two visual u lar weight homologues of bacteriochlorophyll pheo- pigments ( max values approximately 522 and 534 nm), phorbides c or d that are hitherto undescribed. similar to those observed in extract and by MSP. The

Table 2 Expected molecular ions and main fragment ions for bacteriochlorophyll c derivatives (1a–1f) and bacteriochlorophyll d derivatives (1g–1n)a,b

Derivative R8 R12 R20 Conﬁguration at M+ [M–18]+ [M–benzylic-cleavage [M–59]+ [M–73]+ [M–87]+ [M–115]+ C–31 group]+

1a Et Me Me R 580 562 565 521 507 493 465 1b Et Et Me R 594 576 579 535 521 507 479 1c Prn Et Me R 608 590 579, 593 549 535 521 493 1d Prn Et Me S 608 590 579, 593 549 535 521 493 1e Bui Et Me R 622 604 579, 607 563 549 535 507 1f Bui Et Me S 622 604 579, 607 563 549 535 507 1g Et Me H R 566 548 551 507 493 479 451 1h Et Et H R 580 562 565 521 507 493 465 1i Prn Me H R 580 562 551 521 507 493 465 1j Prn Et H R 594 576 565, 579 535 521 507 479 1k Bui Me H S 594 576 551 535 521 507 479 1l Bui Et H S 608 590 565, 593 549 535 521 493 1m Pnneo Me H S 608 590 551 549 535 521 493 1n Pnneo Et H S 622 604565, 607 563 549 535 507

a + + + + + + + + Fragmentations, [M–18] =[M–H2O] ; [M–59] =[M–CO2CH3] ; [M–73] =[M–CH2CO2CH3] ; [M–87] =[M–CH2CH2CO2CH3] ; + + [M–115] =[M–(CH2CH2CO2CH3–CO)] . b Me, methyl; Et, ethyl; Pri, isopropyl; Bui, isobutyl; Pnneo, neopentyl; prn, n-propyl. R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2827 absorbance spectra suggest that these pigments form a far-red light. Unfortunately, there is no tractable rhodopsin/porphyropsin pigment pair with a single method that one could apply to the observed data to opsin bound in some photoreceptors to the chro- model the system and to calculate this efficiency gain. mophore retinal, while in others the same opsin com- Not only does the M. niger retina contain two visual bines with 3,4-dehydroretinal. The presence of a single pigments (which is a complication that could be mod- opsin gene in the M. niger genome that encodes a rod elled) but the empirical data involve the bleaching of opsin was also indicated by molecular and cladistic visual pigments initially with absorbances close to 0.4. analysis. This is too great for the simple modelling of bleaching The rhodopsin pigment identified in the retina of M. kinetics, such as employed by, for example, Makino, niger is longwave-shifted by around 18 nm compared to Taylor & Baylor (1991), which tacitly assume low pig- that of the goldfish, and a comparison of the deduced ment densities and hence enable certain approximations amino acid sequences of the respective opsins identifies to be employed. As made clear by Liebman (1972), a total of six amino acid differences (Ser51Gly, approximations inherent in the classic bleaching equa- -E Asp82Ser, Asn83Asp, Ser168Ala, Tyr208Phe, and tion Pt =P0e (where Pt is the pigment concentration Tyr261Phe) that involve non-conservative or polar/non- at time t, P0 is that before bleaching, E the total light polar changes at potential tuning sites. Tyr261 is also exposure, and the photosensitivity) are only valid for present in the longwave-shifted rod pigments of shallow a limited range of absorbances less than approximately water species of cottoid fish in Lake Baikal (Hunt et al., 0.05. Such approximations are appropriate for single 1996); mutagenesis and in vitro expression has shown cells (e.g. Makino et al., 1991) but not for the high that this substitution results in an 8 nm longwave shift pigment densities reported here. Furthermore, Dart- in fish rhodopsin (Yokoyama et al., 1995). In contrast, nall’s analytical method of visual pigment bleaching, Asn83 in M. niger would be expected to result in a the method of photometric curves, also cannot be used, spectral shift in the opposite direction (i.e. shortwave) as it is specifically invalidated by the presence of cata- of around 6 nm (Nathans, 1990b; DeCaluwe´ et al., lysts or photosensitisers (Dartnall, 1972). 1995), requiring therefore a longwave-shift of around We are thus unable to quantify the efficiency of the 16 nm as the aggregate effect of substitutions at the photosensitiser except by comparison with data ob- other four sites. tained from similar experiments conducted on species lacking a retinal photosensitiser. Such data are pre- 4.2. E6idence for a retinal photosensitiser sented in Fig. 5, in which the rate of bleaching u Chauliodus sloani visual pigment (a rhodopsin with max The outer segments of M. niger photoreceptors con- 485 nm: Douglas & Partridge, 1997) is shown. This tain not only visual pigments but also one or more comparison reveals two main points: Firstly, the data pigments that give rise to the unusual multipeaked from C. sloani, in contrast to those of M. niger, con- appearance of retinal extracts (Fig. 1A; Bowmaker et form to a straight line. This is to be expected because al., 1988) and MSP records (Partridge et al., 1989). C. sloani has only one retinal visual pigment, whereas Since this pigment complex has an absorption maxi- the M. niger retina contains two. Secondly, the rate of mum around 667 nm (Fig. 6B), which is closer to M. bleaching of C. sloani visual pigment increases as the u niger’s own far red bioluminescence (emission maxi- wavelength of bleaching approaches the max of the mum approximately 705 nm; Widder et al., 1984) (Fig. visual pigment, which for wavelengths close to the 9) than the absorption maxima of its visual pigments photosensitiser absorption peak, is not the case for M. u ( max values approximately 520 and 540 nm), it is niger. possible that this pigment, as suggested by Bowmaker The three genera of stomiid that produce far red et al. (1988), acts as a photosensitiser, absorbing light at bioluminescence have thus evolved two distinct ways of its absorption peak in the far red and indirectly bleach- enhancing their sensitivity to this illumination. The ing the shorter wave sensitive visual pigments. That this retinae of A. tittmanni and P. microdon contain, in is likely to be the case is shown by the fact that addition to the two longwave shifted visual pigments exposure to 671 nm illumination, which is close to the found in all three genera, further extreme longwave wavelength of maximum absorption of the putative sensitive pigments (Partridge & Douglas, 1995; Douglas photosensitiser, is more effective at bleaching the visual et al., 1998a). M. niger, on the other hand, enhances its pigments than a 654 nm stimulus (Fig. 4). If the stimu- longwave sensitivity by coupling its two visual pig- lating light was bleaching the visual pigments directly, ments, which on their own are relatively insensitive to as is the case for C. sloani (Fig. 5), the shorter wave the red bioluminescence, to a longwave absorbing, stimulus would be the more effective. chlorophyll-derived, photosensitising pigment. Ideally, the increased rate of bleaching attributable to Thus, three members of the family stomiidae have the M. niger photosensitiser should be quantified to evolved two different solutions to a common problem. ascertain its effectiveness in enhancing the visibility of Interestingly, however, the species distribution of these 2828 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832

Fig. 9. (a) Photograph of the anterior half of Malacosteus niger. The large, red, tear-drop shaped photophore below the eye that produces longwave bioluminescence is clearly visible, as is the post orbital photophore that produces shortwave light. The red colour of the eye is due to its longwave reﬂecting tapetum (courtesy Dr T. Frank). (b) Summary of absorbance spectra of Malacosteus niger retinal pigments and emission u spectra of its bioluminescence. The yellow and red curves represent two visual pigment templates: a rhodopsin with max 515 nm and a u porphyropsin with max 540 nm, respectively. The emission spectra for the shortwave postorbital photophore (dotted blue) and the longwave emitting suborbital photophore (dotted red) are taken from Widder et al. (1984). The absorption spectrum of the presumed photosensitiser (green) is represented by a puriﬁed diethyl ether extract of a retinal suspension. two methods of perceiving far red illumination does not (Nelson, 1994). It is interesting that, in line with their relate to the accepted phylogeny of this family. While retinal specialisations, and again at odds with their the genera Aristostomias and Malacosteus are placed phylogeny, the photophore structure of Aristostomias within the same subfamily (Malacosteinae), Pachysto- and Pachystomias sp. is also very similar and quite mias sp. is grouped within the distinct Melanostomiinae distinct from that of M. niger (Herring pers. com.). R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 2829

4.3. Source of the photosensitiser 132-hydroxypheophorbide a. It is therefore perhaps not as far fetched as it might at first sight appear to suggest The photosensitising pigment located in the outer that dietary chlorophyll derivatives have a photosensi- segments of the M. niger retina is composed of several tising role in a deep-sea fish eye. Chlorobium pheophorbides; that is a mixture of defarnesylated and demetallated derivatives of Chlorobium 4.4. Mechanism of photosensitisation chlorophylls 660 (bacteriochlorophylls c) and Chloro- bium chlorophylls 650 (bacteriochlorophylls d), with Although the photoreceptors of some invertebrates the latter predominating. contain opsin-bound carotenoid-derived chromophores We know of no instance of chlorophyll derivatives (retinol or 3-hydroxyretinol) which act as photosensitis- being synthesised de novo in vertebrates. Most likely, ers (Kirschfeld, Franceschini & Minke, 1977; Kirschfeld the photosensitiser is, like the visual pigment chro- & Vogt, 1986; Vogt, 1989), this is the first demonstra- mophores, derived from dietary sources. M. niger,in tion of a pigment with a similar function in a verte- fact, has a most unusual diet compared with its close brate. Apart from the identity of the photosensitising relatives. The stomiid dragon fish are a relatively large pigment, there is, however, a major difference between family of deep-sea fishes and most, including Aristosto- the invertebrate system and that proposed here. In mias and Pachystomias, eat mainly myctophids (deep- invertebrates high energy shortwave photons are ab- sea lantern fish). In contrast, M. niger feeds primarily sorbed by the photosensitiser and excite longer wave on euchaetid and aetideid copepods (Sutton & Hop- sensitive visual pigments, while in M. niger relatively kins, 1996), a diet for which it morphologically appears low energy longwave photons result in excitation of most unsuited, having the largest gape of any stomiid shorter wave visual pigments. and lacking gill rakers as an adult (Sutton & Hopkins, At present we can only speculate as to the mecha- 1996). These copepods in turn prey on smaller cope- nism of energy transfer from the photosensitiser to the pods which ingest photosynthetic organisms. The two visual pigments. In photosynthetic light-harvesting sys- principal modifications to chlorophylls required to pro- tems, the predominant mode of energy transfer involves duce the photosensitising pigment, hydrolysis of the the migration of singlet excited states between chloro- farnesyl group and demetallation, can easily take place phyll species (Clayton, 1980). However, such a mecha- in the alimentary tracts of organisms. Indeed such nism would not work in this case because the pheophorbides are known to exist in the guts of cope- singlet-singlet energy transfer from the pheophorbide to pods and are widely used as an assay of zooplankton the visual pigment must be highly endothermic. It is ingestion rates (Mackas & Bohrer, 1976; Boyd, Smith & possible, however, that the mechanism involves inter-

Cowles, 1980; Dagg & Grill, 1980; Head, 1992). system crossing to generate a triplet state (T1)ofthe However, the chlorophyll derivatives found in the pheophorbide which could then transfer energy to the retina of M. niger are not derivatives of chlorophylls a ground state of the 11-cis chromophore of the visual and b, which are commonly found in plankton, but are pigment to generate its triplet excited state (Turro, derived from bacteriochlorophylls c and d, which are 1978). Such a triplet state of the visual pigment chro- known to occur only in photosynthetic green sulphur mophore has in fact been proposed as an intermediate bacteria (order Rhodospirillales; suborder Chlorobi- of photoisomerisation (Jensen, Wilbrandt & Bensasson, ineae, families Chlorobiaceae; bacteriochlorophlls c and 1989; Tahara, Toleutaev & Hamaguchi, 1994). The d and Chloroﬂexaceae; bacteriochlorophyll c) (Pfennig, energy transfer from the triplet excited state of the

1978; Tru¨per & Pfennig, 1978). Since these organisms pheophorbide a derivative (ET =30.7 kcal/mol: Seely, are obligate anaerobes living in subtidal marine sedi- 1978; Krasnovsky, Neverov, Egorov & Roeder, 1988; ments, it is unclear how they are incorporated into the Krasnovsky, Neverov, Egorov, Roeder & Levald, 1990) open-ocean food chain leading to M. niger. Interest- to the triplet excited state of the 11-cis chromophore of B ingly, however, anaerobic purple sulphur bacteria pos- the visual pigment (ET of 11-cis retinal is 38 kcal/ sessing bacteriochlorophyll a have recently been mol: Jensen et al., 1989) might still be endothermic but described in the guts of some pelagic copepods (Proc- the long lifetime (1 ms) of the triplet state pheophor- tor, 1997). bide and the high quantum yield of its formation Dietary derived pheophorbide a has also been shown (approximately 0.8) would, nevertheless, make the to be incorporated into mouse skin (Weagle, Paterson, triplet photosensitisation mechanism highly probable.

Kennedy & Pottier, 1988). Furthermore, similar chloro- Besides, the ET of the protonated Schiff base 11-cis phyll derivatives are known to be potent photosensitis- chromophore of the visual pigment is unknown to our ers for mammals (Kimura, 1987; Spikes & Bommer, knowledge and could be considerably lower than 38 1991) inducing, for instance, photodermatitis in hu- kcal/mol, which would imply exothermicity of the en- mans as a consequence of eating processed foods con- ergy transfer. However, the triplet-triplet energy trans- taining pheophorbide a, pyropheophorbide a or fer can only take place by the electron exchange 2830 R.H. Douglas et al. / Vision Research 39 (1999) 2817–2832 mechanism (also called collisional mechanism), which Proctor gave much useful advice and allowed access to requires, to be efficient, a short distance (B1.5 nm) unpublished data. between the energy donor and the acceptor (Turro, 1978). An interesting possibility is the ground-state complex formation between the pheophorbide and the References protonated Schiff-base 11-cis chromophore of the visual pigment, to ensure such a short distance. Asenjo, A. B., Rim, J., & Oprian, D. D. (1994). Molecular determi- nants of human red/green color discrimination. Neuron, 12, 1131– 1138. 4.5. Mismatch between photosensitising pigment Baldwin, J. (1993). The probable arrangement of the helices in G absorbance and bioluminescent emission protein-coupled receptors. EMBO Journal, 12, 1693–1703. Bowmaker, J. K., Dartnall, H. J. A., & Herring, P. J. (1988). Despite the fact that the identified photosensitiser, Longwave-sensitive visual pigments in some deep sea fishes: segre- with its absorption maximum around 667 nm, is more gation of paired rhodopsins and porphyropsins. Journal of Com- parati6e Physiology A, 163, 685–698. longwave sensitive than the two visual pigments of M. Bownds, D. (1967). Site of attachment of retinal in rhodopsin. u niger ( max values approximately 520 and 540 nm), the Nature, 216, 1178–1181. match with the far-red bioluminescence (wavelength of Boyd, C. M., Smith, S. L., & Cowles, T. J. (1980). Grazing patterns maximum emission 705 nm; Widder et al., 1984) is still of copepods in the upwelling system off Peru. Limnology and not perfect (Fig. 9). Two options would appear to be Oceanography, 25(4), 583–596. Budzikiewicz, H. (1978). Mass spectra of porphyrins and related available to these animals to increase sensitivity to their compounds. In D. Dolphin (Ed.), The Porphyrins, Vol. 3 part A own bioluminescence even further; develop a more (chapter 9). New York: Academic Press. longwave sensitive photosensitiser, or shorten the wave- Clayton, R. K. (1980). Photosynthesis: physical mechanisms and chem- length of their bioluminescence. However, neither of ical patterns. Cambridge: Cambridge University. these options is desirable: a photosensitiser with an Crescitelli, F. (1989). The visual pigments of a deep-water Mala- costeid fish. Journal of the Marine Biological Association of the absorption maximum shifted towards longer wave- United Kingdom, 69, 43–51. lengths will be less efficient, since energy transfer would Crescitelli, F. (1991). Adaptations of visual pigments to the photic become more endothermic. The second alternative, environment of the deep-sea. Journal of Experimental Zoology shortening the wavelength of their bioluminescent emis- (Supplement), 5, 66–75. sion, appears more feasible, since the light produced Dagg, M. J., & Grill, D. W. (1980). Natural feeding rates of Cen- tropages typicus females in the New York Bight. Limnology and within the suborbital photophore of M. niger is rich in Oceanography, 25(4), 597–609. shortwave photons which are selectively eliminated by Dartnall, H. J. A. (1972). Photosensitivity. In H. J. A. Dartnall, an absorbing filter within the photophore (Widder et Handbook of sensory physiology, vol. VII/1 (pp. 122–145). Berlin: al., 1984; Denton et al., 1985). Given a less dense filter, Springer-Verlag. the bioluminescence maximum could quite easily be Dartnall, H. J. A., & Lythgoe, J. N. (1965). The spectral clustering of visual pigments. Vision Research, 5, 81–100. shifted to shorter wavelengths to match the photosensi- DeCaluwe´, G. L. J., Bovee-Geurts, P. H. M., Rath, P., Rothschild, tiser’s absorption more closely. However, this would K. J., & de Grip, W. J. (1995). Effect of carboxyl mutations on inevitably increase the visibility of this light to other functional properties of bovine rhodopsin. Biophysical Chemistry, animals within the deep-sea. Presumably an adaptive 56, 79–87. compromise has been reached in which the photosensi- Denton, E. J., Gilpin-Brown, J. B., & Wright, P. G. (1970). On the filters in the photophores of mesopelagic fish and on a fish tiser gives M. niger adequate visual sensitivity to its emitting red light and especially sensitive to red light. Journal of own far red bioluminescence while maintaining the Physiology (London), 208, 72–73. invisibility of this signal to other animals. Denton, E. J., & Herring, P. (1971). Report to the council. Journal of the Marine Biological Association of the United Kingdom, 51, 1035. Denton, E. J., Herring, P. J., Widder, E. A., Latz, M. F., Case, J. F. (1985). The roles of filters in the photophores of oceanic animals Acknowledgements and their relation to vision in the oceanic environment. Proceed- ings of the Royal Society of London B, 225, 63–97. This work was funded by grants from the Natural Denton, E. J. (1990). Light and vision at depths greater than 200 m. Environment Research Council (R.H. Douglas, J.C. In P. J. Herring, A. K. Campbell, M. Whitfield, & L. Maddock, Partridge, D.M. Hunt) and the Royal Society (J.C. Light and life in the sea (pp. 127–148). Cambridge: Cambridge University. Partridge). We would like to thank the officers and Douglas, R. H., Partridge, J. C., & Marshall, N. J. (1998a). The crew of RRS Challenger, A.T. Schilling and G.N. visual systems of deep-sea fish I—optics, tapeta, visual and Teagh for support at sea, A.J. Hope for assistance with lenticular pigmentation. Progress in Retinal and Eye Research, the photosensitivity experiments, and H. Bjo¨rk (VER- 17(4), 597–636. IFIN Project researcher, PO Box 55, A.I. Virtasenaukio Douglas, R. H., Partridge, J. C., & Hope, A. J. (1995). Visual and lenticular pigments in the eyes of demersal deep-sea fishes. Journal 1, FIN-00014 University of Helsinki, Finland) for the of Comparati6e Physiology A, 177, 111–122. measurement of the mass spectra. Drs Peter Herring, Douglas, R. H., & Partridge, J. C. (1997). On the visual pigments of Edie Widder, Tammy Frank, Tracey Sutton and Lita deep-sea fish. 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