Longwave-sensitive visual pigments in some deep-sea fishes: segregation of ‘paired’ rhodopsins and porphyropsins

Summary Microspectrophotometric examination of individual rods, and partial bleaching of visual pigment extracts from three species of deep-sea fish,Aristostomias grimaldii, Malacosteus niger andPachystomias microdon, suggest the presence of a rhodopsin-porphyropsin system of paired pigments.Aristostomias andMalacosteus have a P552₂-P517₁ pair, whilePachystomias has a P544₂-P513₁ pair. In contrast to most pigmentpair systems, each pigment is restricted to a single class of rod, thus giving the fish two spectrally distinct classes of photoreceptor. It is suggested that the longwave sensitive rods are an adaptation enabling these species to perceive their own deep-red bioluminescence. The astaxanthin-based red tapetum inMalacosteus, and a chlorin-like pigment, possibly acting as a photosensitizer and found in the rod outer segments ofMalacosteus, may also act to increase longwave sensitivity.     

On the visual pigments of deep-sea fish

The retinal visual pigments of 52 species of deep-sea fish were measured by partial bleaching of detergent extracts. The retinae of 45 species contained only a single rhodopsin with maximum absorbance (λ_max) at a wavelength between 474 and 490 nm, matching both the region of highest intensity downwelling sunlight and the maximum emission of most deep-sea bioluminescence. Seven species were shown to have more than one visual pigment within their retinae and these had λ_max values that generally fell outside the usual range. One of these, Bonapartia pedaliota , was particularly interesting as, unlike most such multipigment species, it had one rhodopsin and one porphyropsin pigment, apparently based on different opsins. The relative proportions of the visual pigments in the seven multipigment species are presented.     

Genetic analyses of visual pigments of the pigeon (Columba livia)

We isolated five classes of retinal opsin genes rh1(Cl), rh2(Cl), sws1(Cl), sws2(Cl), and lws(Cl) from the pigeon; these encode RH1(Cl), RH2(Cl), SWS1(Cl), SWS2(Cl), and LWS(Cl) opsins, respectively. Upon binding to 11-cis-retinal, these opsins regenerate the corresponding photosensitive molecules, visual pigments. The absorbance spectra of visual pigments have a broad bell shape with the peak, being called lambdamax. Previously, the SWS1(Cl) opsin cDNA was isolated from the pigeon retinal RNA, expressed in cultured COS1 cells, reconstituted with 11-cis-retinal, and the lambdamax of the resulting SWS1(Cl) pigment was shown to be 393 nm. In this article, using the same methods, the lambdamax values of RH1(Cl), RH2(Cl), SWS2(Cl), and LWS(Cl) pigments were determined to be 502, 503, 448, and 559 nm, respectively. The pigeon is also known for its UV vision, detecting light at 320-380 nm. Being the only pigments that absorb light below 400 nm, the SWS1(Cl) pigments must mediate its UV vision. We also determined that a nonretinal P(Cl) pigment in the pineal gland of the pigeon has a lambdamax value at 481 nm.     

The cone visual pigments of an Australian marsupial,the tammar wallaby (Macropus eugenii): sequence,spectral tuning,and evolution

Studies on marsupial color vision have been limited to very few species. There is evidence from behavioral, electroretinographic (ERG), and microspectrophotometric (MSP) measurements for the existence of both dichromatic and trichromatic color vision. No studies have yet investigated the molecular mechanisms of spectral tuning in the visual pigments of marsupials. Our study is the first to determine the mRNA sequence, infer the amino acid sequence, and determine, by in vitro expression, the spectra of the cone opsins of a marsupial, the tammar wallaby (Macropus eugenii). This yielded some information on mechanisms and evolution of spectral tuning of these pigments. The tammar wallaby retina contains only short-wavelength sensitive (SWS) and middle-wavelength sensitive (MWS) pigment mRNAs. This predicts dichromatic color vision, which is consistent with conclusions from previous behavioral studies ( Hemmi 1999). We found that the wallaby has a SWS1 class pigment of 346 amino acids. Sequence comparison with eutherian SWS pigments predicts that this SWS1 pigment absorbs maximally (lambdamax) at 424 nm and, therefore, is a blue rather than a UV pigment. This (lambdamax) is close to that of the in vitro-expressed wallaby SWS pigment (lambdamax of 420 +/- 2 nm) and to that determined behaviorally (420 nm). The difference from the mouse UV pigment (lambdamax of 359 nm) is largely accounted for by the F86Y substitution, in agreement with in vitro results comparing a variety of other SWS pigments. This suggests that spectral tuning employing F86Y substitution most likely arose independently in the marsupials and ungulates as a result of convergent evolution. An apparently different mechanism of spectral tuning of the SWS1 pigments, involving five amino acid positions, evolved in primates. The wallaby MWS pigment has 363 amino acids. Species comparisons at positions critical to spectral tuning predict a lambdamax near 530 nm, which is close to that of the in vitro-expressed pigment (529 +/- 1 nm), but quite different from the value of 539 nm determined by microspectrophotometry. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

The visual pigments of a deep-sea myctophid fish Myctophum nitidulum Garman; an HPLC and spectroscopic description of a non-paired rhodopsin–porphyropsin system

Unusually for a deep-sea fish, the retina of the myctophid (lanternfish) Myctophum nitidulum was found to contain two visual pigments, shown by extract spectrophotometry to be maximally sensitive at 468 and 522 nm, respectively, giving this species one of the broadest spectral ranges of all deep-sea fishes. High performance liquid chromatography (HPLC) indicated that the retina contained both A₁- and A₂-based chromophores. Surprisingly, the maximum absorbance (λ_max) values of the two visual pigments were too far apart to form a rhodopsin–porphyropsin 'pigment pair', suggesting they were based on distinct opsins each linked to a different chromophore. This might be an adaptation to the detection of both long-wave bioluminescence and residual shorter-wave surface illumination, and could be related to this animal's tendency to migrate towards surface waters at night.     

Spectral tuning of obelin bioluminescence by mutations of Trp92

The Ca(2+)-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambdamax=390 nm) and monomodal fluorescence (lambdamax=425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity.     

Retinal pigments and carotenoids of the light-shading "spectacles" of the greenling Hexagrammus octogrammus]

Three light-sensitive pigments having lambdamax of 480, 505 and 540 nm which contain retinal as a chromophore were found in the digitonine extracts from the retina of H. octogrammus. In summer time, only one pigment (lambdamax equals 480 nm) was found, whereas during autumn and winter periods the other two pigments (lambdamax equals 505 and 540 nm) could be also observed together with the first one. The lambdamax 480 pigment is easily degraded when being exposed to light, although it is resistant to the effect of hydroxylamine. The other two pitments are less sensitive to the light, but are readily bleached by hydroxylamine. The yellow-orange coloured cells of the light-shading "spectacles" contain a mixture of beta-carotenoids. When extracted by petroleum ether, these beta-carotenoids display lambdamax at 425, 445 and 476 nm. Column chromatography on aluminium oxide revealed 6 fractions in the extracted carotenoids: light-yellow, dark-yellow, brown, reddish-brown, pink and pinkish ones. In the range from yellow to pink fractions, the contribution of the lambdamax 475 nm band increases, while that of two other ones-decreases.     

Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.     

Does the chromophore's ring move after photoexcitation of rhodopsin?

By comparing the shift of the absorption maxima when a visual pigment is converted to its lumirhodopsin photointermediate for two classes of pigments, we can infer whether or not the pigment's beta-ionone ring has left its binding site. We compare this shift for the long-wavelength sensitive visual pigment of chicken iodopsin (lambdamax = 571 nm), which has polar residues in the ring binding site that interact with the ring, with that for three pigments, which do not. We conclude that by the time the Lumi product of the pigment is formed, the ring has moved away from the ring binding site.     

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambdamax=469 nm) and obelin (lambdamax=482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambdamax=453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambdamax=501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection.     

Spectral tuning and the visual ecology of mantis shrimps

The compound eyes of mantis shrimps (stomatopod crustaceans) include an unparalleled diversity of visual pigments and spectral receptor classes in retinas of each species. We compared the visual pigment and spectral receptor classes of 12 species of gonodactyloid stomatopods from a variety of photic environments, from intertidal to deep water (> 50 m), to learn how spectral tuning in the different photoreceptor types is modified within different photic environments. Results show that receptors of the peripheral photoreceptors, those outside the midband which are responsible for standard visual tasks such as spatial vision and motion detection, reveal the well-known pattern of decreasing lambdamax with increasing depth. Receptors of midband rows 5 and 6, which are specialized for polarization vision, are similar in all species, having visual lambdamax-values near 500nm, independent of depth. Finally, the spectral receptors of midband rows 1 to 4 are tuned for maximum coverage of the spectrum of irradiance available in the habitat of each species. The quality of the visual worlds experienced by each species we studied must vary considerably, but all appear to exploit the full capabilities offered by their complex visual systems.     

A visual pigment from chicken that resembles rhodopsin: amino acid sequence, gene structure, and functional expression.

The amino acid sequence of a rhodopsin-like visual pigment from chickens has been determined by isolating and sequencing its gene. The predicted sequence is between 70% and 80% identical to bovine, human, and chicken rhodopsins and between 40% and 50% identical to human blue, green, and red cone pigments, the chicken red cone pigment, and cavefish long-wave cone pigments. The encoded pigment, produced by transfection of cDNA into cultured cells, absorbs maximally at 495 nm as determined from photobleaching difference spectra and reacts at 20 degrees C with 50 mM hydroxylamine with a half-time of 16 min. These properties, together with a high pI predicted from the amino acid sequence, suggest that this cloned gene encodes the chicken green pigment previously identified by biochemical and spectroscopic studies. This sequence defines a new branch of the visual pigment gene family.     

Effects of organic solvent vapors on the protochlorophyllic complex from etiolated leaves. Conditions for reversible and irreversible destruction]

The spectral properties and the ability of etyolated leaves pigments treated with organic solvent vapours (OS) for phototransformations were studied by measuring low temperature fluorescence spectra (-196 degrees C). Under the effects of OS the fluorescence at 655 nm was gradually decreased and that at 630--640 nm was increased. The effects of OS depended on the partial pressure of OS. The ability of the pigments for phototransformations was decreased with an increase in fluorescence at 630 nm. The emission maximum of fluorescence of the pigment formed in the light was shifted by 10--18 nm towards the shortwave region. Partial reversibility of the destroying effects of diethyl ester was found. A removal of the ester vapours resulted in a relative increase of fluorescence in the etyolated leaves at 640--645 nm and a decrease of the amount of "photo-inactive" pigment. The maximum of fluorescence of the pigment formed in the light was shifted towards the long-wave region (approximately 5 nm) as compared to the leaves irradiated in the presence of the ester. Partial functional reconstitution indicates that at least part of the pigment molecules are able to form a protochlorophyllide (protochlorophyll) -- protein complex, similar to the native one.     

Mechanisms of wavelength tuning in the rod opsins of deep-sea fishes.

The main object of this study was to investigate the molecular basis for changes in the spectral sensitivity of the visual pigments of deep-sea fishes. The four teleost species studied, Hoplostethus mediterraneus, Cataetyx laticeps, Gonostoma elongatum and Histiobranchus bathybius, are phylogenetically distant from each other and live at depths ranging from 500 to almost 5000 m. A single fragment of the intronless rod opsin gene was PCR-amplified from each fish and sequenced. The wavelength of peak sensitivity for the rod visual pigments of the four deep-sea species varies from 483 nm in H. mediterraneus and G. elongatum to 468 nm in C. laticeps. Six amino acids at sites on the inner face of the chromophore-binding pocket formed by the seven transmembrane a-helices are identified as candidates for spectral tuning. Substitutions at these sites involve either a change of charge, or a gain or loss of a hydroxyl group. Two of these, at positions 83 and 292, are consistently substituted in the visual pigments of all four species and are likely to be responsible for the shortwave sensitivity of the pigments. Shifts to wavelengths shorter than 480 nm may involve substitution at one or more of the remaining four sites. None of the modifications found in the derived sequences of these opsins suggest functional adaptations, such as increased content of hydroxyl-bearing or proline residues, to resist denaturation by the elevated hydrostatic pressures of the deep sea. Phylogenetic evidence for the duplication of the rod opsin gene in the Anguilliform lineage is presented.     

Comparative study of spectral and antioxidant properties of pigments from the eyes of two Mysis relicta (Crustacea, Mysidacea) populations, with different light damage resistance

Retinal visual and screening pigments of two populations (one marine and the other freshwater) of the opossum shrimp Mysis relicta Lovén (Crustacea, Mysidacea), which have different ocular tolerance to light, was investigated. Visual pigments were extracted by detergent and their bleaching difference spectra were determined. The difference between the visual pigment absorption maximum of the two populations correlated with their difference in spectral sensitivity. Using buffer or neutral methanol, a yellow pigment was extracted which had absorption maxima at 440 nm and 325 nm and bright blue fluorescence (λ_max 415 nm). A screening pigment (ommochrome) with maximum at 525 nm was extracted by acid methanol, and was probably related to the group of ommines. The eyes of the lake population had 1.8–2.7 times less of this pigment than the eyes of the sea population. The sea population is more resistant to photo-induced accumulation of thiobarbituric acid-reactive substances in eye tissues. This resistance may be due to the higher ommochrome content. Accepted: 8 December 1998     

Photochromic pigments in akinetes and pigment characteristics of akinetes in comparison with vegetative cells of Anabaena variabilis

Since akinete germination is triggered by light and the action spectrum for this process has features in common with the spectra of the two photochromic pigments, phycochromes b and d, a search was made for the presence of these phycochromes in akinetes of the blue-green alga. Anabaena variabilis Kützing. Allophycocyanin-B was also looked for, since the action spectrum for akinete germination points to a possible participation of this pigment too. Isoelectric focusing was used for purification of the pigments. The different fractions were investigated for phycochromes b and d by measuring the absorbance difference spectra: for phycochrome b. 500 nm irradiated minus 570 nm irradiated, and for phycochrome d, 650 nm irradiated minus 610 nm irradiated. For determination of allophycocyanin-B. fourth derivative analysis of absorption spectra was made for some of the fractions from the isoelectric focusing column. Phycochrome b was also assayed for by measuring in vivo absorption difference spectra. The assays were positive for all three pigments. The complete photosynthetic pigment systems were also studied by in vivo fluorescence measurements on both akinetes and vegetative cells of Anabaena variabilis. Fluorescence emission and excitation spectra at selected emission wavelengths were measured at room temperature and liquid nitrogen temperature. The energy transfer from phycoerythrocyanin to phycocyanin is very efficient under all conditions, as is the energy transfer from phycocyanin to allophycocyanin at room temperature. At low temperature, however, phycocyanin is partly decoupled from allophycocyanin, particularly in the akinetes; the energy transfer from allophycocyanin to chlorophyll a is less efficient at low temperature in both types of cells, but especially in akinetes. Delayed light emission was measured for both types of cells and found to be very weak in akinetes compared to vegetative cells. From this study it would seem that akinetes lack an active photosystem II, although the 691 nm peak in the 570 nm excited low temperature fluorescence emission spectrum proves the presence of photosystem II chlorophyll, and also its energetic connection to the phycobilisomes.     

The molecular genetics of red and green color vision in mammals.

To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red pigment of goldfish (Carassius auratus), which has a lambdamax value at 559 +/- 4 nm. Multiple linear regression analyses show that S180A, H197Y, Y277F, T285A, and A308S shift the lambdamax values of the red and green pigments in mammals toward blue by 7, 28, 7, 15, and 16 nm, respectively, and the reverse amino acid changes toward red by the same extents. The additive effects of these amino acid changes fully explain the red-green color vision in a wide range of mammalian species, goldfish, American chameleon (Anolis carolinensis), and pigeon (Columba livia).     

The visual pigments of four deep-sea crustacean species

Summary The visual pigments of four mesopelagic crustacean species were studied at sea by means of microspectrophotometry. The absorbance maxima obtained for the visual pigments and their metarhodopsins, respectively, were: 493 nm and 481 nm (Systellaspis debilis), 485 nm and 480 nm (Acanthephyra curtirostris), 491 nm and 482 nm (A. smithi), and 495 nm and 487 nm (Sergestes tenuiremis). The spectral characteristics of the rhodopsins and metarhodopsins permit high photosensitivity and facilitate photoregeneration in a nearly monochromatic environment. Photic regeneration of rhodopsins from the deep-sea environment was demonstrated, and data were obtained which are consistent with the occurrence of dark regeneration. Specific optical density of the observed visual pigments was calculated for two species.     

Visual pigments in the individual rods of deep-sea fishes

Summary The visual pigments in the rods of 15 species of deep-sea fish were examined by microspectrophotometry. In 13 species a single visual pigment was found. The max of these pigments, which ranged from 475 nm to 488 nm, suggest they give the fish maximum sensitivity to the ambient light in the deep, blue ocean waters where they live. In two species two visual pigments were found in separate rods.Bathylagus bericoides had rhodopsins of max 466 nm and 500 nm andMalacocephalus laevis had two rhodopsins of max 478 nm and 485 nm. It is noted that the species with two visual pigments tend to be dark in colour and live in deeper, darker, water.