Trichromacy in Australian marsupials.

Vertebrate color vision is best developed in fish, reptiles, and birds with four distinct cone receptor visual pigments. These pigments, providing sensitivity from ultraviolet to infrared light, are thought to have been present in ancestral vertebrates. When placental mammals adopted nocturnality, they lost two visual pigments, reducing them to dichromacy; primates subsequently reevolved trichromacy. Studies of mammalian color vision have largely overlooked marsupials despite the wide variety of species and ecological niches and, most importantly, their retention of reptilian retinal features such as oil droplets and double cones. Using microspectrophotometry (MSP), we have investigated the spectral sensitivity of the photoreceptors of two Australian marsupials, the crepuscular, nectivorous honey possum (Tarsipes rostratus) and the arhythmic, insectivorous fat-tailed dunnart (Sminthopsis crassicaudata); these species are representatives of the two major taxonomic divisions of marsupials, the diprotodonts and polyprotodonts, respectively. Here, we report the presence of three spectrally distinct cone photoreceptor types in both species. It is the first evidence for the basis of trichromatic color vision in mammals other than primates. We suggest that Australian marsupials have retained an ancestral visual pigment that has been lost from placental mammals.

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart).

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.

Cone topography and spectral sensitivity in two potentially trichromatic marsupials, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus).

The potential for trichromacy in mammals, thought to be unique to primates, was recently discovered in two Australian marsupials. Whether the presence of three cone types, sensitive to short- (SWS), medium- (MWS) and long- (LWS) wavelengths, occurs across all marsupials remains unknown. Here, we have investigated the presence, distribution and spectral sensitivity of cone types in two further species, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus). Immunohistochemistry revealed that SWS cones in the quokka are concentrated in dorso-temporal retina, while in the quenda, two peaks were identified in naso-ventral and dorso-temporal retina. In both species, MWS/LWS cone spatial distributions matched those of retinal ganglion cells. Microspectrophotometry (MSP) confirmed that MWS and LWS cones are spectrally distinct, with mean wavelengths of maximum absorbance at 502 and 538 nm in the quokka, and at 509 and 551 nm, in the quenda. Although small SWS cone outer segments precluded MSP measurements, molecular analysis identified substitutions at key sites, accounting for a spectral shift from ultraviolet in the quenda to violet in the quokka. The presence of three cone types, along with previous findings in the fat-tailed dunnart and honey possum, suggests that three spectrally distinct cone types are a feature spanning the marsupials.

The rod opsin pigments from two marsupial species, the South American bare-tailed woolly opossum and the Australian fat-tailed dunnart.

Rod visual pigment genes have been studied in a wide range of vertebrates including a number of mammalian species. However, no marsupials have yet been examined. To correct this omission, we have studied the rod pigments in two marsupial species, the nocturnal and frugivorous bare-tailed woolly opossum, Caluromys philander, from Central and South America, and the arhythmic and insectivorous fat-tailed dunnart, Sminthopsis crassicaudata, from Australia. Phylogenetic analysis establishes that the cloned opsin sequences are orthologues of rod opsin genes from other vertebrate species. The deduced amino acid sequences show that both possess glutamate at residue 122, a feature of rod opsins, and the corresponding gene follows the typical vertebrate rod opsin pattern of five exons separated by four introns. Compared to other vertebrates, a stretch of five residues near the C-terminus is deleted in the rod opsin of both marsupials and all eutherian mammals. From microspectrophotometric measurements, the pigments in the two species show an 8 nm difference in peak absorbance; the molecular basis for this spectral shift is discussed and two candidate substitutions are identified.

Retinal characteristics of the ornate dragon lizard, Ctenophorus ornatus.

The retina of a diurnal insectivorous lizard, Ctenophorus ornatus (Agamidae) was investigated using microspectrophotometry and light and electron microscopy. A prominent broad yellow band was observed that extended across the mid-retina. The yellow coloration was found to originate from both oil droplets and diffuse pigmentation within cone inner segments. Microspectrophotometric analysis revealed yellow oil droplets with variable absorption of wavelengths below 520 nm and transparent oil droplets with no detectable absorptance between 350 and 750 nm. Cones with transparent oil droplets lacked the diffuse yellow pigmentation. The mean wavelengths of maximum absorbance of visual pigments in the isolated cone outer segments were at 440, 493, and 571 nm. The retina was found to possess a deep convexiclivate fovea located within the yellow band, slightly dorsotemporal of the retinal midpoint. The topography of the retinal ganglion cells revealed that the fovea was contained within an area centralis. Photoreceptors were either single (80%) or unequal double (20%) cones. Within the region of the fovea, the cones were approximately 20% the diameter of those in the peripheral retina. Colored oil droplets and yellow pigment may increase visual acuity by absorbing short wavelength light scattered either by the atmosphere or the optical structures of the eye. The presence of a fovea containing slender cone photoreceptors and three visual pigments suggests that the lizard has high acuity and the potential for color vision.

The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri.

The correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aalpha and beta, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aalpha, contrasting with SWS2A, Rh2Abeta and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wild-caught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.

Spectral sensitivities of the seahorses Hippocampus subelongatus and Hippocampus barbouri and the pipefish Stigmatopora argus.

The Syngnathidae are specialized diurnal feeders that are known to possess a retinal fovea and use independent eye movements to locate, track, and strike individual planktonic prey items. In this study, we have investigated the spectral sensitivities of three syngnathid species: a pipefish and two seahorses. We used spectrophotometry to measure the spectral transmission properties of ocular lenses and microspectrophotometry to measure the spectral absorption characteristics of visual pigments in the retinal photoreceptors. The pipefish, Stigmatopora argus, together with the seahorse Hippocampus subelongatus, is found in "green-water" temperate coastal seagrass habitats, whereas the second seahorse, H. barbouri, originates from a "blue-water" tropical coral reef habitat. All species were found to possess short wavelength absorbing pigment(s) in their lenses, with the 50% cut-off point of S. argus and H. subelongatus at 429 and 425 nm respectively, whereas that of H. barbouri was located at 409 nm. Microspectrophotometry of the photoreceptors revealed that the rods of all three species contained visual pigment with the wavelength of maximum absorption (lambda(max)) at approximately 500 nm. The visual pigment complement of the cones varied between the species: all possessed single cones with a lambda(max) close to 460 nm but H. barbouri also possessed an additional class of single cone with lambda(max) at 430 nm. Three classes of visual pigment were found in the double cones, the lambda(max) being approximately 520, 537, and 560 nm in the two seahorses and 520, 537, and 580 nm in the pipefish. The spectral sensitivities of the syngnathids investigated here do not appear to conform to generally accepted trends for fishes inhabiting different spectral environments. The influence of the specialized feeding regime of the syngnathids is discussed in relation to our findings that ultra-violet sensitivity is apparently not necessary for zooplanktivory in certain habitats.

Morphology and spectral absorption characteristics of retinal photoreceptors in the southern hemisphere lamprey (Geotria australis).

The morphology and spectral absorption characteristics of the retinal photoreceptors in the southern hemisphere lamprey Geotria australis (Agnatha) were studied using light and electron microscopy and microspectrophotometry. The retinae of both downstream and upstream migrants of Geotria contained two types of cone photoreceptor and one type of rod photoreceptor. Visual pigments contained in the outer segments of these three photoreceptor types had absorbance spectra typical of porphyropsins and with wavelengths of maximum absorbance (downstream/upstream) at 610/616 nm (long-wavelength-sensitive cone, LWS), 515/515 nm (medium-wavelength-sensitive cone, MWS), and 506/500 nm (medium-wavelength-sensitive rod). A "yellow" photostable pigment was present in the myoid region of all three types of photoreceptor in the downstream migrant. The same short-wavelength-absorbing pigment, which prevents photostimulation of the beta band of the visual pigment in the outer segment, was present in the rods and LWS cones of the upstream migrant, but was replaced by a large transparent ellipsosome in the MWS cones. Using microspectrophotometric and anatomical data, the quantal spectral sensitivity of each photoreceptor type was calculated. Our results provide the first evidence of a jawless vertebrate, represented today solely by the lampreys and hagfishes, with two morphologically and physiologically distinct types of cone photoreceptors, in addition to a rod-like photoreceptor containing a colored filter (a cone-like characteristic). In contrast, all other lampreys studied thus far have either (1) one type of cone and one type of rod, or (2) a single type of rod-like photoreceptor. The evolution or retention of a second type of cone in adult Geotria is presumably an adaptation to life in the brightly lit surface waters of the Southern Ocean, where this species lives during the marine phase of its life cycle. The functional significance of the unique visual system of Geotria is discussed in relation to its life cycle and the potential for color vision.

Vision in the southern hemisphere lamprey Mordacia mordax: spatial distribution, spectral absorption characteristics, and optical sensitivity of a single class of retinal photoreceptor.

The dorso-laterally located eyes of the southern hemisphere lamprey Mordacia mordax (Agnatha) contain a single morphological type of retinal photoreceptor, which possesses ultrastructural characteristics of both rods and cones. This photoreceptor has a large refractile ellipsosome in the inner segment and a long cylindrical outer segment surrounded by a retinal pigment epithelium that contains two types of tapetal reflectors. The photoreceptors form a hexagonal array and attain their peak density (33,200 receptors/mm2) in the ventro-temporal retina. Using the size and spacing of the photoreceptors and direct measures of aperture size and eye dimensions, the peak spatial resolving power and optical sensitivity are estimated to be 1.7 cycles deg-1 (minimum separable angle of 34'7'') and 0.64 microm2 steradian (white light) and 1.38 microm2 steradian (preferred wavelength or lambdamax), respectively. Microspectrophotometry reveals that the visual pigment located within the outer segment is a rhodopsin with a wavelength of maximum absorbance (lambdamax) at 514 nm. The ellipsosome has very low absorptance (<0.05) across the measured spectrum (350-750 nm) and probably does not act as a spectral filter. In contrast to all other lampreys studied, the optimized receptor packing, the large width of the ellipsosome-bearing inner segment, together with the presence of a retinal tapetum in the photophobic Mordacia, all represent adaptations for low light vision and optimizing photon capture.

Developmental changes in the cone visual pigments of black bream Acanthopagrus butcheri.

The spectral absorption characteristics of the visual pigments in the photoreceptors of the black bream Acanthopagrus butcheri Munro (Sparidae, Teleostei), were measured using microspectrophotometry. A single cohort of fish aged 5-172 days post-hatch (dph), aquarium-reared adults and wild-caught juveniles were investigated. During the larval stage and in juveniles younger than 100 dph, two classes of visual pigment were found, with wavelengths of maximum absorbance (lambda(max)) at approximately 425 nm and 535 nm. Following double cone formation, from 40 dph onwards, the short wavelength-sensitive pigment was recorded in single cones and the longer wavelength-sensitive pigment in double cones. From 100 dph, a gradual shift in the lambda(max) towards longer wavelengths was observed in both cone types. By 160 dph, and in adults, all single cones had a lambda(max) at approximately 475 nm while the lambda(max) in double cones ranged from 545 to 575 nm. The relationships between the lambda(max) and the ratio of bandwidth:lambda(max), for changes in either chromophore or opsin, were modelled mathematically for the long-wavelength-sensitive visual pigments. Comparing our data with the models indicated that changes in lambda(max) were not mediated by a switch from an A(1) to A(2) chromophore, rather a change in opsin expression was most likely. The shifts in the lambda(max) of the visual pigments occur at a stage when the juvenile fish begin feeding in deeper, tanninstained estuarine waters, which transmit predominantly longer wavelengths, so the spectral sensitivity changes may represent an adaptation by the fish to the changing light environment.