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Mammalians process their photoreceptions through lateral eyes; however, non-mammalian vertebrates and invertebrates possess additional extraretinal photoreceptors over their bodies to detect light stimuli. Chromatophores, i.e. dermal specialized pigment cells, play important roles in the regulation of body patterns. Since chromatophores derive from neural crest, they share the common embryonic origin with retina. Recent evidence shows that they are light-sensitive due to opsin expression. In the present study, the expression of seven cone opsins was detected in tilapia caudal fin tissues. Moreover, distinct photoresponses were found in two chromatophore types. Regardless of stimulating wavelengths, melanophores tend to disperse and maintain cell shape at dispersion stage by shuttling pigment granules. Conversely, erythrophores respond to light in a wavelength-dependent manner. The opsin expression profiles of melanophores and erythrophores imply SWS1 and RH2 group genes may play important roles in chromatophore photoresponses. Through measuring photosensitivity, I suggest the two opsins play opposite roles in light-induced translocations of pigment granules within erythrophores: SWS1 for aggregations at UV and short wavelength regions and RH2b for dispersion in middle/long wavelengths. An antagonistic interaction occurs in the overlapping of the absorbance spectra of the two opsins. I also found that the photoresponses take place along with the occurrence of the change of cell membrane potential. In addition, the effect of different light backgrounds (broad spectrum, short wavelength-rich, and red-shifted light conditions) on the photosensitivity of tilapia erythrophores was investigated. I found that the major opsin classes (SWS1 and RH2b) responsible for photoresponses remain constant in three groups of erythrophores. Together, I postulate that melanophores may serve as a light filter in integumentary tissues, and the chromatically antagonistic mechanism enables tilapia erythrophores to sense the subtle change of environmental photic condition and to fine-tune pigmentation. I also investigated the ontogenetic change of photoresponses of rainbow trout melanophores. Distinct photoresponses were found in parrs and smolts. Furthermore, smolt melanophores responded to light in a wavelength-dependent manner. Since the change of coloration and visual system during smoltification of salmonids is regulated by thyroid hormone (TH), I suggest that the development of melanophore photosensitivity is associated to TH as well. / Thesis (Ph.D, Biology) -- Queen's University, 2013-08-27 09:57:22.907

Mammalians process their photoreceptions through lateral eyes; however, non-mammalian vertebrates and invertebrates possess additional extraretinal photoreceptors over their bodies to detect light stimuli. Chromatophores, i.e. dermal specialized pigment cells, play important roles in the regulation of body patterns. Since chromatophores derive from neural crest, they share the common embryonic origin with retina. Recent evidence shows that they are light-sensitive due to opsin expression. In the present study, the expression of seven cone opsins was detected in tilapia caudal fin tissues. Moreover, distinct photoresponses were found in two chromatophore types. Regardless of stimulating wavelengths, melanophores tend to disperse and maintain cell shape at dispersion stage by shuttling pigment granules. Conversely, erythrophores respond to light in a wavelength-dependent manner. The opsin expression profiles of melanophores and erythrophores imply SWS1 and RH2 group genes may play important roles in chromatophore photoresponses. Through measuring photosensitivity, I suggest the two opsins play opposite roles in light-induced translocations of pigment granules within erythrophores: SWS1 for aggregations at UV and short wavelength regions and RH2b for dispersion in middle/long wavelengths. An antagonistic interaction occurs in the overlapping of the absorbance spectra of the two opsins. I also found that the photoresponses take place along with the occurrence of the change of cell membrane potential. In addition, the effect of different light backgrounds (broad spectrum, short wavelength-rich, and red-shifted light conditions) on the photosensitivity of tilapia erythrophores was investigated. I found that the major opsin classes (SWS1 and RH2b) responsible for photoresponses remain constant in three groups of erythrophores. Together, I postulate that melanophores may serve as a light filter in integumentary tissues, and the chromatically antagonistic mechanism enables tilapia erythrophores to sense the subtle change of environmental photic condition and to fine-tune pigmentation. I also investigated the ontogenetic change of photoresponses of rainbow trout melanophores. Distinct photoresponses were found in parrs and smolts. Furthermore, smolt melanophores responded to light in a wavelength-dependent manner. Since the change of coloration and visual system during smoltification of salmonids is regulated by thyroid hormone (TH), I suggest that the development of melanophore photosensitivity is associated to TH as well. / Thesis (Ph.D, Biology) -- Queen's University, 2013-08-27 09:57:22.907

As corujas formam um grupo diversificado, estando presentes em diversos habitats ao redor do globo e têm diferentes padrões de atividade, com espécies diurnas, noturnas e crepusculares. Os fotorreceptores encontrados em corujas são os bastonetes e três classes de cones, levando potencialmente à tricromacia, e as demais camadas da retina mantém a mesma organização de outras aves. O gene LWS tem sido estudado em aves e o pico de absorção espectral da opsina expressa por esse gene está entre 560-570nm. Exceções foram reportadas no melro-preto (P557), pinguim Humboldt (P543) e na corujado- mato (Strix aluco). Entre esses três gêneros, somente as corujas apresentam espécies com diferentes hábitos circadianos. Dessa forma é possível que diferentes adaptações visuais possam ser encontradas em associação com o padrão circadiano. Neste trabalho foi investigada a morfologia da retina e a genética do pigmento visual LWS de cinco espécies de corujas com diferentes ritmos circadianos: Asio clamator, Megascops choliba, Tyto alba (noturnas), Athene cunicularia e Glaucudium brasilianum (diurnas). Um indivíduo de cada espécie foi utilizado nos experimentos. Foi realizada a extração de RNA a partir de uma retina homogeneizada de cada espécie e o RNA mensageiro (mRNA) foi convertido em DNA complementar (cDNA). Partes do gene LWS foram amplificadas utilizado a reação em cadeia da polimerase (PCR) e sequenciadas utilizando a metodologia de Sanger. Cinco sítios importantes para o ajuste espectral da opsina LWS (164,181, 261, 269 e 292) foram analisados e comparados com a sequência de outras aves e da rodopsina bovina, a qual foi referência para determinar as posições dos aminoácidos. No estudo morfológico, foram realizados cortes transversais em criostato de uma retina de cada espécie de coruja. Para a reação de imunohistoquímica foi utilizado o anticorpo Rabbit anti opsin (AB5405) para marcar cones L/M e DAPI marcando núcleos celulares. Também foi realizada a coloração de Hematoxilina-Eosina (HE) para visualizar a organização da retina. A partir das análises morfológicas foi possível observar a presença de cones nas retinas das cinco espécies de corujas, bem como uma organização laminar semelhante a de outros vertebrados. Para todas as espécies estudadas, os resultados da análise de sequência da opsina LWS foram: A164, H181, Y261, T269 e A292. Ao menos para o gene LWS, não foram encontradas diferenças entre espécies diurnas e noturnas de corujas / The owls forms a diverse group present in many habitats around the world and they have different activity patterns, with diurnal, nocturnal and crepuscular species. Photoreceptors found in owls are the rods and three classes of cones that potentially provide trichromacy, and the other retinal layers maintain the same organization of other birds. The LWS gene has been studied in birds and the peak spectral absorption of opsin expressed by this gene is between 560- 570nm. Exceptions were reported on blackbird (P557), Humboldt penguin (P543) and tawny owl (Strix aluco). Among these three genera, only owls have species with different circadian habits. It is therefore possible that different visual adaptations can be found in association with the circadian pattern. In this study the retinal morphology and the genetics of LWS visual pigment of five owl species with different circadinan habits were investigated: Asio clamator, Megascops choliba, Tyto alba (nocturnal), Athene cunicularia e Glaucudium brasilianum (diurnal). One individual of each species was used in the experiments. RNA extraction was performed from a homogenized retina of each species and messenger RNA (mRNA) was converted into complementary DNA (cDNA). Parts of the LWS gene were amplified using the polymerase chain reaction (PCR) and sequenced using the methodology of Sanger. Five important sites for the spectral tuning of the LWS opsin (164, 181, 261, 269 and 292) were analyzed and compared to the sequence of other birds and bovine rhodopsin, which was referenced to determine amino acid positions. In the morphological study, cross - sections were performed in cryostat of a retina of each owl species. For the immunohistochemistry reaction, the rabbit anti-opsin antibody (AB5405) was used to label L / M cones and DAPI labeling cell nuclei. Hematoxylin-Eosin (HE) staining was also performed to visualize the organization of the retina. From the morphological analyzes it was possible to observe the presence of cones in the retinas of the five species of owls, as well as a laminar organization similar to that of other vertebrates. For all species studied, the results of LWS opsin sequence analysis were: A164, H181, Y261, T269 and A292. At least for the LWS gene, no differences were found between diurnal and nocturnal species of owls

Photosensitive behaviors and circadian rhythms are well documented in reef-building corals and their larvae, yet photoreceptive structures and opsins have not been described in these organisms. Here I provide evidence for red sensitivity in several species of coral larvae. Behavioral experiments with two Caribbean corals, Porites astreoides and Acropora palmata demonstrated that larvae settle and metamorphose at a greater frequency on red substrata than on similar substrata of other colors. Attachment to red substrata was not observed when larvae were maintained in the dark, suggesting that red sensitivity was responsible for the observed behavior. Extracellular recordings confirm photosensitivity and indicate that the peak sensitivity of coral photoreceptors are shifted towards the orange-red region of the visible light spectrum, similar to the spectra (fluorescence and reflectance) of preferred artificial (plastic) and natural (crustose coralline algae) settlement substrata. Using Blast analyses and a PCRbased approach, I have identified, sequenced and cloned two full-length opsin cDNAs from A. palmata larvae. One cDNA (Acropsin 1) encodes an opsin protein that is similar to a vertebrate melanopsin; the second (Acropsin 2) encodes a protein that is most similar to cephalopod rod opsin. I have successfully developed synthetic peptide antibodies against each Acropsin 1 and Acropsin 2. Western blots of adult A. palmata and A. cervicornis protein detect a 37kDa and 40kDa band, corresponding to the predicted molecular weights of Acropsins 1 and 2, respectively. Immunohistochemistry confirms expression of both opsins in A. palmata larvae. Staining of sectioned larvae demonstrates that Acropsin 1 is localized in the larval gastroderm while Acropsin 2 is localized in solitary epithelial cells, scattered throughout the larval ectoderm but with a polarized distribution and higher concentration in the aboral epidermis. This research provides several lines of evidence to support the existence, and demonstrate one potential ecological function, of opsin-based photosensitivity in corals.

Primate habitats differ dramatically in the intensity and spectral quality (color) of ambient light. However, little research has explored the effects of habitat variation in ambient light on primate and mammalian visual systems. An understanding of variation in nocturnal light environments is particularly lacking, considering the significance of nocturnality and vision in primate evolutionary hypotheses. In this dissertation, I explored effects of habitat variation in light environments on primate visual evolution in three studies. First, I examined how variation in ambient light intensity influenced visual morphology in 209 mammals. Second, I analyzed effects of variation in nocturnal light environments on color vision in nocturnal primates and mammals. For this second objective, I first identified factors influencing variation in nocturnal light environments within and between habitats in Madagascar and explored how nocturnal light spectral quality has influenced mammalian visual pigment spectral tuning. I then analyzed selection acting on the SWS1 opsin gene (coding for blue-sensitive cone visual pigments) between nocturnal lemurs from different habitat types to explore whether nocturnal light environments affect selection for dichromatic color vision. The results of all three studies suggest that habitat variation in light environments has had a significant influence on primate and mammalian visual evolution. In the first study, I found that day-active mammals from forested habitats exhibited larger relative cornea size compared to species from open habitats, reflecting an adaptation to increase visual sensitivity in diurnal forests. The results of the second study revealed that forest and woodland habitats share a yellow-green dominant nocturnal light environment and that nocturnal vertebrates exhibit visual pigments tuned to maximize photon absorption in these environments. Additionally, I observed a potential effect of diet on long-wavelength-sensitive cone spectral tuning among nocturnal mammals. In the third study I sequenced the SWS1 opsin gene in 106 nocturnal lemurs (19 species). Both population genetic and phylogenetic analyses identified clear signatures of differential selection on the gene by habitat type, suggesting that nocturnal light environments has influenced selection for nocturnal dichromacy in nocturnal lemurs. Finally, I discussed the implications of these results for nocturnal primate visual ecology and evolution. / text

Sexual selection and the maintenance of species diversity in Lake Malawi cichlid fishes are greatly dependent on optical communication, which is influenced by environmental, physiological and endocrinological factors. The diversity in spectral sensitivity of cichlids has been partially attributed to differences in opsin gene expression, with each species preferentially expressing a subset of seven possible genes. Hormones such as gonadotropin releasing hormone (GnRH) can mediate changes in gene expression and the presence of GnRH immunoreactive fibers and GnRH receptors throughout the retinal layers make it an excellent candidate for mediating changes in visual processes. Effects of exogenous GnRH administration on the visual system of zebra cichlids (Metriaclima zebra) via prolonged release cholesterol implants and intubation was investigated using electroretinogram (ERG) recordings, quantitative real-time RT-PCR and in situ hybridization. Three week and ten week sampling periods were used in the intubation study. No obvious differences in spectral sensitivity were evident when looking at a-wave, b-wave and d-wave components of the ERG waveform in any of the treatment groups. A multiple mechanism model was used to describe the cone mechanisms mediating spectral sensitivity and this analysis showed that the activity of cones was shaped by opponent and non opponent cone interactions based on subsets of five opsin genes previously described in cichlids (SWS1, SWS2b, RH2b, RH2aβ, and RH2aα). Although differences in the spectral sensitivity between control and GnRH-treated fish were not evident on a functional level, there were changes in the gonadosomatic index in the intubation group. Quantitative real-time RT-PCR (qRT-PCR) and in situ hybridization demonstrated that treatment with a synthetic GnRH3 analogue using the oral intubation delivery system resulted in statistically significant changes in opsin gene expression in both three week and ten week treatment groups, specifically the upregulation of RH2b and the downregulation of RH2a opsin genes. Moreover, in situ hybridization analysis showed that the pattern of labeling for the RH2a and RH2b riboprobes corroborated the changes in opsin gene expression found in the qRT-PCR data. In contrast, GnRH treatment using the cholesterol implant delivery system did not result in significant changes in spectral sensitivity or opsin gene expression. / Thesis (Master, Biology) -- Queen's University, 2011-01-05 22:57:11.308

The signalling cascade of rods and cones use different but related protein components. Rods and cones, emerged in the common ancestor of vertebrates around 500 million years ago around when two whole genome duplications took place, named 1R and 2R. These generated a large number of additional genes that could evolve new or more specialised functions. A third event, 3R, occurred in the ancestor of teleost fish.  This thesis describes extensive phylogenetic and comparative synteny analyses of the opsins, transducin and phosphodiesterase (PDE6) of this cascade by including data from a wide selection of vertebrates. The expression of the zebrafish genes was also investigated. The results show that genes for these proteins duplicated in 1R and 2R as well as some in 3R. Expression analyses of the zebrafish genes revealed additional specialisations for the 3R gene duplicates. The transducin beta subunit genes, gnb1a and gnb1b, show co-localisation in rods but are expressed at different levels. Gnb3a and gnb3b show different expression in the adult retina with low expression of gnb3a and expression of gnb3b in cones of the dorso-medial retina. The transducin gamma subunit genes gngt2a and gngt2b are expressed in the ventral and dorso-medial retina respectively. The both of PDE6 gamma subunit genes, pde6ga and pde6gb are both expressed in rods but pde6ga shows rhythmic changes of expression with low daytime levels. Pde6ha and pde6hb are expressed in cones however pde6ha show high daytime expression. All investigated transducin and PDE6 subunit genes, but gnb1b, were also expressed in the adult pineal complex or at some point during development. These results provide compelling evidence that the 1R and 2R genome duplications facilitated the evolution of rods and cones by generating gene duplicates that could evolve distinct expression and function. This supports existence of colour vision before the origin of vertebrates, elaboration of this in the early vertebrate ancestor, along with origin of the black-and-white dim-light vision of rods. Furthermore, the different expression patterns observed in the zebrafish retina for teleost 3R duplicates demonstrate multiple additional specialisations.

No description available.

Light energy is converted into an electrical signal by a set of proteins in the phototransduction cascade in photoreceptors. In this work, I focus on two critical elements of the phototransduction cascade in canine, the opsin molecules and CNG channels of cone photoreceptors. Canines are dichromats possessing two types of cone photoreceptors with different opsin molecules that detect either long and medium wavelengths (L/M) or short wavelengths (S). The L/M- and S-opsin genes were cloned from an Alaskan Malamute and used to investigate key amino acids that are responsible for tuning the spectral properties of the 11-cis retinal chromophore. Cone CNG channels are composed of CNGA3 and CNGB3 subunits. I characterized antibodies to detect cone CNG channel subunits to investigate expression of mutations in CNGB3 subunit on two canine models for achromatopsia. One model contains a missense CNGB3 mutation D262N (CNGB3m/m) and the other is a complete deletion of all exons of the CNGB3 gene (CNGB3-/-). Studies presented in this thesis show CNGB3 is expressed later in cone during retinal development compared to CNGA3. It also presents evidence for the necessity of CNGB3 in cone outer segment targeting of CNGA3. / Biology