Tesis sobre el tema "Visual opsin"

Fireflies are some of the most captivating organisms on the planet. Because of this, they have a rich history of study, especially concerning their bioluminescent and visual behavior. Among insects, opsin copy number variation has been shown to be quite diverse. However, within the beetles, very little work on opsins has been conducted. Here we look at the visual system of fireflies (Coleoptera: Lampyridae), which offer an elegant system in which to study visual evolution as it relates to their behavior and broader ecology. They are the best-known case of a terrestrial organism that communicates through the use bioluminescence. The molecular basis for this communication is relatively simple: one gene-family (opsins) controls the detection of the signal, and one gene family (luciferase) controls the production of the signal. We use a transcriptomic approach to sample for and investigate opsin evolution in fireflies. We also present the first total evidence approach using both an extensive molecular matrix and a robust morphological matrix to reconstruct the lampyrid phylogeny. We then use this phylogeny to assess the hypothesis that adult use of bioluminescence occurred after the origin of Lampyridae. We find evidence for only two expressed opsin classes in each of the nine firefly species studied, one in the ultra-violet sensitive and one in the long-wavelength sensitive areas of the visible spectrum. Despite the need for most adult fireflies to respond to a clearly sexual and colorful visual signal (bioluminescence) to maximize fitness, their visual system is relatively simple, and does not match the trend for opsin duplication found in other insect groups. All subfamilies except for Lampyrinae are recovered as monophyletic; Pterotinae and Ototretinae are recovered within the Lampyridae. The ancestral state of adult bioluminescence is suggested to be non-bioluminescent, with at least three gains and at least three losses.

Understanding how individual photoreceptor cells factor in the spectral sensitivity of a visual system is essential to explain how they contribute to the visual ecology of the animal in question. Existing methods that model the absorption of visual pigments use templates which correspond closely to data from thin cross-sections of photoreceptor cells. However, few modeling approaches use a single framework to incorporate physical parameters of real photoreceptors, which can be fused, and can form vertical tiers. Akaike’s information criterion (AIC c ) was used here to select absorptance models of multiple classes of photoreceptor cells that maximize information, given visual system spectral sensitivity data obtained using extracellular electroretinograms and structural parameters obtained by histological methods. This framework was first used to select among alternative hypotheses of photoreceptor number. It identified spectral classes from a range of dark-adapted visual systems which have between one and four spectral photoreceptor classes. These were the velvet worm, Principapillatus hitoyensis , the branchiopod water flea, Daphnia magna , normal humans, and humans with enhanced S-cone syndrome, a condition in which S-cone frequency is increased due to mutations in a transcription factor that controls photoreceptor expression. Data from the Asian swallowtail, Papilio xuthus , which has at least five main spectral photoreceptor classes in its compound eyes, were included to illustrate potential effects of model over-simplification on multi-model inference. The multi-model framework was then used with parameters of spectral photoreceptor classes and the structural photoreceptor array kept constant. The goal was to map relative opsin expression to visual pigment concentration. It identified relative opsin expression differences for two populations of the bluefin killifish, Lucania goodei . The modeling approach presented here will be useful in selecting the most likely alternative hypotheses of opsin-based spectral photoreceptor classes, using relative opsin expression and extracellular electroretinography.

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.

The duplication of genes followed by selection is perhaps the most prominent way in which molecular biological systems gain multiplicity, diversity and functional complexity in evolution. Whole genome duplications (WGDs) therefore have the potential of generating an extraordinary amount of evolutionary innovation. It is now accepted that the vertebrate lineage has gone through two rounds of WGD in its early stages, after the divergence of invertebrate chordates and before the emergence of jawed vertebrates. These basal vertebrate WGDs are called 2R for two rounds of whole genome duplication. An additional WGD called 3R occurred early in the evolution of teleost fishes, before the radiation of this species-rich group. This thesis describes the evolution of several endocrine and neuronal gene families in relation to the vertebrate WGDs, through a comparative genomic approach including both phylogenetic analyses and chromosomal location data across a wide range of vertebrate taxa. These results show that numerous endocrine gene families have expanded in 2R and in several cases also in 3R. These include the gene families of oxytocin and vasopressin receptors (OT/VP-R), somatostatin receptors (SSTR) and insulin-like growth factor binding proteins (IGFBP). For the OT/VP-R and SSTR families, previously undescribed subtypes were identified. The protein hormone family that includes growth hormone (GH), prolactin (PRL) and somatolactin (SL) acquired a new PRL gene in 2R, however the origins of GH, PRL and SL likely predate 2R. The corresponding family of receptors diversified during different time periods through a combination of local duplications and 3R. Neuronal gene families of the visual system have also expanded in 2R and 3R. The results presented here demonstrate that the vertebrate repertoire of visual opsin genes arose in 2R as part of chromosomal blocks that also include the OT/VP-R genes. The gene families including the transducin alpha, beta and gamma subunits also arose in 2R, hinting at the importance of these events in the diversification and specialization of phototransduction cascades for rods and cones. Thus, the whole genome duplications have been important contributors to the evolution of both vision and endocrine regulation in the vertebrates.

Serpentes habitam grande diversidade de habitats na maior parte do planeta. Tamanha variedade ambiental implica o desempenho de distintos nichos ecológicos e padrões comportamentais, muitas vezes relacionados a diferentes adaptações de seus sistemas visuais. Não apenas a dispersão destes animais oferece oportunidades privilegiadas de investigação, os diversos e particulares históricos evolutivos neste grupo demarcam transições ambientais convenientes para esclarecer a influência da cena visual sobre a organização de sistemas visuais, comportamentos e a filogenia. A análise comparativa da retina destes animais traz informações a respeito de adaptações comportamentais e ecológicas relativas ao ambiente e padrões circadianos de atividade. Neste trabalho foi realizada a análise dos genes de fotopigmentos visuais e da morfologia dos fotorreceptores de duas espécies de serpentes da família Viperidae, Bothrops jararaca e Crotalus durissus terrificus. Três indivíduos de cada espécie foram obtidos junto ao laboratório de Herpetologia do Instituto Butantan. O RNA total foi extraído a partir de retinas homogeneizadas e convertido em cDNA por meio da reação de transcriptase reversa. Os genes de interesse foram amplificados com uso de primers específicos por meio de reação em cadeia de polimerase (PCR). Após purificação dos produtos de PCR foi realizado o sequenciamento dos genes de opsinas visuais expressos nas retinas das duas espécies, lws, rh1 e sws1. Cada opsina, maximamente sensível a uma banda espectral específica e presente em diferentes populações de fotorreceptores da retina, teve o seu pico de absorção estimado com base na estrutura proteica revelada. Adicionalmente, foi conduzida análise dos tipos celulares de células fotorreceptoras da retina das duas espécies de viperídeos, por meio da técnica de imunohistoquímica, visando caracterização morfológica dos fotorreceptores em que estão compreendidas cada classe de opsina. Os resultados obtidos apontam para os mesmos grupos morfológicos de fotorreceptores e a mesma sensibilidade espectral dos respectivos pigmentos visuais, das duas espécies analisadas: cones simples e cones duplos com o fotopigmento LWS, e pico de sensibilidade espectral (max) estimado em ~555nm; cones simples com o fotopigmento SWS1, e max estimado em ~360nm; e bastonetes, com o fotopigmento RH1, com max de ~500nm. Desta forma conclui-se similaridade do nicho ecológico e do histórico natural das duas espécies, que apontam para adaptações ao habito noturno. Isto demonstra o sucesso evolutivo e a versatilidade proporcionada pela disposição de retina duplex dominada por bastonetes. Em B. jararaca e C. d. terrificus, serpentes da subfamília Crotalinae, estas características de organização do sistema visual são somadas à capacidade de detecção de comprimentos de onda infravermelhos, o que aponta para similaridades em relação a serpentes do grupo Henophidia, consideradas evolutivamente mais primitivas, e consagra novamente a vantagens obtidas na manutenção deste padrão sensorial
Serpents inhabit a great diversity of habitats around the planet. Such environmental variability implies the performance of distinct ecological niches and behavior patterns that are related to different visual system adaptations. The diversity of environments inhabited by snakes and their evolutionary history provides a privileged investigative opportunity on the adaptive organization of the visual systems, specific behaviors and phylogeny. The comparative analysis of the retina of those animals provide many information concerning behavior and ecological adaptations related to their respective environment and circadian rhythm patterns. In this study, we performed genetic analysis of the opsin genes and morphological analysis of the photoreceptors of two snakes from the Viperidae family, Bothrops jararaca and Crotalus durissus terrificus. Three subjects of each species were collected at the Butantan Institute. Total RNA was extracted from homogenized retinas, and mRNA was converted to cDNA by reverse transcriptase reaction. The opsin genes lws, rh1 and sws1 were amplified by polymerase chain reactions (PCR), using specific primers. Each opsin is expressed in a different photoreceptor population and is maximally responsive to a determined spectral absorption peak (max) that was inferred according to the protein structure. Additionally, photorreceptor cell populations were analyzed using immunohistochemistry technique. Results point out to the same morphological cell populations and the same absorption peak in their respective opsins in the two species: double and single cones with the LWS photopigment and estimated max at ~555nm; single cones with the SWS1 photopigment and max at ~360nm; and rods with the rhodopsin RH1 photopigment and max at ~500nm. In this way, great similarity of ecological niche and natural history was concluded for both species, which present adaptations to the nocturnal habit. This should demonstrate the great evolutionary success and versatility attained by the rod-domminated duplex retina. In B. jararaca and C. d. terrificus, snakes from the Crotalinae subfamily, those retinal features are summed to the capability of infra-red detection, which point out to similarity with snakes from the basal Henophidia group

Visual opsins are G protein-coupled receptors that function as light photoreceptors in the vertebrate retina. Rhodopsin is the visual pigment located in the rod photoreceptor cells specialized in scotopic vision. Bovine and mouse rhodopsins have been thoroughly used as in vitro and in vivo models for physiological and biochemical characterization. In the last years, different lines of evidence point to significant functional differences among rhodopsins of different species. In this thesis bovine, murine and human rhodopsins were immunopurified and biochemically characterized, revealing differences in their thermal stabilities and retinal release rates. Besides, the Y102H RP-like rhodopsin mutation was introduced in the human and bovine backgrounds to bring up potential phenotypic differences. Therefore, keeping in mind that a large body of studies on human genetic retinal degenerative diseases related with opsins (e.g. retinitis pigmentosa) have used these models, our results suggest that using human rhodopsin for future studies would be advised. The most important biochemical differences were observed between the diurnal (human and bovine) versus nocturnal (mouse) species, especially in their retinal release rates. In addition, we also found a novel relevant amino acid position that appears to be significantly correlated with rhodopsin molecular adaptation to the nocturnal (L290) and the diurnal (I290) niches throughout terrestrial therian mammals. Previous studies suggested that L290 is present in the inferred therian ancestor rhodopsin in agreement with mammalian “nocturnal bottleneck” theories. Thus, the L290I substitution could have an important role in mammal rhodopsin molecular evolution and adaptation as it is likely to be the result of independent analogous changes, a fact that can be well-appreciated in the primate and rodent orders. This hypothesis was experimentally confirmed by the L290I mutation in murine rhodopsin that resulted in a Meta II decay rate similar to that of bovine rhodopsin. These results provide support for a role of the Meta II decay rate in rhodopsin evolution, beyond the well-studied ¿max spectral shift used by animal species to adapt to different light environments. Moreover, a novel mechanism is proposed involving a compromise between improving rod protection under bright light in nocturnal species by means of a stabilized Meta II conformation, and a faster dark adaption that occurs under dim-light conditions in diurnal species by means of a faster retinal reléase. Our statistical analysis found three new candidate positions for positive selection in the mammal therian branch. The reverse mutations (F13M, Q225R and A346S) were introduced into bovine rhodopsin and the expressed proteins were immunopurified to functionally and biochemically characterize the consequences of these ancestral changes. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Position 13 was shown to be very important for the proper folding and glycosylation of rhodopsin as only in the engineered thermally stable double Cys mutant (N2C/N282C) background was able to be regenerated with 11-cis-retinal. Similarly a double Cys mutation (W90C/A169C) previously proposed for the green cone opsin was biochemically analyzed confirming the formation (at least partially) of this bond. Finally, a recently detected interaction between the membrane protein peripherin-2 and rhodopsin was functionally studied, showing reduced G-protein activation, by rhodopsin, in presence of peripherin-2 when the two proteins were in a partially solubilised system. These results could have physiological implications in the desensitization process involving rhodopsin on the rim of discs of photoreceptor cells.
Los opsinas visuales son receptores acoplados a proteína G que funcionan como fotoreceptores en retinas de vertebrados. La rodopsina es el pigmento visual de los bastones, células fotoreceptoras especializadas en la visión escotópica. Las rodopsinas bovina y murina han sido ampliamente usadas como modelos para caracterización bioquímica y fisiológica. En esta tesis, las rodopsinas bovina, murina y humana fueron inmunopurificadas y caracterizadas bioquímicamente, revelando diferencias en su estabilidad térmica y en la tasa de salida de retinal. Además, la mutación tipo RP Y102H se introdujo en las rodopsinas humana y bovina para revelar potenciales diferencias fenotípicas. Teniendo en cuenta que una gran parte de estudios en enfermedades genéticas degenerativas de la retina humana relacionadas con opsinas (Ej. Retinitis Pigmentosa) han usado estos modelos, los resultados sugieren que el uso de rodopsinas humanas en estudios futuros sería aconsejable. Las mayores diferencias bioquímicas fueron observadas entre especies diurnas (humano y vaca) en comparación con la nocturna (ratón), especialmente en las tasas de salida de retinal. Además, se encontró una nueva y relevante posición aminoacídica que parece estar significativamente correlacionada con la adaptación molecular de la rodopsina a la nocturnidad (L290) y a la diurnidad (I290) a lo largo de los mamíferos terios terrestres. Estudios previos sugieren que L290 estaba presente en la rodopsina ancestrales inferidas, en concordancia con las teorías del ?cuello de botella nocturno?en mamíferos. La substitución L290I podría haber tenido un importante papel en la adaptación y la evolución molecular de las rodopsina de mamíferos al ser probablemente el resultado de cambios análogos independientes, hecho que puede ser apreciado en los órdenes de primates y roedores. Esta hipótesis fue confirmada experimentalmente mediante la mutación L290I en rodopsina murina que resultó en una tasa de decaimiento del Meta II similar al de rodopsina bovina. Estos resultados dan apoyo al papel de la tasa de decaimiento del Meta II en la evolución de la rodopsina, más allá del bien estudiado desplazamiento espectral de ¿max relacionado con la adaptación a diferentes niveles de luz ambiental. Además, se propone un nuevo mecanismo que implica un compromiso entre la protección en bastones ante luz brillantes en especies nocturnas mediante una estabilización de la conformación Meta II, y una adaptación a la oscuridad más rápida bajo condiciones de luz tenue en especies diurnas mediante una salida de retinal más rápida. Análisis estadístico encontraron tres nuevas posiciones candidatas a haber sido positivamente seleccionadas en la rama de los mamíferos terios. Las mutaciones reversas (F13M, Q225R y A346S) se introdujeron en la rodopsina bovina y se inmunopurificaron para caracterizar estos cambios ancestrales. 225 aparenta ser importante para la funcionalidad de la proteína afectando el proceso de activación de la proteína G, y 346 regularía también la funcionalidad mediante la mejora de la activación de la proteína G y presumiblemente afectando la fosforilación por parte de la rodopsina kinasa. La posición 13 es muy importante para el correcto plegamiento y glicosilación de la rodopsina al solo poder ser regenerada con 11-cis-retinal al insertar la doble mutación de Cys (N2C/N282C) termalmente estable. De manera similar una doble mutación de Cys (W90C/A169C) previamente propuesta para la opsina verde de conos fue analizada bioquímicamente confirmando la formación (al menos parcialmente) de este enlace. Finalmente, la interacción entre la periferina-2 y la rodopsina se estudió funcionalmente. Se detectó una reducción en la activación de la proteína G por la rodopsina, cuando las dos proteínas están en un sistema parcialmente solubilizado. Estos resultados podrían tener implicaciones fisiológicas en el proceso de desensibilización que implica la rodopsina en el borde de los discos de las células fotorreceptoras.

A visão de cores em vertebrados necessita de pelo menos duas classes de cones, (fotorreceptores presentes na retina) e a existência de um substrato neural para que os fótons de luz sejam comparados, processados e posteriormente resultar na sensação da cor. Primatas do Velho Mundo, incluindo humanos, apresentam visão de cor tricromata, enquanto que primatas do Novo Mundo apresentam um polimorfismo nos genes dos pigmentos visuais e, entre os primatas, são os únicos que podem apresentar indivíduos com visão dicromata ou tricromata. O polimorfismo encontrado em primatas do Novo Mundo ocorre devido à variabilidade dos genes que expressam as opsinas responsáveis por absorver comprimentos de onda médios ou longos. Os estudos genéticos das opsinas são essenciais para compreensão do processamento e da sensação de cores nesses animais, e podem ajudar a entender a evolução da visão de cores nos Primatas. O objetivo deste trabalho é caracterizar a diversidade dos pigmentos visuais (LWS/MWS e SWS1) das espécies de primatas do Novo Mundo através de análises genéticas e descrever a sequência de aminoácidos observados para estimar o pico de sensibilidade espectral das opsinas. Foram coletadas amostras de sangue, fezes e/ou pelo de seis gêneros de primatas provenientes de diferentes regiões do Brasil (Pará, Rio de Janeiro, Rio Grande do Norte e São Paulo) e pertencentes às espécies Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth e Brachyteles arachnoides e posteriormente foram analisados os genes que expressam as opsinas nesses indivíduos. As sequências de aminoácidos encontradas nas posições importantes do gene SWS1 (52, 86, 93, 114 e 118) foram diferentes para algumas espécies. No gene SWS1 as espécies C. apella, L. lagotricha, A. belzebuth e B. arachnoides apresentam a sequência de aminoácidos LLPAT e as espécies C. jacchus, A. caraya e A. clamitans apresentaram a sequência de aminoácidos LLPGT. Foi descoberto que variações de aminoácidos na posição 50 do gene SWS1 em primatas do Novo Mundo podem ser importantes na determinação do pico de absorção espectral dos pigmentos expressos por este gene. Os genes LWS e MWS de indivíduos da espécie C. jacchus foram estudados e os aminoácidos localizados nas posições 180, 277 e 285 das opsinas foram identificados. Os resultados dos alelos encontrados nesses grupos tiveram cinco combinações diferentes (SFT, SYA, SYT, AYA e AYT), os alelos AYA e SYA foram descritos pela primeira vez neste grupo e a partir do resultado genético foi inferido o pico de absorção espectral da opsina. Este trabalho preencheu algumas lacunas da bibliografia e trouxe novas informações a respeito da diversidade genética dos pigmentos visuais em primatas do Novo Mundo
Color vision in vertebrates requires the presence of at least two different classes of cones in the retina, and a neural substrate capable to compare the activation of the different photoreceptors, which ultimately leads to color perception. Old World Monkeys (OWM), including humans, have trichromatic color vision, whereas New World Monkeys (NWM) have visual pigment genes polymorphism and among primates, are the only group with dichromatic or trichromatic individuals in the same species. This polymorphism in NWM occurs due to the variability of genes that express the opsins responsible for absorbing medium or long wavelengths. The genetic studies of color vision are fundamental for the comprehension of color perception in these animals and it could help to understand the color vision evolution in Primates. The aim of this work is to characterize the visual pigment diversity (LWS/MWS and SWS1) in NWM species by genetic analysis and estimate the opsin spectral absorption peak, based on the amino acid sequence. Blood, feces and hair were collected from six primate genres from different regions of Brazil (Pará, Rio de Janeiro, Rio Grande do Norte and São Paulo): Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth and Brachyteles arachnoides. The amino acid sequences found in important positions of the SWS1 gene (52, 86, 93, 114 and 118) were different among some species. In C. apella, L. lagotricha, A. belzebuth and B. arachnoides was found the amino acid sequence LLPAT. In C. jacchus, A. caraya and A. clamitans the amino acid sequence was LLPGT. It was observed in previous studies that residue 50 of the SWS1 gene in the New World primates is important to determining the spectral absorption peak of the visual pigments expressed by this gene. The LWS and MWS genes of C. jacchus have been studied and the amino acids located at positions 180, 277 and 285 have been identified. Five different combinations were found among the individuals analyzed: SFT, SYA, SYT, AYA and AYT. Two alleles, AYA and SYA, were described for the first time in this species. The present study filled some gaps in the literature and brought new information on the genetic diversity of visual pigments in New World primates

Guppies exhibit color based sexual dimorphism and females generally prefer the most colorful males. It has also recently been found that guppies possess a large opsin repertoire. As opsins are the receptors responsible for color vision, this ten gene repertoire might have contributed to the evolution of extravagant male coloration in this species. My study starts by characterizing the opsin repertoire of Jenynsia onca, a noncolorful relative of the guppy belonging to the family Anablepidae (sister group to Poeciliidae, of which the guppy is a member). A PCR based survey indicated that J. onca had a very similar opsin repertoire to the guppy; J. onca had nine genes including orthologs of all but one of the guppy opsins. To gain further insight into the origin of the guppy repertoire, a bioinformatics based survey of ray-finned fish opsins was undertaken. This revealed that large opsin repertoires are common in ray-finned fish and are the product of gene duplication events, spanning the age of the taxon Teleostei. Given that the large opsin repertoire of the guppy did not appear to be perfectly correlated with the evolution of color based sexual selection in this lineage, I turned to investigating the expression of this opsin repertoire. In situ hybridization was used to characterize the pattern of opsin expression across the surface of the retina of adult male and female guppies. In situ hybridization demonstrated that most opsin genes had distinct expression profiles. These expression patterns also indicated that sensitivity and discrimination in the dorsal retina might differ from the ventral retina; the ventral retina appears to be tuned to middle-wavelength light (green), while the dorsal retina is predicted to have exceptional wavelength discriminatory ability and broad spectral sensitivity. This expression data was then used to evaluate models of sexual selection in the context of the predicted visual capacity of the guppy.
Graduate

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.
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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

Ray-finned fish from a diversity of distantly related lineages have remarkably large visual opsin repertoires. Starry flounder (Platichthys stellatus) development, morphology, life history, and behavior make this species especially suitable for experiments designed to determine why fish have so many opsins. Human and bird colour vision uses three and five opsins, respectively. Fish often have many more opsins. We sequenced an eye transcriptome to determine the starry flounder opsin repertoire, and used high performance liquid chromatography to determine the chromophore content of the retina. We found eight visual opsins that utilize only 11-cis-retinal (vitamin A1). This species’ entire visual opsin toolkit appears to be functional. The number of distinct cone and rod cell absorbance profiles determined using microspectrophotomery are consistent with the number of visual opsins in the transcriptome. RH2 transcripts were more abundant and SWS1 and SWS2 transcripts were less abundant in the dorsal retina, where cone density was highest, outer segments the longest, and where we observed double cones with outer segments that differed in their wavelength of maximum absorbance. Regions of fish retinas appear to be specialized and I predict that this fine-tuning is enhanced by photoreceptor plasticity and opsin gene duplication and divergence. Studies that compare opsin expression patterns among individuals, populations, or species typically assume that the differences observed influence vision. Direct connections between opsin expression and quantitative behaviours are rare. This thesis aimed to test whether varying opsin expression affects vision by modifying opsin expression and characterizing vision in starry flounder. We held starry flounder in aquaria exposed to either broad spectrum sunlight or green-filtered light. We tested vision by quantifying the visually-mediated camouflage response and we measured opsin expression using digital-PCR. Granularity analysis of photographs of the camouflage response revealed higher overall pattern energy at each of the seven spatial frequency bands in fish exposed to broad spectrum sunlight compared to the green-filtered fish. However, no statistical difference in typical measurements of pattern or contrast (e.g., maximum filter size, the standard deviation of pattern energy, and the proportional power) was observed between the two groups. Opsin expression was different between fish held in the green light environment compared to those exposed to broad spectrum light. SWS1 (UV sensitive) and SWS2B (blue sensitive) were significantly down regulated in response to the green light environment. Surprisingly, this difference was lost after only three hours under a white LED light, suggesting rapid changes in opsin expression in response to the light environment. We found tantalizing, albeit not statistically significant evidence that fish with higher expression of UV- and blue-wavelength sensitive opsins could see more contrast in colour on blue-green checkerboards.
Graduate

Visual pigments are light sensitive receptors in the eye that form the basis of sensory visual transduction. This thesis presents three studies that explore visual pigment proteins in vertebrates using a number of computational and experimental methods in an evolutionary framework. The objective is not only to identify, but also to experimentally investigate the functional consequences of genetic variation in vertebrate visual pigments. The focus is on great bowerbirds (Chlamydera nuchalis), which are a model system in visual ecology due to their spectacular behaviour of building and decorating courtship bowers. There are 4 chapters: Chapter 1 introduces background information on visual pigments and vision in birds. Among visual pigment types, the short-wavelength-sensitive (SWS1) pigments have garnered particular interest due to the broad spectral range among vertebrates and the importance of UV signals in communication. Chapter 2 investigates the evolutionary history of SWS1 in vertebrates with a view toward its utility as a phylogenetic marker. Chapter 3 investigates SWS1 evolution and short-wavelength vision in birds, with particular focus on C. nuchalis and its SWS1. The evolution of spectral tuning mechanisms mediating UV/violet vision in passerines and parrots is elucidated in this chapter using site-directed mutagenesis, protein expression, and phylogenetic recreation of ancestral opsins. While cone opsins mediate colour vision in bright light, the rhodopsin visual pigment contained in rod photoreceptors is critical for dim light vision. Detailed characterization of rhodopsin function has only been conducted on a few model systems. Chapter 4 examines C. nuchalis RH1 using a number of functional assays in addition to absorbance spectra, including hydroxylamine sensitivity and the rate of retinal release. This chapter includes an investigation into the role of amino acid mutations typical of dim-light adapted vertebrates, D83N and A292S, in regulating functional properties of bovine and avian RH1s using site-directed mutagenesis. Together these chapters describe naturally occurring mutations in visual pigments and explore the way they can influence visual perception. These represent one of the few investigations of visual pigments from a species that is not a model lab organism and form a significant contribution to the field of visual pigment biochemistry and evolution.

The overall objective of this thesis was to understand better the mechanisms that shape the diversity in colour vision of fish, and to explore the adaptive significance of this divergence. Among the vertebrates, teleost fish show the greatest diversity in colour vision systems. The cichlid model system illustrates that the visual system of fish may differ among species, sexes, individuals, and life stages of individuals. The large number of available cone opsin genes, which have resulted from multiple opsin gene duplications, facilitates this high degree of variation in the mechanisms of colour vision. In general, cichlids possessed complements of four to five cone pigments, and these complements varied across species, sexes, and individuals. Additionally, lens transmission, cone pigment expression, post-receptoral sensitivity, and retinal circuitry differed across life stages of individuals. My results suggest that the diversification of colour vision across species and across life stages of individuals contributes to sensory adaptations that enhance both the contrast of zooplanktonic prey, and the detection of optical signals from conspecifics. Therefore, both natural and sexual selection may have worked in concert to shape colour vision in fish. Since light is more complex under water than on land, fish required four to six cone classes to reconstruct the colour signals reflected from aquatic objects. This suggests that the large number of cone pigments in fish have likely evolved to enhance the reconstruction of the complex colour-signals in aquatic environments. Taken together, these findings improve our understanding of the variable nature of fish colour vision, and, more generally, help unravel the evolution of photoreceptors and colour vision.
Thesis (Ph.D, Biology) -- Queen's University, 2012-05-14 13:16:50.276

For many species, vision contributes to a number of fitness-related tasks, including mating and the detection of prey and predators. Selection on the visual system should therefore be strong, especially when ecological or genomic changes open new avenues for evolutionary changes. Visual system proteins are thus attractive systems for molecular evolutionary analyses. This thesis presents a collection of evolutionary studies on two gene families, opsins and crystallins. Opsin proteins determine the wavelengths of light detected by the retina, while crystallin proteins contribute to lens transparency and refractory power. My studies focus on teleost fishes, because teleost visual ecology is exceptionally diverse and because gene duplication is common in this group. In Chapter One, I outline the relevance of protein variation to organismal evolution and describe the analytical methods employed throughout this thesis. Chapter Two considers the long-wavelength sensitive (LWS) opsins of the guppy (Poecilia reticulata). The guppy is shown to possess multiple LWS opsins that have accumulated differences at functionally important amino acid sites since duplicating. Chapter Three focuses on the guppy’s main predator, the pike cichlid Crenicichla frenata, which is shown to have a greater capacity for short-wavelength vision than previously believed. However, this cichlid possesses three fewer opsins than closely-related African cichlids, a difference partly due to duplication of a green-sensitive (RH2) opsin in African cichlids. In Chapter Four, this RH2 duplication event is studied in greater depth; variation in selective constraint is documented following gene duplication and between species from different lakes. Some of the analytical methods employed in Chapter Four were newly developed, as detailed in Chapter Five, where a test for functional divergence among clades is evaluated and then improved upon through the presentation of a new null model that better accommodates among-site variation in selection. In Chapter Six, phylogenetic relationships within the βγ lens crystallin superfamily are clarified, and the functionally distinct γN family is shown to have evolved conservatively compared to other crystallin families. The thesis concludes with suggestions for future directions for evolutionary research on opsins and crystallins, and summarizes recent work that has built on these studies.