In the literature, few studies investigated the retinas of less than a handful of scolecophidians and reported they have only rods and express only the rod photopigment (RH1). However, in this action, I was able to differentiate rods containing rhodopsin and cones containing the long-wavelength sensitive opsin (LWS), in retinas of five species from three families, including the two main branches of Scolecophidia. In three species, I also identified cones expressing a second photopigment, the short-wavelength sensitive opsin, SWS1. This retina organization, with rods and two types of cones, is the typical setup of nocturnal alethinophidians. In one particular species, the retina was dominated by LWS cones, the SWS1 cones were numerous, and the RH1 highly coexpressed with cone opsins in cone-like photoreceptors, similar to diurnal caenophidian (“advanced”) snakes. Therefore, my results reveal considerable morphological diversity of the retinas of scolecophidians and contradicts a long-established paradigm that all species from this group have all-rod retinas, with no cone opsins, and would be too reduced to represent the retina blueprint of ancestral snakes. From the perspective of retinal anatomy and visual opsins, my data bring new lines of evidence to support the fossorial hypotheses for snake origins and to place scolecophidians as the most ancestral representatives of modern groups.
I also aimed to elucidate, for the first time, aspects of the neural circuitry of the snake retina. Several studies have described the types of photoreceptors and visual opsins in many species. However, nothing was known about the downstream neurons that receive the light information from the photoreceptors, and their wiring. For that, I used immunohistochemistry with different markers, some of which showed labeling patterns that varied according to the species phylogeny, and others revealed types of neurons that varied between diurnal and nocturnal snakes. In the retinas of nocturnal snakes, with rods and cones, I identified a bipolar cell that contacts exclusively rods, a groundbreaking finding described to date only for mammals. In the “all-cone” retinas of diurnal snakes (without typical rods), this rod bipolar cell is absent. Therefore, this project has brought extremely relevant and innovative results on the retina circuitry in snakes.
Overview of the work per Work Package (WP): Samples were adequately collected (WP1), and I was able to analyze the retinas of five scolecophidian species, in addition to a wide variety of aletinophidians. This allowed a broad comparative approach within a phylogenetic framework. For Immunohistochemistry Studies (WP2), retinas were appropriately processed, and I was able to generate a large amount of data that revealed the diversity of patterns of scolecophidian retinas, the presence of the three visual opsins, as well as documenting the retinas of new species of alethinophidians, showing the diversity of opsin expression patterns. For Electron Microscopy (WP3), the retinas of 10 caenophidians were properly processed for analysis of the ultrastructure of photoreceptors and interneurons. For Retinal Circuitry Studies (WP4), the retinas were processed for immunohistochemistry with different antibodies, and for the first time, different types of bipolar cells were identified in snakes, and aspects of ‘wiring’ of photoreceptors and bipolar cells were discovered. The results were interpreted (WP5) in a phylogenetic and ecological context, to infer ancestral states and test evolutionary hypotheses. My results help to resolve important conflicts about the origin of snakes: based on the presence of rods and cones, and three visual opsins in scolecophidians, this group can be positioned as ancestors of current snakes. The data was presented in three conferences (WP6), thus achieving great visibility in the scientific community.
Overview of the exploitation and dissemination: 3 conferences (1 invited talk)
