Heethaka, Krishantha Sameera De Zoysa (2023) Octopus Neuroethology (Octopus vulgaris): Octopus Senses & RNA Editing Mechanisms. [Tesi di dottorato]

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Octopuses, particularly the common octopus (Octopus vulgaris), exhibit intriguing sensory capabilities and intricate RNA editing mechanisms. This Ph.D. thesis aims to explore the neuroethology of O. vulgaris, specifically focusing on evidence for smell-by-touch behavior and molecular evidence for olfactory receptor genes in arm suckers, as well as photoreceptor genes in the optic lobe. Furthermore, it investigates the RNA editing mechanism of Egr1 (Early Growth Response 1) and its implications for protein diversification in O. vulgaris. The investigation of photoreceptor genes and their localization was conducted using molecular techniques and WMISH in the optic lobe of O. vulgaris. Molecular approaches identify the expression and localization of various photoreceptors, including Retina Rhodopsin, Retinochrome, Melanopsin Rhodopsin, Rhabdomeric, and Rhodopsin kinase in the optic lobes. These findings suggest distinct roles for these photoreceptors in light perception and visual processing, contributing to our understanding of cephalopod vision and octopus’ vision. To investigate O. vulgaris' sensory systems, including smell-by-touch behavior and olfactory receptor genes, a comprehensive approach has been employed. Molecular techniques and behavioral experiments have been used to examine the evidence supporting the detection of odors through tactile contact by octopus’s suckers. The respective genes have been localized using the whole-mount in-situ hybridization (WMISH) technique, indicating the presence of a "smell by touch" behavior. The expression of selected G-protein-coupled receptors (GPCRs) in the suckers of octopuses provides direct evidence of the presence of olfactory receptors in contact chemoreception, among other varieties of GPCRs. Behavioral experiments have revealed that octopuses can recognize water-insoluble compounds and distinguish them, showing clear interest. The time spent analyzing the compounds has been quantified using BORIS software. The process of effectively anesthetizing invertebrates, particularly O. vulgaris, during animal experiments presents a significant challenge, with the goal of reducing stress on these animals while minimizing the use of anesthetics. The research explores the limitations of various substances used for anesthesia and underscores the need to achieve muscle relaxation, pain relief, unconsciousness, and amnesia with a single substance. In this study, it is demonstrated that the clinical anesthetic isoflurane (1%), commonly used by veterinarians, can rapidly and efficiently anesthetize octopuses when employed in combination with 1% magnesium chloride, which serves as a muscle relaxant. Recovery is similarly swift, and the entire process, from anesthetization to recovery, is completed in 30 to 35 minutes, thus minimizing animal stress. This research development showcases the effectiveness of this approach in reducing stress on the animals and reducing the quantity of anesthetics required, making a valuable contribution to the field of invertebrate anesthesia. In addition to sensory systems, the last chapter investigated the RNA editing mechanism of Egr1 in O. vulgaris. By employing molecular techniques and bioinformatic analyses, the RNA editing events within the Egr1 gene were examined. RNA editing mechanisms, particularly in the Egr1 gene, were explored in O. vulgaris. RNA editing was found to be prevalent in cephalopods, with a high percentage of editing events occurring in the nervous system. The investigation compared A-to-G(I) RNA editing levels in the Egr1 gene between wild and controlled O. vulgaris, revealing variations likely due to different environmental conditions and stressors. Although the modifications in the Egr1 gene does not result in changes to the amino acid sequence, further research is needed to understand the timing, duration, and impact of RNA editing in cephalopods. Understanding these specific editing patterns and their functional consequences will shed light on the regulation of neural plasticity and adaptive responses in O. vulgaris. Overall, this Ph.D. thesis provides comprehensive insights into O. vulgaris' sensory capabilities and RNA editing mechanisms. The investigation of photoreceptor genes in the optic lobe enhances our understanding of cephalopod vision and its adaptation to visual surroundings. The evidence for smell-by-touch behavior and GPCRs in arm suckers highlights the multi-modal sensory system of octopuses. The use of clinical anesthetics provides a standardized method for efficiently anesthetizing octopuses, reducing the entire process from anesthesia to recovery and minimizing animal stress. Furthermore, the exploration of RNA editing mechanisms and the Egr1 gene sheds light on the regulatory processes underlying neural plasticity in O. vulgaris. The findings from this research contribute to our broader understanding of cephalopod neuroethology and have implications for sensory biology, neurobiology, and potential applications in therapeutic strategies and artificial visual systems.

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