Biological sciences

Abstract

Abstract Although pigment cells in fish species play a crucial role in forming the distinctive colour patterns of each species, these cells also have species-specific physiological characteristics. Our model Arabian killifish, Aphanius dispar, possesses distinctive pigmented cells where in addition to black pigment cells (melanophores), this species has extremely fluorescent pigment cells that have been named fluoroleucophores, which contain two types of pigments: pteridine fluorescent pigment and the white pigment urea. A. dispar is tolerant to high temperatures and thrives in environments with intense light, which makes it a suitable model for studying the effects of ultraviolet (UV) radiation. Understanding the molecular mechanisms of UV-induced DNA and cellular protection is crucial due to increasing levels of UV radiation resulting from climate change, which can impact aquatic ecosystems. From this perspective, this study investigated the responses of pigment cells in A. dispar to UV radiation and the molecular mechanisms involved in UV-induced DNA and cellular protection. This was achieved by using three genetic lines of A. dispar, wild type, gch mutant and gch/tyr double mutant lines. Gch mutant does not have pteridine fluorescent pigment, and gch/tyr line lacks the fluorescent pigment and melanin pigment. The PhD project started with the generation of the gch-/-tyr-/- mutant line using CRISPR/Cas9 genome editing technology, in which the tyr gene was knocked out in the previously established gch mutant line. The results showed an induced albino phenotype in the founder at a rate of 83%, indicating the high efficiency of the CRISPR/Cas9 technique in A. dispar. Subsequently, embryos of these three strains were exposed to varying doses of UV radiation, and the expression of critical genes involved in DNA repair, stress response, and cell cycle control were analysed. The results showed varied responses to UV light among the three lines, as indicated by differences in survival rates, hatching rates, and gene expression. Compared to the wild type, the albino (gch-/-tyr-/-) line displayed the highest UV sensitivity, significantly increasing mortality and altering gene expression levels. Additionally, the gch line showed slightly higher sensitivity to UV than the wild type. To effectively preserve newly generated genetic lines, I have developed a cryopreservation method for A. dispar spermatozoa, utilizing ultra-freezing techniques 2 | Pa g e at -150°C as an alternative to liquid nitrogen. The study evaluated the quality of fresh and frozen sperm in in vitro fertilization. According to the results, fertilization tests using stored spermatozoa showed a fertilization rate of 59 %, compared to 87 % using fresh sperm (unfrozen). The following analyses have been conducted to test the quality of sperm: evaluating the speed of sperm for both fresh and thawed sperm, the viability of frozen sperm, and fertilization rates by storing sperm after different durations of cryopreservation: one hour, one week, two weeks, one month, and three months. The data showed an effective cryopreservation and fertilisation rate. Therefore, the method can be widely used to preserve the genetic strain of A. dispar. In summary, I have generated a very useful transgenic mutant A. dispar line, gch-/-tyr-/-. The line would be a valuable model for studying and imaging internal organs with fluorescently labelled cells. These pigment mutants are highly useful for studying mechanisms of UV toxicity. From the survival studies and gene expression analyses, we have identified the crucial role of melanin and pteridine pigments in UV protection. All these lines are effectively stored and can be used by the research community for broader purposes in the future.