UV-induced DNA damage in coral reef fish: Damage levels and protection mechanisms

Ultraviolet radiation (UVR) reaching the surface of earth has been recognized as a major environmental stressor for marine organisms due to the potential of UVB (280 315 nm) to induce DNA damage such as cyclobutane pyrimidine dimers (CPDs) that can lead to cell death. Despite inhabiting a UV-rich environment, the levels of DNA damage in reef fish and factors influencing these levels are unknown. Whether reef fish are able to avoid UVR and repair UV-induced DNA damage is unclear despite the importance of these protection mechanisms in other animals. The presence of UV-absorbing Mycosporine-like Amino Acids (MAAs) in the external mucus of reef fish has been confirmed, however the efficiency of these compounds in preventing CPDs has not been studied. The aim of this PhD was to assess the impact of ambient and elevated levels of UVR on reef fish in terms of UV-induced DNA damage, and to evaluate the protective mechanisms available to fish with and without UV vision. In order to determine the net level of DNA damage in skin samples of 15 species of fish from the reefs surrounding Lizard Island, a CPD-specific antibody was used in an ELISA. Analysis using a boosted regression tree shows that the most important factors governing CPDs were species and size, with higher damage being detected in smaller individuals. Other factors such as family, depth and the presence or absence of UV vision contributed the least to the variation in damage. The length of exposure to natural levels of UVR over the course of a day was found to have no significant influence on net DNA damage levels, which were relatively low in situ, compared to levels that were detected in later experiments using elevated UVR. The first protection mechanism, behavioural avoidance to UVR, was tested using behavioural experiments in which fish with (Pomacentrus amboinensis) and without UV vision (Thalassoma lunare) were given a choice between UV-protected and UV-exposed compartments. Additionally, foraging behaviour of settlement-stage larvae of P. amboinensis was determined under ambient levels of UVR. Automated analysis of video footage using MatLab shows that neither species showed a specific avoidance response to varying levels of UVB. Although P. amboinensis showed a preference for deeper sections of the experimental tanks, fish spent equal amounts of time in exposed and protected compartments. The foraging activity and distance to shelter of P. amboinensis that were exposed to UVR were significantly reduced compared to fish that were observed under light conditions that lacked UVR. Next, the efficiency of natural sunscreens, MAAs, in preventing CPDs was tested in P. amboinensis and T. lunare. The levels of MAAs in the mucus of the two species were either reduced or maintained during captivity before exposure to a short pulse of high UVR. Spectrometric measurements of light transmission through mucus samples collected after irradiation were used to quantify the amount of MAAs available for protection. In both species, DNA damage levels in skin samples from UVR exposed individuals was higher than in control groups that were exposed to light lacking the UV component. Spectrometric measurements of external mucus of both species revealed a clear link between higher mucus absorbance, i.e. MAA levels, and lower DNA damage levels. Furthermore, a significant increase in mucus absorption was observed in P. amboinensis after UV exposure. The last protection mechanism, the ability to revert DNA damage via photoreactivation and dark repair was investigated in four species (P. amboinensis, Pomacentrus moluccensis, Lethrinus variegatus and Siganus corallinus). All species examined showed significant increases of DNA damage after exposure to elevated UVR levels. Interspecific variation in the susceptibility to UVR was observed, with L. variegatus showing the highest damage levels. Significant reductions in DNA damage levels were found in P. moluccensis and L. variegatus that were exposed to photoreactivating light after the initial damage accumulation. Individuals of P. moluccensis that were shielded from any light exposure post UVR exposure also showed less DNA damage at the end of the experiment. This is the first study to address levels of UV-induced DNA damage in reef fish under natural conditions as well as under elevated doses of UVR that could occur in a changing climate. The relatively low levels of CPDs in a diverse group of reef fish indicate that current levels of UVR pose only low threat and underline the importance of protection mechanisms against UVR. Increases in UVR could have an impact not only on adult fish capable of adjusting their protection mechanisms, but also settlement stage larvae which show some of the highest levels of DNA damage in situ as well as mortality during slight increases of UVR. Whether these and other effects such as decreased foraging activity have broader implications on the recruitment and reproduction of reef fish and the community structure on coral reefs needs to be examined in the future. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Publications during candidature Peer reviewed papers Siebeck, U. E., O'Connor, J., Braun, C., & Leis, J. M. (2014). Do human activities influence survival and orientation abilities of larval fishes in the ocean? Integrative zoology (in press). Conference abstracts Braun, C., Reef, R. & Siebeck U.E. (2011). The effect of UV radiation on reef fish: avoidance, DNA damage and DNA repair. 3MT ® presentation, International Postgraduate Symposium, School of Biomedical Sciences, Brisbane, Australia. Eckes, M., Siebeck, U. E., Dunn, S., Braun, C. & Dove, S. (2012). Prevention and repair of ultraviolet induced DNA damage in fish. Oral presentation, International Coral Reef Symposium, Cairns, Australia. Braun, C., Reef, R. & Siebeck, U.E. (2012). How do reef fish cope with harmful UVB radiation? Poster presentation, International Postgraduate Symposium, School of Biomedical Sciences, Brisbane, Australia. Braun, C., Reef, R. & Siebeck, U.E. (2013). Mycosporine-like Amino Acids provide a rapid mechanism for UV protection in reef fish. Poster presentation, International Postgraduate Symposium, School of Biomedical Sciences, Brisbane, Australia & Australian Coral Reef Society conference, Sydney, Australia. Braun, C., Reef, R. & Siebeck, U.E. (2014). Natural sunscreens in coral reef fish prevent UVinduced DNA damage. Oral presentation, Otago University Postgraduate Symposium, Dunedin, New Zealand. Braun, C., Reef, R. & Siebeck, U.E. (2014). Natural sunscreens really do work: slip, slop, slap on the Great Barrier Reef. Oral presentation, Australian Marine Sciences Association student conference, Dunwich, Australia. Braun, C., Reef, R. & Siebeck, U.E. (2014). Natural sunscreens really do work: UV protection in reef fish. Oral presentation, International Conference on the Biology of Fish, Edinburgh, Scotland. Siebeck, U. E., Parker, A. & Braun, C. (2014). The impact of UVR on reef fish – the good and the bad. Australian Coral Reef Society conference, Brisbane, Australia. Publications included in this thesis No publications included. Contributions by others to the thesis U. E. Siebeck was significantly involved in the conception and planning of experiments and design of the whole study, catching of reef fish for chapter 2 and helped with revisions and interpretations of all chapters. R. Reef assisted with establishing the assay for the DNA damage analysis, provided resources and technical assistance as well as feedback and revisions for chapters 1, 2, 4 and 5. R. Reef also helped with measurements of light intensity in chapter 2 and 5. N. Rosic provided the measurements of MAAs in algae and fish food in chapter 4 as well as discussion and feedback on the manuscript. Y. Reshitnyk wrote and improved the MatLab script for the analysis of video data in chapter 3, and provided feedback and drafts for the method section of chapter 3. A. Parker, C. Newport and S. Van-Eyk assisted in aquarium maintenance and fish keeping, as well as discussions and feedback on this thesis. J. Leis donated larvae for chapters 2 and 5, and gave feedback and discussion on chapter 1. S. Blomberg provided assistance in analysing the data of chapters 3 and 4. Statement of parts of the thesis submitted to qualify for the award of another degree