Physiological responses to daily temperature variation and ultraviolet radiation in amphibian larvae Pippa Kern B An Vet Bio Sci (Hons 1) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Biological Sciences 1 ABSTRACT Understanding how environmental variability influences species fitness is important for predicting species persistence in changing environments. Environments are highly variable and organisms have discrete physiological limits which determine the range of conditions they can tolerate. Daily thermal fluctuations (DTFs) impact the capacity of ectotherms to maintain performance and energetic demands due to thermodynamic effects on physiological rates. However, organisms can also flexibly alter their phenotype in order to maintain fitness in variable environments. Ectotherms experiencing DTFs would benefit from mechanisms which reduce the thermal sensitivity of physiological traits and buffer energetic costs. Furthermore, energetic consequences of DTFs may be exacerbated by the presence of additional stressors. Exposure to ultraviolet radiation (UVR) can increase energy requirements due to the repair of UVR induced damage. The costs of UVR exposure may cause energy trade-offs which exacerbate consequences of DTFs, and reduce the capacity to flexibly alter phenotypes. Importantly, temperature and UVR are also linked in their effects on cellular function as exposure to temperature extremes and UVR causes cellular damage that induces common protective mechanisms. Interactive effects of these environmental drivers on mechanistic traits may explain whole animal responses. Understanding what drives responses to temperature variation and the interactive effects between environmental stressors will broaden our understanding of how animals respond to environmental variation. The overarching aim of this thesis was to investigate physiological mechanisms underlying responses to DTFs and exposure to UVR in amphibian larvae. The capacity to flexibly alter the thermal sensitivity of traits in response to DTFs may depend on the degree of environmental DTF species experience. The first specific aim of this thesis was to investigate whether plasticity in response to DTFs is influenced by the thermal variability of a species’ habitat (Chapter 2). Tadpoles of three related species whose habitats vary in the degree of DTF were raised in stable temperatures and conditions in which temperatures fluctuated daily. Plasticity of upper thermal limits and the thermal sensitivity of swimming performance, resting metabolic rate and metabolic enzyme activity were examined. Environmental variation in species habitats did not predict the capacity to alter physiological rate processes. Tadpoles were unable to reduce the thermal sensitivity of physiology traits, which led to smaller body sizes and altered the length of development in a species specific fashion. DTFs increased upper thermal limits which may i buffer tadpoles from the lethal consequences of temperature extremes. The effects of DTFs on growth and development however, will likely negatively impact individual fitness. The second specific aim of this thesis was to investigate whether UVR exposure alters the physiological response to DTFs (Chapter 3). Tadpoles were raised in stable or daily fluctuating temperature conditions under high or low UVR levels, and the plasticity of upper thermal limits and the thermal sensitivity of swimming performance and resting metabolic rate were examined. Temperature and UVR exposure had an interactive effect, with DTFs reducing survival and increasing upper thermal limits only in tadpoles exposed to UVR. Tadpoles (Platypletrum ornatum) which inhabit ephemeral pools characterised by large DTFs and high UVR were inherently thermally insensitive for burst swimming performance and metabolic rate at high temperatures, and these traits were not affected by temperature or UVR treatments. Inherent thermal insensitivity may buffer development from the energetic challenges of such variable environments. Interactive effects of temperature and UVR exposure may reflect responses of lower level mechanistic traits. The third specific aim of this thesis was to investigate the interactive effects of temperature and UVR on upper thermal limits, heat shock protein (Hsp) abundance, antioxidant activity and oxidative damage (Chapter 4). Tadpoles were exposed to cold, warm or daily fluctuating temperature treatments in the presence or absence of UVR. Upper thermal limits were determined by an interaction between temperature and UVR. Ultraviolet radiation increased upper thermal limits in cold acclimated tadpoles and reduced upper thermal limits in warm acclimated tadpoles. This interactive effect was not explained by the relative abundance of Hsp70. Oxidative damage and antioxidant activity were influenced by UVR and temperature respectively, however there were no interactive effects. Understanding the interactive effects of multiple stressors on thermal tolerance is important to predict responses to environmental variation. Thermal tolerance is however, a complex trait and may not be readily explained by a subset of molecular and cellular mechanisms. This research has contributed to a broader understanding of the physiological consequences of DTFs and UVR in amphibian larvae. The results highlight that responses to DTFs may be species specific which may influence survival in variable environments, and that consequences of DTFs are exacerbated by exposure to UVR. Importantly, interactive effects between stressors determined physiological limits. Responses to these ii environmental drivers are complex and likely determined by interactions among a range of mechanistic traits. Physiological responses to different scales of temperature variation and the interactive effects of multiple stressors determine how animals function, and may ultimately determine population persistence in changing environments. iii 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. iv Publications during candidature Peer-reviewed publications Kern, P., Cramp, R. L. and Franklin, C. E. (2014). Temperature and UV-B insenstive performance in tadpoles of the ornate burrowing frog: an ephemeral pond specialist. Journal of Experimental Biology 217, 1246-1252. Kern, P., Cramp, R. L. and Franklin, C. E. (2015). Physiological responses of ectotherms to daily temperature variation. Journal of Experimental Biology 218, 3068-3076. Kern, P., Cramp, R. L., Seebacher, F., Ghanizadeh Kazerouni, E. and Franklin, C. E. (2015). Plasticity of protective mechanisms only partially explains interactive effects of temperature and UVR on upper thermal limits. Comparative Biochemistry and Physiology Part A 190, 75-82. Conference abstracts – oral presentations Kern, P., Cramp, R. L. and Franklin, C. E. (2014) Temperature and UV-B insensitive performance in tadpoles of the ornate burrowing frog. In: 50th Meeting of The Australian Society of Herpetologists, Greenhills Conference Centre, Canberra, Australia. Kern, P., Cramp, R. L., Seebacher, F and Franklin, C. E. (2014) Temperature does not affect oxidative damage from UV-B radiation in Limnodynastes peronii tadpoles. In: 31st Meeting of the Australian New Zealand Society for Comparative Physiology and Biochemistry, The University of New England (UNE), Armidale, Australia. Kern, P., Cramp, R. L., Seebacher, F. and Franklin, C. E. (2015) Development in variable environments: can developmental plasticity alter the costs of short-term temperature fluctuations and UV-B exposure in tadpoles. In: Main Meeting of the Society of Experimental Biology, the Clarion Congress Hotel, Prague, Czech Republic. v Publications included in this thesis Kern, P., Cramp, R. L. and Franklin, C. E. (2015). Physiological responses of ectotherms to daily temperature variation. Journal of Experimental Biology 218, 3068-3076. Incorporated as Chapter 2 with minor changes. Contributor Statement of contribution Pippa Kern (Candidate) Concept and design (60%),