Biochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research Oliver R. B. Thomas https://orcid.org/0000-0002-8497-670X Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy December 2018 School of BioSciences The University of Melbourne 1 ABSTRACT All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been i identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry. ii DECLARATION This is to certify that: The thesis comprises only my original work towards the PhD except where otherwise indicated. Due acknowledgement has been made in the text to all other material used. The thesis is fewer than 100 000 words in length, exclusive of tables, maps, bibliographies and appendices. Oliver R. B. Thomas December 2018 Cover image: Sagittal otolith from juvenile black bream (Acanthopagrus butcheri), D. Chamberlain, 2016 iii PREFACE I am the primary author and principle contributor on all chapters presented in this thesis. My primary supervisor, Stephen E. Swearer, is a co-author on all chapters. My supervisor Blaine R. Roberts is a co-author on Chapters 3 and 4. My third supervisor, Gregory P. Jenkins is a co-author on Chapter 5. Article publication status and co-author contributions Chapter Two: Unpublished material not yet submitted for publication. Co-authored by Stephen E. Swearer. ORBT and SES conceived and designed the review; ORBT read, assessed and selected manuscripts to be included in the review; ORBT wrote the manuscript; SES provided editorial comments. Contributions: ORBT 85%, SES 15% Chapter Three: Published in Metallomics on March 2017. Co-authored by Stephen E. Swearer, Katherine Ganio and Blaine R. Roberts. BRR, SES and ORBT conceived and designed the experiments; ORBT conducted the experiments and collected data with assistance from KG and BRR; ORBT analysed the data and wrote the manuscript; SES, KG and BRR provided editorial comments. Contributions: ORBT 75%, KG 5%, BRR 10%, SES 10% Chapter Four: Accepted for publication in FEBS Journal, December 2018. Co-authored by Stephen E. Swearer, Eugene A. Kapp, Po Peng, Gerry Q. Tonkin-Hill, Anthony Papenfuss, Anne Roberts, Pascal Bernard and Blaine R. Roberts. ORBT, BRR and SES conceived and designed the experiments. ORBT conducted the experiments and collected data with assistance from GQT, AP, AR and PB. ORBT, EAK and PP analysed data and designed workflows. ORBT wrote the manuscript. SES, EAK and BRR provided editorial comments. Contributions: ORBT 75%, SES 5%, EAK 5%, PP 3%, GQT 3%, AP 1%, AR 2%, PB 1%, BRR 5% Chapter Five: Unpublished material not submitted for publication. Co-authored by Gregory P. Jenkins, Katherine V. Thomas and Stephen E. Swearer. ORBT, GPJ and SES conceived and designed the study. ORBT conducted the experiments and collected data with assistance from GPJ and SES. KVT provided GIS expertise. ORBT wrote the manuscript. SES and GPJ provided editorial comments. Contributions: ORBT 75%, GPJ 10%, KVT 5%, SES 10% iv This research was funded by grants from the Holsworth Wildlife Research Endowment (Chapter Five), the Australian Research Council (Chapters Three, Four and Five), the Research on the Ecology and Evolution (REEF) Lab (all chapters), and the University of Melbourne’s John and Allen Gilmour Award (Chapter Four). All animal research was conducted in accordance with the animal ethics requirements of the University of Melbourne (Chapters Three, Four, Five). Permits were obtained from the Victorian state government for collection of fish (Chapters Three, Four and Five; RP 1204, 1135 and 1141). v ACKNOWLEDGMENTS Firstly, a huge thank you to my supervisors for all the support and friendship that they have provided over the past years. Greg, I have really appreciated your insight, advice and guidance in the field! Blaine, thank you for taking the plunge and joining the world of fish inner ears and, in turn, welcoming me into the Metalloproteomics group. Things would’ve been very different if you hadn’t agreed to collaborate with us! Steve, thank you for taking the gamble on me back in 2013. You’re a great supervisor, mentor and friend. I owe you much. My colleagues and mates in BioSciences, the Florey and the REEF, NCCC and Metalloproteomics laboratory groups (past and present) provided great advice and feedback on many of my manuscripts and presentations. Not only have they been fantastically collegial, they’ve also been stalwart friends! Of special note are those that made me feel part of everything: Po, Luke, Rob, Valeriya, Cleves, Sievers, Joao, Juanma, Matt, Fletch, Dean, Nicole, Kath, Jess, Eric, Soumya, Stephan, Larissa, Katie, Anne and Eugene. Many people were generous in sharing their time with me, volunteering to help me in the laboratory or the field: Dean Chamberlain, Luke Barrett, Michael Sievers, Jessica French, Kathryn Hassell, Anne Roberts, Soumya Mukherjee, Akiva Gebler, Ryan Woodland, Fiona Warry, David Brehm. Apologies if I’ve omitted anyone here! A special mention goes to my collaborators Po Peng and Eugene Kapp who co-designed a bioinformatics workflow with me and provided consistent support and friendship. My family and friends have been hugely supportive of me, giving me the space to write and think…and talk, talk, talk. My sister, Kath, provided expert advice and counsel. (She also made some fantastic maps!) My children, Jasmine, Frederick and Dashiell have all inadvertently become experts on fish ears through patiently listening to me. My mother and father, first introduced me to the amazing marine world through SCUBA – thank you! Finally, to Carolyn, my best friend. I couldn’t have done this without you. You’ve made more sacrifices than anyone so that I could follow my passion. Thank you. vi DEDICATION For Carolyn