Reproductive physiology of Arapaima gigas (Schinz, 1822) and development of tools for broodstock management Lucas Simon Torati, BSc, MSc July 2017 A Thesis Submitted for the Degree of Doctor of Philosophy Institute of Aquaculture University of Stirling Scotland Declaration This thesis has been composed in its entirety by the candidate. Except where specifically acknowledged, the work described in this thesis has been conducted independently and has not been submitted for any other degree. Candidate Name: Lucas Simon Torati Signature: ……………………………………………….. Date: ……………………………………………….. Supervisor Name: Professor Hervé Migaud Signature: ……………………………………………….. Date: ……………………………………………….. III Lucas Torati Abstract Abstract Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the V Lucas Torati Abstract female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning. VI Lucas Torati Acknowledgements ACKNOWLEDGEMENTS I would like to express my gratitude to my main supervisor Dr. Hervé Migaud, for his enthusiastic guidance, friendship, support and encouragement along this study both in Scotland and Brazil. I thank also Dr. John Taylor for his co-supervision, all support and corrections provided. I am also very grateful to Dr. Amaya Albalat for being so positive with her guidance, and advising me with the proteomic and peptidomic analyses and chapter corrections. Likewise, I’m grateful to Dr. John B. Taggart for his assistance with ddRAD library construction, MiSeq sequencing, genomic data processing, for providing a comparative sequencing dataset on other teleosts, advice and corrections in the chapter on population genomics. I also thank colleagues and friends who helped me with histology (Dr. Debbie Faichney and Dr. Luciana Ganeco), microscopy (Dr. James Bron), molecular biology, lab techniques and data analyses (Dr. Andrew Davie, Dr. Sarah- Louise Counter, Dr. Robyn Harris, Dr. Stefanie Wehner, Dr. Kate Howie, Dr. Rogelio Flores, Dr. Nicole Rhody, Dr. Rosiana Rodrigues). I wish to thank Dr. Ben Clokie also for precious help with R programming and for all his aid in Stirling during my studies. I would also like to thank EMBRAPA for the funding and education opportunity, herein represented by Dr. Carlos Magno C. Rocha, Dr. Ariovaldo Luchiari, Dr. Eric Routledge and Dr. Alexandre A. Freitas. Very special thanks to Dr. Adriana Lima, Dr. Eduardo Varela and Dra. Luciana N. Ganeco for supporting the project in Brazil, and for all productive discussions we’ve had along this PhD. I thank Dr. Marta E. Ummus for elaborating maps in ArcGIS used to illustrate this thesis. Whereas in EMBRAPA Tropical Agroindustry (Fortaleza), I thank logistical and laboratorial support and advice received from Dr. Guilherme Zucolo, Dr. Edy S. Brito, Dr. Kirley Canuto, Dr. Lorena Maia, Dr. Tigressa Rodrigues and Dr. Patricia Bordallo. Special thanks also to Dr. Celli Muniz and VII Lucas Torati Table of Contents Dr. Luciana Ganeco for aid with SEM analyses of eggs. Still in Fortaleza, I was aided by Dr. Arlindo Moura and Dra. Paula Rodriguez who helped with preliminary proteomic analyses in Federal University of Ceará. Broodstock and infrastructure used along this thesis were provided by DNOCS, particularly Dr. Pedro Eymard C. Mesquita; and Mirador farm, represented by Marcelo Borba and Juarez Santos, to whom I owe my gratitude. I also thank Dr. Juliana Araripe and Dr. Eduardo S. Varela for providing samples which enabled the genomic analyses of Arapaima wild populations (Chapter 2). Also, slow-release implants used in Chapter 3 were made available thanks to Dr. Yonathan Zohar and Dr. John Stubblefield. I wish to thank also H. Strattner, Karl Storz and Ana Paula Varges for providing the endoscopy system used in Chapter 4, and Dr. Adriana Lima who strongly supported the experiments performed in Chapter 4 – Parts B and C with also important ideas and discussions on Arapaima cannulation. Also, thanks to Dr. Willian Mullen (University of Glasgow), Dr. Mary K. Doherty (University of the Highlands and Islands) and Justyna Siwy (Mosaiques Diagnostics GmbH) who provided their analytical facilities and support with CE-MS and LC-MS/MS analyses. I thank also important help received from very skilled colleagues along fish netting and handling, namely Rogério Miranda Araújo, Francisco Valdécio M. de Sousa, Valberto M. de Sousa, Luiz Oliveira, Agenor Galvão, Joacir Xavier da Silva, Tiago Vieira da Costa, Emilio Pinho and staff from Mirador farm. I acknowledge and thank financial support received from EMBRAPA, MAPA- CNPq (Grants. N. 457465/2012-3 and 434400/2016-5) and SEBRAE (FAPTO Grant N. 2538/2012). Finally, I wish to thank all my family for supporting me along this project. I’m deeply grateful to my beloved wife Melina Cambi for her constant and unconditional VIII Lucas Torati Acknowledgements support throughout this journey, accepting to move from a city to another (and from a job to another), for aiding me in moments of stress, and for facing the new challenges together in her joyful manner. IX Lucas Torati Table of Contents Table of Contents Abstract ........................................................................................................................... V ACKNOWLEDGEMENTS ....................................................................................... VII LIST OF ABBREVIATIONS ...................................................................................... 15 LIST OF SPECIES ....................................................................................................... 21 LIST OF FIGURES AND TABLES............................................................................ 23 1. Chapter 1 ................................................................................................................... 35 GENERAL INTRODUCTION .................................................................................... 35 1.1. Biology of Arapaima gigas and prospects for aquaculture ......................................... 37 1.1.1. Natural history ........................................................................................................... 37 1.1.2. General physiology and life-history .......................................................................... 40 1.1.3. Prospects for aquaculture .......................................................................................... 43 1.2. Physiology of fish reproduction ...................................................................................