Environmental effects during gonadal development in fish: role of epigenetics Memòria presentada per Alejandro Valdivieso Muñoz per optar al grau de Doctor per la Universitat de Barcelona Programa de Doctorat de Genètica, Microbiologia i Estadística Tesi desenvolupada al Departament de Recursos Marins Renovables de l’Institut de Ciències del Mar (ICM -CSIC) de Barcelona Director de la tesi : Co -directora de la tesi: Dr. Francesc Piferrer Circuns Dra. Laia Ribas Cabezas Tutora de la tesi : Doctorand : Dra. Montserrat Coromina s Guiu Alejandro Valdivieso Muñoz Barcelona, gener de 2020 Preface Preface This Ph.D. thesis was carried out at the Group of Biology of Reproduction (GBR) of the Department of Renewable Marine Resources, Institute of Marine Sciences (ICM-CSIC) in Barcelona, under the supervision of Dr. Francesc Piferrer and Dr. Laia Ribas, and under the Ph.D. Genetics Program of the Universitat de Barcelona during the years 2015 to 2020. The aim of this thesis was to contribute to our understanding of the genetic and environmental factors on sex differentiation in zebrafish ( Danio rerio ). The thesis is structured as follows: Block A: Density effects on zebrafish Chapter 1. Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response. Chapter 2. Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations. Block B: Temperature effects on zebrafish Chapter 3 . Fish reproductive tissues as heat recorders: DNA methylation epigenetic marks in zebrafish gonads correctly recapitulate past thermal history. Chapter 4 . Hot sex in wild zebrafish: can the natural genetic sex determination mechanism buffer environmental effects on population sex ratios? Chapter 5 . Family-dependent variation in the multigenerational effects on sex ratios in zebrafish exposed to elevated temperature: changes in the testicular epigenome of unexposed offspring. iii Preface Each chapter corresponds to an article either published, to be submitted or soon to be published: Ribas, L., Valdivieso, A., Díaz, N., Piferrer, F., 2017. Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response. Journal of Experimental Biology , 220: (6), 1056 –1064. doi: 10.1242/jeb.144980. Valdivieso, A., Ribas, L., Piferrer, F., 2019. Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution , 332: (3 – 4), 55 –68. Valdivieso A., Anastasiadi, D., Ribas L., Piferrer F. Fish reproductive tissues as heat recorders: DNA methylation epigenetic marks in zebrafish gonads correctly recapitulate past thermal history. Epigenetics & Chromatin . To be submitted. Valdivieso, A., Wilson, C., Amores, A., Rodrigues, M.D.S., Nóbrega, R.H., Ribas, L., Postlethwait, J.H., Piferrer, F. Hot sex in wild zebrafish: can the natural genetic sex determination mechanism buffer environmental effects on population sex ratios? In preparation. Valdivieso, A., Ribas, L., Monleon, A., Orban, L. Piferrer, F. Family-dependent variation in the multigenerational effects on sex ratios in zebrafish exposed to elevated temperature: changes in the testicular epigenome of unexposed offspring. In preparation. During the realization of this thesis, Alejandro Valdivieso also contributed in the following articles: Ribas, L., Valdivieso, A., Díaz, N., Piferrer, F., 2017. Response to “The importance of controlling genetic variation –remarks on ‘Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response’”. Journal of Experimental Biology , 220: (6), 4079 –4080. doi: 10.1242/jeb.167437. Note: this paper contains data presented in chapter 3. Piferrer, F., Anastasiadi, D., Valdivieso, A., Sánchez-Baizán, N., Moraleda-Prados, J., Ribas, L, 2019. The Model of the Conserved Epigenetic Regulation of Sex. Frontiers in Genetics , 10, 857. doi:10.3389/fgene.2019.00857. Note: this paper is related to chapter 2. iv Preface The results obtained in this thesis have been presented at the following scientific meetings: Alejandro Valdivieso, Laia Ribas, Francesc Piferrer . Gene expression analysis in gonads of zebrafish (Danio rerio) subjected to masculinization treatments . Avenços en Recerca en Aqüicultura. Societat Catalana de Biologia (SCB), 12 June 2015. Barcelona (Spain). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer. Effect of high rearing density on sex differentiation in zebrafish (Danio rerio): sex ratio and gene expression analysis. 10° Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), 23 –25 June 2015. Castellón (Spain). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer . Transgenerational effects by Temperature in zebrafish (Danio rerio). XVI Jornada de Biologia de la Reproducció. Societat Catalana de Biologia (SCB), 2 July 2015. Universitat Autònoma de Barcelona (UAB), Barcelona (Spain). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer. Thermal transgenerational and rearing density effects in laboratory strains of zebrafish (Danio rerio). Epiconcept Conference. COST European Cooperation in Science and Technology: COST Action FA1201, Epiconcept, 6 –7 October 2015. Creta (Greece). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer. Temperature and density masculinize a laboratory strain of zebrafish (Danio rerio). Epiconcept Workshop. COST European Cooperation in Science and Technology: COST Action FA1201, Epiconcept, 18 –19 May 2016. Velingrad (Republic of Bulgaria). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer. DNA methylation levels of sex- related genes involved in the gonadal development of zebrafish (Danio rerio). VIII Jornada de Cromatina i Epigenética. Societat Catalana de Biologia (SCB), 16 March 2018. Barcelona (Spain). Alejandro Valdivieso, Laia Ribas, Francesc Piferrer. DNA methylation of genes promoters involved in sexual differentiation and stress of zebrafish (Danio rerio) subjected to high temperature during the early gonadal development. 5th International Symposium on Genomics in Aquaculture (GIA), 21 –23 March 2018. Faro, Algarve (Portugal). Alejandro Valdivieso, Javier Moraleda-Prados, Dafni Anastasiadi, Laia Ribas, Francesc Piferrer. Metilació de cyp19a1a i dmrt1, dos marcadors del desenvolupament gonadal en el peix zebra (Danio rerio) exposats a diferents perturbacions ambientales . XVI Jornada de Biologia de la Reproducció. Societat Catalana de Biologia (SCB), 19 May 2019. Barcelona (Spain). v Preface Alejandro Valdivieso, Dafni Anastasiadi, Laia Ribas, Francesc Piferrer. Persistent epigenetic changes due to elevated temperature in mature zebrafish gonads . Epigenetic Inheritance: Impact for Biology and Society. University of Zurich (UZH), 26 –28 August 2019. Zurich (Switzerland). The research presented here was supported by AGL2013-41047-R (EPIFARM) and AGL2016-78710-R (EPIMARK) grants from the Ministerio de Economía y Empresa and Ministerio de Ciencia, Innovación y Universidades, respectively, both awarded to Dr. Francesc Piferrer. Alejandro Valdivieso was supported by a scholarship (BES-2014 –069051) from the Government of Spain (September 2015 –August 2019) and a contract linked to the project Consumer-driven production: Integrating innovative approaches for competitive and sustainable performance across the Mediterranean aquaculture value chain (PERFORMFISH, EU2020 code 727610). (December 2019 – present). During the course of this research, Alejandro Valdivieso did two stays of three months each during 2017 and 2018 at the Institute of Neuroscience (ION) at the University of Oregon, USA under the supervision of Dr. John Postlethwait. Travel and living expenses were supported by the Spanish grants: EEBB-I-17 –12342 and EEBB-I-18 –12947. vi Agradecimientos / Acknowledgments Agradecimientos / Acknowledgments Esta gran aventura empezó hace ya casi cinco años de forma inesperada. Realizar un doctorado no se dibujaba en mi horizonte más cercano, pero paulatinamente se fue gestando en mí la posibilidad de llevar a cabo un proyecto de esta magnitud. Creía, por aquel entonces, ser consciente del compromiso que adquiría en relación al nivel de esfuerzo y dedicación que requería semejante objetivo. Pero sin duda alguna, la realidad superó cualquier idea inicial. La elaboración de esta tesis ha significado una de las etapas de mi vida más desafiante e intensa. Y también, por qué no decirlo, dura, en la que he debido poner en marcha mis estrategias de resistencia y superación. Pero por encima de todo, ha sido apasionante e enriquecedora. Porque al final lo que importa, no es la meta sino el camino. Y a lo largo de este camino, he crecido profesional y personalmente. Durante este viaje por el Institut de Ciències del Mar (ICM) me han acompañado personas que han aportado, cada uno en su medida, un valioso apoyo en la materialización de esta tesis. Por ello, quiero expresar mi más sincero agradecimiento a todas ellas, por haber formado parte de este camino y sin las cuales no hubiera transitado por él tal y como lo he hecho. En primer lugar, me gustaría dar las gracias por su dirección, sugerencias y apoyo constante a mis dos directores de tesis, el Dr. Francesc Piferrer y la Dra. Laia Ribas. Sin la confianza que depositaron en mí y sin su dedicación este proyecto no sería hoy una realidad. A Francesc le quiero agradecer muy especialmente el haberme dado la oportunidad de hacer mi tesis doctoral en su laboratorio, haberme transmitido su pasión por la ciencia