Proteomics and Transcriptomics of Venomous Animals Dany Domínguez Pérez PhD Thesis presented to the Faculty of Sciences of the University of Porto Biology 2017 D Proteomics and Transcriptomics of Venomous Animals Dany Domínguez Pérez Biology Faculty of Sciences 2017 Supervisor D Prof. Agostinho Antunes, Faculty of Sciences of the University of Porto Co-supervisor Prof. Vitor Vasconcelos, Faculty of Sciences of the University of Porto “Look deep into nature, and then you will understand everything better” Albert Einstein Dedicated to my son and my wife ii iii Acknowledgements I acknowledge the Portuguese Fundação para a Ciência e a Tecnologia (FCT) for financial support of my PhD project (SFRH/BD/80592/2011). This study was funded in part by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845), and by the Structured Programs of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), CORAL NORTE (NORTE-01-0145-FEDER-000036), and MarInfo—Integrated Platform for Marine Data Acquisition and Analysis (NORTE-01-0145- FEDER-000031), and funded by the Northern Regional Operational Program (NORTE2020) through the ERDF. I want to give a special acknowledgment to my Supervisor Prof. Agostinho Antunes and Co- supervisor Prof. Vítor Vasconcelos for letting me come to work with them and their teams, for providing me all the resources needed in the execution of the work presented in this thesis, for always show kindness, respect, and trust. Thanks to my colleagues from EGB and BBE, CIIMAR, for facilitating me the integration inside the groups; specially to Guillermin Aguero Chapin for his orientation during the process, to Bárbara Frazão, Tibisay Escalona, Imran Khan, Jorge Neves, Micaela Vale, João Morais, Pratheepa Moorthy, Dina Gomes, Aldo Barreiro, Sofia Costa, Raquel Castelo Branco, Margarida Costa, Vitor Ramos, Pedro Leão, Cristiana Moreira, Marisa Silva, Anoop Alex and Cidália Gomes, for their useful help at the beginning. To those colleagues who participated directly in the research work with me: Carlos Manlio Diaz Garcia, Neivys García Delgado, Yusvel Sierra Gómez, Olga Castañeda, Carlos Varela, Armando Alexei Rodríguez, Hugo Osorio, Joana Azevedo, Alexandre Campos, Maria V Turkina, Tiago Ribeiro, Isabel Cunha, Ralph Urbatzka, Jordi Durban, Javier Torres Lopez, Reinaldo Molina Ruiz, Tito Mendes, Emanuel Maldonado, Filipe Silva, Bruno Reis and Juan J. Calvete. iv Thanks to SASUP for provide me a comfortable lodging, specially to the workers of the residence Residência Universitária Campo Alegre 695, (Pólo III): Angela Braga, and for give me the opportunity to meet people from many countries and culture who shared their experiences, which has allowed me to understand the story much better and leave as a result many bonds of friendship worldwide, only real and unique reason to feel proud. To those colleagues who helped in the thesis preparation: Daniela Almeida and João Paulo Machado. Thanks to faculty staff, specially to Rosária Seabra and Ana Isabel Barreira. I want to thank those special people who were always there, who helped me many times or who welcomed me in their family circle at least once: Fernando Cagide Fajin and José Luis Cagide Fajin Bros, Doris Decoro Rojas and his husband and great Portuguese friend Antonio Luis Lopes de Sousa Castro, Robert Carcasses and Yuselis Castaño, Yonni Romaguera and Lisa Benamati, Rudy and Yaya, Gerardo González jr, Tibysay Escalona, Alexandre Campos, Quiaoquio Chen and Carlos Gustavo Moraes Castro. Finally, thanks to all my family member and relatives: To my maternal grandparents who represent "the theory of everything", Reymundo Pérez (I hope you feel happy and a bit proud wherever you are) and Inocencia Rodríguez: the effort, Altruism, gentleness and humility. To my mother Maritza Pérez Rodríguez for the education and for giving me her infinite strength to overcome all obstacles. To my sister Mayté Domínguez and my brother-in-law Héctor González, because all the help and support they have given me. Specially, to my wife Yudermys Moya Chaviano and to my son Eiden Fabián Domínguez Moya, thank you for all the love, understanding, affection, for the endless sacrifice of watching time pass, while we stay away from each other. All this work is dedicated to you. The fruit harvested is yours, and if it would produce more, it will also be yours. I just hope that the knowledge and experiences that come to our home enrich our daily life and trace the path for my son to the truth. Thanks to Portugal and its people for welcoming me throughout this period which has been a great experience. v Abstract The presence of toxins is a feature that confers significant advantages to venomous animals in the struggle for survival. Throughout evolution, many group of animals have been independently developing specialized tissues coupled to a delivery system like fangs, needles, harpoons, to produce and inoculate venoms. Indeed, more than 100,000 venomous species are distributed among different taxa. The poison contains what we refer by toxins, but venoms are essentially a mixture of many compounds including proteins, peptides, salts, organic molecules, amino acids, and neurotransmitters-like molecules that produce a synergically toxic effect. In general, the mechanism of action involves hydrolytic enzymes that degrade tissues, allowing other toxins to diffuse up on their targets mainly in the nervous or cardiovascular system. Among these targets we can highlight, membrane receptors, ion channels and enzymes that regulate the metabolism of excitable cells. Such toxins usually act at very low concentrations on their targets, causing a drastic change in important physiological functions that eventually lead to death. Toxins are widely distributed among metazoans and there are some venomous lineages both in vertebrates and invertebrates. Within vertebrates, snakes represent one of the major sources of toxins, and have been so far studied due to its powerful toxins and biomedical interest. By contrast, Cnidarians, which are grouped in the largest phylum of venomous animals, remain still unexplored. The species of the phylum Cnidaria commonly possess specialized stinging cells called nematocyst that produce and inject into prey or predator a mixture of toxins, whilst snakes possess maxillary venom glands coupled to front or rear fangs. Many toxins like enzymes, protease inhibitors, ion channels modulators, have been isolated and characterized from both groups. Venoms often contain a group of peptide/protein toxins with neurotoxic and cardiotoxic activities. However, Cuban and Portuguese cnidarians represent a rich source of toxins but remain mostly underexplored. Similarly, there are no studies addressing the production of toxins in snakes from Cuba, even though clinical symptoms have been reported after bites of some colubrids. The main goal of this project is to perform the proteomic characterization of toxins from Cuban and Portuguese cnidarians, and to profile the Harderian gland transcriptome from Cuban snakes. The generated information will increase the information about such toxins and its protein-encoding genes. Moreover, the characterization of novel toxins may allow us to discover novel cell excitability modulators as a source of new pharmacological tools or therapeutic products. In addition, the new findings will provide insight into the evolutionary history of the molecular diversification of toxins and its venom-encoding genes. The phylum Cnidaria is an ancient group of venomous animals, specialized in the production and delivery of toxins. Many species belonging to the class Anthozoa have been vi studied and their venoms often contain a group of peptides of less than 10 kDa that act upon ion channels. These peptides and their targets interact with high affinity producing neurotoxic and cardiotoxic effects, and even death, depending on the dose and the administration pathway. Zoanthiniaria (Cnidaria) is an order of the Subclass Hexacorallia, class Anthozoa, and unlike sea anemone (order Actiniaria), neither its diversity of toxins nor the in vivo effects of the venoms has been exhaustively explored. Unlike sea anemones, proteomics studies aiming toxins discovering from the order Zoanthidea are scarce. There are only few reports about the toxicological properties of its members and their toxins composition is scarce. In CHAPTER 2, some toxicological tests on mice with a low molecular weight fraction obtained by gel filtration in Sephadex G-50 from Zoanthus sociatus crude extract were assessed. The toxicological effects of the studied fraction seem to be mostly autonomic and cardiotoxic, causing death in a dose dependent manner with a LD50 of 792 μg/kg. Moreover, at a sub-lethal dose the active fraction accelerated the KCl-induced lethality in mice. Information obtained in the CHAPTER 2 shed light about the molecular mass composition of the fraction from Z. sociatus, which resulted lethal to mice. However, the identification and nature of the components of such fraction remains unknown. Therefore, in CHAPTER 3, a mass spectrometry analysis of a low