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Immobilization of Phaeobacter 27-4 in Biofilters As a Strategy for the Control of Vibrionaceae Infections in Marine Fish Larval Rearing

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Immobilization of Phaeobacter 27-4 in Biofilters As a Strategy for the Control of Vibrionaceae Infections in Marine Fish Larval Rearing INSTITUTO DE ACUICULTURA CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS INSTITUTO DE INVESTIGACIÓNS MARIÑAS Immobilization of Phaeobacter 27-4 in biofilters as a strategy for the control of Vibrionaceae infections in marine fish larval rearing Ph.D. Thesis presented by: María Jesús Prol García To achieve the philosophiae doctor degree in: Marine Biology and Aquaculture April, 2010 Don José Pintado Valverde, Científico Titular no Instituto de Investigacións Mariñas (CSIC), Don Miquel Planas Oliver, Científico Titular no mesmo instituto do CSIC e Dona Ysabel Santos Rodríguez, Profesora Titular na Facultade de Bioloxía - Instituto de Acuicultura da Universidade de Santiago de Compostela INFORMAN: Que a presente Tese de Doutoramento titulada: “Immobilization of Phaeobacter 27-4 in biofilters as a strategy for the control of Vibrionaceae infections in marine fish larval rearing” presentada por dona María Jesús Prol García para optar ó título de Doutora en Bioloxía Mariña e Acuicultura realizouse baixo a dirección de José Pintado Valverde e Miquel Planas Oliver no Instituto de Investigacións Mariñas (CSIC), e considerándoa concluída autorizan a súa presentación para que poida ser xulgada polo tribunal correspondente. E para que así conste expídese a presente, en Vigo a 25 de xaneiro de 2010. María Jesús Prol García (Doutoranda) José Pintado Valverde Miquel Planas Oliver Ysabel Santos Rodríguez (Director) (Codirector) (Titora) v The work presented in this Ph.D. Thesis was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria – Ministerio de Ciencia e Innovación (Project: Modificación de la microbiota bacteriana asociada a la utilización de bacterias probióticas en el cultivo de larvas de rodaballo. Reference: ACU03-003) and the Consejo Superior de Investigaciones Científicas (Project: Modo de acción y optimización del uso de Roseobacter 27-4 como probiótico en el cultivo de larvas de rodaballo. Reference: 2006 7 01 067) During its development, I was granted by the I3P program of CSIC, which is co-funded by the European Social Fund and included funds for the development of three research short stays at National Institute of Aquatic Resources (Denmark), Centre for Marine Bio- Innovation - School of Biotechnology and Biomolecular Sciences (Australia) and Institute for Chemistry and Biology of the Marine Environment (Germany). vii Agradecementos/Acknowledgements Despois de catro anos de duro traballo, esforzo e moita paciencia chega o final desta etapa que non é mais que o principio da miña carreira profesional como investigadora. Neste punto, gustaríame darlle as grazas ós meus directores de Tese, José Pintado Valverde e Miquel Planas Oliver por apostar por min e darme a oportunidade de desenvolver o traballo incluído nesta Tese de Doutoramento. Moitas grazas polo voso apoio e dedicación nestes catro anos. Á profesora Teresa Pérez Nieto porque grazas á súa confianza en min fai catro anos esta Tese de Doutoramento é unha realidade. Grazas de todo corazón. I would like to thank Prof. Lone Gram at DTU Aqua for accepting my short stay, her hospitality and showing me that everything works when you are organized and careful in everything you do. I am also grateful to everyone at seafood research group, especially to Jesper for guiding me through real-time PCR and share his enthusiasm for research with me and to Cisse for being so nice with me during my stay at DTU. It has been a pleasure to work with all of you. Thank you! I would like to thank Prof. Staffan Kjelleberg at BABS & CMB for accepting my short stay, Suhelen Egan and Torsten Thomas for teaching me on fluorescent labelling of marine bacteria and Nidhi and Ana María for being my friends during my stay in Australia. I shared good times with you girls. I am grateful to Prof. Meinhard Simon and Dr. Thorsten Brinkhoff for giving me the opportunity to work at ICBM, their hospitality and making me feel like a real member of their research group. I would also like to thank Renate, Birgit, Martine, Dagmar, Sarah, Daniela, Sebastian, Ines, Thomas, Olaf, Lina, Carla, Christine, Andrea, Rolf, Heike, Marco, Helena and Yvonne for showing me how everything worked in the lab and being really nice partners. Thank you for making me feel like home. A Carmen G. Sotelo, Ana, Maribel e Susana, do grupo de bioquímica dos alimentos no IIM, por permitirme usar a PCR a tempo real, o transiluminador e o servizo de secuenciación. A Miguel Anxo Murado e todo o grupo de reciclado no IIM por permitirme usar o seu material de filtración e as incubadoras orbitais. A Marta viii López Cabo do grupo de teconoloxía de produtos pesqueiros no IIM. A Suso Méndez e Inés Pazos, do CACTI na Universidade de Vigo, polo procesamento das mostras de microscopía electrónica. A David Chavarrías de Insuíña Pescanova (Mougás) por facilitarme larvas de rodaballo para a realización dos experimentos na planta piloto do IIM. Ó meu Pedriño por estar sempre ó meu carón, tirar de min nos momentos de desánimo, aturar os meus momentos de “mala leche”, axudarme na preparación de tódolos meus traballos de investigación para que vaian o máis perfectos posible, pero sobre todo por crer en min e na miña capacidade para desenvolver o meu traballo. Pero, ¿sabes que? Se son como son e son o que son é en gran parte grazas a ti. Quérote! Moitísimas grazas meu Sol! Ós meus pais por ter fe cega en min e apoiarme sempre. Ós meus irmáns, Isa e Toño por facerme rir e compartir comigo os bos e malos momentos. Á miña familia política, en especial a Esther, por ser unha amiga excepcional e escoitarme sempre. A Begoña, amiga coa que compartín bos e malos momentos. En nada xa che fago compaña no club de Doutores. Ós meus compañeiros de laboratorio Álex, Patri, Sonia, Andreu e Tomás por estar sempre aí para escoitarme e apoiarme nos momentos de baixón. Gustaríame darlle as grazas de xeito especial a Álex por estar sempre dispoñible para resolverme dúbidas e propoñerme ideas para o desenvolvemento dos experimentos en planta piloto. A tódolos compañeiros que xa non están no laboratorio María, Silvia, Mariana, Cynthia, José Luis e Alicia Abalo. Ós estudantes de prácticas Alicia Bechler, Eva, Marina, Lorenzo, Laura, Cristina e Lara cos que compartín momentos moi bos e me deron a oportunidade de poñer en práctica as miñas dotes de “profesora”. Ó CSIC e ó INIA por ofrecerme a oportunidade de gozar dunha bolsa predoutoral e polo financiamento do material e equipamento empregado nesta Tese de Doutoramento. A tódala xente que dalgún xeito colaborou no desenvolvemento desta Tese de Doutoramento, GRAZAS! / To everyone that has participated in the development of this Ph.D. Thesis, THANK YOU! ix Ó meu Pedriño Ós meus pais e irmáns Index xi INDEX 1. INTRODUCTION: NEW STRATEGIES FOR THE CONTROL OF BACTERIAL INFECTIONS IN MARINE FISH LARVAL REARING 3 1.1. BACTERIA IN FISH LARVAE 4 1.2. NEW STRATEGIES FOR BACTERIAL INFECTIONS CONTROL IN FISH LARVAE 5 1.2.1. Acting on the microbial communities of the system 6 1.2.1.1. Promoting beneficial bacteria in the system 6 1.2.1.2. Adding beneficial bacteria 9 1.2.1.3. Inhibiting or inactivating detrimental bacteria in the system 15 1.2.2. Acting on host-microbe interactions 17 1.3. STRATEGIES BASED ON THE FOOD CHAIN 19 1.4. NEW MOLECULAR TOOLS 20 1.5. CONCLUSIONS AND PERSPECTIVES 22 1.6. REFERENCES 23 2. BACKGROUND: PHAEOBACTER 27-4 AS PROBIOTIC AGAINST VIBRIONACEAE INFECTIONS IN TURBOT LARVAE 37 2.1. REFERENCES 41 3. OBJECTIVES 49 4. CHAPTER I: REAL-TIME PCR DETECTION AND QUANTIFICATION OF THE FISH PROBIOTIC PHAEOBACTER STRAIN 27-4 AND TWO FISH PATHOGENIC VIBRIONACEAE IN MICROALGAE, ROTIFER, ARTEMIA AND FIRST FEEDING TURBOT (PSETTA MAXIMA) LARVAE 55 4.1. INTRODUCTION 55 4.2. MATERIALS AND METHODS 57 4.2.1. Bacterial strains and culture conditions 57 4.2.2. Preparation of cultures for quantification 57 4.2.3. Microalgae, rotifer, Artemia and turbot larvae culturing 58 xii Index 4.2.4. Mixing of samples and DNA extraction 59 4.2.5. Design of primers for quantitative real-time PCR 59 4.2.6. Quantitative real-time PCR 61 4.2.7. Standard curves and quantification of strain mixtures using quantitative real-time PCR 62 4.2.8. Quantification by real-time PCR of bacteria experimentally introduced in rotifers and transferred to seawater and turbot larvae 62 4.3. RESULTS 63 4.3.1. Verification of primers specificity 63 4.3.2. Standard curves for quantitative real-time PCR 64 4.3.3. Real-time PCR quantification of known concentrations of the target bacteria 66 4.3.4. Real-time PCR quantification of target bacteria experimentally introduced in rotifers (bioencapsulation) and transferred to seawater and to turbot larvae in a challenge trial 68 4.4. DISCUSSION 69 4.5. REFERENCES 72 5. CHAPTER II: DIFFERENT COLONIZATION AND RESIDENCE TIME OF LISTONELLA ANGUILLARUM AND VIBRIO SPLENDIDUS IN THE ROTIFER BRACHIONUS PLICATILIS DETERMINED BY REAL- TIME PCR AND DGGE 79 5.1. INTRODUCTION 79 5.2. MATERIALS AND METHODS 81 5.2.1. Bacterial strains and culture conditions 81 5.2.2. Infection of rotifers with the pathogens 81 5.2.3. Preparation of samples for microbiological analysis 82 5.2.4. DNA extraction 83 5.2.5. Quantitative real-time PCR 83 5.2.6. PCR-DGGE 84 5.2.7. Analysis of DGGE profiles 85 5.2.8. Sequencing of DNA from PCR fragments and bacterial isolates 86 5.3. RESULTS 87 5.3.1. Colonization and residence time of Listonella anguillarum 90- 11-287 and Vibrio splendidus DMC-1 in rotifers 87 Index xiii 5.3.2. Effect of pathogens colonization on bacterial communities associated with rotifers 91 5.3.2.1.
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