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Rearing of the Seahorse Hippocampus Guttulatus: Key Factors Involved in Growth and Survival

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Rearing of the Seahorse Hippocampus Guttulatus: Key Factors Involved in Growth and Survival Rearing of the seahorse Hippocampus guttulatus: Key factors involved in growth and survival ANDREU BLANCO CARTAGENA Marine Ecology doctorate programme PhD Thesis 2014 Dr. Miquel Planas Oliver, scientific researcher from the department of Marine Ecology and Biodiversity at the Institute of Marine Research of the National Council of Scientific Research, certifies that: The present thesis, entitled “Rearing of the seahorse Hippocampus guttulatus: Key factors involved in growth and survival” is a novel work and has been fully written by Andreu Blanco Cartagena and carried out under the advisement of Dr. Miquel Planas Olvier. The University tutor, Dr. Gabriel Moyà Niell allows its defence to the Department of Biology, responsible of the doctorate programme of Marine Ecology. University tutor, Doctorand, Thesis director, Dr. Gabriel Moyà Niell Andreu Blanco Cartagena Dr. Miquel Planas Oliver Professor and researcher Researcher University of the Balearic Marine Research Institute Islands (UIB) (IIM-CSIC) Rearing of the seahorse Hippocampus guttulatus: Key factors involved in growth and survival PhD Thesis presented by: ANDREU BLANCO CARTAGENA to obtain the degree in: Marine Ecology Palma de Mallorca, 2014 “Y no pienses, recuerda” Guillermo Cartagena Tello, 2003 “Hombre libre, ¡siempre adorarás la mar!” Charles Baudelaire A mi familia, os quiero. Index Acknowledgements XI Abstract / Resum XV 1. Introduction 1 2. Objectives 31 3. General Material and Methods 35 3.1. Seahorse Collection 37 3.2. Broodstock captive conditions 38 3.3. Rearing of juveniles 41 3.4. Production of live food 43 3.4.1. Microalgae 43 3.4.2. Rotifer, Brachionus plicatillis 44 3.4.3. Brine shrimp, Artemia sp. 45 3.4.4. Calanoid copepod, Acartia tonsa 45 3.5. Juvenile sampling 46 3.6. Proximal biochemical composition 47 3.6.1. Total Lipid content 47 3.6.2. Total Protein content 48 3.6.3. Soluble Carbohydrate content 48 3.6.4. Ash content 48 CHAPTER 1: Implications of physical key factors in the early rearing of the long-snouted seahorse Hippocampus guttulatus 49 1.1. Introduction 52 1.2. Materials and methods 54 1.2.1. Broodstock 54 1.2.2. Rearing system 55 1.2.3. Experiment 1: Aquaria Design and Photoperiod 56 1.2.4. Experiment 2: Aeration Intensity 58 1.2.5. Sampling and data analysis 59 VII 1.3. Results 60 1.3.1. Experiment 1: Aquaria Design and Photoperiod 60 1.3.2. Experiment 2: Aeration Intensity 63 1.4. Discussion 63 1.5. Conclusions 69 CHAPTER 2: Temperature-induced changes of growth and survival in the early development of the seahorse Hippocampus guttulatus 71 2.1. Introduction 74 2.2. Materials and methods 75 2.2.1. Broodstock 75 2.2.2. Rearing system 76 2.2.3. Experiment 1: Feeding conditions 77 2.2.4. Experiment 2: Starvation conditions 77 2.2.5. Sampling and data analysis 78 2.3. Results 80 2.3.1. Feeding juveniles 80 2.3.2. Starving juveniles 84 2.4. Discussion 85 CHAPTER 3: Mouth growth and prey selection in juveniles of the European long-snouted seahorse Hippocampus guttulatus 91 3.1. Introduction 94 3.2. Materials and methods 95 3.2.1. Seahorse culture 95 3.2.2. Prey culture 96 3.2.3. Experimental procedures 96 3.2.4. Sampling and data analysis 98 3.3. Results 100 3.4. Discussion 105 3.5. Conclusions 111 VIII CHAPTER 4: Improvements in the rearing of the seahorse Hippocampus guttulatus Cuvier 1829 by the use of copepods 113 4.1. Introduction 116 4.2. Materials and methods 118 4.2.1. Seahorse culture 118 4.2.2. Rearing system 118 4.2.3. Prey culture 119 4.2.4. Experimental design 119 4.2.5. Seahorse sampling 120 4.2.6. Proximal biochemical composition 121 4.2.7. Data analysis 121 4.3. Results 122 4.4. Discussion 123 4.5. Conclusions 130 CHAPTER 5: Ontogeny of digestive enzyme activities in juvenile long snouted seahorses Hippocampus guttulatus fed on different live diets 131 5.1. Introduction 134 5.2. Materials and methods 135 5.2.1. Broodstock 135 5.2.2. Rearing system 136 5.2.3. Feeding conditions 137 5.2.4. Seahorse sampling 138 5.2.5. Enzyme activities determination 139 5.2.6. Data analysis 141 5.3. Results 141 5.4. Discussion 146 5.5. Conclusions 151 IX CHAPTER 6: Closing the life cycle of the European long-snouted seahorse Hippocampus guttulatus Cuvier 1829 153 6.1. Introduction 156 6.2. Materials and methods 157 6.2.1. Broodstock 157 6.2.2. Rearing system 158 6.2.3. Feeding conditions 158 6.2.4. Prey culture 159 6.2.5. Experimental procedure 160 6.2.6. Data analysis 161 6.3. Results 161 6.4. Discussion 165 6.5. Conclusions 168 4. Summary 169 5. Conclusions 177 6. References 183 7. Annexes 219 X Acknowledgments ACKNOWLEDGMENTS Esta es mi aventura: la historia de un chaval con una mochila en una ciudad nueva. Pero esta aventura no empieza en esa nueva ciudad, sino en Mallorca (“Sa roqueta”), ese pedacito de tierra en medio del mar que me vio nacer y crecer. Rodeado por mar, mi pasión por él y sus habitantes fue in crescendo desde pequeño. Con el inestimable apoyo de mi familia hice y deshice para empezar carrera en la investigación marina, la cual comencé en 2006 en el laboratorio de la Dra. Salud Deudero, a quien debo mis inicios, muchas gracias. De ahí, mi aventura se materializó con el beneplácito del Dr. Miquel Planas quien confió en mí y me brindó la oportunidad de salir de mi isla y conocer mundo, quien me apoyó durante momentos de flaqueza y me permitió poder realizar un sueño; sin tu respaldo jamás hubiera podido hacer todo lo que he hecho. Por el lado burocrático-administrativo quisiera agradecer al Dr. Biel Moyà, gracis per fer-me de pont entre institucions. Quisiera agradecer también al Dr. Pepe Pintado, cuya aportación profesional y personal ha permitido mejorar mi orden ambiental y, consecuentemente, mental. Tengo que agradecer a Sonia Valladares su ayuda en la búsqueda de caballitos en los mares de Galicia. Este mar gallego me ha enseñado tanta diversidad que no ha dejado de asombrarme desde la primera inmersión. A Patricia Ruiz me gustaría agradecerle su constante sonrisa y su capacidad logística. Especialmente quisiera agradecer al resto del equipo: A Tomás Hermelo, galego onde os haxa; moitas grazas por falarme galego dende o inicio e ensinarme os costumes da terra, dende as agrolimpiadas e o licor café ata o carnaval do Hio e os petroglifos, entre outros moitos, e grazas por presentarme a túa xente do Morrazo. A la Dra. Patricia Quintas, Patri, quien me facilitó todo desde el primer día, por tu amistad y ayuda en todo, mil gracias. A Alex Chamorro, a quien prácticamente debo esta tesis. Me enseñaste de todo, desde el cultivo en acuarios hasta cómo hacer una mesa, siempre a tope y dispuesto a echarme una mano con lo que fuera, muchísimas gracias. A Claudia Ofelio, quien además de compañera de laboratorio ha sido una gran amiga y un buen apoyo en momentos duros. No sólo te has ocupado de los cultivos durante mis últimos fines de semana, sino que, además, me has sacado muchas sonrisas. A todos estos compañeros os lo agradezco de corazón, cuando las cosas se me pusieron realmente difíciles siempre me lo facilitasteis todo. Mis primeros pinitos en la ciencia los compartí con el Dr. Toni Box, la Dra. Silvia Tejada, el Dr. Antoni Sureda y el Dr. Guillem Mateu, a quienes agradezco mis inicios, junto a otros compañeros. Además, no quisiera olvidarme de quienes, de un modo u otro XI Acknowledgments otro, han puesto su granito de arena en la realización de esta tesis ayudándome en cultivos, experimentos y rutinas: Juanpi, Fernando, Mariana, Jarintzin, Vanessa, Tania, David, Rita, Irene, Lucía, Alba, Diana, Manuel, Rachel, Jose, Beto, Alex y algún otro que haya volado de mi cabeza cual pájaro al viento. A todos aquellos que habéis hecho que el IIM no fuera sólo un lugar de trabajo, haciéndolo lugar de risas y divagación intelectual: Fiona, Isabelinha, Silvinha, Marcelo, Alba, Maruxa, Lorena, Marcos, Jose, Miguel, Raquel, personal de conserjería y otros tantos que han amenizando mi paso por el centro. Los inicios en una nueva ciudad pueden hacerse difíciles, pero al final, cuando haces lo que te apasiona y lo haces rodeado de gente buena, todo es mucho más llevadero. A mi arribada me reencontré con Laura y Judit, que hicieron que Galicia tuviera alguna razón para convertirse en mi destino y en mi hogar, gracias desde el minuto 0. Al inicio de mi sendero no pude cruzarme con mejor compañía que la de JuanPi, con quien recorrí tierra firme en busca de conciertos, ciudades y demás lugares, gracias pana! Cris e Isa, mis coleguiñas, salieron a mi paso y desde entonces han compartido esta experiencia conmigo; llevándome a crear, junto a Jorge, Noha y Sergio, el grupo de apoyo a doctorandos marinos mediante el uso terapéutico del humor gráfico, y así conseguir arrancarme sonrisas durante las largas horas de trabajo. Bego, has sido un apoyo indispensable en este paseo gallego, un bastón que me ha permitido avanzar cuando flaqueaba, gracias. A Peque, Iago y Marisa, por disfrutar conmigo de los placeres de vuestra tierra y pasar mi única noche vieja gallega a mi lado. Jari y nuestra niñita, Vane, con quienes pasé a un estado de euforia neurótica de risoterapia asistida tanto dentro como fuera de nuestro hogar; buenos momentos de un verano memorable. La cronología de los acontecimientos me lleva a recordar con cariño y apego a mucha gente que ha compartiendo conmigo techo, lar y microaventuras paralelas: Juanpi, Victorine, Justyna, Edyta, Dani, Arturo, Lois, Manu, Clau, Paolo y Alex.
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