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Hypsipyla Grandella (Zeller 1848) Cedrela Odorata En Colombia

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Hypsipyla Grandella (Zeller 1848) Cedrela Odorata En Colombia Hypsipyla grandella (Zeller 1848) (Lepidoptera: Pyralidae: Phycitinae) en plantaciones de Cedrela odorata en Colombia Luz Miryam Gómez Piñerez Bacterióloga - M.Sc. Entomología Universidad Nacional de Colombia sede Medellín Facultad de Ciencias Agrarias, Departamento de Ciencias Agrarias Medellín, Colombia 2014 Hypsipyla grandella (Zeller 1848) (Lepidoptera: Pyralidae: Phycitinae) en plantaciones de Cedrela odorata en Colombia Luz Miryam Gómez Piñerez Bacterióloga - M. Sc. Entomología Trabajo de investigación presentado como requisito parcial para optar al título de: Doctor en Ciencias Agropecuarias Director (a): PhD. Sandra Uribe Soto MSc. Doctorado en Ciencias Agropecuarias – Línea Agraria Grupo de investigación Sistemática Molecular Universidad Nacional de Colombia Sede Medellín Medellín, Colombia 2014 A mi madre Agradecimientos En este camino un poco más solitario, más personal, quiero agradecer a la ayuda desinteresada, a la curiosidad de las personas sencillas…. A cada una de las personas con la que de una u otra forma tuve contacto durante estos últimos años, y que me colaboraron con una palabra, una pregunta, o una acción, para continuar por este camino. Agradezco a Carlos López Vaamonde por generar el interrogante frente a la especie objeto de este estudio, por su voluntad, su acogida y colaboración. A David Giron y su equipo de trabajo en el Institut de Recherche sur la Biologie de l´ Insecte - IRBI, a su director Jean Paul Monge y en especial a Wilfried Kaiser por su orientación inicial para el trabajo en el laboratorio. A el Dr. Luko Hilje del Centro Agronómico Tropical de Investigación y Enseñanza por su orientación sobre el insecto, y a su colaborador Arturito, con su particular gracia y sabiduría me enseño sobre su cría de Hypsipyla grandella. A Colciencias por darme un apoyo que permitió que este trabajo se llevara a cabo y que además me enriqueciera al permitir conocer parajes del viejo mundo Agradezco a la profesora Sandra Uribe por brindarme un espacio después de tanto, a los integrantes del Grupo Sistemática Molecular por oír una y otra vez A Didier Chavarría por brindarme su apoyo y acompañamiento en salida de campo y establecimiento de contactos con personas vinculadas a la industria forestal en nuestro país. A Carlos Mario Ospina – CENICAFE, agradezco igualmente por brindarme ayuda desinteresada y contactarme con sitios a visitar. A los profesores Alejandro Madrigal e Ignacio de Valle por orientarme sobre el estado de las plantaciones de cedro en el país y la problemática del insecto a nivel nacional. A las personas dueñas y trabajadores de las fincas San José, La cascada, El Diamante, La Caquetá, Villa Lourdes, Parcelas los Riscales, Jardín Botánico de Medellín. A Du, con sabores y sinsabores A Paula Sepúlveda mi Paulina….por ser compañera y amiga, por compartir esta lucha, comprender las dificultades por las que he pasado en este camino dadas las condiciones particulares que nos han tocado en estos años, gracias por servir como apoyo en todo momento A Gonzalo Abril por estar presente A el Black por su orientaciones para la identificación de insectos inmaduros y por su compañía. A Andrés López y Alba Marín por escuchar…. A mis amigas más antiguas, Natalia Ramírez y Ana María Castro por estar atentas a mí trasegar A mi familia por el solo hecho de preguntar A Dora, Ferley Pino, Isabel Cadavid y Carolina Álzate, aunque no caminamos juntos hasta el final me aportaron su colaboración y ganas de aprender. A las miradas curiosas a esas sonrisas blancas en rostros de tez negra…..Llenos de dulzor y alegría. A los acompañantes bajo el sopor de un medio día ardiente llenos de curiosidad de saber qué pasa con aquellos pequeños que tienen como morada los árboles que ellos plantaron. A impulsores, vitales para poder culminar, Profesor Rodrigo Vergara y Dra. Lilian Gómez. A Inés, por ser un gran soporte, por su apoyo incondicional. Agradezco igual a la tristeza…… aquella que me llevo a refugiarme en este mundo y que permitió que aprendiera vivir con otras metas, la que permitió que mis ojos se llenaran de nuevos paisajes y que conociera gente maravillosa que enriqueció mi vida. Tabla de contenido. Resumen. ..............................................................................................................................................................1 Introducción. ........................................................................................................................................................3 Hipótesis. .............................................................................................................................................................6 Sección 1. Observaciones del ciclo de vida de Hypsipyla grandella (Zeller 1849). ..................................................7 Sección 2. Presencia y distribución de Hypsipyla grandella (Zeller 1849) en plantaciones de Cedrela odorata L. 1759 en Colombia. .......................................................................................................................................................... 19 Sección 3A. Extracción de ADN a partir de larvas de Hypsipyla grandella. ............................................................ 36 Sección 3B. Taxonomía molecular de Hypsipyla grandella (Zeller 1849) con base en secuencias de ADN del gen Citocromo Oxidasa I (COI). .............................................................................................................................. 45 Sección 3C. Variabilidad molecular, distribución geográfica de haplotipos y flujo de genes de Hypsipyla grandella (Zeller) asociada a plantaciones de Cedrela odorata L. en Colombia. ..................................................................... 56 Sección 4. Resumen de resultados. .................................................................................................................... 89 5. Referencias Bibliográficas............................................................................................................................... 93 | Lista de tablas. Tabla 1. Duración de los estados del ciclo de vida de Hypsipyla grandella (Zeller) a temperatura promedio de 28 °C y humedad relativa promedio de 70%.................................................................................................................................. 13 Tabla 2. Ubicación de plantaciones de Cedrela odorata y porcentajes ataque por Hypsipyla grandella. ........................ 27 Tabla 3. Secuencias de los cebadores utilizados para la amplificación de fragmentos de los genes Histona 3 (H3), citocromo oxidasa I (COI), Citocromo B (Cyt B), ARN de trasferencia para serina (ARNtser), NAD deshidrogenasa 1 (ND1) y Factor de Elongación 1 alfa (EF-1alfa). ............................................................................... 39 Tabla 4. Condiciones de PCR para los fragmentos de los genes COI, CytB, RNAtser-IGS2, ND, EF1-alfa y H3. .................................................................................................................................................................................................... 40 Tabla 5. Comparación de porcentaje de éxito entre los protocolos Collins et al. 1987 y DNeasy® Kit (QIAgen) en la amplificación de los fragmentos de los genes CytB-RNAtser-ND1, COI, EF1-alfa y H3. ............................. 42 Tabla 6. Número de larvas presuntivamente de Hypsipyla grandella por plantación de Cedrela odorata. ..................... 49 Tabla 7. Polimorfismos de los fragmentos mitocondriales COI COI, CytB, RNAtser, IGS2 y ND1 en H. grandella asociada a plantaciones de Cedrela odorata en Colombia, según la jerarquía geográfica.. ............................... 66 Tabla 8. Polimorfismos en los fragmentos de mitocondriales asociados a la jerarquía geográfica. ........................ 67 Tabla 9. Polimorfismos de los fragmentos de genes H3- EF1alfa, en H. grandella en plantaciones de Cedrela odorata asociados con la jerarquía geográfica. ................................................................................................................................... 74 Tabla 10. Polimorfismos de análisis conjunto d H3- EF1alfa genes asociado con la jerarquía geográfica. n= número de muestras; S = sitios segregantes; Nh =número de haplotipos; h =diversidad haplotipica; Pi = diversidad nucleotídica; k = promedio de diferencias nucleotídicas ............................................................................. 75 Tabla 11. Estimativos de estructuración (Fst) y flujo génico (Nm) para delos fragmentos COI, Cyt B, RNAtser y ND1para la totalidad de ejemplares de Hypsipyla grandella en Cedrela odorata en Colombia......................................... 77 Tabla 12. Comparaciones valores de diferenciación (Fst) y flujo génico (Nm) de Hypsipyla grandella entre plantaciones de Cedrela odorata en Colombia a partir de los fragmentos mitocondriales COI-CytB-RNAtser-IG2- ND1 y nucleares EF1-alfa H3. .............................................................................................................................................. 78 Lista de figuras. Figura 1. A. Montículos compuestos por residuos de alimentación, excretas y seda dejados por larvas de H. grandella tras su alimentación B. Larva de quinto instar de H. grandella localizada en el interior del tallo. ................. 11 Figura 2. A. Vista dorsal de larva de Hypsipyla grandella de cuarto instar sobre hoja de Cedrela odorata. B. Larva de H grandella de quinto instar sobre hoja de C. odorata. ........................................................................................................
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