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Haemonchus Contortus UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MORELOS DOCTORADO EN CIENCIAS NATURALES CENTRO DE INVESTIGACIÓN EN BIOTECNOLOGÍA CEIB-UAEM Caracterización taxonómica y actividad depredadora de hongos nematófagos, evaluación y clasificación química de los compuestos con actividad nematicida contra Haemonchus contortus T E S I S QUE PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS NATURALES ÁREA TERMINAL: BIOTECNOLOGÍA PRESENTA: M. en B. Ana Yuridia Ocampo Gutiérrez Director de tesis: Dr. Víctor Manuel Hernández Velázquez Codirector: Dr. Pedro Mendoza de Gives Cuernavaca, Morelos México 2021 I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII Universidad Autónoma del Estado de Morelos Doctorado en Ciencias Naturales Centro de Investigación en Biotecnología CEIB-UAEM Caracterización taxonómica y actividad depredadora de hongos nematófagos, evaluación y clasificación química de los compuestos con actividad nematicida contra Haemonchus contortus Cuernavaca, Morelos México, 2021 XVIII Agradecimientos: Al Consejo Nacional de Ciencias (CONACYT) por la beca otorgada durante el proceso de doctorado. Quiero aprovechar este espacio para agrader brevemente a mis directores de tesis Dr. Víctor Manuel Hernández Velázquez y Dr. Pedro Mendoza de Gives por la paciencia, por las tantas estrategias profesionales aprendidas y sobre todo por haberme otorgado su amistad y confianza durante la realización del presente proyecto. A mi comté tutorial por los comentarios muy acertados hacia mi proyecto, ya que en conjunto logramos mejorar cada vez los experimentos y presentación de los resultados. Al Dr. Alejandro Zamilpa y al Dr. Manasés González por la experiencia profesional, conocimientos y apoyo otorgado en el área química. A la Dra. Maria Eugenia López Arellano por el apoyo profesional en el laboratorio, gracias por los consejos laborales y muchas muchas gracias por su amistad. A todas las personas involucradas en la presente tesis, gracias al Dr. Caleb C. Lormendez por el apoyo prestado en el tema de bioprocesos, a la Maestra Laura Lina por la ayuda, además de todo el tiempo y paciencia que me otorgó, al Dr. Jesús Hernández por el asesoramiento obtenido, a la Dra. Manuela Reyes Estebanez por el material bilógico que nos otorgó y por las revisiones de escritorio de documentos. Muchas gracias a todos A todo el personal que me apoyó en los laboratorios: Laboratorio de control biológico, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Laboratorio de Helmintología, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, INIFAP Centro de Investigación Biomédica del Sur, CIBIS-IMSS. Infinitas gracias ”Mejores son dos que uno; porque tienen mejor paga de su trabajo. Porque si cayeren, el uno levantará a su compañero; pero ¡ay del solo! que cuando cayere, no habrá segundo que lo levante. También si dos durmieren juntos, se calentarán mutuamente; más ¿cómo se calentará uno solo? Y si alguno prevaleciere contra uno, dos le resistirán; y cordón de tres dobleces no se rompe pronto.” (Eclesiastés 4:9-12) XIX A mi equipo “familia”: a mi papi Luis, mami Mari y hermanas Gabi, Maye, sobrino Joshua y cuñados, gracias por estar conmigo siempre y por todos los días de junta informativa y bebidas ocasionales que compartimos. Las más grandes gracias a mi hijo Jorge Milton por esperar a su mami pacientemente en casa, por creer en mí y por poseer increiblemente y como por arte de magia todo el poder necesario para hacer de mí una mejor persona y a la vez una mejor profesionista, acuérdate hijo que todo es en pro de nosotros. XX Contenido temático Resumen ...................................................................................................................................... 1 Abstract ......................................................................................................................................... 2 1. Introducción .............................................................................................................................. 3 2. Antecedentes ............................................................................................................................ 5 2.1. Parasitosis gastrointestinal.................................................................................................... 5 2.2. Características del parásito Haemonchus contortus ............................................................ 7 2.3. Ciclo biológico de Haemonchus contortus ............................................................................ 7 2.4. Productos químicos utilizados como control parasitario ....................................................... 9 2.5. Impacto de los antihelmínticos en el ambiente ................................................................... 11 2.6. Resistencia antihelmíntica................................................................................................... 12 2.7 Alternativas de control de nematodos gastrointestinales .................................................... 12 2.7.1. Partículas de óxido de cobre ............................................................................................ 13 2.7.2. Medicina tradicional .......................................................................................................... 13 2.7.3. Bacterias ........................................................................................................................... 13 2.7.4. Ácaros nematófagos ....................................................................................................... 14 2.7.5. Hongos nematófagos ....................................................................................................... 14 2.8. Hongos nematófagos: clasificación, actividad depredadora y uso antihelmíntico ............. 14 2.8.1 Clasificación ...................................................................................................................... 14 2.8.2. Forma de alimentación ..................................................................................................... 15 2.8.3. Desarrollo de órganos de captura .................................................................................... 15 2.8.4. Depredación Hongo-Nematodo ....................................................................................... 16 2.8.5. Mecanismos moleculares involucrados en la depredación de nematodos ..................... 16 2.8.6. Evaluación del uso de hongos nematófagos en el control de nematodos ...................... 17 2.9. Metabolitos secundarios producidos por hongos nematófagos ......................................... 18 2.10. Cultivo de hongos nematófagos y producción de metabolitos secundarios .................... 21 3. Justificación ............................................................................................................................ 25 4. Hipótesis ................................................................................................................................. 26 5. Objetivo General ..................................................................................................................... 27 6.1. Objetivos particulares .......................................................................................................... 27 6. Metodología ............................................................................................................................ 28 6. 1. Metodología sección I: ....................................................................................................... 29 XXI 6.1.1. Activación de cepas ......................................................................................................... 29 6.1.2. Obtención de larvas infectantes de Haemonchus contortus ........................................... 31 6.1.3. Lavado de larvas de Haemonchus contortus .................................................................. 31 6.1.4. Obtención de huevo de Haemonchus contortus .............................................................. 31 6.1.5. Obtención del nematodo de vida libre Panagrellus redivivus. ......................................... 32 6.1.6. Identificación fúngica a través de taxonomía tradicional (sistema morfométrico) ........... 32 6.1.7. Identificación molecular .................................................................................................... 33 6.1.8. Análisis múltiple y elaboración de árbol filogenético ....................................................... 33 6.1.9. Evaluación de la actividad depredadora .......................................................................... 34 6.1.10. Producción de cepas en medio líquido estático. ........................................................... 35 6.1.11. Producción de cepas en medio líquido agitado ............................................................. 35 6.1.12. Producción a escala de los cultivos de hongos nematófagos seleccionados en condiciones estáticas y agitadas ................................................................................................ 35 6.1.13. Separación de líquido filtrado y micelio ......................................................................... 36 6.1.14. Elaboración de extractos ................................................................................................ 36 6.2. Sección metodológica II ...................................................................................................... 37 6.2.1. Realización de bioensayos para evaluar
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