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Tesis José Luis Duarte De Jesús 27 Nov 2020.Pdf Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California Maestría en Ciencias en Ciencias de la Vida con orientación en Microbiología Celular y Molecular Identificación de secuencias genómicas virales en el piojo harinoso de la vid (Planococcus ficus Signoret, 1875) presente en viñedos de Ensenada, Baja California Tesis para cubrir parcialmente los requisitos necesarios para obtener el grado de Maestro en Ciencias Presenta: José Luis Duarte de Jesús Ensenada, Baja California, México 2020 Tesis defendida por José Luis Duarte de Jesús y aprobada por el siguiente Comité Dra. Jimena Carrillo Tripp Directora Miembros del comité Dr. Miguel Ángel Martínez Mercado Dra. Clara Elizabeth Galindo Sánchez Dr. Jorge Alberto Cáceres Martínez Dra. Patricia Juárez Camacho Coordinadora del Posgrado en Ciencias de la Vida Dra. Rufina Hernández Martínez Directora de Estudios de Posgrado José Luis Duarte de Jesús © 2020 Queda prohibida la reproducción parcial o total de esta obra sin el permiso formal y explícito del autor y director de la tesis. ii Resumen de tesis que presenta José Luis Duarte de Jesús como requisito parcial para la obtención del grado de Maestro en Ciencias en Ciencias de la Vida con orientación en Microbiología Celular y Molecular. Identificación de secuencias genómicas virales en el piojo harinoso de la vid (Planococcus ficus Signoret, 1875) presente en viñedos de Ensenada, Baja California Resumen aprobado por: _____________________ Dra. Jimena Carrillo Tripp Directora de tesis La vid, Vitis vinifera L., es la especie de vid más cultivada para la explotación de su fruto en las regiones de Europa, África y América. En México, Baja California (B.C.) es la entidad con mayor aporte a la producción nacional de uva con fines de uso industrial. Actualmente, la plaga de insecto más importante de los cultivos de Vitis vinifera L. en México y otros países es el piojo harinoso de la vid, Planococcus ficus (Signoret, 1875). En las últimas décadas, se ha explorado la diversidad de virus que infectan exclusivamente a insectos, con la finalidad de esclarecer sus interacciones ecológicas y encontrar, en última instancia, alternativas para el control de las poblaciones de insectos. Dicha prospección de virus específicos a P. ficus no había sido abordada. Por lo tanto, mediante técnicas de secuenciación masiva (RNA-seq) y análisis bioinformáticos, en el presente estudio se realizó la búsqueda e identificación de secuencias de virus con genoma de RNA o DNA en muestras de P. ficus colectadas en 14 viñedos de Ensenada, B.C. Dentro de los resultados de virus con genoma de RNA, se identificaron dos genomas casi completos de virus putativos relacionados con las familias Tymoviridae y Dicistroviridae. Adicionalmente, se encontraron fragmentos de genomas de virus putativos correspondientes a las familias Tymoviridae, Reoviridae, Rhabdoviridae, Tombusviridae, Iflaviridae, y al orden Picornavirales. Por otro lado, se logró la detección de secuencias relacionadas a virus de dsDNA de las familias Baculoviridae, Poxviridae y Polydnaviridae. Complementario al análisis bioinformático, se realizaron enriquecimientos de partículas tipo virus (VLPs) a partir de tejido de P. ficus y se analizaron por microscopía electrónica. Esto reveló la presencia de agregados de partículas que, en algunos casos, asemejan en tamaño y/o forma a los cuerpos de oclusión producidos por algunos virus de insecto durante la infección. La evidencia conjunta obtenida de los análisis realizados indica la presencia de uno o más virus de RNA putativos que pudieran asociarse específicamente con P. ficus. Este trabajo sienta las bases para futuros estudios de caracterización de virus específicos de P. ficus y virus de vid asociados a este insecto. Resumen en español Palabras clave: Planococcus ficus, Vitis vinifera L., secuencias virales, virus específicos de insecto, control biológico, virómica, metagenómica. iii Abstract of the thesis presented by José Luis Duarte de Jesús as a partial requirement to obtain the Master of Science degree in Life Sciencies with orientation in celular and molecular microbiology. Identification of viral genome sequences in the vine mealybug (Planococcus ficus, Signoret 1875) present in vineyards of Ensenada, Baja California Abstract approved by: _____________________ Dr. Jimena Carrillo Tripp Thesis director The grapevine, Vitis vinifera L., is the most worldwide-spread species cultivated for grape production in Europe, Africa, and America. In Mexico, Baja California (B.C.) is the region with the highest grape production for industrial purposes. The vine mealybug, Planococcus ficus (Signoret, 1875), is the most important insect pest in grapevine growing areas in Mexico and other countries. Recently, the diversity of viruses infecting insects has been broadly explored to elucidate further ecological viral-hots interactions in many insect species, which in some cases has resulted in the application of virus-based biological control agents for insect pests. However, a prospection of viruses associated with P. ficus had not been addressed yet. In the present study, P. ficus individuals collected through 14 different vineyards in Ensenada, B.C., were analyzed by RNA metagenomics. Several sequences of putative RNA viruses were found, including two nearly complete genomes related to the Dicistroviridae and the Tymoviridae families. Moreover, several smaller sequences related to viral genomes of the Tymoviridae, Reoviridae, Rhabdoviridae, Tombusviridae, and the Iflaviridae families, as well as with Picornavirales order, were identified. A group of sequences related to dsDNA viral genomes of the Baculoviridae, Poxviridae, and the Polydnaviridae families was also found. As a complement to these bioinformatic results, virus-like particle (VLPs) enrichment and electron microscopy analyses were carried out. These experiments revealed the presence of some particles that resemble viral occlusion bodies in shape and size. The overall obtained evidence points to the presence of one or more putative RNA viruses that could specifically infect P. ficus. This study provides the first insight into the P. ficus virome. It sets the foundation to further studies aimed to characterize those viruses potentially infecting P. ficus, in addition to grapevine viruses associated with this mealybug. Resumen en inglés Keywords: Planococcus ficus, Vitis vinifera L., viral sequences, insect specific viruses, biological control, viromics, metagenomics. iv Dedicatoria A Catalina, por supuesto. El haber llegado a este lugar fue tu último regalo para mí, y lo hiciste sin que tú o yo lo supieramos; como dar un beso con amor mientras se está dormido, sólo una veladora de sueños como tú sería capaz. “Oh, be wiser you! Instructed that true knowledge leads to love” -William Wordsworth Lines left upon a Seat in a Yew-tree v Agradecimientos Al Consejo Nacional de Ciencia y Tecnología por haberme beneficiado con la beca No. 911868 que me permitió sustentarme durante el desarrollo de mis estudios de maestría. Al CICESE y al personal académico (especialmente del Depto. de Microbiología) por su contribución tan importante a mi formación de posgrado, y al personal administrativo, por ser tan eficientes y profesionales en su labor. A cada uno de los profesores que me dio clase o laboratorio y me enseñaron lo que saben, especialmente a los “docs” Edgardo, Jimena, Carolina, Rafael, Leobardo, Miguel y Edgar, además de muchos otros doctores, posdocs y técnicos de CICESE, por sus clases tan instructivas, bien estructuradas y amenas. Ahora, hay tantas personas a las que quiero agradecer (en tantas maneras) por haber llegado hasta aquí, que debo expresar mi inconformidad por tan breve espacio para hacerlo; pero ciertamente un texto como el presente no les haría justicia. Y si por descuido omito a alguien, sepan que cuando caiga en cuenta se los haré saber. A la Dra. Jimena Carrillo Tripp, por su incansable esfuerzo de principio a fin en transmitirme su disciplina y su conocimiento, pero sobre todo por haber siempre exigido de mí lo mejor y siempre alentarme. Además, quiero agradecerle por haber puesto todo su empeño y estar siempre al pie del cañón buscando que este proyecto fuera una realidad (fue complicado muchas veces, pero lo sacamos ), y por haberme dado su confianza para comunicarle las buenas y las malas noticias. La admiro en lo personal y en lo académico. Al Dr. Miguel Á. Martínez Mercado, por haber sido mi mentor en bioinformática, pero sobre todo, por tener siempre tanta disposición y paciencia para enseñar. Eres un gran profesional que me motivo y me oriento en cada etapa. Sin tu ayuda este trabajo simplemente no hubiera sido posible; más de una vez me ayudaste a brincar las bardas de callejones sin salida. Gracias además por todos los tips y consejos personales y académicos. A la Dra. Clara E. Galindo Sánchez y al Dr. Jorge Cáceres Martínez, por su valioso tiempo, orientación, apoyo y comentarios, que me permitieron pulir y dar detalle a este trabajo. Además, le agradezco a la Dra. Clara por haberme permitido trabajar en el laboratorio de Genómica Funcional del Dpto. de Biotecnología Marina. Quiero agradecer muy especialmente (pero de verdad mucho) a la M. en C. Anaid Saavedra Flores, por tu apoyo, enseñanza, orientación y mucha (pero de verdad mucha) disposición en la construcción de las vi bibliotecas para la secuenciación. Dicen que el primer paso es el más importante de toda empresa, y gracias a tu pericia y destreza llevamos a cabo con éxito esa etapa. A mis amigas
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