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Redalyc.REPRODUCCIÓN DE HETERORHABDITIS INDICA EN Fitosanidad ISSN: 1562-3009 [email protected] Instituto de Investigaciones de Sanidad Vegetal Cuba Valdés Vázquez, Yirina; Lobaina Audevert, Antonio A.; Márquez Gutiérrez, María E.; Gómez Pacheco, Maylen; Escobar Hernández, Mercedes REPRODUCCIÓN DE HETERORHABDITIS INDICA EN CULTIVOS BIDIMENSIONALES ELABORADOS CON PROTEÍNA ANIMAL Fitosanidad, vol. 10, núm. 1, marzo, 2006, pp. 29-31 Instituto de Investigaciones de Sanidad Vegetal La Habana, Cuba Disponible en: http://www.redalyc.org/articulo.oa?id=209116158005 Cómo citar el artículo Número completo Sistema de Información Científica Más información del artículo Red de Revistas Científicas de América Latina, el Caribe, España y Portugal Página de la revista en redalyc.org Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto FITOSANIDAD vol. 10, no. 1, marzo 2006 REPRODUCCIÓN DE HETERORHABDITIS INDICA EN CULTIVOS BIDIMENSIONALES ELABORADOS CON PROTEÍNA ANIMAL Yirina Valdés Vázquez,1 Antonio A. Lobaina Audevert,1 María E. Márquez Gutiérrez1, Maylen Gómez Pacheco2 y Mercedes Escobar Hernández1 1 Instituto de Investigaciones de Sanidad Vegetal Calle 110 no. 514 e/ 5a. B y 5a. F, Playa, Ciudad de La Habana, CP 11600, [email protected] 2 Instituto de Investigaciones de Fruticultura Tropical. Calle 7a. no. 3005 e/ 30 y 32, Playa, Ciudad de Control biológico La Habana, [email protected] RESUMEN ABSTRACT La reproducción in vivo de nematodos entomopatógenos es alta- In vivo reproduction of entomopathogenic nematodes is highly cost mente costosa y laboriosa, lo que trae aparejado una gran necesi- and laborious, so it necessary to develope new methodologies for dad de elaboración de metodologías para su reproducción in vitro high level in vitro reproduction. The objective of this project was to a gran escala. Con este objetivo se evaluó la reproducción y desarro- evaluate the reproduction and development of infective juveniles of llo de juveniles infectivos de Heterorhabditis indica [Poinar, Heterorhebditis indica [Poinar, Karunakar y David, 1992] in four Karunakar y David, 1992] sobre diferentes medios de cultivo bidimentional culture media. These culture media were elaborated bidimensionales, que se elaboraron por la combinación de híga- combining chicken liver with starch and pork liver with molasses, do de pollo con almidón, así como hígado de cerdo con melaza, starch and rice powder. Every culture medium allowed the reproduction almidón y polvo de arroz. Los cuatro medios de cultivos permitie- of the nematodes primarily the variant with pork liver-rice powder due ron la reproducción de los nematodos. Se destaca la variante con to the combination of fatty acids and carbohydrates present in this hígado de cerdo-polvo de arroz, que evidenció que la combinación culture medium were more effective than in the rest culture media. entre la fuente de ácidos grasos y carbohidratos fue más eficaz que en el resto de las variantes. Key words: Heterorhabditis indica, nematodes, reproduction Palabras clave: Heterorhabditis indica, nematodos, reproducción INTRODUCCIÓN Los nematodos entomopatógenos ocupan un lugar muy dos de reproducción in vitro se deben ajustar a la dis- importante en la lucha biológica como una alternativa ponibilidad de materias primas, y que a su vez permi- más dentro de los métodos de control de plagas [Kaya tan una eficiente reproducción de los nematodos. Con y Gaugler, 1993] debido a que el uso indiscriminado de este objetivo se evaluó en este trabajo la reproducción los plaguicidas químicos causa grandes afectaciones en y desarrollo de juveniles infectivos de Heterorhabditis la diversidad biológica. A pesar de haberse estudiado indica [Poinar, Karunakar y David, 1992] sobre cuatro significativamente desde 1980 y de emplearse con bue- medios de cultivo bidimensionales. nos resultados en cultivos como cítricos, arroz, maíz y hortalizas [Arteaga, et al., 1996], aún su producción está muy limitada y no satisface la demanda. MATERIALES Y MÉTODOS La reproducción de estos organismos sobre larvas de Se elaboraron cuatro medios de cultivos nutritivos insectos es muy costosa [Bedding, 1984], por lo que bidimensionales en combinaciones de hígado de pollo existen numerosas patentes que se refieren fundamen- con almidón e hígado de cerdo con melaza, almidón y talmente a tecnologías para la reproducción masiva de polvo de arroz. Por cada una de estas variantes se pre- forma artificial, como son el cultivo bidimensional, el pararon cuatro réplicas que se inocularon primeramente tridimensional y la fermentación líquida. Estos méto- con la bacteria simbionte Photorhabdus luminescens fitosanidad/29 Valdés y otros [Boemare, Akhurst y Mourant, 1993]. A las 48 h se veniles infectivos se extrajo un gran número de hem- realizaron dos perforaciones de 1 cm de diámetro en el bras en estado de gravidez. centro de cada placa, donde se inocularon 6 000 juveni- Se conoce que cuando el nematodo llega al hemocele del les infectivos de H. indica, previamente esterilizados insecto, sale de su estado de juvenil infectivo para con hipoclorito de sodio, y finalmente todo se incubó a reiniciar su desarrollo, proceso que es denominado re- o 28 C. cuperación. La mayoría de las señales para la ocurren- El diseño empleado en este experimento fue completa- cia de este evento se producen en la hemolinfa del hos- mente aleatorizado. A partir de las 24 h se realizaron pedero; pero Grewal et al. (1997) demostraron que la observaciones diarias, y a los 13 días comenzó la reco- bacteria simbionte también produce señales que con- lección de los juveniles mediante el lavado de las placas trolan el desarrollo del nemátodo. Muchas de estas se- con agua destilada esterilizada. Esta operación se repi- ñales deben estar altamente relacionadas con la pro- tió cuatro veces a intervalos de 24 h. Se contó la canti- ducción de metabolitos, pues cuando el simbionte dad de nemátodos extraídos de cada uno de los medios bacteriano se cultiva artificialmente produce diferen- de cultivo y se analizaron los datos mediante la dócima tes combinaciones de enzimas durante la etapa post- de comparación múltiple de Newman-Keuls con 5% de exponencial del crecimiento [Thaler et al., 1998], y en el caso específico de la especie P. luminescens, Daborn et probabilidad de error. al. (2001) plantearon que también produce una metaloproteasa en esta misma fase del cultivo. RESULTADOS Y DISCUSIÓN Todo este fenómeno antes descrito explica por qué la Los cuatro medios evaluados permitieron la reproduc- reproducción de H. indica sobre medios de cultivo ela- ción de H. indica. Se observó que los juveniles inocula- borados con hígado de pollo ocurrió con tanto tiempo dos en las placas se ubicaron en áreas con abundante de diferencia con respecto a la reproducción sobre híga- crecimiento bacteriano, es decir, alrededor de las colo- do de cerdo. Como estas fuentes de proteína tienen di- nias de P. luminescens. Esto representa la gran relación ferente composición, el hígado de pollo resultó de más que existe entre los nematodos y sus simbiontes, pues difícil degradación para el simbionte bacteriano, la fase ellas son las responsables de transformar el sustrato y postexponencial de crecimiento se alcanzó más tarde, convertirlo en una fuente asequible para su alimentación se retardó la producción de metabolitos y consigo la [Forst y Clarke, 2002]. Como organismos microvívoros, señal alimenticia que desencadenaría la recuperación estos nematodos pueden además alimentarse de la de los juveniles infectivos. biomasa bacteriana, y esto garantiza que las nuevas ge- En la Fig. 1 se evidencian las diferencias significativas neraciones también presenten la bacteria simbionte alo- de la reproducción de H. indica. En las variantes con jada en su intestino, lista para ser liberada en el interior hígado de cerdo el número de juveniles obtenidos fue de otro insecto hospedero [Boemare, 2002]. superior que con hígado de pollo. Otros autores plantean también la necesidad que tiene Este resultado también está relacionado con la el nematodo de su simbionte bacteriano, fundamental- asincronía observada en el desarrollo de los juveniles, mente para el género Heterorhabditis, donde no se ha lo- causado por su pobre recuperación. Gaugler y Han grado la reproducción de nematodos axénicos [Gerritsen (2002) plantearon que esto conlleva la obtención de bajos y Smits, 1993; Lunau et al., 1993], es decir, que no con- rendimientos en las producciones. tengan la bacteria simbionte en su intestino. Otra causa importante es que el contenido lipídico en A las 72 h de incubación se observaron hembras gigan- el hígado de cerdo es superior que en el de pollo. En tes en todos los medios elaborados con hígado de cerdo, este sentido Yoo et al. (2000) enfatizaron la importan- lo que evidenció el desarrollo de los juveniles infectivos cia de los lípidos ricos en ácidos grasos monoinsaturados a estado adulto. Durante la recolección de las nuevas para la elaboración de un medio de cultivo para generaciones de juveniles infestados (JI) la presencia Heterorhabditis bacteriophora. Hatab y Gaugler (1997) de adultos fue mínima; sin embargo, en la combinación plantearon además que los lípidos metabolizados por hígado de pollo-almidón las primeras hembras gigan- los nematodos le proporcionan el 60% del total de su tes aparecieron a los 13 días, y el ciclo reproductivo se energía, de manera que su presencia es primordial en extendió hasta los 24. Cuando se recolectaron además los medios de cultivo que se elaboren artificialmente los nemátodos de las placas, conjuntamente con
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