Journal of Entomology and Nematology Vol. 5(1), pp. 1-9, March 2013 Available online at http://www.academicjournals.org/JEN DOI: 10.5897/JEN12.015 ISSN 2006-9855 ©2013 Academic Journals Full Length Research Paper Infection, colonization and extrusion of Metarhizium anisopliae (Metsch) Sorokin (Deuteromycotina: Hyphomycetes) in pupae of Diatraea saccharalis F. (Lepidoptera: Crambidae) L. C. L. Schneider, C. V. Silva, J. A. Pamphile and H. Conte* Department of Cell Biology and Genetics, CCB, Maringá University State, Avenida Colombo 5790, Bl. H67, Maringá PR. Brazil, CEP - 87020-900, Brazil. Accepted 6 August, 2012 The strains MT and E9 of Metarhizium anisopliae were found to be effective in causing the morphological changes in pupae of Diatraea saccharalis of different ages. However, during the treatment with the two strains, it was observed that there was a decrease in the longevity of old pupae and adults that emerged from old and young pupae. The adults that emerged died 24 h later and were totally covered by the pathogen’s mycelium. Regarding the mortality of pupae of different ages, but in relation to the abnormalities, the pre-pupae were found to be more susceptible. The morphological alterations observed in the cuticle and in the internal tissues of the pupae were analyzed using a stereo- microscope, a light microscope and a scanning electron microscope. The adhesion stage and formation of the appressorium happened 18 h after the infection, occasioning black spots and depressions in the cuticle of young and old pupae. In pre-pupae, the sclerotization was not observed and the fungus acted faster and more effectively, thereby, achieving 100% mortality. The colonization of the pathogen was observed in young pupae between 48 and 120 h. The alterations were observed in the fat body, digestive system and musculature; moreover, several vacuoles were noticed inside the pupae. The extrusion happened after 120 h, presenting the mycelial growth upon the pupae cuticle and further sporulation upon the corpses. The morphological alterations that took place during the development of M. anisopliae suggest the possible causes for the mortality of the pupae of D. saccharalis. Key words: Biological control, entomopathogenic fungi, pupal stage, sugarcane borer. INTRODUCTION The mycopathogen Metarhizium anisopliae (Metsch.) Sorokin and other entomopathogenic fungi are widely known and used as biological control agents for several agricultural plagues of considerable economical expression. This pathogen presents infection and colonization mechanisms similar to those found in the arthropods in their several stages of development and the stages are as follows: adhesion, germination, formation of the appressorium, penetration, extrusion and sporulation upon the corpse (Vicentini and Magalhães, *Corresponding author. E-mail: hconte@uem.br. Tel: 55-44- 3261-4466. Fax: 55 – 44-3011-4346. 1996; Alves et al., 2002; Garcia et al., 2005; Bai et al., 2010). During the colonization, the fungus caused physiological disturbances inside the insect which started on the integument and later reached the circulatory, reproductive, respiratory, nervous, and digestive systems, and interacted with the insect’s immune mechanisms; this interaction was very complex and specific (Hajek and Leger, 1994; Hegedus and Khachatourians, 1996; Alves and Pereira, 1998). The penetration of entomopathogenic fungus through the insect’s cuticle presents advantages over other pathogens such as virus and bacteria whose infection occurs through oral pathway, mesenteron (Ferron, 1978; Alves, 1998). In the pupal stage, the insect does not eat