The importance of the insect cuticle as a primary protective barrier against entomopathogens has long been noted. In the present study, we addressed this issue by utilizing an experimental infection system composed of the model beetle T. castaneum and two entomopathogenic fungal species, Beauveria bassiana and Metarhizium anisopliae. The pupae were relatively susceptible to these fungi by the natural route of infection, with some refractoriness developed with age, while the adults exhibited much higher refractoriness. Whereas M. anisopliae exhibited seemingly higher infectivity to the pupae compared to B. bassiana when the natural conidium infection was employed, direct inoculation of cultured hyphal body cells into the hemocoel was found highly and equally virulent in the pupae for the both fungal species. These results collectively suggest an important role of the cuticular integument in antifungal host defense, and we subsequently conducted the knockdown of chitin synthase 1 gene (CHS1). We targeted the prepupal and mid-pupal peaks of its expression respectively by using injection of the dsRNA at very low dosages to avoid lethality. The resulting pupae looked normal, but the adults showed a mild phenotype with dimpled/wrinkled elytra. The CHS1 gene knockdown compromised significantly host defense against the fungal infection via the natural route, except the configuration of knockdown pupae and M. anisopliae, suggesting an indispensable role of CHS1. (C) 2016 Elsevier Inc. All rights reserved.