In vitro culture and low temperature incubation tolerance of staged embryos of the silkworm, Bombyx mori

Abstract

Development of cryopreservation for the silkworm, Bombyx mori, has been anticipated for a long time, and now two methods are available. One uses frozen sperm (Takemura and Kanda, 1999; Takemura et al., 2000) and the other uses frozen ovaries (Mochida et al., 2003, Banno et al., 2013). These methods are already in practice for most silkworm strains, however, a small number of strains are not available due to their low tolerance to low temperature. It is important to develop a greater variety of methods for safe preservation of the large collections of silkworm bio-resources. Recently, larval hatching from cryopreserved embryos kept in liquid nitrogen was reported in Drosophila melanogaster (Mazur et al., 1992) and in Pectinophora gossypiella (Rajamohan et al., 2013). These reports present a potential alternative to those previously developed for the silkworm. To perform in vitro culture of B. mori embryos, penetration of cryoprotectant into the embryo is necessary. But silkworm embryos are enveloped with chorions (egg shells), which prevents the penetration of the cryoprotectant. To improve the permeability of embryos, first, removal of chorions is essential, and these dechorionated eggs should be cultured in vitro. The possibility of using silkworm embryos for long-term preservation was studied twenty years ago by Imanishi et al. (1996). According to their report, embryos died when they were kept at −196°C. However, their experiments were limited by the selection of the stage and basic procedures, and more detailed experimentation is needed to determine the possibility of embryo use for cryopreservation. In this study, optimal developmental stages for in vitro culture of embryos were investigated. The tolerance of the embryos for low temperature incubation was tested as the next step, along with detailed observations of their morphological features and developmental stages. MATERIALS AND METHODS