A Simple Method for the Decapsulation of Dormant Resting Eggs for Nuclear Staining in the Monogonont Rotifer, Brachionus plicatilis Muller

Abstract

The bdelloid rotifer Brachionus plicatilis Müller is an aquatic invertebrate that predominately populates freshwater lakes and ponds. It is a pseudocoelomate which consists of approximately 1000 cells. B. plicatilis exhibits both asexual reproduction in which parthenogenetically derived eggs quickly mature into new progeny (~12 hours) or sexual reproduction in which fertilized eggs diapause for 2-3 months before reinitiating progression into organismic development based on environmental cues, such as water conditions and sunlight. Because B. plicatilis shares many of the conserved genes responsible for dormancy survival such as genes that code for late embryogenesis abundant (LEA), trehalose metabolism and small heat shock proteins (sHSP), it has become an excellent model organism for studies investigating dormancy and developmental biology. Several recent reports have developed procedures in quiescent B. plicatilis resting eggs for molecular manipulation of gene transcription, such as RNAi. However, the dechorionation procedure employed for those previous studies was not effective in our hands to penetrate dessicated resting eggs for nuclei staining. Therefore, we developed a procedure described in this manuscript for decapsulating encysted resting eggs for staining with Hoechst dye. This method may ultimately be used to track phenotypic aspects of entry into and exit from dormancy by determining whether there is a correlation in nuclei number and developmental progress in the Monogonont Rotifer, Brachionus plicatilis Müller.