Double-copulated introduction of ejaculate with dominant larval phenotype to maintain Bombyx mori mutant with dysfunctional apyrene sperm

Abstract

 Advanced lepidopteran males produce two types of sperm; nucleated eusperm called eupyrene sperm, and anucleated parasperm called apyrene sperm. Apyrene sperm is necessary for successful eupyrene sperm fertilization, and apyrene dysfunction results in sterility. In a previous study, we generated Bombyx mori Sex-lethal (Bm-Sxl) mutants, a phenotype mediated by transcription activator-like effector nuclease (TALEN), and found that the Bm-Sxl gene is essential for apyrene spermatogenesis. One of the Bm-Sxl mutant strains, Sxl^In3-homo (Sxl^In3/Sxl^In3), possessed a 3-bp insertion in the highly conserved RNA binding domain. The sterility of Sxl^In3-homo can be rescued by administration of apyrene sperm from triploid males with defective eupyrene sperm. The Bm-Sxl mutants can be maintained using the triploid. However, the production of triploid males is labor-intensive. Considering the logistical challenges of that method, we developed an alternative double copulation method to maintain the mutant using a diploid strain carrying a dominant larval phenotype to preempt the challenging and labor-intensive production of triploid silkworms. In this study, we successfully maintained our Sxl^In3-homo strain by double copulation of the female with a striped (p^S/p^S) and a Sxl^In3-homo male.