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

摘要

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 insterility. In a previous study, we generated Bombyx mori Sex-lethal (Bm-Sxf) 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 eupyrenesperm. 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.