BmHen1 plays an essential role in the regulation of eupyrene sperm development in Bombyx mori

In lepidopteran insects, sperm polymorphism is a remarkable feature, in which males exhibit two different types of sperms. Both sperm morphs are essential for fertilization as eupyrene (nucleate) sperm carries DNA and fertilizes the egg, while apyrene (anucleate) sperm is necessary for transporting eupyrene sperm into females. To date, the functional genetic study on dichotomous spermatogenesis has been limited. It is known that, in the model species including mice, worms, and flies, the components in piRNA biogenesis pathway play an important role in gonad development. In this study, we characterize BmHen1 as a new critical component involved in the regulation of eupyrene sperm development in B. mori. We generated the loss-of-function mutant of BmHen1 ({Delta}BmHen1) through CRISPR/Cas9-based gene editing, and found that it is both female- and male-sterile.{Delta} BmHen1 females lay significantly fewer eggs than wild-type, which display morphological defects. Fluorescence staining assays show that the{Delta} BmHen1 eupyrene sperms exhibit severe defects in nuclei formation, while its apyrene sperms are normal. We then constructed the loss-of-function mutants of Siwi and BmAgo3 ({Delta}Siwi and{Delta} BmAgo3) through CRISPR/Cas9-based gene editing, which encode PIWI proteins acting as the core elements in piRNA biogenesis, and explored whether they might be involved in spermatogenesis. To our surprise,{Delta} Siwi and{Delta} BmAgo3 mutants develop normal male reproduction system, indicating that they dont participate in sperm development. As the activity of BmHen1 depends on BmPnldc1 during piRNA biogenesis, and{Delta} BmHen1 and{Delta} BmPnldc1 mutants display similar defects in sperm development, we performed RNA sequencing analysis to look for the genes that might be co-regulated by BmHen1 and BmPnldc1. Our results indicate that the defects in{Delta} BmHen1 and{Delta} BmPnldc1 eupyrene sperms could be attributed to dysregulated genes involved in energy metabolism and cell differentiation. Furthermore, we found that the piRNA biogenesis is inhibited in{Delta} BmHen1 and{Delta} BmPnldc1 sperm bundles, whereas the transposon activity was induced. Taken together, our findings suggest that BmHen1 is a new crucial component regulating eupyrene sperm development in B. mori, whereas the PIWI proteins Siwi and BmAgo3 are not involved in this process. Our results may provide a potential gene target for genetic modification of sterility in B. mori.