The Development of Eupyrene Sperm Is Dependent on Sperm-Leucylaminopeptidase in Bombyx mori
1
Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
2
Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang 212100, China
*
Author to whom correspondence should be addressed.
Insects 2026, 17(4), 389; https://doi.org/10.3390/insects17040389
Submission received: 24 February 2026
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Revised: 31 March 2026
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Accepted: 1 April 2026
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Published: 3 April 2026
(This article belongs to the Section Insect Physiology, Reproduction and Development)
Simple Summary
While the role of leucine aminopeptidase (LAP) in reproductive development is well-established in mammals and Diptera, its function in the male reproductive system of lepidopteran insects remains largely unexplored. In this study, we performed functional analysis of Bombyx mori sperm-Leucineaminopeptidase (BmS-LAP), the sole homolog of Drosophila melanogaster S-LAPs in B. mori, using CRISPR/Cas9 system. Depletion of BmS-LAP resulted in complete male sterility while the fertility of female silkworms remained unaffected. The male sterility phenotype was caused by developmental defects of eupyrene sperm, visualized as nuclear mislocalization and a failure of flagellar elongation. Furthermore, morphological observation and double copulation assays revealed that the BmS-LAP mutation had no significant effect on apyrene sperm development. This study provided the first evidence that LAP is implicated in dimorphic spermatogenesis.
Abstract
Sexual reproduction is the predominant mode of reproduction in animals, and spermatogenesis is the fundamental step in this process. As the model organism for lepidopteran, the silkworm Bombyx mori exhibits typical dichotomous spermatogenesis, producing both nucleated (eupyrene) and anucleate (apyrene) sperm. Leucylaminopeptidases (LAPs), members of the M17 metalloprotease family, are characterized by their ability to cleave leucine residues from the N-terminus of peptides. In addition to this canonical function, they have been implicated in male fertility in mammals and Diptera. Nevertheless, whether LAPs are required for dimorphic spermatogenesis in Lepidoptera remains to be clarified. Here, we demonstrated that Sperm-Leucylaminopeptidase (S-LAP) plays vital roles in the silkworm eupyrene sperm development. Similar to the testis-specific expression pattern of eight S-LAPs in Drosophila melanogaster, BmS-LAP was also predominantly expressed in testis. Depletion of BmS-LAP via CRISPR/Cas9 system resulted in male sterility, while the fertility of female mutant was unaffected. Notably, male mutants displayed severe defects in the formation and migration of eupyrene sperm, whereas apyrene sperm development appeared normal. In addition, RNA-seq and qRT-PCR analyses demonstrated that spermatogenesis defects were associated with energy metabolism and flagellar assembly. Our study provides the first evidence that LAP is necessary for dimorphic spermatogenesis in Lepidopteran, offering new insights into the molecular basis of male infertility.
