A testis-specific heat shock protein gene plays a critical role in male fertility in the fall armyworm.

Heat shock proteins (Hsps) are critical for stress responses and multiple physiological processes, yet their function in insect reproduction remains underexplored. In this study, we identified 36 Hsp genes in the fall armyworm (Spodoptera frugiperda), of which LOC118269601 (designated as SfHsp68A) exhibited robust testis-specific expression, peaking during pupal testis development. CRISPR/Cas9-mediated knockout of SfHsp68A resulted in recessive male sterility, significantly reduced testis size, sperm counts, and egg hatch rates. Transcriptomic analysis of mutant testes revealed substantial downregulation of genes linked to mitochondrial function, oxidative phosphorylation, energy metabolism, and reactive oxygen species (ROS) metabolism. These results demonstrate that SfHsp68A is essential for male fertility in S. frugiperda, likely by maintaining mitochondrial integrity and metabolic homeostasis during spermatogenesis. These studies reveal that testis-specific Hsps function in male fertility and highlight SfHsp68A as a potential genetic target for developing sterile insect technology, offering an environmentally sustainable strategy for controlling this globally invasive pest.