Pigment-dispersing factor is not essential for eclosion rhythm and inducing the photoperiodic diapause in the silkworm, Bombyx mori

The neuropeptide pigment-dispersing factor (PDF) functions as a key circadian clock output that regulates circadian rhythmicity at physiological and behavioral levels in the fruit fly Drosophila melanogaster. Similarly, the involvement of PDF in circadian behavioral rhythmicity and photoperiodism has been demonstrated across a wide range of insect orders. However, in Lepidoptera, the second largest insect order, the function of PDF remains poorly elucidated. The silkworm Bombyx mori, which exhibits clear circadian rhythmicity and photoperiodism, represents a suitable model for exploring the role of PDF in lepidopterans. Herein, we investigated whether PDF acts as a circadian clock output regulating circadian behavior and photoperiodic diapause induction in B. mori. Using CRISPR/Cas9-mediated mutagenesis, we introduced a frameshift mutation into pdf in a bivoltine strain. The mutated allele was predicted to encode a protein lacking the mature PDF region due to a premature stop codon. However, the pdf mutant retained the circadian eclosion rhythm and the ability to induce diapause in response to photoperiodic conditions during the larval stage. Comparative analysis of PDF sequences from multiple insect orders retrieved from the NCBI database revealed that lepidopteran PDF amino acid sequences are less conserved than those of other insect orders. These findings suggest that the PDF of lepidopterans has undergone divergent evolution, and at least in B. mori, it lost function in circadian clock-related output. To the best of our knowledge, this is the first study to investigate the function of PDF in Lepidoptera using reverse genetics.