Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas9 and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior. Author summary The fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model, Drosophila melanogaster, the sex determination cascade genes fruitless and doublesex are involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth, Bombyx mori. Our results support the presence of two genetic pathways in B. mori, named Bmdsx-BmOR1-bombykol and Bmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.