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.

Illumina sequencing as perfomered as our previous study [47], total RNA was isolated from 10 Bmfru mutant and WT antennas using TRIzol (Invitrogen, Carlsbad, CA, USA), and the residual DNA was removed with RNase-free DNase I (New England BioLabs, Ipswich, MA, USA) for 30 min at 37 °C. For RNA-seq, library construction and sequencing using an Illumina HiSeq 2000 were conducted by BGI Genomic Services (Shenzhen, China), briefly described as follows. The mRNA was enriched using oligo (dT) magnetic beads samples were mixed with a fragmentation buffer, and the mRNA reduced to short fragments (~200 bp). The first strand of the cDNA was synthesized using random hexamer primers,buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand, and the double-stranded cDNA was purified with magnetic beads followed by performing end repair and 3'-end single nucleotide adenine addition. Finally, sequencing adaptors were ligated to the fragments which were enriched by PCR amplification, and an Agilent 2100 Bioanaylzer and an ABI Step One Plus Real-Time PCR System were used to quantify libraries. The library products were sequenced using an Illumina HiSeq 2000 (BGI Biotech Co. Ltd.). The raw sequencing data were qualified, filtered, and mapped to the reference silkworm genome database (http://silkworm.genomics.org.cn/) using tophat/bowtie2.

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