Two neuropeptide signalings regulate post-mating refusal behavior and reproductive system in male crickets

After mating, insects always perform mating refusal behavior, termed post-mating refractoriness, due to physiological restrictions. Male crickets, Gryllus bimaculatus , characteristically exhibit 1-hour post-mating refractory stage, controlled by terminal abdominal ganglion. The molecular mechanisms underlying the male-specific precisely timed refractory stage remain elucidated. Here we show that among 28 neuropeptide precursors expressed in the terminal abdominal ganglion, DH31 , myosuppressin , allatotropin , and sNPF exhibited male-specific expression based on RT-qPCR and in situ hybridization. However, RNA interference experiments showed that only knockdown of allatotropin and sNPF changed the duration of the refractoriness. Furthermore, allatotropin and sNPF knockdown influenced functions of male reproductive system by inhibiting seminal fluid secretion from male accessory gland and decreasing sperm storage in seminal vesicles, respectively. Knockdown of their receptors caused similar phenotypes. In conclusion, this study demonstrated the regulation of post-mating refusal behavior and reproductive system by Allatotropin and sNPF signalings in male crickets.