Identification and Functional Analysis of the fruitless Gene in a Hemimetabolous Insect, Nilaparvata lugens .

The fruitless ( fru ) gene functions as a crucial "tuner" in male insect courtship behavior through distinct expression patterns. In Nilaparvata lugens , our previous research showed doublesex ( dsx ) influencing male courtship songs, causing mating failures with virgin females. However, the impact of fru on N. lugens mating remains unexplored. In this study, the fru homolog ( Nlfru ) in N. lugens yielded four spliceosomes: Nlfru-374-a/b , Nlfru-377 , and Nlfru-433 , encoding proteins of 374aa, 377aa, and 433aa, respectively. Notably, only Nlfru-374b exhibited male bias, while the others were non-sex-specific. All NlFRU proteins featured the BTB conserved domain, with NlFRU-374 and NlFRU-377 possessing the ZnF domain with different sequences. RNAi-mediated Nlfru or its isoforms' knockdown in nymph stages blocked wing-flapping behavior in mating males, while embryonic knockdown via maternal RNAi resulted in over 80% of males losing wing-flapping ability, and female receptivity was reduced. Nlfru expression was Nldsx -regulated, and yet courtship signals and mating success were unaffected. Remarkably, RNAi-mediated Nlfru knockdown up-regulated the expression of flightin in macropterous males, which regulated muscle stiffness and delayed force response, suggesting Nlfru 's involvement in muscle development regulation. Collectively, our results indicate that Nlfru functions in N. lugens exhibit a combination of conservation and species specificity, contributing insights into fru evolution, particularly in Hemiptera species.