A Distinct Fruitless Homolog Participates in Reproductive Maturation and Pairing Behavior in the Brine Shrimp Artemia...

Fruitless (fru) is a conserved transcriptional regulator of sex-specific traits and mating behavior in insects; however, its functions in crustaceans remain largely unknown. In the brine shrimp Artemia franciscana, we identified and characterized a novel fru homolog, which we named Afrfru. The Afrfru gene encodes a BTB-C2H2 zinc-finger transcription factor. Phylogenetic analysis placed the Afrfru protein (AfrFRU) in close proximity to other branchiopod fru homologs. Expression profiling revealed predominant expression in the second antenna of males, while transcript levels peaked during middle oogenesis in females. Knockdown of Afrfru via RNA interference (RNAi) in females disrupted oocyte development and led to abnormal oocyte accumulation. In males, Afrfru RNAi knockdown reduced locomotor velocity and markedly decreased pairing success. To investigate downstream mechanisms, we conducted sex-specific RNA-seq after Afrfru-RNAi. In knockdown females, differentially expressed genes (DEGs) were enriched in glutathione and arachidonic acid metabolism pathways; expression of genes associated with insect hormone biosynthesis and ecdysone synthesis was upregulated. In Afrfru-RNAi males, DEGs predominantly exhibited downregulated expression, particularly among genes involved in cytochrome P450-mediated xenobiotic/drug metabolism, glutathione metabolism, and fatty acid metabolic processes. Together, these results indicate that Afrfru contributed to reproductive maturation and pairing behavior in A. franciscana, likely through sex-specific metabolic and hormonal programs. Our study expands the understanding of reproductive behavior regulation in Artemia and extends the known functional scope of fru beyond insect models.