Functional Insights into Bpcreb1 in Bellamya purificata: Expression Dynamics and regulatory Roles during gonadal development.

Cyclic AMP response element-binding protein 1 (CREB1), a conserved bZIP transcription factor, is crucial for animal reproduction. Therefore, this study investigated its role in gonadal development of the freshwater snail Bellamya purificata using qRT-PCR, ISH, RNAi, and DNA methylation analysis. Results revealed that the creb1 CDS (867 bp, 288 aa) features a highly conserved BRLZ domain. Significantly, tissue expression showed markedly higher creb1 transcript levels in testes versus ovaries (p < 0.01). ISH localized enriched mRNA in ovarian oocytes/follicular walls and testicular spermatogonia/sperm. Functionally, efficient RNAi (max. 71.98% silencing) caused acute downregulation of sex genes: female β-catenin, cyp17a1 and sox9 expression fell 27%-34%, while male expression dropped 58%-77%. Critically, prolonged interference induced testicular defects, namely reduced mature sperm and increased spermatogonia, implicating Bpcreb1 in spermatogenesis-potentially via tssk and sox9 regulation. Furthermore, epigenetic analysis found consistently low Bpcreb1 promoter methylation (<5%) in both sexes, yet RNAi did not alter this hypomethylation, hinting at non-methylation regulatory mechanisms. In summary, creb1 demonstrably regulates gonadal development in B. purificata, with a pivotal role in spermatogenesis. Consequently, these findings provide a theoretical foundation for studying its gonadal development and monosex breeding strategies.