A piRNA modulates the levels of 20-hydroxyecdysone in the ovary of the German cockroach

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs, typically 26 to 31 nucleotides long, originally known for silencing transposable elements (TEs), thus maintaining genomic stability. However, recent research has revealed additional regulatory roles. In this study, we investigate piRNA-305221, which is highly expressed in the ovaries of the German cockroach, Blattella germanica, to understand its involvement in oogenesis and reproduction. piRNA-305221 is found in germinal and somatic cells during the gonadotropic cycle, and is maternally provided to the egg. Its expression correlates with critical ovarian events, such as endoreplication and follicular cell differentiation, suggesting regulatory functions beyond TE silencing. Functional knockdown using antisense oligonucleotides (ASOs) resulted in delayed oviposition, malformed oothecae, and reduced offspring viability. Gene expression analysis revealed that the reduction of piRNA-305221 decreased shade (Cyp314a1) mRNA levels, impairing the conversion of ecdysone to its active form, 20-hydroxyecdysone, and a concomitant increase in expression of upstream steroidogenic genes (spook (Cyp307a1), phantom (Cyp306a1), disembodied (Cyp302a1)). These results indicate that piRNA-305221 may regulate steroidogenesis through direct or indirect control of mRNA targets. This study highlights the broader regulatory functions of piRNAs and demonstrates the utility of ASO-mediated knockdown in functional studies of non-coding RNAs.