Dynamic Regulation of Gonadal Transposons and Pseudogenes via PIWI/piRNA Pathway in Gynogenetic Japanese Flounder...

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that interact with PIWI proteins and play essential roles in genome stability, gonadal development, and gametogenesis in animals. The Japanese flounder (Paralichthys olivaceus) is an important marine culture teleost in North Asia, showing pronounced sexual size dimorphism, where gynogenetic induction of all-female cohorts can markedly enhance production. However, the PIWI/piRNA pathway in gynogenetic diploid P. olivaceus, which often exhibit gonadal dysgenesis, poor gamete quality, and low fertilization rates, remains poorly understood. In this study, RNA-seq and small RNA-seq data from 11 tissues and 6 developmental stages of common P. olivaceus, as well as the gonads of gynogenetic P. olivaceus, were analyzed to characterize the PIWI/piRNA pathway and its roles in transposon and gene regulation within the germline. The results showed that PIWI/piRNA genes were predominantly expressed in gonads and early embryogenesis in common P. olivaceus, with the highest expression in testis. Clustered piRNAs were identified in the testis and early embryos of common P. olivaceus, which targeted multiple transposon and gene families. Intriguingly, gynogenetic P. olivaceus gonads harbored abundant clustered piRNAs not only in the testes but also in the ovaries, both targeting similar transposon families as that in common P. olivaceus. Notably, the DNA transposon Tc1/Mariner family and pim genes were the most heavily targeted by piRNAs in gynogenetic P. olivaceus, with testis-biased expression. Expanded pim genes were identified in P. olivaceus, overlapping with piRNA clusters, and the in vitro test in P. olivaceus testes revealed that the expanded pim genes may be pseudogenes as a piRNA cluster reference to generate piRNAs regulating the conventional pim members. These unique features of the PIWI/piRNA pathway in gynogenetic diploid P. olivaceus may underline their impaired reproductive ability, and have important theoretical and practical implications for teleost gynogenetic breeding.