Dynamic Regulation of Gonadal Transposons and Pseudogenes via PIWI/piRNA Pathway in Gynogenetic Japanese Flounder (Paralichthys olivaceus)

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 (<i>Paralichthys olivaceus</i>) 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 <i>P. olivaceus</i>, 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 <i>P. olivaceus</i>, as well as the gonads of gynogenetic <i>P. olivaceus,</i> 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 <i>P. olivaceus</i>, with the highest expression in testis. Clustered piRNAs were identified in the testis and early embryos of common <i>P. olivaceus</i>, which targeted multiple transposon and gene families. Intriguingly, gynogenetic <i>P. olivaceus</i> gonads harbored abundant clustered piRNAs not only in the testes but also in the ovaries, both targeting similar transposon families as that in common <i>P. olivaceus</i>. Notably, the DNA transposon <i>Tc1/Mariner</i> family and <i>pim</i> genes were the most heavily targeted by piRNAs in gynogenetic <i>P. olivaceus</i>, with testis-biased expression. Expanded <i>pim</i> genes were identified in <i>P. olivaceus</i>, overlapping with piRNA clusters, and the <i>in vitro</i> test in <i>P. olivaceus</i> testes revealed that the expanded <i>pim</i> genes may be pseudogenes as a piRNA cluster reference to generate piRNAs regulating the conventional <i>pim</i> members. These unique features of the PIWI/piRNA pathway in gynogenetic diploid <i>P. olivaceus</i> may underline their impaired reproductive ability, and have important theoretical and practical implications for teleost gynogenetic breeding.