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

## TL;DR

The study explores how the PIWI/piRNA pathway regulates transposons and pseudogenes in gynogenetic Japanese flounder, revealing differences in gene expression and reproductive issues.

## Contribution

The study identifies unique features of the PIWI/piRNA pathway in gynogenetic Japanese flounder, linking them to reproductive impairments.

## Key findings

- Clustered piRNAs in gynogenetic flounder ovaries show testis-like patterns, differing from common flounder.
- Expanded pim genes in gynogenetic flounder may act as pseudogenes to generate piRNAs regulating conventional pim members.
- The PIWI/piRNA pathway in gynogenetic flounder targets transposons like Tc1/Mariner and pim genes, contributing to reproductive issues.

## Abstract

To characterize the PIWI/piRNA pathway and its role in transposon and gene regulation within the germline, RNA-seq and small RNA-seq data were analyzed from different tissues and developmental stages of common P. olivaceus, as well as the gonads of gynogenetic P. olivaceus, often exhibiting gonadal dysgenesis, poor gamete quality, and low fertilization rates. Clustered piRNAs were mainly detected in the testes and early embryos of common P. olivaceus, whereas the ovaries of gynogenetic P. olivaceus harbored abundant testis-like piRNA clusters, representing a marked divergence from common P. olivaceus and suggesting that piRNA-mediated regulation may play a distinct role in the gynogenetic lineage. In gynogenetic P. olivaceus, pim genes were heavily targeted by piRNAs, exhibiting male-biased expression and conspicuous expansion across the teleost lineage. Expanded pim genes in P. olivaceus gonads overlapped with piRNA clusters, and the in vitro assay showed that overexpression of an expanded pim-related piRNA cluster significantly reduced the expression of conventional pims in P. olivaceus testes, supporting the model that the expanded pim genes may be putative pseudogenes as a piRNA cluster reference to generate piRNAs regulating the conventional pim members.

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.