# Histological Observation and Functional Gene Expression Analysis of Gonadal Development in Ruditapes philippinarum Under Artificial Culture Conditions

## TL;DR

This study shows how artificial conditions can speed up the reproductive maturity of Manila clams and identifies specific genes involved in the process.

## Contribution

The study reveals sex-specific gene expression patterns linked to accelerated gonadal maturation in clams under artificial culture conditions.

## Key findings

- Manila clams reached full gonadal maturity within 28 days under controlled food and temperature conditions.
- Sex-specific gene expression patterns were observed, with genes like GnRH, ERR, 17β-HSD, CYP17, and Dmrt4-like showing distinct temporal activity.
- Gene expression changes closely matched histological changes in reproductive tissue development.

## Abstract

This study investigated how to improve the reproductive efficiency of the Manila clam, a popular seafood species, for aquaculture purposes. Controlling reproduction timing is key to improving yield. To induce gonadal maturation, we maintained the clams in an artificial system with regulated food and temperature. This treatment prompted them to reach full reproductive maturity within 28 days. To understand how gonadal maturation was accelerated under these artificial conditions, we monitored the activity of key reproductive genes. We found clear, sex-specific patterns: some genes were active early in males, while others peaked later in females, and these genetic changes matched the physical development of the reproductive tissue. This work helps explain the biological process behind accelerated clam maturation and provides a scientific basis for future methods to make clam farming more stable and productive.

This study employed an artificial greenhouse-based system to enhance gonadal development in the Manila clam, Ruditapes philippinarum. The dynamics of gonadal development and expression patterns of five regulatory genes were analyzed through histological sections and quantitative PCR. Under the controlled conditions (Chlorophyll a: 18.61 ± 1.36 μg/L; temperature: 27.3 ± 2.0 °C), the clams’ gonads reached full maturity within approximately one month, underscoring the critical importance of appropriate food availability and temperature. Gene expression analysis revealed sex- and stage-specific patterns. The gonadotropin-releasing hormone (GnRH) gene was more expressed in males at day 7, reaching a significant peak at day 7. In contrast, females exhibited synchronized expression peaks for both estrogen-related receptor (ERR) and 17β-hydroxysteroid dehydrogenase (17β-HSD) at day 14 (p < 0.05). Cytochrome P450 17 (CYP17) exhibited a dynamic, sex-specific profile, with significantly higher levels in males at day 7 but elevated expression in females at day 14 and day 21. Furthermore, doublesex and mab-3-related transcription factor 4-like (Dmrt4-like) showed clear sex-dependent temporal patterns: it peaked early during maturation in males, while in females, the peak occurred significantly later, at day 21 (p < 0.05). These dynamic changes in gene expression were closely synchronized with histological alterations, providing mechanistic insights into the regulation of gonadal maturation in R. philippinarum.