# Molecular Identification of HR97 in the Swimming Crab Portunus trituberculatus and Its Potential Involvement in Ovarian Development

## TL;DR

This study identifies HR97, a nuclear receptor gene in crabs, and shows it plays a role in ovarian development, with arachidonic acid affecting its activity.

## Contribution

The study identifies and characterizes HR97 in the crab Portunus trituberculatus and reveals its role in ovarian development and gene regulation.

## Key findings

- HR97 is predominantly expressed in crab ovaries and is linked to ovarian development.
- Arachidonic acid suppresses HR97 expression and partially overlaps with its transcriptional responses.
- Reducing HR97 activity disrupts ovarian development and affects genes related to transport, signaling, and transcription.

## Abstract

Nuclear receptors are DNA-binding gene regulators that can be influenced by hormones or fats. This study aimed to identify and explore the function of HR97, a nuclear receptor gene found in many arthropods but not in insects, in an economically important marine crab. HR97 was most active in the ovary, and temporarily reducing its activity in living crabs disrupted ovarian development and altered genes involved in nutrient transport, cell surface signaling, and nuclear transcriptional control. We also found that arachidonic acid, an important unsaturated fatty acid in crustacean ovaries, reduced HR97 gene expression. However, arachidonic acid and HR97 reduction produced only partially overlapping transcriptional responses, suggesting that arachidonic acid can active additional pathways. These findings advance understanding of crab reproduction and may inform future broodstock management.

Nuclear receptor HR97 is considered as a non-insect arthropod–specific receptor, but its roles in decapod reproduction remain poorly understood. Here, we identified and characterized an HR97 ortholog from the swimming crab Portunus trituberculatus (PtHR97) and verified its placement within the NR1L nuclear receptor family by phylogenetic analysis. PtHR97 encodes a canonical nuclear receptor with a conserved DNA-binding domain (DBD) and ligand-binding domain (LBD). Quantitative PCR revealed predominant PtHR97 expression in the ovary and stage-dependent changes during ovarian development. Using an ovarian explant culture system, we found that arachidonic acid (AA) consistently suppressed PtHR97 transcript levels, while methyl farnesoate (MF) and pyriproxyfen (P) had no significant effect, indicating a potential inhibitory role for AA in PtHR97 expression. RNA interference of HR97 caused significant changes in ovarian development, including reduced GSI, smaller oocytes, and uneven eosinophilic granule distribution. Transcriptomic profiling of HR97-silenced ovaries indicated that the major responses involved genes associated with substrate transport/exchange, cell boundary–related signaling and transduction, and disturbed nuclear transcriptional regulation. Short-term in vivo perturbations (HR97 RNAi and AA treatment) further supported these expression changes and revealed that AA- and HR97 RNAi–elicited transcriptional responses only partially overlapped. Taken together, these results suggest that HR97 contributes to ovarian development, potentially through broad transcriptional responses related to transport, signaling, and gene regulation. Although AA may suppress HR97 expression, HR97 does not fully explain AA-mediated regulation of ovarian development.