Tissue-Specific IAG Interactome in Cherax quadricarinatus Gonads

Insulin-like androgenic gland (IAG) hormone is a central regulator of sex differentiation in decapod crustaceans. However, the tissue-specific molecular interaction networks governing its gonadal functions remain poorly characterized. Through protein pull-down assays, 279 candidate IAG-interacting proteins were captured from gonadal tissues (androgenic gland [AG], testis and ovary) of Cherax quadricarinatus followed by GO and KEGG enrichment analyses. Subsequently, 32 high-confidence interactors were identified via dimeric complex structures prediction using a composite reliability threshold (ipTM + pTM > 0.75). For eight key candidates, interacting amino acid residues and binding energies were further predicted with AlphaFold3. The AG harbored the largest number of interactors, showing significant enrichment in metabolic pathways. Notably, interactions with ubiquitin-like modifier-activating enzyme 1 (UBA1), heat shock protein (HSP) and histone-lysine N-methyltransferase (HKMT) in male gonadal tissues (AG and testis) suggest potential epigenetic regulatory roles for IAG. Conversely, in the ovary, IAG interacted with endoribonuclease Dicer (DICER1) and casein kinase II (CK2), implying putative post-transcriptional and post-translational mechanisms for inhibiting female maturation. This study reveals the tissue-specific complexity of the IAG interactome in Cherax quadricarinatus gonads, elucidating its potential dual roles in regulating metabolic processes in the AG and modulating post-transcriptional/post-translational events in the ovary. These findings provide novel insights into the molecular mechanisms of crustacean sex development and identify new targets for sex-control strategies in Cherax quadricarinatus.