Molecular insights into the interconnections in the eyestalk-androgenic gland-testis axis in crayfish Cherax quadricarinatus

In crustaceans, the neuroendocrine organ located in the eyestalk plays a critical role in regulating development and reproduction. Evidence has demonstrated that a specialized mechanism, referred to as the “eyestalk-androgenic gland-testis” axis, governs male reproductive function. However, the molecular mechanisms underlying this process remain poorly understood. In this study, we investigated the expression profiles of the androgenic gland and eyestalk in male Cherax quadricarinatus across three developmental stages to identify co-expression patterns and explore the interconnections within this axis. Our analysis revealed considerable variation in the differential expression of genes throughout the development of both the eyestalk and androgenic gland, involving distinct regulatory pathways. However, forty-seven genes were consistently differentially expressed across all stages. Furthermore, we found 2111 genes in six clusters exhibited both positive and negative correlations between the eyestalk and androgenic gland, suggesting the regulatory complexity in the axis. We further retrieved genes showing strong co-regulation with known reproductive-related genes, including the most explored insulin-like androgenic gland hormone ( IAG ) gene. Several germline determining genes including membrane-anchored androgenic gland-specific factor, doublesex and mab-3-related transcription factor 3 ( DMRT3 ), probable ATP-dependent RNA helicase vasa-like, sex peptide receptor-like were detected. We also used dsRNA of IAG to test interaction between two sexual related genes DMRT3 and forkhead box l2-like ( FOXL2) . However, no significant transcriptional changes were observed in both androgenic gland and testis. These findings provide new insights into the molecular mechanisms of reproductive development in crustaceans and offer valuable guidance for advancing artificial breeding techniques in these species. • The interconnection in the “eyestalk-androgenic gland-testis” axis was investigated based on co-expression and co-regulation analysis. • The development of eyestalk and androgenic gland largely involve distinct regulatory pathways, but share process related to the myofibril. • Both positive and negative regulation between eyestalk and androgenic gland were detected, with genes involving in different functions. • Genes showing strong co-expression with sexual related functions were identified, providing extensive candidates for further studies.