Genomic and regulatory basis of visual opsin genes in the cleaner wrasse behavior: features, expression, and enhancer...

Cleaner wrasse (Labroides dimidiatus) undergoes sex reversal to male, and the two sexes exhibit distinctive cleaning behavior mediated by vision. As a keystone cleaner species in coral reef ecosystems, this fish plays an indispensable ecological role. Yet, the visual regulatory mechanisms underlying its sex-specific cleaning behaviors remain unknown.

Through genome annotation and phylogenetic analysis, visual opsin genes were identified in the cleaner wrasse genome. Gene expression levels were quantified by RT-qPCR to examine sexual dimorphism. Additionally, 4 C-seq coupled with dual-luciferase reporter assays was employed to identify and validate putative enhancers regulating opsin gene expression.

Eight visual opsin genes in five families were identified: rhodopsin (RH1), green-sensitive (RH2), ultraviolet-sensitive (SWS1), blue-sensitive (SWS2), and red-sensitive (LWS). Notably, RH2 has undergone diversification into four genes, indicating a high degree of functional specialization in hue resolution. All opsins maintain high homology in conserved motifs and functional domains, while also possessing distinct regions that may facilitate adaptation to complex photic environments. RT-qPCR revealed significant sexual dimorphism in opsin expression. Females exhibited higher expression of SWS1, SWS2B and RH2 family genes, consistent with their primary role as cleaners, which requires precise visual discrimination of client fish coloration and ectoparasites. In contrast, males showed significantly higher expression of RH1 and LWS, likely reflecting adaptation for territorial patrol and social competition following sex change. Furthermore, using 4 C-seq coupled with dual-luciferase reporter assays, we identified and validated active enhancers upstream of RH1 and RH2-1 genes. RH1 was found to be regulated by multiple enhancers acting in concert, suggesting a capacity for robust environmental responsiveness and developmental stage-specific modulation. In contrast, RH2-1 appears to be under the control of a single enhancer binding to transcription factors.

These findings help elucidate the molecular basis of visual system adaptation in cleaner wrasse and provide preliminary insights into the potential links between visual opsin gene regulation, cleaning behavior, and ecological adaptation.