Egg-laying hormone expression in identified neurons across developmental stages and reproductive states of the nudibranch Berghia stephanieae

Neuropeptides play essential roles in coordinating reproduction. Egg-laying hormone (ELH) is conserved in genetic sequence and behavioral function across molluscs, where neuronal clusters secrete ELH to modulate and induce egg-laying. Here we investigated ELH in the nudibranch mollusc, Berghia stephanieae. ELH preprohormone gene orthologs, which showed clade-specific differences at the C-terminus of the predicted bioactive peptide, were identified in brain transcriptomes across several nudipleuran species, including B. stephanieae. Injection of synthesized B. stephanieae ELH peptide into mature individuals induced egg-laying. ELH gene expression in the brain and body was mapped using in-situ hybridization chain reaction. Across the adult brain, 300-400 neurons expressed ELH. Twenty-one different cell types were identified in adults, three of which were located unilaterally on the right side, which corresponds to the location of the reproductive organs. Ten cell types were present in pre-reproductive juvenile stages. An asymmetric cluster of approximately 100 small neurons appeared in the right pedal ganglion of late-stage juveniles. Additional neurons in the pleural and pedal ganglia expressed ELH only in adults that were actively laying eggs and sub-adults that were on the verge of doing so, implicating their direct role in reproduction. Outside the brain, ELH was expressed on sensory appendages, including in presumptive sensory neurons. ELH shares deep homology with the corticotropin-releasing hormone gene family, which has roles broadly in stress response. Its widespread expression in the nudibranch B. stephanieae suggests that ELH plays a role beyond reproduction in gastropod molluscs. HighlightsO_LIEgg-laying hormone (ELH) preprohormone sequences were identified in the transcriptomes of several nudipleuran molluscs. C_LIO_LIMany ELH-expressing neurons and clusters in the brain could be individually identified based on soma position and morphology. C_LIO_LISome identified ELH-expressing neurons were not observed until later juvenile stages. C_LIO_LISome neurons expressed ELH in adults only when they were actively laying eggs. C_LIO_LIMany hundreds of ELH-expressing neurons were present in peripheral appendages. C_LI