Molecular investigation of reproductive processes in the sea cucumber, Holothuria leucospilota.

Paul Wright

doi:10.25907/01135

Aspidochirotid sea cucumbers are ecologically important deposit feeders that contribute to sediment turnover, nutrient cycling and benthic ecosystem function, yet many commercially valuable species are under increasing pressure from overexploitation. Aquaculture and restocking offer alternatives to wild harvest, but reliable broodstock selection and controlled spawning remain constrained by limited understanding of the molecular processes that regulate reproductive maturation. This thesis investigated reproductive regulation in the tropical sea cucumber Holothuria leucospilota, with the aim of developing a molecular framework for assessing gonad maturation, identifying candidate broodstock markers and resolving key components of the neuroendocrine pathway controlling final oocyte maturation. First, RNA sequencing was used to characterise gene expression across five histologically defined ovarian maturation stages: Recovery, Growing, Mature, Partially Spawned and Spent. Ovarian transcriptomes broadly reflected reproductive stage, with the strongest transcriptional shifts occurring during the Recovery-to-Growing and Partially Spawned-to-Spent transitions. These patterns indicate that early ovarian rebuilding and post-spawning remodelling are major molecular transitions in the reproductive cycle, whereas Mature and Partially Spawned ovaries are less sharply separated at the whole-transcriptome level. Matched radial nerve cord (RNC) transcriptomes showed weaker stage-associated structure but identified secretory-pathway candidates that may contribute to reproductive signalling. Second, a proteomics-led marker-discovery workflow was developed to identify candidate sex and maturation markers in gonad tissue and coelomic fluid. Gonad proteomics provided the clearest tissue-level separation of sex- and stage-associated protein profiles, whereas coelomic fluid provided a more practical matrix for future minimally invasive broodstock screening. Candidate sex-associated proteins were linked to gamete structure and chromatin organisation, while maturation-associated candidates were consistent with gonad development, extracellular matrix remodelling, cell turnover and post-spawning tissue change. Third, the relaxin-like gonad-stimulating peptide receptor was investigated as a key upstream component of the reproductive neuroendocrine pathway. Comparative sequence analysis identified candidate RGP receptors in multiple sea cucumber species, with conserved features typical of relaxin-family G protein-coupled receptors. Functional assays showed that the H. scabra receptor responded specifically and showed a dose-dependent response to sea cucumber RGP, providing functional evidence for an RGP receptor in holothurians. An RGP receptor homologue was also detected in the H. leucospilota ovary across all reproductive stages, although expression did not differ significantly among stages. Finally, the downstream maturation-inducing hormone pathway was examined using bioassay-guided fractionation and LC-QTOF-MS analysis of ovarian tubule incubation products. Secretome products from RGP-treated ovarian tubules induced germinal vesicle breakdown in isolated immature oocytes, with the strongest activity recovered from the 40% acetonitrile C18 Sep-Pak eluate. LC-MS analysis prioritised an m/z 649.34 feature associated with bioactive ovarian fractions, although MS/MS and SIRIUS analysis provided only putative annotations and did not establish a definitive molecular identity. Together, these findings support a model in which RGP acts upstream of the oocyte and enhances the release of a separate ovarian maturation-inducing activity, rather than acting as the direct maturation-inducing hormone. Overall, this thesis advances our understanding of reproductive regulation in H. leucospilota by linking ovarian maturation, candidate broodstock markers, RGP receptor signalling and downstream MIH-associated activity within a single molecular framework. These findings provide a foundation for future validation of reproductive biomarkers and for the development of more targeted spawning-induction strategies in sea cucumber aquaculture and restoration.

Molecular investigation of reproductive processes in the sea cucumber, Holothuria leucospilota.

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