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The surging market demand for sea cucumbers (Apostichopus japonicus) has fueled a thriving growth in the sea cucumber breeding industry. Pheromones are molecules that evolve signals in defined proportions in the case of multicomponent pheromones, emitted by one individual and received by a second individual of the same species to cause a specific response. It remains an enigma as to how the species of sea cucumber, devoid of visual, auditory, and olfactory senses, aggregate and communicate within their groups, as well as coordinate gamete release during the breeding period. We hypothesize that pheromones may serve as the medium for facilitating these behaviors and propose conducting experiments centered around pheromonal investigation. In this study, we aimed to analyze common metabolites of male A. japonicususing LC-MS/MS technology and bioinformatics. Coelomic fluid and ejaculate fluid were analyzed, with aquaculture water serving as the control. Eight target pheromones were analyzed, and eight of metabolite analogs were tested for aggregation and spawning behaviors. Results showed that β-sitosterol at a concentration of 10 μg·L−1significantly promoted aggregation (21.25 % average rate) and induced a significant increase in spawning rate (58 %) of A. japonicus. The significance of this study lies in the fact that by collecting and utilizing pheromones of A. japonicus, farmers can accurately determine the reproductive cycle and optimal breeding season for A. japonicus. This enables fish farmers to implement effective breeding management strategies at the appropriate time, thereby increasing the success rate of reproduction and providing valuable technical support for the cultivation of A. japonicusseedlings.

AbstractTechniques for in vitrooocyte maturation are important for studying maturation regulatory mechanisms, cell differentiation, and embryonic development. In this study, after Apostichopus japonicusgonads were dissected, oocytes, together with gonadal fluid extracted from gonads, were dried in shade. The effects of three temperatures (17, 21, and 25 °C) on oocyte maturation and following development were studied. The results showed that germinal vesicle breakdown (GVBD) of most oocytes occurred after desiccation, and those oocytes with GVBD could be fertilized and develop normally. There were no significant differences in the percentages of GVBD and individuals in blastula and gastrula stages between 17 and 21 °C (p > .05). The percentages of GVBD reached a maximum (17 °C: 98.6%, 21 °C: 97.1%) after 2 h of desiccation. The percentages of blastulae and gastrulae reached the maximum of more than 80% after 2.5–3.5 h of desiccation. At 25 °C, the percentages of GVBD, blastulae, and gastrulae reached a maximum of 76.7, 61.0, and 59.9% after 3.5 h of desiccation, respectively, and were significantly lower than that at 17 and 21 °C (p < .01). This study not only provided an effective and simple technique for in vitroinduction of A. japonicusoocyte maturation, but also furthered understanding of gamete biology of this species.

Gonads play a critical role in the reproduction of sea cucumbers, and their development is regulated by a large number of genes. To date, the ideal reference genes (RGs) for studying sea cucumber gonadal development have not been reported. In this study, we selected ten RGs and conducted quantitative real-time PCR analysis to identify the most stable among Apostichopus japonicusgonads at four different stages of gonadal development. We used ∆Cq, GeNorm, NormFinder, BestKeeper, and RefFinder to evaluate the stability of candidate genes. EF1βand Grb2together were the preferred combination of stable RGs among all of the gonad samples and for the ovaries alone. UBQcombined with RPS4was the best combination of stable RGs in the testes. This study aimed to provide a basis for analysis of differences in gene expression related to gonadal development in sea cucumbers, and the results provide valuable information for further studies of the molecular mechanisms involved in sea cucumber gonadal development.