Background At present, domestic and foreign scholars have used morphology, spectroscopy, mass spectrometry, molecular biology and other technologies to identify sea cucumber from the morphology, chemical substances contained, nucleic acid differences and other aspects of sea cucumber, but considering the technical requirements and time cost, morphology is still the main identification method of sea cucumber.

Objective The dermis surface layer of sea cucumbers contains ossicles, which constitute the distinguishing feature of this class. The types and relative proportions of these ossicles can facilitate rapid identification of different sea cucumber species.

Methods In this study, NaClO and protease K methods were employed to extract and process tissues of sea cucumbers (Apostichopus japonicus) aged 1 to 3 years from the Pingtan sea area (Fujian, China). Tissue samples included spines, body walls, tentacles, tube feet, longitudinal muscles, respiratory trees, and intestines. Ossicle images obtained through these methods were compared, and the relative proportions of each type of ossicle were analyzed using SPSS software.

Results The results revealed seven types of ossicles: table-shaped, button-shaped, rod-shaped, rosette-shaped, long hole-like body ossicles, composite discoid ossicles, and C-shaped ossicles, observable under both light and electron microscopes, with significant differences (P<0.05). Comparing the images obtained by NaClO and protease K methods, NaClO-extracted ossicles displayed clearer types and approximate numbers, free from tissue attachment, but were brittle and prone to damage, resulting in incomplete structures. In contrast, protease K-extracted ossicles showed better structural integrity under the microscope, facilitating more accurate counting and analysis, although they retained some tissue. Additionally, the relative proportions of ossicle types significantly differed (P<0.05) across different ages and tissues. For instance, in one-year-old specimens, button-shaped ossicles constituted the highest proportion in the body wall 83.28%±9.33%, while table-shaped ossicles dominated in the body cavity 35.02%±10.84%, and long hole-like body ossicles were most prevalent in tentacles 7.23%±2.50%. Relative proportions in the body wall and tube feet at one year (20.28%±7.29% and 35.02%±10.84%, respectively) were significantly higher than in the second year (3.80%±1.35% and 17.21%±1.87%, respectively) and third year (3.40%±1.43% and 11.23%±1.01%, respectively) (P<0.05).

Conclusion The results show that the relative proportion of ossicle types of sea cucumber have a difference in different ages and tissues, and can be obtained by various methods stability, which is an important index to identify sea cucumber species.

Significance This study provides the basic data for the biological identification of the A. japonicus and the reference for the age identification.