Non-specific immune factors differences in coelomic fluid from polian vesicle and coelom of Apostichopus japonicus, and their early response after evisceration.

Coelomic fluid contains a population of coelomocytes, enzymes, nutrients and kinds of molecules that could be essential for Apostichopus japonicus live. The coelom and polian vesicle are the main tissues that hold the most coelomic fluid in the animal, but whether there exists any immunological difference of the coelomic fluid from the two tissues remains unknown. In this study, we first extracted the coelomic fluid both from the coelom and polian vesicle, and compared their non-specific immune factors. It was found that the ACP and AKP activities in the polian vesicle were significantly higher than those in the coelom, but it was contrary for the SOD and CAT. Meanwhile, the expression levels of several immune-related genes including AjC3-2, AjMKK3/6, AjTLR3 and AjToll in the polian vesicle were significantly lower than those in the coelom. Besides, the early changes of non-specific immune factors were further monitored after eviscerated. During 7 days post evisceration, the immunoenzymes activities of ACP, AKP, SOD and CAT were decreased first and then recovered gradually in the coelomic fluid from the coelom. In the polian vesicle, the ACP and AKP activities showed a similar trend with the coelom, while the SOD and CAT activities showed a transitory increase during 2 h post evisceration (hpe) to 12 hpe. Moreover, the expression profiles of the immune-related genes in the coelom reached the peak at 3 days post evisceration (dpe), while their expression levels in the polian vesicle reached the peak at 7 dpe. All the results suggested that the immunocompetence of coelomic fluid differed in the coelom and polian vesicle, and thus may exert their respective immunological functions. It was likely that the respond speed in the coelom would be faster than that in the polian vesicle after evisceration. Our data will provide a basis for better understanding of the immune defense mechanism of A. japonicus.

Regeneration of coelomocytes after evisceration in the sea cucumber, Apostichopus japonicus.

Sea cucumber, Apostichopus japonicus, is one of the most important holothurian species cultured in China. Severe evisceration induced by various natural and artificial factors commonly occurs during transport and culture of A. japonicus. Evisceration causes higher mortality and lower yield. Along with the visceral regeneration process, sea cucumbers also regenerate coelomocytes in order to recover immune function. In this study, evisceration of A. japonicus was induced by intracoelomic injection of 0.35 M KCl. Regeneration of coelomocytes was investigated by time course cell counting as well as detection of DNA replication by the EdU labeling technique. Coelomic fluid volume was restored to the pre-evisceration level within 2 h after evisceration. Total coelomocyte count (TCC) reached a peak at 6 h post-evisceration, followed decreased and then increased with a slight fluctuation, restored to the pre-evisceration level at 35 d post-evisceration. The change in different subtypes of coelomocytes was consistent with that of total coelomocytes. However, there were some variations in the regeneration of coelomocyte subtypes. At the end of the study, only the counts of amoebocytes and morula cells recovered to the pre-evisceration level. DNA replication assay showed EdU-positive cells accounted for 9.5% before evisceration and 4.7% at 6 h post-evisceration. However, the percentage of EdU-positive cells significantly increased, reaching 18.6% at 3 d after evisceration, then declined. Therefore, we analyzed the observed increase in coelomocytes at 6 h post-evisceration, which may be due to coelomocyte migration from the water-vascular system into the coelom rather than de novo cell proliferation.