BAG2 mediates coelomocyte apoptosis in Vibrio splendidus challenged sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are closely related to the occurrence, development, and immune response of diseases. BCL2-associated athanogene 2 (BAG2) is a member of the BAG family that functions in diverse cellular processes, including cell death, differentiation, and cell division. In this study, we cloned the cDNA full-length of sea cucumber (Apostichopus japonicus) BAG2 (AjBAG2) and confirmed it is an anti-apoptotic protein in vitro and in vivo during Vibrio splendidus infection. Moreover, we identified a perfect complementarity between miR-375 and the 3'-untranslated region (UTR) sequence of AjBAG2. The miR-375 expression decreased the luciferase activity dose-dependently when co-transfected with the AjBAG2 3'-UTR-luciferase reporter containing the miR-375 target site in epithelioma papulosum cyprini (EPC) cells. This inhibition was partially recovered by a miR-375 specific inhibitor. The mRNA and protein levels of AjBAG2 were opposite to that of coelomocytes in challenged sea cucumber when treated with miR-375 mimics or inhibitors. Additionally, miR-375 expression induced coelomocytes apoptosis and blocked the anti-apoptotic activity of AjBAG2. Our data demonstrated that AjBAG2 is an anti-apoptotic protein during V. splendidus infection and this function can be inhibited by miR-375 in sea cucumbers.

Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber.

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Production, characterization and application of monoclonal antibody to spherulocytes: a subpopulation of coelomocytes of Apostichopus japonicus.

One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA), Western blotting and fluorescenceactivated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136 kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus edulis was compared and the result showed that none of them was positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5-7 microm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong positive signals on cytoplasm of some spherical cells with diameter of 7-8 microm. Some other cells with higher nucelo-plasmic ratio, about 5-6 microm in diameter showed weak positive signals on membrane. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive.

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber.

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.

Identification and expression characterization of WntA during intestinal regeneration in the sea cucumber Apostichopus japonicus.

Wnt genes encode secreted glycoproteins that act as signaling molecules; these molecules direct cell proliferation, migration, differentiation and survival during animal development, maintenance of homeostasis and regeneration. At present, although the regeneration mechanism in Apostichopus japonicus has been studied, there is a little research on the Wnt signaling pathway in A. japonicus. To understand the potential role of the Wnt signaling pathway in A. japonicus, we cloned and sequenced the WntA gene in A. japonicus. Protein localization analysis showed that WntA protein was ubiquitously expressed in epidermal cells, the muscle and submucosa of the intestinal tissue. After stimulation and evisceration, the dynamic changes in expression of the WntA gene and protein showed that WntA was constitutively expressed during different stages of intestine regeneration in A. japonicus, with higher levels during the early wound healing stage and late lumen formation in the residual and nascent intestinal tissues, indicating its response to intestinal regeneration. Simultaneously, cell proliferation and apoptosis analysis showed that the patterns of cell proliferation were similar to the patterns of WntA protein expression during different intestinal regeneration stages in this organism. Taken together, these results suggested that WntA might participate in intestinal regeneration and may be connected with cell proliferation, apoptosis in different intestinal layers. This research could establish a basis for further examination of WntA functions in A. japonicus and Wnt genes in other echinoderms.

Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

Nervous system development of the sea cucumber Stichopus japonicus.

The nervous system development of the sea cucumber Stichopus japonicus was investigated to explore the development of the bilateral larval nervous system into the pentaradial adult form typical of echinoderms. The first nerve cells were detected in the apical region of epidermis in the late gastrula. In the auricularia larvae, nerve tracts were seen along the ciliary band. There was a pair of bilateral apical ganglia consisted of serotonergic nerve cells lined along the ciliary bands. During the transition to the doliolaria larvae, the nerve tracts rearranged together with the ciliary bands, but they were not segmented and remained continuous. The doliolaria larvae possessed nerves along the ciliary rings but strongly retained the features of auricularia larvae nerve pattern. The adult nervous system began to develop inside the doliolaria larvae before the larval nervous system disappears. None of the larval nervous system was observed to be incorporated into the adult nervous system with immunohistochemistry. Since S. japonicus are known to possess an ancestral mode of development for echinoderms, these results suggest that the larval nervous system and the adult nervous system were probably formed independently in the last common ancestor of echinoderms.