Shigen Ye

Comparison of cells free in coelomic and water-vascular system of sea cucumber, Apostichopus japonicus

The sea cucumber, Apostichopus japonicus possesses a variety of cells populating in both the coelomic (cells in the coelomic are called coelomocytes) and water-vascular system. In this study, we compared cells in these two systems of A. japonicus on total cell number, cell types and surface antigens through monoclonal antibodies against coelomocytes. The results demonstrated that the cell types observed in coelomic also could be found in water-vascular system, but the total cell number and percentages of each type were different. The total number of coelomocytes was 2-3 times of that in water-vascular system. Lymphoid cells were numerically dominant in coelomic system, while spherulocytes with pseudopods in water-vascular system. Results of indirect immunofluorescence assay technique showed that both coelomocytes and cells in water-vascular system could be recognized by the corresponding MAbs, and the distribution of its positive signals was not different. In conclusion, cell types and surface antigens in coelomic and water-vascular system were same, but the total cell number and percentages of each type were different. And further researches are needed on whether there are differences in functions of the different composition.

Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens.

Enhanced immune response and resistance to edwardsiellosis following dietary chitooligosaccharide supplementation in the olive flounder (Paralichthys olivaceus)

This study was conducted to evaluate the effects of dietary chitooligosaccharide (COS) supplementation on peripheral leukocyte count, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, serum lysozyme activity, and immune protection in Paralichthys olivaceus. A total of 300 flounder with an average body weight of 80-100 g were randomly assigned into four dietary groups: (I) basic diet (control), basic diet containing (II) 0.5% COS, and (III) 1% COS, fed continuously for 28 d, and (IV) basic diet containing 1% COS fed in 14 d intervals. Continuous feeding of 0.5% and 1% COS diets for 28 d significantly increased the number of peripheral leukocytes, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, and serum lysozyme activity (P < 0.05 or P < 0.01). After a 10 d Edwardsiella tarda challenge, the immune protection rates in the 0.5% and 1% COS groups were 30% and 60%, respectively. No control fish survived the E. tarda challenge treatment. Most immune indices were slightly lower after removal of COS from the diet for 14 d, but all immune indices were observed to recover after another 14 d of COS supplementation. This study demonstrates that supplementation of a basic diet with COS enhances the non-specific immune response and improves survival rates following infection with E. tarda in P. olivaceus. An optimized interval feeding strategy with diets containing 1% COS may have potential applications in the prevention of disease in aquacultured P. olivaceus.

Ontogenesis of coelomocytes in sea cucumber (Apostichopus japonicus) studied with probes of monoclonal antibody

Monoclonal antibodies (MAbs) specifically against coelomocytes of Apostichopus japonicus were employed to study the ontogenesis of coelomocytes by indirect immunofluorescence assay technique (IIFAT). Different developmental stages were identified by histochemical staining method. Stages including blastula, gastrula, auricularia (small-auricular larvae, mid-auricular larvae and big-auricular larvae), doliolaria, pentactula and juvenile were examined. The positive reactions with both MAb1C2 against all the types of coelomocytes and MAb3F6 specific to spherulocytes, were observed firstly at the blastula stage of the embryos. The positive reaction with MAb1E2 against lymphoid cells was observed from the big-auricular larvae, which indicated that lymphoid cells may not be progenitor cells or stem cells for A. japonicus. An increase of fluorescence intensity for each cell may imply a possible enhancement of the innate defensive mechanism as the embryogenesis progress.

Mitochondrial genome sequencing and analysis of scuticociliates (Uronema marinum) isolated from Takifugu rubripes

Abstract Scuticociliates are dangerous parasitic pathogens for in worldwide mariculture. Scuticociliates cause high mortality to marine fish. After an outbreak of scuticociliatosis in Takifugu rubripes in Liaoning Province, northern China, Uronema marinum, a scuticociliate, was identified. In this study, using Illumina MiSeq next-generation sequencing, the mitochondrial genome of U. marinum was assembled and analysed phylogenetically using mitochondrial genomes of other scuticociliates. The complete U. marinum mitochondrial genome was 39,845 bp; it contained two rRNAs, six tRNAs, and 39 protein-coding genes (PCGs). From the 39 PCGs, 15 PCGs were located on the heavy strand, and 24 PCGs on the light strand of U. marinum mitogenome. The phylogenetic tree showed that there were two main clades, Oligohymenophorea and Spirotrichea. Nine ciliate species were clustered together within Oligohymenophorea; Uronema marinum was a separate cluster sharing a relatively close ancestry with Hymenostomatida. The results of this study will help advance the systematics, and studies of evolution and molecular epidemiology of scuticociliates.

Localization and characterization of hematopoietic tissues in adult sea cucumber, Apostichopus japonicus

Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus.

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

&NA; 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. HighlightsAll the coelomocytes were recovered at 35d after evisceration.Regeneration stage of coelomocytes was divided into two periods.Sharp increase period might be due to coelomocyte migration from the water vascular system of A. japonicus.