CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus.

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.

Cloning, expression analysis and functional characterization of an interleukin-1 receptor-associated kinase 4 from Apostichopus japonicus.

Interleukin 1 receptor-associated kinase 4 (IRAK4) is a key factor in TLR-mediated host immune function. In this study, an IRAK4 homologue molecule was identified from Apostichopus japonicus (designated as AjIRAK4) by RACE approach. The full-length cDNA of AjIRAK4 was of 2024 bp containing an open reading frame of 1311 bp encoding a 436-amino-acid (aa) residue polyprotein with the typical death domain (10-113aa) and the kinase domain (160-426aa). The mRNA transcripts of AjIRAK4 displayed constitutively expressed in all examined tissues with highest expression in the muscles (7.20-fold increase compared to the coelomocytes). The pathogen Vibrio splendidus challenge and LPS exposure could both significantly up-regulate the mRNA expression of AjIRAK4. Silencing AjIRAK4 in vitro and in vivo could inhibit the expression of TLR members at mRNA and protein levels, suggesting AjIRAK4 was an important component of TLR cascade in sea cucumber. More importantly, knockdown of AjIRAK4 by specific siRNA resulted in the significant promotion of coelomocyte apoptosis by 1.82-fold increase in vitro and 1.95-fold in vivo. Taken together, all our results suggested that AjIRAK4 might be served as coelomocyte apoptosis regulator via TLR cascade.

Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus.

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P < 0.05) inhibition of growth of V. splendidus VS-ABTNL compared with untreated V. splendidus VS-ABTNL with the cocktail being superior to any of the 3 phages used individually. The lytic capability of the 3 phages was subsequently determined with a Spot Assay Technique performed with 4 isolates of V. splendidus, 3 other Vibrio species and 2 environmental isolates. Both PVS-1 and PVS-2 were lytic to all 4 isolates of V. splendidus while PVS-3 only inhibited the growth of 3 of them. V. splendidus VS-ABTNL was more susceptible to phage PVS-2 than the other 2 phages. In an in vivo performance trial, 360 sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 10(6) CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P < 0.05) increased compared with those fed the control diet. However, no significant differences (P > 0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid of the sea cucumbers. The control was injected with 1 ml of sterilized seawater while the untreated group and the test group were injected with the same volume of V. splendidus-ABTNL culture (3 × 10(5) CFU/mL). Then, the test group was injected with 1 ml of the 3 phage cocktail (MOI = 10). After 48 h, the activities of lysozyme, acid phosphatase and superoxide dismutase were elevated in the untreated group while the levels of these enzymes in the test group were similar to the blank control. After 10-day observation, the survival rate of the sea cucumber was 100% for the blank control, 80% for the test group and 20% for the negative control. The overall results of this experiment indicate that phage therapy increased the survival of sea cucumber infected with V. splendidus VS-ABTNL. The above results demonstrate that using phages, especially a combination of different phages, may be a feasible way to control Vibrio infection in the sea cucumber industry.

[Etiology of off-plate syndrome in cultured larval Apostichopus japonicus at attachment stage and analysis of reservoir of pathogens].

OBJECTIVE: To clarify pathogens and sources of the off-plate syndrome at the attachment stage in the larval culture of Apostichopus japonicus, and further to find out effective medicines for this disease. METHODS: Etiological analysis was performed on larvae with typical off-plate syndrome from three larvae culture factories. Suspicious pathogens were used for artificial infection test, and were identified through morphological, physiological and biochemical tests, and 16S rDNA sequence analysis. Quantitative bacterial analysis was done on the culture systems of the three factories, including water sources, rearing water, ordure (in the pond floor), attachments and feeds. Finally, drug-sensitive tests were done against the pathogens. RESULTS: A common dominant bacterium strain was isolated from all ill larvae included in the study. Artificial infection test showed it was the causative pathogen associated with the disease, and the artificially infected sea cucumbers had same syndromes to the naturally ill ones. The bacterium was identified as Vibrio sp. Bacterial quantity of water sources was in the qualified range (<50 cfu/mL), while out of the standard range in others (> 1 x 10(5) cfu/mL). The sources of the pathogen were complicated, since pathogens were discovered in the water sources, rearing water, ordure, attachments and feeds. However, the density of causative bacteria was the highest in the feeds, middle in the attachments, and lowest in the water sources. Twelve antibiotics could inhibit growth of the pathogens. CONCLUSION: The possible pathogen for off-plate syndrome was Vibrio sp. Feeds may be the main source of the pathogen. Twelve antibiotics besides nalidixic acid could be applied for disease prevention and treatment of Apostichopus japonicus.