Novel lncRNA lncRNA001074 participates in the low salinity-induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis.

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3'-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.

miR-10 involved in salinity-induced stress responses and targets TBC1D5 in the sea cucumber, Apostichopus japonicas.

The sea cucumber is an economically important aquaculture species in China, where it encounter hypo-saline conditions caused by freshwater outflow from rivers and rainfall. MicroRNAs (miRNA) are small noncoding RNAs of about 22 nucleotides, which are crucial regulators of gene expression at the post-transcriptional level and are involved in a variety of physiological and pathophysiological processes. miR-10 is differentially expressed in salinity acclimation, and has a seed-region match with TBC1D5. The expression profiles of miR-10 and TBC1D5 indicate that miR-10 negatively regulates the expression of TBC1D5 in coelomocytes and sea cucumbers with a miR-10 agomir or antagomir. During salinity acclimation, up-regulation of miR-10 was induced after transfection in coelomocytes with a miR-10 inhibitor, while down-regulation of TBC1D5 was induced. The miR-10 expression maximum in coelomocytes appeared at 48 h post-transfection with a miR-10 inhibitor, was later than that of in sea cucumbers, which appeared 24 h after miR-10 antagomir injection. There was no longer a negative relationship between miR-10 and TBC1D5 expression in coelomocytes and sea cucumbers with miR-10 mimics or agomir during salinity acclimation. The miR-10 antagomir or agomir only affected sodium and NKA enzyme activities, and has little effect on other chloride and potassium ions. Our results demonstrate miR-10 directly regulates TBC1D5 by targeting its 3'-UTR, and that miR-10 suppression substantially increases TBC1D5 mRNA levels in vivo and in vitro. Furthermore, miR-10 and TBC1D5 fluctuating expression patterns after treatment with a miR-10 inhibitor or mimics during salinity acclimation may indicate that they are required for adaptation to salinity stress caused by environmental change. Especially, the miR-10 up-regulation in coelomocytes with miR-10 inhibitor during salinity acclimation indicated that they are required for adaptation to salinity stress caused by environmental change. We propose that miR-10 participates in a regulatory circuit that allows for rapid gene program transitions in response to osmotic stress.

Salinity stress-induced differentially expressed miRNAs and target genes in sea cucumbers Apostichopus japonicus.

Environmental salinity is an important abiotic factor influencing normal physiological functions and productive performance in the sea cucumber Apostichopus japonicus. It is therefore important to understand how changes in salinity affect sea cucumbers in the face of global climate change. In this study, we investigated the responses to salinity stress in sea cucumbers using mRNA and miRNA sequencing. The regulatory network of mRNAs and miRNAs involved in salinity stress was examined, and the metabolic pathways enriched for differentially expressed miRNAs and target mRNAs were identified. The top 20 pathways were involved in carbohydrate metabolism, fatty acid metabolism, degradation, and elongation, amino acid metabolism, genetic information processing, metabolism of cofactors and vitamins, transport and catabolism, and environmental information processing. A total of 22 miRNAs showed differential expression during salinity acclimation. The predicted 134 target genes were enriched in functions consistent with the results of gene enrichment based on transcriptome analysis. These results suggested that sea cucumbers deal with salinity stress via changes in amino acid metabolism, ion channels, transporters, and aquaporins, under stimulation by environmental signals, and that this process requires energy from carbohydrate and fatty acid metabolism. Salinity challenge also induced miRNA expression. These results provide a valuable genomic resource that extends our understanding of the unique biological characteristics of this economically important species under conditions of salinity stress.

Establishment of lysozyme gene RNA interference systemand its involvement in salinity tolerance in sea cucumber (Apostichopus japonicus).

The lysozyme gene was silenced using RNA interference (RNAi) to analyze the function of lysozyme in sea cucumber under salt stress. The interfering efficiency of four lysozyme RNAi oligos ranged from 0.55 to 0.70. From the four oligos, p-miR-L245 was used for further interfering experiments because it had the best silencing efficiency. Peristomial film injection of p-miR-L245 (10 µg) was used for further interfering experiments. The lowest gene expression, determined by RT-PCR assay, in muscle, coelomic fluid, and parapodium occurred 48 h after p-miR-L245 injection, while that of body wall and tube foot was 96 h and 24 h, respectively. Lysozyme activity in muscle and body wall was significantly lower than the controls. The lowest lysozyme activity in muscle, body wall and parapodium, was found at 48, 72, and 48 h, respectively, which was consistent with the transcription expression of lysozyme. The lowest point of lysozyme activity was at 96 h in coelomic fluid and tube foot, which was laid behind lysozyme expression in transcription level. The expression profile of the lysozyme transcription level and lysozyme activity in the body wall and tube foot increased at 12 h after p-miR-L245 injection before the interference effect appeared. NKA gene expression was expressed at a high level in the positive control (PC) and negative control (NC) groups at 12, 24, and 48 h, while NKA was expressed at low levels in the lysozyme RNAi injection group at 12 and 24 h. The level of NKA gene expression recovered to the level of the PC and NC group at 48, 72, and 96 h after the lysozyme RNAi injection. NKCC1 gene expression was high in the PC and NC groups at 96 h, while the NKCC1 was expressed at a low level 96 h after lysozyme RNAi injection. The results suggest that lysozyme decay involves NKA and NKCC1 gene expression under salinity 18 psµ. The K+ and Cl- concentration after lysozyme RNAi injection was lower than in the PC and NC group.

Effect of acute salinity stress on ion homeostasis, Na+/K+-ATPase and histological structure in sea cucumber Apostichopus japonicus.

BACKGROUND: Sea cucumbers (Apostichopus japonicus) are an imperiled fauna exposed to a variety of environmental condition such as salinity and studies are urgently needed to assess their effects to guide aquaculture efforts. The effects of acute salinity stress on coelomic fluid osmotic pressure, ion concentrations, the activity of Na+/K+-ATPase in respiratory trees and the histological variations were measured to evaluate the salinity tolerance of sea cucumbers. RESULTS: Significant correlations in osmotic pressure were observed between coelomic fluid and ambient environmental salinity. In coelomic fluid, Na+ concentration was observed fluctuated during salinity 18 psu and the inflection point presented at the 6 h. The Na+/K+-ATPase activity in respiratory trees indicated the "U-shaped" fluctuant change and the change trend was opposite with the Na+ concentration. The ions (K+, Cl-) concentration decreased and showed the same tendency at salinity 40 psu with salinity 18 psu. The total coelomocytes counts and phagocytosis of coelomic fluid Na+/K+-ATPase activity indicated fluctuating changes under different salinity stress. Histological variation revealed a negative relation between decreasing salt concentration and tissue integrity. Tissue damages were significantly observed in intestines, muscles and tube feet under low salinity environment (18, 23 and 27 psu). The connective tissue in intestines of A. japonicus exposed to 18 and 23 psu damaged and partly separated from the mucosal epithelium. The significant variations occurred in tube feet, which presented the swelling in connective tissue and a fracture in longitudinal muscles under low salinity (18 psu). The morphological change of tube feet showed the shrinkage of connective tissue under high salinity (40 psu). The amount of infusoria in the respiratory trees decreased or even disappeared in salinity treatment groups (18 and 23 psu). CONCLUSION: The results inferred that osmoconformity and ionoregulation were seen in sea cucumbers, which contributed to understand the salinity regulatory mechanisms of A. japonicus under acute salinity stress.