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Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the digestive tract. From the mesentery, a rudiment grows and gives rise to a new intestine within a few weeks. In the last decades, the cellular events that occur during intestinal regeneration have been characterized, including apoptosis, cell proliferation, and muscle cell dedifferentiation. Nevertheless, their contribution to the formation and early growth of the rudiment is still unknown. Furthermore, these cellular events' relationship and potential interdependence remain a mystery. Using modulators to inhibit apoptosis and cell proliferation, we tested whether rudiment growth or other regenerative cellular events like muscle cell dedifferentiation were affected. We found that inhibition of apoptosis by zVAD and cell proliferation by aphidicolin and mitomycin did not affect the overall size of the rudiment seven days post-evisceration (7-dpe). Interestingly, animals treated with aphidicolin showed higher levels of muscle cell dedifferentiation in the distal mesentery, which could act as a compensatory mechanism. On the other hand, inhibition of apoptosis led to a decrease in cell proliferation in the rudiment and a delay in the spatiotemporal progression of muscle cell dedifferentiation throughout the rudiment-mesentery structure. Our findings suggest that neither apoptosis nor cell proliferation significantly contributes to early rudiment growth during intestinal regeneration in the sea cucumber. Nevertheless, apoptosis may play an essential role in modulating cell proliferation in the rudiment (a process known as apoptosis-induced proliferation) and the timing for the progression of muscle cell dedifferentiation. These findings provide new insights into the role and relationship of cellular events during intestinal regeneration in an emerging regeneration model. [Display omitted] • Neither apoptosis nor cell proliferation play a critical role in rudiment formation. • Apoptosis-induced proliferation occurs during holothurian intestinal regeneration. • Apoptosis regulates the spatiotemporal progression of muscle cell dedifferentiation. • Muscle cell dedifferentiation acts as a compensatory mechanism during regeneration. [ABSTRACT FROM AUTHOR]

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus , named AjLysM-1 , using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens , and Shewanella baltica , implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms. • A novel LysM-containing gene in Apostichopus japonicus , AjLysM-1 , were uncovered. • AjLysM-1 is an intracellular non-secreted protein with pathogen-recognition abilities. • AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. • AjLysM-1 was proven to be interacted with tyrosine protein kinase and filamin-B. [ABSTRACT FROM AUTHOR]

Pacifastin family proteins play a crucial role in regulating innate immune responses such as phagocytosis in invertebrates. However, the function of the Ajpacifastin-like counterpart in the sea cucumber Apostichopus japonicus remains elusive. In this study, the pacifastin gene of A. japonicus was cloned, characterized and named Ajpacifastin-like. The open reading frame of Ajpacifastin-like is 1497 bp in length and encodes a polypeptide containing 498 amino acid residues. Structural analysis revealed that the protein encoded by Ajpacifastin-like contains two pacifastin light chain domains (amino acids 287–322 and amino acids 376–407). Real-time reverse transcriptase PCR showed that Ajpacifastin-like mRNA is ubiquitously expressed in all tissues examined, with the highest expression in muscle. Ajpacifastin-like mRNA expression was significantly upregulated to 3.27-fold after challenge with Vibrio splendidus for 24 h. To explore the function of the Ajpacifastin-like protein in the immune response of A. japonicus , dsRNA interference with Ajpacifastin-like expression and with the expression of its postulated target gene was performed. Flow cytometry analysis showed that the rate of phagocytosis by coelomocytes increased to 1.21-fold in individuals treated with specific Ajpacifastin-like siRNA. However, rate of phagocytosis by coelomocytes decreased to 86% in individuals treated with Ajphenoloxidase siRNA. These results show that the Ajpacifastin-like gene is ubiquitously expressed in almost all tissues and that Ajpacifastin-like protein acts as an immunomodulatory factor via phenoloxidase to mediate phagocytosis by coelomocytes in pathogen-challenged A. japonicus. • One pacifastin-like gene was cloned from Apostichopus japonicus. • It ubiquitously expressed in different tissues and responded to pathogen challenge. • It negatively regulate phagocytosis of coelomocyte through phenoloxidase. [ABSTRACT FROM AUTHOR]

Despite being one of the bilaterians, the body plan of echinoderms shifts from bilateral symmetry to five-fold radial, or pentaradial symmetry during embryogenesis or their metamorphosis. While the clarification of the developmental mechanism behind this transition will be a basis for understanding their unique body plan evolution, it is still poorly understood. With this regard, the hydrocoel, a mesodermal coelom formed on the left side of bilateral larva, would be a clue for understanding the mechanism as it is the first pentaradial structure that appears before metamorphosis and develops into the water vascular system of adults. By analyzing the development of a sea cucumber, Apostichopus japonicus , we found that the hydrocoel expresses genes related in muscle and neural formation such as myosin heavy chain , tropomyosin , soxC, and elav , implying that cells of the hydrocoel contributes to muscle and neural structures in the adult. Furthermore, ablation of one of the hydrocoel lobes led to incomplete development of adult pentameral structures. The ablation of primary hydrocoel lobes resulted in the reduction in tentacles and the ablation of secondary hydrocoel lobes resulted in the reduction in water vascular canals and nerve cords. Our findings suggest that the hydrocoel lobes may serve as a potential organizing center for establishing the pentaradial body plan in echinoderms. [Display omitted] • The origin of cells that form adult structures in a sea cucumber was investigated. • The hydrocoel is suggested to supply adult nervous and muscular cells. • Laser-ablation of a hydrocoel lobe causes incomplete formation of pentamery. • The hydrocoel may act in organizing the pentaradial adult structures. [ABSTRACT FROM AUTHOR]

The immune regulatory roles of microRNAs (miRNAs) have recently attracted considerable attention. Bioinformatics prediction revealed that both let-7 and miR-210 provide potential binding sites for the Akt (rac-alpha serine/threonine-protein kinase) gene sequence in the sea cucumber Apostichopus japonicus (termed AjAkt). In this study, we first used a dual-luciferase reporter assay and functional validation techniques to verify the interactions between these two miRNAs (let-7 and miR-210) and AjAkt , and then investigated the functions of the validated miRNA/mRNA pair as part of the innate immune response against Vibrio splendidus infection. We found that AjAkt interacts with miR-210 rather than let-7, and miR-210 negatively regulates the expression of AjAkt. From 8 to 48 h after infection with V. splendidus , opposite trends were observed in the expression levels of miR-210 and Aj Akt (mRNA and protein) in coelomocytes, suggesting that the miR-210/ AjAkt pair is involved in immune regulation during this period after infection. Both AjAkt silencing and miR-210 overexpression enhanced the phagocytic capacity and reduced the infectivity of A. japonicus after pathogen infection, suggesting that the miR-210/ AjAkt pair may regulate the innate immune response of A. japonicus by altering phagocytic capacity. The findings of this study enrich our knowledge of the role of miRNA/mRNA pairs in immune regulation in sea cucumbers and provide insights into the molecular mechanisms of the innate immune response in marine echinoderms. • MiR-210, not let-7, interacts with Akt in Apostichopus japonicus. • MiR-210 overexpression or Akt silencing reduced the prevalence of Vibrio splendidus infection in A. japonicus. • The miR-210/ AjAkt pair regulates the innate immune response of A. japonicus by altering phagocytic capacity. [ABSTRACT FROM AUTHOR]

When organisms are exposed to external stimuli, misfolded proteins accumulate continuously, resulting in endoplasmic reticulum (ER) stress. Autophagy is of great significance for eliminating aggregated proteins and maintaining cellular homeostasis. However, the molecular mechanism of activating autophagy in response to ER stress in sea cucumber is remain unclear. In the current study, we demonstrated that the pathogen Vibrio splendidus can cause ER stress in Apostichopus japonicus coelomocytes and identified a Ca2+ binding partner calreticulin (designated as AjCRT), which increased with the occurrence of ER stress. The nucleotide sequence analysis showed that the open reading frame of AjCRT was 1242 bp and encoded a 413-amino-acid residue polyprotein with calreticulin domains. The spatial expression analysis revealed that AjCRT was ubiquitously expressed in all examined tissues with large magnitude in the coelomocytes and was minimally expressed in muscle. Furthermore, silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus and resulted in the significant reduction of coelomocyte autophagy. These findings indicate a calreticulin-based mechanism that positively regulates autophagy in response to ER stress induced by pathogen infection. The results will provide a basis for understanding the way of host alleviating ER stress through autophagy, and pharmacological approaches may have potential for managing ER stress induced by pathogen and related cellular disorders. • Complete ORF sequence of calreticulin was identified in Apostichopus japonicus. • Vibrio splendidus can cause endoplasmic reticulum stress in sea cucumber coelomocytes. • Apostichopus japonicus calreticulin (designated as AjCRT) was increased with the occurrence of endoplasmic reticulum stress. • Silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus. • Silencing AjCRT in vivo resulted in the significant reduction of coelomocyte autophagy. [ABSTRACT FROM AUTHOR]

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes. • A phosphomevalonate kinase (PMK) gene was identified in sea cucumber Apostichopus japonicus. • AjPMK is involved in sea cucumber saponin biosynthesis. • AjPMK might be regulated by multiple immune processes. [ABSTRACT FROM AUTHOR]

As an important aquaculture species, improving the immunity of cultured Apostichopus japonicus (A. japonicus) is vital for its health in aquaculture farming. It has been shown that ARA is an important metabolite for A. japonicus infected by Vibrio splendidus. In this study, we aimed to determine the effects of dietary exogenous ARA on healthy sea cucumber cultures, including assessments of immunity, growth, and fatty acid content. Five experimental diets containing 0.01%, 0.29%, 0.46%, 0.70%, and 1.09% ARA were tested. The specific growth rate (SGR) of sea cucumbers did not be significantly affected by exogenous ARA diet groups. The results showed that dietary ARA between 0.49 and 1.09% notably improved the survival rate of sea cucumbers infected by Vibrio splendidus compared with the control group without exogenous ARA. The results also showed the effects of dietary ARA on immune-related genes, enzymes, and oxidation indices; most of the exogenous ARA significantly upregulated the mRNA expression of the genes NFκB, TLR, TLR3, TRAF6, Toll, and MyD88. The activities of ACP, AKP, and lysozyme increased in the 0.49–1.09% ARA groups, especially the dietary 0.49% ARA group. The SOD1 and NOS activities were enhanced by dietary ARA between 0.29 and 0.70%. Compared with the control, the MDA content increased, but the 0.49% ARA-diet group had a lower MDA content. Based on these data, 0.49–0.70% ARA significantly enhanced immunity in cultured A. japonicus. Exogenous 0.49% and 0.70% ARA also elevated the ARA, total PUFA and n-6 PUFA in the body walls. In conclusion, the appropriate exogenous ARA (0.49%–0.70%) in diets could improve immunity and fatty acid content considerably. The results provide basic evidence that ARA can serve a useful immune enhancer for A. japonicus aquaculture. • The dietary ARA (0.49–1.09%) improved the survival rate of sea cucumbers infected by Vibrio splendidus. • The dietary moderate ARA increased the immune-related genes, enzymes, and oxidation indices. • The dietary ARA elevated the ARA and n-6 PUFA in the body walls. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus. • The genome-wide map of m6A circRNAs profiles was sequenced in sea cucumbers. • More than 62% of modified circRNAs harbor only a single m6A peak. • V. splendidus challenge upregulated most of m6A peaks in circRNAs in both SUS-diseased and SUS-resistant groups. • GO analysis indicated circRNAs with dysregulated m6A peaks were both mainly enriched in the adhesion pathway. • More than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels. [ABSTRACT FROM AUTHOR]

Fas-associated protein with death domain (FADD) is a pivotal adaptor protein that functions in mediating cell death, cell cycle regulation, and particular in innate immunity by the main death receptors. In this study, a second FADD gene in sea cucumber Apostichopus japonicus (termed AjFADD-2) was cloned and its potential function in the innate responses was analyzed. The full-length cDNA of AjFADD-2 consists of 2405 bp and contains a 47 bp 5′-untranslated region (UTR), a 1629 bp 3′-UTR, and a 729 bp ORF encoding 242 amino acids. AjFADD-2 possesses two conserved domains of intracellular N-terminal death effector domain and an extracellular C-terminal death domain, which is different from the first cloned FADD gene in A. japonicus that only possesses the death domain. AjFADD-2 was examined in all sampled six tissues and was significantly induced in V. splendidus -challenged sea cucumbers and LPS-exposed coelomocytes. Subcellular localization detection showed that AjFADD-2 was primarily observed in the coelomocyte cytoplasm, and transferred to the nucleus post V. splendidus challenge. Consistently, AjFADD-2 knockdown significantly inhibited apoptosis in V. splendidus -challenged sea cucumbers and LPS-exposed coelomocytes. Taken together, our results provided evidence that AjFADD functioned as a positive regulator of coelomocytes apoptosis in response to pathogen V. splendidus challenge. • A novel FADD homolog was cloned and characterized in sea cucumber Apostichopus japonicus. • AjFADD-2 was mainly distributed in the cytoplasm and transferred into the coelomocytes nucleus post pathogen challenge. • AjFADD-2 knockdown significantly decreased the apoptosis levels in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Improving the immune ability and guiding healthy culture for sea cucumber by purposefully screening the significant differential metabolites when Apostichopus japonicus (A. japonicus) is infected by pathogens is important. In this study, 35 types of significant differential metabolites appeared when A. japonicus were infected by Vibrio splendens (VSI group) compared with the control A. japonicus group (CK group) by using liquid chromatography–mass spectrometry (LC–MS/MS)-based untargeted metabolomics. Based on that finding, the 10 types of key metabolic pathways were analyzed by MetPA. The "arachidonic acid (ARA) metabolism" pathway, which was screened by three elevated biomarkers: ARA, prostaglandin F2α and 2-arachidonoyl glycerol, had an important impact on immune stress in A. japonicus. Due to the similar changes in several metabolites in its metabolic pathway, the ARA metabolic pathway was selected for further study. The activities of ACP, AKP and lysozyme, which are important innate immune-related enzymes, the survival rates of A. japonicus infected with V. splendidus and the relative content of ARA in the body wall detected by GC–MS were all upregulated significantly by exogenous daily 0.60% and 1.09% ARA consumption over a short period of approximately 7 days. These results demonstrated that ARA and its metabolic pathway indeed played important roles in the immunity of A. japonicus infected by the pathogen. The findings also provide novel insights for the effects of metabolites in A. japonicum healthy culture. • The significant differential metabolites of A. japonicus infected by V. splendens were screened. • Liquid chromatography–mass spectrometry (LC–MS/MS)-based untargeted metabolomics was used. • Exogenous ARA elevate the immunity of A. japonicus. • The arachidonic acid (ARA) and its metabolism pathway were important for A. japonicus immunity. [ABSTRACT FROM AUTHOR]

Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine β-sheets, Cys30-Cys67, Cys38-Cys58, Cys53-Cys90, Cys56-Cys66, and Cys81-Cys102 participating in the formation of five pairs of disulfide bonds. In vitro experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 μmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 μmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation. • AjPSPLPs contain a strict CXXCXC motif and eleven conserved Cys residues. • A novel A. japonicus derived protein AjPSPLP-3 is heterogeneously expressed. • AjPSPLP-3 contributes to the HaCaT cells proliferation and migration. • MBP fused to the N-terminal of AjPSPLP-3 enhances the activity. [ABSTRACT FROM AUTHOR]

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (Aj NCOA4) has not been reported until now. In this study, we identified and characterized Aj NCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of Aj NCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between Aj NCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that Aj NCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus , the coelomocytes exhibited a significant increase in Aj NCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant Aj NCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between Aj NCOA4 and Aj Ferritin in coelomocytes. RNA interference-mediated knockdown of Aj NCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of Aj NCOA4 reduced the occurrence of ferritinophagy. These findings suggest that Aj NCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus. • Aj NCOA4 was characterized and validated in Apostichopus japonicus. • The recombinant Aj NCOA4 was expressed in E. coli and purified. • Aj NCOA4 interacted with Aj Ferritin in coelomocytes. • Knockdown of Aj NCOA4 reduced intracellular Fe2+ levels. • Aj NCOA4 promoted ferritinophagy induced by V. splendidus. [ABSTRACT FROM AUTHOR]

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl− concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5′-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress. [Display omitted] • Transcriptomics and metabolomics reveal hypo-osmotic stress effects on sea cucumber. • Coelomic fluid ion concentrations were highly correlated with environment. • Inorganic ions, amino acids, nucleotides, and their derivatives play crucial osmoregulatory roles. • ROS scavenging and glycerophospholipid metabolism were activated to resist oxidative stress. • Acyl-CoA-binding protein activity was inhibited, resulting in blocked energy metabolism. [ABSTRACT FROM AUTHOR]

Bisphenols are emerging endocrine disrupting pollutant, and several studies have reported that they are already ubiquitous in various environmental matrices and intend to deposit in sediment. The primary sources of bisphenols are river and sewage discharge. Sea cucumber (Apostichopus japonicus), a typical deposit feeder, is one of the most important commercial marine species in Aisa. However, the effects of the bisphenol A (BPA) and its analogues bisphenol AF (BPAF) on sea cucumber was unclear. In this study, we carried out field survey in major sea cucumber farming areas in northern China, with the aim of determining which bisphenol analogue is the major bisphenol contamination in this aquaculture area. The results showed that the presence of BPAF was detected in four sampling sites (Dalian, Tangshan, Laizhou, and Longpan). The mean level of BPAF in Laizhou sediment samples was the highest which reached to 9.007 ± 4.702 μ g/kg. Among the seawater samples, the BPAF only have been detected in the samples collected at Longpan. (0.011 ± 0.003 μ g/L). Furthermore, we conducted an experiment to evaluate the single and combined toxicity of BPA and BPAF on sea cucumbers. The concentrations were informed by the findings based on the results of field research. (0.1, 1.0, and 10 μ g/L). After exposure, the body weight gain, and specific growth rate showed no significant changes (P > 0.05). We observed the histological alterations in respiratory tree of treated sea cucumbers including the fusion and detachment of lining epithelial tissue, and increase of lumen space. However, the catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) activity was not significantly changed (P > 0.05). We evaluated the effects of BPA and BPAF through calculating the integrated biomarker response index (IBR), and the results indicated that the toxicity of combined treatment was higher than single treatment. Additionally, BPAF exposure to A. japonicus was more toxic than BPA. [Display omitted] • The presence of bisphenols along the Shandong peninsula was investigated. • The impact of long-term exposure to BPA and BPAF at environmental level on Apostichopus japonicus was explored. • Single and combined treatment of BPA and BPAF caused histological alterations of respiratory tree in A. japonicus. • The immune enzyme activity was not significantly changed after the exposure to the bisphenols. • Adverse effects caused by BPA and BPAF was evaluated through calculating the integrated biomarkers response index. [ABSTRACT FROM AUTHOR]

Ferroptosis, a kind of programmed cell death, is characterized with iron-dependent lipid ROS buildup, which is considered as an important cellular immunity in resisting intracellular bacterial infection in mammalian macrophages. In this process, lipid ROS oxidizes the bacterial biofilm to inhibit intracellular bacteria. However, the function of ferroptosis in invertebrate remains unknown. In this study, the existence of ferroptosis in Apostichopus japonicus coelomocytes was confirmed, and its antibacterial mechanism was investigated. First, our results indicated that the expression of glutathione peroxidase (Aj GPX4) was significantly inhibited by 0.21-fold (p < 0.01) after injecting A. japonicus with the ferroptosis inducer RSL3, and the contents of MDA (3.93-fold, p < 0.01), ferrous iron (1.40-fold, p < 0.01), and lipid ROS (3.10-fold, p < 0.01) were all significantly increased under this condition and simultaneously accompanied with mitochondrial contraction and disappearance of cristae, indicating the existence of ferroptosis in the coelomocytes of A. japonicus. Subsequently, the contents of ferrous iron (1.40-fold, p < 0.05), MDA (2.10-fold, p < 0.01), ROS (1.70-fold, p < 0.01), and lipid ROS (2.50-fold, p < 0.01) were all significantly increased, whereas the mitochondrial membrane potential and GSH/GSSG were markedly decreased by 0.68-fold (p < 0.05) and 0.69-fold (p < 0.01) under Vibrio splendidus (AJ01) infection. This process could be reversed by the iron-chelating agent deferoxamine mesylate, which indicated that AJ01 could induce coelomocytic ferroptosis. Moreover, the results demonstrated that the intracellular AJ01 load was clearly decreased to 0.49-fold (p < 0.05) and 0.06-fold (p < 0.01) after treating coelomocytes with RSL3 and ferrous iron, which indicated that enhanced ferroptosis could inhibit bacterial growth. Finally, subcellular localization demonstrated that ferrous iron efflux protein ferroportin (Aj FPN) and intracellular AJ01 were co-localized in coelomocytes. After Aj FPN interference (0.58-fold, p < 0.01), the signals of ferrous iron and lipid ROS levels in intracellular AJ01 were significantly reduced by 0.38-fold (p < 0.01) and 0.48-fold (p < 0.01), indicating that Aj FPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via Aj FPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection. • Ferroptosis program was conservative in sea cucumber Apostichopus japonicus. • Ferroptosis might play an important role in AJ01 infection. • A. japonicus coelomocytes can introduce ferroptosis-induced oxidative pressure into intracellular AJ01 via Aj FPN to suppress intracellular AJ01 growth during infection. [ABSTRACT FROM AUTHOR]

Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5′-untranslated region (UTR), and a 154 bp 3′-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function. • AjAkirin2 and Aj14-3-3ζ act as positive regulator involved in mediating innate immune responses in A. japonicus. • AjAkirin2 directly interacts with Aj14-3-3ζ and positively regulates the expression of Aj14-3-3ζ in A. japonicus. • AjAkirin2 and Aj14-3-3ζ positively regulates the NF-κB pathway and the expression of inflammatory cytokines in A. japonicus. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are novel functional non-coding RNAs which engaged in many aspects of biological processes. N6-methyladenosine (m6A) as a kind of abundant epitranscriptomic modification in eukaryotes, plays important roles in regulation of gene expression for various physiological functions. Our previous study demonstrated that sea cucumber lncRNAs were differentially expressed during bacterial infection. However, whether the post-transcriptional regulation of lncRNAs influenced by m6A modification in sea cucumbers with different stages of skin ulceration syndrome (SUS) are largely unknown. Here, we generated the genome-wide map of m6A lncRNAs in SUS-diseased and SUS-resistant sea cucumbers for the first time, revealed that m6A levels in lncRNAs were mainly upregulated in SUS-resistant group. Intriguingly, most of the m6A lncRNAs showed a positive correlation between the expression levels and m6A levels based on conjoint analysis, suggesting that m6A modification on a lncRNA may contribute to its RNA stability. Furthermore, the host genes of lncRNAs with dysregulated m6A peaks were enriched in immune pathway. More importantly, methyltransferase METTL3 was required for m6A methylation modification and played positive roles in lncRNA expression. Collectively, this study presents the comprehensive characters of m6A lncRNAs in marine invertebrate. These m6A modified lncRNAs may be served as potential regulators associated with SUS and provide a promising avenue for disease therapy through targeting METTL3. • m6A methylation are enriched within the sea cucumber lncRNAs. • m6A methylation of lncRNA showed differential expressed in the SUS-diseased and SUS-resistant group. • m6A lncRNAs mainly showed a positive correlation between the expression levels and m6A levels. • METTL3 involved in m6A regulated expression of lncRNAs in SUS-resistant sea cucumbers. [ABSTRACT FROM AUTHOR]

Immune cells have many efficient ways to participate in the host immunity, including phagocytosis, which is an important pathway to eliminate pathogens. Only β-integrin-mediated phagocytosis pathways have been confirmed in Apostichopus japonicus. The Src family kinases (SFKs), a class of non-receptor tyrosine kinases plays an important role in the regulation of phagocytic signals in invertebrates. However, the SFK-mediated phagocytic mechanism is largely unknown in A. japonicus. In this study, a novel SFK homologue (AjSrc) with a conservative SH 3 domain, an SH 2 domain, and a tyrosine kinase domain was identified from A. japonicus. Both gene and protein expression of AjSrc and phosphorylation levels increased under Vibrio splendidus challenged, reaching the highest level at 24 h. Knock-down of AjSrc could depress coelomocytes' phagocytosis by 25% compared to the control group. To better understand the mechanism of AjSrc-mediated phagocytosis, focal adhesion kinase (FAK) was identified by a Co-immunoprecipitation experiment to be verified as an interactive protein of AjSrc. The phagocytosis rates of coelomocytes were decreased by 33% and 37% in AjFAK and AjSrc + AjFAK interference groups compared with the control group, respectively. Furthermore, the phosphorylation level of AjFAK was increased and reached the maximum level at 24 h post V. splendidus infection, as the same as that of AjSrc. Our results suggested that AjSrc could mediate V. splendidus- induced coelomocytes' phagocytosis via interacting with AjFAK and co-phosphorylation. This study enriched the mechanism of phagocytosis in echinoderm and provided the new theoretical foundation for disease control of sea cucumber. • A novel SFK homologue (AjSrc) is identified from Apostichopus japonicus. • AjSrc promotes the coelomocytes phagocytosis in Vibrio splendidus challenged A. japonicus. • AjSrc participates in Src/FAK signaling pathway regulating coelomocytes phagocytosis via interacting with AjFAK. [ABSTRACT FROM AUTHOR]

In the present study, a potential probiotic Bacillus subtilis D1-2 with antibacterial activity was isolated from the gut of Apostichopus japonicus. The purpose of this experiment was to assess the effect of B. subtilis D1-2 at different concentrations (C: 0 CFU/g, BL: 105 CFU/g, BM: 107 CFU/g and BH: 109 CFU/g) on the growth performance, digestive enzyme activity, immune ability and intestinal flora of A. japonicus. After the 56-day feeding trial, the final body weight and weight gain rate of juvenile sea cucumber A. japonicus fed B. subtilis D1-2 were significantly increased, especially in the BM group. Additionally, the lipase activity of the intestine was significantly increased in the BM and BH groups. Enhanced immunity was also found in sea cucumbers supplemented with B. subtilis D1-2. Alpha diversity indices showed that the B. subtilis D1-2-supplemented groups had higher intestinal microbial richness and diversity than the control group. The beta diversity analysis indicated that the bacterial communities in the B. subtilis D1-2-supplemented groups were quite similar but different from the bacterial communities in the control group. Dietary supplementation with B. subtilis D1-2 increased the relative abundance of some potential probiotic-related genera, including Lactobacillus, Clostridium , Lactococcus , Bifidobacterium and Streptococcus. In conclusion, dietary addition of B. subtilis D1-2 could effectively promote the growth of A. japonicus , improve its digestion and immunity capacity to a certain extent, and actively regulate the intestinal microflora of A. japonicus. • Bacillus subtilis D1-2 was isolated from the gut of Apostichopus japonicus. • B. subtilis D1-2 had positive effects on the growth, immunity and intestinal microflora of A. japonicus. • The optimum dietary supplementation of B. subtilis D1-2 for A. japonicus was 107 CFU/g. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and β-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, β-1,3-glucan, Vibrio splendidus , Pseudoalteromonas nigrifaciens , Shewanella baltica , Bacillus cereus , Escherichia coli , and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway. • AjMMP-16 was able to be induced by diverse microbial polysaccharides. • AjMMP-16 might be associated with MyD88-dependent TLR signaling pathway. • AjMMP-16 had broad-spectrum pathogen recognition ability. [ABSTRACT FROM AUTHOR]

Transglutaminases (TGases) are widely known to play critical roles in innate immunity, in particular, TGase2, which involves in autophagy process to help degrade protein aggregates under stressful conditions in mammals. Nevertheless, the function of the TGase2 counterpart whether involves in invertebrate autophagy is largely unknown. In this study, a novel TGase2-like homologous gene from the sea cucumber Apostichopus japonicus (named as AjTGase2-like) was cloned using RACE technology and its biological functions were also investigated. The AjTGase2-like gene encoded a peptide of 750 amino acids with the representative domains of Transglut_N domain, TGc domain, and two Transglut_C domains, which exhibited highly conservative with vertebrate TGase2. Multiple sequence alignments and phylogenetic analysis both supported that Aj TGase2-like belonged to a new member of TGase2 subfamily. AjTGase2-like was pervasively expressed in all examined tissues, with the largest transcription in muscle, followed by respiratory trees, and intestine. After immersion infection with Vibrio splendidus , the mRNA and protein levels of Aj TGase 2-like were both significantly induced and reached the highest levels at 24 h, indicating AjTGase2-like plays a key role in immune response. Further functional analysis showed that the ubiquitinated protein level was significantly increased by 1.65-fold (p < 0.01) after silencing of AjTGase2-like , and the protein levels of Aj LC3-II/I and Aj Beclin1 were both obviously decreased by 0.49-fold (p < 0.01) and 0.64-fold (p < 0.01) at the same time, while the authophagy receptor of Aj p62 was signally up-regulated by 1.40-fold (p < 0.01) under same condition. Moreover, the immunofluorescence signals of Aj LC3 and Aj p62 were consistent with their protein levels, suggesting knockdown of AjTGase2-like causes a blockage in autophagy. More importantly, the Aj LC3 positive signal was not increased after adding with chloroquine in the case of AjTGase2-like interference, indicating AjTGase 2-like might play pivotal role in the early step of autophagosome formation. Besides, our results showed that the fluorescence signal of Aj TGase2-like was largely co-localized with Aj p62 around the cytoplasm in vivo , and r Aj p62 could directly combine with r Aj TGase2-like in vitro , indicating Aj TGase2-like interacts with Aj p62 during autophagy. Overall, our findings supported that Aj TGase2-like served as a positive regulator in sea cucumber authophay. • A novel gene of transglutaminase 2-like was identified in Apostichopus japonicus. • AjTGase2-like was ubiquitously expressed in all examined tissues with the highest level in muscle. • AjTGase2-like was involved in mediating sea cucumber innate immune response and coelomocytes autophagy. • AjTGase 2-like might play important role in the early step of autophagosome formation. [ABSTRACT FROM AUTHOR]

Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/β-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of β-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/β-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration. [Display omitted] • Wnt target genes are differentially expressed during intestinal regeneration. • DsiRNA-targeting β-catenin was tested in gut rudiment explants. • Dedifferentiation and apoptosis are unaffected by β-catenin DsiRNA. • β-catenin DsiRNA affects cell proliferation during intestinal regeneration. [ABSTRACT FROM AUTHOR]

Echinoderms are marine deuterostomes with fascinating adaptation features such as aestivation and organ regeneration. However, post-transcriptional gene regulation by microRNAs (miRNAs) underlying these features are largely unexplored. Here, using homology-based and de novo approaches supported by expression data, we provided a comprehensive annotation of miRNA genes in the sea cucumber Apostichopus japonicus. By linkage and phylogenic analyses, we characterized miRNA genomic organization, evolutionary history and expression regulation. The results showed that sea cucumbers evolved a large number of new miRNAs, which tended to form polycistronic clusters via tandem duplication that had been especially active in the echinoderms. Most new miRNAs were weakly expressed, but miRNA clustering increased the expression level of clustered new miRNAs. The most abundantly expressed new miRNAs were organized in a single tandem cluster (cluster n2), which was activated during aestivation and intestine regeneration. Overall, our analyses suggest that clustering of miRNAs is important for their evolutionary origin, expression control, and functional cooperation. • Evolution of sea cucumber miRNAs is a dynamic and active process, which has given rise to numerous new miRNA families. • New miRNAs are enriched in the tube feet or the regenerating intestine and involved in regulation of these specific organs. • The key new miRNAs for regulation of aestivation and regeneration are organized in a single tandem cluster (cluster n2). • miRNA clustering increased the expression level of new miRNAs, which might enhance their functionality. • miRNAs in cluster n2 may collaboratively activate Wnt signaling by suppressing different inhibitors of the Wnt pathway. [ABSTRACT FROM AUTHOR]

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named Aj AIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of Aj AIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of Aj AIF - 1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. Aj AIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, Aj AIF-1 and Aj importin β were mainly co-localized around the nucleus in vivo and silencing Aj importin β significantly inhibited the nuclear translocation of Aj AIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of Aj AIF-1 will likely mediate coelomocyte apoptosis through an importin β-dependent pathway in sea cucumber. • Apoptosis-inducing factor 1 was identified from Apostichopus japonicus. • The mRNA expression of Aj AIF-1 was significantly increased after Vibrio splendidus challenge. • Silencing of Aj AIF - 1 was found to significantly inhibit coelomocyte apoptosis. • Aj importin β significantly inhibited the nuclear translocation of Aj AIF-1 and suppressed coelomocyte apoptosis. [ABSTRACT FROM AUTHOR]

Ready-to-eat sea cucumbers (RSC) are prone to autolysis when stored at room temperature. The objective of this study was to compare the effects of high pressure steam sterilization (HPSS) and high temperature boiling (HTB) on the biochemical, physiochemical, and structural changes of RSC stored at 25°C. After undergoing thermal treatment by HPSS and HTB, the shelf life of RSC was significantly extended to 35 and 63 days, respectively, compared to the control (CT) group. The results from TVB-N, microbial, and pH measurements indicated that both HPSS and HTB effectively maintained the quality of RSC by decreasing protein breakdown and lactic acid accumulation through glucose decomposition, thereby inhibiting microbial growth. The HPSS group showed a significant increase in soluble protein content during storage, reaching a final value of 25.48±0.36 mg/100 g on day 35, while the HTB group recorded a respective value of 12.97±0.03 mg/100 g. Furthermore, Van Gieson's staining and scanning electron microscope images revealed a tight arrangement and structural integrity of collagen fibers in the RSC of the HTB group on day 35. These findings suggested that HTB treatment was more effective in extending the shelf life of RSC and improving RSC processing compared to HPSS. [Display omitted] • The quality of RSC was minimally affected by HTB treatment. • Thermal treatments were found to prolong the shelf life of RSC. • HTB was effective in inhibiting the growth and reproduction of microorganisms in RSC. • HTB inhibited the reduction of RSC freshness and degradation of collagen fibers. [ABSTRACT FROM AUTHOR]

CO 2 -induced ocean acidification and warming pose ecological threats to marine life, especially calcifying species such as echinoderms, who rely on biomineralization for skeleton formation. However, previous studies on echinoderm calcification amid climate change had a strong bias towards heavily calcified echinoderms, with little research on lightly calcified ones, such as sea cucumbers. Here, we analyzed the embryo-larval development and their biomineralization–related gene expression of a lightly calcified echinoderm, the sea cucumber (Apostichopus japonicus), under experimental seawater acidification (OA) and/or warming (OW). Results showed that OA (– 0.37 units) delayed development and decreased body size (8.58–56.25 % and 0.36–19.66 % decreases in stage duration and body length, respectively), whereas OW (+3.1 °C) accelerated development and increased body size (33.99–55.28 % increase in stage duration and 2.44–14.41 % enlargement in body length). OW buffered the negative effects of OA on the development timing and body size of A. japonicus. Additionally, no target genes were expressed in the blastula stage, and only two biomineralization genes (colp3α , cyp2) and five TFs (erg , tgif , foxN2/3 , gata1/2/3, and tbr) were expressed throughout the embryo-larval development. Our findings suggest that the low calcification in A. japonicus larvae may be caused by biomineralization genes contraction, and low expression of those genes. Furthermore, this study indicated that seawater acidification and warming affect expression of biomineralization-related genes, and had an effect on body size and development rate during the embryo-larval stage in sea cucumbers. Our study is a first step toward a better understanding of the complexity of high p CO 2 on calcification and helpful for revealing the adaptive strategy of less-calcified echinoderms amid climate change. [Display omitted] • A skeletogenic GRN in early development of holothurians was speculated. • Ocean acidification and warming affect biomineralization-related genes expression. • Acidification delayed development and shortened body length. • Warming mitigated negative effects of acidification. [ABSTRACT FROM AUTHOR]

Sulfamethoxazole (SMZ) is a frequently detected antibiotic in the environment, and there is a growing concern about its potential toxic effects on aquatic organisms. sea cucumber (Apostichopus japonicas) is a benthic invertebrate whose gut acts as a primary immune defense and serves critical protective barrier. In this study, growth performance, histology, gut microbiota, and metabolomics analyses were performed to investigate the toxic response in the intestine of sea cucumber effects caused by SMZ stress for 56 d by evaluating with different concentrations of SMZ (0, 1.2×10−3, and 1.2 mg/L). The weight gain rate of sea cucumbers under SMZ stress showed significant decrease, indicating that the growth of sea cucumbers was hindered. Analysis of the intestinal morphological features indicated that SMZ stimulation resulted in atrophy of the sea cucumber gut. In the 1.2×10−3 mg/L concentration, the thickness of muscle and mucosal layers was reduced by 12.40% and 21.39%, while in the 1.2 mg/L concentration, the reductions were 35.08% and 26.98%. The abundance and diversity of sea cucumber intestinal bacteria decreased significantly (P < 0.05) under the influence of SMZ. Notably, the intestinal bacteria of sea cucumber became homogenized with the increase in SMZ concentration, and the relative abundance of Ralstonia reached 81.64% under the stress of 1.2 mg/L concentration. The SMZ stress significantly impacted host metabolism and disrupted balance, particularly in L-threonine, L-tyrosine, neuronic acid, piperine, and docosapentaenoic acid. SMZ leads to dysregulation of metabolites, resulting in growth inhibition and potential inflammatory responses that could adversely affect the normal activities of aquatic organisms. Further metabolic pathway enrichment analyses demonstrated that impaired biosynthesis of unsaturated fatty acids and aminoacyl-tRNA biosynthesis metabolic pathway were major reasons for SMZ stress-induced intestinal bacteria dysbiosis. This research aims to provide some theoretical evidence for the ecological hazard assessment of antibiotics in water. [Display omitted] • The toxic effects of sulfamethoxazole (SMZ) on sea cucumbers were assessed. • Exposure to SMZ inhibits growth. • SMZ exposure significantly alters the gut microecological environment. • The metabolic process was disrupted by SMZ exposure. • Exposure to SMZ induced inflammatory responses in gut cells. [ABSTRACT FROM AUTHOR]

The sea cucumber Apostichopus japonicus is dioecious, with seasonal reproduction. G protein-coupled receptor (GPCR)-mediated signaling systems might play critical roles in the reproductive control of A. japonicus. Here, we classified GPCR from the genome in silico and used transcriptomic analyses to further mine those that function in gonadal-development control. Totally, 487 GPCRs were predicted from A. japonicus , and 183 of these were further annotated to molecular pathways. Transcriptome analysis revealed 327 GPCRs expressed in gonads, and these were classified into four families and 19 subfamilies. Three pathways were apparently associated with reproduction, including neuroactive ligand-receptor interaction, the mTOR and Wnt signaling pathways. Seven and eight ovary- and testis-specific GPCRs were filtered, and the gene expression profiles were determined in multiple tissues and gonads at different developmental stages by qPCR. These results provide new insights into the discovery of GPCR-mediated signaling control in sea cucumber reproduction, especially in gonadal development control. • 487 G protein-coupled receptors are predicted and classified from Apostichopus japonicus. • A variety of G protein-coupled receptors are detected to be expressed in the gonads of Apostichopus japonicus. • Gonad-specific G protein-coupled receptors have been screened out. [ABSTRACT FROM AUTHOR]

The influence of ocean acidification (OA) is particularly significant on calcifying organisms. The sea cucumber Apostichopus japonicus is an important cultured calcifying organism in the northern China seas. Little was known about the effects of OA on this economically important species. In this study, individuals from embryo to juveniles stage of A. japonicus , cultured in different levels of acidified seawater, were measured their enzymes activities, including five metabolic enzymes and three immune enzymes. The activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) was significantly lower in the severely acid group (pH 7.1), while the content of lactate dehydrogenase (LDH) was significantly higher. Superoxide dismutase (SOD) and catalase (CAT) were significantly lower in the severely acid group. The multivariate statistical results showed that the significant difference of enzyme assemblage existed among three experimental groups. This study indicated that OA could reduce the biomineralization capacity, influence the anaerobic metabolism and severely affect the immune process of A. japonicas. More researches are needed in the future to reveal the mechanisms of enzyme regulation and expression of A. japonicas underlying mixture environmental stress. • The long-time impact of ocean acidification on enzyme activity of sea cucumbers were studied. • The significant difference of enzyme assemblage existed among three experimental groups by the multivariate statistical results. • The acidic environment has a great effect on immune process by influencing the elimination of reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

Probiotics play vital roles in controlling diseases, enhancing specific and non-specific immunity and stimulating growth in the aquaculture industry. However, the effect of fermentation of feed by probiotics on the immune ability of sea cucumber has not been reported to date. Here, three candidate probiotic strains (Bacillus species) were isolated from the culture seawater and sediment of sea cucumber, and fishmeal and scallop mantle fermented by the candidate probiotic strains were used to feed sea cucumber. The results showed that the free amino acid and small peptide contents of the fishmeal and scallop mantle were significantly increased after fermentation for 72 h. However, the weight gain (WG) and specific growth rate (SGR) of sea cucumber showed no significant differences among the fermented fishmeal, fermented scallop mantle and control groups. Scallop mantle fermented by the three candidate probiotics could increase the coelomocyte number and respiratory burst activity. The immune-related enzymatic activity was increased after consuming the fermented fishmeal and scallop mantle, while the activity of antioxidant enzymes was reduced. The expression levels of immune- and antioxidant-related genes were changed after consuming the fermented fishmeal and scallop mantle. Taken together, our results suggest that probiotics could increase the immunocompetence of sea cucumber, and fermented scallop mantle might be a potential substitute for fishmeal during feed preparation. Our results lay a foundation for further understanding the relationship between probiotics and the non-specific immunity of sea cucumber. • Three candidate probiotic strains were isolated from the culture seawater and sediment of sea cucumber. • Growth of sea cucumber was no difference after feeding fermented scallop mantle. • Coelomocyte number of sea cucumber increased after dietary fermented scallop mantle. • Enzymatic activity of immune genes changed after dietary fermented scallop mantle. • Expression level of immune genes changed after dietary fermented scallop mantle. [ABSTRACT FROM AUTHOR]

MiR-210 plays a crucial role in cell survival, migration, and regeneration in vertebrates. In our previous work, the expression of miR-210 was considerably induced in diseased Apostichopus japonicus with skin ulcer syndrome (SUS). To further explore the mechanism of miR-210 in regulating the SUS, this study identified E2F transcription factor 3 (E2F3), a candidate target of miR-210, from the sea cucumber A. japonicus via RNA-seq and RACE (designated as AjE2F3). A 1992 bp fragment representing the full-length cDNA of AjE2F3 was obtained, which includes an ORF of 1194 bp encoding a polypeptide of 398 amino acids with a molecular weight of 44.43 kDa. Expression profiling analysis suggested that the expression of AjE2F3 decreased while that of miR-210 increased in Vibrio splendidus -challenged sea cucumber coelomocytes. Dual-luciferase reporter assay revealed that miR-210 targeted AjE2F3 via binding to the 3ʹUTR region from 108 nt to 128 nt. MiR-210 overexpression in cultured coelomocytes repressed AjE2F3 at the mRNA level and reduced cell proliferation in vitro. Consistently, AjE2F3 overexpression significantly promoted coelomocyte proliferation, as assessed by MTT in vitro. Overall, our results indicated that miR-210 can suppress coelomocyte proliferation by targeting AjE2F3 in pathogen-challenged sea cucumbers. ∙ The full-length cDNA of E2F transcription factor 3 was identified in Apostichopus japonicus. ∙ AjE2F3 was decreased in Vibrio splendidus -challenged coelomocytes, which was contrary to the expression of miR-210. ∙ Dual-luciferase reporter assay revealed that miR-210 targeted AjE2F3 via binding 3′UTR region. ∙ MiR-210 overexpression in cultured coelomocytes repressed AjE2F3 on mRNA levels, and reduced cell proliferation in vitro. ∙ Overexpression of AjE2F3 significantly promoted coelomocyte proliferation by MTT assay. [ABSTRACT FROM AUTHOR]

The intestinal microbiota may play important roles in regenerating intestine of the sea cucumber Apostichopus japonicus , the underlying mechanism remains unclear. In the present study, a germ-free sea cucumber model was developed, and the intestinal microbial differentiation of faster and slower regenerating A. japonicus individuals during intestine regeneration was analyzed. The results revealed that depletion of the intestinal microbiota resulted in elevated abundance of the potential key players Flavobacteriaceae and Rhodobacteraceae during intestine regeneration and thus promoted the intestine regeneration rate of A. japonicus. Metagenomic analysis revealed that the increased abundance of Flavobacteriaceae elevated the enrichment of genes associated with carbohydrate utilization, whereas the abundant Rhodobacteraceae-enriched genes were associated with polyhydroxybutyrate production. We identified microbiota abundance as a key driver of microbial community alterations, especially beneficial microbiota members, in the developing intestine of A. japonicus. This study provides new insights into the mechanism of host-microbiota interactions related to organ regeneration. • The depletion of the intestinal microbiota resulted in elevated the abundance of the keystone taxa. • Flavobacteriaceae and Rhodobacterales act as the key players and promote the intestine regeneration rate of A. japonicus. • Flavobacteriaceae elevated the enrichment of genes associated with carbohydrate utilization. • Rhodobacteraceae-enriched genes were associated with polyhydroxybutyrate production. [ABSTRACT FROM AUTHOR]

The inhibitory kappa B kinase (IKK) is a critical regulator for the nuclear factor-κB (NF-κB) pathway. In this study, an IKKβ named as HLIKKβ was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKβ is 4246 bp in size, containing a 132 bp 5′-untranslated region (UTR), a 1783 bp 3′-UTR and a 2331 bp open reading frame (ORF) encoding a protein of 776 amino acids with a deduced molecular weight of 89.66 kDa. HLIKKβ contains a kinase domain (KD) at its N-terminal, a leucine zipper (LZ) and a helix-loop-helix (HLH) motif at its C-terminal. In the KD, a conserved active loop (SXXXS) were identified. The results of luciferase reporter assay and ELISA assay showed that over-expressed HLIKKβ in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1β. When HLIKKβ was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKβ. Moreover, the up-regulation of HLIKKβ mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKβ might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections. • We identified an IKKβ from the tropical sea cucumber Holothuria leucospilota. • The HLIKKβ mRNA was detected in all the examined tissues, organs and cells. • HLIKKβ mRNA was up-regulated by immune challenges in the sea cucumber coelomocytes. • Over-expressed HLIKKβ in HEK293T cells could activate NF-κB signaling pathway. • The anti-apoptotic function of HLIKKβ was found by RNA interference. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus. • WGBS were conducted on the healthy and SUS-infected body wall of Apostichopus japonicus. • A total of 116,522 DMRs were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs. • The integration of methylome and transcriptome revealed that 10,499 DMRs had a negative correlation with 496 DEGs. • The variations of methylation in mTOR and NLK were associated with SUS infection and immune regulation. [ABSTRACT FROM AUTHOR]

Autophagy mediated by mTOR pathway is a particularly important immune defense mechanism in the pathogens infected mammals. However, the role of TOR in echinoderm autophagy is largely unknown. Here, a cDNA encoding TOR protein was cloned and characterized from sea cucumber Apostichopus japonicus (designated as AjTOR) and its biological functions were also investigated. The AjTOR gene encoded a peptide of 2499 amino acids with the representative domains of DUF3385, FAT, FRB, PI3Kc, and FATC, which exhibited highly conservation with vertebrate orthologs. Phylogenetic analysis supported that AjTOR belonged to a new member of TOR family. Moreover, tissues distribution analysis indicated that AjTOR was ubiquitously expressed in all the tested tissues, with the highest transcription in muscle. Vibrio splendidus infection in vivo and LPS challenge in vitro could both significantly down-regulate the mRNA expression of AjTOR. What's more, transmission electron microscopy observations showed that rapamycin treatment resulted in rapid formation of autophagosomes in coelomocytes both at 3 and 6 h, however, injection with mTOR activator of MHY1485 showed an inhibitory effect on autophagosomes formation compared to the control, suggesting blocking the expression of AjTOR could accelerates autophagy signals. Our findings supported that AjTOR served as a negative regulator in sea cucumber authophay. • A novel target of rapamycin homologous gene was identified from Apostichopus japonicus. • AjTOR transcript was ubiquitous expressed in all detected tissues with the highest level in muscle. • Vibrio splendidus infection and LPS challenge could both significantly down-regulate the mRNA expression of AjTOR. • The mTOR inhibitor of rapamycin could markedly induce the formation of autophagosomes in coelomocytes. • Autophagosomes formation was significantly down-regulated by the mTOR activator of MHY1485 treatment. [ABSTRACT FROM AUTHOR]

Winter is a high incidence period of skin ulceration syndrome in the sea cucumber Apostichopus japonicus. Disease control during the overwintering of sea cucumber can help increase yield and reduce losses. The purpose of this study was to study the effect of the low temperature-resistant probiotic Bacillus baekryungensis MS1 on the growth and immune parameters of sea cucumbers and preliminarily investigate the molecular mechanism of the effects. A low temperature-resistant bacterium, B. baekryungensis MS1, was isolated from a sea cucumber pond in winter and used for culture experiments. After 10 days of prefeeding, the experiment was divided into the control group (fed with commercial diet) and the MS1 group (fed with diet containing B. baekryungensis MS1 at 107 cfu g−1) for a total of 60 days. The specific growth rate was measured at the end of the culture period to evaluate the growth performance of the sea cucumber. Samples were taken on days 30 and 60 to determine the immune parameters (including superoxide dismutase activity, catalase activity, alkaline phosphatase activity, acid phosphatase activity, nitric oxide synthetase activity, phagocytosis and respiratory burst), aquaculture water microbiota and gut microbiota of the sea cucumber. Finally, transcriptome sequencing and qRT-PCR verification of the two groups of sea cucumbers were performed to study the mechanism of B. baekryungensis MS1 to improve the immunity of the sea cucumber. The results showed that after 60 days of feeding, B. baekryungensis MS1 significantly improved the growth performance and immune enzyme activity and formed a healthier structure of the gut microbiota in the sea cucumber. The challenge test showed that B. baekryungensis MS1 significantly reduced the mortality of sea cucumbers infected with Vibrio splendidus. Transcriptome and gene expression analysis indicated that B. baekryungensis MS1 activated the ubiquitin-mediated proteolysis pathway and inhibited the mTOR signaling pathway to regulate the immunity of the sea cucumber. In summary, the low temperature-resistant bacterium B. baekryungensis MS1 could be applied for the aquiculture of sea cucumber in winter to improve health status and resist pathogenic bacteria such as V. splendidus. • Bacillus baekryungensis MS1 improved the immune parameters of Apostichopus japonicus. • The gut microbiota of sea cucumbers in MS1 groups differed from that in control groups. • The expression of TLR and UBE2G2 were up-regulated, and Ras , TNFα and SOS were down-regulated.in MS1 groups. • Ubiquitin mediated proteolysis pathway was activated while mTOR signaling pathway was inhibited in MS1 groups. [ABSTRACT FROM AUTHOR]

As a wide distribution molecule, 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) catalyzes the second step in the tyrosine catabolism pathway. This process commonly occurs in all aerobic life forms. The broad distribution of these metabolites suggests that they have an important role in many organisms. A portion of the 4-HPPD homology sequence was also identified in Apostichopus japonicus transcriptome. However, the functional roles of A. japonicus 4-HPPD remain unclear. In the current study, a 4-HPPD homolog was cloned from A. japonicus (designated as AjHPPD). The nucleotide sequence analysis showed that the open reading frame of AjHPPD was 1149 bp and encoded a 382-amino-acid residue polyprotein with glyoxalase_4 (residues 20–133) and glyoxalase (residues 180–335) domains. The spatial expression analysis revealed that AjHPPD was ubiquitously expressed in all examined tissues with large-magnitude in the respiratory tree and was minimally expressed in coelomocytes. Compared with a control group, the significant increase in transcription of AjHPPD mRNA in the Vibrio splendidus -challenged sea cucumber was 2.10-fold (p < 0.01) at 48 h and returned to the normal level at 72 and 96 h. Similarly, compared with a control group, the significant increase in the transcription of AjHPPD mRNA was 3.36-fold (p < 0.01) at 24 h after stimulation with 10 mg mL−1 of LPS. On the one hand, silencing AjHPPD in vitro could inhibit the expression of pentose phosphate pathway (PPP) flux enzyme glucose-6-phosphate dehydrogenase (G6PD) at the mRNA level and prevent the clearance of reactive oxygen species (ROS) in sea cucumbers. On the other hand, interference of AjHPPD by using specific siRNA can result in the significant promotion of coelomocyte apoptosis with a 1.61-fold increase in vitro. AjHPPD negatively regulated ROS levels by modulating tyrosine catabolism on AjG6PD expression and coelomocyte apoptosis in response to pathogen infection. • Complete ORF sequence of 4-hydroxyphenylpyruvate dioxygenase was identified in Apostichopus japonicus. • AjHPPD mRNA transcripts could be induced in Vibrio splendidus challenged sea cucumber or LPS exposed coelomocytes. • Silencing AjHPPD in vitro could inhibit the expression of glucose-6-phosphate dehydrogenase at mRNA level. • Silencing AjHPPD in vitro could prevent the clearance of reactive oxygen species. • AjHPPD knockdown by siRNA could accelerate the coelomocytes apoptosis. [ABSTRACT FROM AUTHOR]

Desiccation is a commonly stressful situation experienced by sea cucumber during transportation without/less water. The present study was conducted to investigate the effects of aerial exposure on the survival, oxidative damage, antioxidant capacity, immune-related response and gene expression of Apostichopus japonicus at different low temperatures. After acclimation, sea cucumbers were randomly divided into 3 groups, which were exposed to 5 °C, 10 °C and 15 °C in the closed laboratory condition, respectively. Each group has three parallel replicates. During the experiment, coelomic fluid and respiratory tree of A. japonicus were sampled at the time points of 0, 3, 6, 12, 24 and 48 h post-desiccation for further analysis. The results showed that the survival rates of sea cucumber significantly decreased as time prolonged, and those of 5 °C at 6–48 h of desiccation were significantly higher than 15 °C. Most oxidative damage parameters (e.g., O 2 − production, MDA, LPO and PC contents) significant increased after 6–12 h of desiccation. Antioxidant enzyme activities and T-AOC in coelomic fluid firstly increased and then decreased during aerial exposure, indicating that sea cucumber could adjust antioxidant defense to reduce the concentrations of ROS and MDA as a strategy for protecting organisms from oxidative damage in the early stage (0–6 h) of desiccation. The relative expression levels of Hsp90 and Hsp70 mRNA in respiratory tree of sea cucumber exhibited similar rise-fall trends with antioxidant parameters, while immune enzyme activities of ACP, AKP, LSZ and T-NOS, and gene expression of TLR, Rel and p105 all significantly decreased as time prolonged. Overall, low temperature postponed the process of ROS formation and the depression of antioxidant and non-specific immune responses of sea cucumber within a certain extent, which implied that it might play a positive role in improvement of desiccation tolerance. This study not only contribute to better understand the adaption mechanisms of A. japonicus to desiccation stress, but also provide valuable information for sea cucumber culture and transportation. • Oxidative stress and immune responses of A. japonicus to desiccation stress were detected at 5, 10 and 15 °C. • Sea cucumber could adjust antioxidant defense to protect from oxidative damage at 0–6 h of desiccation. • Immune-related enzyme activities and genes expression were significantly regulated. • Low temperature contributed to the improvement of desiccation tolerance. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system via specific recognizing peptidoglycan, a unique component of bacterial cell wall. In the present study, a homologous gene encoding PGRP-S was identified and characterized from Apostichopus japonicus and designated as AjPGRP-S. The open reading frame of AjPGRP-S is 756 bp encoding a polypeptide of 251 amino acids (aa) with a signal peptide (1–24 aa) and a typical PGRP domain (37–178 aa). Phylogenetic analysis and sequence alignment revealed that AjPGRP-S is a member of the PGRP-S family. In healthy sea cucumbers, AjPGRP-S was expressed in all examined tissues with the highest distribution in body wall, muscle, and intestine. In Vibrio splendidus -infected sea cucumbers, AjPGRP-S was remarkably induced in coelomocytes. The recombinant AjPGRP-S (rAjPGRP-S) was shown to possess the highly amidase activity in the presence of Zn2+. Moreover, rAjPGRP-S exhibited agglutination abilities and strong bacteriostatic activities against V. splendidus , V. harveyi , V. parahaemolyticus , Staphylococcus aureus , and Micrococcus luteus. Furthermore, the agglutination ability can be enhanced in the presence of Zn2+. In conclusion, our results suggested that AjPGRP-S serves as a pattern recognition molecule involved in the immune response towards various pathogenic infections. • Complete cDNA sequence of S-type PGRP was identified in Apostichopus japonicus. • Vibrio splendidus challenge could significantly induce AjPGRP-S mRNA expression. • RAjPGRP-S was shown to possess the highly amidase activity in the presence of Zn2+. • RAjPGRP-S displayed both the agglutination abilities and bacteriostatic activities against examined bacteria. [ABSTRACT FROM AUTHOR]

As a ubiquitously expressed protein, cyclophilin A (CypA) is involved in a variety of pathological process, including immune suppression, inflammation, cell apoptosis, viral infection and stress response. However, the functional roles of CypA were largely unknown in economic marine animals. In this report, a novel CypA gene from sea cucumber Apostichopus japonicus (designated as AjCypA) was cloned and its function roles in immune responses were explored. The full-length cDNA of AjCypA was 1297 bp containing an open reading frame of 489 bp encoding a putative protein of 162 amino acids (aa). A conserved cyclophilin-like domain (CLD) with PPIase signature was located from 5 to 155 aa sequences in AjCypA, in which five necessary aa residues was totally conserved. In healthy sea cucumbers, AjCypA was expressed in all detected tissues, with highly expressed in muscles and weakly expressed in coelomocytes. AjCypA transcripts was significantly induced 8.08-fold and 5.65-fold in coelomocytes when sea cucumbers challenged with Vibrio splendidus in vivo and LPS in vitro , respectively. The expression pattern is similar with the expression of AjRel in the same condition. Moreover, GST pull-down and immunofluorescence analysis both revealed that AjCypA might be interacted with AjRel. Furthermore, AjCypA knockdown not only inhibited the expression of inflammation cytokines, but also suppressed the translocation of AjRel in nucleus induced by LPS. Taken together, our results suggested that AjCypA play key roles in V. splendidus mediated immune responses via suppressing the nuclear translocation of AjRel activity in sea cucumber. • AjCypA homologue was cloned and characterized in sea cucumbers. • AjCypA was expressed in all detected tissues, with highly expressed in muscles. • AjCypA transcripts showed similar expression profiles to that of AjRel in Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes. • GST pull-down and immunofluorescence analysis both revealed that AjCypA might be interacted with AjRel. • AjCypA knockdown suppressed the translocation of AjRel in nucleus induced by LPS. [ABSTRACT FROM AUTHOR]

Myelocytomatosis viral oncogene (MYC), a multifunctional transcription factor, (TF) exerts various physiological and pathological effects on animals. AjMYC could induce coelomocyte apoptosis in Apostichopus japonicus , but the underlying molecular mechanism remains poorly understood. In this study, the promoter sequence of apoptosis regulator Bcl-2-associated X (Bax) was cloned by genomic walking. The AjBax promoter region spaning 1189 bp, containing several transcription factor binding sites, included four potential E-boxes (−1030 bp to −1019 bp, −785 bp to −774 bp, −570 bp to −559 bp, −100 bp to −89 bp), two P53 binding sites (−439 bp to −430 bp, −845 bp to −836 bp), and one NF-κB site (−191 bp to −182 bp). Transient transfection of EPC cells with 5′–deletion constructs linked to luciferase reporter revealed that the region −1189/+454 contributed importantly to the expression of the AjBax. In addition, the AjBax promoter was induced by LPS, PGN or MAN. The four potential MYC binding sites were cotransfected with AjMYC in EPC cell whether AjMYC could activate AjBax expression as a transcriptional factor. Only P1 (−1189/+454) fragment containing the first MYC binding site transfection increased the luciferase activity by 2.08-fold (p < 0.01) compared with the control. The first MYC binding site −1030/−1019 was essential to induce AjBax transcription. Further functional assay indicated that AjBax was significantly induced by 3.54-fold increase (p < 0.01) after AjMYC overexpression in sea cucumber coelomocytes. All our findings supported that AjMYC could regulate coelomocyte apoptosis by directly targeting AjBax expression in A. japonicus. • Promoter region of Bcl-2-associated X was identified in Apostichopus japonicus. • Luciferase assay indicated the 5′-flanking region −1189/+454 contributed importantly to expression of the AjBax. • The first MYC binding site (−1030 bp to −1019 bp) was crucial to induce AjBax transcription. • Overexpression AjMYC upregulated the expression of AjBax and coelomocytes apoptosis. [ABSTRACT FROM AUTHOR]

Polian vesicles are considered as the site of coelomocyte formation, and play crucial roles in the inflammatory reaction in sea cucumber. After evisceration, coelomocytes and internal organs except polian vesicles are excreted. Our previous study found that the total number of coelomocytes was rapidly recovered at 6 h post-evisceration in sea cucumber Apostichopus japonicus , and this regeneration of coelomocytes might be closely related to polian vesicles. To further investigate the related-gene expression pattern of the polian vesicles at 6 h post-evisceration, the transcriptome analysis of polian vesicles was carried out. A total of 2752 differentially expressed genes (DEGs) were identified, including 1,453 up-regulated genes and 1299 down-regulated genes. Gene Ontology (GO) enrichment showed that most of the DEGs were classified under Regulation of transcription, Regulation of RNA metabolic process, Regulation of nucleic acid-templated transcription. Meanwhile, 11 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified. Among them, Wnt, TGF-β and Endocytosis pathways are well-related with cell proliferation and differentiation, which may be involved in the regeneration of coelomocytes in A. japonicus after evisceration. In addition, FoxO signaling pathway plays important roles in immunoregulation, in which the expression levels of the DEGs were significantly up-regulated, inferring that polian vesicles could not only participate in the coelomocyte regeneration process, but also undertake a certain immune defense function in A. japonicus after evisceration. These findings will be beneficial for understanding the mechanisms of coelomocyte regeneration and immune defense of A. japonicus after evisceration. • This is the first report on transcriptome in polian vesicles of sea cucumber Apostichopus japonicus. • A total of 2752 DEGs were identified in polian vesicles at 6 h post-evisceration. • Part of DEGs were significantly enriched in Wnt, TGF-β and endocytosis pathways. • Evisceration activated the FoxO signaling pathway in polian vesicles. [ABSTRACT FROM AUTHOR]

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/β-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro , using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of β-catenin activity was determined using an antibody against phosphorylated β-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated β-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined. • The effects of putative Wnt pathway modulators on intestine regeneration in Holothuria glaberrima were evaluated. • LiCl and iCRT14, presumed Wnt pathway modulators, modify the gut rudiment growth in vivo. • Cell proliferation appears to be mediated by the Wnt pathway during early intestinal regeneration. • Muscle dedifferentiation appears to be modulated by a Wnt-independent signaling via GSK-3. • Cell dedifferentiation can be decoupled from cell proliferation during intestinal regeneration. [ABSTRACT FROM AUTHOR]

The sea cucumber is one of the most economically significant echinoderms. The immunity against exogenous stimulation of sea cucumber is of great academic and economic importance. MicroRNAs (miRNAs) are a class of short endogenous non-coding RNAs (ncRNAs) that are considered as vital regulators of both innate and adaptive immune responses in most eukaryotes. In sea cucumbers, some miRNAs (such as miR-133, miR-137, and miR-2008, among others) that participate in the regulation of innate immunity have been recently identified and characterized. This review focuses on those known miRNAs and their corresponding target genes that participate in the regulation of the complement system, Toll-like receptor (TLR) pathway, reactive oxygen species (ROS) production and apoptosis pathways in sea cucumbers. Moreover, we cover immune-related miRNA investigations in sea cucumbers that provide insights into developing more miRNA-based biomarkers and therapeutic strategies for sea cucumber diseases. • Summarizes the microRNAs (miRNA) involved in immunity regulation in the sea cucumber. • Provides new insights into the immune regulation mechanisms of the sea cucumber. • Enriches our basic knowledge of the regulation of miRNAs expression in aquatic animals. [ABSTRACT FROM AUTHOR]

Temperature and dissolved oxygen concentration are important abiotic factors that can limit the growth and survival of sea cucumbers by affecting their immune systems. As global warming intensifies, sea cucumbers are increasingly exposed to adverse environmental conditions, which can cause severe economic losses and limit the sustainable development of sea cucumber aquaculture. It is therefore important to better understand how sea cucumbers respond to environmental stress, especially with regard to its effects on immunity. In the present study, the time series of immunity-related gene expression in sea cucumbers under thermal and hypoxic stresses were analyzed separately. The expression trends of 17 genes related to the nuclear factor κB (NF-κB) pathway, the protease family, the complement system, heat shock proteins (HSPs) and the transferrin family during exposure to two stresses at eight time points were concluded. These genes have interconnected roles in stress defense. The expression levels of genes relating to the NF-κB pathways and HSPs were strongly affected in the sea cucumber thermal stress response, while melanotransferrin (Mtf) , ferritin (Ft) and mannan-binding C-type lectin (MBCL) were affected by hypoxia. In contrast, complement factor B (Bf) , myosin V (Mys) and serine protease inhibitor (SPI) were not that sensitive during the initial period of environmental stress. Similar expression patterns under both thermal and hypoxic stress for certain genes, including an increase in Hsp90 and decreases in lysozyme (Lys) , major yolk protein (MYP) and cathepsin C (CTLC) were observed in sea cucumbers. Conversely, NF-κB and Hsp70 were differentially affected by the two stress treatments. Lysozyme-induced immune defense was inconstant in sea cucumbers coping with stress. A gene ontology (GO) analysis of the selected genes revealed that the most co-involved terms related to immunity and iron ion. Our analysis suggests that sea cucumbers demonstrate complex and varied immune responses to different types of stresses. This dynamic image of the immune responses and stress tolerance of sea cucumbers provides new insights into the adaptive strategies of holothurians in adverse environments. • Expressed trends of 17 immune genes under two stresses at 8 time points were concluded. • Genes related to Nf-κB pathway, protease, complement system, HSP and transferrin family were coefficient to defense stress. • Sea cucumber showed complex and different immune pattern towards different stresses. • These dynamic images provide new insights into adaptive strategies in sea cucumber tolerance to adverse environment. [ABSTRACT FROM AUTHOR]

Vibrio splendidus is one of the main pathogens caused diseases with a diversity of marine cultured animals, especially the skin ulcer syndrome in Apostichopus japonicus. However, limited virulence factors have been identified in V. splendidus. In this study, one aerA Vs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerA Vs was analyzed. Western blotting was used to study of the secretion pathway of proaerolysin, and it showed that the proaerolysin was secreted via both outer membrane vehicles and classical secretion pathways. Since no active protein of aerolysin was obtained, one aerolysin surface displayed bacterium DH5α/pAT- aerA was constructed, and its haemolytic activity and virulence were determined. The results showed that the AerA Vs displayed on the surface showed obvious haemolytic activity and cytotoxic to the coelomocyte of A. japonicus. Artificial immerse infection separately using the DH5α/pAT or DH5α/pAT- aerA was conducted. The result showed that the mortality percent of sea cucumber A. japonicus challenged with DH5α/pAT- aerA was 38.89 % higher than that challenged with the control strain DH5α/pAT, and earlier death occurred. Combined all the results indicates that aerolysin with the haemolytic activity and cytotoxic activity is a virulence factor of V. splendidus. • One aerA Vs gene, coding aerolysin, was cloned from a pathogenic V. splendidus. • The DH5α/pAT- aerA with AerA Vs displayed on the cell surface was constructed. • AerA Vs possessed haemolytic and cytotoxic activities contributing to virulence. [ABSTRACT FROM AUTHOR]

Sea cucumbers are important sources of human food and pharmacological compounds. However, few studies have evaluated the potential differences between the sexes or organs in a particular species to explore new commercial opportunities. In this study, the proximate composition and amino acid profile of the body wall, the saponin content in the organs of Athyonidium chilensis males and females, and their cytotoxicity against colon cancer cells were evaluated. There were no significant differences in the proximate composition and amino acid profile of the body wall between the sexes. Protein content differed significantly between organs and sexes, fluctuating between 36.29 % and 72.98 % (dry weight). Saponin content was significantly higher in the gonads and lower in the longitudinal musculature. Cell viability was significantly lower (9.50 ± 2.23 %) with the digestive system saponin extract than with 40 % (94.75 ± 2.65 %). This work demonstrates the great potential of A. chilensis as a food and source of saponins with pharmacological importance. • The viscera of A. chilensis are an important source of proteins. • The viscera of A. chilensis are a source of saponins, especially in the gonads. • Saponin extracts from A. chilensis organs have a cytotoxic effect on HCT-116 cells. • HCT-116 showed morphological changes and reduction in volume after extract exposure. [ABSTRACT FROM AUTHOR]

Pattern recognition receptors (PRRs) are the first line of immune defense in invertebrates against pathogen infection; they recognize pathogens and transmit signals to downstream immune pathways. Among these, peptidoglycan recognition proteins (PGRPs) are an important family in invertebrates that generally comprise of complicated isoforms. A comprehensive understanding of PGRPs in evolutionarily and economically important marine invertebrates, such as the sea cucumber, Apostichopus japonicus, is crucial. Previous studies have identified two PGRPs in sea cucumber, AjPGRP-S and AjPGRP-S1 , and another novel short-type PGRP, AjPGRP-S3 , was additionally identified here. The full-length cDNA sequence of AjPGRP-S3 was obtained here by PCR-RACE, followed by which showed its gene expression analyses by in situ hybridization that showed it to be relatively highly expressed in coelomocytes and tube feet. Based on an analysis of the recombinant protein, rAjPGRP-S3, a board-spectrum pathogen recognition ability was noted that covered diverse Gram-negative and -positive bacteria, and fungi. Moreover, according to the results of yeast two-hybridization, it was suggested that rAJPGRP-S3 interacted with multiple immune-related factors, including proteins involved in the complement system, extracellular matrix, vesicle trafficking, and antioxidant system. These findings prove the important functions of AjPGRP-S3 in the transduction of pathogen signals to downstream immune effectors and help explore the functional differences in the AjPGRP isoforms. • A novel short-type peptidoglycan recognition gene in sea cucumber, AjPGRP-S3 , were uncovered. • AjPGRP-S3 is strongly expressed in coelomocytes with unique polysaccharide recognition specificity. • AjPGRP-S3 interacts with complement system and extracellular matrix proteins. • AjPGRP-S3 transduces the pathogen invasion-related signal to activate phagocytosis. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of Bacillus cereus LS2 on the growth performance, innate immunity, intestinal microbiota, and disease resistance of sea cucumber Apostichopus japonicus. After feeding with LS2 for 30 days, results showed that dietary with LS2 had a significant improvement in the growth rate and immune parameters (including total coelomocytes counts, phagocytosis, respiratory burst, and immune-related enzymes) of juvenile sea cucumbers. Subsequently, transcriptome sequencing and qRT-PCR verification were performed to analyze the potential mechanism of LS2 diet and thus improve the immune response of A. japonicus. GO and KEGG pathway analysis indicated that LS2 can primarily activate the "Lectins" and "complement and coagulation cascades" pathways to modulate the innate immunity of the sea cucumbers. Furthermore, 16S rRNA sequencing was used to analyze the intestinal microbial composition of sea cucumbers after dietary with LS2. Results showed that Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were the most prevalent phyla in A. japonicus intestinal microbiota. The abundance of Actinobacteria (46.20%) and Bacteroidetes (12.80%) were significantly higher in the LS2 group, whereas the relative abundance of Proteobacteria (49.98%) and Firmicutes (14.97%) were higher in the control group. The LDA scores of Nocardiaceae and Rhodococcus were also the highest taxa after the dietary administration of LS2, indicating that Actinobacteria phylum played a pivotal role in the intestinal microbial function of A. japonicus. Overall, these results suggested that feeding with Bacillus LS2 may be beneficial for A. japonicus farming. • Dietary B. cereus LS2 improved growth and innate immunity parameters of sea cucumber. • The differentially expressed genes in LS2 group was enriched in Lectins pathway. • The gut microbiota community of sea cucumbers were different between control and LS2. • Dietary LS2 increased survival rate of sea cucumbers after V. splendidus infection. [ABSTRACT FROM AUTHOR]