AjTGFß alleviates V. splendidus-induced inflammation through SMADs pathway in Apostichopus japonicus.

The inhibition of inflammatory response is an essential process to control the development of inflammation and is an important step to protect the organism from excessive inflammatory damage. As a pleiotropic cytokine, transforming growth factor beta (TGF-ß) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-ß in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-ß gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-ß. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-ß mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-ß was expressed in all tissues with the highest expression in the body wall. When AjTGF-ß was knocked down by using small interfering RNA (siRNA-KD) to 0.45-fold, AjSMAD 2/3 and AjSMAD6 were downregulated to 0.32- and 0.05-fold compared with the control group, respectively. Furthermore, when the damaged sea cucumber was challenged by V. splendidus co-incubated with rAjTGF-ß, the damage area had no extensive inflammation, and damaged repair appeared at 72 h compared with the Vs + BSA group, in which the expression of AjSMAD 2/3 was upregulated by 1.35-fold. Under this condition, AjSMAD 2/3 silencing alleviated rAjTGF-ß-induced damage recovery. Moreover, rAjTGF-ß slightly induced the collagen I expression from 6.13 ng/mL to 7.84 ng/mL, and collagen III was upregulated from 6.23 ng/mL to 6.89 ng/mL compared with the Vs + BSA group. This finding indicates that AjTGF-ß negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression.

MiR-7 Regulates Pathogen-Induced Immune Response via PAK1 in the Sea Cucumber Apostichopus japonicus.

MicroRNA-7 (miR-7) is a highly conserved short non-coding RNA involved in various bioprocesses via the regulation of multiple target genes. To enrich our knowledge of the functions of miR-7 in innate immune regulation in echinoderms, we first investigated the targeting relationship between miR-7 and PAK1 in the sea cucumber Apostichopus japonicus and then explored the functions of miR-7, the PAK1 gene, and the miR-7/PAK1 axis in the pathogen-induced immune response of A. japonicus. Our results showed that miR-7 can bind to the 3'UTR of PAK1 and negatively regulate the expression of PAK1 in A. japonicus. Overexpression and inhibition of miR-7 and inhibition of the expression of PAK1 can alter phagocytosis, cellular agglutination, and lysozyme contents in A. japonicus. Both miR-7 and the PAK1 gene are involved in immune defense against Vibrio splendidus infection; the miR-7/AjPAK1 axis showed immune regulatory function at 48 to 72 h post-infection (hpi) after V. splendidus infection in A. japonicus. In summary, the results of this study established that miR-7 regulates the pathogen-induced immune response by targeting PAK1 in A. japonicus.

Apostichopus japonicus matrix metalloproteinase-16 might act as a pattern recognition receptor.

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 ß-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, ß-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.

IL-17/IL-17 Receptor Pathway-Mediated Inflammatory Response in Apostichopus japonicus Supports the Conserved Functions of Cytokines in Invertebrates.

Inflammation participates in host defenses against infectious agents and contributes to the pathophysiology of many diseases. IL-17 is a well-known proinflammatory cytokine that contributes to various aspects of inflammation in vertebrates. However, the functional role of invertebrate IL-17 in inflammatory regulation is not well understood. In this study, we first established an inflammatory model in the Vibrio splendidus-challenged sea cucumber Apostichopus japonicus (Echinodermata). Typical inflammatory symptoms, such as increased coelomocyte infiltration, tissue vacuoles, and tissue fractures, were observed in the V. splendidus-infected and diseased tissue of the body wall. Interestingly, A. japonicus IL-17 (AjIL-17) expression in the body wall and coelomocytes was positively correlated with the development of inflammation. The administration of purified recombinant AjIL-17 protein also directly promoted inflammation in A. japonicus Through genome searches and ZDOCK prediction, a novel IL-17R counterpart containing FNIII and hypothetical TIR domains was identified in the sea cucumber genome. Coimmunoprecipitation, far-Western blotting, and laser confocal microscopy confirmed that AjIL-17R could bind AjIL-17. A subsequent cross-linking assay revealed that the AjIL-17 dimer mediates the inflammatory response by the specific binding of dimeric AjIL-17R upon pathogen infection. Moreover, silencing AjIL-17R significantly attenuated the LPS- or exogenous AjIL-17-mediated inflammatory response. Functional analysis revealed that AjIL-17/AjIL-17R modulated inflammatory responses by promoting A. japonicus TRAF6 ubiquitination and p65 nuclear translocation and evenly mediated coelomocyte proliferation and migration. Taken together, our results provide functional evidence that IL-17 is a conserved cytokine in invertebrates and vertebrates associated with inflammatory regulation via the IL-17-IL-17R-TRAF6 axis.

Effects of chronic dietary hexavalent chromium on bioaccumulation and immune responses in the sea cucumber Apostichopus japonicus.

Sea cucumbers Apostichopus japonicus (3.54 ± 0.01 g of wet weight) were exposed to five concentrations of dietary hexavalent chromium (Cr6+) [0 (control), 100, 200, 400, and 800 mg Cr6+/kg dry weight] amended with K2Cr2O7 for 30 days. The bioaccumulation and immune responses [antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT); hydrolytic enzymes: acid phosphatase (ACP) and alkaline phosphatase (AKP)] of sea cucumbers were subsequently evaluated. This study found that the order of Cr accumulation in the experimental tissues was respiratory tree > intestine > body wall. Significantly lower SOD activities occurred in the 400 mg/kg group compared to that in the control group. Higher dietary Cr6+ exposure (400 and 800 mg Cr6+/kg dry weight) did not negatively alter the CAT activities, but significantly inhibited CAT activities in 100 mg/kg group, compared to control group. ACP activities in groups 200, 400 and 800 mg/kg were significantly lower than those in control group, while no significant differences occurred in AKP activities among groups. The present study provides important information into the bioaccumulation and immune responses of the sea cucumber A. japonicus in response to chronic dietary Cr6+ exposure.

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

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

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

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

Distinct microbiota assembly mechanisms revealed in different reconstruction stages during gut regeneration in the sea cucumber Apostichopus japonicus.

Apostichopus japonicus is a useful model for studying organ regeneration, and the gut microbiota is important for host organ regeneration. However, the reconstruction process and the mechanisms of gut microbiota assembly during gut regeneration in sea cucumbers have not been well studied. In the present study, gut regeneration was induced (via evisceration) in A. japonicus, and gut immune responses and bacterial diversity were investigated to reveal gut microbiota assembly and its possible mechanisms during gut regeneration. The results revealed that bacterial community reconstruction involved two stages with distinct assembly mechanisms, where the reconstructed community was initiated from the bacterial consortium in the residual digestive tract and tended to form a novel microbiota in the later stage of reconstruction. Together, the results of immunoenzyme assays, community phylogenetic analysis, and source tracking suggested that the host deterministic process was stronger in the initial stage than in the later stage. The bacterial interactions that occurred were significantly different between the two stages. Positive interactions dominated in the initial stage, while more complex and competitive interactions developed in the later stage. Such a dynamic bacterial community could provide the host with energetic and immune benefits that promote gut regeneration and functional recovery. The results of the present study provide insights into the processes and mechanisms of gut microbiota assembly during intestinal regeneration that are valuable for understanding gut regeneration mechanisms mediated by the microbiota.

mTORC2/Rictor is essential for coelomocyte endocytosis in Apostichopus japonicus.

Endocytosis plays an important role in the immune defence systems of invertebrates through the interaction between the mechanical target of rapamycin complex 2 (mTORC2) and the AGC kinase family. Rictor is the most important unique subunit protein of mTORC2 and is thought to regulate almost all functions of mTORC2, including endocytosis. In the present study, a novel invertebrate Rictor homologue was identified from Apostichopus japonicus (designated as AjRictor) via the rapid amplification of cDNA ends (RACE). Spatial expression analysis indicated that AjRictor is ubiquitously expressed in all the examined tissues and has the highest transcript level in coelomocytes. Vibrio splendidus challenge in vivo and lipopolysaccharide (LPS) exposure in vitro could remarkably up-regulate the messenger RNA (mRNA) expression of AjRictor compared with the control group. AjRictor knockdown by 0.49- and 0.69-fold resulted in the significant decrease in endocytosis rate by 0.53- (P < 0.01) and 0.59-fold (P < 0.01) in vivo and in vitro compared with the control group, respectively. Similarly, the treatment of coelomocytes with rapamycin for 24 h and the destruction of the assembly of mTORC2 markedly decreased the endocytosis rate of the coelomocytes by 35.92% (P < 0.05). We detected the expression levels of endocytosis-related molecular markers after AjRictor knockdown and rapamycin treatment to further study the molecular mechanism between mTORC2 and endocytosis. Our results showed that AGC kinase family members (PKCα and Pan1) and the phosphorylation level of AktS473 were remarkably decreased after reducing mTORC2 activity; thus, mTORC2/Rictor plays a key role in the immune regulation of endocytosis in coelomocytes. Our current study indicates that mTORC2/Rictor is involved in the coelomocyte endocytosis of sea cucumber and plays an essential regulation role in defending pathogen invasion.

Enhanced protein phosphorylation in Apostichopus japonicus intestine triggered by tussah immunoreactive substances might be involved in the regulation of immune-related signaling pathways.

The sea cucumber Apostichopus japonicus is an economically important species owing to its high nutritive and medicinal value. In order to avoid the pollution resulting from the overuse of antibiotics in A. japonicus aquaculture, various immunostimulants have been used as an alternative to improve the efficiency of A. japonicus farming. Our previous proteomic investigation has shown that several proteins participating in the immune-related physiology of A. japonicus were differentially expressed in the intestinal tissue in response to tussah immunoreactive substances (TIS). This study further explored the immunostimulation mechanism of TIS in A. japonicus. Phosphoproteomics technology was used to investigate the effect of TIS on protein phosphorylation in the intestine of A. japonicus following feeding with a TIS-supplemented diet. A total of 213 unique phosphoproteins were detected from 225 unique phosphopeptides. KEGG pathway analysis showed that majority of the phosphoproteins are involved in endocytosis, carbon metabolism and spliceosome functional group. Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS and 12 of these were found to associate with biological functions. Of these 12 phosphoproteins, eight exhibited enhanced phosphorylation while four displayed reduced phosphorylation. These 12 proteins were further analyzed and all were found to play a role in regulating some aspects of the immune system and the growth of sea cucumbers, especially in phagocytosis, energy metabolism and disease resistance. The findings of this study could therefore shed new light on the immune pathways of sea cucumber that are affected by TIS. This could help us to better understand the underlying mechanism linked to the immunoenhancement of A. japonicus in response to TIS, one that is associated with the change in protein phosphorylation.

The effect of ocean acidification on the enzyme activity of Apostichopus japonicus.

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.

Isolation of probiotics and their effects on growth, antioxidant and non-specific immunity of sea cucumber Apostichopus japonicus.

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.

MiR-210 regulates coelomocyte proliferation through targeting E2F3 in Apostichopus japonicus.

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 3'UTR 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 iron-sulfur protein subunit of succinate dehydrogenase is critical in driving mitochondrial reactive oxygen species generation in Apostichopus japonicus.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme with the unique ability to participate in both the tricarboxylic acid cycle and the electron transport chain to produce reactive oxygen species (ROS). The B subunit of SDH is required for succinate oxidation, which is critical for pro-inflammatory response. In this study, we cloned the iron-sulfur protein subunit of SDH from Apostichopus japonicus (denoted as AjSDHB) via RACE technology and explored its role in the immune system as a response to pathogen infection. The full-length cDNA of AjSDHB was 1442 bp with a complete open reading frame of 858 bp encoding 286 amino acids. Simple modular architecture research tool analysis revealed that AjSDHB contained two conserved domains, including a 2Fe-2S iron-sulfur cluster binding domain and a 4Fe-4S dicluster domain, without a signal peptide. Multiple sequence alignment demonstrated that AjSDHB shared a high degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. Phylogenetic analysis supported the finding that AjSDHB is a new member of the SDHB protein subfamily. Tissue distribution analysis revealed that AjSDHB was expressed in all examined tissues and particularly highly expressed in the muscles. AjSDHB transcripts were markedly induced in coelomocytes both by Vibrio splendidus challenge in vivo and lipopolysaccharide exposure in vitro. Function analysis showed that siRNA-mediated AjSDHB knockdown could substantially reduce the mitochondrial membrane potential (ΔΨm) and further decrease mitochondrial ROS production in A. japonicus coelomocytes. By contrast, AjSDHB overexpression considerably increased ΔΨm and mitochondrial ROS production of A. japonicus coelomocytes. These results supported the idea that AjSDHB is involved in the innate immunity of A. japonicus through its participation in mitochondrial ROS generation.

Target of rapamycin signaling inhibits autophagy in sea cucumber Apostichopus japonicus.

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.

Bacillus baekryungensis MS1 regulates the growth, non-specific immune parameters and gut microbiota of the sea cucumber Apostichopus japonicus.

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.

Effects of aerial exposure on oxidative stress, antioxidant and non-specific immune responses of juvenile sea cucumber Apostichopus japonicus under low temperature.

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., O2- 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.

A novel ß-catenin from Apostichopus japonicus mediates Vibrio splendidus-induced inflammatory-like response.

ß-catenin, an adaptor molecule in Wnt/ß-catenin signaling pathway, is associated with different physiological processes such as intestinal immune, apoptosis, and inflammation-associated response. However, the function of ß-catenin is still largely unknown in Apostichopus japonicus. In the present study, we cloned and characterized ß-catenin gene from A. japonicus by RNA-seq and RACE approaches. The complete sequence of Ajß-catenin consisted of a 5' UTR of 166 bp, a 3' UTR of 501 bp and an ORF of 2433 bp encoding a protein of 810 amino acids. Ajß-catenin has a GSK-ß consensus phosphorylation site of 21 amino acids located at N-terminal region and twelve Armadillo/ß-catenin-like repeat (ARM) domains from 145 to 671 aa. Spatial expression analysis revealed that Ajß-catenin mRNA levels displayed higher abundance in intestine. For Vibrio splendidus challenged sea cucumber, Ajß-catenin transcripts reached their peak at 6 h and remained at higher levels until 24 h post infection in comparison with that of the control group. GSK-3ß inhibitor treatment could induce both Ajß-catenin and the inflammatory factors expression. Ajß-catenin silencing could also down-regulate inflammatory factors expression. These results collectively suggested that Ajß-catenin was a novel molecule mediate V. splendidus-induced immune response of A. japonicus via regulating the inflammatory factors expression.

4-Hydroxyphenylpyruvate dioxygenase from sea cucumber Apostichopus japonicus negatively regulates reactive oxygen species production.

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.

Fas-associated death domain (FADD) in sea cucumber (Apostichopus japonicus): Molecular cloning, characterization and pro-apoptotic function analysis.

Fas-associated death domain (FADD) is an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In this study, a full-length cDNA of FADD homologue in sea cucumber Apostichopus japonicas (AjFADD) was cloned and characterized, and its functional roles in apoptosis investigated. In healthy sea cucumbers, AjFADD was expressed in all detected tissues, with higher levels in coelomocytes and intestine. AjFADD mRNA and protein levels were significantly expressed in coelomocytes after exposed with LPS or poly (I:C) in vitro, and challenged with Vibrio splendidus in vivo. Moreover, siRNA-mediated AjFADD knockdown in coelomocyte much decreased AjFADD mRNA and protein levels as well as the coelomocytes apoptosis levels. Furthermore, over-expression of the expression plasmid pcDNA3.1 encoding AjFADD (pcAjFADD) significantly increased the apoptosis levels in HEK293 cells. Taken together, our results support that AjFADD is a novel pro-apoptotic protein that might play key roles in defensing the bacterial and virus invasion in sea cucumber.