Importance of the ECM-receptor interaction for adaptive response to hypoxia based on integrated transcription and translation analysis.

Low dissolved oxygen (LO) conditions represent a major environmental challenge to marine life, especially benthic animals. For these organisms, drastic declines in oxygen availability (hypoxic events) can trigger mass mortality events and thus, act as agents of selection influencing the evolution of adaptations. In sea cucumbers, one of the most successful groups of benthic invertebrates, the exposure to hypoxic conditions triggers adaptive adjustments in metabolic rates and behaviour. It is unclear, however, how these adaptive responses are regulated and the genetic mechanisms underpinning them. Here, we addressed this knowledge gap by assessing the genetic regulation (transcription and translation) of hypoxia exposure in the sea cucumber Apostichopus japonicus. Transcriptional and translational gene expression profiles under short- and long-term exposure to low oxygen conditions are tightly associated with extracellular matrix (ECM)-receptor interaction in which laminin and collagen likely have important functions. Finding revealed that genes with a high translational efficiency (TE) had a relatively short upstream open reading frame (uORF) and a high uORF normalized minimal free energy, suggesting that sea cucumbers may respond to hypoxic stress via altered TE. These results provide valuable insights into the regulatory mechanisms that confer adaptive capacity to holothurians to survive oxygen deficiency conditions and may also be used to inform the development of strategies for mitigating the harmful effects of hypoxia on other marine invertebrates facing similar challenges.

Cloning and characterization of an aerolysin gene from a marine pathogen Vibrio splendidus.

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 aerAVs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerAVs 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 AerAVs 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.

Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities.

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 ß-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.

Ferroptosis resists intracellular Vibrio splendidus AJ01 mediated by ferroportin in sea cucumber Apostichopus japonicus.

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 (AjGPX4) 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 (AjFPN) and intracellular AJ01 were co-localized in coelomocytes. After AjFPN 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 AjFPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via AjFPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus.

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5'-untranslated region (UTR), and a 233 bp 3'-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37-335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Calreticulin from Apostichopus japonicus relieves endoplasmic reticulum stress induced by Vibrio splendidus through autophagy.

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.

MiR-210, not let-7, regulates Akt gene expression against Vibrio splendidus infection in the sea cucumber Apostichopus japonicus.

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 AjAkt (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.

Akirin2 enhances antibacterial ability via interacting with 14-3-3ζ in V. splendidus-challenged Apostichopus japonicus.

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.

Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin ß dependent nuclear translocation in Apostichopus japonicus.

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 AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-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. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

A novel short-type peptidoglycan recognition protein with unique polysaccharide recognition specificity in sea cucumber, Apostichopus japonicus.

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.

Effects of seawater acidification and warming on morphometrics and biomineralization-related gene expression during embryo-larval development of a lightly-calcified echinoderm.

CO2-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 pCO2 on calcification and helpful for revealing the adaptive strategy of less-calcified echinoderms amid climate change.

Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads.

Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 µmol·L-1, HDWWKER with an IC50 value of 583.6 µmol·L-1, and THDWWKER with an IC50 value of 1291.8 µmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

Sulfamethoxazole stress endangers the gut health of sea cucumber (Apostichopus japonicus) and affects host metabolism.

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.

Chemical substances and their activities in sea cucumber Apostichopus japonicus: A review.

Apostichopus japonicus, also known as Stichopus japonicus, with medicinal and food homologous figures, is a globally recognized precious ingredient with extremely high nutritional value. There is no relevant review available through literature search, so this article selects the research articles through the keywords "sea cucumber" and "Apostichopus japonicus (Stichopus japonicus)" in six professional databases, such as Wiley, PubMed, ScienceDirect, ACS, Springer, and Web of Science, from 2000 to the present, summarizing the extraction, isolation, and purification methods for the four major categories (polysaccharides, proteins and peptides, saponins, and other components) of the A. japonicus chemical substances and 10 effective biological activities of A. japonicus. Included are anticoagulation, anticancer/antitumor activities, hematopoiesis, regulation of gut microbiota, and immune regulatory activities that correspond to traditional efficacy. Literature support is provided for the development of medicines and functional foods and related aspects that play a leading role in future directions.

TMT-Based Quantitative Proteomic Analysis Reveals the Key Role of Cell Proliferation and Apoptosis in Intestine Regeneration of Apostichopus japonicus.

Sea cucumbers are widely known for their powerful regenerative abilities, which allow them to regenerate a complete digestive tract within a relatively short time following injury or autotomy. Recently, even though the histological changes and cellular events in the processes of intestinal regeneration have been extensively studied, the molecular machinery behind this faculty remains unclear. In this study, tandem mass tag (TMT)-based quantitation was utilized to investigate protein abundance changes during the process of intestine regeneration. Approximately 538, 445, 397, 1012, and 966 differential proteins (DEPs) were detected (p < 0.05) between the normal and 2, 7, 12, 20, and 28 dpe stages, respectively. These DEPs also mainly focus on pathways of cell proliferation and apoptosis, which were further validated by 5-Ethynyl-2'-deoxyuridine (EdU) or Tunel-based flow cytometry assay. These findings provide a reference for a comprehensive understanding of the regulatory mechanisms of various stages of intestinal regeneration and provide a foundation for subsequent research on changes in cell fate in echinoderms.

Vibrio splendidus flagellin C binds tropomodulin to induce p38 MAPK-mediated p53-dependent coelomocyte apoptosis in Echinodermata.

As a typical pathogen-associated molecular pattern, bacterial flagellin can bind Toll-like receptor 5 and the intracellular NAIP5 receptor component of the NLRC4 inflammasome to induce immune responses in mammals. However, these flagellin receptors are generally poorly understood in lower animal species. In this study, we found that the isolated flagellum of Vibrio splendidus AJ01 destroyed the integrity of the tissue structure of coelomocytes and promoted apoptosis in the sea cucumber Apostichopus japonicus. To further investigate the molecular mechanism, the novel intracellular LRR domain-containing protein tropomodulin (AjTmod) was identified as a protein that interacts with flagellin C (FliC) with a dissociation constant (Kd) of 0.0086 ± 0.33 µM by microscale thermophoresis assay. We show that knockdown of AjTmod also depressed FliC-induced apoptosis of coelomocytes. Further functional analysis with different inhibitor treatments revealed that the interaction between AjTmod and FliC could specifically activate p38 MAPK, but not JNK or ERK MAP kinases. We demonstrate that the transcription factor p38 is then translocated into the nucleus, where it mediates the expression of p53 to induce coelomocyte apoptosis. Our findings provide the first evidence that intracellular AjTmod serves as a novel receptor of FliC and mediates p53-dependent coelomocyte apoptosis by activating the p38 MAPK signaling pathway in Echinodermata.

Whole-genome resequencing reveals genetic differences and the genetic basis of parapodium number in Russian and Chinese Apostichopus japonicus.

BACKGROUND: Apostichopus japonicus is an economically important species in the global aquaculture industry. Russian A. japonicus, mainly harvested in the Vladivostok region, exhibits significant phenotypic differentiation, including in many economically important traits, compared with Chinese A. japonicus owing to differences in their habitat. However, both the genetic basis for the phenotypic divergence and the population genetic structure of Russian and Chinese A. japonicus are unknown. RESULT: In this study, 210 individuals from seven Russian and Chinese A. japonicus populations were sampled for whole-genome resequencing. The genetic structure analysis differentiated the Russian and Chinese A. japonicus into two groups. Population genetic analyses indicated that the Russian population showed a high degree of allelic linkage and had undergone stronger positive selection compared with the Chinese populations. Gene ontology terms enriched among candidate genes with group selection analysis were mainly involved in immunity, such as inflammatory response, antimicrobial peptides, humoral immunity, and apoptosis. Genome-wide association analysis yielded eight single-nucleotide polymorphism loci significantly associated with parapodium number, and these loci are located in regions with a high degree of genomic differentiation between the Chinese and Russia populations. These SNPs were associated with five genes. Gene expression validation revealed that three of these genes were significantly differentially expressed in individuals differing in parapodium number. AJAP08772 and AJAP08773 may directly affect parapodium production by promoting endothelial cell proliferation and metabolism, whereas AJAP07248 indirectly affects parapodium production by participating in immune responses. CONCLUSIONS: This study, we performed population genetic structure and GWAS analysis on Chinese and Russian A. japonicus, and found three candidate genes related to the number of parapodium. The results provide an in-depth understanding of the differences in the genetic structure of A. japonicus populations in China and Russia, and provide important information for subsequent genetic analysis and breeding of this species.

CHOP promotes coelomocyte apoptosis through p38-MAPK pathway in Vibrio splendidus-challenged sea cucumber Apostichopus japonicus.

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) belongs to the C/EBP family of transcription factors that has been proven to regulate apoptosis in many vertebrate species. However, the functional role of CHOP in invertebrates is largely unknown. In this paper, the open reading frame of CHOP was cloned and characterized in the sea cucumber Apostichopus japonicus (AjCHOP). The deuced amino acid of AjCHOP shared a conserved RTP801_C domain from 63 to 171 aa. Phylogenetic analysis indicated that AjCHOP clustered with CHOPs from Lytechinus variegatus and Strongylocentrotus purpuratus. To confirm the immune function of AjCHOP, the time-course expression profiles of AjCHOP were investigated, and the findings revealed AjCHOP was significantly induced in coelomocytes at mRNA and protein levels after Vibro splendidus challenge. Furthermore, knockdown of AjCHOP in coelomocyes by siRNA transfection significantly decreased the apoptosis level induced by V. splendidus. Mechanically, AjCHOP-mediated apoptosis was dependent on the activation of p38-MAPK pathway but not JNK/ERK-MAPK. Overall, our results supported that V. splendidus triggers apoptosis among the coelomocytes, whereas AjCHOP mediates through the p38-MAPK pathway in A. japonicus.

CircRNA254 functions as the miR-375 sponge to inhibit coelomocyte apoptosis via targeting BAG2 in V. splendidus-challenged Apostichopus japonicus.

Circular RNAs (circRNAs) function as immune regulators in many biological processes in mammals, while their function and underlying mechanisms in invertebrates are largely unexplored. In this study, the competing endogenous RNA (ceRNA) mechanism of circRNA that sponges miR-375 and thus regulates AjBAG2-mediated coelomocyte apoptosis was evaluated in Apostichopus japonicus. The results showed that circRNA254 (circ254) was significantly down-regulated in the intestines and coelomocytes after Vibrio splendidus challenge or Lipopolysaccharide exposure, which matched the RNA-seq results in A. japonicus within skin ulceration syndrome. Dual-luciferase and RNA FISH assays indicated that circ254 could directly combine with miR-375, in which circ254 possesses three binding sites of miR-375. Moreover, circ254 knockdown significantly promoted the coelomocyte apoptosis levels upon pathogen infection in vivo and in vitro. Furthermore, circ254 silencing could also down-regulate AjBAG2 expression and thereby promoting the levels of coelomocyte apoptosis levels and the expression of caspase 3, which the phenomenon could be reversed by treatment with miR-375 inhibitors. Taken together, our results confirmed that circ254 functions as a ceRNA of AjBAG2 by sponging miR-375, resulting in the inhibition of coelomocyte apoptosis in A. japonicus.

Dietary Bacillus cereus LS2 protects juvenile sea cucumber Apostichopus japonicus against Vibrio splendidus infection.

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.