Vibrio splendidus infection promotes circRNA-FGL1-regulated coelomocyte apoptosis via competitive binding to Myc with the deubiquitinase OTUB1 in Apostichopus japonicus.

Circular RNAs (circRNAs) are involved in various physiological and pathological processes in both vertebrates and invertebrates. However, most studies on circRNAs have focused on their roles as endogenous competitive RNAs. Here, we report a novel function of circRNA derived from the Fibrinogen-like protein 1 gene (circ-FGL1) that inhibits coelomocyte apoptosis via competing with the deubiquitinase AjOTUB1 to bind AjMyc in Apostichopus japonicus during Vibrio splendidus infection. The results showed that circ-FGL1 is significantly downregulated in coelomocytes of V. splendidus-induced A. japonicus and negatively regulates coelomocyte apoptosis through the AjBax-AjCyt c pathway. Mechanistically, the deubiquitinase AjOTUB1 and circ-FGL1 could interact with the transcription factor protein AjMyc in the same region with circ-FGL1/AjMyc having greater affinity. Under normal conditions, high levels of circ-FGL1 bind directly to AjMyc, inhibiting the deubiquitylation of AjMyc by AjOTUB1 and leading to the degradation of AjMyc. After V. splendidus infection, AjMyc disassociates from the depressed expression of circ-FGL1, promoting its deubiquitylation by binding to the induced deubiquitinase AjOTUB1 to inhibit its degradation. AjMyc is then transferred to the nucleus and promotes the transcription of AjCyt c and AjBax to induce coelomocyte apoptosis. The new finding will expand our present outstanding on the functional role of circRNAs and suggest new therapeutic targets for the treatment of echinoderms during bacterial invasion.

YTHDC1/CRM1 Facilitates m6A-Modified circRNA388 Nuclear Export to Induce Coelomocyte Autophagy via the miR-2008/ULK Axis in Apostichopus japonicus.

N 6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic RNA, was able to mediate circular RNA (circRNA) function in many immune processes. Nevertheless, the functional role of m6A-modified circRNAs in innate immunity of invertebrates remained unclear. In this study, we identified m6A-modified circRNA388 from cultured sea cucumber (Apostichopus japonicus) coelomocytes, which was mainly detected in cytoplasm after Vibrio splendidus infection. A knockdown assay indicated that cytoplasm circRNA388 promoted coelomocyte autophagy and decreased the number of intracellular V. splendidus. Mechanistically, the circRNA388 in the cytoplasm directly sponged miR-2008 to block its interaction with Unc-51-like kinase 1 from A. japonicus (AjULK) and further promoted autophagy to resist V. splendidus infection. More importantly, we found that m6A modification was vital to circRNA388 nuclear export with YTH domain-containing protein 1 from A. japonicus (AjYTHDC1) as the reader. AjYTHDC1 facilitated the nuclear export of m6A-modified circRNA388 via interaction with exportin-1 (chromosomal maintenance 1) from A. japonicus (AjCRM1). Knockdown of AjCRM1 could significantly decrease the content of cytoplasm circRNA388. Overall, our results provide the first evidence that nuclear export of m6A-modified circRNA388 is dependent on the novel AjCRM1 to our knowledge, which was further promoted coelomocyte autophagy by miR-2008/AjULK axis to clear intracellular V. splendidus.

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.

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.

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.

The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

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 (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 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 AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 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 AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

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.

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.

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.

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.

Comparative Analysis of Gut Bacterial Community Composition in Two Tropical Economic Sea Cucumbers under Different Seasons of Artificial Environment.

With the continuous rise of the sea cucumber aquaculture industry in China, the tropical sea cucumber aquaculture industry is also improving. However, research on the gut microorganisms of tropical sea cucumbers in captivity is scarce. In this study, high-throughput sequencing methods were used to analyze the gut microbial composition of Stichopus monotuberculatus and Holothuria scabra in the dry season and wet season of artificial environments. The results showed that 66 phyla were obtained in all samples, of which 59 phyla were obtained in the dry season, and 45 phyla were obtained in the wet season. The Tax4Fun analysis showed that certain gut bacterial communities affect the daily metabolism of two sea cucumber species and are involved in maintaining gut microecological balance in the gut of two sea cucumber species. In addition, compared with differences between species, PCoA and UPGMA clustering analysis showed the gut prokaryotes of the same sea cucumber species varied more in different seasons, indicating that the influence of environment was higher than the feeding choices of sea cucumbers under relatively closed conditions. These results revealed the gut bacterial community composition of S. monotuberculatus and H. scabra and the differences in gut bacterial structure between two sea cucumber species in different seasons were compared, which would provide the foundation for tropical sea cucumber aquaculture in the future.

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.

Ajpacifastin-like is involved in the immune response of Apostichopus japonicus challenged by Vibrio splendidus.

Pacifastin proteins are previously found to regulate the phenoloxidase system in invertebrates and arthropods. In this study, the immune response that was regulated by Ajpacifastin-like in the sea cucumber Apostichopus japonicus was determined. RNA interference was used to knock down the expression of the Ajpacifastin-like gene in A. japonicus, followed by challenge with Vibrio splendidus, and the colony count showed that the survival of V. splendidus in the si-Ajpacifastin group increased 4.64-fold compared to that of the control group. The purified recombinant Ajpacifastin-like showed an inhibitory effect on the extracellular protease activity of the supernatant collected from the V. splendidus culture. Consequently, a comparative transcriptome analysis of the coelomocytes from the control group and the si-Ajpacifastin group was performed to explore the global regulatory effect of the Ajpacifastin-like. A total of 1486 differentially expressed genes (DEGs) were identified, including 745 upregulated genes and 741 downregulated genes. GO enrichment showed that the DEGs were mainly enriched in translation, cytosolic ribosomal subunit and structural constituent of ribosome. KEGG analysis showed that the DEGs were significantly enriched in the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor-kappa B signaling pathway and other immune-related pathways. Furthermore, real-time reverse transcriptase PCR was used to determine the RNA levels of six DEGs, i.e., cathepsinB, CYLD, caspase8, TRAF6, hsp90 and FADD, to verify the RNA-seq results. Overall, our results specified the immune response and pathways of A. japonicus in which Ajpacifastin-like was involved in.

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.

NEDD4 activates mitophagy by interacting with LC3 to restrain reactive oxygen species and apoptosis in Apostichopus japonicus challenged with Vibrio splendidus.

Mitophagy, the selective degradation of damaged mitochondria by autophagy, plays a crucial role in the survival of coelomocytes in Apostichopus japonicus following Vibrio splendidus infection by suppressing the generation of reactive oxygen species (ROS) and attenuating cell apoptosis. A recent study revealed that reducing the expression of the neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), an enzyme 3 (E3) ubiquitin ligase, significantly affects mitochondrial degradation. Prior to the present study, the functional role of NEDD4 in marine invertebrates was largely unexplored. Therefore, we investigated the role of NEDD4 in the activation of mitophagy, modulation of ROS levels, and induction of apoptosis in A. japonicus infected with V. splendidus. The results demonstrated that V. splendidus infection and lipopolysaccharide (LPS) challenge significantly increased the mRNA levels of NEDD4 in A. japonicus coelomocytes, which was consistent with changes in mitophagy under the same conditions. Knockdown of AjNEDD4 using specific small interfering RNAs (siRNAs) impaired mitophagy and caused accumulation of damaged mitochondria, as observed using transmission electron microscopy (TEM) and confocal microscopy. Furthermore, AjNEDD4 was localized to the mitochondria in both coelomocytes and HEK293T cells. Simultaneously, coelomocytes were treated with the inhibitor indole-3-carbinol (I3C) to confirm the regulatory role of AjNEDD4 in mitophagy. The accumulation of AjNEDD4 in the mitochondria and the level of mitophagy decreased. Subsequent investigations demonstrated that AjNEDD4 interacts directly with the microtubule-associated protein light chain 3 (LC3), a key regulator of autophagy and mitophagy, indicating its involvement in the mitophagy pathway. Moreover, AjNEDD4 interference hindered the interaction between AjNEDD4 and LC3, thereby impairing the engulfment and subsequent clearance of damaged mitochondria. Finally, AjNEDD4 interference led to a significant increase in intracellular ROS levels, followed by increased apoptosis. Collectively, these findings suggest that NEDD4 acts as a crucial regulator of mitophagy in A. japonicus and plays a vital role in maintaining cellular homeostasis following V. splendidus infection. NEDD4 suppresses ROS production and subsequent apoptosis by promoting mitophagy, thereby safeguarding the survival of A. japonicus under pathogenic conditions. Further investigation of the mechanisms underlying NEDD4-mediated mitophagy may provide valuable insights into the development of novel strategies for disease control in aquaculture farms.

PPARα alleviates inflammation via inhibiting NF-κB/Rel pathway in Vibrio splendidus challenged Apostichopus japonicus.

Organisms trigger pro-inflammatory responses to resist the invasion of foreign pathogens in the early infection stage. However, excessive or chronic inflammation can also cause several diseases. We previously validated IL-17 from sea cucumbers mediated inflammatory response by the IL-17R-TRAF6 axis. But the anti-inflammatory effect was largely unknown in the species. In this study, the conserved PPARα gene was obtained from Apostichopus japonicus by RNA-seq and RACE approaches. The expression of AjPPARα was found to be significantly induced at the late stage of infection not only in Vibrio splendidus-challenged sea cucumbers, but also in LPS-exposed coelomocytes, which was negative correlation to that of AjIL-17 and AjNLRP3. Both silencing AjPPARα by specific siRNA and treatment with AjPAPRα inhibitor MK-886 could significantly upregulate the transcriptional levels of pro-inflammatory factors the AjIL-17 and AjNLRP3. The infiltration of inflammatory cells and tissues damage were also detected in the body walls in the same condition. In contrast, AjPAPRα agonist of WY14643 treatment could alleviate the V. splendidus-induced tissue injury. To further explore the molecular mechanism of AjPPARα-mediated anti-inflammatory in A. japonicus, the expression of the transcriptional factors of AjStat5 and AjRel (subunit of NF-κB) were investigated under AjPPARα aberrant expression conditions and found that AjRel exhibited a negative regulatory relationship to AjPPARα. Furthermore, silencing AjRel was led to down-regulation of AjIL-17 and AjNLRP3. Taken together, our results supported that AjPPARα exerted anti-inflammatory effects through inhibiting AjRel in response to V. splendidus infection.

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.

Exosomal microRNAs regulate the heat stress response in sea cucumber Apostichopus japonicus.

Exosomes are small extracellular vesicles that contain nucleic acids such as microRNAs and may participate in important biological processes. We made the initial report of exosomes from sea cucumber Apostichopus japonicus, that were classically cup-shaped and had an average size of 74.65 nm, and identified specific exosome biomarkers (HSP70, TSG101, and CD9). We explored changes in the global expression of microRNAs in exosomes from the commercially important A. japonicus under normal conditions and heat-stressed conditions for 3 and 7 d. We found that heat stress increased exosome production and modified the expression profiles of the microRNAs that they contained. Novel_mir31, novel_mir132, novel_mir26, miR-92_1, and novel_mir27 were commonly found to be differentially expressed in three comparison groups, indicating their importance in the heat stress response. The microRNA expression levels were validated by qPCR. Function analysis of the target genes of these microRNAs indicated they were involved mainly in replication and repair in the initial response of A. japonicus to heat stress exposure. Conversely, during acclimation to the high temperature conditions, the target genes of the differentially expressed microRNAs were primarily involved in metabolism adjustments. Our results will contribute to a better understanding of the regulatory roles of exosomes in sea cucumber, and provide insights into the functions of sea cucumber exosome-shuttled microRNAs against environmental stresses exacerbated by global warming.

Single-Cell Transcriptome and Pigment Biochemistry Analysis Reveals the Potential for the High Nutritional and Medicinal Value of Purple Sea Cucumbers.

The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals.