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.

Novel secreted STPKLRR from Vibrio splendidus AJ01 promotes pathogen internalization via mediating tropomodulin phosphorylation dependent cytoskeleton rearrangement.

We previously demonstrated that the flagellin of intracellular Vibrio splendidus AJ01 could be specifically identified by tropomodulin (Tmod) and further mediate p53-dependent coelomocyte apoptosis in the sea cucumber Apostichopus japonicus. In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for internalization remains unclear. Here, we identified a novel AJ01 Type III secretion system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) domain, which could specifically interact with tropomodulin domain of AjTmod. Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 (S52) to reduce the binding stability between AjTmod and actin. After AjTmod dissociated from actin, the F-actin/G-actin ratio decreased to induce cytoskeletal rearrangement, which in turn promoted the internalization of AJ01. The STPKLRR knocked out strain could not phosphorylated AjTmod and displayed lower internalization capacity and pathogenic effect compared to AJ01. Overall, we demonstrated for the first time that the T3SS effector STPKLRR with kinase activity was a novel virulence factor in Vibrio and mediated self-internalization by targeting host AjTmod phosphorylation dependent cytoskeleton rearrangement, which provided a candidate target to control AJ01 infection in practice.

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.

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.

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.

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.

Investigation of copper-induced intestinal damage and proteome alterations in Takifugu rubripes: Potential health risks and environmental toxicology detection.

Copper is one of the predominant water pollutants. Excessive exposure to copper can cause harm to animal health, affecting the central nervous system and causing blood abnormalities. Cuproptosis is a novel form of cell death that differs from previous programmed cell death methods. However, the impact of copper on the intestines remains unclear. Therefore, we investigated the effects of different concentrations of copper exposure on the intestinal proteome of Takifugu rubripes (T. rubripes). Relevant biomarkers were used to detect cuproptosis. We revealed the crosstalk relationship between cuproptosis and self-rescue at different concentrations, and discussed the feasibility of using potential cuproptosis indicators as anti-infection factors. We observed intestinal damage in the three copper exposure groups, especially in T. rubripes treated with 100 and 500 µg/L copper, with shedding and breakage of intestinal villus and fuzzy and loose structure of intestinal mucosa. The presence of copper stress not only causes cuproptosis but also oxidative damage caused by reactive oxygen species (ROS). The results of quantitative proteomics by TMT showed that compared to the 50 and 100 µg/L copper exposure groups, the expression of glutaminase, pyruvate kinase, and skin mucus lectin in the 500 µg/L group was significantly increased. The positive mediators COX5A and CTNNB1, as well as the negative mediators CD4 and FDXR, were found to be differentially expressed. Using the protein expression trends of cuproptosis indicator factors FDX1 and DLAT to indicate the concentration of copper ions in the environment. In addition, we found a new effect of promoting ferroptosis: providing additional copper ions can activate the phenomenon of ferroptosis. Our results expand our understanding of the potential health risks of copper in T. rubripes. At the same time, it is of great significance for the process of copper poisoning and the development of new environmental toxicology detection reagents.

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.

A Genome-Wide Analysis of the Jasmonic Acid Biosynthesis Gene Families in Peanut Reveals Their Crucial Roles in Growth and Abiotic Stresses.

Abiotic stress is a limiting factor in peanut production. Peanut is an important oil crop and cash crop in China. Peanut yield is vulnerable to abiotic stress due to its seeds grown underground. Jasmonic acid (JA) is essential for plant growth and defense against adversity stresses. However, the regulation and mechanism of the jasmonic acid biosynthesis pathway on peanut defense against abiotic stresses are still limitedly understood. In this study, a total of 64 genes encoding key enzymes of JA biosynthesis were identified and classified into lipoxygenases (AhLOXs), alleno oxide synthases (AhAOSs), allene oxide cyclases (AhAOCs), and 12-oxo-phytodienoic acid reductases (AhOPRs) according to gene structure, conserved motif, and phylogenetic feature. A cis-regulatory element analysis indicated that some of the genes contained stress responsive and hormone responsive elements. In addition to proteins involved in JA biosynthesis and signaling, they also interacted with proteins involved in lipid biosynthesis and stress response. Sixteen putative Ah-miRNAs were identified from four families targeting 35 key genes of JA biosynthesis. A tissue expression pattern analysis revealed that AhLOX2 was the highest expressed in leaf tissues, and AhLOX32 was the highest expressed in shoot, root, and nodule tissues. AhLOX16, AhOPR1, and AhOPR3 were up-regulated under drought stress. AhLOX16, AhAOS3, AhOPR1, and AhAOC4 had elevated transcript levels in response to cold stress. AhLOX5, AhLOX16, AhAOC3, AhOPR1, and AhOPR3 were up-regulated for expression under salt stress. Our study could provide a reference for the study of the abiotic stress resistance mechanism in peanut.

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.

Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms.

In marine environments, organisms are confronted with numerous microbial challenges, although the differential regulation of xenophagy in response to different pathogenic bacterial species remains relatively unknown. Here, we addressed this issue using Apostichopus japonicus as a model. We identified 39 conserved autophagy-related genes by genome-wide screening, which provided a molecular basis for autophagy regulation in sea cucumbers. Furthermore, xenophagy of two Gram-negative bacteria, Vibrio splendidus and Escherichia coli, but not a Gram-positive bacteria, Micrococcus luteus, was observed in different autophagy assays. Surprisingly, a significantly higher autophagy capacity was found in the E. coli-challenged group than in the V. splendidus-challenged group. To confirm these findings, two different lipopolysaccharides, LPSV. splendidus and LPSE. coli, were isolated; we found that these LPS species differentially activated coelomocyte xenophagy. To explore the molecular mechanism mediating differential levels of xenophagy, we used an siRNA knockdown assay and confirmed that LPSV. splendidus-mediated xenophagy was dependent on an AjTLR3-mediated pathway, whereas LPSE. coli-mediated xenophagy was dependent on AjToll. Moreover, the activation of different AjTLRs resulted in AjTRAF6 ubiquitination and subsequent activation of K63-linked ubiquitination of AjBeclin1. Inversely, the LPSV. splendidus-induced AjTLR3 pathway simultaneously activated the expression of AjA20, which reduced the extent of K63-linked ubiquitination of AjBeclin1 and impaired the induction of autophagy; however, this finding was no t evident with LPSE. coli. Our present results provide the first evidence showing that xenophagy could be differentially induced by different bacterial species to yield differential autophagy levels in echinoderms.

A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin.

Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the ß-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-ß-actin-Arp2/3 complex-lysosome pathway.

Metabacillus arenae sp. nov., isolated from seashore sand.

A Gram-stain-positive, non-motile, rod-shaped, facultatively anaerobic bacterium, designated as IB182487T, was isolated from a seashore sand sample collected from Zhaoshu Island, PR China. Strain IB182487T grew at pH 6.0-10.0 (optimum, pH 8.0), 4-45 °C (optimum, 25-30 °C) and with 0-17 % (w/v) NaCl (optimum, 2-10 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IB182487T belonged to the genus Metabacillus and was closely related to Metabacillus idriensis SMC 4352-2T, (96.6 %), Metabacillus indicus LMG 22858T (96.5 %), Metabacillus niabensis DSM 17723T (96.3 %) and Metabacillus halosaccharovorans DSM 25387T (96.1 %). Strain IB182487T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and contained menaquinone MK-7 as the predominant isoprenoid quinone. Its polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and three unidentified glycolipids. The major cellular fatty acids of strain IB182487T were iso-C15 : 0 and anteiso-C15 : 0. The whole genome average nucleotide identity and digital DNA-DNA hybridization analysis between the isolate and its closely related type strains demonstrated that the strain significantly differed from other Metabacillus species. The genomic DNA G+C content of strain IB182487T was 37.4 mol%. On the basis of phenotypic and chemotaxonomic properties, phylogenetic relatedness as well as genomic characteristics, strain IB182487T represents a novel species of the genus Metabacillus, for which the name Metabacillus arenae sp. nov. is proposed. The type strain of M. arenae is IB182487T (=MCCC 1K04629T=JCM 34523T).

Phenotypic and functional characterization of two coelomocyte subsets in Apostichopus japonicus.

The hemocytes of invertebrates are composed of different cell subsets with different morphologies and structures. Different cell subsets have different immune functions, which play an important role in innate immune response against pathogens. However, the understanding of the classification of Apostichopus japonicus coelomocytes and the molecular basis of immune function of different cell subsets is very limited. In this study, two coelomocyte subpopulations of A. japonicus were isolated by Percoll density gradient centrifugation. They were identified from their morphological and structural characteristics, namely, spherical cells with a size of 10-12 µm spherical in shape and a large number of small granules inside; lymphocyte-like cells with a size of 4-5 µm spherical or oval in shape, and 1-3 filopodia. Functionally, the phagocytic capacity and lysosomal activity in spherical cells were significantly greater than those in lymphocyte-like cells. The results suggest that spherical cells may play a more critical role in the immune responses. Meanwhile, transcriptome sequencing analysis was performed to further clarify the functional differences between the two cell subsets. The data indicated significantly different gene expression patterns in them. Spherical cells tend to participate in immune defense, whereas lymphocyte-like cells tend to participate in energy metabolism. In addition, lymphocyte-like cells may convert oxidative phosphorylation to glycolysis by changing the manner of energy metabolism to quickly adapt to the energy demand of external stimuli. Spherical cells may respond to LPS stimulation through phagocytosis, and their response time is slower than that of lymphocyte-like cells. The expression of genes involved in endocytosis, phagocytosis, and lysosomal and humoral immunity in spherical cells was significantly higher than that in lymphocyte-like cells. These data provide valuable information for understanding the molecular basis of cellular and humoral immunity in A. japonicus.

ATGL-mediated lipophagy balances cholesterol-induced inflammation in pathogen infected Apostichopus japonicus coelomocytes.

Cholesterol metabolism can be dynamically altered in response to pathogen infection that ensure proper macrophage inflammatory function in mammals. However, it is unclear whether the dynamic between cholesterol accumulation and breakdown could induce or suppress inflammation in aquatic animal. Here, we aimed to investigate the cholesterol metabolic response to LPS stimulation in coelomocytes of Apostichopus japonicus, and to elucidate the mechanism of lipophagy in regulating cholesterol-related inflammation. LPS stimulation significantly increased intracellular cholesterol levels at early time point (12 h), and the increase in cholesterol levels is associated with AjIL-17 upregulation. Excessive cholesterol in coelomocytes of A. japonicus was rapidly converted to cholesteryl esters (CEs) and stored in lipid droplets (LDs) after 12 h of LPS stimulation and prolonged for 18 h. Then, increased colocalization of LDs with lysosomes was observed at late time point of LPS treatment (24 h), accompanied by elevated expression of AjLC3 and decreased expression of Ajp62. At the same time, the expression of AjABCA1 rapidly increased, suggesting lipophagy induction. Moreover, we demonstrated that AjATGL is required for induction of lipophagy. Inducing lipophagy by AjATGL overexpression attenuated cholesterol-induced AjIL-17 expression. Overall, our study provides evidence that cholesterol metabolic response occurs upon LPS stimulation, which is actively involved in regulating the inflammatory response of coelomocytes. AjATGL-mediated lipophagy is responsible for cholesterol hydrolysis, thereby balancing cholesterol-induced inflammation in the coelomocytes of 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.

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.

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.

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.