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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 Ca 2+ 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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine β-sheets, Cys 30 -Cys 67 , Cys 38 -Cys 58 , Cys 53 -Cys 90 , Cys 56 -Cys 66 , and Cys 81 -Cys 102 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The sea cucumber Apostichopus japonicus can expel internal organs under stress and regenerate them subsequently. However, growth is delayed during regeneration, significantly impacting the industry. Circular RNAs (circRNAs) are single-stranded circular RNA molecules produced through alternative splicing of mRNA precursors. They play crucial roles in regulating gene expression via the ceRNA mechanism. In this study, circRNA profiles of control and regenerated intestines were constructed. A total of 15,874 circRNAs were identified, with a length of 300-350 nucleotides (nt) being the most abundant. Sanger sequencing confirmed the circular structure of circRNA398. Compared with the normal intestine, 50 and 83 differentially expressed circRNAs (DE-circRNAs) were identified in the regenerated intestine at 1 and 3 days post evisceration (dpe), respectively. Gene ontology (GO) terms for signal transduction and development regulation were most significantly enriched in 1dpeVScon and 3dpeVScon treatments, respectively. The dual-luciferase assay revealed that circRNA8388 functions as a sponge for miR-2392, participating in the remodeling of the extracellular matrix (ECM). In conclusion, these findings will contribute to the enhancement of the non-coding RNA database for echinoderms and lay the groundwork for future investigations into circRNA regulation during intestinal regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 10 7  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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β were mainly co-localized around the nucleus in vivo and silencing Ajimportin β 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 β-dependent pathway in sea cucumber., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Caspase (CASP) is a protease family that plays a vital role in apoptosis, development, and immune response. Herein, we reported the identification and characterization of two CASPs, AjCASPX1 and AjCASPX2, from the sea cucumber Apostichopus japonicus , an important aquaculture species. AjCASPX1/2 share similar domain organizations with the vertebrate initiator caspases CASP2/9, including the CARD domain and the p20/p10 subunits with conserved functional motifs. However, compared with human CASP2/9, AjCASPX1/2 possess unique structural features in the linker region between p20 and p10. AjCASPX1, but not AjCASPX2, induced marked apoptosis of human cells by activating CASP3/7. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2 were able to bind to a wide range of bacteria, as well as bacterial cell wall components, and inhibit bacterial growth. AjCASPX1, when expressed in Escherichia coli , was able to kill the host bacteria. Under normal conditions, AjCASPX1 and AjCASPX2 expressions were most abundant in sea cucumber muscle and coelomocytes, respectively. After bacterial infection, both AjCASPX1 and AjCASPX2 expressions were significantly upregulated in sea cucumber tissues and cells. Together, these results indicated that AjCASPX1 and AjCASPX2 were initiator caspases with antimicrobial activity and likely functioned in apoptosis and immune defense against pathogen infection.

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.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 μg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti- A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

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., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Steroids play a vital role in animal survival, promoting growth and development when administered appropriate concentration exogenously. However, it remains unclear whether steroids can induce gonadal development and the underlying mechanism. This study assessed sea cucumber weights post-culturing, employing paraffin sections and RNA sequencing (RNA-seq) to explore gonadal changes and gene expression in response to exogenous steroid addition. Testosterone and cholesterol, dissolved in absolute ethanol, were incorporated into sea cucumber diets. After 30 days, testosterone and cholesterol significantly increased sea cucumber weights, with the total weight of experimental groups surpassing the control. The testosterone-fed group exhibited significantly higher eviscerated weight than the control group. In addition, dietary steroids influenced gonad morphology and upregulated genes related to cell proliferation，such as RPL35, PC, eLF-1, MPC2, ADCY10 and CYP2C18. Thees upregulated differentially expressed genes were significantly enriched in the organic system, metabolism, genetic information and environmental information categories. These findings imply that steroids may contribute to the growth and the process of genetic information translation and protein synthesis essential for gonadal development and gametogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Morphologically cryptic and pseudo-cryptic species pose a challenge to taxonomic identification and assessments of species diversity and distributions. Such is the case for the sea cucumber Stichopus horrens, commonly confused with Stichopus monotuberculatus. Here, we used mitochondrial cytochrome oxidase subunit I (COI) and microsatellite markers to examine genetic diversity in Stichopus cf. horrens throughout the Philippine archipelago, to aid species identification and clarify species boundaries. Phylogenetic analysis reveals two recently diverged COI lineages (Clade A and Clade B; c. 1.35-2.54 Mya) corresponding to sequence records for specimens identified as S. monotuberculatus and S. horrens, respectively. Microsatellite markers reveal two significantly differentiated genotype clusters broadly concordant with COI lineages (Cluster 1, Cluster 2). A small proportion of individuals were identified as later-generation hybrids indicating limited contemporary gene flow between genotype clusters, thus confirming species boundaries. Morphological differences in papillae distribution and form are observed for the two species, however tack-like spicules from the dorsal papillae are not a reliable diagnostic character. An additional putative cryptic species was detected within Clade B-Cluster 2 specimens warranting further examination. We propose that these lineages revealed by COI and genotype data be referred to as Stichopus cf. horrens species complex., (© 2024. The Author(s).)

Activator protein-1 subfamily member c-Fos wields significant influence over cellular activities, such as regulation of cell growth and division, cell death, and immune responses under various extracellular situations. In this study, the full-length c-Fos of sea cucumber, Apostichopus japonicus (Ajfos) was successfully cloned and analyzed. The anticipated 306 amino acid sequences of Ajfos displayed a basic-leucine zipper (bZIP) domain, similar to invertebrate counterparts. In addition, the qPCR results suggested Ajfos expressed in all tissues, with the highest level in coelomocytes from polian vesicle (vesicle lumen cells), followed by coelomocytes from coelom (coelomocytes). Moreover, the expression levels of Ajfos in the coelomocytes and vesicle lumen cells of sea cucumber showed significant changes after the Vibrio splendidus challenge, especially reaching a peak at 6 h. Compared with the silencing negative control RNA interference (siNC) group, silencing Ajfos (siAjfos) in vivo decreased the downstream proliferation-related gene expression of vesicle lumen cells after infection with V. splendidus while no significant influence was observed on coelomocytes. Furthermore, the proliferation proportion of vesicle lumen cells in the siAjfos group was significantly reduced under pathogen stimulation conditions. Finally, based on the fluctuation trend of total coelomocyte density (TCD) from coelom and polian vesicle previously discovered, it is evident that Ajfos played a critical role in facilitating the swift proliferation of vesicle lumen cells in response to V. splendidus stimulation. Altogether, this research provided an initial reference of the function of Ajfos in echinoderms, unveiling its participation in host coelomocyte proliferation of sea cucumbers during bacterial challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Vibrio splendidus is one of the main pathogens caused diseases with a diversity of marine cultured animals, especially the skin ulcer syndrome in Apostichopus japonicus. However, limited virulence factors have been identified in V. splendidus. In this study, one aerA Vs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerA Vs was analyzed. Western blotting was used to study of the secretion pathway of proaerolysin, and it showed that the proaerolysin was secreted via both outer membrane vehicles and classical secretion pathways. Since no active protein of aerolysin was obtained, one aerolysin surface displayed bacterium DH5α/pAT-aerA was constructed, and its haemolytic activity and virulence were determined. The results showed that the AerA Vs displayed on the surface showed obvious haemolytic activity and cytotoxic to the coelomocyte of A. japonicus. Artificial immerse infection separately using the DH5α/pAT or DH5α/pAT-aerA was conducted. The result showed that the mortality percent of sea cucumber A. japonicus challenged with DH5α/pAT-aerA was 38.89 % higher than that challenged with the control strain DH5α/pAT, and earlier death occurred. Combined all the results indicates that aerolysin with the haemolytic activity and cytotoxic activity is a virulence factor of V. splendidus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sea cucumber Apostichopus japonicus displays the typical circadian rhythms. This present study investigated the molecular regulation of clock genes, as well as monoamines and melatonin, in multiple tissues of A. japonicus, responding to the photoperiod. In order to determine their pivotal role in circadian rhythms, the crucial clock genes, namely AjClock, AjArnt1, AjCry1, and AjTimeless, were identified and a comprehensive analysis of their expressions across various tissues in adult A. japonicus was conducted, revealing the potential existence of central and peripheral oscillators. Results demonstrated that the tissues of polian vesicle and nerve ring exhibited significant clock gene expression associated with the orchestration of circadian regulation, and that environmental light fluctuations exerted influence on the expression of these clock genes. However, a number of genes, such as AjArnt1 and AjCry1, maintained their circadian rhythmicity even under continuous light conditions. Moreover, we further investigated the circadian patterns of melatonin (MT), serotonin (5-HT), and dopamine (DA) secretion in A. japonicus, data that underscored the tissue-specific regulatory differences and the inherent adaptability to dynamic light environments. Collectively, these findings will provide the molecular mechanisms controlling the circadian rhythm in echinoderms and the candidate tissues playing the role of central oscillators in sea cucumbers., Competing Interests: Declaration of Competing Interest All authors disclosed no relevant relationships., (Copyright © 2023. Published by Elsevier Inc.)

The microRNA novel-3 (miRn-3) is a 23-nt small endogenous noncoding RNA of unknown function. To enrich our knowledge of the regulatory function of miRn-3 in the process of wound healing, the sea cucumber Apostichopus japonicus was used as a target model in this study. Gelsolin (AjGSN), a potential target gene of miRn-3, was cloned and characterized, and the interaction between miRn-3 and AjGSN was verified. The function of the miRn-3/AjGSN axis in regulating cutaneous wound healing was explored in the sea cucumber A. japonicus. The results showed that 1) the full-length cDNA of AjGSN was 2935 bp, with a high level of sequence conservation across the echinoderms; 2) miRn-3 could bind to the 3'UTR of AjGSN and negatively regulate the expression of AjGSN; 3) overexpression of miRn-3 and inhibition of the expression of AjGSN suppressed cutaneous wound healing in A. japonicus. In general, all observations of this study suggest that miRn-3 plays an important role in the early process of cutaneous wound healing by negatively targeting AjGSN, and that it may be a potential biomarker in wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The sea cucumber Apostichopus japonicus is an economically important marine species in China, and understanding the mechanisms underlying its gonad development is crucial for successful reproduction and breeding. In this study, we performed transcriptome comparisons and analyses of A. japonicus gonadal and non-gonadal tissues to identify genes and molecular pathways associated with gonadal development. We also supplemented the annotation of the A. japonicus genome. Collectively, results revealed a total of 941 ovary-specific genes and 2499 testis-specific genes through different expression analysis and WGCNA analysis. The most enriched pathways in ovary and testis were "DNA replication" and "purine metabolism", respectively. Additionally, we identified key candidate gene modules that control gonad development and germ cell maturation, with CDT1 and DYNC2LI1 serving as hub genes. Our findings provide important insights into the gonadal development system of A. japonicus and offer valuable references for further research on reproductive biology in this marine invertebrate species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Salinity is important abiotic factor influencing sea cucumber aquaculture. This study aimed to identify and functional study of a novel transient receptor potential cation channel subfamily A member 1 (TRPA1) involved in salinity stress through interaction with miR-2013 in the sea cucumber. The full-length cDNA sequence was 1369 bp in length and encoded 138 amino acids. The TRPA1 homolog protein was a hydrophilic protein without a signal peptide and was predicted to a spatial structure of seven helices and eight random coils and two major ANK functional domains. Bioinformatic analysis and luciferase reporter assays confirmed TRPA1 as a target gene of miR-2013. Quantitative PCR revealed that miR-2013 was induced upregulation after salinity stress, while TRPA1 showed upregulated expression with maximum expression at 24 h. The expression of miR-2013 and TRPA1 was negatively regulated. Transfection experiments were conducted to validate the role of miR-2013 and TRPA1 in salinity response. The results showed that miR-2013 was upregulated and TRPA1 was downregulated after transfection with miR-2013 mimics, while miR-2013 was downregulated and TRPA1 was upregulated after transfection with miR-2013 inhibitor. Transfection with si-TRPA1 homolog resulted in upregulation of miR-2013 and downregulation of TRPA1 homolog. These findings suggest that miR-2013 can regulate the expression of TRPA1 under salt stress, and highlight the importance of miR-2013 and TRPA1 in salt stress response. miR-2013 mimics improved the survival rate, while miR-2013 inhibitor and si-TRPA1 reduced it. These findings suggest that miR-2013 and TRPA1 play important roles in sea cucumbers adaptation to salinity changes., (© 2023. The Author(s), under exclusive licence to Cell Stress Society International.)

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

Sea cucumber is a morphologically diverse and ecologically important clade of echinoderms. The sea cucumber Apostichopus japonicus is the most economically valuable species of sea cucumber. The initial assembly of the A. japonicus genome was released in 2017. However, this genome assembly is fragmented and lacks relative position information of genes on chromosomes. In this study, we produced a high-quality chromosome-level genome of A. japonicus using Pacbio HiFi long-reads and Hi-C sequencing data. The assembled A. japonicus genome spanned 671.60 Mb with a contig N50 size of 17.20 Mb and scaffold N50 size of 29.65 Mb. A total of 99.9% of the assembly was anchored to 23 chromosomes. In total, 19,828 genes were annotated, and 97.2% of BUSCO genes were fully represented. This high-quality genome of A. japonicus will not only aid in the development of sustainable aquaculture practices, but also lay a foundation for a deeper understanding of their genetic makeup, evolutionary history, and ecological adaptation., (© 2023. The Author(s).)

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&#95;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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Polian vesicle is thought to produce coelomocytes and contribute to the sea cucumber's immune system. Our previous work has indicated that polian vesicle was responsible for cell proliferation at 72 h post pathogenic challenge. However, the transcription factors related to the activation of effector factors and the molecular process behind this remained unknown. In this study, to reveal the early functions of polian vesicle in response to the microbe, a comparative transcriptome sequencing of polian vesicle in V. splendidus-challenged Apostichopus japonicus, including normal group (PV 0 h), pathogen challenging for 6 h (PV 6 h) and 12 h (PV 12 h) was performed. Compared PV 0 h to PV 6 h, PV 0 h to PV 12 h, and PV 6 h to PV 12 h, we found 69, 211, and 175 differentially expressed genes (DEGs), respectively. KEGG enrichment analysis revealed the DEGs, including several transcription factors such as fos, FOS-FOX, ATF2, egr1, KLF2, and Notch3 between PV 6 h and PV 12 h were consistently enriched in MAPK, Apelin and Notch3 signaling pathways related to cell proliferation compared with that in PV 0 h. Important DEGs involved in cell growth were chosen, and their expression patterns were almost the same as the transcriptome profile analysis by qPCR. Protein interaction network analysis indicated that two DEGs of fos and egr1 were probably significant as key candidate genes controlling cell proliferation and differentiation in polian vesicle after pathogenic infection in A. japonicus. Overall, our analysis demonstrates that polian vesicles may play an essential role in regulating proliferation via transcription factors-mediated signaling pathway in A. japonicus and provide new insights into hematopoietic modulation of polian vesicles in response to pathogen infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

N6-Methyladenosine (m6A) modification is one of the most abundant post-transcriptional modifications that can mediate autophagy in various pathological processes. However, the functional role of m6A in autophagy regulation is not well-documented during Vibrio splendidus infection of Apostichopus japonicus. In this study, the inhibition of m6A level by knockdown of methyltransferase-like 3 (AjMETTL3) significantly decreased V. splendidus-induced coelomocyte autophagy and led to an increase in the intracellular V. splendidus burden. In this condition, Unc-51-like kinase 1 (AjULK) displayed the highest differential expression of m6A level. Moreover, knockdown of AjULK can reverse the V. splendidus-mediated autophagy in the condition of AjMETTL3 overexpression. Furthermore, knockdown of AjMETTL3 did not change the AjULK mRNA transcript levels but instead decreased protein levels. Additionally, YTH domain-containing family protein (AjYTHDF) was identified as a reader protein of AjULK and promoted AjULK expression in an m6A-dependent manner. Furthermore, the AjYTHDF-mediated AjULK expression depended on its interaction with translation elongation factor 1-alpha (AjEEF-1α). Altogether, our findings suggest that m6A is involved in resisting V. splendidus infection via facilitating coelomocyte autophagy in AjULK-AjYTHDF/AjEEF-1α-dependent manner, which provides a theoretical basis for disease prevention and therapy in A. japonicus., (© 2023. The Author(s).)

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., Competing Interests: Declaration of interest statement We declare that we have no conflict of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. The sea cucumbers A. japonicus were commercially cultured animals, and all the experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study protocol was approved by the Experimental Animal Ethics Committee of Ningbo University, China., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Alternative splicing (AS) is an essential post-transcriptional regulation mechanism for sex differentiation and gonadal development, which has rarely been reported in marine invertebrates. Sea cucumber (Apostichopus japonicus) is an economically important marine benthic echinoderm with a potential XX/XY sex determination mechanism, whose molecular mechanism in the gonadal differentiation has not been clearly understood. In this study, we analyzed available RNA-seq datasets of male and female gonads to explore if AS mechanism exerts an essential function in sex differentiation and gonadal development of A. japonicus. In our results, a total of 20,338 AS events from 7219 alternatively spliced genes, and 189 sexually differential alternative splicing (DAS) events from 156 genes were identified in gonadal transcriptome of sea cucumber. Gene Ontology analysis indicated that these DAS genes were significantly enriched in spermatogenesis-related GO terms. Maximal Clique Centrality (MCC) was then applied for protein-protein interaction (PPI) analysis to search for protein interactions and hub DAS gene. Among all DAS genes, we identified 10 DAS genes closely related to spermatogenesis and (or) sperm motility and a hub gene dnah1. Thus, this study revealed that alternative isoforms were generated from certain genes in female and male gonads through alternative splicing, which may provide direct evidence that alternative splicing mechanisms participate in female and male gonads. These results suggested a novel perspective for explaining the molecular mechanisms underlying gonadal differentiation between male and female sea cucumbers., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Polyploid breeding can produce new species with a faster growth rate, higher disease resistance, and higher survival rate, and has achieved significant economic benefits. This study investigated the protein differences in the body wall of triploid Apostichopus japonicus and diploid A. japonicus using isotope-labeled relative and absolute quantitative Tandem Mass Tag technology. A total of 21,096 independent peptides and 4621 proteins were identified. Among them, there were 723 proteins with significant expression differences, including 413 up-regulated proteins and 310 down-regulated proteins. The differentially expressed proteins (DEPs) were enriched in 4519 Gene Ontology enrichment pathways and 320 Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Twenty-two key DEPs related to important functions such as growth and immunity of triploid A. japonicus were screened from the results, among which 20 were up-regulated, such as cathepsin L2 cysteine protease and fibrinogen-like protein A. Arylsulfatase A and zonadhesin were down-regulated. The up-regulated proteins were mainly involved in oxidative stress response, innate immune response, and collagen synthesis in triploid A. japonicus, and the down-regulated proteins were mainly associated with the sterility of triploid A. japonicus. In addition, the transcriptome and proteome were analyzed jointly to support proteome data. In this study, the differences in protein composition between triploid and diploid A. japonicus were analyzed for the first time, and the results revealed the underlying reasons for the growth advantage of triploid A. japonicus., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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-β) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-β in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-β gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-β. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-β mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-β was expressed in all tissues with the highest expression in the body wall. When AjTGF-β 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-β, 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-β-induced damage recovery. Moreover, rAjTGF-β 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-β negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The mitogen-activated protein kinase family plays an important role in cell differentiation, growth, proliferation, and survival. However, the current research on the mitogen-activated protein kinase (MAPK) family in invertebrates is limited to the individual gene, and the analysis has not been conducted at the family level. In the present study, echinoderm MAPK family was identified by genomic screening, and five members, including three ERK subfamily members, one c-Jun N-terminal kinase (JNK) subfamily, and one p38-MAPK member were detected. Phylogenetic analysis showed that three MAPK subfamilies were separated into three separated clusters, and ERK subfamily appeared earlier than the other two subfamilies. Synteny analysis revealed that the p38 subfamily might be derived from the continuous gene duplication events of MAPK14 subfamily in invertebrates, which displayed genome expansion via gene duplication in vertebrates. The role of MAPK family in echinoderm immune defense was determined by investigating the expression profiles of MAPKs in Vibrio splendidus-challenged Apostichopus japonicus and LPS-exposed coelomocytes. The result showed that five MAPK members displayed induced expression profiles both in vitro and in vivo, and the peak expression was detected at different time points. Our study provides new insights into the evolutionary history of the MAPK family and show the similar immune function among MAPK members., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes' phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

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&#95;mir31, novel&#95;mir132, novel&#95;mir26, miR-92&#95;1, and novel&#95;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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ever-increasing consumer demand for sea cucumbers mainly leads to huge damage to wild sea cucumber resources, including Stichopus monotuberculatus , which in turn exerts negative impacts on marine environments due to the lack of ecological functions performed by sea cucumbers. Aquaculture of sea cucumbers is an effective way to meet consumer demand and restore their resources. Unsynchronous growth is a prominent problem in the aquaculture of sea cucumbers which has concealed unelucidated molecular mechanisms until now. In this study, we carried out an integrative analysis of transcriptomics and metabolomics on fast-growing (SMF) and slow-growing (SMS) groups of S. monotuberculatus cultured in the same environmental conditions. The results revealed that a total of 2054 significantly differentially expressed genes (DEGs) were identified, which are mainly involved in fat digestion and absorption, histidine metabolism, arachidonic acid metabolism, and glutathione metabolism. 368 differential metabolites (DMs) were screened out between the SMF group and the SMS group; these metabolites are mainly involved in glycerophospholipid metabolism, purine metabolism, biosynthesis of unsaturated fatty acids, pyrimidine metabolism, arachidonic acid metabolism, and other metabolic pathways. The integrative analysis of transcriptomics and metabolomics of S. monotuberculatus suggested that the SMF group had a higher capacity for lipid metabolism and protein synthesis, and had a more frequent occurrence of apoptosis events, which are likely to be related to coping with environmental stresses. The results of this study provide potential values for the aquaculture of sea cucumbers which may promote their resource enhancement.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand&#95;7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

The functional characteristics of neuropeptides in marine invertebrates have attracted significant attention recently although functional studies of luqin-type neuropeptides are still very limited, especially in deuterostomes. The sea cucumber, Apostichopus japonicus, is a representative species of deuterostomian Holothurian invertebrates. The species has high nutritional and medicinal value in China. In this study, we report the first comprehensive histological, biochemical and pharmacological characterization of luqin-type neuropeptide signaling in the sea cucumber A. japonicus. The A. japonicus luqin-like neuropeptide precursor (AjLQP) contains a single typical deuterostomian luqin-like neuropeptide AjLQ with an xFxRWamide motif. AjLQ was identified as the ligand for a luqin-type neuropeptide receptor AjLQR, that was previously predicted to be a tachykinin-type receptor, and triggers a rapid intracellular mobilization of Ca 2+ , followed by receptor internalization and a transient increase in ERK1/2 phosphorylation. In situ hybridization, immunohistochemistry and qRT-PCR analysis revealed extensive expression of AjLQP and AjLQ in A. japonicus tissues, especially in locomotion-related organs. In vitro pharmacological tests revealed that AjLQ caused 12.69% ± 1.99% (p < 0.01) relaxation of longitudinal muscle preparations at 10 -7 M concentration. Furthermore, we observed significantly increased expression of AjLQP (about 17.63 fold, p < 0.01) in intestine of deeply aestivating sea cucumbers, which suggests that AjLQ might be involved in feeding inhibition during aestivation. The present study provides a first insight into the experimental characterization of luqin-type neuropeptide signaling in a sea cucumber. The results will broaden our understanding of the potential function of neuropeptides during important biological processes in marine invertebrates and provide theoretical support for optimizing sea cucumber aquaculture technology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m 6 A modification has been found in circRNA, and m 6 A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m 6 A levels are largely unknown. Here, we firstly investigated the genome-wide map of m 6 A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m 6 A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m 6 A peak and about 55% of m 6 A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m 6 A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m 6 A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m 6 A circRNAs showed a positive correlation between the circRNAs expression and m 6 A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m 6 A modification was constructed based on conjoint analysis. Our results indicated that several selected m 6 A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m 6 A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m 6 A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m 6 A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

N 6 -methyladenosine (m 6 A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m 6 A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m 6 A and the proportion of the m 6 A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m 6 A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m 6 A methylation level and mRNA abundance, indicating a positive regulatory role of m 6 A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m 6 A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m 6 A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m 6 A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m 6 A methylation in Resistant group and lower mRNA level in Later group. The levels of m 6 A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m 6 A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m 6 A in mRNA in marine invertebrates., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

MicroRNA-7 (miR-7) is a highly conserved short non-coding RNA involved in various bioprocesses via the regulation of multiple target genes. To enrich our knowledge of the functions of miR-7 in innate immune regulation in echinoderms, we first investigated the targeting relationship between miR-7 and PAK1 in the sea cucumber Apostichopus japonicus and then explored the functions of miR-7, the PAK1 gene, and the miR-7/ PAK1 axis in the pathogen-induced immune response of A. japonicus . Our results showed that miR-7 can bind to the 3'UTR of PAK1 and negatively regulate the expression of PAK1 in A. japonicus . Overexpression and inhibition of miR-7 and inhibition of the expression of PAK1 can alter phagocytosis, cellular agglutination, and lysozyme contents in A. japonicus . Both miR-7 and the PAK1 gene are involved in immune defense against Vibrio splendidus infection; the miR-7/ AjPAK1 axis showed immune regulatory function at 48 to 72 h post-infection (hpi) after V. splendidus infection in A. japonicus. In summary, the results of this study established that miR-7 regulates the pathogen-induced immune response by targeting PAK1 in A. japonicus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Ren, Li, Liu, Zou, Yan, Zhan and Chang.)

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

DNAJ proteins function as co-chaperones of HSP70 and play key roles in cell physiology to promote protein folding and degradation, especially under environmental stress. Based on our previous study on HSP70, a systematic study of DNAJ was performed in sea cucumber Apostichopus japonicus using the transcriptomic and genomic data, identifying 43 AjDNAJ genes, including six AjDNAJA genes, eight AjDNAJB genes, and 29 AjDNAJC genes. Slight expansion and conserved genomic structure were observed using the phylogenetic and syntenic analysis. Differential period-specific and tissue-specific expression patterns of AjDNAJs were observed between adult and juvenile individuals during aestivation. Strong tissue-specific expression correlations between AjDNAJ and AjHSP70 genes were found, indicating that the involvements of AjHSP70IVAs in the aestivation of sea cucumbers were regulated by AjDNAJs. Several key genes with significant expression correlations, such as AjDNAJB4L and AjHSP70IVAs, were suggested to function together under heat stress. Together, these findings provide early insight into the involvement of AjDNAJs in the aestivation and their roles as co-chaperones of AjHSP70s., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Improving the immune ability and guiding healthy culture for sea cucumber by purposefully screening the significant differential metabolites when Apostichopus japonicus (A. japonicus) is infected by pathogens is important. In this study, 35 types of significant differential metabolites appeared when A. japonicus were infected by Vibrio splendens (VSI group) compared with the control A. japonicus group (CK group) by using liquid chromatography-mass spectrometry (LC-MS/MS)-based untargeted metabolomics. Based on that finding, the 10 types of key metabolic pathways were analyzed by MetPA. The "arachidonic acid (ARA) metabolism" pathway, which was screened by three elevated biomarkers: ARA, prostaglandin F2α and 2-arachidonoyl glycerol, had an important impact on immune stress in A. japonicus. Due to the similar changes in several metabolites in its metabolic pathway, the ARA metabolic pathway was selected for further study. The activities of ACP, AKP and lysozyme, which are important innate immune-related enzymes, the survival rates of A. japonicus infected with V. splendidus and the relative content of ARA in the body wall detected by GC-MS were all upregulated significantly by exogenous daily 0.60% and 1.09% ARA consumption over a short period of approximately 7 days. These results demonstrated that ARA and its metabolic pathway indeed played important roles in the immunity of A. japonicus infected by the pathogen. The findings also provide novel insights for the effects of metabolites in A. japonicum healthy culture., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Sex determination mechanisms are complicated and diverse across taxonomic categories. Sea cucumber Apostichopus japonicus is a benthic echinoderm, which is the closest group of invertebrates to chordate, and important economic and ecologically aquaculture species in China. A. japonicus is dioecious, and no phenotypic differences between males and females can be detected before sexual maturation. Identification of sex determination locus will broaden knowledge about sex-determination mechanism in echinoderms, which allows for the identification of sex-linked markers and increases the efficiency of sea cucumber breeding industry., Results: Here, we integrated assembly of a novel chromosome-level genome and resequencing of female and male populations to investigate the sex determination mechanisms of A. japonicus. We built a chromosome-level genome assembly AJH1.0 using Hi-C technology. The assembly AJH1.0 consists of 23 chromosomes ranging from 22.4 to 60.4 Mb. To identify the sex-determination locus of A. japonicus, we conducted genome-wide association study (GWAS) and analyses of distribution characteristics of sex-specific SNPs and fixation index F ST . The GWAS analysis showed that multiple sex-associated loci were located on several chromosomes, including chromosome 4 (24.8%), followed by chromosome 9 (10.7%), chromosome 17 (10.4%), and chromosome 18 (14.1%). Furthermore, analyzing the homozygous and heterozygous genotypes of plenty of sex-specific SNPs in females and males confirmed that A. japonicus might have a XX/XY sex determination system. As a physical region of 10 Mb on chromosome 4 included the highest number of sex-specific SNPs and higher F ST values, this region was considered as the candidate sex determination region (SDR) in A. japonicus., Conclusions: In the present study, we integrated genome-wide association study and analyses of sex-specific variations to investigate sex determination mechanisms. This will bring novel insights into gene regulation during primitive gonadogenesis and differentiation and identification of master sex determination gene in sea cucumber. In the sea cucumber industry, investigation of molecular mechanisms of sex determination will be helpful for artificial fertilization and precise breeding., (© 2022. The Author(s).)

Heat stress poses an increasing threat for the marine invertebrate Apostichopus japonicus. Histone lysine acetylation is a central chromatin modification for epigenetic regulation of gene expression during stress response. In this study, a genome-wide characterization for acetylated lysine 9 on histone H3 (H3K9ac) binding regions in normal temperature (18 °C) and heat-stress conditions (26 °C) via ChIP-seq were carried out. The results that revealed H3K9ac was an extensive epigenetic modulation in A. japonicus. The GO terms "regulation of transcription, DNA-templated" and "transcription coactivator activity" were significantly enriched in both groups. Particularly, various transcriptional factors (TFs) families showed notable modification of H3K9ac. Differentially acetylated regions (DARs) with H3K9ac modification under heat stress were identified with 24 hyperacetylated and 23 hypoacetylated peaks, respectively. We further examined the transcriptional expression for 13 genes with dysregulated H3K9ac level in the promoter regions by qRT-PCR. Combined H3K9ac ChIP-seq characteristics with the transcriptional expression, 5 up-up genes (ZCCHC3, RPA70, MTRR, β-Gal and PHTF2) and 2 down-down genes (PRPF39 and BSL78&#95;10147) were identified. Surprisingly, the increasing mRNA expression of NECAP1 under heat stress was negatively related to the decreasing H3K9ac level in its promoter region. Our research is the first genome-wide characterization for the epigenetic modification H3K9ac in A. japonicus, and will help to advance the understanding of the roles of H3K9ac in transcriptional regulation under heat-stress condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The mitochondrial genome is widely used to study the molecular evolution of and perform phylogenetic analyses on animals. In this study, the complete mitochondrial genome (mitogenome) of Stichopus naso was sequenced. The mitogenome was 16,239 bp in length and contained 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes (rRNAs). The genome composition showed positive AT-skew (0.023) and negative GC-skew (-0.158). The order of the mitochondrial genes was consistent with those from the Stichopus and Isostichopus species, whereas it was different from those of other species of Aspidochirotida. The phylogenetic analysis, based on the nucleotide sequences of 13 PCGs through the methods of Bayesian inference (BI) and maximum likelihood (ML), indicated that S. naso has close relationships with S. horrens and S. monotuberculatus , and belongs to a member of Stichopodidae. Our study provides a reference mitogenome for further molecular evolution studies and phylogenetic research on sea cucumbers.

A Gram-stain negative, aerobic, rod-shaped bacterium, designated 126 T , was isolated from the intestinal content of a sea cucumber, Apostichopus japonicus, in China. Strain 126 T was found to grow optimally at 25-28 °C and pH 7.5-8.0 in marine 2216 E medium, with tolerance of 1-7% (w/v) NaCl. Strain 126 T is motile by means of one to several polar flagella. The dominant fatty acids of strain 126 T were identified as C 16:1 ω7c/C 16:1 ω6c (29.5%), C 18:1 ω7c/C 18:1 ω6c (19.8%) and C 16:0 (16.7%). The respiratory quinone was found to be Q-8. The polar lipid profile was found to be mainly composed of phosphatidylglycerol and phosphatidylethanolamine. The total length of the draft genome is approximately 4.2 × 10 6  bp, encoding 3655 genes and 3576 coding sequences. The G + C content of the genomic DNA is 48.0%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 126 T belongs to the genus Neiella and is closely related to Neiella marina J221 T (96.5%). Genomic comparisons of 126 T to N. marina J221 T revealed that they had similar genome size, G + C content and complement of clusters of orthologous groups. However, average nucleotide identity and digital DNA-DNA hybridization values between strains126 T and N. marina J221 T was 75.5% and 19.7%, which could distinguish the strains. On the basis of these phenotypic and genotypic data, strain 126 T is concluded to represent a novel species, for which the name Neiella holothuriorum sp. nov. is proposed. The type strain is 126 T (= GDMCC 1.2530 T  = KCTC 82829 T )., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Organisms produce high levels of reactive oxygen species (ROS) to kill pathogens or act as signaling molecules to induce immune responses; however, excessive ROS can result in cell death. To maintain ROS balance and cell survival, mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates. In marine invertebrates, however, mitophagy and its functions remain largely unknown. In the current study, Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential (ΔΨm) and mitophagosome formation. The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide (LPS) treatment increased mitophagy flux. To explore the regulatory mechanism of mitophagy, we cloned Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a common mitophagy receptor, from sea cucumber Apostichopus japonicus ( AjBNIP3 ) and confirmed that AjBNIP3 was significantly induced and accumulated in mitochondria after V. splendidus infection and LPS exposure. At the mitochondrial membrane, AjBNIP3 interacts with microtubule-associated protein 1 light chain 3 (LC3) on phagophore membranes to mediate mitophagy. After AjBNIP3 interference, mitophagy flux decreased significantly. Furthermore, AjBNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide (H 2 O 2 ), ROS scavengers, and ROS inhibitors. Finally, inhibition of BNIP3-mediated mitophagy by AjBNIP3 small interfering RNA (siRNA) or high concentrations of lactate increased apoptosis and decreased coelomocyte survival. These findings highlight the essential role of AjBNIP3 in damaged mitochondrial degradation during mitophagy. This mitophagy activity is required for coelomocyte survival in A. japonicus against V. splendidus infection.