Characterization of the Apoptotic and Antimicrobial Activities of Two Initiator Caspases of Sea Cucumber Apostichopus japonicus.

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.

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

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

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

Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5'-untranslated region (UTR), and a 154 bp 3'-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function.

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

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

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.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

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.

Pseudocryptic diversity and species boundaries in the sea cucumber Stichopus cf. horrens (Echinodermata: Stichopodidae) revealed by mitochondrial and microsatellite markers.

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.

Dietary steroids promote body weight growth and induce gametogenesis by increasing the expressions of genes related to cell proliferation of sea cucumber (Apostichopus japonicus).

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.

Molecular cloning, biological description, and functional analysis of Ajfos transcription factor in pathogen-induced Apostichopus japonicus.

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.

Circadian rhythm regulation in the sea cucumber Apostichopus japonicus: Insights into clock gene expression, photoperiod susceptibility, and neurohormone signaling.

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.

miRn-3 inhibits cutaneous wound healing by targeting gelsolin in the sea cucumber Apostichopus japonicus.

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.

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

Vibrio splendidus is one of the main pathogens caused diseases with a diversity of marine cultured animals, especially the skin ulcer syndrome in Apostichopus japonicus. However, limited virulence factors have been identified in V. splendidus. In this study, one aerAVs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerAVs was analyzed. Western blotting was used to study of the secretion pathway of proaerolysin, and it showed that the proaerolysin was secreted via both outer membrane vehicles and classical secretion pathways. Since no active protein of aerolysin was obtained, one aerolysin surface displayed bacterium DH5α/pAT-aerA was constructed, and its haemolytic activity and virulence were determined. The results showed that the AerAVs displayed on the surface showed obvious haemolytic activity and cytotoxic to the coelomocyte of A. japonicus. Artificial immerse infection separately using the DH5α/pAT or DH5α/pAT-aerA was conducted. The result showed that the mortality percent of sea cucumber A. japonicus challenged with DH5α/pAT-aerA was 38.89 % higher than that challenged with the control strain DH5α/pAT, and earlier death occurred. Combined all the results indicates that aerolysin with the haemolytic activity and cytotoxic activity is a virulence factor of V. splendidus.

Identification and functional characterization of a novel TRPA1 gene from sea cucumber Apostichopus japonicus and interaction with miR-2013 in response to salt stress.

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.

Comparative transcriptome analysis reveals key genes and pathways related to gonad development in the sea cucumber Apostichopus japonicus.

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.

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

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

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

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

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

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

Chromosome-level genome assembly of the sea cucumber Apostichopus japonicus.

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.

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.

N6-methyladenosine helps Apostichopus japonicus resist Vibrio splendidus infection by targeting coelomocyte autophagy via the AjULK-AjYTHDF/AjEEF-1α axis.

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.