Biosynthesis of saponin defensive compounds in sea cucumbers.

Soft-bodied slow-moving sea creatures such as sea stars and sea cucumbers lack an adaptive immune system and have instead evolved the ability to make specialized protective chemicals (glycosylated steroids and triterpenes) as part of their innate immune system. This raises the intriguing question of how these biosynthetic pathways have evolved. Sea star saponins are steroidal, while those of the sea cucumber are triterpenoid. Sterol biosynthesis in animals involves cyclization of 2,3-oxidosqualene to lanosterol by the oxidosqualene cyclase (OSC) enzyme lanosterol synthase (LSS). Here we show that sea cucumbers lack LSS and instead have two divergent OSCs that produce triterpene saponins and that are likely to have evolved from an ancestral LSS by gene duplication and neofunctionalization. We further show that sea cucumbers make alternate sterols that confer protection against self-poisoning by their own saponins. Collectively, these events have enabled sea cucumbers to evolve the ability to produce saponins and saponin-resistant sterols concomitantly.

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

Pattern recognition receptors (PRRs) are the first line of immune defense in invertebrates against pathogen infection; they recognize pathogens and transmit signals to downstream immune pathways. Among these, peptidoglycan recognition proteins (PGRPs) are an important family in invertebrates that generally comprise of complicated isoforms. A comprehensive understanding of PGRPs in evolutionarily and economically important marine invertebrates, such as the sea cucumber, Apostichopus japonicus, is crucial. Previous studies have identified two PGRPs in sea cucumber, AjPGRP-S and AjPGRP-S1, and another novel short-type PGRP, AjPGRP-S3, was additionally identified here. The full-length cDNA sequence of AjPGRP-S3 was obtained here by PCR-RACE, followed by which showed its gene expression analyses by in situ hybridization that showed it to be relatively highly expressed in coelomocytes and tube feet. Based on an analysis of the recombinant protein, rAjPGRP-S3, a board-spectrum pathogen recognition ability was noted that covered diverse Gram-negative and -positive bacteria, and fungi. Moreover, according to the results of yeast two-hybridization, it was suggested that rAJPGRP-S3 interacted with multiple immune-related factors, including proteins involved in the complement system, extracellular matrix, vesicle trafficking, and antioxidant system. These findings prove the important functions of AjPGRP-S3 in the transduction of pathogen signals to downstream immune effectors and help explore the functional differences in the AjPGRP isoforms.

Chromosome-level genome assembly of sea scallop Placopecten magellanicus provides insights into the genetic characteristics and adaptive evolution of large scallops.

Placopecten magellanicus (Gmelin, 1791), a deep-sea Atlantic scallop, holds significant commercial value as a benthic marine bivalve along the northwest Atlantic coast. Recognizing its economic importance, the need to reconstruct its genome assembly becomes apparent, fostering insights into natural resources and generic breeding potential. This study reports a high-quality chromosome-level genome of P. magellanicus, achieved through the integration of Illumina short read sequencing, PacBio HiFi sequencing, and Hi-C sequencing techniques. The resulting assembly spans 1778 Mb with a scaffold N50 of 86.71 Mb. An intriguing observation arises - the genome size of P. magellanicus surpasses that of its Pectinidae family peers by 1.80 to 2.46 times. Within this genome, 28,111 protein-coding genes were identified. Comparative genomic analysis involving five scallop species unveils the critical determinant of this expanded genome: the proliferation of repetitive sequences recently inserted, contributing to its enlarged size. The landscape of whole genome collinearity sheds light on the relationships among scallop species, enhancing our broader understanding of their genomic framework. This genome provides genomic resources for future molecular biology research on scallops and serves as a guide for the exploration of longevity-related genes in scallops.

Performance enhancement, bacterial communities optimization and emerging pollutants elimination by microalgal-bacterial consortium for treating aquaculture pond sediments.

Aquaculture pond sediments have a notable influence on the ecosystem balance and farmed animal health. In this study, microalgal-bacterial immobilization (MBI) was designed to improve aquaculture pond sediments via synergistic interactions. The physicochemical characteristics, bacterial communities, and the removal efficiencies of emerging pollutants were systematically investigated. The consortium containing diatom Navicula seminulum and Alcaligenes faecalis was cultivated and established in the free and immobilized forms for evaluating the treatment performance. The results indicated that the immobilized group exhibited superior performance in controlling nutrient pollutants, shaping and optimizing the bacterial community compositions with the enrichment of functional bacteria. Additionally, it showed a stronger positive correlation between the bacterial community shifts and nutrient pollutants removal compared to free cells. Furthermore, the immobilized system maintained the higher removal performance of emerging pollutants (heavy metals, antibiotics, and pathogenic Vibrios) than free group. These findings confirmed that the employment of immobilized N. seminulum and A. faecalis produced more synergistic benefits and exerted more improvements than free cells in ameliorating aquaculture pond sediments, suggesting the potential for engineering application of functional microalgal-bacterial consortium in aquaculture.

Temporal stability and assembly mechanisms of gut microbiota in sea cucumbers response to nanoplastics treatment.

Aquaculture provides essential food for humans, and the health of farmed species is particularly important for the aquaculture industry. Aquaculture environment could be a sink of plastic debris (PDs) due to the enclosed character and heavy use of plastics. Gut microbiota of aquaculture species could respond to the exogenous pollutants and regulate the health of hosts. Here, variations in gut microbiota of Apostichopus japonicus induced by the ingested nanoplastics (NPs) were investigated by a lab experiment. We selected a NPs concentration gradient of 100 mg/kg and 500 mg/kg to simulate microplastic pollution to A. japonicus, and the significant differences in gut microbiota composition after 21 days of NP exposure were evaluated. According to the high-throughput sequencing from time series samples, a decrease of diversity in gut microbiota of A. japonicus with dietary NPs was observed. In addition, the gut microbiota compositions of sea cucumbers with and without NPs exposure were also distinct, expressing as enrichment of Bacteroidota while reducement of Proteobacteria under NPs stresses. Combined the results of network analysis, the less complexity and stability of gut microbiota in sea cucumbers with dietary NPs were proved. Based on the neutral community model, the ingested NPs elevated the contribution of stochastic processes for the gut microbiota assembly in sea cucumbers. Our study showed that substantial variations in gut microbiota of A. japonicus under NPs stresses, and also explored the underlying mechanisms regulating these changes. This research would offer new meaningful insights into the toxicity of NPs on sea cucumbers, contributing a solid fundament to improve the health of sea cucumbers under NPs stresses.

Exploiting the gut microbiota to predict the origins and quality traits of cultured sea cucumbers.

Nowadays, the true economic and nutritional value of food is underpinned by both origin and quality traits, more often expressed as increased quality benefits derived from the origin source. Gut microbiota contribute to food metabolism and host health, therefore, it may be suitable as a qualifying indicator of origin and quality of economic species. Here, we investigated relationships between the gut microbiota of the sea cucumber (Apostichopus japonicus), a valuable aquaculture species in Asia, with their origins and quality metrics. Based on data from 287 intestinal samples, we generated the first biogeographical patterns for A. japonicus gut microbiota from origins across China. Importantly, A. japonicus origins were predicted using the random forest model that was constructed using 20 key gut bacterial genera, with 97.6% accuracy. Furthermore, quality traits such as saponin, fat and taurine were also successfully predicted by random forest models based on gut microbiota, with approximately 80% consistency between predicted and true values. We showed that substantial variations existed in the gut microbiota and quality variables in A. japonicus across different origins, and we also demonstrated the great potential of gut microbiota to track A. japonicus origins and predict their quality traits.

Apostichopus japonicus matrix metalloproteinase-16 might act as a pattern recognition receptor.

Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and ß-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, ß-1,3-glucan, Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway.

Cloning and characterization of a phosphomevalonate kinase gene that is involved in saponin biosynthesis in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.

Functions of lysin motif (LysM)-containing protein in antibacterial responses of sea cucumbers, Apostichopus japonicus.

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus, named AjLysM-1, using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens, and Shewanella baltica, implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms.

rAj-Tspin, a novel recombinant peptide from Apostichopus japonicus, suppresses the proliferation, migration, and invasion of BEL-7402 cells via a mechanism associated with the ITGB1-FAK-AKT pathway.

Purpose Our study aimed to investigate the antitumor effects of rAj-Tspin on BEL-7402 hepatocellular carcinoma cells and to explore the underlying mechanism. Method For the in vivo experiment, BEL-7402 cells were inoculated subcutaneously into the axilla of nude mice to generate a BEL-7402 cell-bearing model, and model mice were then treated with different doses of rAj-Tspin. A CCK-8 assay was used to evaluate the in vitro viability of BEL-7402 and LO2 cells after treatment with different concentrations of rAj-Tspin. The effects of rAj-Tspin on BEL-7402 cell apoptosis, migration and invasion were evaluated by AO/EB and Hoechst fluorescent staining and by scratch and Transwell assays, and the tumor-suppressive mechanism of rAj-Tspin was explored by Western blotting. Results rAj-Tspin suppressed the proliferation of BEL-7402 cells with an IC50 of 0.89 µM. The results of both microscopic analysis and Western blotting suggested that rAj-Tspin induced the apoptosis of BEL-7402 cells through a mitochondria-dependent pathway. Furthermore, rAj-Tspin disrupted EMT; this disruption ultimately caused BEL-7402 cells to lose their shape and decreased their migration and invasion. Moreover, rAj-Tspin might inhibit the proliferation and metastasis of BEL-7402 cells through the ITGB1-FAK-AKT pathway. Conclusion rAj-Tspin exerts an antitumor effect through the ITGB1-FAK-Akt signaling pathway and exhibits low toxicity at an effective dose.

Cloning, Expression and Inhibitory Effects on Lewis Lung Carcinoma Cells of rAj-Tspin from Sea Cucumber (Apostichopus japonicus).

In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber (Apostichopus japonicus), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it's effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.

Insights into the DNA methylation of sea cucumber Apostichopus japonicus in response to skin ulceration syndrome infection.

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus.

Gender specific differences of immune competence in the sea cucumber Apostichopus japonicus before and after spawning.

The gender differences of immunity have been elucidated in many vertebrates and invertebrates. However, the information of this difference was still not clear in the sea cucumber Apostichopus japonicus, which is one of the most valuable aquaculture species and susceptible to diseases caused by pathogen infection. In the present study, the transcriptome of coelomocytes from female and male A. japonicus before and after spawning was obtained by RNA-sequencing technology. A total of 4,538 and 8,248 differentially expressed genes were identified between female and male A. japonicus before and after spawning, respectively, indicating that the gender differences of gene expression profiles in A. japonicus were more remarkable after spawning. Further KEGG enrichment analyses were conducted for both male and female up-regulated genes before and after spawning. The results revealed that the capacity to kill pathogens in female A. japonicus might be more powerful than that in males no matter before and after spawning; the antioxidant ability in male A. japonicus was probably stronger than that in females after spawning; the complement system in male A. japonicus might be more effective than that in females after spawning; and the apoptosis was likely to be more serious in male A. japonicus before spawning. Moreover, we speculated that the fatty acid composition might be one of the inducements for gender specific immune differences of A. japonicus. Overall, the results of our study illustrated the global gender specific immune differences of A. japonicus and contributed to understanding of the molecular mechanisms underlying sea cucumber immune regulation.

Comparative expression analysis of immune-related factors in the sea cucumber Apostichopus japonicus.

In order to preliminarily explore the joint involvement of different immune-related factors during the same immune process in Apostichopus japonicus, the transcriptional expression of Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT), c-type lysozyme (c-LYZ), i-type lysozyme (i-LYZ), cathepsin D, melanotransferrin (MTF), Toll, c-type lectin (c-LCT) and complement 3 (C3) during the development from fertilized eggs to juveniles and after challenging the juveniles with Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica and Bacillus cereus, respectively, was measured using the method of quantitative real-time PCR (qRT-PCR), and then the correlations among different immune-related factors were analyzed. The results showed that the selected immune-related factors were expressed at all of the determined developmental stages and significantly up-regulated at doliolaria stage, suggesting the selected factors are indispensable immune components and the immune system might be broadly activated at doliolaria stage in A. japonicus. After challenged with four pathogenic bacteria, Cu/Zn-SOD, CAT, i-LYZ, cathepsin D, MTF, Toll, C3 were all significantly down-regulated at 4 h, indicating that some components of A. japonicus immune system might be inhibited at the beginning of pathogenic bacteria invasion. The immune-responsive analysis also showed that the significant regulation in Toll after challenged with four tested bacteria, that in MTF after challenged with S. baltica and that in C3 after challenged with P. nigrifaciens were all minus, suggesting Toll, MTF and C3 are probably the primary targets of pathogenic bacteria attack. Furthermore, the correlation analysis indicated that, all of the selected immune-related factors except cathepsin D might be in the same immune regulatory network during A. japonicus development, while all of the selected immune-related factors except c-LYZ might be in the same responsive regulatory network after challenged with four pathogenic bacteria. Altogether, A. japonicus immune system exhibited high complexity in regulation during organism development and after bacterial challenges.

In-depth profiling of miRNA regulation in the body wall of sea cucumber Apostichopus japonicus during skin ulceration syndrome progression.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A. japonicus. A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA-mRNA pairs were identified by target prediction and 314 miRNA-mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti-correlated miRNA-mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune-related processes. Deep analysis of miR-31c-5p, miR-29b-3p, NF-kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation.

Rapid response to changing environments during biological invasions: DNA methylation perspectives.

Dissecting complex interactions between species and their environments has long been a research hot spot in the fields of ecology and evolutionary biology. The well-recognized Darwinian evolution has well-explained long-term adaptation scenarios; however, "rapid" processes of biological responses to environmental changes remain largely unexplored, particularly molecular mechanisms such as DNA methylation that have recently been proposed to play crucial roles in rapid environmental adaptation. Invasive species, which have capacities to successfully survive rapidly changing environments during biological invasions, provide great opportunities to study molecular mechanisms of rapid environmental adaptation. Here, we used the methylation-sensitive amplified polymorphism (MSAP) technique in an invasive model ascidian, Ciona savignyi, to investigate how species interact with rapidly changing environments at the whole-genome level. We detected quite rapid DNA methylation response: significant changes of DNA methylation frequency and epigenetic differentiation between treatment and control groups occurred only after 1 hr of high-temperature exposure or after 3 hr of low-salinity challenge. In addition, we detected time-dependent hemimethylation changes and increased intragroup epigenetic divergence induced by environmental stresses. Interestingly, we found evidence of DNA methylation resilience, as most stress-induced DNA methylation variation maintained shortly (~48 hr) and quickly returned back to the control levels. Our findings clearly showed that invasive species could rapidly respond to acute environmental changes through DNA methylation modifications, and rapid environmental changes left significant epigenetic signatures at the whole-genome level. All these results provide fundamental background to deeply investigate the contribution of DNA methylation mechanisms to rapid contemporary environmental adaptation.

Identification, expression analysis, and the regulating function on C/EBPs of KLF10 in Dalian purple sea urchin, Strongylocentrotus nudus.

Accumulating evidence indicates that Krüppel-like factors (KLFs) play important roles in fat biology via the regulation of CCAAT/enhancer binding proteins (C/EBPs). However, KLFs and C/EBPs have not been identified from Strongylocentrotus nudus, and their roles in this species are not clear. In this study, the full-length cDNA of S. nudus KLF10 (SnKLF10) and three cDNA fragments of S. nudus C/EBPs (SnC/EBPs) were obtained. Examination of tissue distribution and expression patterns during gonadal development implied that SnKLF10 and SnC/EBPs play important roles in gonadal lipogenesis. The presence of transcription factor-binding sites (TFBSs) for KLFs in SnC/EBPs, and the results of an over-expression assay, revealed that SnKLF10 negatively regulates the transcription of SnC/EBPs. In addition, the core promoter regions of SnC/EBPs were determined, and multiple TFBSs for transcription factor (TFs) were identified, which are potential regulators of SnC/EBP transcription. Taken together, these results suggest that SnC/EBP genes are potential targets of SnKLF10, and that SnKLF10 plays a role in lipogenesis by repressing the transcription of SnC/EBPs. These findings provide information for further studies of KLF10 in invertebrates and provide new insight into the regulatory mechanisms of C/EBP transcription.

Comparative study of three phenoloxidases in the sea cucumber Apostichopus japonicus.

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus, the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3'- and 5'-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052, named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs.

Comparative analysis of immunocompetence between females and males in the sea cucumber Apostichopus japonicus.

In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus, the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native-PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell-free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native-PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus, the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus, the coelomocytes might take more immune responsibility in comparison with CCF.

RNA sequencing analysis to capture the transcriptome landscape during skin ulceration syndrome progression in sea cucumber Apostichopus japonicus.

BACKGROUND: Sea cucumber Apostichopus japonicus is an important economic species in China, which is affected by various diseases; skin ulceration syndrome (SUS) is the most serious. In this study, we characterized the transcriptomes in A. japonicus challenged with Vibrio splendidus to elucidate the changes in gene expression throughout the three stages of SUS progression. RESULTS: RNA sequencing of 21 cDNA libraries from various tissues and developmental stages of SUS-affected A. japonicus yielded 553 million raw reads, of which 542 million high-quality reads were generated by deep-sequencing using the Illumina HiSeq™ 2000 platform. The reference transcriptome comprised a combination of the Illumina reads, 454 sequencing data and Sanger sequences obtained from the public database to generate 93,163 unigenes (average length, 1,052 bp; N50 = 1,575 bp); 33,860 were annotated. Transcriptome comparisons between healthy and SUS-affected A. japonicus revealed greater differences in gene expression profiles in the body walls (BW) than in the intestines (Int), respiratory trees (RT) and coelomocytes (C). Clustering of expression models revealed stable up-regulation as the main pattern occurring in the BW throughout the three stages of SUS progression. Significantly affected pathways were associated with signal transduction, immune system, cellular processes, development and metabolism. Ninety-two differentially expressed genes (DEGs) were divided into four functional categories: attachment/pathogen recognition (17), inflammatory reactions (38), oxidative stress response (7) and apoptosis (30). Using quantitative real-time PCR, twenty representative DEGs were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the 20 DEGs ranged from 0.811 to 0.999, which confirmed the consistency and accuracy between these two approaches. CONCLUSIONS: Dynamic changes in global gene expression occur during SUS progression in A. japonicus. Elucidation of these changes is important in clarifying the molecular mechanisms associated with the development of SUS in sea cucumber.