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Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

In order to study the optimal use of Lactobacillus plantarum in sea cucumber ( Apostichopus japonicus ), 49 days feeding trial was conducted to determine the influence of immersion bathing in different concentrations of Lactobacillus plantarum CLY-05 on body weight gain rate and non-specific immune activities. The potential effect of CLY-05 on gut microbiota was also analyzed during the immersion bathing at the optimum concentration. The results showed that the body weight growth rate of all bathing groups was higher than that of control. The highest specific growth rate (4.58%) and weight gain rate (25.35%) was achieved at the bacterial concentration of 1×10 3 CFU/mL. The activities of non-specific immune enzymes (ACP, AKP, SOD and LZM) of all bathing groups increased after immersion bathing, and the enzyme activities of groups bathed with the bacterium at 1×10 3 and 1×10 4 CFU/mL reached the highest. Therefore, 1×10 3 CFU/mL was considered as the optimum concentration of L . plantarum CLY-05 for A . japonicus pond culture. The results of gut microbiota analysis showed that the gut microbiota changed with the addition of L . plantarum CLY-05, and the richness and diversity of the gut microbiota peaked on day 14 and day 21, respectively. The correlation analysis revealed that the non-specific immune enzyme activities were significantly correlated to some gut bacteria (in the phyla Proteobacteria, Firmicutes) after immersion bathing in L . plantarum CLY-05. These findings provide the theoretical foundation for probiotic application in sea cucumber farming.

Perilipins (PLINs) are lipid droplet-associated proteins that regulate lipid storage, mobilization, and metabolism, yet their roles in invertebrates remain poorly characterized. This study aimed to investigate the evolutionary conservation and functional adaptation of PLIN2 in the sea cucumber . Phylogenetic analysis placed PLIN2 within the PLIN2 clade, forming an echinoderm-specific branch distinct from vertebrate PLIN2s. Structural prediction revealed an N-terminal PAT domain containing an amphipathic helix that was required for lipid droplet targeting, as deletion of this region abolished its localization to lipid droplet. Functionally, PLIN2 abundance positively correlated with lipid droplet formation, and its knockdown reduced triacylglycerol accumulation while upregulating lipolysis-related genes. Pull-down and co-immunoprecipitation assays identified interactions between PLIN2 and the endoplasmic reticulum protein ERP44, as well as the mitochondrial protein TRXR2, suggesting a role in lipid droplet-organelle coupling. Consistently, disruption of the PLIN2-TRXR2 module impaired fatty acid transfer from lipid droplets to mitochondria, leading to suppressed β-oxidation and decreased ATP production. In addition, PLIN2 mediates the protective role of lipid droplets against -induced ER stress. Together, these findings establish PLIN2 as a multifunctional lipid droplet-associated protein that coordinates lipid droplet stability with organelle communication, energy metabolism, and ER homeostasis.

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.

Recently, sea cucumber has been an increasingly important marine invertebrate in the fields of EcoDevo and fisheries studies. After the discovery of Sulfitobacter pontiacus strain BL28, which is a growth-promoting probiotic bacteria isolated from blastula of Apostichopus japonicus, host-microbes interaction has become a major new focus of study to be unveiled. Previous genome assessments of the strain BL28 hypothesized involvements of cobalamin/B12 biosynthesis, type IV secretion system, polyhydroxybutyrate production, and sulfate metabolism for host growth promotions. Here, we performed A. japonicus host transcriptome after five weeks of supplementation with S. pontiacus BL28. Unexpectedly, the host transcriptome revealed significant gene regulations of the BL28 supplementation on the host lipid and chondroitin metabolisms and transcriptionally \"rest\" status of heat shock protein genes un-expressed. Physiological and environmental crossover by the S. pontiacus BL28 supplementation may trigger stress-free growth promotion in the host sea cucumber. These symbiotics-type probiotics could help herald the future eco-friendly aquaculture of sea cucumber.

The Philippines is a renowned marine biodiversity hotspot, home to several sea cucumber species with unusual biological traits. Among these, Stichopus cf. horrens is notable for its ability to undergo rapid body wall liquefaction when stressed, coupled with remarkable regenerative abilities. In contrast, Holothuria scabra has one of the most robust body walls in sea cucumbers and thrives in many regimes in the tropics. Despite their intriguing traits, the neurobiology and chemical diversity of these species remain underexplored. Neuropeptides are important components of an animal’s neurobiological toolkit that underlie various physiological and behavioral processes. Thus, the discovery of neuropeptides is a crucial step for understanding the molecular underpinnings of unique traits in sea cucumbers. Leveraging the throughput and sensitivity of tandem mass spectrometry, we obtained an unbiased view of the endogenous peptidomes of radial nerve cord tissues of non-model sea cucumber species, H. scabra and S. cf. horrens . In this work, we sequenced 60 mature peptides from S. cf. horrens that were derived from 22 precursor proteins, and 43 peptides originating from 25 precursor proteins in H. scabra nervous tissues. A total of seven previously unannotated and uncharacterized neuropeptide precursors were identified, thereby expanding the known animal neuropeptide repertoire. Furthermore, we discovered consistent structural features in mature neuropeptides based on the type of post-translational modifications while pushing forward potentially novel proteolytic processing sites during peptide maturation based on the enriched flanking amino acid residues. Collectively, our results provide preliminary data that expand our understanding of echinoderm neurobiology through neuropeptide discovery, potentially paving the way for innovative solutions to address the global demand for echinoderms.

Currently, global climate change and oil pollution are two main environmental concerns for sea cucumber (Apostichopus japonicus) aquaculture. However, no study has been conducted on the combined effects of elevated temperature and oil pollution on sea cucumber. Therefore, in the present study, we treated sea cucumber with elevated temperature (26 °C) alone, water-accommodated fractions (WAF) of Oman crude oil at an optimal temperature of 16 °C, and Oman crude oil WAF at an elevated temperature of 26 °C for 24 h. Results showed that reactive oxygen species (ROS) level and total antioxidant capacity in WAF at 26 °C treatment were higher than that in WAF at 16 °C treatment, as evidenced by 6.03- and 1.31-fold-higher values, respectively. Oxidative damage assessments manifested that WAF at 26 °C treatment caused much severer oxidative damage of the biomacromolecules (including DNA, proteins, and lipids) than 26 °C or WAF at 16 °C treatments did. Moreover, compared to 26 °C or WAF at 16 °C treatments, WAF at 26 °C treatment induced a significant increase in cellular apoptosis by detecting the caspase-3 activity. Our results revealed that co-exposure to elevated temperature and crude oil could simulate higher ROS levels and subsequently cause much severer oxidative damage and cellular apoptosis than crude oil alone on sea cucumber.

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.

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus , markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Background: Tissue regenerative capacity following evisceration, potentially influenced by environmental contaminants and intestinal microflora, is essential for the financial success of Apostichopus japonicus farming. However, the morphological structure, gut microbiome composition, and genes expression pattern of the regenerated gut after exposure to high levels of TBBPA remain poorly unclear. Methods: In this research, the effect of TBBPA exposure on tissue regeneration in A. japonicus was investigated through a comprehensive multi-omics approach. Results: Our results showed that the integrity, the intestinal wall thickness, and the villi length of the regenerated intestines in A. japonicus decreased after treatment with high levels of TBBPA. The findings from PCoA and NMDS analyses revealed that the microbial community composition was significantly altered following exposure to high concentrations of TBBPA in the regenerated intestines of A. japonicus. The KEGG pathway enrichment analysis indicated that the DEGs (differentially expressed genes) were predominantly enriched on metabolism and immunity-related signaling pathways after exposure to high levels of TBBPA. These included pathways involved in the PPAR signaling pathway, ECM receptor interaction, glycerolipid metabolism, and fatty acid degradation. Interestingly, the results have demonstrated that there are 77 transcript factors that were significantly different after exposure to TBBPA. Conclusions: These results suggested that high levels of exposure to TBBPA induces an imbalance of the metabolic homeostasis by regulating the expression levels of transcription factors in the regenerated intestines of A. japonicus.