A peek into the population genetics of white teatfish (Holothuria fuscogilva) of Kenya's south coast.

BACKGROUND: The white teatfish, Holothuria fuscogilva, is widely distributed in coastal areas, including waters around coral reefs and seagrasses in the Indo-Pacific. In Kenya, the species is distributed in shallow reefs with higher landings reported from the Vanga-Shimoni-Gazi seascape on the Kenyan south coast. Despite its high exploitation for export and its vulnerable and endangered statuses under IUCN and CITES respectively, Kenya's H. fuscogilva populations and how they may have been impacted by the fishing pressure have not been studied. METHODS: We estimated the genetic diversity and structure of H. fuscogilva population conveniently sampled from three sites in Kenyan south coast using the mitochondrial cytochrome oxidase subunit I (COI) gene sequences. We recorded 30 haplotypes with 43 polymorphic sites across the population. Furthermore, we estimated an overall high haplotype diversity and low nucleotide diversity of estimates of h = 0.970 ± 0.013 and π = 0.010 ± 0.001 respectively. CONCLUSIONS: These preliminary findings suggest several population outcomes, among them a fit population, which require confirming with more comprehensive study to inform strategies for the sustainable exploitation and management of the species.

Transcriptomic analysis reveals the early body wall regeneration mechanism of the sea cucumber Holothuria leucospilota after artificially induced transverse fission.

BACKGROUND: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. RESULTS: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-ß, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. CONCLUSION: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

Barcoding (COI) Sea Cucumber Holothuria mammata Distribution Analysis: Adriatic Rare or Common Species?

The overexploitation of the western Pacific Ocean has expanded the sea cucumber fishery into new regions to supply the Asian market. In 2013, sea cucumbers were removed from the Croatian marine protected species list, and commercial fishery took place for a short period (2017-2018) in the Eastern Adriatic Sea. However, holothuroid species are difficult to distinguish. Holothuria mammata is a species that has rarely been reported in this region and strongly resembles the common species Holothuria tubulosa. This is the first study to assess the genetic diversity of sea cucumbers in the Adriatic Sea using genetic barcoding of the mitochondrial gene cytochrome c oxidase subunit 1 (COI). Specimens for barcoding were collected from the northern and central Adriatic, along with a specimen that had been previously identified as H. sp. cf. mammata based on its morphological characteristics. While genetic analyses showed identified this specimen as H. tubulosa, 30% of the collected specimens were genetically identified as H. mammata. These results call into question the historically accepted sea cucumber assemblage in the Adriatic Sea, which regarded H. mammata as a rare species and generally disregarded its presence in large census studies. Such species distribution data are extremely important in developing and monitoring a sustainable fishery.

Metabolome and Transcriptome Association Analysis Reveals the Link Between Pigmentation and Nutrition Utilization in the Juveniles of Sea Cucumber Holothuria leucospilota.

The sea cucumber Holothuria leucospilota is an economically and ecologically important tropical species. Following development into juveniles, H. leucospilota undergoes a color change from white to black, involving a pigmentation process for over a period of several months. In this study, a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Next-Generation sequencing (NGS) were employed to investigate the changes in metabolomic and transcriptomic profiles during pigmentation in H. leucospilota juveniles. The metabolomic analysis identified a total of 341 metabolites, of which 52 were found to be differentially regulated (P < 0.05 and VIP > 1), with 27 being upregulated in white individuals and 25 in black individuals. Additionally, 632 differentially expressed genes (DEGs) were identified, with 380 genes upregulated in white samples and 252 genes upregulated in black samples. Interestingly, the melanin content and tyrosinase transcript levels did not display significant differences between the two groups. Metabolomic data suggested the involvement of the linoleic acid metabolic pathway in pigmentation. Transcriptomic analysis, coupled with realtime PCR validation, revealed a decrease in the transcript levels of digestive enzymes like α-amylase, maltase-glucoamylase, and trehalase after the juveniles changed to black. Furthermore, the mRNA expressions of major yolk proteins showed a decline, indicating a shift in the accumulation of protein nutrient sources. Overall, our findings suggest that during the pigmentation process in H. leucospilota, no significant changes were observed in the classical melanin pathway, while notable alterations were observed in their nutritional status. This study provides valuable insights into the regulatory mechanisms of pigmentation in marine organisms.

Applications of Environmental DNA (eDNA) in Monitoring the Endangered Status and Evaluating the Stock Enhancement Effect of Tropical Sea Cucumber Holothuria Scabra.

The tropical sea cucumber Holothuria scabra is naturally found in the Indo-West Pacific. However, due to their commercial value, natural H. scabra populations have declined significantly in recent years, resulting in its status as an endangered species. Surveys of H. scabra resource pose a challenge due to its specific characteristics, such as sand-burrowing behavior. To overcome this problem, our study established a convenient and feasible method for assessing H. scabra resources using environmental DNA (eDNA) monitoring technology. First, H. scabra-specific TaqMan primers and probe were designed based on its cox1 gene, followed by the development of an eDNA monitoring method for H. scabra in two separate sea areas (Xuwen and Daya Bay). The method was subsequently employed to investigate the distribution of H. scabra and assess the effects of aquaculture stock enhancement through juvenile releasing in the Weizhou Island sea area. The H. scabra eDNA monitoring approach was found to be more appropriate and credible than traditional methods, and a positive impact of stocking on H. scabra populations was observed. In summary, this is the first report to quantify eDNA concentration in a Holothuroidea species, and it provides a convenient and accurate method for surveying H. scabra resources. This study introduces novel concepts for eDNA-based detection of endangered marine benthic animals and monitoring their population distribution and abundance.

The Holothuria leucospilota genome elucidates sacrificial organ expulsion and bioadhesive trap enriched with amyloid-patterned proteins.

Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-ß structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.

Isolation and characterization of 21 microsatellite loci for the sea cucumber Holothuria (Halodeima) atra (Echinodermata, Holothuroidea) reveal low asexual propagation through time in Reunion Island (southwestern Indian Ocean).

BACKGROUND: Holothuria (Halodeima) atra Jaeger, 1833 is a tropical sea cucumber usually harvested for the "bêche-de-mer" trade market. It has been reported to reproduce both sexually, through gamete spawning, or asexually, through fission. To date, no study has ever investigated clonality, nor genetic connectivity, among its populations, using microsatellite markers. METHODS AND RESULTS: We isolated the first 21 microsatellite loci specific for H. atra, which were then used to investigate clonal diversity, genetic structure and diversity among 44 H. atra individuals sampled in Reunion Island (southwestern Indian Ocean), over two seasons. All 21 loci were polymorphic, with number of alleles per locus ranging from 2 to 10. No repetitive multi-locus genotype (MLG) and few clonal lineages (MLL) were found. Observed heterozygosities per locus and season ranged from 0.000 to 0.909, while expected heterozygosities ranged from 0.290 to 0.882. Four loci were at Hardy-Weinberg equilibrium for both seasons, all others presenting a deficit of heterozygotes in one or both seasons. Meanwhile, no genetic differentiation was detected between seasons, according to assignment tests and global FST. CONCLUSIONS: These results suggest low asexual propagation in this population. These loci represent useful tools to better understand reproductive strategies and population connectivity of H. atra, and thus provide relevant knowledge for efficient management.

De novo genome assembly and annotation of Holothuria scabra (Jaeger, 1833) from nanopore sequencing reads.

BACKGROUND: Holothuria scabra is a costly gourmet and traditional Chinese tonic medicine. However, the lack of high-quality genome information hinders the genetic, phylogenetic, and bioactivator researches. OBJECTIVE: To construct high-quality genomic data of H. scabra and conduct genome-wide phylogenetic analysis. METHODS: The whole genome of a male H. scabra was sequenced based on Nanopore MinION platform, and the sequence was assembled by wtdbg2. Transcriptome sequencing was used to aid the gene annotation. Repeat sequences, non-coding RNA, pseudogene and gene functional annotation were analyzed. 750 single-copy gene families from ten species were applied to construct phylogenetic tree for evolutionary analysis by using the ML method. RESULTS: The H. scabra genome of 1.18 Gb (N50 = 1557,492 bp) with 500.42 Mb of putative repetitive sequences was assembled from a male H. scabra individual, and 16,642 protein-coding genes, 951 pseudogenes, 1791 motifs and 45,400 domains from the generated assembly were identified. The divergence time between H. scabra and its ancestor was estimated approximately 192.6 million years ago. H. scabra and A. japonicas joined together while sea urchin and sea star diverged about 440 Mya ago. Some key genes involved in notochord and gill slits development, skeleton degeneration and nervous system, as well as homeobox genes differ between H. scabra and Apostichopus japonicas. CONCLUSION: We report the first whole genome of H. scabra with expectation that this will be a valuable resource for genetic, phylogenetic, breading, molecular biology and bioactivator studies of sea cucumbers and other invertebrates.

Saving the sea cucumbers: Using population genomic tools to inform fishery and conservation management of the Fijian sandfish Holothuria (Metriatyla) scabra.

The sea cucumber Holothuria (Metriatyla) scabra, known as sandfish, is a high-value tropical echinoderm central to the global bêche-de-mer (BDM) trade. This species has been heavily exploited across its natural range, with overharvesting and ineffective fishery management leaving stocks in the Pacific region heavily depleted. In Fiji, sandfish stocks have not recovered since a 1988 harvest ban, with surveys reporting declining populations and recruitment failure. Therefore, to inform fishery management policy for the wild sandfish resource and to guide hatchery-based restocking efforts, a high-resolution genomic audit of Fijian populations was carried out. A total of 6,896 selectively-neutral and 186 putatively-adaptive genome-wide SNPs (DArTseq) together with an independent oceanographic particle dispersal model were used to investigate genetic structure, diversity, signatures of selection, relatedness and connectivity in six wild populations. Three genetically distinct populations were identified with shallow but significant differentiation (average Fst = 0.034, p≤0.05), comprising (1) Lakeba island (Lau archipelago), (2) Macuata (Vanua Levu), and (3) individuals from Yasawa, Ra, Serua island and Kadavu comprising the final unit. Small reductions in allelic diversity were observed in marginal populations in eastern Fiji (overall mean A = 1.956 vs. Lau, A = 1.912 and Macuata, A = 1.939). Signatures of putative local adaptation were also discovered in individuals from Lakeba island, suggesting that they be managed as a discrete unit. An isolation-by-distance model of genetic structure for Fijian sandfish is apparent, with population fragmentation occurring towards the east. Hatchery-based production of juveniles is promising for stock replenishment, however great care is required during broodstock source population selection and juvenile releases into source areas only. The successful use of genomic data here has the potential to be applied to other sea cucumber species in Fiji, and other regions involved in the global BDM trade. While preliminary insights into the genetic structure and connectivity of sandfish in Fiji have been obtained, further local, regional and distribution-wide investigations are required to better inform conservation efforts, wild stock management and hatchery-based restocking interventions for this valuable invertebrate.

Holothuria Leucospilota polysaccharides alleviate hyperlipidemia via alteration of lipid metabolism and inflammation-related gene expression.

Hyperlipemia is becoming a chronic disease that threatens human health. At the same time, people pay more and more attention to hyperlipemia. Holothuria Leucospilota polysaccharide (HLP) has been reported to ameliorate hyperlipidemia in high-fat diet-induced rats. Therefore, this study aimed to explore further metabolomics' role in improving liver function and reveal its mechanism. After oral administration of HLP for 4 weeks, total cholesterol (TC) and triglycerides (TG) levels of the liver in 100 and 200 mg/kg HLP groups were both decreased significantly (p < .05). The results showed that serum AST and ALT activity decreased by professing to be convinced of HLP. HLP also exerted antioxidant activities and up-regulated the expression of ACC, CD36, TNF-α and NF-κB in the liver of diabetic rats. Six potential biomarkers were recognized by UPLC-Q-TOF/MS and OPLS-DA. HLP alleviated liver injury by regulating the contents of metabolic end products in the serum of hyperlipidemic rats, such as nadolol and glycodeoxycholic acid. The results indicated that HLP effectively relieved HFD-induced hyperlipidemia by regulating metabolic disorders. PRACTICAL APPLICATIONS: As a chronic disease, hyperlipidemia has attracted more and more attention. Studies have shown that HLP regulates dyslipidemia, oxidative damage and inflammation to relieve hyperlipidemia. It mainly improved the liver damage caused by hyperlipidemia by inhibiting the expression of hepatic lipogenesis, oxidative stress and inflammatory factors. At the same time, we also detected six metabolites, among which high GDCA content indicated serious liver damage. Therefore, in the future, it can be suggested that HLP may be used as a functional, active substance in health products to assist in relieving hyperlipidemia, and GDCA may be used as an essential metabolic marker for the degree of liver injury.

Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota.

Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota, are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 (Hl-CASP9) cDNA in the sea cucumber, Holothuria leucospilota. Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H2O2), nitric oxide (•NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo. Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates.

Transcriptome analysis of growth variation in early juvenile stage sandfish Holothuria scabra.

The sandfish Holothuria scabra is a high-value tropical sea cucumber species representing a major mariculture prospect across the Indo-Pacific. Advancements in culture technology, rearing, and processing present options for augmenting capture production, stock restoration, and sustainable livelihood activities from hatchery-produced sandfish. Further improvements in mariculture production may be gained from the application of genomic technologies to improve performance traits such as growth. In this study, we performed de novo transcriptome assembly and characterization of fast- and slow-growing juvenile H. scabra from three Philippine populations. Analyses revealed 66 unigenes that were consistently differentially regulated in fast-growing sandfish and found to be associated with immune response and metabolism. Further, we identified microsatellite and single nucleotide polymorphism markers potentially associated with fast growth. These findings provide insight on potential genomic determinants underlying growth regulation in early juvenile sandfish which will be useful for further functional studies.

Comparative transcriptome analysis reveals changes in gene expression in sea cucumber (Holothuria leucospilota) in response to acute temperature stress.

Ambient temperature is an important abiotic factor that influences growth performance and physiological functions in sea cucumbers. To understand the molecular responses of the sea cucumber Holothuria leucospilota to acute temperature stress, we performed a de novo transcriptome analysis of body wall tissue from H. leucospilota exposed to 2 hoursh of acute heat (35 ± 1 °C) and cold stress (15 ± 1 °C). A total of 99,015 unigenes were obtained after assembly of the sequenced reads. Compared with a control group maintained at 25.0 ± 1 °C, 1169 differentially expressed unigenes (DEGs) were identified after heat stress, 781 were up-regulated and 388 were down-regulated. After cold stress, 1464 DEGs were identified; 900 were up-regulated and 564 were down-regulated. The annotation of DEGs revealed that heat shock proteins play important roles in protecting H. leucospilota from high temperature stress. Furthermore, KEGG pathway enrichment analysis showed that the categories: "Ribosome" and "Protein processing in endoplasmic reticulum" were strongly affected by heat stress. These two pathways are associated with biosynthesis and processing of proteins, and refolding of misfolded proteins. The lipid metabolism pathways "Sphingolipid metabolism" and "Ether lipid metabolism", were affected by cold stress. The RNA-Seq results for eight selected DEGs were verified the expression by quantitative real-time PCR analysis. Our results will improve the understanding of the molecular response mechanisms of H. leucospilota to ambient temperature stress.

Transcriptomic analysis of early stages of intestinal regeneration in Holothuria glaberrima.

Echinoderms comprise a group of animals with impressive regenerative capabilities. They can replace complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied. The molecular machinery behind this faculty, however, remains to be understood. Here we assembled and annotated a de novo transcriptome using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 h and 3 days regenerating intestine, revealing a large number of differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with mechanisms involving developmental and cancer-related activity that could be related to the regenerative process. Our study demonstrates the broad and diversified gene expression profile during the early stages of the process using the mesentery as the focal point of intestinal regeneration. It also establishes the genes that are the most important candidates in the cellular processes that underlie regenerative responses.

Molecular characterization, inducible expression and functional analysis of an IKKß from the tropical sea cucumber Holothuria leucospilota.

The inhibitory kappa B kinase (IKK) is a critical regulator for the nuclear factor-κB (NF-κB) pathway. In this study, an IKKß named as HLIKKß was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKß is 4246 bp in size, containing a 132 bp 5'-untranslated region (UTR), a 1783 bp 3'-UTR and a 2331 bp open reading frame (ORF) encoding a protein of 776 amino acids with a deduced molecular weight of 89.66 kDa. HLIKKß contains a kinase domain (KD) at its N-terminal, a leucine zipper (LZ) and a helix-loop-helix (HLH) motif at its C-terminal. In the KD, a conserved active loop (SXXXS) were identified. The results of luciferase reporter assay and ELISA assay showed that over-expressed HLIKKß in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1ß. When HLIKKß was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKß. Moreover, the up-regulation of HLIKKß mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKß might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections.

Microscopic ossicle analyses and the complete mitochondrial genome sequence of Holothuria (Roweothuria) polii (Echinodermata; Holothuroidea) provide new information to support the phylogenetic positioning of this sea cucumber species.

Sea cucumbers (Holothuroidea) are ecologically important organisms for their bioturbation and alkalinization activities of the seabed. These species are extensively fished as they are considered luxury food. Sea cucumbers are also relevant for biomedical studies and the production of bioactive compounds. A few initiatives are recently evaluating sea cucumbers as novel aquaculture species. The aim of this study was to provide morphological and genetic information useful for the identification of Holothuria polii, the white spot sea cucumber (a common species of the Mediterranean Sea). We generated the complete sequence of the mitochondrial DNA (mtDNA) genome of this species and combined it with a detailed ossicle characterization of the sequenced specimen by scanning electron microscopic analysis. Ossicles (known also as sclerites) are anatomical features that can discriminate Holothuroidea species, including the closely related ones of the genus Holothuria. The complete mitochondrial genome was assembled, functionally annotated and then used to evaluate the phylogenetic relationship of H. polii against the other few Holothuroidea species for which the whole mtDNA was available. The 15,907 bp H. polii mtDNA sequence has the same gene order already reported for H. scabra, H. forskali and other species of the same class. Cox1 and 16S gene sequences were informative for species identification across the genus and could be used for the authentication of commercialized Holothuria spp. The mitochondrial genome sequence presented here provides the basis to a future analysis of the variability of H. polii populations in the Mediterranean region.

The first cloned echinoderm tumor necrosis factor receptor from Holothuria leucospilota: Molecular characterization and functional analysis.

In this study, an echinoderm tumor necrosis factor receptor named HLTNFR-16 was first cloned from the tropical sea cucumber Holothuria leucospilota. The full-length cDNA of HLTNFR-16 is 3675 bp in size, containing a 415 bp 5'-untranslated region (UTR), a 2024 bp 3'-UTR and a 1236 bp open reading frame (ORF) encoding a protein of 411 amino acids with a deduced molecular weight of 45.63 kDa. The HLTNFR-16 protein contains a signal peptide, four TNFR domains (the last three were identified as extracellular cysteine-rich domains), a transmembrane region and a death domain. Phylogenetic analysis showed that HLTNFR-16 was clustered into a clade with TNFR-16s in other species, indicating that this echinoderm TNFR may be a new member of the TNFR-16 subfamily. The results of TUNEL assay showed that the over expression of HLTNFR-16 could induce apoptosis in HEK293T cells. When HLTNFR-16 was silenced by siRNA, the apoptosis of sea cucumber coelomocytes induced by inactivated Vibrio harveyi was suppressed significantly, indicating that HLTNFR-16 is important for apoptosis induction. Additionally, luciferase reporter assay exhibited that the over-expressed HLTNFR-16 in HEK293T cells could activate the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Moreover, the secretion of proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-18 in HEK293T cells was increased by the over-expression of HLTNFR-16. This study provides evidences for the potential roles of sea cucumber TNFR in the innate immunity.

Identification of neuropeptides in the sea cucumber Holothuria leucospilota.

Neuropeptides play important roles in the regulation of physiological processes such as growth, metabolism and reproduction. In sea cucumbers (Phylum Echinodermata), numerous neuropeptides have been identified and some are attributed to reproductive processes. In this study, our goal was to gain a better understanding of the neuropeptide repertoire for the black sea cucumber Holothuria leucospilota, a species that has been severely overfished from the wild due to human consumption. We applied in silico transcriptome analysis of the adult H. leucospilota radial nerve cord, gonad and body wall to elucidate 35 neuropeptides that are conserved throughout the Bilateria. Then, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of radial nerve cord was employed and showed an additional 8 putative novel neuropeptide precursors, whose predicative cleaved peptides do not share sequence similarity with any reported neuropeptides. These data provide an important basis for experimental approaches to manipulate H. leucospilota broodstock reproduction and growth in culture, which will hopefully re-establish population numbers.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis.

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5'-untranslated region (UTR), a 1334-bp 3'-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.