Holothuria Leucospilota Research Articles

Molecular cloning, expression and functional analysis of NF-kB1 p105 from sea cucumber Holothuria leucospilota

The nuclear factor-κB (NF-κB) family is evolutionary conserved and plays key roles in the regulation of numerous basic cellular processes. In this study, a sea cucumber Holothuria leucospilota NF-κB1 p105 named HLp105 was first obtained. The full-length cDNA of HLp105 is 6564 bp long, with a 219 bp 5′ untranslated region (UTR), a 2979 bp 3′ UTR, and a 3366 bp open reading frame (ORF) encoding for 1121 amino acids with a deduced molecular weight of 123.92 kDa and an estimated pI of 5.31. HLp105 protein contains the conserved domain RHD, IPT, ANK and DEATH. HLp105 mRNA can be detected in all tissues examined, with the highest level in the intestine, followed by the transverse vessel, rete mirabile, coelomocytes, respiratory tree, bolishiti, cuvierian tubules, body wall, oesophagus and muscle. Challenged by LPS or poly (I:C), the transcription level of HLp105 was apparently up-regulated in the tissues examined. Besides, Over-expression of HLp105 in HEK293T cells, the apoptosis was inhibited, and the cytokines IL-1β and TNF-α were activated. The results are important for better understanding the function of NF-κB1 p105 in sea cucumber and reveal its involvement in immunoreaction.

Checklist of Marine Fungi and Yeasts Associated with Holothuria (Mertensiothuria) leucospilota from Pangkor Island

Marine microorganisms such as fungi and yeasts can adapt to extreme marine environment conditions and play different roles especially in the nutrient cycling and as bioindicator of ocean change. This study was carried out to isolate and identify fungi and yeasts associated with Holothuria (Mertensiothuria) leucospilota from Pangkor Island, Perak, Malaysia in order to determine their species richness. Two specimens of H. leucospilota were collected from Giam Island and Teluk Nipah Beach of Pangkor Island. Nine samples of fungi and ten samples of yeasts were isolated from the internal and external parts of the H. leucospilota specimens such as cuticle, tentacle, coelomic fluid, cloaca, cuvierian tubules, and surrounding sediment and seawater. Polymerase Chain Reaction (PCR) and DNA sequencing of the Internal Transcribed Spacer (ITS) region were applied for species identification of the microorganisms. Sequence analyses of the ITS region resulted in the identification of five genera of fungi i.e. Cladosporium, Curvularia, Polyporaceae, Acremonium, and Penicillium; and four genera of yeasts i.e. Sterigmatomyces, Pichia, Debaryomyces, and Candida with some of them could be identified up to the species level. The findings have significantly contributed to the recentinformation on the checklist of fungi and yeasts isolated from the H. leucospilota specimens from Pangkor Island.

Molecular characterization, inducible expression and functional analysis of an IKKβ 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 IKKβ named as HLIKKβ was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKβ 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. HLIKKβ 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 HLIKKβ in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1β. When HLIKKβ was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKβ. Moreover, the up-regulation of HLIKKβ mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKβ might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections.

EXPLORATION OF SEA CUCUMBER INTESTINAL SYMBIONT MICROBE AS PROBIOTIC MICROBE CANDIDATE IN HEALTHCARE PRODUCTS

Natural ingredients from animals and plants generally have potential in pharmaceutical applications. However, there are still relatively little exploration made on the active ingredients from marine life. Sea cucumbers inhabit seabed and are filter feeders, consuming all microorganisms within their vicinity, which opens the prospect of medicinal applications of sea cucumber intestinal symbiont microbes. This study aims to obtain sea cucumber intestinal symbiont microbes with potential pharmaceutical application as probiotic microbe candidate. The study was carried out by collecting samples, microbe isolation, antimicrobial screening, identification of potential probiotic microbe candidate. Characteristics of the microbe were studied by biochemical screening, and molecular identification . The study found 21 microbial isolates from Holothuria atra and 30 microbial isolates from Holothuria leucospilota . Antimicrobial activity screening results against pathogens of Bacillus cereus dan Pseudomonas aeruginosa found 13 microbial isolates with positive activity. Four species were identified by molecular identification, namely Bacillus aquimaris , Bacillus maritimus , Bacillus toyonensis , and Virgibacillus chiguensis, whereas the other nine were identified by biochemical screening. Four microbe genus, namely Rothia sp., Listeria sp., Micrococcus sp., and Staphylococcus sp, were found to be the most viable candidate for probiotics. It was concluded that Bacillus sp., Rothia sp., Micrococcus sp., and Staphylococcus sp. Exhibited the most potential as probiotic microbes.

Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge

BackgroundThe sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq.ResultsSea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 μg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 μg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h.ConclusionA de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 μg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.

Antifouling and antibacterial activities of bioactive extracts from different organs of the sea cucumber Holothuria leucospilota

In this study, antifouling and antibacterial properties of polar, semi polar and non-polar bioactive compounds derived from the sea cucumber Holothuria leucospilota were investigated. A series comprising n-hexane, ethyl acetate and methanol extracts from four different organs of sea cucumber (body wall, gonad, digestive tract and respiratory tree) were investigated for their antibacterial and antifouling activity against two species of microalgae, larvae of barnacle Amphibalanus amphitrite and a non-target organism: larvae of Artemia salina. Results showed that the highest antibacterial activity was found in the ethyl acetate extract of body wall against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 0.250 mg/mL. The MIC of ethyl acetate extract of body wall ranged between 0.062 and 0.250 mg/mL against two microalgal strains. All extracts showed moderate to low toxicity against larvae of barnacles. The most pronounced anti-barnacle activity was found in the ethyl acetate extract of body wall with median lethal concentration (LC50) value of 0.049 mg/mL. Among the twelve extracts, the ethyl acetate extracts of body wall showed the maximum antibacterial and antifouling activities. The bioactive compounds of the ethyl acetate extract of the sea cucumber body wall were determined using gas chromatography coupled with mass spectrometry. Fatty acids and terpenes were the major compounds, which may be responsible for antibacterial and antifouling activity of the ethyl acetate extract of body wall of the sea cucumber H. leucospilota.

Angiotensin-Converting Enzyme (ACE) Inhibitory Activities of Protein Hydrolysates from Indonesian Sea Cucumbers

Hypertension is one of the risk factors that contribute significantly to the prevalence of cardiovascular diseases in Indonesia. Peptides have been known to exhibit anti-hypertension activities, thus are potential as an alternative treatment of hypertension. In this study, we collected three species of under-utilized sea cucumbers from Lampung and Gorontalo provinces to explore their hydrolysates as angiotensin-converting enzyme (ACE)-inhibitors. The identification of the collected samples was conducted by a molecular approach. The sea cucumber hydrolysates were produced by using bromelain, alcalase, or the combination of both enzymes. The hydrolysis process was carried out using a 50 mM phosphate buffer at pH 7 with E/S ratio of 1% at 45 °C for 24 h. The fractionation of the most active protein hydrolysate was conducted using ultrafiltration membranes with 3, 5, and 10 kDa cut-offs. The results showed that the collected sea cucumbers were identified as Holothuria atra, Holothuria leucospilota, and Bohadschia marmorata, respectively. The ACE inhibitory analysis revealed that the alcalase hydrolysates of H. atra (AHHa) contain the most active ACE inhibitor with an IC50 value of 0.32 mg/mL. Fraction AHHa IV (< 3 kDa) showed the highest inhibition toward ACE compared to the other fractions. The kinetic studies of unfractionated AHHa and AHHa IV indicated that both peptides inhibited ACE in a mixed-inhibition mode. The lower Ki value of unfractionated AHHa peptides than that of AHHa IV, suggesting that the former binds stronger to ACE than the latter.

Community Structure of Sea Cucumber (Holothuroidea) In Pombo Island Conservation Area, Central Maluku

Sea cucumbers (Holothuroidea) are marine animals that is highly nutritious for human consumption, commercially important, and serve a useful role in the marine ecosystem Less optimal monitoring in Pombo Island Conservation Area and the activity of catching sea cucumbers by the surrounding community, then the aim of this research is to find out community structure of sea cucumber in Pombo Island Conservation Area, Central Maluku. It is a descriptive quantitative research. The research was conducted from October 28, to November 28, 2018 by using linear quadratic transect method. Based on the result of the research, it can be concluded that there were 208 individuals which consists of 1 family (Holothuroidea) and 3 species (Holothuria atra, Holothuria leucospilota, Holothuria nobilis). The most observed species is Holothuria atra with a total of 111 individuals whereas the least observed species is Holothuria nobilis with a total of 42 individuals. Furthermore, Holothuria atra was the species with highest density, abundance, frequency of occurance, and important value index while the lowest is Holothuria nobilis. The diversity index value is in medium category at 1.0086, while the dominance index value is close to 0 (0.3385) or no species dominates.

Characteristics of environment and habitat of sea cucumbers in Pane Island, Tapanuli Tengah Regency

Pane Island is one of the tourist destinations in Tapanuli Tengah Regency, visually Pane Island has some of marine ecosystems such as seagrass, mangrove, seaweed, and dead coral ecosystems. Sea cucumbers are invertebrates, it’s have high commercial value and has ecological functions. In this study, sea cucumbers identification, abundance and seagrass cover was investigated in Pane Island. The results showed that 1 sea cucumber species were found, Holothuria leucospilota. Sea cucumber abundance ranged from 0 to 1.2 ind/m2. Based on Principal Components Analysis (PCA), sea cucumber abundance is positively related to temperature, current, pH, salinity, total nitrogen, phosphate, and C-organic content. Furthermore, sea cucumber abundance is negatively related to the parameters of water depth, water transparency, DO and percentage of seagrass cover.

Antioxidant Activity of Bioactive Peptides Extracted from Sea Cucumber (Holothuria leucospilata)

Sea cucumber (Holothuria leucospilota) is a native species of Echinodermata that has long been used as traditional Asian medicine. This study was conducted for the evaluation of the antioxidant property of hydrolyzed protein with molecular weight of < 30-KDa extracted from H. leucospilota using Alcalase and Flavourzyme enzymes. Antioxidant activity was evaluated by DPPH radical scavenging assay and ferric reducing antioxidant power. The results of antioxidant experiments showed that with increasing concentration of hydrolyzed protein (both enzymes), the percentage of DPPH scavenging and ferric reducing increased and there was a significant difference between the different concentrations studied (P < 0.05). The inhibitory effects of the peptides extracted by the Alcalase were higher than that of Flavourzyme. It can be concluded that a hydrolyzed protein with a molecular weight < 30-KDa can be used as a natural source of antioxidant compounds in the pharmaceutical and food industries.

Species Distribution, Diversity, and Abundance of Sea Cucumbers in Tropical Intertidal Zones of Aurora, Philippines

The distribution, diversity, and abundance of sea cucumbers in intertidal zones of Aurora were studied from February to June 2013 to obtain baseline data. A one-hour timed-search survey covering an approximate one kilometer over a ten-meter wide area was conducted in six coastal municipalities to obtain a comprehensive list of sea cucumber species found in the zones. Only four sampling stations (Baler, Dipaculao, Dilasag, and Dingalan) were selected for belt transect survey, 50 × 50 m, based on the availability of coralline, sandy, muddy sand, and rocky habitats which were laid perpendicular and parallel to each intertidal zone. Timed-search survey revealed a total of 15 species of sea cucumbers distributed among two Orders (Order Aspidochirotida and Order Apodida) and four Families (Family Holothuriidae, Family Stichopodidae, Family Synaptidae, and Family Chirotidae) were recorded. Synapta maculata is most common and has widespread distribution. Belt-tranect surveys showed Holothuria leucospilota is among the most frequently occurring species both during day time (58%) and night time (75%). The Dilasag sampling station had the most diverse species (0.71) while the Dipaculao sampling station had the densest species of Holothuria leucospilota (10,014 in∙ha−1) and abundant (95.91%) species.

Chemotaxonomic study of the most abundant Egyptian sea-cucumbers using ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS)

Actinopyga mauritiana, Bohadschia marmorata, Holothuria leucospilota, H. edulis, H. atra and H. polii are the abundant sea-cucumbers inhabiting either Red or Mediterranean Sea shore in Egypt. The aim of this study was to elucidate the metabolic content in the tegument of the selected sea-cucumbers using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC/HRMS). The identified metabolites included sulfated saponins such as holothurin B1 and 24-dehydroechinoside A and non-sulfated saponins such as bivittoside C and D. Our results showed that B. marmorata is substantially different from all the other species and occupies alone one taxon. Consequently, it can be suggested to reclassify the family Holothuriidae into three subfamilies. The first suggested comprising the genera, Holothuria and Actinopyga, the second includes only genus Bohadschia, whereas the third includes only genus Pearsonothuria. Lipidomic analysis of sea-cucumbers showed high variation in saturated and unsaturated fatty acids (USFAs) between the collected species. H. edulis and B. marmorata showed the highest amount of all identified SFAs. Among the USFAs, palmitoleic, docosatrienoic and linoleic acid were detected in varied amount in most species.

Holothuroidea as a Constituent of Benthic Communities in the Seagrass Ecosystems at Bira Island Islands

Abstact. Seagrass ecosystem has an important ecological role for the life of marine organisms and as one of the suitable habitats for the holothuroidea group, known as sea cucumbers. One of the distribution areas of Holothuroidea in Indonesia is Pulau Bira Kepulauan Seribu. this study were undertaken to investigate the composition, abundance and species diversity of the holothuroidea as constituents of benthic communities, and to determine the quality of waters in the seagrass ecosystem. Descriptive method with survey design was used in this study. Data analysis was carried out descriptively based on the calculation of abundance, composition, and diversity, while the analysis of environmental parameters affecting holothuroidea diversity was carried out by principal component analysis (PCA). The results showed as many as 3 species were identified as Holothuriidae, with the highest to lowest abundance consecutive namely Holothuria atra (1.77 ind/m2), Holothuria leucospilota (0.16 Ind/m2), and Holothuria fuscocinerea (0.10 Ind/m2) with the composition of 86.96%, 7, 90% and 5, 14% respectively. Species diversity is classified as low criteria, while the environmental parameters that affect species diversity are dissolved oxygen, turbidity, orthophosphate and Biologycal Oxygen Demand (BOD). Although some environmental parameters were still in the normal condition, analysis of the community structure of Holoturoidea indicates a decline in water quality.

Biotechnological Potential of Bacteria Isolated from the Sea Cucumber Holothuria leucospilota and Stichopus vastus from Lampung, Indonesia.

In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS1) as well as product/daughter ions (MS2) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential.

Saponin-rich extracts from Holothuria leucospilota mediate lifespan extension and stress resistance in Caenorhabditis elegans via daf-16.

Saponins are secondary metabolite compounds that can be found in sea cucumbers (Holothuroidea spp.). However, little is known about how saponin-rich extracts from Holothuria leucospilota can delay and prolong the lifespan of the whole organism. In this study, anti-aging effects of H. leucospilota extracts were studied on Caenorhabditis elegans. NMR analysis revealed that body wall n-butanol-extract of H. leucospilota (BW-BU) is saponin-rich. BW-BU extracts exhibited antioxidant activities by 2,2'-diphenyl-2-picrylhydrazyl assay (EC50 =10.23±0.12mg/ml) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid assay (EC50 =3.91±0.04mg/ml). BW-BU extracts increased lifespan of L4 and L1 C. elegans (5.92% and 15.76%, respectively), which also increased worm growth, stress resistance, and reduced biomarkers for aging. BW-BU extracts activated DAF-16 nuclear localization and upregulated daf-16 and DAF-16 target genes expression. Taken together, this study revealed the evidences on anti-aging activities of saponin-rich extracts from H. leucospilota, which can extend lifespan of C. elegans via daf-16. PRACTICAL APPLICATIONS: In recent years, age-associated chronic diseases have had a significant impact on quality of life. Many natural compounds exhibit anti-aging activities, especially in sea cucumber, H. leucospilota. Our results indicated that H. leucospilota is good for health. Extracts from H. leucospilota contain a bioactive compound that can be potentially used to promote longevity and disease prevention in aging.

Functional characterisation of Holothuria leucospilota Fas-associated death domain in the innate immune-related signalling pathways.

In this study, the functions of Holothuria leucospilota Fas-associated death domain (HLFADD) in the innate immune-related signalling pathways were investigated. The results showed that over-expression of HLFADD in HEK293T cells could activate the transcription factors NF-κB and activator protein-1 (AP-1), and induce the secretion of downstream pro-inflammatory cytokines IL-6, IL-8 and IL-18, suggesting the involvement of the sea cucumber FADD in activating the NF-κB and c-Jun NH2-terminal kinase-dependent pathways. On the other hand, HLFADD could down-regulate the activations of NF-κB and AP-1 that induced by over-expression of H. leucospilota myeloid differentiation factor 88 (HLMyD88), which is supposed to be mediated through its interaction with HLMyD88 to keep the MyD88-dependent TLR signalling at a proper magnitude. The interaction of HLFADD and HLMyD88 were further supported by a co-immunoprecipitation assay. Moreover, HLFADD could activate transcription factor IFN regulatory factor-3 and induced the secretion of downstream IFN-α and IFN-β, indicating that the sea cucumber FADD may also activate the antiviral IFN signalling pathway. In summary, our study may give new insights on the functions of sea cucumber FADD in the innate immune-related signalling pathways.

Holothuria Leucospilota Polysaccharides Ameliorate Hyperlipidemia in High-Fat Diet-Induced Rats via Short-Chain Fatty Acids Production and Lipid Metabolism Regulation.

Holothuria leucospilota polysaccharides (HLP) are expected to become potential resources for the treatment of hyperlipidemia because of their various bioactivities. In the study, the treatment of HLP on improving hyperlipidemia in rats was explored. Oral administration of HLP at 100 or 200 mg/kg body weight effectively alleviated serum lipid levels and liver histological abnormalities in high-fat-diet rats. HLP regulated abnormal mRNA, lipogenesis-related hormones and inflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interleukin-12) levels. HLP improved the ability of gut microbiota to produce short-chain fatty acids (SCFAs). SCFAs have been found to ameliorate liver lesions. Therefore, HLP alleviated hyperlipidemia by improving the levels of SCFAs to regulate lipid metabolism. These results indicated that HLP could be used as beneficial polysaccharides to alleviate hyperlipidemia.

Distribution of sea cucumbers in relation to sediment characteristics in coral reef lagoons and adjacent waters around Ishigaki Island, southern Japan

AbstractTo elucidate which environmental factors affect lagoonal‐scale sea cucumber distributions in Ishigaki Island, Okinawa, Japan, intertidal and subtidal areas of three coral reef lagoons were classified into several ground divisions by bottom characteristics, and sea cucumber densities therein were compared with the composition of sediment cover, grain size and organic content, and coverage of macroalgae, seagrass, and massive corals. Holothuria atra, Holothuria leucospilota, Stichopus chloronotus, and Synapta maculata had highest densities in the nearshore areas but were rare in reef flats, probably because of wave disturbance and low areal cover of sand sediment as potential feeding environments. No relationship was observed between sea cucumber densities and sediment organic content or grain size. Thus, even if these sea cucumbers have selectivity for habitats with a high sediment organic content, the effect of such selectivity on the distribution seems to be limited to relatively small areas. The sea cucumber distributions can be classified by bottom sediment/biota composition into bedrock (H. leucospilota), sand (H. atra), and lagoonal types (St. chloronotus and Sy. maculata). These habitat selections were possibly related to various aspects of sea cucumber ecology such as refuge from predators or turbulence, or settlement and nursery place, which have implications for importance of the complexity of lagoonal‐scale topography and sediment/biota conditions for the coexistence of various holothurian species.

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)-1β, 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.