Sea Cucumber Stichopus Horrens Research Articles

Alginate – Gamat Gel Hydrogel Films as the Potential Wound Dressing

Alginate is a biopolymer that is biodegradable, non-antigenic, non-toxic, and has high biocompatibility. Gamat gels of sea cucumber Stichopus horrens are used to treat pain, wounds, and other ailments. Victims suffering from burn wounds frequently complain of pain and discomfort while being bandaged. This study aims to analyze the characteristics of alginate as the base biomaterial for hydrogel films and its interaction with gamat gels bioactive components. Antioxidant activity assay was done to the gamat gels using DPPH assay. Hydrogel films were prepared with different alginate concentrations of 1% w/v, 3% w/v, and 5% w/v. Film degradation was analyzed using stereo microscope. Mechanical characteristics were evaluated based on tensile strength, brittleness, elasticity, adhesion capacity, and the ease when handling the films. Hydrolytic stability was evaluated based on swelling capacity and matrix stability. Antibacterial activity assay was carried out using disk diffusion on Staphylococcus aureus. Results show that gamat gels have DPPH inhibition capacity of IC50 = 2937.5 ppm. The alginate concentrations treatment resulted in films with different morphology. Films with 5% w/v alginate concentration exhibit a rough surface, the better tensile strength, capability to conform to various surfaces, good adhesion (average detachment time of 60 seconds), swelling capacity of 11,96 ± 13,80%, and matrix stability with the average weight loss of 33,26%. FTIR spectroscopy confirmed that alginate was able to bond with Ca2+ ions and the gamat gels. Gamat gels show inhibition zone of ≤ 5 mm. Films with 5% w/v alginate concentration were able to release bioactive compounds.

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

Morphologically cryptic and pseudo-cryptic species pose a challenge to taxonomic identification and assessments of species diversity and distributions. Such is the case for the sea cucumber Stichopus horrens, commonly confused with Stichopus monotuberculatus. Here, we used mitochondrial cytochrome oxidase subunit I (COI) and microsatellite markers to examine genetic diversity in Stichopus cf. horrens throughout the Philippine archipelago, to aid species identification and clarify species boundaries. Phylogenetic analysis reveals two recently diverged COI lineages (Clade A and Clade B; c. 1.35–2.54 Mya) corresponding to sequence records for specimens identified as S. monotuberculatus and S. horrens, respectively. Microsatellite markers reveal two significantly differentiated genotype clusters broadly concordant with COI lineages (Cluster 1, Cluster 2). A small proportion of individuals were identified as later-generation hybrids indicating limited contemporary gene flow between genotype clusters, thus confirming species boundaries. Morphological differences in papillae distribution and form are observed for the two species, however tack-like spicules from the dorsal papillae are not a reliable diagnostic character. An additional putative cryptic species was detected within Clade B-Cluster 2 specimens warranting further examination. We propose that these lineages revealed by COI and genotype data be referred to as Stichopus cf. horrens species complex.

Potential of Epidermal Growth Factor-like Peptide from the Sea Cucumber Stichopus horrens to Increase the Growth of Human Cells: In Silico Molecular Docking Approach.

The sea cucumber is prominent as a traditional remedy among Asians for wound healing due to its high capacity for regeneration after expulsion of its internal organs. A short peptide consisting of 45 amino acids from transcriptome data of Stichopus horrens (Sh-EGFl-1) shows a convincing capability to promote the growth of human melanoma cells. Molecular docking of Sh-EGFl-1 peptide with human epidermal growth factor receptor (hEGFR) exhibited a favorable intermolecular interaction, where most of the Sh-EGFl-1 residues interacted with calcium binding-like domains. A superimposed image of the docked structure against a human EGF–EGFR crystal model also gave an acceptable root mean square deviation (RMSD) value of less than 1.5 Å. Human cell growth was significantly improved by Sh-EGFl-1 peptide at a lower concentration in a cell proliferation assay. Gene expression profiling of the cells indicated that Sh-EGFl-1 has activates hEGFR through five epidermal growth factor signaling pathways; phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), phospholipase C gamma (PLC-gamma), Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Ras homologous (Rho) pathways. All these pathways triggered cells’ proliferation, differentiation, survival and re-organization of the actin cytoskeleton. Overall, this marine-derived, bioactive peptide has the capability to promote proliferation and could be further explored as a cell-growth-promoting agent for biomedical and bioprocessing applications.

New hosts and morphological data for the Star pearlfish Carapus mourlani (Ophidiiformes: Carapidae) from collections made in the North Pacific coast of Costa Rica

Introduction: The family Carapidae includes about 40 species of marine fishes distributed in coastal habitats worldwide. The family includes some free-living species, however, most of them are found as commensal inquilines or parasites of marine invertebrates, including several echinoderm species. In the Eastern Tropical Pacific, the biology and host use of the representatives of the Carapidae is relatively poorly known.
 Objective: The present study reports the occurrence of the Star pearlfish Carapus mourlani within three previously unknown hosts in the region: the sea stars Nidorellia armata, Phataria unifascialis, and the sea cucumber Stichopus horrens. Some ecological implications and considerations regarding such symbiotic relationships are raised and discussed. Additional morphometric and meristic data for the fish and the echinoderms are also provided and discussed.
 Methods: Echinoderms were collected, from 25 localities along the North Pacific coast of Costa Rica, and were carefully examined searching for commensal/parasitic fishes. Echinoderms and fishes were identified and characterized in accordance with the specialized literature.
 Results: A total of 497 echinoderms, including about 60 species, were collected and examined. Commensal/parasitic fish (a single species represented by 13 specimens) were found in three echinoderm specimens/species.
 Conclusions: The list of echinoderm hosts for this carapid fish, through its whole distribution range, rises to 12 species (six sea stars and six sea cucumbers) and that could be a consequence of its wide geographic distribution, its generalist feeding habits and opportunistic commensal behavior.

Evaluation of microplastics ingested by sea cucumber Stichopus horrens in Pulau Pangkor, Perak, Malaysia.

The widespread presence of microplastics has caused significant concerns on their potential effects on marine ecosystems. Microplastics are classified as plastic products of less than 5 mm in size and are known to be one of the most dangerous aquatic debris to marine species. Sea cucumbers are deposit feeders living in sediment regions and benthic zone that may collect microplastics in low-energy environments. This research aims to evaluate the types of microplastic isolated from the intestine of Stichopus horrens in Malaysia. This species is selected because it is mainly found in this island. Pulau Pangkor is one of the leading tourism destinations on the small islands is facing increasing numbers of tourists and development and it is useful for sustainable study. A total of 20 S. horrens samples collected from Pulau Pangkor, Malaysia were dissected, and their intestines were collected for digestion. Microplastics were extracted using NaOH and filtered using filter paper. Microplastic identification was conducted on the based of the physical characteristics (colour, shape, size) and chemical characterisation was evaluated using FTIR for polymer functional groups. A total of 1446 unit of microplastics were found in S. horrens. Among various types and colours, fibre (90%) and black (59%) were dominant amongst the various particles identified. The majority of microplastics sizes were 0.51 μm and 1-2 μm. Two polymer materials were identified, namely polyethylene and poly(methyl methacrylate). As a conclusion, the findings of the study will serve as primary data for pollution indicators in respective islands. The microplastics could impact the immune system and upset gut's balance of human.

ISOLATION, PURIFICATION AND STRUCTURAL CHARACTERISTICS OF GLYCOSAMINOGLYCANS FROM SEA CUCUMBER STICHOPUS HORRENS

Sea cucumber glycosaminoglycans have been well known as potential anticoagulant and antithrombin agents. In this investigation, glycosaminoglycans were isolated from sea cucumber Stichopus horrens by papain enzymatic digestion. Crude glycosaminoglycans were fractionated and purified by using anion-exchange chromatography on the DEAE-Macro Prep column to give two fractions of fucosylated chondroitin sulfate (FCS1) and fucan sulfate (FS2). Structural characteristics of F1 and F2 fractions were elucidated using chemical and IR, NMR spectroscopic methods. The results showed that the monosaccharide compositions of FCS1 consist of N-Acetyl-Galactosamine (GlcNAc), D-Glucuronic acid (GlcA) and Fucose (Fuc) residues with different molar ratios, while FS2 content only fucose residues. Sulfate contents of FCS1 and FS2 were 47.4% and 48.1%, respectively. FCS1 and FS2 fractions were different in the pattern of sulfation of N-Acetyl-Galactosamine and fucose residues. IR and NMR spectra of two frations showed that sulfate groups were primarily occupied at C4 of pyranose residues in FS2 and C6, C2 and/or C3 of pyranose residues in FCS1 fraction. Our results contributed to knowledge on structural types of glycosaminoglycan from sea cucumbers in Vietnam. The establishment of structural features plays an important role in further studies of the structure-bioactivity relationship of sea cucumber glycosaminoglycan.

Screening of vibrio spp. isolated from sea cucumber stichopus horrens

Lack of knowledge about ingestion of raw or undercooked sea cucumber might contribute to the increase of food poisoning cases around the world. This study aim to screen the pathogenic bacteria Vibrio spp. isolated from the sea cucumber Stichopus horrens. An experimental study was conducted by collecting 20 samples of Stichopus horrens from Pangkor Island, Malaysia. The samples were transferred to the lab for microbiology analysis. The bacterial count (CFU/ml) method was utilized to quantify the Vibrio spp. in the samples, followed by bacteriologic culture technique on Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar. Vibrio spp. was further confirmed by biochemical tests and antibiotic sensitivity tests. The outcome showed that total presumptive Vibrio counts of sea cucumber were 11.8 ± 1.79 log CFU/ml. Based on the colony morphology and biochemical tests, the Vibrio species presence were confirmed isolated from the sea cucumber. The most frequently isolated Vibrio spp. were Vibrio cholerae (35%), followed by Vibrio parahemolyticus (15%), Vibrio alginolyticus (5%) and Vibrio vulnificus (5%). These species were susceptible to tetracycline and streptomycine while being resistant to ampicilin. Overall, it was indicated that Vibrio spp. was not detected in (40%) of the sea cucumbers Stichopus horrens while detected in the other (60%).

Effects of traditional fishing techniques on internal organ regeneration, physiology, and biochemistry in the tropical sea cucumber Stichopus horrens

The sea cucumber Stichopus horrens is an important resource in the Kingdom of Tonga. Domestically, there are two fisheries; one at a commercial scale and one for subsistence purposes. Both fisheries involve harvesting all viscera (digestive tract, gonads and respiratory tree) through a lateral body wall incision, although only the digestive tract and gonads are consumed. Under the right conditions the sea cucumber can survive organ harvest and regenerate the viscera. This study investigated the biochemical and physiological consequences of organ regeneration by comparing sham (longitudinal body wall incision without organ removal) and excised (internal tissues removed after incision) sea cucumbers to control (handled but not incised) animals. Biochemical measurements of lactate, glucose, and glycogen showed that body wall energy reserves were depleted in both sham and excised animals relative to controls at the Day 10 measurement. The presence of an energy substrate depletion in sham animals indicated an effect related to loss of body wall integrity, rather than internal organ regeneration. Although the regeneration of digestive and respiratory organs was rapid, oxygen consumption rate and ammonia excretion rate were unchanged in excised and sham animals relative to controls. These data suggest that S. horrens can survive the internal organ harvest associated with subsistence fishing and regenerate excised tissues rapidly. However, this comes at an energetic cost that is likely to affect growth and reproduction. Fisheries management measures such as the implementation of a minimum rest period between harvests may promote the sustainability of this important fishery.

De novo sequencing and transcriptome analysis of Stichopus horrens to reveal genes related to biosynthesis of triterpenoids

As the widespread species in the tropical Indo-Pacific area, the sea cucumber Stichopus horrens is characterized by the presence of appreciable amounts of triterpene glycosides. However, genes involved in biosynthesis of the triterpenoids in sea cucumber have rarely been researched. Here, we aim to explore the triterpenoid biosynthesis of S. horrens collected in the south sea of China. In this study, the de novo sequencing of S. horrens was carried out by Illumina HiSeq 2000. A total of 99,854,932 high quality sequence reads were obtained. These raw data were assembled into 168,151 contigs, and 78,024 unigenes were left after redundance removing. The transcriptomic data clearly showed that triterpenoid biosynthesis was via the MVA pathway in S. horrens. The content of total triterpene glycosides was examined by method of orcinol reaction, and 0.116 ± 0.012 mg/g and 0.016 ± 0.003 mg/g in intestine and body wall, respectively. qRTPCR showed that both oxidosqualene cyclases OSC1 and OSC2 are highly expressed in intestine, suggesting their commitment to triterpenoid biosynthesis. We also reported some potential genes encoding Cytochrome P450 and glycosylations enzymes, which might be involved in the late step of biosynthesis of triterpenoid glycosides. Overall, the present study provided the basic data for future molecular studies on biosynthesis of triterpene glycosides in sea cucumber.

A highly regular fucan sulfate from the sea cucumber Stichopus horrens

A highly regular fucan sulfate SHFS was isolated from the sea cucumber Stichopus horrens by extraction of the body walls in the presence of papain followed by ion-exchange and gel permeation chromatography. SHFS had MW of about 140 kDa and contained fucose and sulfate in the molar ratio of about 1:1. Chemical and NMR spectroscopic methods were applied for the structural characterization of the polysaccharide. SHFS was shown to have linear molecules built up of 3-linked α-l-fucopyranose 2-sulfate residues. Anticoagulant properties of SHFS were assessed in vitro in comparison with the LMW heparin (enoxaparin) and totally sulfated 3-linked α-l-fucan. SHFS was found to have the lowest activity, and hence, both sulfate groups at O-2 and O-4 of fucosyl units seem to be important for anticoagulant effect of sulfated homo-(1 → 3)-α-l-fucans.

Triterpene tetraglycosides from the sea cucumber Stichopus horrens

Using various chromatographic separations, three triterpene tetraglycosides (1–3), including one new compound, namely stichorrenoside E (1) along with thelenotoside B (2) and deacetyl thelenotoside B (3), were isolated from the MeOH extract of the Vietnamese sea cucumber Stichopus horrens. Their structures were confirmed by spectroscopic experiments, such as 1D and 2D NMR and HR-ESI-MS. Deacetylated thelenotoside B (3) is firstly isolated as a natural product. Among these compounds, thelenotoside B (2) showed strong cytotoxicities against five human cancer cell lines as HepG2, KB, LNCaP, MCF7 and SK-Mel2 with the IC50 values from 0.95 ± 0.08 to 1.90 ± 0.13 μM, whereas stichorrenoside E (1) and deacetyl thelenotoside B (3) exhibited significant activities with the IC50 values from 6.87 ± 0.25 to 11.62 ± 1.05 μM.

Effects of waterborne cadmium on energy metabolism in the tropical sea cucumber, Stichopus horrens, and a comparison of tissue-specific cadmium accumulation with the temperate sea cucumber Australostichopus mollis

The sea cucumber Stichopus horrens is an important component of near-shore ecosystems, and in the Kingdom of Tonga it also comprises an important commercial and subsistence fishery. To assess the sensitivity of this species to the toxic trace metal cadmium (Cd), adult S. horrens were exposed for 96h to an environmental (15µgL−1) or effect (765µgL−1) concentration of waterborne Cd. The respiratory tree and intestine accumulated higher concentrations of Cd than the muscle and body wall, but there were no effects of Cd on tissue ions (sodium, potassium, magnesium, calcium). For comparison, Cd accumulation was also examined in the Australasian sea cucumber Australostichopus mollis. This species displayed a similar pattern of tissue-specific accumulation to S. horrens, but exhibited lower tissue Cd burdens, likely a consequence of lower experimental temperature. Effects on gonad ion content were also seen in this species. At the highest Cd exposure concentration, S. horrens showed impaired ammonia excretion rates and an increased molar oxygen:nitrogen ratio (O:N), indicative of a decreased reliance on protein metabolism. Overall, this study suggests that S. horrens is relatively tolerant of Cd exposure, but raises concerns regarding the subsistence fishery practice of consuming the viscera of this species.

Short Communication Isolation and characterization of microsatellite markers of sea cucumber Stichopus horrens

Curry fish (Stichopus horrens) is a tropical holothurian species and is widely distributed in the India-West Pacific. In the present study, 9 polymorphic microsatellite loci were isolated and characterized for S. horrens. These loci were tested in 30 individuals from Hainan Island in China. The number of alleles ranged from 2 to 5. The polymorphism information content ranged from 0.348-0.584. The levels of observed and expected heterozygosities varied from 0.1500-0.8000 and from 0.2014-0.5000, respectively. Most loci were in Hardy-Weinberg equilibrium, except HCS1-27 and HCS2-7, after sequential Bonferroni's correction, and no significant linkage disequilibrium was detected for any pairwise combination of loci. These polymorphic microsatellite loci will be useful for studying population structure and conservation strategy design for S. horrens.

Identification and characterization of microsatellite markers from the tropical sea cucumber, Stichopus horrens (Selenka).

Tropical commercial sea cucumber Stichopus horrens is extensively distributed throughout the tropical Indo-Pacific region, and wild stocks have been severely depleted over the past decade. In this study, we used the microsatellite enrichment library of S. horrens to identify and characterize 13 microsatellite loci, including 11 polymorphic loci and 2 monomorphic loci. Among the 11 polymorphic loci, the number of alleles was 3-8. The observed and expected heterozygosity varied from 0.1364 to 0.8966 and from 0.1653 to 0.7551, respectively. Additionally, all 11 polymorphic loci showed moderate and high polymorphism with the polymorphism information content (0.271-0.7311). A total of 9 polymorphic loci were in Hardy-Weinberg equilibrium, except for 2 loci (adjusted P = 0.004545). Linkage disequilibrium was not detected in any pairs of polymorphic loci. The present study will be useful for studying genetic structure, population conservation, and breeding of wild S. horrens; moreover, our results contribute to the phylogeny and evolutionary research of Holothuroidea.

Spawning, larval development and juvenile growth of the sea cucumber Stichopus horrens

Tropical commercial sea cucumber Stichopus horrens is extensively distributed throughout the tropical Indo-Pacific region, and wild stocks are severely depleted over the past decade. This study firstly presented preliminary results on the spawning, the development of larvae and the growth of the juvenile of S. horrens in Xisha Islands. It was proved that S. horrens followed a predictable lunar spawning periodicity and might reproduce throughout the year in Xisha Islands. Dry stimulation was a simple and efficient way to induce S. horrens to spawn. The mean quantity of spawned eggs was 1.37×106 (s.e.=2.09×106, n=8), and the mean egg diameter was 114μm (s.e.=2.8μm, n=8). S. horrens developed from fertilized eggs to pentactula larval after 19–27days at 25–27°C. From oosperm to settled juvenile, the survival rate of S. horrens was 1.0%–6.83% when enough live microalgae were supplied. The mean growth rate of juveniles was 0.4mm per day and the survival rate was about 23.3%–50.5% during 80days culture after settlement. Then some juveniles of S. horrens were further cultivated in tanks and majority were released in the wild. Ongoing observations showed good survival and growth of juvenile in the wild. The study indicates S. horrens are amenable to culture using standard techniques which may also be applied on the culture of other tropical species. They exhibit a relatively quick growth both in indoor and wild culture. Furthermore, the test results support possible aquaculture and stock restoration using hatchery produced stocks.

Characterization of mitochondrial genome of sea cucumber Stichopus horrens: A novel gene arrangement in Holothuroidea

The complete mitochondrial DNA sequence contains useful information for phylogenetic analyses of metazoa. In this study, the complete mitochondrial DNA sequence of sea cucumber Stichopus horrens (Holothuroidea: Stichopodidae: Stichopus) is presented. The complete sequence was determined using normal and long PCRs. The mitochondrial genome of Stichopus horrens is a circular molecule 16257 bps long, composed of 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes. Most of these genes are coded on the heavy strand except for one protein-coding gene (nad6) and five tRNA genes (tRNA ( Ser(UCN) ), tRNA ( Gln ), tRNA ( Ala ), tRNA ( Val ), tRNA ( Asp )) which are coded on the light strand. The composition of the heavy strand is 30.8% A, 23.7% C, 16.2% G, and 29.3% T bases (AT skew=0.025; GC skew=-0.188). A non-coding region of 675 bp was identified as a putative control region because of its location and AT richness. The intergenic spacers range from 1 to 50 bp in size, totaling 227 bp. A total of 25 overlapping nucleotides, ranging from 1 to 10 bp in size, exist among 11 genes. All 13 protein-coding genes are initiated with an ATG. The TAA codon is used as the stop codon in all the protein coding genes except nad3 and nad4 that use TAG as their termination codon. The most frequently used amino acids are Leu (16.29%), Ser (10.34%) and Phe (8.37%). All of the tRNA genes have the potential to fold into typical cloverleaf secondary structures. We also compared the order of the genes in the mitochondrial DNA from the five holothurians that are now available and found a novel gene arrangement in the mitochondrial DNA of Stichopus horrens.