Sea Cucumber Fishery in Aurora, Philippines

This study was conducted to provide a preliminary assessment of the sea cucumber fishery in Aurora, Philippines. This was conducted from February to June 2013. All sea cucumber gatherers and traders, obtained through snowball sampling, in each identified sea cucumber fishery area in six coastal municipalities (Baler, Dipaculao, Dinalungan, Casiguran, Dilasag and Dingalan) were interviewed using a structured interview schedule and focused group discussions (FGD). Results revealed that gathering sea cucumbers were accomplished by handpicking, skin diving or compressor diving. A total of 15 commercial species were regularly gathered. Out of this, three species are of very high economic value (≥Php 4000/kg) (Holothuria nobilis, Holothuria scabra, and Actinopyga lecanora). The average number of kilograms harvested per hour per trip ranges from 0.21 (Holothuria fuscopunctata) to 2.71 (Holothuria scabra), while the average size of commonly gathered sea cucumber varies among species with Holothuria lecanora showed an average length (29.26 cm) greater than recorded 24 cm. Processing mainly involves washing, slicing, boiling, drying and packing. Processed sea cucumbers or trepan were sold to middlemen or direct buyers who usually offered higher prices (36.36% up to 69.20%) than the former. Problems encountered by sea cucumber gatherers and traders include a decline in total catch, a shift in area of collection from intertidal zones to deeper waters, an increase in the number of hours spent in finding and gathering sea cucumbers, the use of compressors in gathering, the decline of larger species and gathering of smaller sizes in huge amount, the absence of conservation program for sea cucumber, and the lenient implementation of government policies with regards to coastal resources management.

Underwater Sea Cucumber Target Detection Based on Edge-Enhanced Scaling YOLOv4

Sea cucumber detection is widely recognized as the key to automatic culture.The underwater light environment is complex and easily obscured by mud,sand,reefs,and other underwater organisms.To date,research on sea cucumber detection has mostly concentrated on the distinction between prospective objects and the background.However,the key to proper distinction is the effective extraction of sea cucumber feature information.In this study,the edge-enhanced scaling You Only Look Once-v4(YOLOv4)(ESYv4)was proposed for sea cucumber detection.By emphasizing the target features in a way that reduced the impact of different hues and brightness values underwater on the misjudgment of sea cucumbers,a bidirectional cascade network(BDCN)was used to extract the overall edge greyscale image in the image and add up the original RGB image as the detected input.Meanwhile,the YOLOv4 model for backbone detection is scaled,and the number of parameters is reduced to 48%of the original number of parameters.Validation results of 783images indicated that the detection precision of positive sea cucumber samples reached 0.941.This improvement reflects that the algorithm is more effective to improve the edge feature information of the target.It thus contributes to the automatic multi-objective detection of underwater sea cucumbers.

Proteomic Analysis of Sea Cucumber (Holothuria leucospilota) from the South China Sea

[Objectives]Proteomics methods were used to analyze the total proteome of Holothuria leucospilota in the South China Sea,so as to provide a theoretical basis for elucidating the medicinal material basis of sea cucumber proteins and peptides.[Methods]H.leucospilota was collected from the South China Sea.Label-free proteomics methods were used to detect the protein profile of enzymatically hydrolyzed H.leucospilota total protein,and identify sea cucumber protein and peptide sequences,and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)bioinformatics analysis software were used to analyze significantly enriched proteins.The String database was used to construct a protein-protein interaction network of significantly enriched H.leucospilota proteins(PPI network).[Results]Proteomics identified 1200 sea cucumber proteins and 2967 peptides.GO analysis and KEGG analysis showed that the four significantly-enriched sea cucumber proteins hsc70,COX,CYTB and rbbp4 participated in 8 metabolic pathways,and COX1 and CYTB among them were significantly related to cardiac muscle contraction,oxidative phosphorylation and metabolic pathways.The PPI network analysis showed that the 13 significantly-enriched sea cucumber proteins had close protein-protein interaction relationships.[Conclusions]This study used proteomics to conduct preliminary identification and research on the proteins and peptides of H.leucospilota,providing a scientific basis for the application of H.leucospilota proteins and peptides in the fields of functional health food and drug research and development.

Autophagy Plays a Potential Role in the Process of Sea Cucumber Body Wall “Melting” Induced by UV Irradiation

The changes of tissue appearances and structures in the process of UV-induced ＂melting＂ for sea cucumber （Stichopus japonicus） body wall were studied. And the localization and determination of acid phosphatase （ACP）, Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi＇s body and endoplasmic reticulure swelled, curled, and eventually double- or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions, It indicates that autophagy plays a potential role in the ＂melting＂ process for sea cucumber body wall induced by UV irradiation.

A new triterpene glycoside from sea cucumber Holothuria leucospilota

A new triterpene glycoside, leucospilotaside A, along with a known saponin, isolated from sea cucumber Holothuria leucospilota, and its structure was elucidated as 3β-O-[4-O-sodiumsulfate-β-d-quinovopyranosyl-(1 → 2)-β-d-xylopyranosyl]-holosta-22-ketone-9-en-17α,25α-diol (1) by extensive spectroscopic analysis and chemical methods. Leucospilotaside A (1) has a ketone carbonyl group (22) in the aglycon side chain.

An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber(Apostichopus japonicus)

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeIII and size-selected DNA fragments (250-700 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hun-dred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).

Anti-diabetic and anti-hyperlipidemic effects of sea cucumber(Cucumaria frondosa) gonad hydrolysates in type Ⅱ diabetic rats

The present study investigated the anti-diabetic activity and potential mechanisms of sea cucumber gonad hydrolysates(SCGH)in a rat model of type II diabetes induced by streptozotocin(STZ)combined with high-fat diet(HFD).Results showed that SCGH significantly reduced water intake,fasting blood glucose level and glycated hemoglobin level.Moreover,the oral glucose tolerance,insulin resistance and plasma lipid level in diabetic rats were also alleviated.Furthermore,histological analysis showed that SCGH effectively protected the tissue structure of liver.In addition,mechanism studies showed that SCGH improved glucose metabolism via activating the IRS/Akt signaling pathway,and promoted lipid metabolism via activating the AMPK signaling pathway.In summary,these findings suggested that SCGH have potential anti-diabetic effects by improving insulin resistance and lipid metabolism disorders.

Two triterpene glycosides from the sea cucumber Bohadschia marmorata Jaeger

Further studies on the sea cucumber Bohadschia marmorata Jaeger led to the isolation of a new holostan-type triterpene glycoside,Marmoroside C（1）together with a known triterpene glycoside（2）.On the basis of spectroscopic analyses,including two- dimensional NMR techniques,and chemical reactions,the structure of the new triterpene glycoside was elucidated as 3-O-[3-O- methyl-β-D-glucopyranosyl-（1→3）-β-D-glucopyranosyl-（1→4）-β-D-quinovopyranosyl-（1→2）-4-O-sodium-sulfato-β-D-xylo- pyranosyl]-25-acetoxy-22-oxo-9（11）-holostene-3β,12α,17α-triol.

Dynamic changes of peptidome and release of polysaccharide in sea cucumber(Apostichopus japonicus) hydrolysates depending on enzymatic hydrolysis approaches

Enzymatic hydrolysis has been widely used to produce bioactive hydrolysates from sea cucumber body wall.Here,inspired by the clarification of Apostichopus japonicus genome,we investigated the enzymatic hydrolysis of sea cucumber body wall by using the omics strategy.Shared proteins,including major yolk proteins,collagens,extracellular matrix glycoproteins and muscle proteins,were released from the body wall by different hydrolysis condition.A portfolio of 216 shared peptides were detected in the peptidome by papain with different hydrolysis time,while 32 shared peptides were detected in the peptidome by differing proteases.Unshared peptides and the relative abundance distribution profiles of shared peptides changed depending on hydrolysis approaches,indicating dynamic changes of peptidome during hydrolysis.Moreover,release of sulfated fucan and fucosylated chondroitin sulfate changed with the hydrolysis condition.The monitoring of dynamic enzymatic hydrolysis process at a molecular scale would contribute to production and quality control of sea cucumber hydrolysates.

Dietary Supplementation with Sea Cucumber Saponins and Exercise Can Significantly Suppress Adipose Accumulation in Mice Fed with High-Fat Diet

Dietary supplementation with sea cucumber saponins(SCS)and exercise have been confirmed to be effective in pre-venting the development of obesity and its related diseases.However,the combined effectiveness of these interventions has not been explored.Here,we studied whether the beneficial influences of exercise could be further enhanced by dietary supplementation with SCS in high-fat diet-fed KM(Kunming)mice.Mice were randomly divided into four groups,including the high-fat diet group(HF),the SCS group(HF-S),the exercise group(HF-E),and the combination of dietary SCS and exercise group(HF-S+E).There were eight mice in every group.The results demonstrated that the combination of dietary SCS and exercise could synergistically reduce fat accumulation.In particular,white adipose tissue decreased by 63%in the HF-S+E group compared with that in the HF group.SCS supplementation with exercise also improved peripheral markers,such as serum parameters and hepatic TG levels.Further mechani-cal testing indicated that the combined effects of dietary SCS and exercise on inhibiting fat accumulation might be attributed to the inhibition of lipid synthesis in the liver and the activation of lipolysis in white adipose tissue to increase energy consumption.

Triterpenoid saponin biosynthesis genes and their expression patterns during the development of sea cucumber Apostichopus japonicus

Sea cucumbers are echinoderm species with a leathery skin and an elongated body.Natural compounds,including saponins,have been previously isolated from sea cucumber.The majority of saponins derived from this organism typically belong to the triterpene glycosides,which exhibit various biological effects(e.g.,antifungal,cytotoxic,hemolytic,and cytostatic immunomodulatory activities).In the present study,30 saponin biosynthesis enzymes,namely 2 AACTs,2 FPSs,2 HMGSs,2 OSCs,1 SS,2 SEs,and 19 UGTs,were found in the genome of sea cucumber Apostichopus japonicus.Compared with upstream saponin biosynthesis enzymes(i.e.,AACTs,FPSs,HMGSs,OSCs,and SSs),the downstream saponin biosynthesis enzymes(SEs and UGTs)shared lower amino acid sequence similarity with the corresponding genes in other echinoderms.It was proposed that the enzyme structures comprised bothα-helices andβ-sheets.Notably,only a low percentage ofβ-sheets were present in FPSs,OSCs,and SS.The saponin biosynthesis enzymes showed dynamic expression patterns during five critical developmental stages of A.japonicus(fertilized oocytes,blastula,gastrula,doliolaria,and penractula).The present study involved elucidation of putative saponin biosynthesis pathways in sea cucumber and provides a valuable platform for further investigation of saponin biosynthesis in echinoderms.

17-Dehydroxyholothurin A, a New Sulfated Triterpene Glycoside from Sea Cucumber Holothuna impatiens

Abstract： A new sulfated triterpene glycoside named as 17-dehydroxyholothurin A 1 was isolated from the sea cucumber Holothuria impatiens. Its structure was elucidated on the basis of spectroscopic data （2D NMR and MS） and chemical evidence. Compound 1 showed cytotoxic activity against cancer cell lines MKN-28, MFC-7, KB, HL-60 and Hep G2 with IC50 values of 1.98, 4.53, 2.01, 4.69 and 2.80 ×10^-6 tool/L, respectively.

Bacterial community composition in gut content and ambient sediment of two tropical wild sea cucumbers(Holothuria atra and H.leucospilota)

Sea cucumbers play an important role in sediment bioturbation in coral reef and rocky intertidal ecosystems,and bacteria were a commonly-reported component of holothuroid diets.Bacterial community composition in the foregut and hindgut of two common tropical sea cucumbers(Holothuria atra and H.leucospilota)and the ambient surface sediment were investigated using high throughput sequencing based on 16S rRNA gene analysis.A total of 5584 operational taxonomic units(OTUs)were obtained from 25 samples based on a 97%threshold,and more than half of the OTUs(n=3694,66.2%)were shared by the gut contents of two species of sea cucumbers and surrounding sediments.Bacterial richness and diversity in sediment samples were signifi cantly higher than those in the gut content samples(P<0.05).Proteobacteria was the predominant phylum in most samples showing 45.69%±8.61%,70.09%±4.03%,45.88%±5.38%,and 55.19%±0.79%reads in the foregut of H.leucospilota,hindgut of H.leucospilota,hindgut of H.atra,and sediment libraries,respectively,but Bacteroidetes was the predominant phylum with the relative content of 34.98%±5.52%in the foregut of H.atra.Among the dominant genera,reads related to the genera Anderseniella,Ilumatobacter,and Ruegeria were detected in all the gut contents and sediment libraries.A comparison of gut bacteria community between the two species of sea cucumbers suggested that H.atra had stronger feeding preference than H.leucospilota,and the same types of microbes escaped digestion of the two sea cucumber species.Obvious diff erent bacterial community composition in the foreguts of the two species of sea cucumbers and the surrounding sediments might result from the animal’s selective feeding for sediment patches.Bacterial community structure in hindgut contents of H.atra and H.leucospilota both diff ered clearly from adjacent sediments,which indicated feeding activity of deposit-feeding sea cucumbers could change the sedimental bacterial composition.In conclusion,from the perspective of bacteria,sea cucumber H.atra and H.leucospilota had diff erent feeding preferences,yet they could both aff ect bacterial composition in sediments by feeding activity.The motivation for selective feeding and sea cucumber-sediment interaction might be explored in the future.

Development of novel microsatellite markers for Holothurian scabra (Holothuriidae), Apostichopusjaponicas (Stichopodidae) and cross-species testing in other sea cucumbers

Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate(B-Y FDR) correction, whereas six showed statistically significant deviations(CHS2 and CHS11: P <0.014 790; FCS1, FCS6, FCS8 and FCS14: P <0.015 377). Furthermore, four species of plesiomorphous and related sea cucumbers(Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas) were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species.

Proteomic analysis provides insights into the function of Polian vesicles in the sea cucumber A postichopus japonicus post-evisceration

The Polian vesicle is the main accessory structure in the water vascular system of sea cucumbers.It can function to hold water vascular fluid under slight pressure and act as a hematopoiesis,excretory,and inflammatory response organ.Being the only organ to remain after evisceration,the Polian vesicle may function in the survival and regeneration of sea cucumber.We performed Tandem Mass Tag(TMT)-based proteomics to identify how proteins in the Polian vesicle of Apostichopus japonicus respond to evisceration.Among the 8453 proteins identified from vesicle samples before evisceration(PVOh)and at 6-h post-evisceration(PV6h)and 3-d post-evisceration(PV3d),we detected 222 differentially abundant proteins(DAPs).Most of the annotated DAPs were associated with cell growth and proliferation,immune response and wound healing,substance transport and metabolism,cytoskeleton/cilia/flagella,extracellular matrix,energy production and conversion,protein synthesis and modification,and signal recognition and transduction.Compared with PVOh,fewer DAPs were identified at PV6h,and more DAPs were found at PV3d,and these DAPs were widely distributed among multiple biological processes.Our results indicate that a wide range of biological processes was induced in Polian vesicles in response to evisceration.In particular,Polian vesicles may play important roles in the re storation of coelomocyte s,immune defense,and wound healing in sea cucumber.We propose that the Polian vesicle may be involved in visceral regeneration through nutrition and energy supply and by promoting dedifferentiation and migration.Together,these results provided new insights into the function of the Polian vesicle in A.japonicus post-evisceration.

Positive selection analysis reveals the deep-sea adaptation of a hadal sea cucumber ( Paelopatides sp.) to the Mariana Trench

The Mariana Trench,the deepest trench on the earth,is ideal for deep-sea adaptation research due to its unique characters,such as the highest hydrostatic pressure on the Earth,constant ice-cold temperature,and eternal darkness.In this study,tissues of a the hadal holothurian(Paelopatides sp.)were fi xed with RNA later in situ at~6501-m depth in the Mariana Trench,which,to our knowledge,is the deepest in-situ fi xed animal sample.A high-quality transcript was obtained by de-novo transcriptome assembly.A maximum likelihood tree was constructed based on the single copy orthologs across nine species with their available omics data.To investigate deep-sea adaptation,113 positively selected genes(PSGs)were identifi ed in Paelopatides sp.Some PSGs such as microphthalmia-associated transcription factor(MITF)may contribute to the distinct phenotype of Paelopatides sp.,including its translucent white body and degenerated ossicles.At least eight PSGs(transcription factor 7-like 2[TCF7L2],ETS-related transcription factor Elf-2-like[ELF2],PERQ amino acid-rich with GYF domain-containing protein[GIGYF],cytochrome c oxidase subunit 7a,[COX7A],type I thyroxine 5′-deiodinase[DIO1],translation factor GUF1[GUF1],SWI/SNF related-matrix-associated actin-dependent regulator of chromatin subfamily C and subfamily E,member 1[SMARCC]and[SMARCE1])might be related to cold adaptation.In addition,at least nine PSGs(cell cycle checkpoint control protein[RAD9A],replication factor A3[RPA3],DNA-directed RNA polymerases I/II/III subunit RPABC1[POLR2E],putative TAR DNA-binding protein 43 isoform X2[TARDBP],ribonucleoside-diphosphate reductase subunit M1[RRM1],putative serine/threonine-protein kinase[SMG1],transcriptional regulator[ATRX],alkylated DNA repair protein alkB homolog 6[ALKBH6],and PLAC8 motif-containing protein[PLAC8])may facilitate the repair of DNA damage induced by the high hydrostatic pressure,coldness,and high concentration of cadmium in the upper Mariana Trench.

Partial Hydrolysis of the Fucosylated Chondroitin Sulfate from Sea Cucumber Isostichopus badionotus and Its Mechanism

The method for preparing low molecular weight fucosylated chondroitin sulfate from sea cucumber lsostichopus badionotus using partial acid hydrolysis was reported, and its hydrolysis mechanism was also investigated. The sea cucumber chondroitin sulfate FCS was hydrolyzed under different conditions （80℃3 h and 6 h）, then isolated and purified on a Bio-P-4 geltration to prepare low molecular weight fractions （LMWF-FCS）. The chemical compositions of LMWF-FCS showed the branched fucose （Fuc） was cleaved during acid hydrolysis process, whereas the mole ratio of acetyl-galactosamine （GalNAc） and glucuronic acid （GlcA） in the backbone remained the same, which indicated the backbone was a typical chondroitin sulfate structure. The disaccharide composition analysis of LMWF-FCS suggested that the sulfation patterns of GalNAc in the backbone chain changed and the substitution value was reduced. Furthermore, the 1D NMR analysis illustrated the branched-Fuc was cleaved during acid hydrolysis, but their substitution patterns were not influenced, which was distinct from the previous reports that the substitutions of branched-Fuc in FCS were easy to change. Simultaneously, the sulfation pattern of GalNAc in backbone chain changed obviously in the acid hydrolysis process. The anticoagulant activity in vitro illuminated the anticoagulant activity of the degradation products over time in the acid hydrolysis are gradually declined, but still kept good. Therefore, the LMWF-FCS prepared could be developed as a new anticoagulant and antithrombotic drug like low molecular weight heparin.

Effects of different flow velocities on behavior and TRPA1 expression in the sea cucumber Apostichopus japonicas

A water flow simulation device capable of adjusting flow velocity was designed in flow velocity range of 0–30 cm/s,with which an indoor experiment was conducted to simulate the movement and adhesive behaviors of different-sized Apostichopus japonicus under different flow velocities.Observation showed that,in slow flow(~5 cm/s),A.japonicus moved more distance than in still water,and hardly moved in the riptide(~30 cm/s);and the adhesive capacity of A.japonicus was related to the flow velocity and attachment time.A.japonicus were able to attach to the bottom after any attachment time in the slow flow,after 10 s in the medium flow(~15 cm/s),and after 60 s in the riptide(~30 cm/s).In addition,larger A.japonicus were stronger with adhesive ability than smaller ones.The transcriptome data showed that the expression of transient receptor potential cation channel subfamily A,member 1(TRPA1)in the tube feet was increased significantly in a flowing water,but those in the tentacles and tube feet were not significantly changed.Fluorescence in-situ hybridization results showed that TRPA1 was expressed around the watervascular of tentacles,tube feet,body wall,and spines.Therefore,tube feet were important for sea cucumbers to keep themselves stable in relatively swift flow with adhesion ability.

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.

Differential gene expression in the body wall of the sea cucumber(Apostichopus japonicus)under strong lighting and dark conditions

Sea cucumber, Apostichopus japonicus is very sensitive to light changes. It is important to study the influence of light on the molecular response of A. japonicus. In this study, RNA-seq provided a general overview of the gene expression profiles of the body walls of A. japonicus exposed to strong light（＂light＂）, normal light（＂control＂） and fully dark（＂dark＂） environment. In the comparisons of ＂control＂ vs. ＂dark＂, ＂control＂ vs. ＂light＂ and ＂dark＂ vs.＂light＂, 1 161, 113 and 1 705 differentially expressed genes（DEGs） were identified following the criteria of｜log2 ratio｜≥1 and FDR≤0.001, respectively. Gene ontology analysis showed that ＂cellular process＂ and ＂binding＂enriched the most DEGs in the category of ＂biological process＂ and ＂molecular function＂, while ＂cell＂ and ＂cell part＂ enriched the most DEGs in the category of ＂cellular component＂. And the DEGs were mapped to 214, 41 and229 pathways in the Kyoto Encyclopedia of Genes and Genomes database, and 51, 2 and 57 pathways were significantly enriched, respectively. Light-specific DEGs identified in this study will be important targets for further investigation to establish the biochemical mechanisms involved in the adaption of this sea cucumber to changes in the level of environmental light.