Purification and characterization of a novel immunoregulatory peptide from Sipunculus nudus L. protein.

This study aimed to purify and characterize immunoregulatory peptides from Sipunculus nudus L. and to explore the underlying mechanisms. Ultrafiltration, gel filtration chromatography, and reverse phase high-performance liquid chromatography (RP-HPLC) were used to purify the peptide following enzymatic hydrolysis. Rates of lymphocyte proliferation and phagocytosis as well as nitric oxide (NO) production levels were used as indicators of immunoregulatory activity to screen the fractions. The amino acid sequence of the peptide, designated as SNLP, was identified as Arg-Val-Lys-Gly-Lys-Ile-Leu-Ala-Lys-Arg-Leu-Asn (RVKGKILAKRLN) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Treatment with the synthetic SNLP increased the proliferation and phagocytosis of RAW 264.7 macrophages and promoted the secretion of tumor necrosis factor-ɑ (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and NO levels. The mRNA levels of these cytokines and iNOS were also increased by SNLP. Our results provide preliminary evidence suggesting that SNLP acts as a dual immunomodulatory peptide with immunostimulatory and anti-inflammatory activities. In summary, SNLP derived from Sipunculus nudus L. is a potent immunoregulatory peptide and represents a potential functional food or immunoregulatory drug.

Purification and characterization of a glycoprotein from Sipunculus nudus and its immune-enhancing activity to RAW 264.7 macrophages.

Sipunculus nudus, an edible marine invertebrate, has long been used as traditional Chinese medicine in folk remedies. In order to assess the immunoregulatory activity of glycoproteins in Sipunculus nudus and conduct a structure-activity relationship, a glycoprotein (SGP1) with molecular mass of 9.26 kDa was purified from Sipunculus nudus, and its chemical structure as well as immune-enhancing activity was investigated in this study. Structure analysis revealed that SGP1, a protein-dominate glycoprotein with O-glycosidic bonds, contained 92.8 % protein and 3.1 % saccharide. GC-MS result indicated that the saccharide moieties of SGP1 basically consisted of lyxose (Lyx), xylose (Xyl) as well as glucose (Glu) at a molar proportion of 0.87:4.16:1.36. The fourier transform infrared specoscopy (FT-IR) result proved that SGP1 have a typical characteristic of glycoprotein. Besides, circular dichroism (CD) result showed that SGP1 contained 4.1 % α-helix, 42.5 % ß-sheet, 21.4 % ß-turn, and 32.0 % random coil, indicating it's mainly a ß-sheet glycoprotein. The amino acid sequence of SGP1 shared a similarity to the Myohemerythrin (sp|Q5K473|HEMTM) with protein sequence coverage of 28.3 %. Moreover, the activity evaluation results showed that SGP1 exhibited significant immune-enhancing activity to the RAW 264.7 macrophages by promoting macrophages proliferation, enhancing phagocytic capacity, and simultaneously stimulating the secretions of nitric oxide (NO), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) via NF-κB pathways. In this study, SGP1 as a novel glycoprotein had an obvious immune-enhancing activity to macrophages, and thus could be applied in the functional foods as a potential immunopotentiator for the hypoimmune population.

Transcriptome Responses to Different Environments in Intertidal Zones in the Peanut Worm Sipunculus nudus.

The peanut worm (Sipunculus nudus) is an important intertidal species worldwide. Species living in the same aquaculture area might suffer different environmental impacts. To increase knowledge of the molecular mechanisms underlying the response to environmental fluctuations, we performed a transcriptome analysis of S. nudus from different intertidal zones using a combination of the SMRT platform and the Illumina sequencing platform. (1) A total of 105,259 unigenes were assembled, and 23,063 unigenes were perfectly annotated. The results of the PacBio Iso-Seq and IIIumina RNA-Seq enriched the genetic database of S. nudus. (2) A total of 830 DEGs were detected in S. nudus from the different groups. In particular, 33 DEGs had differential expression in the top nine KEGG pathways related to pathogens, protein synthesis, and cellular immune response and signaling. The results indicate that S. nudus from different zones experience different environmental stresses. (3) Several DEGs (HSPA1, NFKBIA, eEF1A, etc.) in pathways related to pathogens (influenza A, legionellosis, measles, and toxoplasmosis) had higher expression in groups M and L. HSPA1 was clearly enriched in most of the pathways, followed by NFKBIA. The results show that the peanut worms from the M and L tidal flats might have suffered more severe environmental conditions. (4) Some DEGs (MKP, MRAS, and HSPB1) were upregulated in peanut worms from the H tidal flat, and these DEGs were mainly involved in the MAPK signaling pathway. These results indicate that the MAPK pathway may play a vital role in the immune response of the peanut worm to the effects of different intertidal flats. This study provides a valuable starting point for further studies to elucidate the molecular basis of the response to different environmental stresses in S. nudus.

A synthetic peptide from Sipunculus nudus promotes bone formation via Estrogen/MAPK signal pathway based on network pharmacology.

The tripeptide Leu-Pro-Lys (LPK), derived from the Sipunculus nudus protein, was synthesized and studied to investigate its potential protective effect on bone formation. The effect and mechanism of LPK were analyzed through network pharmacology, bioinformatics, and experimental pharmacology. The study found that LPK at concentrations of 25 µg/mL and 50 µg/mL significantly increased ALP activity and mineralization in C3H10 cells. LPK also increased the expression of COL1A1 and promoted bone formation in zebrafish larvae. Network pharmacology predicted 148 interaction targets between LPK and bone development, and analysis of the protein-protein interaction network identified 13 hub genes, including ESR1, MAPK8, and EGFR, involved in bone development. Through KEGG enrichment pathways analysis, it was determined that LPK promotes bone development by regulating endocrine resistance, the relaxin signaling pathway, and the estrogen signaling pathway. Molecular docking results showed direct interactions between LPK and ESR1, MAPK8, and MAPK14. Additional verification experiments using western blot assay revealed that LPK significantly upregulated the expression of genes related to bone formation, including COL1A1, OPG, RUNX2, ESR1, phosphorylated MAPK14, and phosphorylated MAPK8 in C3H10 cells. These results suggest that LPK promotes bone formation by activating the estrogen/MAPK signaling pathway.

Hot Air Drying of Sipunculus nudus: Effect of Microwave-Assisted Drying on Quality and Aroma.

The present study aimed to investigate the effect of different microwave pre-drying times under hot-air-drying processes on the quality properties and sensory evaluation of Sipunculus nudus (S. nudus). The colour, proximate analysis, amino acid content, fat oxidation, and volatile components of dried S. nudus were determined. Microwave pre-drying could significantly (p < 0.05) increase the drying rate and shorten the drying time. The results of colour, proximate analysis, and amino acid content indicated that microwave pre-drying could improve the quality of the product, resulting in dried S. nudus with less of a loss in nutrients. The samples that underwent microwave pre-drying had a high degree of fatty acid oxidation and low monounsaturated fatty acid content, which facilitated the formation of volatile components. Additionally, the MAD-2 and MAD-3 groups had high relative contents of aldehydes and hydrocarbons, and the FD group had the highest relative content of esters found in the samples. The relative content of ketones and alcohols did not differ significantly between the different drying groups. The finding of this study has important potential for enhancing the quality and aroma of dry S. nudus products with microwave pre-drying during the drying process.

Fabrication and Characterization of W/O/W Emulgels by Sipunculus nudus Salt-Soluble Proteins: Co-Encapsulation of Vitamin C and ß-Carotene.

W/O/W emulsions can be used to encapsulate both hydrophobic and hydrophilic bioactive as nutritional products. However, studies on protein stabilized gel-like W/O/W emulsions have rarely been reported, compared to the liquid state multiple emulsions. The purpose of this study was to investigate the effect of different oil-water ratios on the stability of W/O/W emulgels fabricated with salt-soluble proteins (SSPs) of Sipunculus nudus. The physical stability, structural characteristics, rheological properties, and encapsulation stability of vitamin C and ß-carotene of double emulgels were investigated. The addition of W/O primary emulsion was determined to be 10% after the characterization of the morphology of double emulsion. The results of microstructure and rheological properties showed that the stability of W/O/W emulgels increased with the increasing concentration of SSPs. Additionally, the encapsulation efficiency of vitamin C and ß-carotene were more than 87%, and 99%, respectively, and still could maintain around 50% retention of the antioxidant capacity after storage for 28 days at 4 °C. The aforementioned findings demonstrate that stable W/O/W emulgels are a viable option for active ingredients with an improvement in shelf stability and protection of functional activity.

Formation of Shelf-Stable Pickering High Internal Phase Emulsion Stabilized by Sipunculus nudus Water-Soluble Proteins (WSPs).

To form a stable emulsion system, the water-soluble proteins (WSPs) of Sipunculus nudus were prepared as the sole effective stabilizer for the high internal phase emulsion (HIPEs), of which the influence of the WSPs concentration and environmental stability was investigated. The HIPEs were fabricated using a simple one-pot homogenization process (10,000 rpm/min, 3 min) that involved blending the WSPs (0.1, 1, 2, 3, 4, and 5 wt%) with soybean oil (60, 65, 70, 75, 80, 85, and 90%). The microstructure and properties of stable HIPEs were characterized by particle size, ζ-potential, visual observations, optical microscopy, and dynamic rheology property measurements. As the concentration of WSPs increases, the mean particle diameter of HIPEs decreases, on the contrary, the apparent viscosity and storage modulus gradually increase. At a given emulsifier concentration (3 wt%), the stable and gel-like HIPEs were formed at the oil internal phase (ϕ) values of 70-75%, all the pH range in values from 3 to 9, and the ionic strength from 100 to 500 mM. Furthermore, the HIPEs that were stabilized formed a gel-like state that was relatively stable to heat and storage (30 days). And there was a new phenomenon that the destabilized HIPE of the freeze-thaw treatments could still return to a gel-like state again after homogenizing. The study results suggest that the WSPs of S. nudus as a natural emulsifier could be widely used in the food industry.

Novel polysaccharide extracted from Sipunculus nudus inhibits HepG2 tumour growth in vivo by enhancing immune function and inducing tumour cell apoptosis.

A novel polysaccharide was extracted from Sipunculus nudus (SNP). The molecular weight (MW) of SNP was determined to be 9223 Da by high-performance gel permeation chromatography analyses, and the structure of the SNP repeat units was determined to be →3,4-ß-D-GlcpNAC (1→ and →4) -α-D-Glcp (1→ in the ratio of 15:1; →2) -α -D-Galp - (1→ as a side chain; and ß-D-Galp-(1→ and α- D-Glcp - (1→ as end groups by GC-MS analysis and NMR assays. The effect of SNP on hepatoma HepG2-bearing mice was analysed to verify its potential in the clinical treatment of liver cancer. A total of 90 male athymic nu/nu mice were divided into therapeutic and preventive groups and fed with different amounts of SNP. The antitumour effect of SNP on HepG2-bearing mice and mechanism of such were studied by analysing the tumour size, spleen index, thymus index, immune factors in the blood, tumour apoptosis factors, etc. The results suggest that SNP not only increased the index of immune organs in the body, but also enhanced the secretion of immune factors, including interleukin-2, interferon gamma and tumour necrosis factor-alpha in the serum. SNP induced the apoptosis of tumour cells via the mitochondrial apoptosis pathway, which upregulated caspase-3, caspase-8, caspase-9 and BCL2-associated X, but downregulated B-cell lymphoma-2 and vascular endothelial growth factor protein expression. In conclusion, SNP inhibited tumour growth by enhancing immune function and inducing tumour cell apoptosis in HepG2-bearing mice. Therefore, SNP may be further investigated as a promising candidate for future antitumour drugs.

Collagen Peptides Derived from Sipunculus nudus Accelerate Wound Healing.

Marine collagen peptides have high potential in promoting skin wound healing. This study aimed to investigate wound healing activity of collagen peptides derived from Sipunculus nudus (SNCP). The effects of SNCP on promoting healing were studied through a whole cortex wound model in mice. Results showed that SNCP consisted of peptides with a molecular weight less than 5 kDa accounted for 81.95%, rich in Gly and Arg. SNCP possessed outstanding capacity to induce human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) cells proliferation and migration in vitro. In vivo, SNCP could markedly improve the healing rate and shorten the scab removal time, possessing a scar-free healing effect. Compared with the negative control group, the expression level of tumor necrosis factor-α, interleukin-1ß and transforming growth factor-ß1 (TGF-ß1) in the SNCP group was significantly down-regulated at 7 days post-wounding (p < 0.01). Moreover, the mRNA level of mothers against decapentaplegic homolog 7 (Smad7) in SNCP group was up-regulated (p < 0.01); in contrast, type II TGF-ß receptors, collagen I and α-smooth muscle actin were significantly down-regulated at 28 days (p < 0.01). These results indicate that SNCP possessed excellent activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to reducing inflammation, improving collagen deposition and recombination and blockade of the TGF-ß/Smads signal pathway. Therefore, SNCP may have promising clinical applications in skin wound repair and scar inhibition.

Proteomic analysis of body wall and coelomic fluid in Sipunculus nudus.

In order to make clear the protein compositions of Sipunculus nudus and investigate its immune-related proteins, proteomic analysis was performed on body wall and coelomic fluid of Sipunculus nudus. A total of 1659 proteins were identified, and 539 proteins were differentially expressed in the coelomic fluid compared to those in the body wall, of which 415 proteins were up-regulated while 124 proteins were down-regulated. Gene Ontology (GO) analysis showed that the GO terms involved in the two parts of Sipunculus nudus were similar, with metabolic processes, catalytic activity and cell occupying the top categories of biological process, molecular function and cellular component, respectively. KEGG pathway analysis showed that 49 pathways in body wall and 48 in coelomic fluid were mapped respectively, and these pathways were mainly related to cellular processes, environmental information processing, genetic information processing and metabolism. The COG analysis showed that 757 proteins from body wall and 889 from coelomic fluid were classified into 26 COG categories, respectively. Pfam annotation revealed the mainly immune-related proteins contained in Sipunculus nudus, such as insulin-like growth factor binding protein, catalase, basement membrane proteoglycan, titin. Our research provides the first proteomic information of Sipunculus nudus, which contributes to the study of functional proteins in Sipunculus nudus and is of great significance for the application of Sipunculus nudus in functional foods and medicines.

[Wound-healing acceleration of mice skin by Sipunculus nudus extract and its mechanism].

In order to explore the effect of Sipunculus nudus extract (SNE) on skin wound healing in mice and its mechanism, hemostasis effect of SNE was measured, the mouse skin wound model was established by full-thickness excision. The morphological changes of the wound were observed after the treatment with SNE and the healing rate was measured. The changes of wound histology were observed by hematoxylin eosin (HE) staining, Masson staining and transmission electron microscope (TEM). The expression of cell factors and related proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that the SNE possessed hemostatic function. SNE could obviously improve the healing rate of wound in mouse and shorten time of scab removal compared with the none-treatment (NT) group ( P < 0.05).The pathological histology analysis results showed complete epidermal regeneration, with remarkable capillary and collagen fiber observed in the SNE group. The expression level of tumor necrosis factor-α (TNF -α), interleukin-1ß (IL-1ß) and transforming growth factor-ß1 (TGF-ß1) in SNE group was significantly lower than that of the NT group on 7 d ( P < 0.05). Moreover, compared with the NT group, the gene expressions level of Smad7 was significantly increased and the level of type II TGF-ß receptors (TGF-ßRII), collagen I (COL1A1) and α-smooth muscle actin (α-SMA) were significantly reduced in the SNE group on 28 d ( P < 0.05), but the difference was not statistically significant compared to Yunnanbaiyao group (PC group) ( P > 0.05). These results indicated that SNE possessed obvious activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to hemostatic function, regulation of inflammatory factors, collagen deposition, collagen fiber remodeling and intervening TGF-ß/Smads signal pathway. Therefore, SNE may have promising clinical applications in skin wound repair and scar inhibition.

Wound-healing acceleration of mice skin by extract and its mechanism / 生物医学工程学杂志

In order to explore the effect of extract (SNE) on skin wound healing in mice and its mechanism, hemostasis effect of SNE was measured, the mouse skin wound model was established by full-thickness excision. The morphological changes of the wound were observed after the treatment with SNE and the healing rate was measured. The changes of wound histology were observed by hematoxylin eosin (HE) staining, Masson staining and transmission electron microscope (TEM). The expression of cell factors and related proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that the SNE possessed hemostatic function. SNE could obviously improve the healing rate of wound in mouse and shorten time of scab removal compared with the none-treatment (NT) group ( 0.05). These results indicated that SNE possessed obvious activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to hemostatic function, regulation of inflammatory factors, collagen deposition, collagen fiber remodeling and intervening TGF-β/Smads signal pathway. Therefore, SNE may have promising clinical applications in skin wound repair and scar inhibition.

Bioturbation of peanut worms Sipunculus nudus on the composition of prokaryotic communities in a tidal flat as revealed by 16S rRNA gene sequences.

To understand the impacts of peanut worms Sipunculus nudus on the prokaryotic community composition in a tidal flat, an onsite investigation was conducted in Suixi in the Beibu Gulf (109.82E, 21.35N) in the burrow sediments, non-burrow sediments and the sediments without peanut worm disturbance (control). The16S rRNA gene Illumina MiSeq sequencing was used to investigate the microbial communities and their response to bioturbation by S. nudus in a sandy tidal flat. A total of 18 bacteria phyla were detected, and Proteobacteria and Cyanobacteria constituted the majority of the prokaryotic community in the samples. The distribution of the relative abundances of genera showed that approximately 6.99%-17% of the reads in the samples were classified into 25 known genera. Sulfate-reducing bacteria (Desulfococcus and Desulfosarcina) were the most abundant taxa, followed by Thermodesulfovibrionaceae LCP-6, indicating that sulfate reduction is the main process in the sandy tidal flat. The abundances of Desulfococcus, LCP-6 and Cyanobacterium in the non-burrow sediment were greater than in the burrow sediment, suggesting that the anoxic condition is more suitable for Desulfococcus and LCP-6 when the activity of S. nudus is absent. The biomass of Cyanobacterium was decreased by the feeding bioturbation of S. nudus. Meanwhile, the relative abundance of the Bacteroidetes Luteimonas in the burrow sediments was significantly greater than in the non-burrow sediment, and there was a strong relationship between S. nudus bioturbation and increased in oxygen contents and oxidation-reduction potentials in the burrow sediment. The abundances of Desulfococcus and LCP-6 were greater in the middle layer (20-30 cm) than in the top layer in the non-burrow sediment. However, the middle and bottom layers (20-30, 30-40 cm) had higher abundances of these genera than did the upper layers (0-10, 10-20 cm) in the burrow sediments. The abundances of the Fusobacteria Propionigenium and the Spirochaetes Spirochaeta were greater in the middle and bottom layers (20-30 cm, 30-40 cm) than in the top layers (0-10, 10-20 cm) in the burrow sediment, but this phenomenon was not found in the non-burrow sediment. This study demonstrates that bioturbation by S. nudus plays an important role in reshaping the bacterial community composition in intertidal regions.

Improvement Effects of Fibrinolytic Enzyme from Sipunculus nudus on Hemorheology Disorder and Vascular Endothelium Injury of Naked Acute Blood Stasis Model Rats / 中国药房

OBJECTIVE： To study the improvement effects of fibrinolytic enzyme from Sipunculus nudus （SNFE） on hemorheology disorder and vascular endothelium injury in naked acute blood stasis model rats. METHODS： SD rats were randomly divided into control group， model group， aspirin group （100 mg/kg） and SNFE high-dose and low-dose groups （2 500， 5 000     U/kg）， with 10 rats in each group. They were given relevant medicine intragastrically once a day， for consecutive 7 d. One hour after the 6th day of administration， except for control group， other groups were given adrenaline hydrochloride 0.8 mg/kg subcutaneously， and then the acute blood stasis model was induced by ice-water bath. Blood was collected from abdominal aorta 2 h after the next day. Blood rheological parameters such as whole blood viscosity （high， medium and low shear rate）， plasma viscosity， hematocrit， erythrocyte aggregation index and erythrocyte deformability index were measured by automatic rheometer. The contents of NO and ET-1 in plasma and their ratio were determined by ELISA， and the damaged degree of vascular endothelium were observed by HE staining. RESULTS： Compared with control group， whole blood viscosity of high， medium and low-shear rate， plasma viscosity， erythrocyte aggregation index and ET-1 content were increased significantly in model group， while erythrocyte deformability index， NO content and NO/ET-1 ratio were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Compared with model group， whole blood viscosity of high， medium and low-shear rate， plasma viscosity， hematocrit， erythrocyte aggregation index and ET-1 content were decreased significantly in SNFE high-dose groups. Erythrocyte deformability index， NO content and NO/ET-1 ratio were increased significantly， with statistical significance （P＜0.05 or P＜0.01）. In SNFE low-dose group， erythrocyte deformability index and NO/ET-1 ratio were increased significantly， while ET-1 content was decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Vascular endothelial staining showed that compared with control group， the structure of aorta layers in model group was loose and disordered， the endothelial defect was incomplete， the vacuoles increased， and the endothelial damage was obvious. The endothelium of rats in each administration group was damaged to varying degrees， but the degree of injury was lighter than in model group. CONCLUSIONS： SNFE can improve hemorheological abnormalities and vascular endothelial injury in rats with acute blood stasis.

A Novel Antithrombotic Protease from Marine Worm Sipunculus Nudus.

Sipunculus nudus, an old marine species, has great potential for use as functional seafood due to its various bioactivities. Its potential antithrombotic activity pushed us to isolate the bio-active components bio-guided by tracking fibrinolytic activity. As a result, a novel protease named as SK (the kinase obtained from S. nudus) was obtained, which possessed a molecular weight of 28,003.67 Da and 15 N-terminal amino acid sequences of PFPVPDPFVWDTSFQ. SK exerted inhibitory effects on thrombus formation through improving the coagulation system with dose-effect relationship within a certain range. Furthermore, in most cases SK got obviously better effect than that of urokinase. With the help of untargeted mass spectrometry-based metabolomics profiling, arachidonic acid, sphingolipid, and nicotinate and nicotinamide mechanism pathways were found to be important pathways. They revealed that the effect mechanism of SK on common carotid arterial thrombosis induced by FeCl3 was achieved by inhibiting vessel contraction, platelet aggregation, adhesion, and release, correcting endothelial cell dysfunction and retarding process of thrombus formation. This study demonstrated SK was a promising thrombolytic agent on the basis of its comprehensive activities on thrombosis, and it should get further exploitation and utilization.

The complete mitochondrial genome of the cryptic species in peanut worm Sipunculus nudus (Sipuncula, Sipunculidae) from Beibu Bay.

The peanut worm Sipunculus nudus is an economic important species in the fishery and aquaculture in Indo-West Pacific. However, it is a species complex consisting of cryptic species, which can be difficult to identify based on morphology. Here, we report the complete mitochondrial genome sequence of S. nudus from Beibu Bay. The mitogenome has 15,375 base pairs and made up of total of 38 genes (13 protein-coding, 23 transfer RNAs, and two ribosomal RNAs), and a putative control region. There were 5948 mutations sites between Chinese and French populations. This study adds a distinct mitogenome of S. nudus, and will provide useful genetic information for future genetic variation identification and genetic diversity evaluation of this economic valuable marine benthic invertebrate.

Effect of polysaccharides extracted from Sipunculus nudus (SNP) on the lifespan and immune damage repair of Drosophila melanogaster exposed to Cd (VI).

The water-soluble polysaccharides extracted from Sipunculus nudus (SNP) was investigated on the lifespan and immune damage repair of Drosophila melanogaster exposed to Cd (VI). SNP increased superoxyde dismutase (SOD), nitrogen monoxide (NO), glutathione peroxidase (GSH-Px) and total anti-oxidation competence (T-AOC), with decreased malondialdehyde (MDA) on D. melanogaster demonstrated that SNP could attenuate oxidative damage of D. melanogaster Exposed to Cd (VI). Real-time PCR and western blot analysis showed that SNP enhanced the gene expression of Diptericin, Drosomycin, Defensin, PGRP-LC and the protein level of Toll, p-JNK and Relish， that suggested the promoting effect of SNP on the immune damage repair of D. melanogaster exposed to Cd (VI). The increased level of Indy, Parkin and AMPK indicated the regulated effect of SNP on the longevity-related pathways through ageing-related moleculars of D. melanogaster exposed to Cd (VI). These results suggested that SNP could also improve the lifespan of D. melanogaster exposed to Cd (VI).

Brachiopods, sipunculans, enteropneusts and metals from two estuarine tidal flats, Pacific, Costa Rica / Braquiópodos, sipuncúlidos, enteropneustos y metales en dos planicies estuarinas de marea, Pacífico, Costa Rica

Abstract:Reports on the abundances and on metal concentrations in intertidal estuarine invertebrates from the Eastern Tropical Pacific are rare. Thus, the objectives of this report are to make accessible data on the abundances (1984-1987, 49 dates; 2013, 12 dates) of sipunculans, brachiopods and hemichordates from a sand-mud flat; and on trace metals (1996, 2000) and abundances (2015, 3 dates) of sipunculans and brachiopods at a sand flat in the Gulf of Nicoya estuary (10o N-85o W). Cores (17.7 cm2) were collected at the sand-mud flat, and quadrats (0.2 m2) at the sand flat. The flats contrasted in their sand (65 % vs 90 %) and silt+clay (31.5 % vs 5.6 %) contents. At the sand-mud flat (1984-87: 1.83 m2) the sipunculans were represented by 13 individuals, the brachiopods by 129 and the acorn worms by 185, with estimated maximum densities of: 5.7, 29, and 40 ind./m2, respectively. Trace metal (Fe, Mn, Ni, Cr, Cd, Zn, and Pb) analysis (Atomic Absorption Spectrometry) were conducted in specimens of Sipunculus nudus (Sipuncula) and Glottidia audebarti (Brachiopoda). Maximum mean concentrations in S. nudus were: For non-depurated worms, Fe (16.0 mg/g dw) > Mn (165 µg/g dw) > Zn (81 µg/g dw) > Cu (26 µg/g dw) > Cr (11 µg/g dw) > Ni (10.4 µg/g dw) > Pb (9.3 µg/g dw) > Cd (1.2 µg/g dw). For 72 hour depurated worms: Fe (5.0 mg/g dw) > Mn (61 µg/g dw) > Zn (39 µg/g dw) > Cu (24 µg/g dw) > Ni (8.4 µg/g dw) > Pb (2.7 µg/g dw) > Cd (0.62 µg/g dw). For G. audebarti: Fe (1.6 mg/g dw-soft parts) > Zn (123.5 µg/g dw-soft parts) > Cu (31.4 µg/g dw-pedicles) > Pb (21.0 µg/g dw-shells) > Cd (5.2 µg/g dw-soft parts) > Cr (4.7 µg/g dw-shells). For sediments; Fe (46 mg/g dw) > Mn (41.3 µg/g dw) > Zn (63 µg/g dw) > Cu (36.2 µg/g dw) > Cr (31.5 µg/g dw) > Pb (21.1 µg/g dw) > Ni (16.1 µg/g dw) > Cd (1.1 µg/g dw). These concentrations were expected for a nonindustrialized estuary. At the sand flat (Area sampled: 10.6 m2 ) 76 individuals of G. audebarti, 112 of G. albida, and 366 of S. nudus were collected in 2015, with estimated maximum densities of: 7.1, 10.5, and 31 ind./m2, respectively. Densities of G. audebarti and G. albida were relatively low, while those of S. nudus were relatively high when compared with other reports. The shell lenght of G. audebarti ranged from 9.0 mm to 38.0 mm and from 6.0 mm to 29.0 mm for G. albida. These ranges were within those found for these lingulides elsewhere. The mean length of S. nudus was 41 mm and the maximum weight was 1.6 g, which are small. No brachiopods were found at the sand-mud flat in 2013, nor enteropneusts at the sand flat in 2015. G. audebarti had a relatively stable presence, while G. albida almost vanished from the samples at the end of 2015. The spatial distributions of the three invertebrates were found aggregated at both intertidal flats. Strong ENSO warming events during 1983 and 2015, and red tides in 1985 may have influenced the abundances. Rev. Biol. Trop. 64 (3): 1311-1331. Epub 2016 September 01.

ResumenSon raros los reportes sobre las abundancias y concentraciones de metales en invertebrados estuarinos de la zona de entre-mareas del Pacífico Este Tropical. Los objetivos de este informe son el hacer accesibles datos sobre las abundancias (1984-1987, 49 fechas; 2013, 12 fechas) de sipuncúlidos, braquiópodos y hemicordados en una planicie arenoso-fangosa y sobre metales traza (1996, 2000) y abundancias (2015, 3 fechas) de sipuncúlidos y braquiópodos en una planicie arenosa en el estuario del Golfo de Nicoya (10o N-85o W). Barrenos (17.7 cm2) fueron recolectados en la planicie arenoso-fangosa y cuadrantes (0.2 m2) en la arenosa. Las planicies contrastaron en sus contenidos de arena (65 % vs 90 %) y de limo + arcilla (31.5 % vs 5.6 %). En la planicie arenoso-fangosa (1984-87: 1.83 m2) los sipuncúlidos estuvieron representados por 13 individuos, los braquiópodos por 129 y los hemicordados enteropneustos por 185, con densidades estimadas de: 5.7, 29, y 40 ind. /m2, respectivamente. Análisis de metales traza (Fe, Mn, Ni, Cr, Cd, Zn, y Pb) por Espectrometría de Absorción Atómica (AAS) fueron hechos en especímenes de Sipunculus nudus (Sipuncula) y Glottidia audebarti (Brachiopoda). Concentraciones máximas promedio en S. nudus fueron: para gusanos no-depurados, Fe (16.0 mg/g dw) > Mn (165 µg/g dw) > Zn (81 µg/g dw) > Cu (26 µg/g dw) > Cr (11 µg/g dw) > Ni (10.4 µg/g dw) > Pb (9.3 µg/g dw) > Cd (1.2 µg/g dw). Para gusanos depurados por 72 horas: Fe (5.0 mg/g dw) > Mn (61 µg/g dw) > Zn (39 µg/g dw) > Cu (24 µg/g dw) > Ni (8.4 µg/g dw) > Pb (2.7 µg/g dw) > Cd (0.62 µg/g dw). Para G. audebarti: Fe (1.6 mg/g dw-partes suaves) > Zn (123.5 µg/g dw-partes suaves) > Cu (31.4 µg/g dwpedículos) > Pb (21.0 µg/g dw-conchas) > Cd (5.2 µg/g dw-partes suaves) > Cr (4.7 µg/g dw-conchas). Para sedimentos; Fe (46 mg/g dw) > Mn (41.3 µg/g dw) > Zn (63 µg/g dw) > Cu (36.2 µg/g dw) > Cr (31.5 µg/g dw) > Pb (21.1 µg/g dw) > Ni (16.1 µg/g dw) > Cd (1.1 µg/g dw). Estas concentraciones fueron esperables para un estuario no industrializado. En la planicie arenosa (Area muestreada: 10.6 m2) 76 individuos de G. audebarti, 112 de G. albida y 366 de S. nudus fueron recolectados en el 2015, con densidades estimadas de: 7.1, 10.5, y 31 ind. /m2, respectivamente. Densidades de G. audebarti y G. albida fueron relativamente bajas, mientras que las de S. nudus fueron relativamente altas cuando se les comparó con otros reportes. La longitud de la concha de G. audebarti varió entre 9.0 mm y 38.0 mm y entre 6.0 mm a 29.0 mm la de G. albida. Estos ámbitos estuvieron dentro de los encontrados para estos lingúlidos en otros sitios. La longitud promedio de S. nudus fue 41 mm y el peso máximo fue de 1.6 g que son pequeños. En la planicie arenoso-fangosa no se encontró braquiópodos en el 2013, ni enteropneustos en la planicie arenosa en el 2015. G. audebarti tuvo una presencia relativamente estable, mientras que G. albida casi desapareció de las muestras al final del 2015. La distribución espacial de las tres especies fue de tipo agregado en ambas planicies. Fuertes eventos ENSO durante 1983 y 2015, así como mareas rojas en 1985, pueden haber influenciado las abundancias.

Morphology, Biology, and Phylogenetic Position of the Bivalve Platomysia rugata (Heterodonta: Galeommatoidea), a Commensal with the Sipunculan Worm Sipunculus nudus.

The bivalve superfamily Galeommatoidea is characterized by its symbiotic associations with other marine invertebrates. However, for many galeommatoideans, the host species remains unknown. Platomysia (Galeommatoidea) is a monotypic genus including a single species P. rugata, which is distinguished from other galeommatoideans in having distinct and evenly spaced commarginal ribs on its shell surface. This species was described based on a single right valve shell collected in Nanao Bay, Japan Sea, by Habe in 1951 and has been known only from Japanese waters. However, the biology of living animals has never been reported. We found that this species lives in the burrows of the sipunculan worm Sipunculus nudus in mud flats in the Seto Inland Sea, Japan. We investigated its host association and described its shell morphology and anatomy. In addition, we performed a phylogenetic analysis using two nuclear (18S and 28S ribosomal RNA) genes to determine its phylogenetic position in Galeommatoidea. The result suggests that this species belongs to the clade of commensal bivalves together with Pseudopythina, Byssobornia, and Pergrinamor. Platomysia rugata and other two groups of sipunculan-associated galeommatoideans were not monophyletic, suggesting that association with sipunculans occurred at least three times in the galeommatoid evolution.

Anti-tumor and anti-virus activity of polysaccharides extracted from Sipunculus nudus(SNP) on Hepg2.2.15.

Many polysaccharides have biological activities and have been investigated for their antitumor effects. In this study, we investigated the anti-tumor activity and anti-virus activity of SNP-the water-soluble polysaccharides extracted from Sipunculus nudus on Hepg2.2.15. Flow cytometry analysis demonstrated that SNP induced dose-dependent cell apoptosis on Hepg2.2.15. Real-time PCR and Western Blot analysis showed that SNP down-regulated the synthesis of HBsAg, HBV-DNA and enhanced the expression of pro-apoptosis proteins TNF-α, caspase-3, and Bax, while decreasing the expression of the anti-apoptosis proteins survivin, Bcl-2, and VEGF. These results suggested that SNP suppressed cell viability of Hepg2.2.15 and that could be a novel anti-tumor and anti-HBV agent.