Evidence of size-dependent toxicity of polystyrene nano- and microplastics in sea cucumber Apostichopus japonicus (Selenka, 1867) during the intestinal regeneration.

Microplastics are ubiquitous pollutants in the global marine environment. However, few studies have adequately explored the different toxic mechanisms of microplastics (MPs) and nanoplastics (NPs) in aquatic organisms. The sea cucumber, Apostichopus japonicus, is a key organism in the marine benthic ecosystem due to its crucial roles in biogeochemical cycles and food web. This study investigated the bioaccumulation and adverse effects of polystyrene micro- and nanoplastics (PS-M/NPs) of different sizes (20 µm, 1 µm and 80 nm) in the regenerated intestine of A. japonicus using multi-omics analysis. The results showed that after 30-day exposure at the concentration of 0.1 mg L-1, PS-MPs and PS-NPs accumulated to 155.41-175.04 µg g-1 and 337.95 µg g-1, respectively. This excessive accumulation led to increased levels of antioxidases (SOD, CAT, GPx and T-AOC) and reduced activities of immune enzymes (AKP, ACP and T-NOS), indicating oxidative damage and compromised immunity in the regenerated intestine. PS-NPs had more profound negative impacts on cell proliferation and differentiation compared to PS-MPs. Transcriptomic analysis revealed that PS-NPs primarily affected pathways related to cellular components, e.g., ribosome, and oxidative phosphorylation. In comparison, PS-MPs had greater influences on actin-related organization and organic compound metabolism. In the PS-M/NPs-treated groups, differentially expressed metabolites were mainly amino acids, fatty acids, glycerol phospholipid, and purine nucleosides. Additionally, microbial community reconstruction in the regenerated intestine was severely disrupted by the presence of PS-M/NPs. In the PS-NPs group, Burkholderiaceae abundance significantly increased while Rhodobacteraceae abundance decreased. Correlation analyses demonstrated that intestinal regeneration of A. japonicus was closely linked to its enteric microorganisms. These microbiota-host interactions were notably affected by different PS-M/NPs, with PS-NPs exposure causing the most remarkable disruption of mutual symbiosis. The multi-omic approaches used here provide novel insights into the size-dependent toxicity of PS-M/NPs and highlight their detrimental effects on invertebrates in M/NPs-polluted marine benthic ecosystems.

Evaluation of the chronic toxicity of bisphenol A and bisphenol AF to sea cucumber Apostichopus japonicus after long-term single and combined exposure at environmental relevant concentration.

Bisphenols are emerging endocrine disrupting pollutant, and several studies have reported that they are already ubiquitous in various environmental matrices and intend to deposit in sediment. The primary sources of bisphenols are river and sewage discharge. Sea cucumber (Apostichopus japonicus), a typical deposit feeder, is one of the most important commercial marine species in Aisa. However, the effects of the bisphenol A (BPA) and its analogues bisphenol AF (BPAF) on sea cucumber was unclear. In this study, we carried out field survey in major sea cucumber farming areas in northern China, with the aim of determining which bisphenol analogue is the major bisphenol contamination in this aquaculture area. The results showed that the presence of BPAF was detected in four sampling sites (Dalian, Tangshan, Laizhou, and Longpan). The mean level of BPAF in Laizhou sediment samples was the highest which reached to 9.007 ± 4.702 µ g/kg. Among the seawater samples, the BPAF only have been detected in the samples collected at Longpan. (0.011 ± 0.003 µ g/L). Furthermore, we conducted an experiment to evaluate the single and combined toxicity of BPA and BPAF on sea cucumbers. The concentrations were informed by the findings based on the results of field research. (0.1, 1.0, and 10 µ g/L). After exposure, the body weight gain, and specific growth rate showed no significant changes (P > 0.05). We observed the histological alterations in respiratory tree of treated sea cucumbers including the fusion and detachment of lining epithelial tissue, and increase of lumen space. However, the catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) activity was not significantly changed (P > 0.05). We evaluated the effects of BPA and BPAF through calculating the integrated biomarker response index (IBR), and the results indicated that the toxicity of combined treatment was higher than single treatment. Additionally, BPAF exposure to A. japonicus was more toxic than BPA.

Effects of chronic dietary hexavalent chromium on bioaccumulation and immune responses in the sea cucumber Apostichopus japonicus.

Sea cucumbers Apostichopus japonicus (3.54 ± 0.01 g of wet weight) were exposed to five concentrations of dietary hexavalent chromium (Cr6+) [0 (control), 100, 200, 400, and 800 mg Cr6+/kg dry weight] amended with K2Cr2O7 for 30 days. The bioaccumulation and immune responses [antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT); hydrolytic enzymes: acid phosphatase (ACP) and alkaline phosphatase (AKP)] of sea cucumbers were subsequently evaluated. This study found that the order of Cr accumulation in the experimental tissues was respiratory tree > intestine > body wall. Significantly lower SOD activities occurred in the 400 mg/kg group compared to that in the control group. Higher dietary Cr6+ exposure (400 and 800 mg Cr6+/kg dry weight) did not negatively alter the CAT activities, but significantly inhibited CAT activities in 100 mg/kg group, compared to control group. ACP activities in groups 200, 400 and 800 mg/kg were significantly lower than those in control group, while no significant differences occurred in AKP activities among groups. The present study provides important information into the bioaccumulation and immune responses of the sea cucumber A. japonicus in response to chronic dietary Cr6+ exposure.

Combined Effects of Elevated Temperature and Crude Oil Pollution on Oxidative Stress and Apoptosis in Sea Cucumber (Apostichopus japonicus, Selenka).

Currently, global climate change and oil pollution are two main environmental concerns for sea cucumber (Apostichopus japonicus) aquaculture. However, no study has been conducted on the combined effects of elevated temperature and oil pollution on sea cucumber. Therefore, in the present study, we treated sea cucumber with elevated temperature (26 °C) alone, water-accommodated fractions (WAF) of Oman crude oil at an optimal temperature of 16 °C, and Oman crude oil WAF at an elevated temperature of 26 °C for 24 h. Results showed that reactive oxygen species (ROS) level and total antioxidant capacity in WAF at 26 °C treatment were higher than that in WAF at 16 °C treatment, as evidenced by 6.03- and 1.31-fold-higher values, respectively. Oxidative damage assessments manifested that WAF at 26 °C treatment caused much severer oxidative damage of the biomacromolecules (including DNA, proteins, and lipids) than 26 °C or WAF at 16 °C treatments did. Moreover, compared to 26 °C or WAF at 16 °C treatments, WAF at 26 °C treatment induced a significant increase in cellular apoptosis by detecting the caspase-3 activity. Our results revealed that co-exposure to elevated temperature and crude oil could simulate higher ROS levels and subsequently cause much severer oxidative damage and cellular apoptosis than crude oil alone on sea cucumber.

The effect of ocean acidification on the enzyme activity of Apostichopus japonicus.

The influence of ocean acidification (OA) is particularly significant on calcifying organisms. The sea cucumber Apostichopus japonicus is an important cultured calcifying organism in the northern China seas. Little was known about the effects of OA on this economically important species. In this study, individuals from embryo to juveniles stage of A. japonicus, cultured in different levels of acidified seawater, were measured their enzymes activities, including five metabolic enzymes and three immune enzymes. The activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) was significantly lower in the severely acid group (pH 7.1), while the content of lactate dehydrogenase (LDH) was significantly higher. Superoxide dismutase (SOD) and catalase (CAT) were significantly lower in the severely acid group. The multivariate statistical results showed that the significant difference of enzyme assemblage existed among three experimental groups. This study indicated that OA could reduce the biomineralization capacity, influence the anaerobic metabolism and severely affect the immune process of A. japonicas. More researches are needed in the future to reveal the mechanisms of enzyme regulation and expression of A. japonicas underlying mixture environmental stress.

Enhanced protein phosphorylation in Apostichopus japonicus intestine triggered by tussah immunoreactive substances might be involved in the regulation of immune-related signaling pathways.

The sea cucumber Apostichopus japonicus is an economically important species owing to its high nutritive and medicinal value. In order to avoid the pollution resulting from the overuse of antibiotics in A. japonicus aquaculture, various immunostimulants have been used as an alternative to improve the efficiency of A. japonicus farming. Our previous proteomic investigation has shown that several proteins participating in the immune-related physiology of A. japonicus were differentially expressed in the intestinal tissue in response to tussah immunoreactive substances (TIS). This study further explored the immunostimulation mechanism of TIS in A. japonicus. Phosphoproteomics technology was used to investigate the effect of TIS on protein phosphorylation in the intestine of A. japonicus following feeding with a TIS-supplemented diet. A total of 213 unique phosphoproteins were detected from 225 unique phosphopeptides. KEGG pathway analysis showed that majority of the phosphoproteins are involved in endocytosis, carbon metabolism and spliceosome functional group. Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS and 12 of these were found to associate with biological functions. Of these 12 phosphoproteins, eight exhibited enhanced phosphorylation while four displayed reduced phosphorylation. These 12 proteins were further analyzed and all were found to play a role in regulating some aspects of the immune system and the growth of sea cucumbers, especially in phagocytosis, energy metabolism and disease resistance. The findings of this study could therefore shed new light on the immune pathways of sea cucumber that are affected by TIS. This could help us to better understand the underlying mechanism linked to the immunoenhancement of A. japonicus in response to TIS, one that is associated with the change in protein phosphorylation.

Bacillus baekryungensis MS1 regulates the growth, non-specific immune parameters and gut microbiota of the sea cucumber Apostichopus japonicus.

Winter is a high incidence period of skin ulceration syndrome in the sea cucumber Apostichopus japonicus. Disease control during the overwintering of sea cucumber can help increase yield and reduce losses. The purpose of this study was to study the effect of the low temperature-resistant probiotic Bacillus baekryungensis MS1 on the growth and immune parameters of sea cucumbers and preliminarily investigate the molecular mechanism of the effects. A low temperature-resistant bacterium, B. baekryungensis MS1, was isolated from a sea cucumber pond in winter and used for culture experiments. After 10 days of prefeeding, the experiment was divided into the control group (fed with commercial diet) and the MS1 group (fed with diet containing B. baekryungensis MS1 at 107 cfu g-1) for a total of 60 days. The specific growth rate was measured at the end of the culture period to evaluate the growth performance of the sea cucumber. Samples were taken on days 30 and 60 to determine the immune parameters (including superoxide dismutase activity, catalase activity, alkaline phosphatase activity, acid phosphatase activity, nitric oxide synthetase activity, phagocytosis and respiratory burst), aquaculture water microbiota and gut microbiota of the sea cucumber. Finally, transcriptome sequencing and qRT-PCR verification of the two groups of sea cucumbers were performed to study the mechanism of B. baekryungensis MS1 to improve the immunity of the sea cucumber. The results showed that after 60 days of feeding, B. baekryungensis MS1 significantly improved the growth performance and immune enzyme activity and formed a healthier structure of the gut microbiota in the sea cucumber. The challenge test showed that B. baekryungensis MS1 significantly reduced the mortality of sea cucumbers infected with Vibrio splendidus. Transcriptome and gene expression analysis indicated that B. baekryungensis MS1 activated the ubiquitin-mediated proteolysis pathway and inhibited the mTOR signaling pathway to regulate the immunity of the sea cucumber. In summary, the low temperature-resistant bacterium B. baekryungensis MS1 could be applied for the aquiculture of sea cucumber in winter to improve health status and resist pathogenic bacteria such as V. splendidus.

Regulation of dietary astragalus polysaccharide (APS) supplementation on the non-specific immune response and intestinal microbiota of sea cucumber Apostichopus japonicus.

Astragalus polysaccharide (APS) plays important roles in antibacterial, antiviral and antiparasitic activities in mammals, birds and aquatic animals. However, the relationship between non-specific immune responses and intestinal microbiota in sea cucumber (Apostichopus japonicus) after dietary APS supplementation has not been reported to date. Here, the effect of dietary APS supplementation on the non-specific immune response and intestinal microbial composition and species distribution of sea cucumber was explored. We found that although there was no significant effect on sea cucumber growth, the enzymatic activity and expression level of immune- and antioxidant-related genes changed after dietary APS supplementation. Furthermore, the intestinal microbial composition and species distribution of sea cucumber were different at the phylum and genus levels after dietary APS supplementation. The phyla Proteobacteria and Bacteroidetes were significantly different between the APS2 group and CK group. The results of PCA and PCoA analysis also showed that the APS2 group was significantly different compared to the other groups. Finally, analysis of the relationship between non-specific immune responses and the intestinal microbiota showed that the expression level of NF-κB was significantly correlated with intestinal microbiota at the genus level. This finding suggests that dietary APS supplementation might affect the non-specific immune response and intestinal microbiota of sea cucumber through the NF-κB signalling pathway; the appropriate added level was 800 mg/kg. Taken together, our results lay a foundation for further understanding the relationship between non-specific immune responses and intestinal microbial of sea cucumber.

The effects of benzo[a]pyrene on the composition of gut microbiota and the gut health of the juvenile sea cucumber Apostichopus japonicus Selenka.

The gut microbiota is essential for health and physiological functions in the host organism. However, the toxicological evaluation of environmental pollutants on the gut microbiota is still insufficient. In the present study, the juvenile sea cucumber Apostichopus japonicus was exposed for 14 days to Benzo[a]pyrene (BaP), which is a model polycyclic aromatic hydrocarbon (PAH), at four different concentrations (0, 0.5, 5, and 25 µg/L). We analyzed the intestinal microbial community of A. japonicas using 16S rRNA gene amplicon sequencing. Our results demonstrate that BaP exposure caused alterations to the microbiome community composition in sea cucumbers. At the phylum level, Planctomycetes were significantly more abundant in BaP exposure groups at 14 d compared with the control group, and the abundance of Proteobacteria and Bacteroidetes increased while the abundance of Firmicutes decreased following BaP exposure. At the genus level, multiple beneficial and autochthonous genera declined in the BaP treatment groups compared to the control, including Lactococcus, Bacillus, Lactobacillus, Enterococcus, Leuconostoc and Weissella; however, a bloom of alkane-degrading bacteria was found in BaP-exposed guts and included Lutibacter, Pseudoalteromonas, Polaribacter, Rhodopirellula and Blastopirellula. Furthermore, histological morphology, enzymatic activity and gene expression analysis revealed that BaP exposure also negatively impacted gut structure and function and presented as inflammation or atrophy, oxidative stress and immune suppression in sea cucumber intestines. Collectively, these findings provide insights into the toxic effects of BaP exposure on A. japonicas associated with intestinal microbiota and health.

The influence of three antibiotics on the growth, intestinal enzyme activities, and immune response of the juvenile sea cucumber Apostichopus japonicus selenka.

The global abuse and misuse of antibiotics in the treatment and prevention of bacterial infections has resulted in the ubiquitous existence of these drugs in aquatic environments, which causes frequent antimicrobial resistance and pollution in ecosystems. However, the chronic effects of antimicrobial agents on aquatic animal growth and health have not been fully evaluated. In the present study, three typical antibiotics (tetracycline, erythromycin, and norfloxacin) were administered orally to juvenile sea cucumbers Apostichopus japonicus for 45 days, to mimic the long-term use of antibiotics. As a result, tetracycline and erythromycin promoted the growth and digestive activity of lipase, pepsin, and trypsin, but norfloxacin did not show significant prompting effect on digestive activity and even retarded the weight gain of the sea cucumbers. The mortality was higher in antibiotic treated groups between the 2nd and 4th days after challenge with Vibrio splendidus. At the same time, lower immune-related parameters were found in antibiotic feeding juveniles, suggesting that the use of antibiotics might weaken the immune defense system of sea cucumbers. This study revealed that antibiotic administration could facilitate the growth of sea cucumbers to varying degrees yet coupled with high risks of impaired immune function and compromised disease resistance.

Dual functions of a 4-hydroxyphenylpyruvate dioxygenase for Vibrio splendidus survival and infection.

Vibrio splendidus is a well-documented pathogenic bacterium that can trigger different diseases, including skin ulcer syndrome in Apostichopus japonicus. In our previous study, a gene named Vshppd encoding a 4-hydroxyphenylpyruvate dioxygenase homologue was cloned from pathogenic V. splendidus, and validated to be responsible for the haemolysis activities of V. splendidus. In this study, Vshppd was determined to participate in the catabolism of tyrosine and promote pyomelanin production in Escherichia coli BL21 (DE3) harboring Vshppd. The purified melanin pigment displayed obvious antimicrobial activity against E. coli and Micrococcus luteus and protective effect on V. splendidus under ultraviolet irradiation. As an important virulence factor, Vshppd was further determined to be cytotoxic to the coelomocyte of A. japonicus and cell viability decreased to approximately 68%, 77%, 54% and 44% when 50, 60, 80 and 100 µL of purified rVshppd was present, respectively. To better understand the potential effect of Vshppd mediated oxidative stress, we injceted A. japonicus with the rVshppd, which showed significantly stimulatory effects on the expression of oxidative stress related genes catalase (cat), glutathione S-transferase (gst), glutathione peroxidase (gpx), heat shock protein 70 (hsp70) of A. japonicus. At 48 h, the expression level of cytochrome P450 (cyp450) was down-regulated compared with that treated with BSA. It was suggested that Vshppd exhibited cytotoxicity via altering the oxidative stress. Our result indicated that Vshppd was not only involved in the self-protection, but also contributed to the pathogenesis of V. splendidus by modulating the oxidative stress imbalance in A. japonicus.

The Effects of Sub-lethal Dietary Mercury on Growth Performance, Bioaccumulation, and Activities of Antioxidant Enzymes in Sea Cucumber, Apostichopus japonicus.

Triplicate groups of sea cucumbers (4.83 ± 0.15 g) were exposed to one of the four nominal concentrations of dietary mercury [0 (control), 67.6, 338, and 676 mg/kg dry weight, and actually total mercury were 17.55, 87.00, 275.50, 468.50 mg/kg, respectively; Table 3] for 21 days. Mercury accumulation in the intestine showed the greatest mercury burden (77.96 ± 1.20 mg Hg/kg tissue wet weight basis). However, survival rate (SR) was not affected. Body weight gain after the 676 mg Hg/kg treatment was significantly lower than the control group. The feed conversion rate of the 676 mg Hg/kg treatment group was significantly higher than the control group. Additionally, the superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of sea cucumbers decreased as the mercury dose increased. SOD, T-AOC and alkaline phosphatase of the 676 mg Hg/kg treatment group were significantly lower than the control group. However, there were no significant differences between the four groups in acid phosphatase and catalase (CAT) activity.

Effects of l-tryptophan on the growth, intestinal enzyme activities and non-specific immune response of sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress.

In order to reveal the effects of l-tryptophan (Trp) on the physiology and immune response of sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress, four density groups of sea cucumbers (i.e. 4, 8, 16 and 32 individuals per 40 L water, represented as L, ML, MH and H) were fed with diets containing 0, 1, 3 and 5% l-tryptophan respectively for 75 days. The results showed that the specific growth rates (SGR) of the sea cucumber fed with diet with 3% Trp (L, 2.1; ML, 1.76; MH, 1.2; H, 0.7) were significantly higher than those fed with basal diet without Trp supplementation (P < .05). Peak amylase activity occurred at H stress density at 3% dietary Trp. Trypsin activity was higher in diet 3% in ML and MH densities than the controls, which increased by 66.4% and 53.8%. However, the lipase activity first increased and then decreased from the stocking density L to H, with highest values of 3% Trp group showed the highest value than other groups. Compared to those fed with the basal diet, sea cucumber fed diets with Trp (3%) had significantly higher phagocytic activities (0.28 OD540/106 cells, H) in coelomic fluid and respiratory burst activities (0.105 OD630/106 cells, MH) (P < .05). The results suggested that Trp cannot improve superoxide dismutase (SOD) activity at L, ML and MH densities. The alkaline phosphatase activity (AKP) significantly decreased at H stress density. Under the experimental conditions, the present results confirmed that a diet supplemented with 3% Trp was able to enhance intestinal enzyme activities, non-specific immune response and higher growth performance of A. japonicus.

Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka.

A feeding experiment was conducted to evaluate the effects of four strains of lactic acid bacteria (LAB) [i.e. Lactobacillus plantarum LL11 (LP), Weissella confuse LS13 (WC), Lactococcus lactis LH8 (LL) and Enterococcus faecalis LC3 (ES)] isolated from marine fish on growth, immune response and expression levels of immune-related gens in body wall of juvenile sea cucumber Apostichopus japonicus. As a result, sea cucumber had better growth performance fed supplementation of LP and ES than the control group (P < .05). Survival rate in each LAB supplementation group was significantly higher than that in control group after Vibrio splendidus challenge (P < .05). In regards to the enzyme activities, LP supplementation significantly imporved the enzyme activities of alkaline phosphatase (AKP) (P < .05), acid phosphatase (ACP) and superoxide dismutase (SOD), ES supplementation significantly imporved AKP activity (P < .05), and WC supplementation significantly imporved ACP activity (P < .05). However, lysozyme (LSZ) activity was not significantly changed in the four LAB supplementation treatments (P > .05). For the gene expression levels, different expression patterns were observed among four groups, heat shock proteins (HSP60, HSP70 and HSP90) and caspase-2 showed dramatic up-regulation at 30 d while NF-kappa-B transcription factor p65 was down-regulated at 15 d and up-regulated at 30 d, and nitric oxide synthase was down-regulated at both timepoints in almost all the four groups. In conclusion, the four LAB strains screened from marine fish supplemented in diets indicated positive effects on immune response for A. japonicus, especially, the L. plantarum LL11 and E. faecalis LC3 indicated better growth performance.

Indole reduces the expression of virulence related genes in Vibrio splendidus pathogenic to sea cucumber Apostichopus japonicus.

Indole is a metabolite of tryptophan that can be synthesized by various bacteria. In the present study, production of indole by Vibrio splendidus Vs was determined using Kovac's reagent, and m/z was further determined by HPLC-MS. Extracellular indole reached a maximum concentration of 160 µM, when OD600 of V. splendidus Vs was approximately 0.9. In addition, glucose could reduce indole level, and 1% (m/v) glucose could reduce the mRNA level of tnaA, the gene encoding tryptophanase, down to 0.2%. To investigate the effects of indole on the mRNA levels of virulence related genes of V. splendidus Vs, mRNA levels of vsm, vsh and ABC respectively related to protease activity, haemolytic activity and ABC transporter ATP-binding protein were determined. Exogenous indole supplemented at a concentration of 125 µΜ could respectively down regulate the mRNA level of vsm, vsh and ABC to 16%, 13% and 11%. Meanwhile, indole could alter the expressions of immune related gene in Apostichopus japonicus. When coelomocytes were co-cultured with exogenous indole at a concentration of 125 µΜ, the mRNA level of Ajp105 and AjLBP/BPI1, were up regulated by 1.6-fold and 2.1-fold, respectively. Combined all the results in our study suggested that indole could alter the expressions of the virulence related genes in pathogenic V. splendidus Vs as well as the immune related genes in A. japonicus.

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.

Quorum sensing-disrupting coumarin suppressing virulence phenotypes in Vibrio splendidus.

In the present study, the effects of an environmental friendly natural reagent coumarin, on the growth and potential virulence factors, as well as its ability to interfere the infection of Vibrio splendidus (Vs), were determined. Coumarin showed no effects on the maximal growth of Vs, and biofilm formation of Vs, while it significantly decreased protease activity and hemolytic activity by 43 and 80%, respectively. Correspondingly, coumarin exhibited an obviously protective effect, with a relative percent survival of 60% upon Apostichopus japonicus from infection by Vs. To preliminarily investigate the mechanism underlining the inhibitory effects, regulation of genes Vsm and Vsh respectively related to protease activity and hemolytic activity by supernatant and supernatant extract containing acyl-homoserine lactones (AHLs) and coumarin was determined. Cell-free supernatant from higher density and its ethyl acetate extract containing AHL signal molecules could respectively upregulate the mRNA level of Vsm by 17.4- and 2.3-fold and Vsh by 7.2- and 5.0-fold, when Vs was at lower cell density. However, coumarin could reduce the stimulatory effects of both the supernatant and its ethyl acetate extract. Combining all the results in our study, it was suggested that coumarin could be considered as an alternative to be used for controlling infection of Vs, downregulating the expression of potential virulence factors through interfering the AHL-mediated pathways.

Toxic effects in juvenile sea cucumber Apostichopus japonicas (Selenka) exposure to benzo[a]pyrene.

To understand the physiological response of sea cucumber, Apostichopus japonicas, were exposed to different concentration of benzo[a]pyrene (B[a]P), and the growth, survival, antioxidant enzyme (SOD and T-AOC) activities were tested. Meanwhile, the quantitative real-time PCR technology was utilized to quantize the expression of immune related genes (i.e. innate immune genes, HSP genes and anti-oxidative genes). In our result, the SOD activity and T-AOC activity were induced at lower level of B[a]P (0.03 µg/L), however, a reduction of SOD activity and T-AOC activity were observed at relatively high B[a]P concentration (3 and 9 µg/L) for A. japonicas. Furthermore, the distinct expression patterns of selected immune-related genes were detected among different concentrations, and a general trend of down-regulation was observed at higher concentration. Especially, lysozyme almost showed the highest down-regulation at all concentrations, followed by NOS. Collectively, the growth, survival and expression signatures of immune related genes reflected an overall suppression of innate immunity in sea cucumber following exposure. Future functional studies should be carried out to characterize the detailed roles of immune genes and their related responses under B[a]P toxicity. Additionally, better understanding of the molecular indicators governing the healthy status under environmental toxicity would facilitate a healthy and sustainable culture program.

Long Non-Coding RNAs (lncRNAs) of Sea Cucumber: Large-Scale Prediction, Expression Profiling, Non-Coding Network Construction, and lncRNA-microRNA-Gene Interaction Analysis of lncRNAs in Apostichopus japonicus and Holothuria glaberrima During LPS Challenge and Radial Organ Complex Regeneration.

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Effects of dietary cadmium on growth, antioxidants and bioaccumulation of sea cucumber (Apostichopus japonicus) and influence of dietary vitamin C supplementation.

The effects of dietary cadmium (Cd) supplementation on growth, antioxidant capacity and accumulation of Cd in tissues (body wall, digestive tracts, and respiratory tree) of sea cucumber, Apostichopus japonicus, exposed to sub-chronic concentrations (0, 10, 50, 100, and 500mg Cd/kg dry weight) of Cd were investigated. In addition, the potential protective effects of vitamin C (L-ascorbic acid, AsA) against the effects of Cd on sea cucumbers were investigated. Sea cucumbers were exposed to dietary Cd for 30 days, after which another group of healthy sea cucumbers was supplied diet supplemented with mixed Cd and AsA for another 30 days. Cd exposure for 30 days resulted in increased Cd accumulation in tissues of sea cucumbers with exposure time and concentration. The order of Cd accumulation in organs was digestive tracts>respiratory tree>body wall. On day 30, the body weight gain (BWG) and specific growth rate (SGR) decreased significantly (P<0.05) in the 500mg Cd/kg treatment. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity and catalase (CAT) activity in the coelomic fluid of sea cucumbers decreased with increasing dietary Cd concentration, but malondialdehyde (MDA) content in the coelomic fluid increased. Providing diet supplemented with Cd and AsA indicated that although sea cucumbers exhibited signs of Cd toxicity, no death occurred in response to 50mg Cd/kg for 30 days. Based on these findings, five treatments were provided: 50mg Cd/kg+0mg AsA/kg, 50mg Cd/kg+ 3000mg AsA/kg, 50mg Cd/kg+ 5000mg AsA/kg, 50mg Cd/kg+10,000mg AsA/kg, and 50mg Cd/kg+15,000mg AsA/kg. The BWG and SGR of sea cucumbers fed the AsA supplemented diet mixed with Cd increased. Additionally, MDA levels in coelomic fluid were negatively correlated with dietary AsA levels, while antioxidant capacities (SOD, GSH-Px and CAT) were positively correlated with dietary AsA levels. Moreover, Cd accumulation in tissues decreased in response to dietary AsA supplementation of treatments. Overall, antioxidant capacity and bioaccumulation in sea cucumber was found to decrease and be induced in response to Cd, but vitamin C mitigated these effects, with 5000mg AsA/kg providing the optimum protection against 50mg/kg Cd.