Mercury disrupts redox status, up-regulates metallothionein and induces genotoxicity in respiratory tree of sea cucumber (Holothuria forskali).

Mercury (Hg) is among the most deleterious contaminant in the aquatic environment and presents a serious risk to humans and ecosystems. This study evaluated the effects of Hg on oxidative stress biomarkers, DNA integrity and histological structure of the respiratory tree of Holothuria forskali exposed to different concentrations of mercury chloride HgCl2 (0.04, 0.08 and 0.16 mg L-1) for 96 h. Exposure of H. forskali to Hg led to oxidative stress with an increase in Malondialdehyde (MDA), hydrogen peroxide (H2O2), advanced oxidation protein product (AOPP) and protein carbonyls (PCO) levels in the treated groups. Alteration of the antioxidant system was also confirmed by the significant increase in glutathione (GSH), nonprotein thiol (NPSH) and vitamin C contents. Moreover, the enzymatic activity of superoxide dismutase (SOD), Glutathione peroxidase (GPX) and Catalase (CAT) increased significantly. Our research revealed that total Metallothionein (MTs) content enhanced in a dose-dependent manner. Interestingly, the exposure to this metal provoked a decrease in Acetylcholinesterase (AChE) activity. Hg genotoxicity was further evidenced by a random DNA degradation that was observed in the treated groups. The histopathological findings confirmed the biochemical results. Overall, our results indicated that mercury-induced genotoxicity, oxidative damage and histopathological injuries in the respiratory tree of H. forskali.

Effects of acute mercury exposure on fatty acid composition and oxidative stress biomarkers in Holothuria forskali body wall.

Mercury is one of the most harmful pollutant that threat marine biota. This study assessed the Hg impact on the fatty acid (FA) composition and the antioxidant statues in Holothuria forskali body wall tissue. Specimens were exposed to HgCl2 graded doses (40, 80 and 160 µg L-1) for 96 h. A decrease in linoleic, arachidonic and eicosapentaenoic acid levels and an increase of docosahexaenoic acid were mainly observed at the nominal tested dose. The exposure to the upper dose promoted oxidative stress with an increase of malondialdehyde, hydrogen peroxide, advanced oxidation protein product, glutathione and non-protein thiols levels. Moreover, a decrease in catalase and an increase in superoxide dismutase and glutathione peroxidase activities were observed. Yet, an increase of the metallothionein level was registered in all treated groups. This study confirmed the Hg toxicity on the redox statue of H. forskali and highlighted the usefulness of the FA composition as an early sensitive bioindicators.

A comparative experimental approach to ecotoxicology in shallow-water and deep-sea holothurians suggests similar behavioural responses.

Exploration of deep-sea mineral resources is burgeoning, raising concerns regarding ecotoxicological impacts on deep-sea fauna. Assessing toxicity in deep-sea species is technologically challenging, which promotes interest in establishing shallow-water ecotoxicological proxy species. However, the effects of temperature and hydrostatic pressure on toxicity, and how adaptation to deep-sea environmental conditions might moderate these effects, are unknown. To address these uncertainties we assessed behavioural and physiological (antioxidant enzyme activity) responses to exposure to copper-spiked artificial sediments in a laboratory experiment using a shallow-water holothurian (Holothuria forskali), and in an in situ experiment using a deep-sea holothurian (Amperima sp.). Both species demonstrated sustained avoidance behaviour, evading contact with contaminated artificial sediment. However, A. sp. demonstrated sustained avoidance of 5mgl-1 copper-contaminated artificial sediment whereas H. forskali demonstrated only temporary avoidance of 5mgl-1 copper-contaminated artificial sediment, suggesting that H. forskali may be more tolerant of metal exposure over 96h. Nonetheless, the acute behavioural response appears consistent between the shallow-water species and the deep-sea species, suggesting that H. forskali may be a suitable ecotoxicological proxy for A. sp. in acute (≤24h) exposures, which may be representative of deep-sea mining impacts. No antioxidant response was observed in either species, which was interpreted to be the consequence of avoiding copper exposure. Although these data suggest that shallow-water taxa may be suitable ecotoxicological proxies for deep-sea taxa, differences in methodological and analytical approaches, and in sex and reproductive stage of experimental subjects, require caution in assessing the suitability of H. forskali as an ecotoxicological proxy for A. sp. Nonetheless, avoidance behaviour may have bioenergetic consequences that affect growth and/or reproductive output, potentially impacting fecundity and/or offspring fitness, and thus influencing source-sink dynamics and persistence of wider deep-sea populations.

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.

Characterization of proteolytic activities during intestinal regeneration of the sea cucumber, Holothuria glaberrima.

Proteolysis carried out by different proteases control cellular processes during development and regeneration. Here we investigated the function of the proteasome and other proteases in the process of intestinal regeneration using as a model the sea cucumber Holothuria glaberrima. This echinoderm possesses the ability to regenerate its viscera after a process of evisceration. Enzymatic activity assays showed that intestinal extracts at different stages of regeneration possessed chymotrypsin-like activity. This activity was inhibited by i) MG132, a reversible inhibitor of chymotrypsin and peptidylglutamyl peptidase hydrolase (PGPH) activities of the proteasome, ii) E64d, a permeable inhibitor of cysteine proteases and iii) TPCK, a serine chymotrypsin inhibitor, but not by epoxomicin, an irreversible and potent inhibitor of all enzymatic activities of the proteasome. To elucidate the role which these proteases might play during intestinal regeneration, we carried out in vivo experiments injecting MG132, E64d and TPCK into regenerating animals. The results showed effects on the size of the regenerating intestine, cell proliferation and collagen degradation. These findings suggest that proteolysis by several proteases is important in the regulation of intestinal regeneration in H. glaberrima.

A novel stiffening factor inducing the stiffest state of holothurian catch connective tissue.

The dermis of sea cucumbers is a catch connective tissue or mutable collagenous tissue that shows large changes in stiffness. Extensive studies on the dermis revealed that it can adopt three different states having different mechanical properties that can be reversibly converted. These are the stiff, standard and soft states. The standard state is readily produced when a dermal piece is immersed in the sea water containing Ca²+, whereas the soft state can be produced by removal of Ca²+. A stiffening protein, tensilin, has been isolated from some sea cucumbers (Cucumaria frondosa and Holothuria leucospilota). Although tensilin converts the state of the dermis from soft to standard, it cannot convert from standard to stiff. In this study, we isolated and partially purified a novel stiffening factor from the dermis of Holothuria leucospilota. The factor stiffened the dermis in normal artificial sea water (ASW) but did not stiffen the soft dermis in Ca²+-free ASW. It also stiffened the dermis that had been converted to the standard state in Ca²+-free ASW by the action of tensilin. These results suggest that the factor produces the stiff dermis from the standard state but cannot work as a stiffener on the soft dermis. Its addition to longitudinal muscles of the sea cucumber produced no effects, suggesting that its effect is specific to the catch connective tissue. Its stiffening activity was susceptible to trypsin, meaning that it is a polypeptide, and its molecular mass estimated from gel filtration chromatography was 2.4 kDa.