Effects of hypo-osmotic stress on osmoregulation, antioxidant response, and energy metabolism in sea cucumber Holothuria moebii under desalination environment.

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl- concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5'-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress.

Post-autotomy regeneration of respiratory tree in sea cucumber Holothuria parva.

The respiratory trees present only in the class Holothuroidea and the rest of the echinoderms lack it. Only some holothurian species have the ability to regenerate their respiratory trees after autotomy. Therefore, respiratory trees could be considered as a suitable model to assess the regeneration mechanisms in animals. In the present study, the respiratory tree regeneration after posterior evisceration were examined in Holothuria parva during 75 days. Since autotomy reduces antioxidant defense in the organisms, in the present study alterations of antioxidant enzymes were also evaluated during the experiment. H. parva is the dominant intertidal species distributed in the north of the Persian Gulf. In the present study, H. parva ejected the left respiratory tree, the digestive tract and supportive mesenteries from the anus, about 1-2 min after potassium chloride injection. The closure of the opening at the posterior ends of the body was the first reaction to the injury. Seven days after evisceration, the small bud formed on the dorsal side of the cloaca which was covered with the coelomic epithelium of cloaca. The coelomic epithelium started to proliferate to undifferentiated cells on the apex of the buds. The primary respiratory tree consisted of the luminal cuboidal epithelium and thin connective tissue surrounded by the slender coelomic epithelium. This preliminary organ was observed at the apex of the buds, 13 days after evisceration. Gradually, myoepithelial cells arranged around a longitudinal axis and formed a circular muscle. The primitive branches of primary respiratory tree started to form 18 days after evisceration. Forty days after evisceration, the luminal epithelium of the respiratory tree had the same appearance as the intact luminal epithelium. The regenerated respiratory tree was histomorphologically very similar to an intact respiratory tree 56 days postevisceration, but unlike that, it was not yet wrapped around the intestine and was completely separate from it. Despite the development of the regenerating respiratory tree, no wrapping around the intestine was observed until the end of the experiment. According to the results, the activity of the catalase (CAT) and superoxide dismutase (SOD) in the muscle homogenate was significantly higher than the control 5 days after evisceration. The CAT and SOD levels gradually decreased in eviscerated animals. The lipid peroxidation level followed a decreasing trend in the eviscerated animals during the experiment. However, its value reduced to the control level at the end of the experiment.

Wnt/ß-catenin signaling pathway regulates cell proliferation but not muscle dedifferentiation nor apoptosis during sea cucumber intestinal regeneration.

Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/ß-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of ß-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of ß-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/ß-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration.

How to Wear a Sandy Coat: Secretory Cells in the Dorsal Epidermis in the Sea Cucumber Holothuria atra (Echinodermata: Holothuroidea).

Holothuria atra is a black sea cucumber commonly found on the sandy bottom of Okinawan coral reefs. The body surface of H. atra is usually covered with sand; however, sand never covers the body of another black congener, Holothuria leucospilota, which is sympatrically distributed with H. atra. The epidermal structures were examined in these two species by means of transmission electron microscopy to determine how sand adheres to the surface of H. atra. While the epidermis was basically composed of support cells bearing microvilli and vacuolated cells probably corresponding to mucus cells, two types of granular cells, type 1 and 2, were also found at the tip of the tube feet. These granular cells were closely similar in structure to secretory cells that have been supposed to secrete adhesive substances in other holothurians. Type 1 granular cells were also found in the dorsal epidermis of H. atra but not in H. leucospilota. Therefore, adhesive secretion by type 1 granular cells probably enables the attachment of sand to the H. atra body.

Transcriptomic analysis of early stages of intestinal regeneration in Holothuria glaberrima.

Echinoderms comprise a group of animals with impressive regenerative capabilities. They can replace complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied. The molecular machinery behind this faculty, however, remains to be understood. Here we assembled and annotated a de novo transcriptome using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 h and 3 days regenerating intestine, revealing a large number of differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with mechanisms involving developmental and cancer-related activity that could be related to the regenerative process. Our study demonstrates the broad and diversified gene expression profile during the early stages of the process using the mesentery as the focal point of intestinal regeneration. It also establishes the genes that are the most important candidates in the cellular processes that underlie regenerative responses.

Humoral responses during wound healing in Holothuria tubulosa (Gmelin, 1788).

Wounds in living organisms trigger tissue-repair mechanisms. The sea cucumber (Holoturia tubulosa) is an excellent model species for achieving a better understanding of the humoral and cellular aspects involved in such healing processes. Consequently, this study assesses data on its morphometric, physiological and humoral responses 1, 2, 6, 24 and 48h after wound induction. In particular, morphometric data on the weight, width, length and coelomic-fluid volume of the species were estimated at different times during our experiments. In addition, the humoral aspects related to the enzymatic activity of esterase, alkaline phosphatase and peroxidase, as well as the cytotoxic activity of cell lysates (CL) and cell-free coelomic fluids (CfCf) are evaluated for the first time. Our results reveal a significant decrease in body length and weight, along with time-dependent, significant changes in the esterase, alkaline phosphatase, peroxidase and cytotoxic activity in both the CL and CfCf. The data obtained lead to the pioneering finding that there is an important time-dependent involvement of morphometric (changes in weight and length) and humoral (enzymatic and cytotoxic) responses in wound healing.

Seasonal variations of digestive enzymes in sea cucumbers (Holothuria tubulosa, G. 1788) under culture conditions.

Sea cucumber, Holothuria tubulosa (Gmelin, 1788), is an economically valuable species due to its rich nutrients content that being exported in Turkey. However, culture of this species is difficult due to a lack of knowledge. The main objective of this study is to investigate seasonal activities of main digestive enzymes (protease, lipase, and amylase) for nutritional requirements. In this sense, sea cucumbers were stocked in the glass aquarium and sediment was 10 cm. It was replaced monthly and enriched (1% of the living individual weight) by dried Sargassum sp. Total protease and amylase enzymes have shown higher activity compared to lipase. All three enzyme activities were maximum in the summer and minimum in the winter. In summer, the highest measured total protease, amylase, and lipase activity values were 6.45 ± 0.66, 6.77 ± 0.72, and 2.78 ± 0.32 U/mg per protein, respectively. In winter, the lowest total protease, amylase, and lipase activity values were measured as 2.03 ± 0.16, 1.14 ± 0.14, and 0.12 ± 0.01 U/mg per protein, respectively. As a conclusion of the study, seasonal expression of the main digestive enzymes was strictly dependent on water temperatures and food abundance and also it was ideal to feed this species with food containing high protein and carbohydrate under appropriate temperatures for commercial culture.

Insights into intestinal regeneration signaling mechanisms.

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/ß-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of ß-catenin activity was determined using an antibody against phosphorylated ß-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated ß-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined.

Acclimation capability inferred by metabolic performance in two sea cucumber species from different latitudes.

The notion that thermal specialists from tropical regions live closer to their temperature limits than temperate eurytherms, seems too generalized. Species specific differences in physiological and biochemical stress reactions are linked to key components of organism fitness, like metabolic capacity, which indicates that acclimation potential across latitudes might be highly diverse rather than simplistic. In this study the exposure of a tropical (Holothuria scabra) and a temperate (Holothuria forskali) sea cucumber species to identical cold- and warm-acclimation stress was compared using the key metabolic parameters, respiration rate, enzyme activity (ETS, LDH, IDH), and energy reserve fractions (lipid, carbohydrate and protein). Results show much broader respiratory adjustments, as response to temperature change, in H. scabra (2-30 µgO2*gww-1*h-1) compared to H. forskali (1.5-6.6 µgO2*gww-1*h-1). Moreover, the tropical species showed clearly pronounced up and down regulation of metabolic enzymes and shifts in energy reserves, due to thermal acclimation, while the same metabolic indicators remained consistent in the temperate species. In summary, these findings indicate enhanced metabolic plasticity in H. scabra at the cost of elevated energy expenditures, which seems to favor the tropical stenotherm in terms of thermal acclimation capacity. The comparison of such holistic metabolic analyses between conspecifics and congeners, may help to predict the heterogeneous effects of global temperature changes across latitudinal gradients.

Metalloproteinase inhibitor GM6001 delays regeneration in holothurians.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

Temperature-induced aerobic scope and Hsp70 expression in the sea cucumber Holothuria scabra.

The Aerobic Scope (AS), which reflects the functional capacity for biological fitness, is a highly relevant proxy to determine thermal tolerance in various taxa. Despite the importance of this method, its implementation is often hindered, due to lacking techniques to accurately measure standard- (SMR) and maximal- (MMR) metabolic rates, especially in sluggish marine invertebrates with low oxygen consumption rates, such as sea cucumbers. In this study the AS concept was modified to define a Temperature-induced Aerobic Scope (TAS), based on metabolic rate changes due to temperature adjustments rather than traditionally used physical activity patterns. Consequentially, temperature dependent peak and bottom O2 consumption rates, defined as Temperature-induced Maximal- (TMMR) and Standard Metabolic Rates (TSMR), respectively, served as MMR and SMR alternatives for the sea cucumber Holothuria scabra. TMMR and TSMR were induced through acute temperature change (2°C per hour; 17-41°C) until critical warm (WTcrit) and cold (CTcrit) temperatures were reached, respectively. In addition, Hsp70 gene expression linked to respiration rates served as synergistic markers to confirm critical threshold temperatures. O2 consumption of H. scabra peaked distinctly at WTcrit of 38°C (TMMR = 33.2 ± 4.7 µgO2 g-1 h-1). A clear metabolic bottom line was reached at CTcrit of 22°C (TSMR = 2.2 ± 1.4 µgO2 g-1 h-1). Within the thermal window of 22-38°C H. scabra sustained positive aerobic capacity, with assumed optimal performance range between 29-31.5°C (13.85-18.7 µgO2 g-1 h-1). Between 39-41°C H. scabra decreased respiration progressively, while gene expression levels of Hsp70 increased significantly at 41°C, indicating prioritization of heat shock response (HSR) and homeostatic disruption. At the cold end (17-22°C) homeostatic disruption was visible through incrementally increasing energetic expenses to fuel basal maintenance costs, but no Hsp70 overexpression occurred. TMMR, TSMR and TAS proved to be reliable metrics, similar to the traditional energetic key parameters MMR, SMR and AS, to determine a specific aerobic performance window for the sluggish bottom dwelling species H. scabra. In addition, the linkage between respiration physiology and molecular defense mechanisms showed valuable analytical synergies in terms of mechanistic prioritization as response to thermal stress. Overall, this study will help to define lethal temperatures for aquaculture and to predict the effects of environmental stress, such as ocean warming, in H. scabra.

Diversity and antimicrobial potential in sea anemone and holothurian microbiomes.

Marine invertebrates, as holobionts, contain symbiotic bacteria that coevolve and develop antimicrobial substances. These symbiotic bacteria are an underexplored source of new bioactive molecules to face the emerging antibiotic resistance in pathogens. Here, we explored the antimicrobial activity of bacteria retrieved from the microbiota of two sea anemones (Anemonia sulcata, Actinia equina) and two holothurians (Holothuria tubulosa, Holothuria forskali). We tested the antimicrobial activity of the isolated bacteria against pathogens with interest for human health, agriculture and aquaculture. We isolated 27 strains with antibacterial activity and 12 of these isolates also showed antifungal activity. We taxonomically identified these strains being Bacillus and Vibrio species the most representative producers of antimicrobial substances. Microbiome species composition of the two sea anemones was similar between them but differed substantially of seawater bacteria. In contrast, microbiome species composition of the two holothurian species was different between them and in comparison with the bacteria in holothurian feces and seawater. In all the holobiont microbiomes Bacteroidetes was the predominant phylum. For each microbiome, we determined diversity and the rank-abundance dominance using five fitted models (null, pre-emption, log-Normal, Zipf and Zipf-Mandelbrot). The models with less evenness (i.e. Zipf and Zipf-Mandelblot) showed the best fits in all the microbiomes. Finally, we tracked (using the V4 hypervariable region of 16S rRNA gene) the relative abundance of these 27 isolates with antibacterial activity in the total pool of sequences obtained for the microbiome of each holobiont. Coincidences, although with extremely low frequencies, were detected only in the microbiome of H. forskali. This fact suggests that these isolated bacteria belong to the long tail of rare symbiotic bacteria. Therefore, more and more sophisticated culture techniques are necessary to explore this apparently vast pool of rare symbiontic bacteria and to determine their biotechnological potentiality.

Molecular characterization and gene expression patterns of retinoid receptors, in normal and regenerating tissues of the sea cucumber, Holothuria glaberrima.

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system.

Wnt and frizzled expression during regeneration of internal organs in the holothurian Eupentacta fraudatrix.

Several genes of the Wnt and Frizzled families in the holothurian Eupentacta fraudatrix are characterized, and the complete coding sequences of wntA, wnt4, wnt6, wnt16, frizzled1/2/7, frizzled4, and frizzled5/8 are obtained. The dynamics of expression of these genes during regeneration of internal organs after evisceration are studied. Evisceration and the associated damages supposedly induce the expression of wnt16 on third day after evisceration. Genes wntA, wnt4, wnt6, and frizzled1/2/7 up-regulate during the period of active morphogenesis (5-7 days after evisceration) and might participate in regulation of tissue and organ formation. The signaling induced via Frizzled5/8 is could be necessary for formation of the anterior (ectodermal) part of the digestive system and development of the calcareous ring on 10th day after evisceration. Our data suggest that the Wnt signaling pathway plays a significant role in the regulation of regeneration of internal organs in holothurians.

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.

Mechanical adaptability of sea cucumber Cuvierian tubules involves a mutable collagenous tissue.

Despite their soft body and slow motion, sea cucumbers present a low predation rate, reflecting the presence of efficient defence systems. For instance, members of the family Holothuriidae rely on Cuvierian tubules for their defence. These tubules are normally stored in the posterior coelomic cavity of the animal, but when the sea cucumber is threatened by a potential predator, they are expelled through the cloacal aperture, elongate, become sticky and entangle and immobilise the predator in a matter of seconds. The mechanical properties (extensibility, tensile strength, stiffness and toughness) of quiescent (i.e. in the body cavity) and elongated (i.e. after expulsion) Cuvierian tubules were investigated in the species Holothuria forskali using traction tests. Important mechanical differences were measured between the two types of tubules, reflecting adaptability to their operating mode: to ease elongation, quiescent tubules present a low resistance to extension, while elongated tubules present a high toughness to resist tractions generated by the predator. We demonstrate that a mutable collagenous tissue (MCT) is involved in the functioning of these organs: (1) some mechanical properties of Cuvierian tubules are modified by incubation in a cell-disrupting solution; (2) the connective tissue layer encloses juxtaligamental-like cells, a cell type present in all MCTs; and (3) tensilin, a MCT stiffening protein, was localised inside these cells. Cuvierian tubules thus appear to enclose a new type of MCT which shows irreversible stiffening.

Effects of thermal stress on the immune and oxidative stress responses of juvenile sea cucumber Holothuria scabra.

Holothuria scabra is the most valued and cultured tropical sea cucumber, given the great demand of this species for human consumption. However, despite its ecological and economic relevance, little is known regarding its immune responses under thermal stress. Here, the main goal was to study the response of sea cucumbers to temperature stress, assessing sub-organismal alterations and acclimation capacities of juveniles to temperature changes. After changing temperature (1 °C/day) for 6 days, organisms were exposed to temperature conditions of 21 °C (cold), 27 °C (control), and 33 °C (warm) over a 30 day period. At each 15-day interval (T0, T15, and T30), six replicates per condition were killed for biochemical analysis. Immune responses were addressed by studying the activity of phenoloxidase (PO) and prophenoloxidase (ProPO) in the coelomic fluid. Antioxidant defence responses-catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) enzymatic activities-were measured in the muscle and respiratory tree tissues, whereas oxidative damage was evaluated by measuring levels of superoxide radicals (ROS), DNA-strand breaks and lipid peroxidation (LPO). Juvenile H. scabra increased SOD and PO activities when temperature was elevated, and revealed low levels of ROS and damage in both cold and warm treatments throughout the experiment, confirming the organism's moderate thermal stress. After the short acclimation period, the immune and antioxidant responses prevented damage and maintained homeostasis. This multi-biomarker approach highlights its usefulness to monitor the health of H. scabra and to gain insight concerning the use of this high-valued species in global-scale aquaculture from different temperature regions.

Lectin-Based Profiling of Coelomocytes in Holothuria scabra and Expression of Superoxide Dismutase in Purified Coelomocytes.

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some minor staining was also detected in spherulocytes. Our results thus provide a fundamental platform for comparing alterations that may happen to the population and SOD contents of coelomocytes when the sea cucumber is subjected to environmental changes that would activate their immune responses.

Expression of pluripotency factors in echinoderm regeneration.

Cell dedifferentiation is an integral component of post-traumatic regeneration in echinoderms. As dedifferentiated cells become multipotent, we asked if this spontaneous broadening of developmental potential is associated with the action of the same pluripotency factors (known as Yamanaka factors) that were used to induce pluripotency in specialized mammalian cells. In this study, we investigate the expression of orthologs of the four Yamanaka factors in regeneration of two different organs, the radial nerve cord and the digestive tube, in the sea cucumber Holothuria glaberrima. All four pluripotency factors are expressed in uninjured animals, although their expression domains do not always overlap. In regeneration, the expression levels of the four genes were not regulated in a coordinated way, but instead showed different dynamics for individual genes and also were different between the radial nerve and the gut. SoxB1, the ortholog of the mammalian Sox2, was drastically downregulated in the regenerating intestine, suggesting that this factor is not required for dedifferentiation/regeneration in this organ. On the other hand, during the early post-injury stage, Myc, the sea cucumber ortholog of c-Myc, was significantly upregulated in both the intestine and the radial nerve cord and is therefore hypothesized to play a central role in dedifferentiation/regeneration of various tissue types.

Cellular responses and HSP70 expression during wound healing in Holothuria tubulosa (Gmelin, 1788).

Wound repair is a key event in the regeneration mechanisms of echinoderms. We studied, at the behavioural, cellular and molecular levels, the wound healing processes in Holothuria tubulosa after injuries to the body wall. The experiments were performed for periods of up to 72 h, and various coelomocyte counts, as well as the expression of heat shock proteins (HS27, HSP70 and HSP90), were recorded. Dermal wound healing was nearly complete within 72 h. In the early stages, we observed the injured animals twisting their bodies to keep their injuries on the surface of the water for the extrusion of the buccal pedicles. At the cellular level, we found time-dependent variations in the circulating coelomocyte counts. After injury, in particular, we observed a significant increase in spherule cells at 2.5 h post-injury. Using the western blot method, we observed and reported that the wounds produced, compared with controls, a significant increase in HSP27 and HSP70 expression in coelomocytes, whereas HSP70 was increased in scar tissue and HSP90 was increased only in cell-free coelomic fluid. These results highlight that the wounds were responsible for the stress condition with the induction of cellular and biochemical responses.