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.

Physiological responses of Holothuria grisea during a wound healing event: An integrated approach combining tissue, cellular and humoral evidence.

Due to their tissue structure similar to mammalian skin and their close evolutionary relationship with chordates, holothurians (Echinodermata: Holothuroidea) are particularly interesting for studies on wound healing. However, previous studies dealing with holothuroid wound healing have had limited approaches, being restricted to tissue repair or perivisceral immune response. In this study, we combined tissue, cellular and humoral parameters to study the wound healing process of Holothuria grisea. The immune responses of the perivisceral coelom were assessed by analyzing the number, proportion and viability of coelomocytes and the volume and protein concentration of the coelomic fluid. Additionally, the morphology of the healing tissue and number of coelomocytes in the connective tissue of different body wall layers were examined over 30 days. Our results showed that perivisceral reactions started 3 h after injury and decreased to baseline levels within 24 h. In contrast, tissue responses were delayed, beginning after 12 h and returning to baseline levels only after day 10. The number of coelomocytes in the connective tissue suggests a potential cooperation between these cells during wound healing: phagocytes and acidophilic spherulocytes act together in tissue clearance/homeostasis, whereas fibroblast-like and morula cells cooperate in tissue remodeling. Finally, our results indicate that the major phases observed in mammalian wound healing are also observed in H. grisea, despite occurring at a different timing, which might provide insights for future studies. Based on these data, we propose a model that explains the entire healing process in H. grisea.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Radial glial cells play a key role in echinoderm neural regeneration.

BACKGROUND: Unlike the mammalian central nervous system (CNS), the CNS of echinoderms is capable of fast and efficient regeneration following injury and constitutes one of the most promising model systems that can provide important insights into evolution of the cellular and molecular events involved in neural repair in deuterostomes. So far, the cellular mechanisms of neural regeneration in echinoderm remained obscure. In this study we show that radial glial cells are the main source of new cells in the regenerating radial nerve cord in these animals. RESULTS: We demonstrate that radial glial cells of the sea cucumber Holothuria glaberrima react to injury by dedifferentiation. Both glia and neurons undergo programmed cell death in the lesioned CNS, but it is the dedifferentiated glial subpopulation in the vicinity of the injury that accounts for the vast majority of cell divisions. Glial outgrowth leads to formation of a tubular scaffold at the growing tip, which is later populated by neural elements. Most importantly, radial glial cells themselves give rise to new neurons. At least some of the newly produced neurons survive for more than 4 months and express neuronal markers typical of the mature echinoderm CNS. CONCLUSIONS: A hypothesis is formulated that CNS regeneration via activation of radial glial cells may represent a common capacity of the Deuterostomia, which is not invoked spontaneously in higher vertebrates, whose adult CNS does not retain radial glial cells. Potential implications for biomedical research aimed at finding the cure for human CNS injuries are discussed.

Organochlorine pollutants in Western Antarctic Peninsula sediments and benthic deposit feeders.

Sediments and benthic deposit feeding holothurians were collected near the Palmer Long Term Ecological Research grid during the austral winter of 2008. Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were measured in Western Antarctic Peninsula continental shelf sediments, porewater, and benthic biota. Concentrations and fluxes in sediments decreased sharply away from the tip of the peninsula toward its interior. Sedimentary PCB fluxes were an order of magnitude lower than reported elsewhere, supporting the notion of a pristiner Antarctic environment. Hexa-chlorinated biphenyls dominated (40-100%) the PCB profiles in the sediments, while trichlorinated biphenyl 28 was the most abundant PCB congener in the porewater. PCB and OCP concentrations in holothurians were comparable to concentrations in other low trophic level biota in the Antarctic food web (i.e., krill). The partitioning of PCBs and OCPs between the sediments and porewater can be explained by a dual-mode model, which included both organic carbon and black carbon as partitioning media. Alternatively, a simpler one-parameter prediction assuming coal tar-like organic carbon performed equally well in explaining porewater concentrations The majorities of PCBs (63-94%) in the Western Antarctic Peninsula sediments were bound to black carbon or recalcitrant tar-like organic carbon, thereby lowering porewater concentrations. PCBs and OCPs in the holothurians were in equilibrium with those in the porewater.

Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrima.

BACKGROUND: Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima. RESULTS: We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes. CONCLUSIONS: Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

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.

Regeneration of the radial nerve cord in the sea cucumber Holothuria glaberrima.

BACKGROUND: Regeneration of neurons and fibers in the mammalian spinal cord has not been plausible, even though extensive studies have been made to understand the restrictive factors involved. New experimental models and strategies are necessary to determine how new nerve cells are generated and how fibers regrow and connect with their targets in adult animals. Non-vertebrate deuterostomes might provide some answers to these questions. Echinoderms, with their amazing regenerative capacities could serve as model systems; however, very few studies have been done to study the regeneration of their nervous system. RESULTS: We have studied nerve cord regeneration in the echinoderm Holothuria glaberrima. These are sea cucumbers or holothurians members of the class Holothuroidea. One radial nerve cord, part of the echinoderm CNS, was completely transected using a scalpel blade. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in the radial nerve cord were analyzed using classical dyes and immunohistochemistry. In addition, the temporal and spatial distribution of cell proliferation and apoptosis was assayed using BrdU incorporation and the TUNEL assay, respectively.We found that H. glaberrima can regenerate its radial nerve cord within a month following transection. The regenerated cord looks amazingly similar in overall morphology and cellular composition to the uninjured cord. The cellular events associated to radial cord regeneration include: (1) outgrowth of nerve fibers from the injured radial cord stumps, (2) intense cellular division in the cord stumps and in the regenerating radial nerve cords, (3) high levels of apoptosis in the RNC adjacent to the injury and within the regenerating cord and (4) an increase in the number of spherule-containing cells. These events are similar to those that occur in other body wall tissues during wound healing and during regeneration of the intestine. CONCLUSION: Our data indicate that holothurians are capable of rapid and complete regeneration of the main component of their CNS. Regeneration involves both the outgrowth of nerve fibers and the formation of neurons. Moreover, the cellular events employed during regeneration are similar to those involved in other regenerative processes, namely wound healing and intestinal regeneration. Thus, holothurians should be viewed as an alternative model where many of the questions regarding nervous system regeneration in deuterostomes could be answered.

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.