On the taxonomic status of Holothuria (Holothuria) tubulosa (s.s.) from the Algerian coast with the description of a new Mediterranean species, Holothuria (Holothuria) algeriensis n. sp. (Echinodermata: Holothuroidea: Holothuriidae).

In this study we redescribe the taxonomy of some holothuriid species collected from different localities of the Algerian coastal waters. Morphological (anatomical and endoskeletal) and previous molecular studies show the presence of two distinct morphotypes of Holothuria (Holothuria) tubulosa "A" "B". Morphotype "A" corresponds to the classical Holothuria (Holothuria) tubulosa Gmelin 1791, described by Koehler (1921) and hereafter referred to as H. (H.) tubulosa (s.s.), while morphotype "B", we believe, represents a new species, herein named H. (Holothuria) algeriensis n. sp. with characteristics significantly different from those of Holothuria (H.) tubulosa (s.s.). Both morphology and statistical analysis (Linear Discriminant Analysis) confirm significant differences between these two morphotypes. To visualize these differences, measurements made on ossicles of Holothuria (H.) tubulosa (s.s.) and Holothuria (H.) algeriensis n. sp. were compared with other species present in our collections, including the north-eastern Atlantic and Mediterranean Holothuria (Roweothuria) arguinensis Koehler Vaney, 1906, recently recorded from Algerian waters, the Mediterranean Holothuria (Roweothuria) poli Delle Chiaje, 1824 and Holothuria (Holothuria) stellati Delle Chiaje, 1824. We conclude that the two morphotypes of H. (H.) tubulosa are significantly different to warrant the recognition of a new species, H. (H.) algeriensis n. sp. and the linear discriminant analysis (LDA) of the five species demonstrate this.

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.

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.

Saponins enriched in the epidermal layer of Holothuria leucospilota body wall.

Saponins are secondary metabolites that provide medicinal benefits in controlling body homeostasis and metabolic functions. Sea cucumber has been consumed in many Asian countries due to their health benefits. Active chemicals found in sea cucumber include natural source of saponins which are enriched in their tissues, including the Cuvierian tubules and the body wall. Tissue origin of the saponin biosynthesis and accumulation is limitedly known. The present study is to indicate major compositions and distributions of saponins in the body wall of Holothuria leucospilota. Structurally, their body wall consisted of the pigmented layer of the epidermis, the dermal connective tissues, and inner muscular layers. Interestingly, release of the pigmented granules from the epidermis was related to detection of epidermal saponins. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed identical mass spectra in the saponin extracts and compared to the known compounds of holothurians. To investigate the release of epidermal saponins, the epidermis dissolved in either butanol or distilled water were analyzed and presented the saponin masses with two prominent masses of m/z 1,243.3 (holothurin A and scabraside B) and 1,259.3 (holothurin A3). MALDI-IMS also demonstrated strong signals of the known saponins which were only localized in the epidermis of the body wall. Taken together, this study shows that granule release from epidermal pigmented cells is somehow related to the amount of epidermal saponins released to surrounding seawater. Hence, the future research in the sea cucumber better focuses on epidermal cells that are the enriched site of saponins, although several active compounds require further investigation.

Densidad poblacional y estructura de talla del pepino de mar Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) en aguas someras del sur del golfo de Morrosquillo, Caribe Colombiano / Density population and size structure of the sea cucumber Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) in shallow waters south of the gulf of Morrosquillo, Colombian Caribbean

Resumen Holothuria grisea, a pesar de no ser considerado un recurso comercial, ha sido objeto de extracciones ilegales en Colombia, hecho que se vuelve más grave debido al vació de información biológica de estos organismos. Por esta razón, se estimó la densidad poblacional y estructura de talla de H. grisea en tres sectores del sur del golfo de Morrosquillo de abril a noviembre 2015. Para ello, se ubicaron mensualmente tres transectos circulares en cada sector de muestreo abarcando un área de 300 m2, donde se contabilizaron y midieron in situ los individuos encontrados. La densidad media de H. grisea en el área estudiada fue 0.4 ind m-2, presentándose los mayores valores en el sector La Ahumadera (1.09 ± 0.11 ind m-2) y los menores valores en el Banco de Arena (0.02 ± 0.004 ind m-2). El análisis de varianza señalo una diferencia significativa en los valores de densidad y talla de H. grisea en los sectores estudiados. La especie presentó una distribución de tallas unimodal, registrándose una talla promedio de 13.5 ± 0.91 cm con una talla mínima de 2.5 y una máxima de 30 cm, en general las tallas intermedias presentaron una mayor representatividad a lo largo de este estudio (77.2 %). Los aspectos biológicos presentados en este documento son de gran importancia para la conservación de esta especie y corresponde a un avance en el conocimiento de la clase Holothuroidea en el Caribe Colombiano.

Abstract Holothuria grisea, despite not being considered a commercial resource has been the subject of illegal extractions in Colombia, a fact that becomes more serious due to the empty of biological information of these organisms. For this reason the density population and size structure of H. grisea were evaluated in three sectors south of the gulf of Morrosquillo from April to November 2015. To this end, three circular transects were located each month in each sampling site covering an area of 300 m2, where individuals were counted and measured in situ. The average density of H. grisea in the study area was 0.4 ind m-2, with the highest values in the La Ahumadera (1.09 ± 0.11 ind m-2) and the lowest values in Banco de Arena (0.02 ± 0.004 ind m-2). The analysis of variance showed a significance in the values of density and size of H. grisea in the studied sectors. The species presented a unimodal distribution of sizes with an average size of 13.5 ± 0.9 cm with a minimum size of 2.5 and a maximum of 30 cm, intermediate sizes generally showed a higher representativeness throughout this study (77.2 %). The biological aspects presented in this paper are of great importance for the conservation of this species and corresponds to an advance in the knowledge of the Holothuroidea class in the Colombian Caribbean. Rev. Biol. Trop. 66(2): 776-787. Epub 2018 June 01.

Holothurian Nervous System Diversity Revealed by Neuroanatomical Analysis.

The Echinodermata comprise an interesting branch in the phylogenetic tree of deuterostomes. Their radial symmetry which is reflected in their nervous system anatomy makes them a target of interest in the study of nervous system evolution. Until recently, the study of the echinoderm nervous system has been hindered by a shortage of neuronal markers. However, in recent years several markers of neuronal and fiber subpopulations have been described. These have been used to identify subpopulations of neurons and fibers, but an integrative study of the anatomical relationship of these subpopulations is wanting. We have now used eight commercial antibodies, together with three antibodies produced by our group to provide a comprehensive and integrated description and new details of the echinoderm neuroanatomy using the holothurian Holothuria glaberrima (Selenka, 1867) as our model system. Immunoreactivity of the markers used showed: (1) specific labeling patterns by markers in the radial nerve cords, which suggest the presence of specific nerve tracts in holothurians. (2) Nerves directly innervate most muscle fibers in the longitudinal muscles. (3) Similar to other deuterostomes (mainly vertebrates), their enteric nervous system is composed of a large and diverse repertoire of neurons and fiber phenotypes. Our results provide a first blueprint of the anatomical organization of cells and fibers that form the holothurian neural circuitry, and highlight the fact that the echinoderm nervous system shows unexpected diversity in cell and fiber types and their distribution in both central and peripheral nervous components.

Novel markers identify nervous system components of the holothurian nervous system.

Echinoderms occupy a key position in the evolution of deuterostomes. As such, the study of their nervous system can shed important information on the evolution of the vertebrate nervous system. However, the study of the echinoderm nervous system has lagged behind when compared to that of other invertebrates due to the lack of tools available. In this study, we tested three commercially available antibodies as markers of neural components in holothurians. Immunohistological experiments with antibodies made against the mammalian transcription factors Pax6 and Nurr1, and against phosphorylated histone H3 showed that these markers identified cells and fibers within the nervous system of Holothuria glaberrima. Most of the fibers recognized by these antibodies were co-labeled with the well-known neural marker, RN1. Additional experiments showed that similar immunoreactivity was found in the nervous tissue of three other holothurian species (Holothuria mexicana, Leptosynapta clarki and Sclerodactyla briareus), thus extending our findings to the three orders of Holothuroidea. Furthermore, these markers identified different subdivisions of the holothurian nervous system. Our study presents three additional markers of the holothurian nervous system, expanding the available toolkit to study the anatomy, physiology, development and evolution of the echinoderm nervous system.

Proteases from the regenerating gut of the holothurian Eupentacta fraudatrix.

Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2-4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed.

The morphology of the digestive tract and respiratory organs of the holothurian Cladolabes schmeltzii (Holothuroidea, Dendrochirotida).

The microanatomy of the digestive and respiratory systems of the holothurian Cladolabes schmeltzii was studied. The digestive tube of C. schmeltzii is divided into seven parts. The pharynx, esophagus, and stomach are lined with cuticular immersed epithelium. In these regions, the epithelial cells are connected via desmosomes, septate junctions, and rivet-like structures. The presence of the cuticle and rivet-like structures suggests an ectodermal origin for these parts of the digestive tube. The luminal intestinal epithelium is formed by vesicular enterocytes, which have different structures in different intestinal regions. Moreover, the epithelium of the first descending part of the intestine contains the granular enterocytes. The respiratory system consists of paired respiratory trees lined by a luminal epithelium that is formed by cells of irregular shape. The apical surface of these epithelial cells has few lamellae. The cells are connected to each other through a system of intercellular junctions, consisting of both desmosomes and well-developed septate junctions. The coelomic epithelium of the intestine and the respiratory trees consists of peritoneal and myoepithelial cells.

Holothuria (Selenkothuria) parvispinea n. sp. (Echinodermata, Hololthuroidea, Holothuriidae) with key to the sub-genus Selenkothuria.

Description of a new species from Australia, belonging to the subgenus Selenkothuria (Holothuria, Aspidochirotida). A dichotomous key of the thirteen valid species included in the subgenus is also given. The species H. perrieri Thandar, 1977 and H. spinea Cherbonnier, 1988 are considered as non valid.

Localization of secondary metabolites in marine invertebrates: contribution of MALDI MSI for the study of saponins in Cuvierian tubules of H. forskali.

BACKGROUND: Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. RESULTS: These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. CONCLUSIONS: All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates.

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.

Identification of nerve plexi in connective tissues of the sea cucumber Holothuria glaberrima by using a novel nerve-specific antibody.

The echinoderm nervous system is one of the least studied among invertebrates, partly because the tools available to study the neurobiology of this phylum are limited. We have now produced a monoclonal antibody (RN1) that labels a nervous system component of the sea cucumber Holothuria glaberrima. Western blots show that our antibody recognizes a major band of 66 kDa and a minor band of 53 kDa. Immunohistological experiments show that, in H. glaberrima, the antibody distinctly labels most of the known nervous system structures and some components that were previously unknown or little studied. A surprising finding was the labeling of nervous plexi within the connective tissue compartments of all organs studied. Double labeling with holothurian neuropeptides and an echinoderm synaptotagmin showed that RN1 labeled most, if not all, of the fibers labeled by these neuronal markers, but also a larger component of cells and fibers. The presence of a distinct connective tissue plexus in holothurians is highly significant since these organisms possess mutable connective tissues that change viscosity under the control of the nervous system. Therefore, the cells and fibers recognized by our monoclonal antibodies may be involved in controlling tensility changes in echinoderm connective tissue.

Ontogeny of the holothurian larval nervous system: evolution of larval forms.

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature.