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.

Population survey of Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) at its limit of geographic distribution in the Western South Atlantic / Estudio poblacional de Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) en su límite de distribución geográfica en el Atlántico Sur Occidental

Abstract Introduction: The sea cucumber Holothuria (Halodeima) grisea Selenka, 1867 is a common echinoderm in intertidal regions along the Brazilian coast, which recently became the focus of unreported and unregulated fisheries. This study was carried out in sandy-rocky substrates at Armação do Itapocoroy, Penha, Santa Catarina (26o47' S; 48o36' W), near its southern limit of geographic distribution. Objective: To determine the densities (individuals*m-2) of Holothuria (H.) grisea within a spatial-temporal perspective as well as to determine biometric and growth characteristics of the population. Methods: Two-meter wide transects perpendicular to the coastline were carried out in winter and spring 2019 and in summer and spring 2020, in periods of spring low-tides. In each sampling occasion the total number of specimens of H. grisea were determined, and a group of 90 organisms was submitted to in situ biometrics (weight, length and width), and immediately returned alive to their habitat. Results: The densities of H. (H.) grisea were significantly higher in the subtidal sector and lower in the upper intertidal sector with no indication of significant differences among sampling campaigns. Depth was the primary factor explaining the observed density patterns and rugosity of the substrate was secondary but also important. The body length ranged from 5.2 to 22.5 cm, whereas the weight varied from 6.0 to 230 g. The mean and modal lengths were 12.54 and 13 cm, respectively. Approximately 75 % of the population sampled was between 10 and 14 cm and the average weight was 60 g. Estimates from von Bertalanffy growth function indicate that the youngest sea cucumber was one year-old, and the oldest had approximately two and a half years. Conclusions: This is the first study to determine biometric parameters for H. (H.) grisea in southern Brazil and the first one to estimate growth and age estimates for a wild population of this species. The densities recorded in the present study were lower than those previously reported for this region, suggesting anthropic influence.

Resumen Introducción: El pepino de mar Holothuria (Halodeima) grisea Selenka, 1867 es un equinodermo común en las regiones intermareales a lo largo de la costa brasileña, que recientemente se convirtió en foco de pesquerías no declaradas y no reguladas. Este estudio se realizó en sustratos arenosos-rocosos en Armação do Itapocoroy, Penha, Santa Catarina (26o47' S; 48o36' W), cerca del límite sur de su distribución geográfica. Objetivo: Determinar las densidades (individuos*m-2) de Holothuria (H.) grisea dentro de una perspectiva espacio-temporal así como determinar las características biométricas y de crecimiento de la población. Métodos: Se realizaron transectos de dos metros de ancho perpendiculares a la línea de costa en invierno y primavera de 2019 y en verano y primavera de 2020, en periodos de bajamar sicigia. En cada ocasión de muestreo se determinó el número total de especímenes de H. (H.) grisea, y se sometió un grupo de 90 organismos a biometría in situ (peso, longitud y ancho), e inmediatamente se los devolvieron vivos a su hábitat. Resultados: Las densidades de H. (H.) grisea fueron significativamente más altas en el sector submareal y más bajas en el sector intermareal superior sin indicios de diferencias significativas entre las campañas de muestreo. La profundidad fue el factor principal que explica los patrones de densidad observados y la rugosidad del sustrato fue secundaria pero también importante. La longitud del cuerpo varió de 5.2 a 22.5 cm, mientras que el peso varió de 6.0 a 230 g. Las longitudes media y modal fueron 12.54 y 13 cm, respectivamente. Aproximadamente el 75 % de la población muestreada midió entre 10 y 14 cm y el peso promedio fue de 60 g. Estimados de la función de crecimiento de von Bertalanffy indican que el ejemplar más joven presentaba un año de edad, mientras el más viejo presentaba cerca de dos años y medio. Conclusiones: Este es el primer estudio que determina parámetros biométricos para una población de H. (H.) grisea en el sur de Brasil y el primero en estimar el crecimiento y edades para una población salvaje de esta especie. Las densidades registradas en el presente estudio fueron inferiores a las reportadas previamente para esta región sugiriendo la ocurrencia de influencia antrópica.

Holothuriophilus trapeziformis Nauck, 1880 (Decapoda: Pinnotheridae) from the Pacific coast of Mexico: taxonomic revision based on integrative taxonomy.

BACKGROUND: Holothuriophilus trapeziformis Nauck, 1880 is a holothurian-dweller pinnotherid crab representing one of the two species of the genus distributed along the Pacific coast of Mexico and Chile, respectively. While the parasitic ecological interaction with its host is well established, the morphology of the male remains unknown, and DNA information for the species is not available. Furthermore, the only morphological trait separating both species of the genus is subjective and corresponds to the presence or absence of a gap between the fingers of the chelae. Our goal is to complete and clarify the taxonomic status of H. trapeziformis and describe the male morphology with the use of the integrative taxonomy, providing additional characters to differentiate this species. METHODS: We collected new biological material in the Pacific coast of Mexico including the topotypes. We also reviewed material from national collections to integrate morphology (based on a complete and detailed description and illustration of the species using light microscopy), ecological data (based on the identification of the host and the place where it was located within the host), and the mtCOI gene information (commonly known as DNA barcode) to differentiate Holothuriophilus trapeziformis from other related crabs. RESULTS: This species presents marked sexual dimorphism only in the primary sexual characters. For the first time we describe morphological variability of traditionally stable characters. In addition to the gap between the fingers of the chelae, Holothuriophilus trapeziformis differs from H. pacificus (Poeppig, 1836) by their ornamentation, the shape of the male abdomen, and the gonopod. Cytocrome Oxidase 1 gene (COI) distance divergence was >3% between both Holothuriophilus species forming a clear clade. DNA barcoding indicates only one taxon, with a maximum divergence of 2.2%. All the specimens have the same Barcode Index Number (BIN; BOLD: ADE9974). All the hosts for H. trapeziformis were identified as Holothuria (Halodeima) inornata Semper, 1868; the presence of the crab in the host's coelomic cavity was confirmed, and for the first time we found it within the intestine. The geographical distribution is the Pacific coast of Mexico. Based on the data presented here, the taxonomic status of Holothuriophilus trapeziformis is now complete.

Feeding habits of Holothuria (Stauropora) fuscocinerea (Echinodermata: Holothuroidea) in a Mexican Pacific reef / Hábitos alimenticios de Holothuria (Stauropora) fuscocinerea (Echinodermata: Holothuroidea) en un arrecife del Pacífico Mexicano

Introduction: The importance of holothuroids in marine ecosystems is related to their feeding activities, which have been little studied in the eastern Pacific. Objective: To describe the feeding habits of the sea cucumber Holothuria (Stauropora) fuscocinerea and their bioturbation potential in La Entrega reef, México. Methods: The population size of H. (Stauropora) fuscocinerea was determined by means of four 20 x 4 m (320 m2) band transects, while the available sediment for the sea cucumbers and their feces were characterized in three stations (A-C) via granulometry and quantification of organic carbon and organic matter. Excretion rate was determined every 4 hr over a 24 hr period and the relationship between sea cucumber length and biomass and the excretion rate evaluated. Results: According to non-parametric permutation, ordination and percentage similarity analyses, sea cucumber feces mostly comprised a large proportion of small grains compared to the available sediment, indicating that H. (Stauropora) fuscocinerea is selective on grain size. Preliminary bioturbation potential of the species may reach up to 8.71 ± 0.17 kg day of sediment in the study area. Conclusion: The spatial distribution of H. (Stauropora) fuscocinerea in the study area is a product of its selective feeding activity, evidencing the preliminary importance of the species in the trophic dynamics of La Entrega reef.

Introducción: La relevancia de los holoturoideos en los ecosistemas marinos está relacionada con sus actividades alimenticias, las cuales han sido escasamente estudiadas en el Pacífico oriental. Objetivo: Describir los hábitos alimenticios del pepino de mar Holothuria (Stauropora) fuscocinerea y su potencial de bioturbación en el arrecife La Entrega, México. Métodos: Se determinó el tamaño poblacional de H. (Stauropora) fuscocinerea mediante cuatro transectos de banda de 20 x 4 m (320 m2), mientras que la caracterización de los sedimentos disponibles y las heces de los pepinos se realizó en tres estaciones (A-C) mediante granulometría y cuantificación de carbono orgánico y materia orgánica. Se determinó la tasa de excreción cada 4 hr durante un periodo de 24 hr y se evaluó la relación entre la talla y la biomasa con la tasa de excreción. Resultados: De acuerdo con un análisis no paramétrico mediante permutaciones, ordenación y similitud, las heces de los pepinos estuvieron compuestas en su mayoría por una gran cantidad de granos pequeños en comparación con el sedimento disponible, lo que sugiere que H. (Stauropora) fuscocinerea es una especie selectiva respecto al tamaño de grano. El potencial de bioturbación preliminar de la especie puede alcanzar 8.71 ± 0.17 kg día en el área de estudio. Conclusiones: La distribución espacial de H. (Stauropora) fuscocinerea en el área de estudio es producto de su selectividad alimenticia, evidenciando la importancia de la especie en la dinámica trófica del arrecife La Entrega.

Caracterización de los ácidos grasos y actividad antimicrobiana del extracto en metanol de Holothuria princeps (Holothuriida: Holothuriidae) / Characterization of fatty acids and antimicrobial activity of the methanol extract of Holothuria princeps (Holothuriida: Holothuriidae)

Introduction: In recent decades, studies related to the search and characterization of bioactive molecules in marine organisms have increased exponentially, demonstrating the enormous wealth of secondary metabolites of diverse structural composition that cannot be found in organisms present in the terrestrial environment. A significant number of the new marine natural compounds discovered have contributed to solving some of the problems of humanity, mainly those related to human health. Objective: The purpose of this research is to evaluate the bactericidal and fungicidal activities of the methanolic extract of sea cucumber Holothuria princeps collect from the bay of Cispatá in the Colombian Caribbean, in addition to chemically identifying its fatty acids. Methods: A methanolic extraction was performed from the collected biological material, by the cold maceration method. The extract obtained was fractionated using chromatographic techniques and the fatty acids were obtained, which were derivatized and identified by means of gas chromatography in coupling with mass spectrometry. The antibacterial and antifungal activities of the methanolic extract of Holothuria princeps was performed through the microdilution method against reference strains and clinical isolates. Results: We found 16 fatty acids present in Holothuria princeps according to the analysis of their mass spectra. Antibacterial activity showed that Enterococcus faecalis was the most susceptible to the extract at low concentrations, while Pseudomonas aeruginosa was the highest at the higher concentrations. In antifungal treatment, the fungus with the highest inhibition was the clinical isolate of Candida albicans (blood sample). Conclusions: Taking into account previous studies in the genus Holothuria, it is considered that the environment plays a fundamental role in the presence and diversity of fatty acids. The evaluation of the antibacterial activity against reference strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Enterococcus faecalis demonstrated the existence of a considerable effect in the reduction of bacterial growth by the extracts applied, mainly at low concentrations (less than 1 000 ppm). On the other hand, the antifungal activity against the reference strain of Candida albicans and the clinical isolates of Candida albicans (blood sample) and Candida krusei (catheter sample), the extract showed that the best results were presented at higher concentrations (above 1 500 ppm).

Introducción: En las últimas décadas los estudios relacionados con la búsqueda y caracterización de moléculas bioactivas en organismos marinos han aumentado de una manera exponencial, lo que demuestra la enorme riqueza de metabolitos secundarios de diversa composición estructural que no pueden ser encontrados en organismos presentes en el medio terrestre. Estas nuevas moléculas halladas poseen numerosas actividades biológicas que ayudan a resolver muchos problemas que ha tenido el hombre a lo largo de su existencia, lo que las convierte en productos de gran importancia para la humanidad. Objetivo: El propósito de este estudio es la identificación de los ácidos grasos presentes en el pepino de mar Holothuria princeps recolectado en costas del Caribe colombiano, además del análisis de las actividades antibacterianas y antifúngicas de su extracto metanólico frente a cepas de referencia y aislados clínicos. Métodos: Del material biológico recolectado se realizó una extracción metanólica usando el método de maceración en frío. El extracto obtenido se fraccionó usando cromatografía en columna y se lograron obtener los ácidos grasos, los cuales fueron derivatizados e identificados por medio de cromatografía de gases en acople con espectrometría de masas. La actividad antibacteriana y antifúngica del extracto metanólico de Holothuria princeps se realizó a través del método de microdilución. Resultados: Los resultados arrojaron la identificación de 16 ácidos grasos presentes en Holothuria princeps de acuerdo con el análisis de sus espectros de masas. La actividad antibacteriana mostró que Enterococcus faecalis fue la bacteria más susceptible al efecto del extracto a bajas concentraciones, mientras que a las más altas lo fue Pseudomonas aeruginosa. A nivel general en el tratamiento antifúngico, el hongo que presentó una mayor inhibición fue el aislado clínico de Candida albicans (muestra de sangre). Conclusiones: Teniendo en cuenta estudios previos en organismos del mismo género, se puede considerar en cuanto a los ácidos grasos identificados, que el entorno juega un papel fundamental en la presencia y composición de estos compuestos. La evaluación de la actividad antibacteriana contra cepas de referencia de Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae y Enterococcus faecalis, demostró la existencia de un efecto considerable en la reducción del crecimiento bacteriano por parte de los extractos utilizados, principalmente a bajas concentraciones (menos de 1 000 ppm). En cuanto

Fucosylated Chondroitin Sulfates from the Sea Cucumbers Paracaudina chilensis and Holothuria hilla: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [→3)-ß-d-GalNAc (N-acethyl galactosamine)-(1→4)-ß-d-GlcA (glucuronic acid)-(1→]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1→2)-Fuc3S4S-(1→ at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.

The nervous system component of the mesentery of the sea cucumber Holothuria glaberrima in normal and regenerating animals.

The mesenterial tissues play important roles in the interactions between the viscera and the rest of the organism. Among these roles, they serve as the physical substrate for nerves connecting the visceral nervous components to the central nervous system. Although the mesenterial nervous system component has been described in vertebrates, particularly in mammals, a description in other deuterostomes is lacking. Using immunohistochemistry in tissue sections and whole mounts, we describe here the nervous component of the intestinal mesentery in the sea cucumber Holothuria glaberrima. This echinoderm has the ability to regenerate its internal organs in a process that depends on the mesentery. Therefore, we have also explored changes in the mesenterial nervous component during intestinal regeneration. Extensive fiber bundles with associated neurons are found in the mesothelial layer, extending from the body wall to the intestine. Neuron-like cells are also found within a plexus in the connective tissue layer. We also show that most of the cells and nerve fibers within the mesentery remain during the regenerative process, with only minor changes: a general disorganization of the fiber bundles and a retraction of nerve fibers near the tip of the mesentery during the first days of regeneration. Our results provide a basic description of mesenterial nervous component that can be of importance for comparative studies as well as for the analyses of visceral regeneration.

Valor nutricional de espécies marinhas comercializadas no mercado informal / Nutritional value of marine species comercialized in the informal market

No Brasil, além das espécies marinhas tradicionais de consumo há captura não reportada e comércio informal de espécies não convencionais, com crescente demanda gastronômica. Este estudo analisou o valor nutricional e rendimento do saquaritá Stramonita haemastomae e do pepino-do-mar Holothuria grisea coletados na Baía de Santos, SP, de fevereiro/2018 a junho/2018. Foram coletados indivíduos das duas espécies e a composição centesimal determinada com metodologias oficiais ou reconhecidas. Os resultados indicaram importante aporte nutricional e o rendimento de 29,72% e 22,57% para o pepino-do-mar e saquaritá, respectivamente. Sendo recomendados estudos e ações de gestão ambiental e sanitária, de forma a assegurar a oferta de um produto seguro para consumo humano, em especial dos equinodermas que não são contemplados na regulação para alimentos.(AU)

Valor nutricional de espécies marinhas comercializadas no mercado informal / Nutritional value of marine species comercialized in the informal market

No Brasil, além das espécies marinhas tradicionais de consumo há captura não reportada e comércio informal de espécies não convencionais, com crescente demanda gastronômica. Este estudo analisou o valor nutricional e rendimento do saquaritá Stramonita haemastomae e do pepino-do-mar Holothuria grisea coletados na Baía de Santos, SP, de fevereiro/2018 a junho/2018. Foram coletados indivíduos das duas espécies e a composição centesimal determinada com metodologias oficiais ou reconhecidas. Os resultados indicaram importante aporte nutricional e o rendimento de 29,72% e 22,57% para o pepino-do-mar e saquaritá, respectivamente. Sendo recomendados estudos e ações de gestão ambiental e sanitária, de forma a assegurar a oferta de um produto seguro para consumo humano, em especial dos equinodermas que não são contemplados na regulação para alimentos.

Characterization of the intestinal microbiota of the sea cucumber Holothuria glaberrima.

High-throughput 16S rRNA gene sequencing has been used to identify the intestinal microbiota of many animal species, but that of marine invertebrate organisms remains largely unknown. There are only a few high-throughput sequencing studies on the intestinal microbiota of echinoderms (non-vertebrate Deuterostomes). Here we describe the intestinal microbiota of the sea cucumber Holothuria glaberrima, an echinoderm, well-known for its remarkable power of regeneration. We characterized the microbiota from the anterior descending intestine, the medial intestine (these two comprise the small intestine) and the posterior descending intestine (or large intestine), using pyrosequencing to sequence the V4 region of the 16S rRNA gene. We compared animals in their natural marine environment and in sea-water aquaria. A total of 8,172 OTU's were grouped in 10 bacterial phyla, 23 classes, 44 orders, 83 families, 127 genera and 1 group of unknown bacteria, present across the digestive tract of 10 specimens. The results showed that the anterior intestine is dominated by Proteobacteria (61%) and Bacteroidetes (22%), the medium intestine is similar but with lower Bacteroidetes (4%), and the posterior intestine was remarkably different, dominated by Firmicutes (48%) and Bacteroidetes (35%). The structure of the community changed in animals kept in aquaria, which had a general dominance of Firmicutes and Bacteroidetes, regardless the intestinal segment. Our results evidence that in the natural sea environment, there is intestinal segment differentiation in the microbiota of H. glaberrima, which is lost in artificial conditions. This is relevant for physiological studies, such as mechanisms of digestive regeneration, which might be affected by the microbiota.

Identification and characterization of microsatellite loci in West Atlantic sea cucumber Holothuria grisea (Selenka 1867).

The sea cucumber Holothuria grisea has become the subject of intense and unregulated fishing in northeastern Brazil due to their growing demand in Asian market. However, there is little knowledge about the dynamics and genetics of H. grisea wild populations on the South American coast. In this study, we present the first set of H. grisea microsatellite markers, identified and characterized using Illumina paired-end reads of whole genome shotgun sequencing. From 50 strictly selected candidates, eight novel microsatellite markers were successfully developed. We then genotyped 30 individuals to evaluate the degree of polymorphism and validate the markers. The number of alleles ranged from three to 14, while observed and expected heterozygotes ranged from 0.156 to 0.906 and from 0.283 to 0.774, respectively. After correcting for multiple tests,we found no evidence of linkage disequilibrium in all pairwise combinations between the loci. One locus (Hgr15607) revealed deviation from the Hardy-Weinberg equilibrium, as well as the presence of null alleles. However, we observed significant differences in frequency distribution between males and females at locus Hgr15607. We believe that the markers describedhere will be useful for conservation efforts and management of H. grisea fisheries and for prospective aquaculture of these organisms.

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.

A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches.

Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in ß-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the ß-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

A revision of Holothuria (Halodeima) kefersteinii (Selenka, 1867) and the revival of Holothuria inornata Semper, 1868 from sea cucumbers collected in Mexico and Central America.

In 1958 Deichmann synonymized Holothuria inornata Semper, 1868 with Holothuria kefersteinii (Selenka, 1867). This nomenclature has been adopted until now. However, some specimens recently collected from the Mexican Pacific matched the original description of H. inornata Semper (1868) whereas others matched the original description by Selenka (1867). A morphological, molecular and ecological study of the specimens was conducted to confirm the identification of these specimens. The morphological characters of our specimens were compared with those contained in the original descriptions and figures of the types of both species. Besides the different color pattern of live specimens of both species, the main differences found are the presence of straight distally perforated rods in the dorsal papillae, and the presence of Holothuriophilus trapeziformis Nauck, 1880 in the cloaca of H. inornata, versus the absence of these kind of rods in the dorsal papillae, and the absence of symbionts in the cloaca in H. kefersteinii. The species identified by Deichmann (1958) as H. kefersteinii is, in fact H. inornata. Both species might have been confused because the morphology of preserved specimens is very similar and the presence of the straight rods in papillae might have been overlooked during identification. A more thorough review of the ossicles from the specimens, together with the comparison of ossicles from the type specimens, has allowed the resurrection of long-forgotten species, H. inornata.

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.

A novel structural fucosylated chondroitin sulfate from Holothuria Mexicana and its effects on growth factors binding and anticoagulation.

Fucosylated chondroitin sulfate (FCS), a structurally distinct glycosaminoglycan from the body wall of sea cucumber, possesses many biological properties and pharmacology functions. The refined structure of FCS isolated from sea cucumber Holothuria Mexicana (FCShm) was characterized by NMR spectra and HILIC-FTMS, which demonstrated four types of branches in FCShm. Among these, two branches were α-L-Fuc-2S4S (where Fuc is fucose and S is sulfo) and α-L-Fuc-4S linked to O-3 of glucuronic acid residues, while others were identified as α-L-Fuc-4S and α-L-Fuc-3S4S attached to O-6 of N-acetylgalactosamine residue. Furthermore, the fucosyl branches were α-1,3-linked with different degree of polymerization from 1 to 5. FCShm exhibited high affinity to fibroblast growth factor 1 and 2, growth factors involved in neovascularization. Moreover, FCShm displayed intrinsic anticoagulant activity and inhibited thrombin and factor Xa activation by antithrombin III. Our results proposed a novel structural FCS and demonstrated its favorable application prospects in anti-angiogenesis and anticoagulation.

Estado poblacional del pepino de mar (Clase Holothuroidea) en el Caribe de Guatemala / Population status of Sea Cucumber (Class Holothuroidea) in the Caribbean of Guatemala

Los holoturoideos o pepinos de mar pertenecen al filo Echinodermata, e incluyen animales con cuerpo vermiforme alargado y blando que viven en los fondos de los mares de todo el mundo. Ecológicamente estos organismos son recicladores eficientes de sedimentos en el ambiente. En los últimos años han cobrado alto valor comercial en el mercado asiático, por lo que su pesquería se ha expandido hacia muchos países. En Guatemala se decretó una veda precautoria en el 2010 por cinco años, al terminar esta se extendió por dos años más, pero con el compromise de obtener información científica durante dicho período. Con este fin, se realizó una evaluación poblacional del pepino de mar a través de transectos lineales de 60 m2, en siete puntos de la bahía de Amatique y la zona expuesta del mar Caribe de Guatemala, durante abril, junio y septiembre del 2016. Se evaluaron 6,300 m2, encontrando un total de 64 individuos de pepinos de mar. La riqueza de holotúridos está representada por siete especies, siendo las más abundantes Isostichopus badionotus (Selenka, 1867) y Holothuria mexicana (Ludwig, 1875). Los sitios con mayor abundancia fueron Faro Rojo y King Fish (p = .004). En base a los resultados de esta evaluación se puede determinar que la explotación comercial de los pepinos de mar en el Caribe guatemalteco no es viable, y que para aprovechar el valor comercial de estas especies se podría impulsar la acuicultura y comercializarlo a través de un sistema eficiente de trazabilidad.

Holothurians or sea cucumbers belong to the filum Equinodermata, which includes animals with long soft vermiform bodies that live on the ocean bottom of the entire world. Ecologically these organisms are efficient recyclers of sediments in the environment. In recent years, they have gained high commercial value in the Asian market, so their fishery has expanded to many countries. In Guatemala, a precautionary ban was decreed in 2010 for five years, when it ended it was extended for two more years, but with the commitment to obtain scientific information during that period. For this purpose, a population assessment of sea cucumber was carried out through linear transects of 60 m2, in seven points of Amatique Bay and the exposed zone of the Caribbean Sea of Guatemala, during April, June and September of 2016. 6300 m2 were evaluated, founding 64 organisms. The holothurians richness is represented by seven species, being the most abundant Isostichopus badionotus (Selenka, 1867) and Holothuria mexicana (Ludwig, 1875). The most abundant sites were Faro Rojo and King Fish (p = .004). Based on the results of this evaluation, it can be determined that the commercial exploitation of sea cucumbers in the guatemalan Caribbean is not viable, and that to take advantage of the commercial value of these species, aquaculture could be promoted and marketed through an efficient traceability system.

Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana.

A novel fucosylated chondroitin sulfate (HmG) was isolated from sea cucumber Holothuria mexicana, the structure of which was characterized by monosaccharide composition, disaccharide composition, IR, 1H and 13C NMR spectrum, additionally with two dimensional NMR spectrum of degraded HmG (DHmG). The backbone of HmG was identified as chondroitin 6-O sulfate, while the major O-4 sulfated fucose branches linked to O-3 position of glucuronic acid in almost every disaccharide unit. The anticoagulant activities of HmG and DHmG were assessed and compared with heparin and low molecular weight heparin. The results indicated that HmG and DHmG both could significantly prolong the activated partial thrombo-plastin time, and the properties were well related to its molecular weight. DHmG showed similar anticoagulant properties to low molecular weight heparin with less bleeding risks, making it a safer anticoagulant drug.

Effects of Tin on Enzyme Activity in Holothuria grisea (Echinodermata: Holothuroidea).

This study evaluated the effect of tin exposure on enzyme activity in the sea cucumber (Holothuria grisea Selenka, 1867). After exposure to 0 (control), 0.04, 0.08, or 0.12 mg L-1 tin, we tested the activities of total cholinesterase in longitudinal muscles, acid phosphatase in gonads and the respiratory tree, as well as alkaline phosphatase in the intestines during a 96-h bioassay. Regression analyses showed that all enzyme activities declined with increasing tin concentrations, except for acid phosphatase in the respiratory tree, which were similarly, inhibited at all tin concentrations. These results indicate that H. grisea is a potential bioindicator for seascape habitat monitoring programs, as its biochemical markers show sensitivity to trace elements that can indicate a rise in pollution levels.

Gametogenesis and weight change of body organs of the sea cucumber Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) in Southern Brazil / Aspectos reproductivos y la relación entre los órganos y la gametogénesis en Holothuria (Halodeima) grisea (Echinodermata: Holothuroidea)

Abstract The sea cucumber Holothuria (Halodeima) grisea is a widely distributed and economically important species that has been harvested in Brazil for several years, where there are no studies on its reproduction. We studied a population near Guaratuba Bay in Southern Brazil. Four sampling campaigns were made, in February, April, June and October 2008. We examined the macroscopic and microscopic features of the gonads to describe the gametogenesis. In addition, we analyzed the weight change of body organs. A total of 94 individuals were examined and no evidence of external sexual dimorphism was found. In April there were more males than females. Biometrically the body wall and gonads are the most important organs. The body wall increases in the June and the gonads during October and February; there might be an exchange of energy between these organs. Mature gonads are elongated and thick (red in females, cream in males). There were mature individuals year round, with an increase of sexual activity in February when oocyte mature diameter ranges from 70 to 160 µm. Spawning was in winter (June). Maturation period and body component variations were similar between sexes. Rev. Biol. Trop. 63 (Suppl. 2): 285-296. Epub 2015 June 01.

Resumen El pepino de mar Holothuria (Halodeima) grisea es una especie ampliamente distribuida y económicamente importante que se ha recolectado en Brasil desde hace varios años. No hay estudios previos realizados sobre la reproducción de esta especie en Brasil. Además, son aún poco conocidos los aspectos bio-ecológicos de esta especie. En este estudio, se investigaron algunos aspectos de la reproducción del pepino de mar Holothuria (Halodeima) grisea en una población cerca de la bahía de Guaratuba en el sur de Brasil. Se hicieron cuatro salidas de muestreo, en febrero, abril, junio y octubre de 2008, abarcando las cuatro estaciones. Examinamos las características macroscópicas y microscópicas de las gónadas para describir la gametogénesis, adicionalmente, se analizó el cambio de peso de cada componente de los órganos del cuerpo (pared corporal, gónadas, sistema haemal, tracto digestivo y árbol respiratorio). Se examinó un total de 94 individuos y no se encontraron evidencias de dimorfismo sexual externo. Los resultados del análisis estadístico de la prueba x² rechazan la hipótesis nula (H0) con una relación de sexos igual a 1, sin embargo, el análisis de cada mes, esta diferencia de género se observó sólo en el mes de abril, cuando el número de los males es mucho mayor que la de las hembras. Las medidas biométricas mostraron que la pared del cuerpo y las gónadas son los órganos más importantes, con aumentos de la pared corporal en el junio y de las gónadas durante octubre y febrero, que pueden indicar que hay un intercambio de energía entre estos órganos. Macroscópicamente, las gónadas maduras muestran túbulos largos, gruesos y ramificados siendo que las hembras presentan coloración rojiza e los machos tonos de crema. Sin embargo, durante el crecimiento y agotado, no se observaron diferencias morfológicas macroscópicas entre los sexos. Microscópicamente, se observó la presencia constante de individuos maduros entre meses, con un aumento de la actividad sexual en febrero, cuando los ovocitos maduros tuvieron un diámetro oscila entre 70 a 160 μ. El período de desove ocurrió en los meses de invierno (junio). El periodo de maduración y la variación del cuerpo de los componentes fueron similares entre los sexos. La similitud en las demandas metabólicas de la población podría estar confirmando una estrategia para maximizar la sincronía reproductiva.