Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts

Abstract Background Sea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects. Methods E. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats. Results One molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10. Conclusions One isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.

Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts

Background Sea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects. Methods E. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats. Results One molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10. Conclusions One isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.(AU)

Dieta del erizo negro Echinometra lucunter (Echinometra: Echinoidea) en el Nororiente de Venezuela / Diet of the black sea urchin Echinometra lucunter (Echinometra: Echinoidea) in the northeast of Venezuela

Resumen Mensualmente entre junio 2010 y agosto 2011 en la ensenada de Turpialito, Golfo de Cariaco, Venezuela, se recolectaron macroalgas e identificaron, y ejemplares de Echinometra lucunter con diámetro de testa 41.04 ± 6.60 mm para examinar el contenido estomacal y el índice de llenado. Se identificaron 65 especies de macroalgas, pertenecientes a los Phylla Rhodophyta, Heterokontophyta y Chlorophyta. La mayor biomasa anual la presentaron Halimeda opuntia (56.5 g.m-2), Acanthophora spicifera (15.94 g.m-2), Sargassum vulgare (15.28 g.m-2) y Centroceras clavulatum (12.26 g.m-2). Los ítems alimenticios más frecuentes fueron las macroalgas filamentosas: Sphacelaria sp. (546) y Herposiphonia sp. (441) y la foliácea Dictyota sp. (410), y entre los invertebrados esponjas (155), moluscos bivalvos (70) y crustáceos cirrípedos (65). El ID osciló entre 0.8 y 6.0% (2.6 ± 1.21 %), con máximos en diciembre 2010 (6.0 ± 2.21 %), abril (4.3 ± 0.34 %) y junio 2011 (4.0 ± 1.79 %), con oscilaciones mensuales asociadas a los períodos de surgencia y estratificación. El conocimiento de las características principales de la dieta, es necesaria para proponer planes de cultivo y de conservación de la especie.

Abstract Echinometra lucunter is relatively abundant in the Venezuela coast, however no details are known about its diet. We compare the stomach contents and repletion index of E. lucunter with macroalgae available in their environment (June 2010-August) and the nutrient cycle at the Golfo de Cariaco. Seventy-one species of macroalgae were identified: Rhodophyta (45), Heterokontophyta (11) and Chlorophyta (15). The mean testa diameter was 41.04 ± 6.60 mm. The food preferences included filamentous, siphonal and calcaeous macroalgae; main food items were the filamentous macroalgae Sphacelaria sp. and Herposiphonia sp., and foliose Dictyota sp., including invertebrates such as sponges, bivalves and barnacle crustaceans. The repletion index was 0.8 - 6.0 % (2.6 ± 1.21 %), maximum values were associated to primary productivity cycle, i.e. December 2010 (6.0 ± 2.21 %), April (4.3 ± 0.34 %), June 2011 (4.0 ± 1.79 %). Food preferences could be associated to the upwelling season and productivity cycle. Rev. Biol. Trop. 63 (Suppl. 2): 233-242. Epub 2015 June 01.

Densidad poblacional y distribución espacial de erizos de mar (Echinodermata: Echinoidea) en la Isla Socorro, Archipiélago de Revillagigedo, México / Population density and spatial distribution of sea urchins (Echinodermata: Echinoidea) at Socorro island, Revillagigedo archipelago, Mexico

Resumen Los estudios sobre los equinodermos de la costa del Pacifico de México se han enfocado en la taxonomía y biogeografía y hay escasos datos sobre ecología comunitaria o poblacional del grupo. En el presente trabajo analizamos los cambios estacionales de la densidad poblacional y la distribución espacial de cuatro especies de erizos regulares (Diadema mexicanum, Echinometra mathaei oblonga. Eucidaris thouarsii y Tripneustes depressus) en tres bahías de la Isla Socorro, Archipiélago de Revillagigedo, México. Las especie más abundante en la isla fue E. mathaei oblonga (3.96 + 0.83 ind m-2; promedio y error típico), seguida de D. mexicanum (2.13 + 0.59 ind m-2), T. depressus y E. thouarsii (entre 0.50 y 0.11 ind m-2, respectivamente). La Bahía Binners tuvo la mayor densidad con 15.98 + 0.43 ind m-2, en gran medida debido a la alta incidencia de E. mathaei oblonga en el sitio. Estacionalmente, T. depressus y E. thouarsii no variaron significativamente su abundancia durante el año, pero D. mexicanum fue más abundante en invierno-primavera, y E. mathaei oblonga en el otoño. En relación al patrón de distribución espacial, dos de las especies (D. mexicanum y E. mathaei oblonga) presentaron una distribución agregada a lo largo del año y en las tres localidades de estudio, pero los otros dos equinoideos se dispusieron de forma azarosa. Hay varias razones para explicar el comportamiento de agregación en erizos de mar, pero para el caso de Isla Socorro aquí se argumenta a favor de que es un modo de defensa contra los abundantes peces depredadores que habitan los arrecifes. En contraste, las especies que no muestran agrupaciones residen en refugios durante el día (E. thouarsii) o bien su morfología y tamaño dificultan su captura por los peces (T. depressus).

Abstract The studies on echinoderms of the Mexican Pacific have focused on taxonomy and biogeography, and there are limited data on their ecology. We used transect to study Diadema mexicanum, Echinometra mathaei oblonga, Eucidaris thouarsii and Tripneustes depressus at three bays in Socorro Island, Revillagigedo Archipelago, Mexico (1989 and 1990). The most abundant species in the island was E. mathaei oblonga (3.96 + 0.83 ind m-2; average and standard error), followed by D. mexicanum (2.13 + 0.59 ind m-2), T. depressus and E. thouarsii (between 0.50 and 0.11 ind m-2, respectively). Binners Bay had the highest density with 15.98 + 0.43 ind m-2, mostly due to the high incidence of E. mathaei oblonga at the site. Seasonally, T. depressus and E. thouarsii did not significantly varied its abundance during the year, while D. mexicanum was more abundant in winter-spring, and E. mathaei oblonga in the fall. Two species (D. mexicanum and E. mathaei oblonga) had an aggregated distribution along the year and in the three study locations, but the other two echinoids followed a random distribution. This may be a defense against the abundant predatory fishes. In contrast, the species that do not aggregate inhabit in refuges during the day (E. thouarsii), or their shape and size makes them difficult to capture by fishes (T. depressus). Rev. Biol. Trop. 63 (Suppl. 2): 221-232. Epub 2015 June 01.

Comunidades de equinodermos en la región de Tunantal, Golfo de Cariaco, Venezuela / Echinoderms communities in the Tunantal area, Gulf of Cariaco, Venezuela

Resumen Los equinodermos son un grupo importante en la estructura de las comunidades bentónicas, tanto por su diversidad como por su relevancia ecológica, sin embargo los trabajos dirigidos al conocimiento de la presencia, abundancia y distribución espacial de este grupo en venezuela aún son escasos. Se comparó la estructura de las comunidades de equinodermos en diferentes hábitats en la región de Tunantal, Golfo de Cariaco, venezuela. Se realizaron muestreos durante septiembre - octubre de 2010 y febrero - abril 2011 mediante buceos cualitativos y con la utilización de cuadrantes de 1 m2, en zonas someras en ambientes de parches de corales, praderas de Thalassia testudimun, sustrato arenoso, rocoso y la desembocadura del Río Tunantal, identificando y contabilizando los equinodermos presentes. Se determinó la densidad, riqueza y diversidad de Shannon, así como los índices de similitud de Bray-Curtis y Sorensen. Se detectó diferencias en la estructura comunitaria entre los hábitats. La riqueza general de equinodermos se estimó en 40 ± 3.02 especies. Los parches coralinos y sustratos rocosos exhibieron mayor riqueza, abundancia de especies, diversidad de Shannon y equidad que el resto de los hábitats, aunque estos últimos resultaron similares en cuanto a equidad y diversidad de Shannon, se detectaron diferencia en las estructura de las comunidades. En los parches de coral la especie dominante fueron Echinometra viridis, Ophiactis savignyi y Ophiothrix angulata. Mientras que en el ambiente rocoso las especies Echinometra lucunter y Ophiocoma echinata mostraron la mayor frecuencia y abundancia seguido por Ophionereis reticulata. Los otros ambientes de la bahía (praderas de T. testudinum, fondo arenoso y desembocadura del río) mostraron muy baja diversidad y abundancia de equinodermos, en algunos casos con ocurrencias ocasionales. Se propone la heterogeneidad de los sustratos como el principal promotor de las diversidades y estructuración de las comunidades de equinodermos en la zona. Por lo que los corales y sustratos rocosos ofrecen los hábitats a las especies dominantes (E. viridis, E. lucunter y O. echinata), pero las diferencias de los ambientes en cuanto a los sustratos, profundidad y presión por oleaje pueden dar a lugar a estas diferencias en las comunidades. La diversidad general de equinodermos es producto de la presencia de gran variedad de ambientes y de los cambios en composición de especies entre los ambientes.

Abstract Echinoderms are relevant in the structure of marine benthic assemblages, both due their diversity and their ecological niche. However, studies related with occurrence, abundance and patterns of distribution of echinoderms in venezuela are still scarce. In the present study we describe the echinoderms in shallow-waters habitats (corals patches, Thalassia beds, sandy bottom, rocky subtidal shore, and mouth of river) at Tunantal bay, Golfo de Cariaco, venezuela, an area that face threats related with the increase of urban development. Samples were performed during September - October 2010 and February - April 2011, using quantitative (1 m2 plots) and qualitative diving observations. Measurements include density, number of species, evenness, Shannon diversity and similarities between habitats based on Bray-Curtis and Sorensen Indexes. Differences in the structure of echinoderm assemblages were detected between habitats. The overall number of echinoderm species in the area was estimated in 40 ± 3.02. Coral patches and rocky shores showed more species and abundance, evenness and Shannon diversity than others habitats. Although the former habitats are similar in evenness and Shannon diversity, differences in the structure of the assemblages were detected. In coral habitats Echinometra viridis, Ophiactis savignyi and Ophiothrix angulata were the dominants species of the assemblages, meanwhile in rocky shores E. lucunter and Ophiocoma echinata were dominants in abundance followed by Ophionereis reticulata. Other habitats of the bay (Thalassia beds, sandy bottom and mouth of river) showed very low diversity and abundance of echinoderms, in some cases only occasional occurrence. Substrate heterogeneity is proposed as the main driving factor of the diversity and structure of the echinoderms assemblages in the bay. Moreover coral patches and rocky shores offer a substratum to dominant species (E. viridis, E. lucunter and O. echinata), but habitat differences in terms of substratum, depth and wave stress could explain the differences in the echinoderm assemblage. The overall echinoderm diversity is promoted by the differences of habitats in the bay and the changes of species composition between habitats. Rev. Biol. Trop. 63 (Suppl. 2): 195-207. Epub 2015 June 01.

Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. Results The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R,A:A,K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. Conclusions E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.

Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. Results The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R-A:A-K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. Conclusions E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.(AU)

Observation of initial clinical manifestations and repercussions from the treatment of 314 human injuries caused by black sea urchins (Echinometra lucunter) on the southeastern Brazilian coast / Observação das manifestações clínicas iniciais e sua influência no tratamento de 314 acidentes causados por ouriços-do-mar pretos (Echinometra lucunter) na região sudeste do Brasil

INTRODUCTION: Injuries caused by sea urchins are the most common caused by marine animals in humans in Brazil, with the black sea urchin (Echinometra lucunter) causing the most injuries to bathers. METHODS: This study observed 314 human wounds with emphasis on the early observation of clinical signs and symptoms and their implications on the recommended treatment. RESULTS: All the injuries were caused by black sea urchins and were observed in bathers. The lesions and the pain were associated with penetration of the spines; there was no early inflammation or pain without pressure on the wounded places. Complications arising from this kind on injury, including infections and foreign body granulomas, are associated with the permanence of the spines in the wounds. CONCLUSIONS: The study confirmed that this kind of injury is the most common accident caused by aquatic animals in Brazil. The main therapeutical recommendation is early removal of the spines to prevent late complications, such as infections and the formation of foreign body granulomas.

INTRODUÇÃO: Os acidentes causados por ouriços-do-mar são as ocorrências por animais marinhos mais comuns no país. O ouriço-do-mar preto (Echinometra lucunter) é a espécie que mais causa ferimentos em banhistas. MÉTODOS: Este trabalho registrou e estudou 314 agravos com ênfase nas manifestações clínicas iniciais observadas e suas implicações na terapêutica recomendada. RESULTADOS: Todos os acidentes foram causados pelo ouriço-do-mar preto e aconteceram em banhistas. As lesões e a dor foram associadas ao trauma causado pela penetração das espículas (não ocorreu inflamação ou dor imediata sem pressão sobre os pontos comprometidos). As complicações deste tipo de acidente, incluindo infecções e granulomas de corpo estranho, estão associadas com a permanência das espículas nos ferimentos. CONCLUSÕES: Foi confirmado o fato do acidente causado por esta espécie ser o mais comum no Brasil e apresentar caráter traumático, sendo a principal recomendação a retirada precoce dos espinhos para prevenção de complicações tardias como as infecções e formação de granulomas de corpo estranho.