Cryptic species and genetic structure in Didemnum granulatum Tokioka, 1954 (Tunicata: Ascidiacea) from the southern Brazilian coast / Espécies crípticas e estrutura genética em Didemnum granulatum Tokioka 1954 (Tunicata: Ascidiacea) na costa sul do Brasil

Didemnum granulatum is a colonial fouling ascidian that lives in subtidal substrates, worldwide. It exhibits two morphotypes, orange and beige. In this study, we verified if the color morphotypes and/or the spatial distribution of specimens in different islands might be associated to patterns of genetic structure of a single species, or if they represent distinct cryptic species. Specimens were collected in four islands, along the coast of the Santa Catarina state. A segment of 490 bp from the mitochondrial gene cytochrome c oxidase subunit 1 (COI) was amplified from 45 samples. Twenty-one haplotypes were identified. The total haplotype diversity (0.912) and the total nucleotide diversity (0.044) were high. The global Fst of the populations analyzed was 0.97, with most of the variation occurring between orange and beige groups (82.19%). The variation found between populations within groups was 15.37%, and 2.45% within populations. Haplotype networks and the neighbor-joining tree showed clear genetic divergence between individuals of distinct colors, and between the islands. These evidences strongly support the presence of a complex of two cryptic species for D. granulatum occupying the studied area. Both species were also highly genetically structured between islands, suggesting that the conservation process of these populations is complex.

Didemnum granulatum é uma ascídia colonial incrustante de substratos consolidados infralitorâneos, e de ampla distribuição mundial. Variação na pigmentação é comum em invertebrados marinhos, e morfotipos de D. granulatum das cores laranja e bege foram detectados na região. Neste estudo, nós verificamos se os morfotipos de cor e/ou a distribuição espacial dos espécimens nas diferentes ilhas podem estar associados aos padrões de estruturação genética de uma única espécie, ou se eles representam espécies crípticas distintas. Os espécimens foram coletados em quatro ilhas ao longo da costa do estado de Santa Catarina. Um total de 45 amostras tiveram amplificados um segmento de 490pb do gene mitocondrial citocromo oxidase subunidade I (COI). Vinte e um haplótipos foram identificados. A diversidade haplotípica total (0.912) e a diversidade nucleotídica total (0.044) foram altas. O Fst global das populações analisadas foi 0.97, e a maior parte da variação ocorreu entre os grupos laranja e bege (82.19%). A variação encontrada entre as populações dentro dos grupos foi 15.37%, e 2.45% dentro das populações. A rede de haplótipos e a árvore de Neighbor-joining mostraram nítidas divergências genéticas entre os indivíduos de cores distintas, e entre as ilhas. Tais evidências sugerem a presença de um complexo de duas espécies crípticas de D. granulatum na região. Ambas as espécies foram fortemente estruturadas geneticamente entre as ilhas, o que reforça a necessidade de que tais atributos sejam considerados em medidas de conservação e proteção do ambiente marinho, mais especificamente nestas ilhas.

Annual abundance of salps and doliolids (Tunicata) around Gorgona Island (Colombian Pacific), and their importance as potential food for green sea turtles / Abundancia anual de salpas y doliolidos (Tunicata) alrededor de la Isla Gorgona (Pacífico colombiano), y su importancia como alimento potencial para las tortugas marinas verdes

Gorgona National Park protects fertile waters that support large vertebrates, including green sea turtles (Chelonia mydas), and for them, gelatinous zooplankton constitute a food resource that can be found year-round in Gorgona Island´s coastal waters. This study was carried out to determine the abundance of salps and doliolids around Gorgona Island over a year, and to determine whether this is a resource that could be used reliably year-round by green turtles and other large plankton-feeding predators. The monthly abundance of salps and doliolids at eight coastal stations around Gorgona Island (Colombian Pacific) was determined between September 2005 and August 2006. Oblique tows were carried out from 50m to the surface, total zooplankton biomass was measured and the number of salps and doliolids per tow, and frequency of occurrence per station and month were determined. Superficial and bottom sea temperature, superficial and bottom salinity, and chlorophyll-a concentration were recorded at each station. There were tunicate abundance peaks in September 2005 and March 2006. The high abundances in March were probably due to a cold water intrusion into the study area, which resulted in colder saltier water and a shallower thermocline. Tunicates were probably advected to the area by currents from the southwest and aggregated due to the underwater topography. In September, the influence of continental river discharge as well as inputs from rainfall over the island could have provided increased nutrients and resulted in higher abundances. The large filter-feeding vertebrates that feed on tunicates include green sea turtle juveniles, which use coastal waters of Gorgona Island as feeding grounds, as part of their migration route in the Eastern Tropical Pacific. These turtles could be using tunicates opportunistically, as a sporadic resource that is available at certain times of the year. Rev. Biol. Trop. 62 (Suppl. 1): 149-159. Epub 2014 February 01.

Se determinó la abundancia de salpas y doliólidos en ocho estaciones costeras alrededor de Isla Gorgona (Pacífico colombiano), entre septiembre 2005 y agosto 2006. Se hicieron arrastres oblicuos desde 50m hasta la superficie; se midió la biomasa zooplanctónica total, el número de salpas y doliólidos por arrastres, y la frecuencia de ocurrencia por estación y por mes. Se registró la temperatura superficial y de fondo, la salinidad superficial y de fondo, así como la concentración de clorofila a en cada estación. Se observaron picos de abundancia de tunicados en septiembre 2005 y marzo 2006. Las altas abundancias en marzo fueron probablemente debidas a una intrusión de agua fría a la zona de estudio, la cual resultó en aguas más frías y saladas, y en una termoclina más somera. Los tunicados fueron probablemente advectados hacia el área por corrientes provenientes del suroeste, y la topografía subacuática causó una agregación de estos organismos. En septiembre, la influencia de las descargas de los ríos continentales, así como aportes por la precipitación sobre la isla pudieron haber provisto mayor cantidad de nutrientes y resultado en mayores abundancias. Los grandes vertebrados marinos filtradores que se alimentan de tunicados incluyen tortugas verdes juveniles, las cuales usan las aguas de Isla Gorgona como zona de forrajeo, como parte de su ruta de migración en el Pacífico Oriental Tropical. Estas tortugas podrían estar utilizando los tunicados oportunistamente, como un recurso esporádico que está disponible en ciertas épocas del año.

Apendicularias (Urochordata) y quetognatos (Chaetognatha) del Parque Nacional Isla del Coco, Costa Rica / Appendicularians (Urochordata) and chaetognaths (Chaetognatha) of Isla del Coco National Park, Costa Rica

Appendicularians and chaetognaths are important members of the zooplancton but little studied in Costa Rica and not at all at Isla del Coco Nacional Park, eastern tropical Pacific. Epipelagic (0-50m) zooplankton samples from the oceanic area off Isla del Coco, were collected in November 2001, and over patch reefs in 2009. We analized the species composition of appendicularians and chaetognaths present in 31 samples. Nine species of appendicularians, and eight species of chaetognaths were identified. Oikopleura longicauda was the most abundant in the surveyed area (217 specimens, 65.56% of total appendicularian numbers), followed by Oikopleura rufescens with 35 (10.57%). All the appendicularian species found in this study are common in warm ocean waters, but six species herein recorded for the first time from waters off Isla del Coco, and two species are new records for Costa Rica. Among the chaetognaths, Flaccisagitta enflata was the commonest species (85 specimens, 50.6% of total numbers) recorded in this study, followed by Serratosagitta pacifica with 38 organisms (20.62%). Zonosagitta bedoti was represented by a single specimen. These species have been recorded from tropical waters of the Eastern Pacific, but six species are new records for Isla del Coco. Data on the distribution and morphology of the species recorded are presented together with keys for the identification of both taxa in the surveyed area.

Las apendicularias y quetognatos son miembros importantes del zooplancton pero poco estudiados en Costa Rica y no del todo en el Parque Nacional Isla del Coco, Pacífico tropical este. Se recolectaron muestras epipelágicas de zooplancton de la zona oceánica (0-50m) de la Isla del Coco, en noviembre de 2001 y sobre parches arrecifales en 2009. Se analizó la composición de especies de apendicularias y quetognatos presentes en 31 muestras. Nueve especies de apendicularias, y ocho especies de quetognatos fueron identificadas. Oikopleura longicauda fue la más abundante en el área estudiada (217organismos, representando el 65.56% del número total de apendicularias), seguida por Oikopleura rufescens, con 35 (10.57%). Todas las especies de apendicularias encontradas en este estudio son comunes en las aguas cálidas de los océanos, seis especies se registran aquí por primera vez en aguas de la Isla del Coco y dos son nuevos registros para Costa Rica. Entre los quetognatos, Flaccisagitta enflata fue la especie más común (85 ejemplares, representando el 50.6% del número total) registrada en este estudio, seguida por Serratosagitta pacifica con 38 organismos (20.62%). Zonosagitta bedoti estuvó representada por un solo espécimen. Estas especies también se han registrado en las aguas tropicales del Pacífico oriental, pero seis especies son nuevos registros para Isla del Coco. Los datos sobre la distribución y morfología de las especies registradas se presentan junto con las claves para la identificación de los taxones en el área estudiada.

New record of Thalia cicar van Soest 1973 (Urochordata: Thaliacea) in the Equatorial Atlantic / Novo registro de Thalia cicar van Soest 1973 (Urochordata: Thaliacea) no Atlântico Equatorial

Thalia cicar van Soest 1973 (Urochordata, Thaliacea) is considered a tropical-subtropical species, registered in the Atlantic Ocean at latitudes between 7-34º S and 6-32º N. This work enlarges the occurrence of this species for Equatorial Atlantic waters. The specimens were found in a shelf break area of the Brazilian northeastern (07º 50'-07º 70' S and 34º 23' W) during the expedition JOPS-II (Joint Oceanographic Projects II) in March 1995; and, in São Pedro e São Paulo arquipelago (0º 55' N and 29º 20' W) in May and June, 2005. These two areas are characterized by the presence of local upwelling that induces the ascent of bottom waters rich in nutrients, generating areas of larger productivity than the typically oligotrophic Equatorial Atlantic waters. The new occurrence of Thalia cicar reported in this work can be related to these more productive waters of Equatorial Atlantic. The species that is most commonly found in the Atlantic Ocean is T. democratica, and the lack of past records of T. cicar might have been caused by the taxonomic difficulties determining of the solitary and aggregate zooids of these two species. This work suggests the potential use of the ratios among tunic lengths as an additional character to differentiate T. cicar and T. democratica oozooids.

Thalia cicar van Soest 1973 (Urochordata, Thaliacea) é considerada uma espécie tropical-subtropical, registrada no oceano Atlântico desde 7 a 34º S e 6 a 32ºN. Este trabalho amplia a ocorrência desta espécie para águas do Atlântico equatorial. Os espécimes foram encontrados na plataforma continental do Nordeste brasileiro (07º 50'-07º 70' S e 34º 23' W) durante a expedição JOPS-II (Joint Oceanographic Projects II) em março de 1995 e no arquipélago de São Pedro e São Paulo (0º 55' N e 29º 20' W) em maio e junho de 2005. Essas duas áreas se caracterizam pela presença de ressurgências locais que induzem a ascensão de nutrientes, gerando locais de alta produtividade, nas águas tipicamente oligotróficas do Atlântico equatorial. O registro de Thalia cicar nesses dois locais pode estar relacionado com essas águas de maior produtividade no Nordeste brasileiro. A espécie de Thalia comumente encontrada no oceano Atlântico é T. democratica e a ausência de registros passados de T. cicar pode decorrer das dificuldades taxonômicas em diferenciar os zoóides solitários e agregados dessas duas espécies. Este trabalho sugere a utilização da proporção entre os comprimentos das projeções da túnica como um caráter adicional para diferenciar os oozoóides destas duas espécies.

Antibiotic, cytotoxic and enzyme inhibitory activity of crude extracts from Brazilian marine invertebrates

Herein we present the results of a screening with 349 crude extracts of Brazilian marine sponges, ascidians, bryozoans and octocorals, against 16 strains of susceptible and antibiotic-resistant bacteria, one yeast (Candida albicans), Mycobacterium tuberculosis H37Rv, three cancer cell lines MCF-7 (breast), B16 (murine melanoma ) and HCT8 (colon), and Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT) enzyme. Less than 15 percent of marine sponge crude extracts displayed antibacterial activity, both against susceptible and antibiotic-resistant bacteria. Up to 40 percent of marine sponge crude extracts displayed antimycobacterial activity against M. tuberculosis H37Rv. Cytotoxicity was observed for 18 percent of marine sponge crude extracts. Finally, less than 3 percent of sponge extracts inhibited L-APRT. Less than 10 percent of ascidian crude extracts displayed antibacterial activity. More than 25 percent of ascidian crude extracts were active against M. tuberculosis and the three cancer cell lines. Only two crude extracts from the ascidian Polysyncraton sp. collected in different seasons (1995 and 1997) displayed activity against L-APRT. Less than 2 percent of bryozoan and octocoral crude extracts presented antibacterial activity, but a high percentage of crude extracts from bryozoan and octororal displayed cytotoxic (11 percent and 30 percent, respectively) and antimycobacterial (60 percent) activities. The extract of only one species of bryozoan, Bugula sp., presented inhibitory activity against L-APRT. Overall, the crude extracts of marine invertebrates herein investigated presented a high level of cytotoxic and antimycobacterial activities, a lower level of antibacterial activity and only a small number of crude extracts inhibited L-APRT. Taxonomic analysis of some of the more potently active crude extracts showed the occurrence of biological activity in taxa that have been previously chemically investigated. These...

No presente estudo apresentamos resultados da triagem biológica realizada com 349 extratos obtidos de esponjas marinhas, ascídias, briozoários e octocorais do Brasil, em testes contra 16 linhagens de bactérias comuns e resistentes à antibióticos, uma levedura (Candida albicans), Mycobacterium tuberculosis H37Rv, três linhagens de células tumorais MCF-7 (mama), B16 (melanoma murínico) e HCT8 (cólon), e de inibição da enzima adenina fosforribosil transferase de Leishmania tarentolae (L-APRT). Menos de 15 por cento dos extratos de esponja marinhas apresentaram atividade antibacteriana, contra linhagens resistentes ou não a antibióticos. Quase 40 por cento dos extratos de esponjas marinhas apresentaram atividade antimicobacteriana contra Mycobacterium tuberculosis H37Rv. Foi observada citotoxicidade para 18 por cento dos extratos de esponjas marinhas. Finalmente, menos de 3 por cento dos extratos de esponjas apresentaram atividade inibitória da enzima L-APRT. Menos de 10 por cento dos extratos de ascídias apresentaram atividade antibacteriana. Mais de 25 por cento dos extratos de ascídias apresentaram atividade contra M. tuberculosis e as três linhagens de células tumorais. Somente dois extratos obtidos da ascídia Polysyncraton sp. coletada em duas diferentes épocas (1995 e 1997) apresentaram atividade contra L-APRT. Menos de 2 por cento dos extratos de briozoários e octocorais apresentaram atividade antibacteriana, mas uma alta percentagem de extratos destes animais apresentaram atividades citotóxica (11 por cento e 30 por cento, respectivamente) e antimicobacteriana (60 por cento). O extrato de somente uma espécie de briozoário, Bugula sp., apresentou atividade inibitória da enzima L-APRT. A análise taxonômica de algumas espécies de invertebrados que forneceram alguns dos extratos mais ativos, indicou a ocorrência de atividade biológica em espécies pertencentes a grupos taxonômicos que já foram anteriormente investigados do ponto de vista...

Alimentación de la ascidia Ecteinascidia turbinata (Ascidiacea: Perophoridae) en dos áreas de manglar de Cuba

Diet of the ascidian Ecteinascidia turbinata (Ascidiacea: Perophoridae) in two mangrove areas of Cuba. Stomach contents of 88 zooids of Ecteinascidia turbinata Herdman 1880 and the qualitative and quantitative composition of phytoplankton in the water column were studied in Santa Fe (North Coast of Havana) and Punta del Este (SW of Cuba). We identified 59 microalgal species and four tintinnids in the stomachs. Cell size was 75-165 µm in length and 2-105 µm in width. There were not significant differences in microalgal diversity in the water column in the two locations. In both locations, the diatoms had the largest number of species and individuals in stomachs and water. In Santa Fe, dinoflagellate biomass was larger in water and stomach contents, while in Punta del Este the contribution of each group to the stomach content was similar to that of the water column. This species filters in a constant and irregular way during the day, independently of food availability. Rev. Biol. Trop. 55 (2): 499-507. Epub 2007 June, 29.

Se estudiaron los contenidos estomacales de 88 zooides de Ecteinascidia turbinata Herdman 1880 y la composición cualitativa y cuantitativa del fitoplancton en la columna de agua en Santa Fe (Litoral Norte de la Habana) y Punta del Este (SW de Cuba). En el contenido estomacal de la ascidia se identificaron 59 especies de microalgas y cuatro tintínidos. El tamaño de las células varió entre 75 y 165 µm de largo y de 2 a 105 µm de ancho. No se encontraron diferencias significativas en la diversidad de especies de microalgas en la columna de agua de las dos localidades. Las diatomeas tuvieron la mayor cantidad de especies y el mayor número de individuos, tanto en los estómagos como en la columna de agua en ambas localidades. La biomasa de dinoflagelados en Santa Fe fue mayor en agua y estómagos. En Punta del Este el aporte de cada grupo al contenido estomacal es similar al encontrado en la columna de agua. Esta especie filtra de forma constante e irregular durante las 24 horas del día, independientemente de la disponibilidad de alimento.

Distribución y abundancia de la ascidia Ecteinascidia turbinata (Ascidiacea: Perophoridae) en Cuba / Distribution and abundance of the ascidian Ecteinascidia turbinata (Ascidiacea: Perophoridae) in Cuba

Permanently submerged mangrove roots (Rhizophora mangle) are the main habitat of the ascidian Ecteinascidia turbinata in Cuba. It was occasionally found on black coral (Antiphates caribeana) between 22 and 38 meters deep. This species exhibits a wide distribution in all the mangrove keys surrounding the Island of Cuba but does not occur in riparian or fringing mangroves. Populations of this species are abundant in Cuba: in 75% of the 58 localities sampled the species was present and in 57% more than 50% of the roots held at least one colony. The highest colony densities were found in the northern coast of Pinar del Rio province with values near one colony per lineal meter of mangrove root. We found the highest density (1.46 col/m) and greatest biomass at Jutías Key, with values between 25 and 660 g/m. The average of wet biomass in the studied mangroves was 73.63 g/m.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10 percent of mammalian heparin and about 5 percent as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5 percent (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4 percent, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13 percent without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Structure and anticoagulant properties of sulfated glycosaminoglycans from primitive Chordates

Dermatan sulfates and heparin, similar to the mammalian glycosaminoglycans, but with differences in the degree and position of sulfation were previously isolated from the body of the ascidian Styela plicata and Ascidia nigra. These differences produce profound effects on their anticoagulant properties. S. plicata dermatan sulfate composed by 2-O-sulfatedalpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. Surprisingly, it has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, A. nigra dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Ascidian heparin, composed by 2-O-sulfated alpha-L-iduronic acid, N- and 6-O-sulfated glucosamine (75 percent) and alpha-L-iduronic acid, N- and 6-O-sulfated glucosamine (25 percent) disaccharide units has an anticoagulant activity 10 times lower than the mammalian heparin, is about 20 times less potent in the inhibition of thrombin by antithrombin, but has the same heparin cofactor II activity as mammalian heparin

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea)

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 ñ SEM = 3.44 ñ 0.84 mg/ml). The <500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68 per cent with 500 mug/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66 per cent of the control value after 24-h incubation with 100 mug/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 ñ SEM = 1.12 ñ 0.02 mglml for a 0.5 per cent erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 mug/ml) increased the EC50 by twofold to 2.86 ñ 0.04 mg/ml, but phosphatidylcholine (80 mug/ml) did not modify hemolysis.

Occurrence of tetrodotoxin and paralytic shellfish toxins in Phallusia nigra (Tunicata, Ascidiacea) from the brazilian coast

We have already shown the presence of guanidine neurotoxins in calcareous red algae and mussels collected in the Säo Sebastiäo channel State of Säo Paulo,Brazil). It is known that these neurotoxins comprise more than 25 analogues such as tetrodotoxin (TTX) and derivatives plus the paralytic shellfish toxins (PST) found in a variety of marine, freshwater and amphibious species. Filter feeding animals generally possess large amounts of these neurotoxins. The tunicates are sessil marine animals with a high rate of sea water filtration. The tunics and siphons of 50 specimens of Phallusia nigra were dissected and the visceral organs were immersed in methanol containing acetic acid 0.02 N ph 5.0. The extract was prepared by homogenization, filtration and the methanolic phase was concentrated under reduced pressure and defatted with chloroform. The polar phase was evaporated and the residue dissolved in deionized water for further purification in ionic-exchange resin column (Bio-Gel P-2) and HPLC analysis. The extract showed paralytic effects on mouse assay (26.9 MU/100mg) and on crustacean isolated nerve preparations. The chemical analysis for TTX and PST revealed toxins with retention times similar to gonyautoxins, saxitoxins and TTX. These findings are important to explain future toxin envenoming outbreaks on the Brazilian coast.