Antileukemic effects of Didemnum psammatodes (Tunicata: Ascidiacea) constituents

Chemical investigation of the methanolic extract of the ascidian Didemnum psammatodes has led to the identification of fourteen known compounds: three methyl esters (methyl myristate, methyl palmitate and methyl stearate), four steroids (cholesterol, campesterol, stigmasterol and beta-sitosterol), two fatty acids (palmitic acid and stearic acid), three glyceryl ethers {(1,2-propanediol, 3-(heptadecyloxy), batyl alcohol and 1,2-propanediol, 3-[(methyloctadecyl)oxy]) and two nucleosides (thymidine and 2`-deoxyguanosine). Their structures were proposed by NMR and comparison with literature data and GC analysis in comparison with authentic sample. The cytotoxic activity of these compounds was evaluated against human leukemia cell line panel using the MTT assay. The mixture of the three methyl esters was the most active group of compounds, showing antiproliferative and cytotoxic effects. Further studies on their mode of action suggest that these activities are connected with inhibition of DNA synthesis and induction of both necrosis and apoptosis. (C) 2007 Elsevier Inc. All rights reserved.

Cytotoxic activity of a dichloromethane extract and fractions obtained from Eudistoma vannamei (Tunicata: Ascidiacea)

This study consists of the bioassay-guided fractionation of the dichloromethane extract from Eudistoma vannamei and the pharmacological characterization of the active fractions. The dried hydromethanolic extract dissolved in aqueous methanol was partitioned with dichloromethane and chromatographed on a silica gel flash column. The anti-proliferative effect was monitored by the MTT assay. Four of the latest fractions, numbered 14 to 17, which held many chemical similarities amongst each other, were found to be the most active. The selected fractions were tested for viability, proliferation and death induction on cultures of HL-60 promycloblastic leukemia cells. The results suggested that the observed cytotoxicity is related to apoptosis induction. (C) 2007 Elsevier Inc. All rights reserved.

Ascidians (Urochordata: Ascidiacea) from Arraial do Cabo, Rio de Janeiro, Brazil

Simple and colonial ascidians were collected at different depths at Arraial do Cabo, Rio de Janeiro, between 2000 and 2003. The collection here presented includes representatives of the families Clavelinidae (Clavelina oblonga), Polycitoridae (Cystodytes dellechiajei), Polyclinidae (Polyclinum constellatum and Polyclinum molle sp. nov.), Holozoidae (Distaplia bermudensis), Ascidiidae (Ascidia sydneiensis and Phallusia nigra), Styelidae (Botrylloides giganteum, Botrylloides nigrum, Symplegma brakenhielmi, Symplegma rubra, Polyandrocarpa anguinea, Eusynstyela floridana, Eusynstyela tincta and Styela plicata), Pyuridae (Herdmania pallida and Microcosmus exasperatus). Didemnids were also collected in the area but were not considered here. Of the 17 species found, one (Polyclinum molle sp. nov.) is a new species in the family Polyclinidae and the others are all species with tropical distribution.

Recruitment and colonization of colonial ascidians (Tunicata: Ascidiacea) on intertidal rocks in Northeastern Brazil

Recruitment and colonization of colonial ascidians, starting on natural scraped substratum, was observed throughout one year, in quadrats marked on sea- and continental-rock faces in the sublittoral fringe of an exposed beach in the Northeast of Brazil. The species of the Didemnidae, Didemnum duplicatum Monniot, 1983, D. psammathodes Sluiter, 1895, and Polysyncraton amethysteum Van Name, 1902, and an unidentified species of Polycitoridae, Eudistoma sp. 1, were recruited. Presenting continuous recruitment of all species, the density of living and dead recruits and colonies was similar on both faces of the rocks. The highest rainfall during winter, affected significantly the recruits density of D. duplicatum and D. psammathodes. Different permanence periods were observed for colonies of each species. The longest permanence periods were assigned to the colonies of P. amethysteum and D. psammathodes, extending up to four and five months, respectively.

Ascídias (Tunicata, Ascidiacea) introduzidas no Arquipélago de Alcatrazes, São Paulo

O Arquipélago de Alcatrazes (24º06'S, 45º42'W) localiza-se a 36 km da costa e constitui uma Estação Ecológica desde 1987. Estando próximo à região portuária de São Sebastião, São Paulo, está sujeito à introdução de espécies exóticas. Com o objetivo de detectar se está ocorrendo ou não introdução de espécies, foram coletados 40 exemplares de ascídias da principal ilha do arquipélago, a Ilha de Alcatrazes, sendo 15 amostras da Baía do Oratório e 25 do Saco do Funil. Foram encontradas 24 espécies em sete famílias. Dentre estas, apenas cinco espécies podem ser consideradas como nativas para o Atlântico; quatro são atlânticas, mas criptogênicas na região devido à distribuição disjunta; cinco são classificadas como criptogênicas de ampla distribuição mundial; e uma pode ser considerada como um caso certo de introdução, Ciona intestinalis (Linnaeus, 1767). Há ainda nove espécies que não puderam ser identificadas, podendo algumas se tratar de espécies novas. A presença de uma espécie exótica e a grande quantidade de espécies criptogênicas com forte evidência de introdução são indícios de que as ilhas estão sendo ameaçadas por uma fauna não nativa. A conservação do Arquipélago deve levar em consideração a presença do porto como fonte de estresse e o controle das espécies introduzidas como parte do plano de manejo da Unidade de Conservação.

Sexual and asexual reproduction in Didemnum rodriguesi (Ascidiacea, Didemnidae)

Sexual and asexual reproduction and associated population dynamics were investigated in the colonial ascidian Didemnum rodriguesi Rocha & Monniot, 1993 (Didemnidae) in southern Brazil. Investment in sexual (production of new individuals) and asexual (colony growth) reproduction was compared between seasons. Permanently marked quadrats were repeatedly photographed to measure changes in colonies. Eggs and larvae were counted monthly in collected colonies. This species alternates seasonally between sexual (summer) and asexual (winter) reproduction. In summer, colonies were smaller, brooded eggs and larvae and recruitment rates were greater, while in winter, colony size was larger and eggs and larvae were absent. There is a relationship between fecundity and colony area. Fragmentation and fusion of colonies were similar in summer and winter, as well as mortality. In conclusion, D. rodriguesi has a lifecycle usual for high latitude ascidians with a limited time length for sexual reproduction and alternate investment in sexual and asexual reproduction along the year.

Lack of COI variation for Clavelina oblonga (Tunicata, Ascidiacea) in Brazil: Evidence for its human-mediated transportation?

Recent studies indicate that ascidians are efficiently dispersed by human transport. We have chosen the mitochondrial gene cytochrome c oxidase subunit I (COI) to address whether Clavelina oblonga is an introduced species in the Brazilian coast. Colonies of C. oblonga were sampled in different localities along Atlantic coasts of USA, Panama, and Brazil. The sequencing of 92 colonies resulted in three haplotypes for the species, two unique to Florida and the other shared by exemplars collected in Brazil and Panama; the latter haplotype is identical to the published sequence of Azores. Our evidence, including the absence of C. oblonga in the country's northern tropical waters, its association with artificial habitats and lack of COI variation suggest that the species has been introduced in the southeastern and southern Brazilian coasts. Previous records (85 years old) suggest that it could be a relatively long-term introduction.

Spatial distribution patterns of ascidians (Ascidiacea: Tunicata) in combination with information on bathymetry, oceanography, chlorophyll-a, and sea-ice on the continental shelves off the northern Antarctic Peninsula during POLARSTERN cruise ANT-XXIX/3

Ascidians (Ascidiacea: Tunicata) are sessile suspension feeders that represent dominant epifaunal components of the Southern Ocean shelf benthos and play a significant role in the pelagic-benthic coupling. Here, we report the results of a first study on the relationship between the distribution patterns of eight common and/or abundant (putative) ascidian species, and environmental drivers in the waters off the northern Antarctic Peninsula. During RV Polarstern cruise XXIX/3 (PS81) in January-March 2013, we used seabed imaging surveys along 28 photographic transects of 2 km length each at water depths from 70 to 770 m in three regions (northwestern Weddell Sea, southern Bransfield Strait and southern Drake Passage), differing in their general environmental setting, primarily oceanographic characteristics and sea-ice dynamics, to comparatively analyze the spatial patterns in the abundance of the selected ascidians, reliably to be identified in the photographs, at three nested spatial scales. At a regional (100-km) scale, the ascidian assemblages of the Weddell Sea differed significantly from those of the other two regions, whereas at an intermediate 10-km scale no such differences were detected among habitat types (bank, upper slope, slope, deep/canyon) on the shelf and at the shelf break within each region. These spatial patterns were superimposed by a marked small-scale (10-m) patchiness of ascidian distribution within the 2-km-long transects. Among the environmental variables considered in our study, a combination of water-mass characteristics, sea-ice dynamics (approximated by 5-year averages in sea-ice cover in the region of or surrounding the photographic stations), as well as the seabed ruggedness, was identified as explaining best the distribution patterns of the ascidians.

Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species (Tunicata, Ascidiacea)

Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.

Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species (Tunicata, Ascidiacea)

Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

Tracking Invasion Histories in the Sea: Facing Complex Scenarios Using Multilocus Data

In recent years, new analytical tools have allowed researchers to extract historical information contained in molecular data, which has fundamentally transformed our understanding of processes ruling biological invasions. However, the use of these new analytical tools has been largely restricted to studies of terrestrial organisms despite the growing recognition that the sea contains ecosystems that are amongst the most heavily affected by biological invasions, and that marine invasion histories are often remarkably complex. Here, we studied the routes of invasion and colonisation histories of an invasive marine invertebrate Microcosmus squamiger (Ascidiacea) using microsatellite loci, mitochondrial DNA sequence data and 11 worldwide populations. Discriminant analysis of principal components, clustering methods and approximate Bayesian computation (ABC) methods showed that the most likely source of the introduced populations was a single admixture event that involved populations from two genetically differentiated ancestral regions - the western and eastern coasts of Australia. The ABC analyses revealed that colonisation of the introduced range of M. squamiger consisted of a series of non-independent introductions along the coastlines of Africa, North America and Europe. Furthermore, we inferred that the sequence of colonisation across continents was in line with historical taxonomic records - first the Mediterranean Sea and South Africa from an unsampled ancestral population, followed by sequential introductions in California and, more recently, the NE Atlantic Ocean. We revealed the most likely invasion history for world populations of M. squamiger, which is broadly characterized by the presence of multiple ancestral sources and non-independent introductions within the introduced range. The results presented here illustrate the complexity of marine invasion routes and identify a cause-effect relationship between human-mediated transport and the success of widespread marine non-indigenous species, which benefit from stepping-stone invasions and admixture processes involving different sources for the spread and expansion of their range.

Systematic UPLC-ESI-MS/MS Study on the Occurrence of Staurosporine and Derivatives in Associated Marine Microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

Rapid assessment survey for exotic benthic species in the São Sebastião Channel, Brazil

The study of biological invasions can be roughly divided into three parts: detection, monitoring, mitigation. Here, our objectives were to describe the marine fauna of the area of the port of São Sebastião (on the northern coast of the state of São Paulo, in the São Sebastião Channel, SSC) to detect introduced species. Descriptions of the faunal community of the SSC with respect to native and allochthonous (invasive or potentially so) diversity are lacking for all invertebrate groups. Sampling was carried out by specialists within each taxonomic group, in December 2009, following the protocol of the Rapid Assessment Survey (RAS) in three areas with artificial structures as substrates. A total of 142 species were identified (61 native, 15 introduced, 62 cryptogenic, 4 not classified), of which 17 were Polychaeta (12, 1, 1, 3), 24 Ascidiacea (3, 6, 15, 0), 36 Bryozoa (17, 0, 18, 1), 27 Cmdana (2, 1, 24, 0), 20 Crustacea (11, 4, 5, 0), 2 Entoprocta (native), 16 Mollusca (13, 3, 0, 0). Twelve species are new occurrences for the SSC. Among the introduced taxa, two are new for coastal Brazil. Estimates of introduced taxa are conservative as the results of molecular studies suggest that some species previously considered cryptogenic are indeed introduced. We emphasize that the large number of cryptogenic species illustrates the need for a long-term monitoring program, especially in areas most susceptible to bioinvasion. We conclude that rapid assessment studies, even in relatively well-known regions, can be very useful for the detection of introduced species and we recommend that they be carried out on a larger scale in all ports with heavy ship traffic.