Coupling astogenic aging in the colonial tunicate Botryllus schlosseri with the stress protein mortalin.

Botryllus schlosseri, a colonial marine invertebrate, exhibits three generations of short-lived astogenic modules that continuously grow and die throughout the colony's entire lifespan, within week-long repeating budding cycles (blastogenesis), each consisting of four stages (A-D). At stage D, aging is followed by the complete absorption of adult modules (zooids) via a massive apoptotic process. Here we studied in Botryllus the protein mortalin (HSP70s member), a molecule largely known for its association with aging and proliferation. In-situ hybridization and qPCR assays reveal that mortalin follows the cyclic pattern of blastogenesis. Colonies at blastogenic stage D display the highest mortalin levels, and young modules exhibit elevated mortalin levels compared to old modules. Manipulations of mortalin with the specific allosteric inhibitor MKT-077 has led to a decrease in the modules' growth rate and the development of abnormal somatic/germinal morphologies (primarily in vasculature and in organs such as the endostyle, the stomach and gonads). We therefore propose that mortalin plays a significant role in the astogeny and aging of colonial modules in B. schlosseri, by direct involvement in the regulation of blastogenesis.

Phylogenomics offers resolution of major tunicate relationships.

Tunicata, a diverse clade of approximately 3000 described species of marine, filter-feeding chordates, is of great interest to researchers because tunicates are the closest living relatives of vertebrates and they facilitate comparative studies of our own biology. The group also includes numerous invasive species that cause considerable economic damage and some species of tunicates are edible. Despite their diversity and importance, relationships among major lineages of Tunicata are not completely resolved. Here, we supplemented public data with transcriptomes from seven species spanning the diversity of Tunicata and conducted phylogenomic analyses on data sets of up to 798 genes. Sensitivity analyses were employed to examine the influences of reducing compositional heterogeneity and branch-length heterogeneity. All analyses maximally supported a monophyletic Tunicata within Olfactores (Vertebrata + Tunicata). Within Tunicata, all analyses recovered Appendicularia sister to the rest of Tunicata and confirmed (with maximal support) that Thaliacea is nested within Ascidiacea. Stolidobranchia is the sister taxon to all other tunicates except Appendicularia. In most analyses, phlebobranch tunicates were recovered paraphyletic with respect to Aplousobranchia. Support for this topology varied but was strong in some cases. However, when only the 50 best genes based on compositional heterogeneity were analysed, we recovered Phlebobranchia and Aplousobranchia reciprocally monophyletic with strong support, consistent with most traditional morphology-based hypotheses. Examination of internode certainty also cast doubt on results of phlebobranch paraphyly, which may be due to limited taxon sampling. Taken together, these results provide a higher-level phylogenetic framework for our closest living invertebrate relatives.

Germ lineage properties in the urochordate Botryllus schlosseri - from markers to temporal niches.

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or ß-estradiol administration affirmed the BS-Vasa(+)BS-DDX1(+)BS-cadherin(+)γ-H2AX(+)phospho-Smad1/5/8(+) population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived 'germ line niches' that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.

Chordate evolution and the three-phylum system.

Traditional metazoan phylogeny classifies the Vertebrata as a subphylum of the phylum Chordata, together with two other subphyla, the Urochordata (Tunicata) and the Cephalochordata. The Chordata, together with the phyla Echinodermata and Hemichordata, comprise a major group, the Deuterostomia. Chordates invariably possess a notochord and a dorsal neural tube. Although the origin and evolution of chordates has been studied for more than a century, few authors have intimately discussed taxonomic ranking of the three chordate groups themselves. Accumulating evidence shows that echinoderms and hemichordates form a clade (the Ambulacraria), and that within the Chordata, cephalochordates diverged first, with tunicates and vertebrates forming a sister group. Chordates share tadpole-type larvae containing a notochord and hollow nerve cord, whereas ambulacrarians have dipleurula-type larvae containing a hydrocoel. We propose that an evolutionary occurrence of tadpole-type larvae is fundamental to understanding mechanisms of chordate origin. Protostomes have now been reclassified into two major taxa, the Ecdysozoa and Lophotrochozoa, whose developmental pathways are characterized by ecdysis and trochophore larvae, respectively. Consistent with this classification, the profound dipleurula versus tadpole larval differences merit a category higher than the phylum. Thus, it is recommended that the Ecdysozoa, Lophotrochozoa, Ambulacraria and Chordata be classified at the superphylum level, with the Chordata further subdivided into three phyla, on the basis of their distinctive characteristics.

A redescription of Syncarpacomposita (Ascidiacea, Stolidobranchia) with an inference of its phylogenetic position within Styelidae.

Two species of styelid colonial ascidians in the genus Syncarpa Redikorzev, 1913 are known from the northwest Pacific. The valid status of the lesser known species, Syncarpacomposita (Tokioka, 1951) (type locality: Akkeshi, Japan), is assessed here. To assess the taxonomic identity of S.composita, we compared one of the syntypes and freshly collected topotypes of S.composita with a syntype of S.oviformis Redikorzev, 1913 (type locality: Ul'banskij Bay, Russia). Specimens of S.composita consistently differed from the syntype of S.oviformis in the number of oral tentacles, the number of size-classes of transverse vessels, and the number of anal lobes. In this paper, S.composita is redescribed as distinct from S.oviformis, and its phylogenetic position inferred within Styelidae based on the 18S rRNA and cytochrome c oxidase subunit I gene sequences. In our phylogenetic tree, Syncarpa formed a well-supported clade together with Dendrodoa MacLeay, 1824. In Syncarpa and Dendrodoa, a single gonad is situated on the right side of the body, which is unique among Styelidae, and thus can be a synapomorphy for this clade.

Bioactive substances with anti-neoplastic efficacy from marine invertebrates: Bryozoa, Mollusca, Echinodermata and Urochordata.

The marine environment provides a rich source of natural products with potential therapeutic application. This has resulted in an increased rate of pharmaceutical agents being discovered in marine animals, particularly invertebrates. Our objective is to summarize the most promising compounds which have the best potential and may lead to use in clinical practice, show their biological activities and highlight the compounds currently being tested in clinical trials. In this paper, we focused on Bryozoa, Mollusca, Echinodermata and Urochordata.

Inventory of ascidians (Tunicata, Ascidiacea) from the National Park La Restinga, Isla Margarita, Venezuela / Inventário de ascídias (Tunicata, Ascidiacea) do Parque Nacional La Restinga, Isla Margarita, Venezuela

Although ascidians form a conspicuous part of sessile assemblages in the Caribbean, no specialized inventories have been developed in Venezuela, except for a list of 15 species reported from Margarita Island (1984). Here we present the results of a taxonomic workshop held in the Universidad de Oriente, Boca del Rio, Margarita Island during April 20-25 of 2009, sponsored by the NaGISA-Caribbean Sea program. La Restinga National park was surveyed and we found 29 species belonging to 19 genera and 10 families. The most abundant colonial species were Clavelina oblonga, Aplidium accarense, Polyclinum constellatum, Distaplia bermudensis, Symplegma rubra, S. brakenhielmi, Botrylloides nigrum and Ecteinascidia turbinata. Among the solitary ascidians Phallusia nigra, Ascidia curvata, Microcosmus exasperatus, Styela canopus, Styela sp.1 and Styela sp. 2 were the most abundant. The ascidian diversity in this lagoon is one of the highest for similar habitats in the Caribbean and seven species are new registers for Venezuela (Ascidia curvata, Ecteinascidia styeloides, Aplidium accarense, Distaplia stylifera, Trididemnum orbiculatum, Symplegma rubra, and S. brakenhielmi). One point of concern for the conservation of la Restinga National Park is the presence of possible introduced species, and some management procedures are suggested.

Apesar das ascídias formarem uma parte conspícua das comunidades incrustantes no Caribe, nenhum inventário especializado foi realizado na Venezuela, com exceção de uma lista de 15 espécies registradas na Ilha Margarita (1984). Aqui apresentamos os resultados de um workshop de taxonomia realizado na Universidad de Oriente, Boca del Rio, Margarita Island em 20-25 de abril de 2009, como parte do programa NaGISA-Caribbean Sea program. O Parque Nacional La Restinga foi estudado e encontradas 29 espécies pertencendo a 19 gêneros e 10 famílias. As espécies coloniais mais abundantes foram Clavelina oblonga, Aplidium accarense, Polyclinum constellatum, Distaplia bermudensis, Symplegma rubra, S. brakenhielmi, Botrylloides nigrum e Ecteinascidia turbinata. Entre as espécies solitárias, Phallusia nigra, Ascidia curvata, Microcosmus exasperatus, Styela canopus, Styela sp.1 e Styela sp. 2 foram as mais abundantes. A diversidade de ascídias nesta laguna é uma das maiores neste tipo de habitat no Caribe e sete espécies representam novos registros para a Venezuela (Ascidia curvata, Ecteinascidia styeloides, Aplidium accarense, Distaplia stylifera, Trididemnum orbiculatum, Symplegma rubra, e S. brakenhielmi). Uma questão de preocupação para a conservação do Parque Nacional La Restinga é a presença de possíveis espécies exóticas, portanto algumas medidas de manejo são sugeridas.

Differentiation of histospecific ultrastructural features in cells of cleavage-arrested early ascidian embryos.

Ultrastructural features of histospecific differentiation were found in early cleavage stage ascidian embryos treated with cytochalasin B and held thereby in cleavagearrest until hatching time. Markers characteristic of tissue differentiation during normal embryonic and larval stages ofCiona intestinalis were expressed in muscle and two brain cell lineages of cleavage-arrested whole embryos and in epidermal and notochordal cell lineages of cleavage-arrested partial embryos. These features were muscle myofilaments and myofibrils, melanosomes of the brain pigment cells, cilium-derived structures present in a "proprioceptive" brain cell, extracellular test material of epidermal cell origin, and the sheath filaments, membrane leaflets, and vacuolar colloid associated with notochord cells. All of these ultrastructural markers of differentiation were blocked in their development by treatment of gastrula stage embryos with actinomycin D, an inhibitor of RNA synthesis, and presumably result from the expression of new gene activity. At the time of cleavage-arrest the five cell lineages studies still contained two or more unsegregated lineage pathways. Subsequent developmental autonomy within the lineages is consistent with the hypothesis of segregation during early development of functionally independent gene regulatory factors.