Community composition and functional prediction of prokaryotes associated with sympatric sponge species of southwestern Atlantic coast.

Prokaryotes contribute to the health of marine sponges. However, there is lack of data on the assembly rules of sponge-associated prokaryotic communities, especially for those inhabiting biodiversity hotspots, such as ecoregions between tropical and warm temperate southwestern Atlantic waters. The sympatric species Aplysina caissara, Axinella corrugata, and Dragmacidon reticulatum were collected along with environmental samples from the north coast of São Paulo (Brazil). Overall, 64 prokaryotic phyla were detected; 51 were associated with sponge species, and the dominant were Proteobacteria, Bacteria (unclassified), Cyanobacteria, Crenarchaeota, and Chloroflexi. Around 64% and 89% of the unclassified operational taxonomical units (OTUs) associated with Brazilian sponge species showed a sequence similarity below 97%, with sequences in the Silva and NCBI Type Strain databases, respectively, indicating the presence of a large number of unidentified taxa. The prokaryotic communities were species-specific, ranging 56%-80% of the OTUs and distinct from the environmental samples. Fifty-four lineages were responsible for the differences detected among the categories. Functional prediction demonstrated that Ap. caissara was enriched for energy metabolism and biosynthesis of secondary metabolites, whereas D. reticulatum was enhanced for metabolism of terpenoids and polyketides, as well as xenobiotics' biodegradation and metabolism. This survey revealed a high level of novelty associated with Brazilian sponge species and that distinct members responsible from the differences among Brazilian sponge species could be correlated to the predicted functions.

Phylogeny and systematics of demospongiae in light of new small-subunit ribosomal DNA (18S) sequences.

The most diverse and species-rich class of the phylum Porifera is Demospongiae. In recent years, the systematics of this clade, which contains more than 7000 species, has developed rapidly in light of new studies combining molecular and morphological observations. We add more than 500 new, nearly complete 18S sequences (an increase of more than 200%) in an attempt to further enhance understanding of the phylogeny of Demospongiae. Our study specifically targets representation of type species and genera that have never been sampled for any molecular data in an effort to accelerate progress in classifying this diverse lineage. Our analyses recover four highly supported subclasses of Demospongiae: Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha. Within Keratosa, neither Dendroceratida, nor its two families, Darwinellidae and Dictyodendrillidae, are monophyletic and Dictyoceratida is divided into two lineages, one predominantly composed of Dysideidae and the second containing the remaining families (Irciniidae, Spongiidae, Thorectidae, and Verticillitidae). Within Myxospongiae, we find Chondrosida to be paraphyletic with respect to the Verongida. We amend the latter to include species of the genus Chondrosia and erect a new order Chondrillida to contain remaining taxa from Chondrosida, which we now discard. Even with increased taxon sampling of Haploscleromorpha, our analyses are consistent with previous studies; however, Haliclona species are interspersed in even more clades. Haploscleromorpha contains five highly supported clades, each more diverse than previously recognized, and current families are mostly polyphyletic. In addition, we reassign Janulum spinispiculum to Haploscleromorpha and resurrect Reniera filholi as Janulum filholi comb. nov. Within the large clade Heteroscleromorpha, we confirmed 12 recently identified clades based on alternative data, as well as a sister-group relationship between the freshwater Spongillida and the family Vetulinidae. We transfer Stylissa flabelliformis to the genus Scopalina within the family Scopalinidae, which is of uncertain position. Our analyses uncover a large, strongly supported clade containing all heteroscleromorphs other than Spongillida, Vetulinidae, and Scopalinidae. Within this clade, there is a major division separating Axinellidae, Biemnida, Tetractinellida, Bubaridae, Stelligeridae, Raspailiidae, and some species of Petromica, Topsentia, and Axinyssa from Agelasida, Polymastiidae, Placospongiidae, Clionaidae, Spirastrellidae, Tethyidae, Poecilosclerida, Halichondriidae, Suberitidae, and Trachycladus. Among numerous results: (1) Spirophorina and its family Tetillidae are paraphyletic with respect to a strongly supported Astrophorina within Tetractinellida; (2) Agelasida is the earliest diverging lineage within the second clade listed above; and (3) Merlia and Desmacella appear to be the earliest diverging lineages of Poecilosclerida.

Molecular sexing of unusually large numbers of Spheniscus magellanicus (Spheniscidae) washed ashore along the Brazilian coast in 2008.

There have been few studies on Magellanic penguins (Spheniscus magellanicus). In 2008, these penguins washed ashore along the Brazilian coast in unusually high numbers, some reaching as far as northeast Brazil. As Magellanic penguins show little sexual dimorphism, sex determination by morphological features is not accurate. Here, we tested a molecular procedure for sexing specimens of S. magellanicus washed ashore along the coasts of Sergipe, Rio de Janeiro and Rio Grande do Sul in 2008, comparing the sex ratio between these localities. Tissue samples were collected from 135 dead, beached specimens. We carried out total genomic DNA extraction and CHD-Z/CHD-W gene amplification by PCR using P2 and P8 primers. Amplicons were separated by 12% acrylamide gel electrophoresis. We found a greater proportion of females (70%). Sex could be determined because females have two intronic regions of CHD gene of different size in the sex chromosomes, visualized as two bands on the gel (380 and 400 bp approximately), while males have only one (400 bp). Therefore, this method proved to be effective and sensitive for sex determination of S. magellanicus individuals. Data on sex ratios are useful for understanding the dynamics and ecology of Magellanic penguin populations.

Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP.

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra- and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

Genotoxic and antigenotoxic evaluation of extracts from Arenosclera brasiliensis, a Brazilian marine sponge.

The marine environment is a rich source of biological active compounds and the sponges can be considered the most productive one. This diversity gives rise to unique chemical compounds with potential pharmacological properties. Our study is focused on the genotoxic and antigenotoxic evaluation of two crude extracts obtained from the Brazilian endemic marine sponge Arenosclera brasiliensis. Salmonella typhimurium reverse mutation test with TA97, TA98, TA100 and TA102 strains were performed. For antimutagenic analysis, a pre-, co-, and post-treatment to evaluate, respectively, intracellular and extracellular reactions and possible modulation on DNA repair. Additionally, in order to verify the influence of the crude extracts on DNA damage induction, a plasmid-DNA treatment was assayed. No mutagenicity was observed in Salmonella reverse mutation test, neither DNA strand induced damage. Antimutagenic activity was observed in pre-, co-, and post-treatment. A significant antigenotoxic effect was observed in the crude extract, which suggests that A. brasiliensis extract has the potential to protect DNA from the action of 4NQO, 2-aminofluorene, sodium azide and mitomycin C.

Cytotoxic, mutagenic and antimutagenic screening of Arenosclera brasiliensis acetone and ethanol extracts.

The marine environment is a rich source of biologically active compounds with pharmacological properties. Marine organisms often produce secondary metabolites with structural features different from those produced by terrestrial ones, and the Phylum Porifera seems to be one of the most productive in this sense. This study was undertaken to provide data on mutagenic and antimutagenic activities from an acetone (Areac) and an ethanol (Areet) extract obtained from Arenosclera brasiliensis, an endemic Brazilian sponge. A qualitative Salmonella reverse mutation test was performed with the TA97, TA98, TA100, and TA102 strains by incubating cells with Areac and Areet in the presence and absence of a known mutagen. A cytotoxic evaluation of the extracts was also performed. A. brasiliensis did not display any mutagenic activity, but Areac showed significant toxicity against test strains. In the antimutagenic assay, a reduction in the number of his+ revertants was observed for the TA97, TA100 and TA102 strains treated with Areac when compared to the positive controls. Areet treatment showed protective activity against DNA lesions only for the TA100. These results are in agreement with those obtained previously with other A. brasiliensis extracts, suggesting an antimutagenic activity.

Cytotoxic, mutagenic and antimutagenic screening of Arenosclera brasiliensis acetone and ethanol extracts

The marine environment is a rich source of biologically active compounds with pharmacological properties. Marine organisms often produce secondary metabolites with structural features different from those produced by terrestrial ones, and the Phylum Porifera seems to be one of the most productive in this sense. This study was undertaken to provide data on mutagenic and antimutagenic activities from an acetone (Areac) and an ethanol (Areet) extract obtained from Arenosclera brasiliensis, an endemic Brazilian sponge. A qualitative Salmonella reverse mutation test was performed with the TA97, TA98, TA100, and TA102 strains by incubating cells with Areac and Areet in the presence and absence of a known mutagen. A cytotoxic evaluation of the extracts was also performed. A. brasiliensis did not display any mutagenic activity, but Areac showed significant toxicity against test strains. In the antimutagenic assay, a reduction in the number of his+ revertants was observed for the TA97, TA100 and TA102 strains treated with Areac when compared to the positive controls. Areet treatment showed protective activity against DNA lesions only for the TA100. These results are in agreement with those obtained previously with other A. brasiliensis extracts, suggesting an antimutagenic activity.

Functional complementation analysis of yeast bc1 mutants. A study of the mitochondrial import of heterologous and hybrid proteins.

Previous complementation studies with yeast bc1 mutants, defective in subunit VII or VIII, using heterologous and hybrid subunits, suggested that the requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins. Prediction algorithms indicate that the N-terminal domain of subunit VII contains all known characteristics of a mitochondrial targeting signal, whereas in subunit VIII such a signal is absent from the N-terminal domain, but possibly present in an internal region of the protein. Despite the fact that the characteristics of a mitochondrial import signal are found in the N-terminus of all known subunit-VII orthologues, in vitro import experiments show that the protein of human origin is not imported into yeast mitochondria. In vitro import can be restored, however, by replacement of the N-terminal part of the human protein by the N-terminus of the Saccharomyces cerevisiae orthologue, indicating a requirement for species-specific elements. Similar experiments were performed with subunit VIII and orthologues thereof, including a hybrid protein in which the N-terminus of the bovine heart orthologue was replaced by that of S. cerevisiae. The ability of yeast mitochondria to import this hybrid protein, in contrast with the bovine subunit-VIII orthologue itself, indicates that for subunit VIII also the N-terminus, in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species-specific elements. Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting sequence and highlight the necessity of performing separate import studies for interpreting complementation studies as long as the species-specific characteristics of the import signals have not been identified.

Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting.

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

The aromatic domain 66(YWYWW)70 of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase is important for both assembly and activity of the enzyme.

The aromatic character of the region 66(YWYWW)70 of the 11-kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase (bc1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex [Hemrika et al. (1994) FEBS Lett. 344, 15-19]. Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc1 complex in the mutant 66(SASAA)70 was restored to nearly 70% of the wild-type level in a phenotypic revertant with the sequence 66(FASAA)70. In the present study, three other site-directed mutants (66(SAYAA)70, 66(SASAW)70 and 66(SWYWW)70) were constructed and analysed. The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity. The results also confirm the relevance of this region of subunit VIII for the formation of the Q(out) reaction domain, as reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601-609]. It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII.

The aromatic nature of residue 66 of the 11-kDa subunit of ubiquinol-cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme.

Transformation of multi- and single-copy plasmids carrying a mutated version (LTN2, region 66-YWYWW-70 replaced by SASAA) of QCR8, the gene encoding the 11-kDa subunit ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae, to a QCR8(0) strain indicated the importance of this aromatic region for the assembly of a functional enzyme. Sequencing of plasmids giving spontaneous restoration of growth to some colonies among the single-copy LTN2 transformants showed that changing the sequence SASAA into the sequence FASAA could, to a large extent, overcome the observed assembly defect, indicating the importance of the aromatic nature of residue 66.