A NGS approach to the encrusting Mediterranean sponge Crella elegans (Porifera, Demospongiae, Poecilosclerida): transcriptome sequencing, characterization and overview of the gene expression along three life cycle stages.

Sponges can be dominant organisms in many marine and freshwater habitats where they play essential ecological roles. They also represent a key group to address important questions in early metazoan evolution. Recent approaches for improving knowledge on sponge biological and ecological functions as well as on animal evolution have focused on the genetic toolkits involved in ecological responses to environmental changes (biotic and abiotic), development and reproduction. These approaches are possible thanks to newly available, massive sequencing technologies-such as the Illumina platform, which facilitate genome and transcriptome sequencing in a cost-effective manner. Here we present the first NGS (next-generation sequencing) approach to understanding the life cycle of an encrusting marine sponge. For this we sequenced libraries of three different life cycle stages of the Mediterranean sponge Crella elegans and generated de novo transcriptome assemblies. Three assemblies were based on sponge tissue of a particular life cycle stage, including non-reproductive tissue, tissue with sperm cysts and tissue with larvae. The fourth assembly pooled the data from all three stages. By aggregating data from all the different life cycle stages we obtained a higher total number of contigs, contigs with blast hit and annotated contigs than from one stage-based assemblies. In that multi-stage assembly we obtained a larger number of the developmental regulatory genes known for metazoans than in any other assembly. We also advance the differential expression of selected genes in the three life cycle stages to explore the potential of RNA-seq for improving knowledge on functional processes along the sponge life cycle.

[Seasonal and geographical variation range of antifungal activity of sponge extracts from the Moroccan Atlantic coasts]. / Variation saisonnière et géographique de l'activité antifongique des extraits de deux éponges marines récoltées sur le littoral atlantique d'El Jadida, Maroc.

Currently, marine organisms have a very important source of new molecules in pharmacology and thus in the development of new bioactive products. The organic and aqueous extracts of two marine sponges, Cinachyrella tarentine collected during two different seasons, winter and summer, and Cliona viridis collected in two different zones on the coast of El Jadida (Morocco) were tested for their antifungal activity using the diffusion method. The C. tarentine sponge collected in January (winter) has a very important activity compared to that collected in August (summer). While the sponge C. viridis collected from Jorf Lasfar port (shallower and polluted area) has a very important activity compared to that collected from the coast of El Jadida (depth and unpolluted area).

Contrasting effects of heavy metals and hydrocarbons on larval settlement and juvenile survival in sponges.

Metals and polycyclic aromatic hydrocarbons (PAHs) contaminate sediments and waters of coastal areas threatening early stages of invertebrate development. Effects on these stages may largely determine the decline and even disappearance of invertebrate populations in polluted environments. Our study aimed to determine the possible influence of metals (Cu and Cd) and PAHs on larval settlement and consecutive survival of two widespread sponges of the Mediterranean: Crambe crambe and Scopalina lophyropoda. Larvae of both species were exposed to Cu and Cd for a short period during 1 week, and settlement and following (6 months) survival of juvenile were monitored. Short exposures to copper and cadmium at the concentrations used did not affect C. crambe settlement compared with SW control, and no effect on consecutive survival of juveniles was observed. In contrast, short pulses of copper and cadmium at the concentrations used enhanced Scopalina lophyropoda settlement and did not affect the consecutive survival of juveniles with respect to SW controls. Furthermore, experiments designed to assess the effects of short exposures to PAHs and the combined effect of contamination by Cu(2+) and PAHs on larval settlement, were conduced during 10 days on C. crambe larvae. Hydrocarbons, differently than copper and cadmium, inhibited the settlement of sponge larvae to a certain extent. The synergetic negative effect of copper and hydrocarbons on C. crambe settlers may cause a decline of populations in areas with both sources of contamination. The present study provides the only available data on toxicity of copper, cadmium and hydrocarbon toxicants on sponge larval settlement.

Response of the Mediterranean sponge Chondrosia reniformis Nardo to copper pollution.

We examined the effects of exposure to copper pollution on the Atlanto-Mediterranean sponge Chondrosia reniformis. We transplanted sponges from an unpolluted control area to a harbour with a moderately high concentration of copper and measured several biological sponge variables. No effect of this habitat was detected on sponge growth, shape, heat-shock protein expression or metal accumulation. However, a decrease in the clearance rate, an increase in the collagen/cell rate (due to a decrease in the cellular components) and a lower survival rate after 4 months of the sponges transplanted to the harbour was observed. We suggest that copper may alter the sponge physiology, by reducing pumping capacity, which may ultimately lead to sponge death. Consequently, copper pollution exerts strong negative effects on this organism.

Molecular and organism biomarkers of copper pollution in the ascidian Pseudodistoma crucigaster.

We studied the effects of pollution in the colonial ascidian Pseudodistoma crucigaster at organismal and suborganismal levels. Our goal was to find early biomarkers to detect some effect of pollution before changes in community structure or species composition occur. We examined the effect of Cu on the production of heat-shock proteins, defence metabolites, growth rates and presence of resistance forms. We performed a transplant experiment to a Cu polluted harbour and observed negative growth and presence of resistance forms but not depressed production of toxic metabolites or an increase in stress proteins (hsp) in the ascidian. In a laboratory experiment, stress proteins were induced only under half the Cu concentration found in the harbour. We conclude that hsp can be used in this ascidian as an early warning system for sublethal pollution but that the response is inhibited above a threshold of the stressing agent, which may vary among species.

Does stress protein induction by copper modify natural toxicity in sponges?

Crambe crambe is a toxic Mediterranean sponge that inhabits the sublittoral rocky bottoms, including some contaminated habitats. We investigated whether contamination by copper induced stress proteins in C. crambe and whether such stress might alter the production of chemical defenses. The monoclonal antibody used cross-reacted with two heat shock proteins (HSP) of 54 and 72 kDa. Both proteins were induced to a greater or lesser extent by copper contamination. The HSP54 accumulated more than HSP72, which, in contrast, appeared to respond faster and be less persistent. In a field experiment, we found a higher accumulation of HSP54 in individuals naturally inhabiting a copper-contaminated site than in those transplanted to this site four months earlier. In contrast, HSP72 was significantly induced only in the individuals transplanted to the contaminated site. In the laboratory, both proteins were induced by copper at 30 microg/L but inhibited at 100 microg/L. The highest mean values of HSP54 and HSP72 corresponded to the sponges, which showed the lowest mean values of toxicity. Thus, toxicity and production of HSP displayed opposite trends, which seems to indicate a preferential investment in cell repair at the expense of toxic molecules under stress conditions.

Silica deposition in Demosponges: spiculogenesis in Crambe crambe.

Transmission electron-microscopy images coupled with dispersive X-ray analysis of the species Crambe crambe have provided information on the process of silica deposition in Demosponges. Sclerocytes (megasclerocytes) lie close to spicules or surround them at different stages of growth by means of long thin enveloping pseudopodia. Axial filaments occur free in the mesohyl, in close contact with sclerocytes, and are triangular in cross section, with an internal silicified core. The unit-type membrane surrounding the growing spicule coalesces with the plasmalemma. The axial filament of a growing spicule and that of a mature spicule contain 50%-70% Si and 30%-40% Si relative to that contained in the spicule wall, respectively. The extracellular space between the sclerocyte and the growing spicule contains 50%-65%. Mitochondria, vesicles and dense inclusions of sclerocytes exhibit less than 10%. The cytoplasm close to the growing spicule and that far from the growing spicule contain up to 50% and less than 10%, respectively. No Si has been detected in other parts of the sponge. The megascleres are formed extracellularly. Once the axial filament is extruded to the mesohyl, silicification is accomplished in an extracellular space formed by the enveloping pseudopodia of the sclerocyte. Si deposition starts at regularly distributed sites along the axial filament; this may be related to the highly hydroxylated zones of the silicatein-alpha protein. Si is concentrated in the cytoplasm of the sclerocyte close to the plasmalemma that surrounds the growing spicules. Orthosilicic acid seems to be pumped, both from the mesohyl to the sclerocyte and from the sclerocyte to the extracellular pocket containing the growing spicule, via the plasmalemma.

Distribution of brominated compounds within the sponge Aplysina aerophoba: coupling of X-ray microanalysis with cryofixation techniques.

The major secondary metabolites of the sponge Aplysina aerophoba are brominated compounds. X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections. To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared the results obtained by conventional aldehyde fixation with those obtained by cryofixation and cryosubstitution with and without cryoembedding. Bromine appeared to be concentrated in two sponge structures, viz. fibres and spherulous cells, when cryofixed material was examined. However, X-ray microanalysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds. Cryofixation plus cryosubstitution methods performed best regarding structural preservation and the immobilization of metabolites. The presence of bromine in the spherulous cells suggests that this cell type is the producer of the secondary metabolites, as described for other sponge species. Nevertheless, the presence of bromine in sponge fibres indicates that they can accumulate metabolic substances, although we have been unable to assess whether the chemicals are in their original form or in a modified state within the fibres. A. aerophoba has both bacterial and cyanobacterial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts.

Phylogenetic Relationships within the Excavating Hadromerida (Porifera), with a Systematic Revision.

This study was performed to ascertain the relationships among clionid and spirastrellid species from the Mediterranean Sea, based on phylogenetic criteria. A matrix of 34 taxa and 20 characters was analysed. Cladistic analysis using PAUP produced 12 equally parsimonious trees of 54 steps (CI=0.648, RI=0.865). Resolution was high in all parts of the strict consensus tree except in those which involved relationships among the most characteristic clionid species: those having only tylostyles as megascleres and, if microscleres are present, these being spirasters. Six monophyletic groups, three of them represented by monospecific clades, appear in the totally resolved part of the trees, which supports their separation into different genera. On the basis of the results obtained, the species at present within the genus Cliona have been allocated to five different genera:Scantilletta, Pione, Volzia, Bernatia and Cliona, while Dotona and Cliothosa are maintained as separate genera.Scantilletta and Pione are genera erected by de Laubenfels (1936 Pap. Tortugas Lab. 30, 1-225) and Gray (1867 Proc. Zool. Soc. Lond. XXXII , 492-558) respectively, and whose diagnoses are amended here.Volzia and Bernatia are proposed as new genera to include Cliona albicans and Cliona rovignensis, and Cliona vermifera, respectively. Thus, the genus Cliona clearly appears in our analysis as a polyphyletic group. It is only maintained provisionally to harbour species whose position cannot be clearly ascertained from the information at hand. Skeletal characters such as spicule types and skeletal arrangement have proven to be useful in discriminating some genera and families but they do not completely resolve the phylogeny of this group of species. Biochemical, genetic, cytological or reproductive information, when available, may help in the resolution of the phylogenetic tree.

Natural variation of toxicity in encrusting spongeCrambe crambe (Schmidt) in relation to size and environment.

The presence of intraspecific variation in toxicity and its relationship with biological or ecological factors were studied in the spongeCrambe crambe. Within-specimen (periphery and central part), between-size (<1000 mm(2) in area, between 1000 and 10,000 mm(2) and >10,000 mm(2)) and between-habitat (well-illuminated and dark communities) variations in toxicity were evaluated by the Microtox bioassay. Quantitative differences were detected that were not attributable to within-specimen variation but to size and habitat effects. Habitat comparisons showed that sponges in the shaded habitat were significantly more toxic than those of the well-illuminated community. Sponges of the smaller size classes displayed significantly less toxicity than the medium-sized specimens. Results are interpreted under the optimal defense theory and their ecological implications are considered.

IgD plasma cell leukemia associated with pyroglobulinemia and pyroglobulinuria.

A 70-yr-old man with multiple myeloma IgD developed a plasma cell leukemia producing a serum IgD monoclonal peak and lambda light chains in the urine. When the serum and the urine were heated at 56 degrees C for 30 min both monoclonal bands disappeared. The precipitate failed to redissolve on heating to 100 degrees C. Ion exchange chromatography with a linear gradient of phosphate buffer, pH 8, 0.020-0.300 mol/l and column electrofocusing showed that the serum pyroglobulin was eluted with buffer concentration between 0.040-0.125 mol/l and had an isoelectric point of 5.02, while the pyroglobulin of the urine was eluted with 0.020-0.033 mol/l and had a pI = 7.16. The serum and urine pyroglobulins had a total antigenic community with their correspondent purified proteins. The isolated lambda chains did not change when they were heated at 56 degrees C for 30 min, nevertheless, the heated purified IgD shows some changes in its isoelectric point, molecular mass and antigenicity. These changes in the purified IgD suggest that the pyroprecipitability could be due to conformational features.

Fanconi's anemia. Clinical study of six cases.

Six cases of constitutional aplastic anemia (Fanconi's anemia), including four boys and two girls who were between 6 and 10 years of age, are presented. Congenital abnormalities were observed in all the patients, especially skin pigmentations, renal anomalies, and growth retardation. The initial symptoms were insidious, mainly related to the anemia. All patients were anemic and thrombocytopenic, but only three were leukopenic. Five cases showed increased levels of fetal hemoglobin, which was marked in two. Bone marrow obtained by aspiration and/or biopsy was hypocellular in all cases. Chromosomal studies showed breaks, ring- or cross-chromosomes, chromosome exchanges, and endoreduplications. Five cases were treated with androgens and a good response was obtained in all of them. Four patients died, and the remaining two are alive and well. The clinical and biological features of these patients are in accordance with those of previous reports. The importance of the androgen therapy is stressed; androgen dependence was observed in all cases.