Characterization of the transcriptome and gene expression of four different tissues in the ecologically relevant sea urchin Arbacia lixula using RNA-seq.

The sea urchin Arbacia lixula is a keystone species in Mediterranean ecosystems that drive landscape changes in littoral communities. However, genomic information available for the whole order Arbacioida is very limited. Using RNA-seq techniques, we have characterized the transcriptome of four different tissue types in A. lixula: the 'somatic' tissues (coelomocytes and digestive tissue) and the 'reproductive' tissues (ovary and testis), from two replicated cDNA libraries for each sample. Additionally, we performed a de novo assembly to build the 'reference' transcriptome, pooling reads of the four tissues, to analyse the differential expression (DE) in pairwise comparisons between tissues. The complete de novo assembly yielded 186,084 transcripts, with a sequence size limit of 100 nt, being 31% of them spliced isoforms. Approximately 21% of the transcripts had blast hits against proteins of metazoans (E < 10(-5) ), being less than 2.2% functionally annotated. Between coelomocytes and digestive, 30,794 transcripts showed DE (~11.8% of them with blast hit), and 19,567 transcripts did so between testis and ovary (~28.5% of them with blast hit). Major GO-term categories upregulated in somatic tissues were those related to muscle contraction and energy generation in digestive, and lipid metabolism associated with immune response in coelomocytes. Between reproductive tissues, the major upregulated GO categories were related to energy generation in testis, and negative regulation of nucleotide metabolism in ovary. We particularly screened for a collection of target genes in each tissue because of their relevance for further studies on evolution and adaptation of echinoids.

Ongoing expansion of the worldwide invader Didemnum vexillum (Ascidiacea) in the Mediterranean Sea: high plasticity of its biological cycle promotes establishment in warm waters.

Non-indigenous ascidians are of particular concern to aquaculture industry and, paradoxically, the activities associated with it represent an important way to translocate these species worldwide. In 2012 a non-indigenous ascidian was found covering the oyster crops in the Ebro Delta (Western Mediterranean). We have identified the ascidian genetically and morphologically as Didemnum vexillum Kott, 2002. This finding indicates that the species is currently expanding its distribution in the Mediterranean Sea, as it has recently been found in the eastern basin (Venice, Adriatic Sea). Introduced populations of D. vexillum are found in temperate and cold waters worldwide, and a successful establishment in the Mediterranean implies a remarkable capacity of adaptability to warm, subtropical conditions. We assessed the life cycle (growth and reproduction) of the ascidian at the studied site. The species has a marked seasonal cycle, with regression in the warmest months and reappearance during winter. In spring D. vexillum reaches its maximum abundance, followed by a peak in reproduction just before regression. This cycle is reversed with respect to the one observed in colder waters, highlighting a plastic biological cycle of this invader and an hitherto unknown ability to establish itself in warm waters. We also analysed the genetic structure of the population of the Ebro Delta and the one established in the Lagoon of Venice using COI sequence data. The low genetic diversity in our samples (three haplotypes) was consistent with what is observed in the introduced populations worldwide. It is likely that the ascidian was introduced with oyster stock from bivalve cultures in the Atlantic French coasts, where the same three haplotypes have been reported. The high boating activity in the Ebro Delta makes further human-mediated transport of the species highly likely, and nearby fishing grounds can be severely affected if invaded. It is urgent to implement measures to prevent the continuous expansion of this ascidian pest in the Mediterranean.

Genetic divergence and assortative mating between colour morphs of the sea urchin Paracentrotus gaimardi.

Some species of sea urchins feature large variation in pigmentation. This variability may be the result of phenotypic plasticity or it may be associated with genetic divergence between morphs. Paracentrotus gaimardi exhibits five colour morphs (pink, brown, green, grey and black), which often occur side by side on the same rock. We studied genetic divergence between these morphs in three populations on the coast of Brazil. A fragment of the region encoding the mitochondrial ATPase 8 and 6 mitochondrial genes, a fragment of the intron of a nuclear histone and the entire nuclear gene coding for the sperm protein bindin were analysed. Mitochondrial DNA was differentiated between the pink and all other morphs, but the histone intron was similar in all colour morphs. In bindin, nine codons were found to be under positive selection and significant differences of allelic frequencies were observed in almost all pairwise comparisons between colour morphs. Although the molecular differentiation in bindin is not large enough to suggest reproductive isolation, some degree of assortative mating within morphs seems to be occurring in this species.

Microsatellite markers reveal shallow genetic differentiation between cohorts of the common sea urchin Paracentrotus lividus (Lamarck) in northwest Mediterranean.

Temporal variability was studied in the common sea urchin Paracentrotus lividus through the analysis of the genetic composition of three yearly cohorts sampled over two consecutive springs in a locality in northwestern Mediterranean. Individuals were aged using growth ring patterns observed in tests and samples were genotyped for five microsatellite loci. No reduction of genetic diversity was observed relative to a sample of the adult population from the same location or within cohorts across years. F(ST )and amova results indicated that the differentiation between cohorts is rather shallow and not significant, as most variability is found within cohorts and within individuals. This mild differentiation translated into estimates of effective population size of 90-100 individuals. When the observed excess of homozygotes was taken into account, the estimate of the average number of breeders increased to c. 300 individuals. Given our restricted sampling area and the known small-scale heterogeneity in recruitment in this species, our results suggest that at stretches of a few kilometres of shoreline, large numbers of progenitors are likely to contribute to the larval pool at each reproduction event. Intercohort variation in our samples is six times smaller than spatial variation between adults of four localities in the western Mediterranean. Our results indicate that, notwithstanding the stochastic events that take place during the long planktonic phase and during the settlement and recruitment processes, reproductive success in this species is high enough to produce cohorts genetically diverse and with little differentiation between them. Further research is needed before the link between genetic structure and underlying physical and biological processes can be well established.

Phylogeny of the families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from mitochondrial and nuclear DNA sequences.

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.

Isolation of nine nuclear microsatellites in the common Mediterranean sea urchin, Paracentrotus lividus (Lamarck).

Nine polymorphic microsatellite loci were isolated and characterized for the edible common sea urchin Paracentrotus lividus. Loci were obtained from two genomic libraries enriched with different di-, tri- and tetranucleotides. Most microsatellites obtained were imperfect dinucleotides. Allelic variation was screened for a total of 56 individuals from two populations from North-Western Mediterranean. Microsatellites showed a number of alleles ranging from 13 to 35. Linkage disequilibrium was not detected between any pair of loci, and all loci showed a significant departure from Hardy-Weinberg equilibrium which is not likely to be the result of null alleles, suggesting that demographic features may be acting upon this commercially interesting species.

Enzymatic hydrolysis of native cellulose nanofibrils and other cellulose model films: effect of surface structure.

Model films of native cellulose nanofibrils, which contain both crystalline cellulose I and amorphous domains, were used to investigate the dynamics and activities of cellulase enzymes. The enzyme binding and degradation of nanofibril films were compared with those for other films of cellulose, namely, Langmuir-Schaefer and spin-coated regenerated cellulose, as well as cellulose nanocrystal cast films. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency and energy dissipation during incubation at varying enzyme concentrations and experimental temperatures. Structural and morphological changes of the cellulose films upon incubation with enzymes were evaluated by using atomic force microscopy. The QCM-D results revealed that the rate of enzymatic degradation of the nanofibril films was much faster compared to the other types of cellulosic films. Higher enzyme loads did not dramatically increase the already fast degradation rate. Real-time measurements of the coupled contributions of enzyme binding and hydrolytic reactions were fitted to an empirical model that closely described the cellulase activities. The hydrolytic potential of the cellulase mixture was found to be considerably affected by the nature of the substrates, especially their crystallinity and morphology. The implications of these observations are discussed in this report.

Temporal variation in the production of four secondary metabolites in a colonial ascidian.

The ecological and applied importance of ascidian secondary metabolites is well reported, but little is known about temporal variation in the production of these compounds. Here, we examined seasonal variation in the production of the four main pyridoacridine alkaloids (shermilamine B, kuanoniamine D, and their deacetylated forms) of the purple morph of the ascidian Cystodytes sp. Five replicate samples were taken monthly from July 2004 to February 2006, and analyzed by high-performance liquid chromatography. Our results showed a high degree of intercolony variability in secondary metabolite concentration and a lack of statistically significant seasonal variation in the production of the four compounds. Minimum values, however, were consistently recorded over the studied years in late summer, after the reproductive period. These minima may be attributable to sexual exhaustion and seasonally varying biotic interactions or abiotic parameters.

Phylogeography and speciation of colour morphs in the colonial ascidian Pseudodistoma crucigaster.

Variation in pigmentation is common in marine invertebrates, although few studies have shown the existence of genetic differentiation of chromatic forms in these organisms. We studied the genetic structure of a colonial ascidian with populations of different colour morphs in the northwestern Mediterranean. A fragment of the c oxidase subunit 1 (COI) mitochondrial gene was sequenced in seven populations of Pseudodistoma crucigaster belonging to three different colour morphs (orange, yellow and grey). Maximum likelihood analyses showed two well-supported clades separating the orange morph from the yellow-grey morphotypes. Genetic divergence between these clades was 2.12%, and gamma(ST) values between populations of the two clades were high (average 0.936), pointing to genetic isolation. Nested clade and coalescence analyses suggest that a past fragmentation event may explain the phylogeographical origin of these two clades. Non-neutral mtDNA evolution is observed in our data when comparing the two clades, showing a significant excess of nonsynonymous polymorphism within the yellow-grey morphotype using the McDonald-Kreitman test, which is interpreted as further support of reproductive isolation. We conclude that the two clades might represent separate species. We compare the population genetic differentiation found with that estimated for other colonial and solitary ascidian species, and relate it to larval dispersal capabilities and other life-history traits.

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.

Strong population structure in the marine sponge Crambe crambe (Poecilosclerida) as revealed by microsatellite markers.

Different categories of molecular markers have been used so far to study the population structure of sponges. However, these markers often did not have the resolution power to address precisely questions on structuring processes, especially at the intrapopulational level. In this study we show that microsatellites fulfil these expectations, allowing a fine description of population structure at different geographical scales in the marine sponge Crambe crambe. Specimens were collected in 11 locations, representing most of the Atlanto-Mediterranean range of the species, and were analysed at six loci. As expected for a sessile invertebrate with lecitotrophic larvae, high levels of between-population structure were found (FST = 0.18) and a significant isolation-by-distance pattern was observed. A strong genetic structure was also found within sampled sites (FIS = 0.21) that may be explained by several factors including inbreeding, selfing and the Wahlund effect. In spite of a sampling design planned to avoid the sampling of clones, genotypically identical individuals for the six loci were found in some locations. The significance of these potential clones is discussed and their effect on the observed pattern of population structure assessed. Patterns of allelic distribution within populations suggest the possibility of a recent colonization of the Atlantic range from the Mediterranean Sea.

Phylogeographical history of the sponge Crambe crambe (Porifera, Poecilosclerida): range expansion and recent invasion of the Macaronesian islands from the Mediterranean Sea.

We studied sequence variation in the nuclear ribosomal internal transcribed spacers (ITS-1 and ITS-2) in 111 individuals from 11 populations/localities of the sponge Crambe crambe across the core species range in the western Mediterranean Sea and Atlantic Ocean. We report the first confirmed instance of intragenomic variation in sponges. Phylogeographical, nested clade and population genetic analyses were used to elucidate the species' evolutionary history. The study revealed highly structured populations affected by restricted gene flow and isolation-by-distance. A contiguous range expansion in the whole distribution area of the sponge was inferred. Phylogenetic analyses indicate a recent origin of most sequence types that could be explained by a recent origin of the species or a by recent bottleneck event in the studied area. A recent expansion of the distribution range to the Macaronesian region from the Mediterranean Sea was also detected, suggesting that C. crambe was recently introduced from the Mediterranean Sea to the Atlantic Ocean via human-mediated transport, and that the pattern observed is not the result of a natural biogeographical relationship between these zones.

Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses.

The effect of low levels of pollution on the growth, reproduction output, morphology and survival of adult sponges and settlers of the sponge Crambe crambe were examined. We transplanted sponges from a control area to a contaminated site and measured the main environmental variables (chemical and physical) of both sites during the study period. Except some punctual differences in particulate organic matter, silicates, nitrates, and water motion, most environmental variables in the water were similar at both sites during the study months. Mainly copper, lead and OM concentrations in the sediment, and water motion were significantly higher at the polluted site and may be implicated in the biological effects observed: decrease in the percentage of specimens with embryos, increase in shape irregularity and decrease in growth rate. Individuals naturally occurring at the polluted site and those transplanted there for four months accumulated ten times more copper than either untouched or transplant controls. Although lead concentration in sediment did not differ between sites, native specimens from the contaminated site accumulated this metal more than untouched controls. Vanadium concentration also tended to increase in the sponges living at or transplanted to the contaminated site but this difference was not significant. C. crambe is a reliable indicator of metal contamination since it accumulates copper, lead and vanadium in high amounts. At the contaminated site, sponge growth, fecundity and survival were inhibited, whereas sponge irregularity ending in sponge fission was promoted. All these effects may compromise the structure and dynamics of the sponge populations in sheltered, metal-contaminated habitats.

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.

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.