Recent ecological studies have shown a strong relation between temperature, echinoids and their grazing effects on macro-algal communities. In this study, we speculate that climate warming may result in an increasingly favourable environment for the reproduction and development of the sea urchin Arbacia lixula. The relationship between increased A. lixula density and the extent of barren grounds in the Mediterranean Sea is also discussed.Keywords: ocean warming; overgrazing; sea urchin fertilisation and development Factors influencing the functioning of ecosystems, such as climate change, inputs of nutrients and/or toxic chemicals, groundwater reduction, habitat fragmentation, harvest of biomasses or loss of biodiversity, are never constant. The state of some ecosystems may respond gradually to such changes, whereas others might remain unresponsive over time (or ranges of conditions), and then respond abruptly when conditions reach a critical level. This implies that, for certain environmental conditions, the ecosystem might have two or more alternative stable states, separated by an unstable equilibrium that marks the border between the 'basins of attraction' of the states (the theory of alternative stable states; ASS) [1,2]. This theory also hypothesised that natural systems are often in persistent, resilient, alternative states: alternative combinations of ecosystem states and environmental conditions that may persist at a particular spatial extent and temporal scale.These states have been shown to be maintained by intrinsic mechanisms involving biotic and abiotic interactions (grazing or predation intensity, storm frequency, pollution, local extinction, invasion, nutrient loading, etc.) which inhibit reversal to the previous community state [3]. In combination with other factors, the loss of a keystone species [4], which results from changes in top-down interactions between (1) predators and herbivores and (2) herbivores and macroalgae, may cause changes in grazing intensity and consequent switches between two or more alternative states in marine ecosystems.For example, in temperate seas, several species of echinoids play a crucial role, and are even more important than other herbivores (i.e. fishes) as controllers of benthic communities [5][6][7][8][9][10][11][12][13].

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 μM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 μM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 μM CdCl2). We found also that, in response to a lethal dose of cadmium (1 μM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

a b s t r a c tThe increasing abundances of the thermophilous black sea urchin Arbacia lixula in the Mediterranean Sea are attributed to the Western Mediterranean warming. However, few data are available on the potential impact of this warming on A. lixula in combination with other global stressors such as ocean acidification. The aim of this study is to investigate the interactive effects of increased temperature and of decreased pH on fertilization and early development of A. lixula. This was tested using a fully crossed design with four temperatures (20, 24, 26 and 27 C) and two pH levels (pH NBS 8.2 and 7.9). Temperature and pH had no significant effect on fertilization and larval survival (2d) for temperature <27 C. At 27 C, the fertilization success was very low (<1%) and all larvae died within 2d. Both temperature and pH had effects on the developmental dynamics. Temperature appeared to modulate the impact of decreasing pH on the % of larvae reaching the pluteus stage leading to a positive effect (faster growth compared to pH 8.2) of low pH at 20 C, a neutral effect at 24 C and a negative effect (slower growth) at 26 C. These results highlight the importance of considering a range of temperatures covering today and the future environmental variability in any experiment aiming at studying the impact of ocean acidification.

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.

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Functional tests, performed by microinjection into Xenopus laevis oocytes, show that a DNA fragment containing the modulator of the early histone H2A gene of Paracentrotus lividus enhances transcription of a reporter gene when located, in the physiological orientation, upstream of the tk basal promoter. Gel retardation and DNase I footprinting assays further reveal that the H2A modulator contains at least two binding sites [upstream sequence elements 1 and 2 (USE 1 and USE 2)] for nuclear factors extracted from sea urchin embryos, which actively transcribe the early histone gene set. Interestingly, USE 1 is highly homologous to a cis-acting element previously identified in the H2A modulator of Psammechinus milians [Grosschedl, R., Machler, M., Rohrer, U. & Birnstiel, M. L. (1983) Nucleic Acids Res. 11, 8123-8136]. Finally, a cloned oligonucleotide containing the USE 1 sequence competes efficiently in Xenopus oocytes with the H2A modulator to prevent enhancement of transcription of the reporter gene. From these results, we conclude that USE 1 and perhaps USE 2 in the H2A modulator are upstream transcriptional elements that are recognized by trans-acting factors common to Xenopus and sea urchin.The sea urchin genome contains several histone gene families that encode the protein subtypes of sperm (S-type) and of cleavage, early, and late stage embryo (1). The synthesis of specific histone protein variants during development is the result of regulatory mechanisms that operate at both the transcriptional and posttranscriptional level (2). The early histone genes in all sea urchin species are tandemly repeated 300-600 times and are organized in quintets (3,4). Transcription of this gene set occurs upon meiotic maturation and soon after fertilization (5-7). Newly synthesized early histone mRNAs accumulate at the 32-to 128-cell stage. Their transcription is shut off in mesenchyme blastula embryos (8-10). The transition from a transcriptionally active to an inactive state of the early histone genes is accompanied by structural alterations of their chromatin arrangement (11-13).Molecular genetic analysis of many polymerase II promoters has revealed that optimal and accurate initiation of transcription requires a cooperative interaction of transacting factors with multiple cis-acting transcriptional elements (14)(15)(16)(17).Recent studies on the factors involved in the transcriptional control of the sea urchin histone H1 and H2B genes have identified basal and ontogenic transcriptional elements involved in the transition from early to late gene expression during development (18,19). Furthermore, they have shown that an embryo-specific repressor element, the CCAAT displacement factor, prevents the interaction of a positive trans-acting factor with the CCAAT box ofthe sperm-specific H2B-1 gene (20) and in so doing may block transcription in the embryo.We have focused our attention on the early H2A gene ofthe sea urchin Paracentrotus lividus for several reasons. The 5' flanking region of this gene contains, upstream...