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Hermaphrodite systems offer unique opportunities to study sexual differentiation, due to their high degree of sexual plasticity and to the fact that, unlike gonochoristic systems, the process is not confined to an early developmental stage. In protandric shrimp species, such as Hippolyte inermis and Pandalus platyceros, male differentiation is followed by transformation to femaleness during adulthood. The mechanisms controlling sexual differentiation have not been fully elucidated in crustaceans, but a key role has been attributed to the insulin-like hormone (IAG) produced by the androgenic gland (AG), a crustacean masculine endocrine organ. To uncover further transcriptomic toolkit elements affecting the sexual differentiation of H. inermis, we constructed eye and whole body RNA libraries of four representative stages during its protandric life cycle (immature, male, young female and mature female). The body libraries contained transcripts related to the reproductive system, among others, while the eye libraries contained transcripts related to the X-organ-sinus gland, a central endocrine complex that regulates crustacean reproduction. Binary pattern analysis, performed to mine for genes expressed differentially between the different life stages, yielded 19,605 and 6,175 transcripts with a specific expression pattern in the eye and body, respectively. Prominent sexually biased transcriptomic patterns were recorded for the IAG and vitellogenin genes, representing, respectively, a key factor within the masculine IAG-switch, and a precursor of the yolk protein, typical of feminine reproductive states. These patterns enabled the discovery of novel putative protein-coding transcripts exhibiting sexually biased expression in the H. inermis body and eye transcriptomes of males and females. Homologs to the above novel genes have been found in other decapod crustaceans, and a comparative study, using previously constructed transcriptomic libraries of another protandric shrimp, P. platyceros, showed similar sexually biased results, supporting the notion that such genes, mined from the H. inermis transcriptome, may be universal factors related to reproduction and sexual differentiation and their control in other crustaceans. This study thus demonstrates the potential of transcriptomic studies in protandric species to uncover unexplored layers of the complex crustacean sex-differentiation puzzle.

Physiological effects of algal metabolites is a key step for the isolation of interesting bioactive compounds. Invertebrate grazers may be fed on live diatoms or dried, pelletized, and added to compound feeds. Any method may reveal some shortcomings, due to the leaking of wound-activated compounds in the water prior to ingestion. For this reason, encapsulation may represent an important step of bioassay-guided fractionation, because it may assure timely preservation of the active compounds. Here we test the effects of the inclusion in alginate (biocompatible and non-toxic delivery system) matrices to produce beads containing two benthic diatoms for sea urchin Paracentrotus lividus feeding. In particular, we compared the effects of a diatom whose influence on P. lividus was known (Nanofrustulum shiloi) and those of a diatom suspected to be harmful to marine invertebrates, because it is often present in blooms (Striatella unipunctata). Dried N. shiloi and S. unipunctata were offered for one month after encapsulation in alginate hydrogel beads and the larvae produced by sea urchins were checked for viability and malformations. The results indicated that N. shiloi, already known for its toxigenic effects on sea urchin larvae, fully conserved its activity after inclusion in alginate beads. On the whole, benthic diatoms affected the embryogenesis of P. lividus, altering the expression of several genes involved in stress response, development, skeletogenesis and detoxification processes. Interactomic analysis suggested that both diatoms activated a similar stress response pathway, through the up-regulation of hsp60, hsp70, NF-κB, 14-3-3 ε and MDR1 genes. This research also demonstrates that the inclusion in alginate beads may represent a feasible technique to isolate diatom-derived bioactive compounds.

Adult sea urchins and their embryos are ideal targets to investigate the medium- and long-term effects of various toxic agents, such as organic and inorganic pollutants, to forecast and mitigate their environmental effects. Similarly, small colonial tunicates such as Botryllid ascidians may reveal acute toxicity processes and permit quick responses for the management of contaminants impacting coastal waters, to preserve the functional integrity of marine ecosystems. This investigation compares the functional responses of two model invertebrates, the sea urchin Paracentrotus lividus and the sea squirt Botryllus schlosseri, to chronic and acute exposures to organic and inorganic toxic agents. Such heavy metals as copper produce both acute and chronic effects on marine biota, while polycyclic aromatic hydrocarbons (PAHs) mainly produce chronic effects at the concentrations ordinarily measured in marine coastal waters. Both models were tested over a range of concentrations of copper and PAHs. Copper triggered a clear effect in both species, producing a delay in the embryo development of P. lividus and a rapid death of sea squirts. B. schlosseri was less sensitive to PAHs than P. lividus. The results on both species may synergistically contribute to assess the toxicity of organic and inorganic compounds at various concentrations and different physiologic levels.

Violet sea squirts are noteworthy model organisms, because they provide insights into various physiologic processes, including cell senescence, ageing, apoptosis and allorecognition. Consequently, their culture is critical to permit experimental studies. Most papers refer to short periods of rearing using various feeds, both living and conserved, missing a formal justification for their use or indications of their actual nutritional value. Here, we use two behavioural responses&mdash, the percentage of open siphons and the frequency of zooid contractions&mdash, as compared to the abundance of suspended microparticles during feeding tests, to identify feeds able to promote filter-feeding. The results will enable to formulate compound diets that maximise positive physiological responses. Our tests demonstrated that plant items, such as dry microalgae and cyanobacteria (Arthrospira platensis, commercially known as Spirulina), along with living planktonic Haptophyta (Isochrysis galbana), trigger clear positive reactions, represented by a higher frequency of zooid contractions and larger proportions of open siphons. These responses correspond to decreases in the concentrations of suspended microparticles during the experiment and indicate higher filter-feeding activity. In contrast, feeds commonly administered to colonies, such as milk powder, dried eggs and artificial plankton, triggered negative behavioural responses, and their intake was lower during the feeding trials.