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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.

The Mediterranean sea urchin Paracentrotus lividus Lamark, 1816 is a resource as food and model organism for scientific research. Increasing market demand, commercial fisheries and destructing harvesting methods produced a dramatic reduction of natural stocks along the Mediterranean coasts. The techniques for in vitro fertilization and growth of larvae and adults are well known but various bottlenecks still hinder the industrial maturity of its aquaculture. In particular, the transition from planktonic larvae to benthic juveniles, as well as their correct development and survival, still represent critical phases, since larval and post-larval experiences are the primary determinant of juvenile fitness. Thus, techniques improving survival and settlement rates in the first two months after fertilization could considerably improve the effectiveness of echinoculture practices. This study was conducted to determine, for the first time, the effect of strictly benthic diatoms on P. lividus post-larval performances, as compared to other micro- and macro-algae. In addition, three larval dietary treatments were applied, to confirm previous findings on the efficacy of algae and formulated diets. In particular, survival and ability to produce competent larvae for settlement were tested in groups subjected to three algal diets (Isochrisis galbana, Rhodomonas baltica and Dunaliella tertiolecta), all integrated with a formulated food. Furthermore, six post-larval feeding regimes, i.e., the benthic diatoms Cocconeis scutellum parva, Diploneis sp., Navicula incerta, Cylindrotheca closterium and Nanofrustulum shiloi, compared with Ulva rigida along with a negative control (absence of food), were tested on larvae deriving from the most effective treatment. Our results confirmed the efficacy of Dunaliella tertiolecta integrated with a formulated food, consistently producing about 60% survival. Furthermore, the most adhesive benthic diatom, Cocconeis scutellum parva, sustained settlement (about 63%) and good survival in the first two months of life of sea urchins.

Dismissed industrial plants with chronic environmental contamination globally affect all levels of biological organization in concert with other natural and anthropogenic perturbations. Assessing the impact of such perturbations and finding effective ways to mitigate them have clear ecological and societal implications. Through indoor manipulative experiments, we assessed here the effects of the temporal regime of reworking of contaminated sediment from the Bagnoli-Coroglio brownfield (Tyrrhenian Sea, Italy) on the fertilization process in Paracentrotus lividus. Adult sea urchins were kept for one month in tanks containing contaminated sediment that was re-suspended according to two temporal patterns of water turbulence differing in the time intervals between consecutive events of agitation (mimicking the storms naturally occurring in the study area) in seawater with natural vs. supersaturated oxygenation levels. At the end of the treatment, gametes were collected and used to test the hypothesis that the regime of contaminated sediment reworking negatively, but reversibly, affects morphological and physiological traits of the fertilized eggs. We found that aggregated events of sediment re-suspension had profound negative effects on gamete interactions and Ca2+ signaling at fertilization. The same experimental condition also inflicted marked ultrastructural changes in eggs. Importantly, however, such detrimental effects were inhibited by increased oxygenation. By contrast, the regime of sediment re-working with a longer interval between consecutive turbulent events had only marginal effects. Thus, the current and predicted changes of climate-related disturbance appear to modulate the biological effects of chronic contamination in post-industrial areas, suggesting that environmental rehabilitation via restoration of habitat-forming primary producers such as seagrasses or algal canopies could alleviate the pollutants' effects on resident biota.

In marinas and harbours, the accumulation of pollutants in sediments, combined with poor exchange of water with the open sea, poses a major environmental threat. The presence of photosynthetic organisms and the related oxygen production, however, may alleviate the negative effects of environmental contamination on heterotrophic organisms, enhancing their physiological defences. Furthermore, possible transgenerational buffer effects may increase the ability of natural populations to face environmental stress. Here we tested the occurrence of transgenerational effects on larvae of the sea urchin Paracentrotus lividus, whose parents were exposed, during the gametogenesis, to contaminated sediments subject to two temporal patterns of water re-suspension events and normal- (90%) vs. super-saturated (200%) levels of O2. The study site was Bagnoli-Coroglio (Gulf of Naples, southern Tyrrhenian Sea), a historically polluted brownfield and Site of National Interest for which environmental restoration options are currently under exploration. Larvae from different adult populations were significantly, although not linearly, affected by the interaction of all factors to which parents were exposed, at both 24h and 48h post fertilization. Specifically, the exposure of larvae to elutriates from contaminated sediments determined a developmental delay, a reduction in size and an increased percentage of abnormalities in all larval populations independently of their parental exposure. On the contrary, larvae from parents exposed to contaminated sediments, when reared in clean filtered sea water, succeeded in developing until the echinopluteus stage after 48h, with size and abundance comparable to those of larvae from control parents. Pre-exposure of parents to contaminated sediments did not successfully buffer the negative effects of elutriates on their offspring, and no positive effects of ‘super-saturated’ levels of O2 in response to contaminants were observed, suggesting that the Bagnoli-Coroglio area is currently not suitable for the re-stocking or re-introduction of this species.

Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world’s ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

The trophic ecology of the sea urchin Paracentrotus lividus, a key species in several shallow benthic communities, has been intensively studied, but the role of various foods in the processes of growth and gonadal maturation is still scarcely understood. This research assessed the effects of two fundamental food items for wild specimens of the sea urchin Paracentrotus lividus, the tissues of the seagrass Posidonia oceanica and of the green alga Ulva rigida, compared to the effect of a commercial compound feed on the somatic growth, gonad development, fertilization success and post-embryonic development. Consumption rates along with the C/N ratios were measured in the feeds and in the faecal pellets. We demonstrated that feeding for three months on U. rigida and P. oceanica did not affect growth and gonadal index of adults, fertilization processes and first cleavage and development, as well as field-collected animals. In contrast, a diet based on formulated pellets triggered a significant increase of gonadal index, but lack of gamete production, due to a follicular hyperthrophy. Our work will be useful for the definition of optimal diets for the production of mature broodstocks of an ecologically important marine model organism.