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The protandric shrimp Hippolyte inermis is the only known marine invertebrate whose sex determination is strongly influenced by the composition of its food. In H. inermis, a sex reversal is triggered by the ingestion of diatoms of the genus Cocconeis associated with leaves of the seagrass Posidonia oceanica. These diatoms contain compounds that promote programmed cell death (PCD) in H. inermis and also in human cancer cells. Transcriptomic analyses suggested that ferroptosis is the primary trigger of the shrimp’s sex reversal, leading to the rapid destruction of the androgen gland (AG) followed by a chain of apoptotic events transforming the testes into ovaries. Here, we propose a molecular approach to detect the effects of compounds stimulating the PCD. An RNA extraction method, suitable for young shrimp post-larvae (five days after metamorphosis; PL5 stage), was established. In addition, six genes involved in apoptosis, four involved in ferroptosis, and seven involved in the AG switch were mined from the transcriptome, and their expression levels were followed using real-time qPCR in PL5 fed on Cocconeis spp., compared to PL5 fed on a basic control feed. Our molecular approach, which detected early signals of sex reversal, represents a powerful instrument for investigating physiological progression and patterns of PCD in marine invertebrates. It exemplifies the physiological changes that may start a few days after the settlement of post-larvae and determine the life destiny of an individual.

The successions of benthic communities over time are strongly influenced by the first colonizers, because surface associations are facilitated by modifications to the adhesive properties promoted by primary colonizers, such as bacteria, protozoans, diatoms, algal propagules, spores, and invertebrate larvae. Bacteria are often the first colonizers on marine submerged surfaces, both organic (e.g., algae, seagrasses and invertebrates) and inorganic. However, they are promptly followed by diatoms and other microorganisms. Consequently, diatoms may represent key elements in the determination of the colonization patterns, although the development of epiphytic communities is a dynamic process influenced by several factors, including nutrient availability, the ability to synthesize and secrete extracellular material, the competition among species and the influence of grazers on individual colonizers. The process may be drastically impacted by global warming and ocean acidification due to the increasing atmospheric levels of CO2. The impact of such global stressors on benthic ecosystems, especially on the primary microphytobenthic assemblages, is still poorly investigated, and may have deleterious consequences for the benthic successions. In this review, we analyze the adhesion patterns of marine microorganisms according to their surface features and the effects of global changes on critical pioneer colonizers, such as the benthic diatoms. The results are remarkable, as they highlight emergent concerns in ecosystem conservation and the prediction of benthic communities.

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.

Cyanobacteria are a diversified phylum of nitrogen-fixing, photo-oxygenic bacteria able to colonize a wide array of environments. In addition to their fundamental role as diazotrophs, they produce a plethora of bioactive molecules, often as secondary metabolites, exhibiting various biological and ecological functions to be further investigated. Among all the identified species, cyanobacteria are capable to embrace symbiotic relationships in marine environments with organisms such as protozoans, macroalgae, seagrasses, and sponges, up to ascidians and other invertebrates. These symbioses have been demonstrated to dramatically change the cyanobacteria physiology, inducing the production of usually unexpressed bioactive molecules. Indeed, metabolic changes in cyanobacteria engaged in a symbiotic relationship are triggered by an exchange of infochemicals and activate silenced pathways. Drug discovery studies demonstrated that those molecules have interesting biotechnological perspectives. In this review, we explore the cyanobacterial symbioses in marine environments, considering them not only as diazotrophs but taking into consideration exchanges of infochemicals as well and emphasizing both the chemical ecology of relationship and the candidate biotechnological value for pharmaceutical and nutraceutical applications.

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.

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—the percentage of open siphons and the frequency of zooid contractions—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.

Malicious cyber activities are on the rise. States and other relevant actors need to constantly adapt to the evolving cyber threat landscape, including by setting up effective deterrence mechanisms. This is what the European Union (EU) has done through the adoption of Common Foreign and Security Policy (CFSP) Decision 2019/797, which allows it to impose targeted sanctions to deter and respond to cyberattacks that constitute an external threat to the EU or its member states. However, in contrast to other horizontal regimes of restrictive measures in force within the EU, foreign governments are not included as potential targets of cyber sanctions. Moreover, the recital of the Decision specifies that the adoption of restrictive measures does not involve attribution of international responsibility for cyber-attacks to a third State. This article aims at identifying the rationale behind the inclusion of these distinctive features. It starts by considering the legal uncertainty that surrounds attribution of international responsibility for cyber operations. Next, it explains why the EU is not well placed to invoke third-State responsibility, and the reasons behind its reluctance to do so. It will then illustrate the risks inherent in the lack of a clear legal framework to attribute the responsibility of cyber-attacks to third countries. This may have serious consequences in terms of legal certainty when a cyber-attack amounts to a breach of the prohibition on the use of force in international relations. Then, we explore recent developments in EU legislation in the area of cyber security and the possibility to strenghten the powers of the European Union Agency for Cybersecurity (ENISA). We draw two conclusions: first, the Union might develop the capacity to attribute cyber attacks to specific actors and there is an interest to do so. However, Member States are probably still reticent to take this step. Two, despite the advantages of establishing a reliable attribution mechanisms, it is submitted that the majority of States prefers to take advantage of a regulative gap that allows them to react to cyber incidents as they see fit.

The role of ecosystem services is interlinked with the growing awareness of research and development funding programmes sensitive to green infrastructure and urban greening strategies. They become one of the structural components for the ecological transition of European urban agendas, in line with the Sustainable Development Goals of Agenda 2030. Ecosystem services are a new parameter for the assessment of urban green infrastructure and re-naturalisation processes, which also aim to counteract the effects of climate change (i.e., flooding, heat islands), or to promote the conservation, resilience and biodiversity of natural habitats. Specifically, urban agriculture and community gardens play an important role, helping to secure various socio-ecological benefits on a local basis and to promote access to healthy, Zero Km sources. In order to encourage good practices within the contemporary agro-urban scenario, the contribution outlines novel ideas on circularity and recycling of waste from across the food chain from the European project Creative Food Cycles.