2D Collagen Membranes from Marine Demosponge Chondrosia reniformis (Nardo, 1847) for Skin-Regenerative Medicine Applications: An In Vitro Evaluation.

Research in tissue engineering and regenerative medicine has an ever-increasing need for innovative biomaterials suitable for the production of wound-dressing devices and artificial skin-like substitutes. Marine collagen is one of the most promising biomaterials for the production of such devices. In this study, for the first time, 2D collagen membranes (2D-CMs) created from the extracellular matrix extract of the marine demosponge Chondrosia reniformis have been evaluated in vitro as possible tools for wound healing. Fibrillar collagen was extracted from a pool of fresh animals and used for the creation of 2D-CMs, in which permeability to water, proteins, and bacteria, and cellular response in the L929 fibroblast cell line were evaluated. The biodegradability of the 2D-CMs was also assessed by following their degradation in PBS and collagenase solutions for up to 21 days. Results showed that C. reniformis-derived membranes avoided liquid and protein loss in the regeneration region and also functioned as a strong barrier against bacteria infiltration into a wound. Gene expression analyses on fibroblasts stated that their interaction with 2D-CMs is able to improve fibronectin production without interfering with the regular extracellular matrix remodeling processes. These findings, combined with the high extraction yield of fibrillar collagen obtained from C. reniformis with a solvent-free approach, underline how important further studies on the aquaculture of this sponge could be for the sustainable production and biotechnological exploitation of this potentially promising and peculiar biopolymer of marine origin.

Marine heatwaves drive recurrent mass mortalities in the Mediterranean Sea.

Climate change is causing an increase in the frequency and intensity of marine heatwaves (MHWs) and mass mortality events (MMEs) of marine organisms are one of their main ecological impacts. Here, we show that during the 2015-2019 period, the Mediterranean Sea has experienced exceptional thermal conditions resulting in the onset of five consecutive years of widespread MMEs across the basin. These MMEs affected thousands of kilometers of coastline from the surface to 45 m, across a range of marine habitats and taxa (50 taxa across 8 phyla). Significant relationships were found between the incidence of MMEs and the heat exposure associated with MHWs observed both at the surface and across depths. Our findings reveal that the Mediterranean Sea is experiencing an acceleration of the ecological impacts of MHWs which poses an unprecedented threat to its ecosystems' health and functioning. Overall, we show that increasing the resolution of empirical observation is critical to enhancing our ability to more effectively understand and manage the consequences of climate change.

Sponge microbiome stability during environmental acquisition of highly specific photosymbionts.

In this study, we used in situ transplantations to provide the first evidence of horizontal acquisition of cyanobacterial symbionts by a marine sponge. The acquisition of the symbionts by the host sponge Petrosia ficiformis, which was observed in distinct visible patches, appeared several months after transplantation and at different times on different sponge specimens. We further used 16S rRNA gene amplicon sequencing of genomic DNA (gDNA) and complementary DNA (cDNA) and metatranscriptomics to investigate how the acquisition of the symbiotic cyanobacterium Candidatus Synechococcus feldmannii perturbed the diverse microbiota associated with the host P. ficiformis. To our surprise, the microbiota remained relatively stable during cyanobacterial symbiont acquisition at both structural (gDNA content) and activity (cDNA expression) levels. At the transcriptomic level, photosynthesis was the primary function gained following the acquisition of cyanobacteria. Genes involved in carotene production and oxidative stress tolerance were among those highly expressed by Ca. S. feldmannii, suggesting that this symbiont may protect itself and its host from damaging light radiation.

Mediterranean Lithophyllum stictiforme (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats.

Lithophyllum species in the Mediterranean Sea function as algal bioconstructors, contributing to the formation of biogenic habitats such as coralligenous concretions. In such habitats, thalli of Lithophyllum, consisting of crusts or lamellae with entire or lobed margins, have been variously referred to as either one species, L. stictiforme, or two species, L. stictiforme and L. cabiochiae, in the recent literature. We investigated species diversity and phylogenetic relationships in these algae by sequencing three markers (psbA and rbcL genes, cox2,3 spacer), in conjunction with methods for algorithmic delimitation of species (ABGD and GMYC). Mediterranean subtidal Lithophyllum belong to a well-supported lineage, hereby called the L. stictiforme complex, which also includes two species described from the Atlantic, L. lobatum and L. searlesii. Our results indicate that the L. stictiforme complex consists of at least 13 species. Among the Mediterranean species, some are widely distributed and span most of the western and central Mediterranean, whereas others appear to be restricted to specific localities. These patterns are interpreted as possibly resulting from allopatric speciation events that took place during the Messinian Salinity Crisis and subsequent glacial periods. A partial rbcL sequence from the lectotype of L. stictiforme unambiguously indicates that this name applies to the most common subtidal Lithophyllum in the central Mediterranean. We agree with recent treatments that considered L. cabiochiae and L. stictiforme conspecific. The diversity of Lithophyllum in Mediterranean coralligenous habitats has been substantially underestimated, and future work on these and other Mediterranean corallines should use identifications based on DNA sequences.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

Silica-induced fibrosis: an ancient response from the early metazoans.

Exposure to crystalline silica particles causes silicosis, an occupational disease leading to an overproduction of collagen in the lung. The first step of this pathology is characterized by the release of inflammatory mediators. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine directly involved in silica-induced pulmonary fibrosis. The marine demosponge Chondrosia reniformis is able to incorporate silica grains and partially dissolve the crystalline forms apparently without toxic effects. In the present work, C. reniformis tissue explants were treated with fine quartz dust and the expression level of fibrogenic genes was assayed by qPCR, demonstrating an overexpression of a fibrillar and a non-fibrillar collagen and of prolyl-4-hydroxylase enzyme. The deposition of new collagen could also be documented in quartz-treated sponge explants. Furthermore, TNF pro-inflammatory cytokine overexpression and involvement in silica-induced sponge collagen biosynthesis was demonstrated in quartz-treated explants as compared with controls by means of specific TNF inhibitors affecting the fibrogenic gene response. As no documentable detrimental effect was observed in treated explants, we conclude that the C. reniformis unique quartz engulfment and erosion is physiological and beneficial to the animal, leading to new collagen synthesis and strengthening of the body stiffness. Thus, we put forward the hypothesis that an ancient physiological behaviour from the lowest of the Metazoa, persisting through evolution via the same molecular mediators such as TNF, may have become the cause of disease in the specialized tissues of higher animals such as mammals.

Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta).

Lithophyllum byssoides is a common coralline alga in the intertidal zone of Mediterranean coasts, where it produces biogenic concretions housing a high algal and invertebrate biodiversity. This species is an ecosystem engineer and is considered a target for conservation efforts, but designing effective conservation strategies currently is impossible due to lack of information about its population structure. The morphological and molecular variation of L. byssoides was investigated using morphoanatomy and DNA sequences (psbA and cox2,3) obtained from populations at 15 localities on the Italian and Croatian coasts. Lithophyllum byssoides exhibited a high number of haplotypes (31 psbA haplotypes and 24 cox2,3 haplotypes) in the central Mediterranean. The psbA and cox2,3 phylogenies were congruent and showed seven lineages. For most of these clades, the distribution was limited to one or a few localities, but one of them (clade 7) was widespread across the central Mediterranean, spanning the main biogeographic boundaries recognized in this area. The central Mediterranean populations formed a lineage separate from Atlantic samples; psbA pair-wise divergences suggested that recognition of Atlantic and Mediterranean L. byssoides as different species may be appropriate. The central Mediterranean haplotype patterns of L. byssoides were interpreted as resulting from past climatic events in the hydrogeological history of the Mediterranean Sea. The high haplotype diversity and the restricted spatial distribution of the seven lineages suggest that individual populations should be managed as independent units.

The pillars of the sea: strategies to achieve successful marine citizen science programs in the Mediterranean area.

Marine ecosystems are facing a dramatic loss of biodiversity worldwide, together with a widespread collapse of habitats and their functionality. In this context, Marine Citizen Science (MCS) can be a powerful tool to monitor these changes over time. The flowering of very well-structured international projects is strengthening the scientific credibility of MCS data, especially when data are collected after specifically designed training programs and shared in public user-friendly repositories. Here we present a new perspective on the use of MCS in the Mediterranean area, along with the main benefits for the stakeholders (i.e., diving centers, trainers, and policymakers) and the users (i.e., divers), resumed in three pillars: Pillar I - MCS as a tool for the site valorization; Pillar II - MCS as a new career opportunity for graduated students; Pillar III - MCS as a business opportunity for diving centers. In the frame of the Quintuple Helix Approach, for which there is a strong need of a socioecological transition of the society and economy, we show how MCS can be a win-win-win solution for all the actors involved, providing the vision for new and highly qualified job and business opportunities for the diving sector.

Mesophotic zone as buffer for biodiversity protection: A promising opportunity to enhance MPA effectiveness.

Coastal areas conservation strategies often left deeper habitats, such as mesophotic ones, unprotected and exposed to anthropogenic activities. In this context, an approach for including the mesophotic zone inside protection plans is proposed, considering 27 Italian Marine Protected Areas (MPAs) as a model. MPAs were classified considering their bathymetries, exposure to marine heat waves (MHWs), mass mortality events (MMEs) and, using a local ecological knowledge (LEK) approach, the estimated resilience of certain sessile species after MMEs. Only 8 MPAs contained considerable mesophotic areas, with stronger MHWs mainly occurring in shallower MPAs, and MMEs mostly affecting coralligenous assemblages. Even with only a 10% response rate, the LEK approach provided useful information on the resilience of certain species, allowing us to suggest that the presence of nearby mesophotic areas can help shallower habitats facing climate change, thus making the "deep refugia" hypothesis, usually related to tropical habitats, applicable also for the Mediterranean Sea.

Cell reactivity to different silica.

The interaction between mineral structures and living beings is increasingly attracting the interest of research. The formation of skeletons, geomicrobiology, the study of the origin of life, soil biology, benthos biology, human and mammalian diseases generated by the inhalation of dust and biomaterials are some examples of scientific areas where the topic has a relevance. In this chapter we focus on cell reactivity to siliceous rocks and to the various forms of silicon dioxide, in particular. The examples here reported carefully review how such minerals may strongly affect different living beings, from simple ones to humans. The biomineralogy concept is explained, focusing on the effects of rocks on cell growth and development. The toxic action of silicon dioxide in mammalian lungs is the oldest evidence of crystalline silica bioactivity. More recently, we could demonstrate that crystalline silica has a deep impact on cell biology throughout the whole animal kingdom. One of the most illustrative case studies is the marine sponge Chondrosia reniformis, which has the amazing ability to incorporate and etch crystalline silica releasing dissolved silicates in the medium. This specific and selective action is due to the chemical reaction of ascorbic acid with quartz surfaces. One consequence of this is an increased production of collagen. The discovery of this mechanism opened the door to a new understanding of silica toxicity for animal cells and mammalian cells in particular. The presence of silica in sea water and substrates also affects processes like the settlement of larvae and the growth of diatoms. The following sections review all such aspects.

Isoswinholide B and swinholide K, potently cytotoxic dimeric macrolides from Theonella swinhoei.

Chemical investigation of an Indonesian specimen of Theonella swinhoei afforded the new dimeric macrolides isoswinholide B (5) and swinholide K (6), along with the known swinholides A (1), B (2) and D (3) and isoswinholide A (4). Isoswinholide B showed an unprecedented 21/19' lactonization pattern, while swinholide K included an sp(2) methylene attached at C-4 and an additional oxymethine group at C-5, whose configuration has been determined through application of J-based configuration analysis. The isolated swinholides (1-6), with the exception of isoswinholide B, showed a cytotoxic activity on HepG2 (hepatocarcinoma cell line) in the nanomolar range.

Natural and semisynthetic analogues of manadoperoxide B reveal new structural requirements for trypanocidal activity.

Chemical analysis of the Indonesian sponge Plakortis cfr. lita afforded two new analogues of the potent trypanocidal agent manadoperoxide B (1), namely 12-isomanadoperoxide B (2) and manadoperoxidic acid B (3). These compounds were isolated along with a new short chain dicarboxylate monoester (4), bearing some interesting relationships with the polyketide endoperoxides found in this sponge. Some semi-synthetic analogues of manadoperoxide B (6-8) were prepared and evaluated for antitrypanosomal activity and cytotoxicity. These studies revealed crucial structure-activity relationships that should be taken into account in the design of optimized and simplified endoperoxyketal trypanocidal agents.

Sponges associated with octocorals in the Indo-Pacific, with the description of four new species.

Sponges are characterised by high levels of phenotypic plasticity, thus allowing the same species to live in different habitats by taking different shapes. Here we describe 28 sponge species associated with the octocorals Carijoa riisei, Paratelesto rosea and Alertigorgia hoeksemai in Indonesia, Hawai'i and Vietnam, including four species that are new to science (Chondropsis subtilis, Hymedesmia (Hymedesmia) spinata, Hymedesmia (Stylopus) perlucida, Mycale (Aegogropila) furcata). Moreover, a large proportion of the described sponge species (21.4%) represent new records for the studied areas (Indonesia and Hawai'i). In total, we have studied 47 colonies of C. riisei associated with 24 sponge species, 5 colonies of P. rosea associated with 4 species and one colony of A. hoeksemai associated with one sponge species. Collectively, these examples of associations highlight the importance of epibiosis as a biodiversity enhancing process.

New tridecapeptides of the theonellapeptolide family from the Indonesian sponge Theonella swinhoei.

Chemical analysis of the organic extract of Theonella swinhoei yielded two new tridecadepsipeptides of the theonellapeptolide family, namely sulfinyltheonellapeptolide, characterized by a methylsulfinylacetyl group at the N-terminus, and theonellapeptolide If, the first member of this class of compounds to show four valine residues. The structures of the compounds, isolated along with the known theonellapeptolide Id, were determined by extensive 2D NMR and MS/MS analyses followed by application of Marfey's method. The isolated peptides exhibited moderate antiproliferative activity against HepG2 cells, a hepatic carcinoma cell line.

Marine beach litter monitoring strategies along Mediterranean coasts. A methodological review.

Marine beach litter (MBL) represents a serious issue for marine life, coastal ecosystems, human health and several economical activities. The Mediterranean Sea is a semi enclosed basin particularly vulnerable to this problem. Its coasts are threatened by critical anthropogenic pressures that sum up with intensive fishing and shipping, and the slow turnover of its waters. In the last decades, several scientific and participative initiatives have been conducted to study, monitor and clean-up shorelines. These studies were generally characterized by differences in timing and frequency of the surveys, as well as in litter sampling, classification and analysis. This paper presents a systematic review of current literature concerning MBL monitoring strategies along the Mediterranean coasts. Scopus indexed studies are analysed to identify discrepancies and similarities among the applied protocols, understand where current gaps lie, and point out what would be needed to develop a basin-scale efficient monitoring for the Mediterranean Sea.

Sponge organic matter recycling: Reduced detritus production under extreme environmental conditions.

Sponges are a key component of coral reef ecosystems and play an important role in carbon and nutrient cycles. Many sponges are known to consume dissolved organic carbon and transform this into detritus, which moves through detrital food chains and eventually to higher trophic levels via what is known as the sponge loop. Despite the importance of this loop, little is known about how these cycles will be impacted by future environmental conditions. During two years (2018 and 2020), we measured the organic carbon, nutrient recycling, and photosynthetic activity of the massive HMA, photosymbiotic sponge Rhabdastrella globostellata at the natural laboratory of Bouraké in New Caledonia, where the physical and chemical composition of seawater regularly change according to the tide. We found that while sponges experienced acidification and low dissolved oxygen at low tide in both sampling years, a change in organic carbon recycling whereby sponges stopped producing detritus (i.e., the sponge loop) was only found when sponges also experienced higher temperature in 2020. Our findings provide new insights into how important trophic pathways may be affected by changing ocean conditions.

Photogrammetry as a promising tool to unveil marine caves' benthic assemblages.

Traditionally, monitoring approaches to survey marine caves have been constrained by equipment limitations and strict safety protocols. Nowadays, the rise of new approaches opens new possibilities to describe these peculiar ecosystems. The current study aimed to explore the potential of Structure from Motion (SfM) photogrammetry to assess the abundance and spatial distribution of the sessile benthic assemblages inside a semi-submerged marine cave. Additionally, since impacts of recent date mussel Lithophaga lithophaga illegal fishing were recorded, a special emphasis was paid to its distribution and densities. The results of SfM were compared with a more "traditional approach", by simulating photo-quadrats deployments over the produced orthomosaics. A total of 22 sessile taxa were identified, with Porifera representing the dominant taxa within the cave, and L. lithophaga presenting a density of 88.3 holes/m2. SfM and photo-quadrats obtained comparable results regarding species richness, percentage cover of identified taxa and most of the seascape metrics, while, in terms of taxa density estimations, photo-quadrats highly overestimated their values. SfM resulted in a suitable non-invasive technique to record marine cave assemblages. Seascape indexes proved to be a comprehensive way to describe the spatial pattern of distribution of benthic organisms, establishing a useful baseline to assess future community shifts.

Not out of the Mediterranean: Atlantic populations of the gorgonian Paramuricea clavata are a separate sister species under further lineage diversification.

The accurate delimitation of species boundaries in nonbilaterian marine taxa is notoriously difficult, with consequences for many studies in ecology and evolution. Anthozoans are a diverse group of key structural organisms worldwide, but the lack of reliable morphological characters and informative genetic markers hampers our ability to understand species diversification. We investigated population differentiation and species limits in Atlantic (Iberian Peninsula) and Mediterranean lineages of the octocoral genus Paramuricea previously identified as P. clavata. We used a diverse set of molecular markers (microsatellites, RNA-seq derived single-copy orthologues [SCO] and mt-mutS [mitochondrial barcode]) at 49 locations. Clear segregation of Atlantic and Mediterranean lineages was found with all markers. Species-tree estimations based on SCO strongly supported these two clades as distinct, recently diverged sister species with incomplete lineage sorting, P. cf. grayi and P. clavata, respectively. Furthermore, a second putative (or ongoing) speciation event was detected in the Atlantic between two P. cf. grayi color morphotypes (yellow and purple) using SCO and supported by microsatellites. While segregating P. cf. grayi lineages showed considerable geographic structure, dominating circalittoral communities in southern (yellow) and western (purple) Portugal, their occurrence in sympatry at some localities suggests a degree of reproductive isolation. Overall, our results show that previous molecular and morphological studies have underestimated species diversity in Paramuricea occurring in the Iberian Peninsula, which has important implications for conservation planning. Finally, our findings validate the usefulness of phylotranscriptomics for resolving evolutionary relationships in octocorals.

Sinularioside, a triacetylated glycolipid from the Indonesian soft coral Sinularia sp., is an inhibitor of NO release.

Chemical analysis of the Indonesian soft coral Sinularia sp. (order Alcyonacea, family Alcyoniidae) afforded a known glucosylcerebroside of the sarcoehrenoside-type and sinularioside (2), a new naturally triacetylated glycolipid containing two α-D-arabinopyranosyl residues and a myristyl alcohol unit. Their complete stereostructures were solved by interpretation of MS and NMR data along with CD analysis of degradation products. Sinularioside proved to moderately inhibit LPS-induced NO release, providing interesting clues into the poorly understood structure-activity relationships for anti-inflammatory glycolipids.

Changes in coral forest microbiomes predict the impact of marine heatwaves on habitat-forming species down to mesophotic depths.

Global warming is causing the increase in intensity and frequency of heatwaves, which are often associated with mass mortality events of marine organisms from shallow and mesophotic rocky habitats, including gorgonians and other sessile organisms. We investigated the microbiome responses of the gorgonians Paramuricea clavata, Eunicella cavolini, and the red coral Corallium rubrum to the episodic temperature anomalies detected in the North Western Mediterranean, during August 2011. Although the investigated corals showed no signs of visible necrosis, the abundance of associated Bacteria and Archaea increased with increasing seawater temperature, suggesting their temperature-dependent proliferation. Coral microbiomes were highly sensitive to thermal anomaly amplitude and exhibited increased bacterial diversity to greater thermal shifts. This effect was explained by the decline of dominant bacterial members and the increase of new, rare and opportunistic taxa, including pathogens, revealing a direct effect of heatwave-induced alteration of the microbiomes and not a secondary consequence of coral necrosis.