Early colonization of sessile megabenthos on electrolytic carbonated structures (Alicante's harbor, Western Mediterranean).

Biofouling of different artificial substrates was studied to determine the differences in biofouling assemblages among different substrates. However, studies on biofouling on natural substrates like electrolytic carbonated ones are lacking. These substrates have a great potential for coral reef restoration in tropical areas and for biofilter construction. Thus, this study was developed to examine the colonization of sessile macrofouling in the port of Alicante (SE Spain, Western Mediterranean) on two types of substrates: electrolytic carbonated and bare steel (as control) over three months of immersion (October 2019-January 2020). The community diversity was studied through different biotic parameters and abundance of assemblages, and preference of organisms according to their status and functional group (active filter feeders). Univariate and multivariate analyses (PERMANOVA and SIMPER) were also applied to examine the differences between carbonate and control substrates. The carbonated substrate had a more structured community and higher abundance, recruitment, and diversity indexes than the bare steel. Moreover, filter feeders (Porifera, Bivalvia, and Ascidiacea) were more abundant, and most of them only appeared in the carbonated substrate. These results show the potential of carbonated structures as biofilters.

The relict population of Pinna nobilis in the Mar Menor is facing an uncertain future.

Pinna nobilis is undergoing one of the most dramatic events suffered by an endangered species. An emerging disease has relegated its populations to coastal lagoons or estuaries with salinities beyond the 36.5-39.7 psu range. The Mar Menor is one of two such locations on the Spanish coastline. Poor environmental conditions and eutrophication and anoxia events, that became critical in 2016, 2019 and 2021, have reduced its population in >99 %. In this work, the spatial distribution of the species within the lagoon and the factors determining its survival along the successive crises of eutrophication are studied using a two-stage (presence/absence estimation and density modelling) Species Distribution Model. A potential area of 200.97 ha and an average density of 1.05 ind.100 m2 is estimated for 2020. The viability of the Mar Menor population depends on management actions designed both for the species and to improve the lagoon environmental state.

Metagenomics Unveils Posidonia oceanica "Banquettes" as a Potential Source of Novel Bioactive Compounds and Carbohydrate Active Enzymes (CAZymes).

Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea. It produces large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." In recent years, interest in the potential uses of these P. oceanica banquettes has increased, and it was demonstrated that biomass extracts showed antioxidant, antifungal, and antiviral activities. The discovery of new compounds through the culture of microorganisms is limited, and to overcome this limitation, we performed a metagenomic study to investigate the microbial community associated with P. oceanica banquettes. Our results showed that the microbial community associated with P. oceanica banquettes was dominated by Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria. Pseudoalteromonas was the dominant genus, followed by Alteromonas, Labrenzia, and Aquimarina. The metagenome reads were binned and assembled into 23 nearly complete metagenome-assembled genomes (MAGs), which belonged to new families of Cyanobacteria, Myxococcota, and Granulosicoccaceae and also to the novel genus recently described as Gammaproteobacteria family UBA10353. A comparative analysis with 60 published metagenomes from different environments, including seawater, marine biofilms, soils, corals, sponges, and hydrothermal vents, indicated that banquettes have numbers of natural products and carbohydrate active enzymes (CAZymes) similar to those found for soils and were only surpassed by marine biofilms. New proteins assigned to cellulosome modules and lignocellulose-degrading enzymes were also found. These results unveiled the diverse microbial composition of P. oceanica banquettes and determined that banquettes are a potential source of bioactive compounds and novel enzymes. IMPORTANCE Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea that forms large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." These banquettes accumulate on the shore, where they can prevent erosion, although they also cause social concern due to their impact on beach use. Furthermore, Posidonia dry material has been considered a source of traditional remedies in several areas of the Mediterranean, and a few studies have been carried out to explore pharmacological activities of Posidonia extracts. The work presented here provides the first characterization of the microbiome associated with Posidonia banquettes. We carried out a metagenomic analysis together with an in-depth comparison of the banquette metagenome with 60 published metagenomes from different environments. This comparative analysis has unveiled the potential that Posidonia banquettes have for the synthesis of natural products, both in abundance (only surpassed by marine biofilms) and novelty. These products include mainly nonribosomal peptides and carbohydrate active enzymes. Thus, the interest of our work lies in the interest of Posidonia "waste" material as a source of new bioactive compounds and CAZymes.

Biogeographic Differences in the Microbiome and Pathobiome of the Coral Cladocora caespitosa in the Western Mediterranean Sea.

The endemic Mediterranean zooxanthellate scleractinian reef-builder Cladocora caespitosa is among the organisms most affected by warming-related mass mortality events in the Mediterranean Sea. Corals are known to contain a diverse microbiota that plays a key role in their physiology and health. Here we report the first study that examines the microbiome and pathobiome associated with C. caespitosa in three different Mediterranean locations (i.e., Genova, Columbretes Islands, and Tabarca Island). The microbial communities associated with this species showed biogeographical differences, but shared a common core microbiome that probably plays a key role in the coral holobiont. The putatively pathogenic microbial assemblage (i.e., pathobiome) of C. caespitosa also seemed to depend on geographic location and the human footprint. In locations near the coast and with higher human influence, the pathobiome was entirely constituted by Vibrio species, including the well-known coral pathogens Vibrio coralliilyticus and V. mediterranei. However, in the Columbretes Islands, located off the coast and the most pristine of the analyzed locations, no changes among microbial communities associated to healthy and necrosed samples were detected. Hence, our results provide new insights into the microbiome of the temperate corals and its role in coral health status, highlighting its dependence on the local environmental conditions and the human footprint.

Vibrio communities in scleractinian corals differ according to health status and geographic location in the Mediterranean Sea.

The increase in seawater temperature associated with global warming is a significant threat to coral health and is linked to increasing mass mortality events and Vibrio-related coral diseases. In the Mediterranean Sea, the endemic Cladocora caespitosa and the invasive species Oculina patagonica are the main scleractinian corals affected by mass mortalities. In this study, culturable Vibrio spp. assemblages associated with healthy and unhealthy colonies of these two shallow coral species were characterized to assess the presence of Vibrio pathogens in tissue necrosis. Vibrio communities associated with O. patagonica and C. caespitosa showed geographical differences, although these became more homogeneous in unhealthy specimens of both species. Furthermore, the number of recovered Vibrio specimens was more than five times higher in unhealthy than in healthy corals. Within these culturable vibrios, the known pathogens Vibrio mediterranei and Vibrio coralliilyticus were present in unhealthy colonies of both coral species in the two localities, suggesting that they could play a role in the health status of C. caespitosa and thus act as generalist pathogens in Mediterranean corals. Nonetheless, a clonal type of V. coralliilyticus detected in C. caespitosa was not associated with disease signs, suggesting that this species could encompass assemblages with different levels of virulence.

Correction: A massive update of non-indigenous species records in Mediterranean marinas.

[This corrects the article DOI: 10.7717/peerj.3954.].

A massive update of non-indigenous species records in Mediterranean marinas.

The Mediterranean Sea is home to over 2/3 of the world's charter boat traffic and hosts an estimated 1.5 million recreational boats. Studies elsewhere have demonstrated marinas as important hubs for the stepping-stone transfer of non-indigenous species (NIS), but these unique anthropogenic, and typically artificial habitats have largely gone overlooked in the Mediterranean as sources of NIS hot-spots. From April 2015 to November 2016, 34 marinas were sampled across the following Mediterranean countries: Spain, France, Italy, Malta, Greece, Turkey and Cyprus to investigate the NIS presence and richness in the specialized hard substrate material of these marina habitats. All macroinvertebrate taxa were collected and identified. Additionally, fouling samples were collected from approximately 600 boat-hulls from 25 of these marinas to determine if boats host diverse NIS not present in the marina. Here, we present data revealing that Mediterranean marinas indeed act as major hubs for the transfer of marine NIS, and we also provide evidence that recreational boats act as effective vectors of spread. From this wide-ranging geographical study, we report here numerous new NIS records at the basin, subregional, country and locality level. At the basin level, we report three NIS new to the Mediterranean Sea (Achelia sawayai sensu lato, Aorides longimerus, Cymodoce aff. fuscina), and the re-appearance of two NIS previously known but currently considered extinct in the Mediterranean (Bemlos leptocheirus, Saccostrea glomerata). We also compellingly update the distributions of many NIS in the Mediterranean Sea showing some recent spreading; we provide details for 11 new subregional records for NIS (Watersipora arcuata, Hydroides brachyacantha sensu lato and Saccostrea glomerata now present in the Western Mediterranean; Symplegma brakenhielmi, Stenothoe georgiana, Spirobranchus tertaceros sensu lato, Dendostrea folium sensu lato and Parasmittina egyptiaca now present in the Central Mediterranean, and W. arcuata, Bemlos leptocheirus and Dyspanopeus sayi in the Eastern Mediterranean). We also report 51 new NIS country records from recreational marinas: 12 for Malta, 10 for Cyprus, nine for Greece, six for Spain and France, five for Turkey and three for Italy, representing 32 species. Finally, we report 20 new NIS records (representing 17 species) found on recreational boat-hulls (mobile habitats), not yet found in the same marina, or in most cases, even the country. For each new NIS record, their native origin and global and Mediterranean distributions are provided, along with details of the new record. Additionally, taxonomic characters used for identification and photos of the specimens are also provided. These new NIS records should now be added to the relevant NIS databases compiled by several entities. Records of uncertain identity are also discussed, to assess the probability of valid non-indigenous status.

Effects of the 2015 heat wave on benthic invertebrates in the Tabarca Marine Protected Area (southeast Spain).

In the late summer of 2015, extensive mortality of scleratinian corals, gorgonians, and sponges was observed in the Marine Protected Area of Tabarca (southeast Spain). Quantitative data indicated that at 25 m depth the sea fan Eunicella singularis was the most affected species (50% of colonies affected by partial mortality); while in shallow waters more than 40% of the endemic scleractinian coral Cladocora caespitosa population showed tissue lesions that affected more than 10% of their surfaces. Other affected species were the scleractinian corals Oculina patagonica and Phyllangia mouchezii, the sea fan Leptogorgia sarmentosa and the sponge Sarcotragus fasciculatus. This mortality event coincided with an abnormal rise in seawater temperature in this region. Microbiological analysis showed a higher abundance of culturable Vibrio species in invertebrates exhibiting tissue lesions, which indicated that these opportunistic pathogens could be a key factor in the process.

Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica.

Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future.

Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture.

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

Eukarya associated with the stony coral Oculina patagonica from the Mediterranean Sea.

Oculina patagonica is a putative alien scleractinian coral from the Southwest Atlantic that inhabits across the Mediterranean Sea. Here, we have addressed the diversity of Eukarya associated with this coral and its changes related to the environmental conditions and coral status. A total of 46 colonies of O. patagonica were taken from Alicante coast (Spain) and Pietra Ligure coast (Italy) and analyzed using denaturing gradient gel electrophoresis (DGGE) of the small-subunit 18S rRNA and 16S plastid rRNA genes, internal transcribed spacer region 2 (ITS 2) analyses, and electron microscopy. Our results show that Eukarya and plastid community associated to O. patagonica change with environmental conditions and coral status. Cryptic species, which can be difficult to identify by optical methods, were distinguished by 18S rRNA gene DGGE: the barnacle Megatrema anglicum, which was detected at two locations, and two boring sponges related to Cliona sp. and Siphonodictyon coralliphagum detected in samples from Tabarca and Alicante Harbour, respectively. Eukaryotic phototrophic community from the skeletal matrix of healthy corals was dominated by Ochrosphaera sp. while bleached corals from the Harbour and Tabarca were associated to different uncultured phototrophic organism. Differences in ultrastructural morphologies of the zooxanthellae between healthy and bleached corals were observed. Nevertheless, no differences were found in Symbiodinium community among time, environments, coral status and location, showing that O. patagonica hosted only one genotype of Symbiodinium belonging to clade B2. The fact that this clade has not been previously detected in other Mediterranean corals and is more frequent in the tropical Western Atlantic, is a new evidence that O. patagonica is an alien species in the Mediterranean Sea.

Thermally tolerant corals have limited capacity to acclimatize to future warming.

Thermal stress affects organism performance differently depending on the ambient temperature to which they are acclimatized, which varies along latitudinal gradients. This study investigated whether differences in physiological responses to temperature are consistent with regional differences in temperature regimes for the stony coral Oculina patagonica. To resolve this question, we experimentally assessed how colonies originating from four different locations characterized by >3 °C variation in mean maximum annual temperature responded to warming from 20 to 32 °C. We assessed plasticity in symbiont identity, density, and photosynthetic properties, together with changes in host tissue biomass. Results show that, without changes in the type of symbiont hosted by coral colonies, O. patagonica has limited capacity to acclimatize to future warming. We found little evidence of variation in overall thermal tolerance, or in thermal optima, in response to spatial variation in ambient temperature. Given that the invader O. patagonica is a relatively new member of the Mediterranean coral fauna, our results also suggest that coral populations may need to remain isolated for a long period of time for thermal adaptation to potentially take place. Our study indicates that for O. patagonica, mortality associated with thermal stress manifests primarily through tissue breakdown under moderate but prolonged warming (which does not impair symbiont photosynthesis and, therefore, does not lead to bleaching). Consequently, projected global warming is likely to cause repeat incidents of partial and whole colony mortality and might drive a gradual range contraction of Mediterranean corals.

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities.

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.

Ammonia-oxidizing Crenarchaeota and nitrification inside the tissue of a colonial ascidian.

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.

Microbial community associated with the colonial ascidian Cystodytes dellechiajei.

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.

Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds.

Many cytotoxic compounds of therapeutic interest have been isolated from marine invertebrates, and some of them have been reported to be of microbial origin. Pyridoacridine alkaloids are the main compounds extracted from the ascidian Cystodytes dellechiajei. Here we describe the in vitro antiproliferative activity against different tumor cell lines of the ascidian extracts and provide some insights on the role of the microbial community associated with the tunicate in the production of these compounds. C. dellechiajei extracts showed remarkably high antiproliferative activity (IC50