ABSTRACT. Corals are iconic marine species that engineer habitat for millions of other organisms. Although their importance in enhancing biodiversity in the marine environment is clear, in many depths and regions around the world, they remain under-described, uncharacterized, often inaccurately identified, and/or yet to be discovered. Still, they are threatened by a variety of anthropogenic stressors-from climate change to resource extraction activities. To successfully mitigate the impacts of these stressors, we need fundamental knowledge on how many species there are, and how and where these species are generated, distributed and connected. Therefore, using genomics and integrative taxonomy, coupled with historical collections and ocean exploration, we are beginning to “rebuild” the coral tree of life. By reconstructing this evolutionary framework, we can better document the extent of coral diversity (taxonomic, genetic morphological, functional), while uncovering the historical and contemporary drivers of that diversity, so that we can better inform the likelihood of persistence into the future.

ABSTRACT. Corals are the foundational species of coral reefs and coralligenous ecosystems. Their success may be linked to their symbioses with microorganisms, which play an important role in the nutrition and health of their host. Therefore, the coral host and its symbionts are considered an entity: the holobiont. But whether there is an evolutionary link between corals and their microbiomes has not been fully investigated. While indications of phylosymbiosis in Scleractinian hexacorals exist, little is known about the holobionts of Alcyonacean octocorals. Here, we used 16S rRNA gene amplicon sequencing to explore the composition of the bacterial communities associated with 14 Alcyonacean species (belonging to 10 genera from 5 (sub)orders) collected from the mesophotic zones of the Mediterranean Sea and Red Sea. The low levels of disturbance in mesophotic ecosystems enabled us to study potential evolutionary links (e.g., phylosymbiosis and cophylogeny) between these corals and their microbiota. Clear differences were, however, observed between corals from the Mediterranean Sea and the Red Sea. The low diversity and consistent dominance of Endozoicomonadaceae and/or Spirochaetaceae in the communities of Mediterranean octocorals, suggested that Mediterranean octocorals could have evolved in parallel with their microbiota. Phylosymbiotic signals were indeed detected and cophylogeny in associations between several bacterial strains belonging to Endozoicomonadaceae or Spirochaetaceae and coral species were identified. Conversely, phylosymbiotic patterns were not evident in Red Sea octocorals, likely due to the high bacterial taxonomic diversity in their microbiota, but cophylogeny in associations between certain coral and bacterial species were observed. Particularly noteworthy were the associations with Endozoicomonadaceae, suggesting a plausible evolutionary link that warrants further investigations with expanded datasets to uncover potential underlying patterns. Overall, our findings emphasize the importance of Endozoicomonadaceae and Spirochaetaceae in coral symbiosis and the significance of exploring host-microbiome interactions in mesophotic ecosystems for a comprehensive understanding of coral-microbiome coevolution.

ABSTRACT. Abstract Aim In the last years an approach based on multiple molecular markers has been increasingly used to in phylogenetic studies. The usage of whole mitochondrial genomes have has already been shown to be of great help in addressing phylogenetic and systematic issues at various levels (e.g. depicting cryptic species, discriminate between subspecies or investigate recent evolutionary phenomena). Phylogenies based on Whole whole mitochondrial genomes-based phylogenies, usually allows for a higher overall support of nodes than traditional phylogenies based on a few loci, and with much lower costs compared to -omics approaches. In this framework annelids are a particularly interesting group, as they were considered to be uniform in terms of mitogenome size and synteny, assumptions that were later challenged by recent studies.

Methods Total DNA material of seven species of Opheliidae (covering the majority of the currently valid genera) was amplified via whole-genome amplification (WGA) method, and then processed via Illumina technology. Obtained reads were assembled with SpADES to obtain the complete mitochondrial genome.

Results Mitochondrial genomes of the family Opheliidae do not show a wide variation in size, being overall between 15,000 and 17,000 base pairs. Conversely, the gene order shows a wide variation, and four main orders can be identified, one of which corresponding to the putative annelid ground pattern. The obtained phylogenetic analysis only partially confirms previous reconstructions, allowing to identify three clades, corresponding to different morphologies and with a coarse correspondence with gene order patterns. Phylogenetic relationships with other annelid families remain uncertain based on this reconstruction.

Main conclusion Mitochondrial genomes revealed themselves a promising and powerful tool to disentangle intra-familiar relationships and solve systematic issues, being able to infer relationships inside the order with good statistical support. Synteny in Opheliidae shows a good correspondence with the main clades retrieved, even if this result cannot be extended to other annelid families.

ABSTRACT. Although cryptic species have routinely been uncovered using barcoding techniques, sympatric cryptic species appearing in the plankton at different times of the year have not yet received adequate attention. This is primarily due to the lack of the necessary temporally dense, long-term sampling that can uncover them. Using barcoding techniques, we analyze the phylogeny of eight species of Hydrozoa (Cnidaria) commonly found in the Gulf of Mexico (GoM) and localities worldwide. We created a unique dataset for each species, combining sequences from specimens collected weekly for a year in the GoM and sequences collected worldwide. Using these datasets, we investigate the presence of sympatric cryptic species appearing in seasonal/temporal gradients (within the GoM) and cryptic species worldwide. We show that eight species of Hydrozoa with wide geographical distribution are composed of cryptic species that span geographically across the globe and seasonally within the GoM. This bi-dimensional cryptic diversity - both in time and space – is essential for a deeper understanding of processes of evolution, biogeography, and dispersal in the seas and more realistic biodiversity assessments.

ABSTRACT. Aim Retinoic acid (RA) plays several essential roles during embryogenesis and throughout adult life in Metazoan. In vertebrates, RA controls the transcription of target genes through binding to retinoic acid receptors (RAR). To date, very little is known about RA function in invertebrates. Recent findings reported that Lophotrochozoan RAR lost the ability to bind RA due to the accumulation of multiple mutations in the ligand binding domain (LBD). Here, we use the bivalve Mytilus galloprovincialis (Mg) to provide new insights on the role and evolution of RA signalling in mollusc.

Methods Genomic screen and phylogenetic inference, were used to identify ortholog sequence to vertebrate RA related proteins. Later, a detailed analysis of the LBD domain of the MgRAR revealed key aminoacidic changes with vertebrates. Molecular docking and dynamics simulations together with functional assay were conducted to test MgRAR ability to respond to RA. Finally, embryos were treated with different doses of RA and with specific inhibitors.

Results RA machinery toolkit genes are conserved with Vertebrates. However, functional in vitro luciferase assay revealed the lack of activation of MgRAR in the presence of RA, even if, in vivo treatments of embryos with this compound or with specific inhibitors resulted in many developmental defects.

Main Conclusions Although our preliminary results seem to suggest that Mytilus RAR is unable to bind RA, we cannot fully exclude the contrary given the limitation of our Luciferase assay system. Therefore, currently, we are using other approaches before to confirm the inability of the MgRAR to be responsive to RA. Furthermore, ongoing RNAseq analyses will help to better explore the potential RA role during Mg embryogenesis.

ABSTRACT. Aim: Transition metals such as iron, zinc, copper, molybdenum, nickel, manganese, tungsten and cobalt play crucial roles in microbial metabolism. They function as essential cofactors for critical enzymes involved in electron transport, and can affect cell growth and viability by disrupting cell components integrity. Microorganisms have developed complex transport systems to meet their demands for each metal, simultaneously maintaining the homeostasis of multiple cations within a cell. The present study aims at describing the diversity and distribution of microbial metal transport systems across diverse geothermal environments, with a specific focus on shallow water hydrothermal vents and terrestrial deeply sourced seeps. In these ecosystems microbial diversity and metabolism are tightly linked to the elements provided by the water-rock interactions, thus providing an excellent model system in which to investigate the diversity of microbial metal transport systems.

Methods: We performed shotgun metagenomics on geofluids from around 200 thermal features across Costa Rica, Chile, Argentina, Panama, Iceland, and Italy, following a modified version of KBase and a custom workflow. The diversity of microbial metal transport genes was assessed via functional annotation of sequencing reads using mi-faser with a tailored database including metal transport genes sequences.

Results: The terrestrial and marine environments in this study present wide ranges of physico-chemical variables, and the diversity of genes encoding microbial metal transport systems appears to be influenced by this geochemistry.

Main conclusion: The study of the diversity of metal transport systems is crucial to investigate the interactions between microorganisms and ecosystems, and hence their metabolic evolution.

ABSTRACT. Aim Ascidians (Chordata: Tunicata) account for more than 3,000 species worldwide, showing an impressive, albeit often understudied, diversity. The Mediterranean ascidian fauna is commonly considered as well-characterized, although it has been only partially investigated through modern approaches. We hereby carried out the first integrative study focusing on the family Pyuridae (Ascidiacea: Stolidobranchia) in the Mediterranean Sea, clarifying the taxonomic status and systematic position of the investigated species within the complex stolidobranch phylogeny.

Methods Used An integrative approach based on morphological examination and single and multi-locus phylogenetic reconstructions was done on specimens collected from various localities in the Mediterranean Sea. In particular, the mitochondrial genes COI and 16S rRNA, and the nuclear genes 18S rRNA and 28S rRNA, were used. A preliminary phylogenetic reconstruction encompassing Pyuridae and its related groups (Styelidae and Molgulidae) was carried out on the basis of available loci mined from transcriptomic and WGS published data.

Results The family Pyuridae resulted to be paraphyletic, in agreement with published literature. Taxonomic mismatches and cryptic diversity were first highlighted in the genera Microcosmus Heller, 1877 and Pyura Molina, 1782.

Main conclusions This contribution provides a first baseline for a general revision of the family Pyuridae and its representatives in the Mediterranean Sea. As these species include important ecosystem engineers, commercially valuable species, renowned worldwide invaders, and important sources of bioactive marine natural products, it also highlights the need of multipronged approaches to investigate their diversity and ecology.

Thematic Areas 1st choice: Biodiversity 2nd choice: Populations and Species

ABSTRACT. Aim Halla parthenopeia is a borrowing, poisonous, clam-predating polychaeta often used as bait. Besides potential biotechnological applications of the toxins, the feeding apparatus of the worm can offer important insights on the species’ adaptation to its habitat. Here we describe the anatomy of H. parthenopeia in relation to its lifestyle, chemical defenses and feeding behavior, which rely on the production of different types of mucus.

Methods Multiple histological and histochemical analyses, both in paraffin and in resin sections, were performed to produce a comprehensive image of the animal’s microanatomy.

Results The tegument is overlayed by a cuticle and is formed by a mucocytes-rich pseudostratified epithelium. Skin mucocytes secrete the mucus that facilitates locomotion and stabilizes the burrow. Between mucocytes we noticed cells with a round soma and dark cytoplasmatic granules. These cells are likely involved in the storage and secretion of hallachrome, the toxic pigment responsible for the purple staining of defensive mucus. The pharynx bears jaws and mandibles that are connected to a single, relatively large, salivary gland. This gland extends from below the esophagus up to one third the length of the animal. This gland produces the feeding mucus, secreted to facilitate opening and digesting clams. The unusual structure of the esophagus, full of different types of mucocytes, may also be associated to the production of gut-lubricating mucins during the feeding.

Main conclusion The abilities to dig burrows into the sediment, to prey on bivalves and to fence off predators and parasites by H. parthenopeia seems to rely on a unique combination of highly specialized anatomical structures. These permit the secretion of multiple type of mucins that convey toxins and enzymes. These features are aligned with the species’ complex behavior, altogether representing efficient adaptations ambush preying in shallow marine benthic environments.

ABSTRACT. A curated, high-quality annotation of mitochondrial genomes is a key step for manifold downstream analyses, such as phylogenetic inference, mitotranscriptomics, and population structure studies. Although good annotations of mitochondrial genomes are nowadays available for many taxa across the tree of life, this does not hold true for the class Bivalvia. Notably, mitochondria account for some odd peculiarities in this class, including the Doubly Uniparental Inheritance. Therefore, we set up a comparative framework to reconsider the annotation of mitochondrial proteins in bivalves, with special reference to gene boundaries. We used three datasets of mitochondrial genomes, containing species with increasing phylogenetic distance. We sought for reasons of the observed disparities in annotations using different approaches. Here we present some insights into our effort towards a reappraisal of mitochondrial genomics of bivalves, with the double aim of providing a robust mitochondrial gene feature overview of this group as well as suggesting good practices for future annotations.

ABSTRACT. Abstract

Aim

Tunicates, the sister group of vertebrates, exhibit stem cell-based processes as adaptation to environmental changes. Stem cells are able to self-renew and differentiate into different cell types, allowing growth and repair of damaged tissues. In the colonial tunicate Botryllus schlosseri, stem cells are involved in asexual reproduction and regeneration, and home in adult zooid niches. A colony, composed of adult individuals and their buds, cyclically undergoes a generational change: adult zooids are resorbed and substituted by their buds. During this phase, stem cells leave their niches to colonize those of the new adults. Here, we describe the dynamics of the cell islands, a B. schlosseri niche, throughout the asexual cycle. Cell islands are groups of phagocytes and stem cells, located in the ventral body wall.

Methods

Colonies fixed at different phases were used to count cell islands in each adult, and analyse their location. Moreover, 39 adults from three colonies were studied in vivo throughout the asexual cycle. Escherichia coli fluorescent bioparticles were microinjected into their vasculature. Circulating phagocytes turned fluorescent after bioparticle ingestion and homed in cell islands, making them detectable. Cell islands were counted in each individual every 24 hours and their location were annotated. Data were statistically analysed to infer the dynamicity of cell islands during the asexual cycle and in different physiological conditions.

Results

Cell islands exhibited varying locations and differed in number in genetically identical individuals. In single individuals, their number showed a positive correlation with the physiological condition and changed throughout the asexual cycle.

Main Conclusion

Cell islands are a dynamic niche, varying in relation to the asexual cycle. Healthy zooids with more buds show also more cell islands. The variations observed in genetically identical individuals suggest that environmental factors play a role in determining the number and position of cell islands.

ABSTRACT. Abstract

Aim Chlorophyll fluorescence is today used as a sensitive, in vivo probe of photosynthetic function and it is commonly used in different fields as well as to investigate the effect of climate change in marine habitats. It is a fast, easy, and non-invasive method but despite its simplicity, it has never been used in Mediterranean Heterobranchia to explore diet and symbiosis, even if these molluscs are known for their symbiotic relationships with unicellular algae or their ability to incorporate into their own cells the chloroplasts from the algae they feed on. For these reasons, this technique was applied to different Heterobranchia species characterized by own ecology and trophic strategies.

Methods Six Sacoglossa and seven Nudibranchia were collected using scraping and scuba dive techniques from different localities along the Salento peninsula (Apulia). All the specimens were observed and photographed using a Nikon SMZ25 stereomicroscope fitted with NIS-Elements imaging software and the fluorescence investigated using Nikon intensilight C-HGFI fiber optic fluorescence.

Results A high presence of fluorescence was reported in all the Sacoglossa analysed except Placida cremoniana (Trinchese, 1892). Regarding nudibranchs, results showed a more complex scenario that perfectly reflects the heterogeneous diet characterizing each nudibranch family. However, the case of Favorinus branchialis (Rathke, 1806) is noteworthy since it was known to feed on nudibranch’s eggs but the discover of unicellular algae in its tissues suggests it may feed also on other unreported prey containing zooxanthellae.

Main conclusion Fluorescence revealed powerful to highlight the presence of chlorophyll and other fluorescing pigments in heterobranchs. Additionally, it allowed to distinguish between symbiosis based on chloroplasts or zooxanthellae. Finally, results on the pattern of distribution of these exogenous organelles or cells in the body of the hosts can fill some gaps of knowledge still existing on these interesting but yet poorly known biological abilities.

Thematic Areas Adaptation, Biodiversity

ABSTRACT. Aim Marine microalgae are photosynthetic eukaryotic organisms that contribute at 40% of the global productivity. They are characterized by a great biodiversity in terms of species able to live in extremely different habitats thanks to their adaptation versatility. Adaptation strategies include the production of a series of molecules with defensive capacities. These compounds have been shown to exert activities useful against some human pathologies. Loss of biodiversity will also impact chemical biodiversity and loss of possible human medications. Due to the high incidence of cancer, infectious diseases, antibiotic resistance and the insurgence of other human pathologies, the aim of this study is to look for bioactive marine microalgae with possible antiproliferative activity for human health applications. Methods Six freeze-dried microalgal pellets (Klebsormidium cf. nitens, Pleurosigma cf. intermedium, Pleurosigma aff. rigidum, Navicula cf. salinicola, Halamphora sp., Hemiselmis cf. andersenii) were extracted with a methanolic extraction and the organic extracts were fractionated with Solid Phase Extraction chromatography method. Raw extracts and fractions were tested on human melanoma and lymphoma cells. Results For each extract, five fractions named fraction A-E, enriched of specific compounds, were obtained. Primary metabolites extracted in each fraction were amminoacids and saccharides (fraction A), nucleosides (fraction B), glycolipids and phospholipids (fraction C), sterols and free fatty acid (fraction D) and triglycerides (fraction E), respectively. Specific extracts and fractions showed antiproliferative activity, without affecting normal cells used as control. Main conclusion These promising extracts will be further considered for successive analyses, such as chemical identification of bioactive components and characterization of the mechanism of action at gene and protein levels. The cell targets of marine compounds and their mechanism of action are, in fact, often completely unknown and further research is necessary to unlock the biotechnological potential of marine organisms.

ABSTRACT. Aim Mediterranean Sea is a hot spot of Heterobranchia diversity; in fact, more than 363 taxa are reported for this semi-closed basin, 281 of which are present in the Italian waters. Even if an increasing effort have been done to study the Mediterranean fauna, almost all the available checklists focused on coastal natural areas while artificial marine sites as ports and marinas, potentially interesting also for the early detection of non-indigenous species (NIS), remain unexplored yet. In fact, most of the previous studies reported few species inhabiting these environments but the list of associated fauna is still lacking. To fulfil this gap of knowledge, the sea slugs inhabiting ports and marinas along the Salento peninsula were investigated and the species list produced.

Methods Brushing and scuba diving techniques were used to collect heterobranchs from eight stations along the Salento peninsula. The benthic detritus was observed under the stereomicroscope and sorting of the species carried out. Individuals collected were photographed and stored in EtOH 96%.

Results A total of 158 specimens was recorded belonging to at least 49 species. Among these, only two are considered NIS while for nine taxa further analysis are needed to correctly assign them at the species level. However, the presence of Stiliger cf. auarita is noteworthy as new species and genus for the entire basin. Furthermore, 49 representative drawings were produced per each species.

Main conclusion Studying the Heterobranchia associated to ports and marinas revealed useful to unravel rare and neglected species and could constitute a valid support for studying Heterobranchia diversity and for the early warning of NIS. Furthermore, bridging some knowledge gaps on these highly impacted environments may constitute an opportunity to investigate the adaptive dynamics shown by these molluscs to respond to the anthropic impacts acting on these sites.

Thematic Areas Biodiversity, Conservation

ABSTRACT. Aim The deep sea is the largest biome on earth and yet the least known. Our poor knowledge of its remote habitats reduces the possibility to understand and protect such an important environment. In this study, molecular tools and bioinformatic pipelines were implemented to unravel nekton biodiversity in the poorly investigated Dohrn Canyon in the Tyrrhenian Sea, with a focus on cephalopods and fish species. An eDNA-based metabarcoding approach has been applied to water and sediment samples collected at different depths in the Dohrn Canyon to study cephalopods and fish assemblages.

Methods Total eDNA was successfully extracted and amplified for the 18S (De Jonge et al. 2021), 12S and COI (Valentini et al. 2016, Miya et al. 2015). Cleaned PCR products were used for library preparation and sequenced. Data derived from the water and sediment matrices (between ca. 280,000 and 17,356,008 raw reads) were cleaned, filtered, and assigned to the lowest possible taxonomic level.

Results Among the main cephalopod taxa detected Heteroteuthis sp., Loligo sp. and Eledone cirrhosa were the most abundant across all the stations, while concerning teleost, preliminary results highlighted the presence of some deep-sea fishes like Lampanyctus pusillus, Mobula sp. and Electrona sp., among others.

Main conclusion Despite some methodological constraints, this study represents one of the few attempts applying the emerging eDNA approaches to a deep-sea environment in the Mediterranean Sea. Most importantly, we described the Dohrn Canyon megafaunal biodiversity, with an important and up-to-date focus on the cephalopods community.

Thematic Areas Biodiversity, Conservation

ABSTRACT. Aim Deep-sea coral habitats host a complex associated fauna that has not been fully revealed yet. The nudibranch Tritonia callogorgiae Chimienti, Furfaro & Taviani, 2020, was recently described from deep Callogorgia verticillata (Pallas, 1766) forests and nowadays found exclusively associated to this octocoral. This tritoniid shows an orange background colour scattered with white and orange gills with white/transparent apical parts. In 2021, during the Gombessa 6 expedition, specimens resembling T. callogorgiae but showing an atypical coloration were observed and photographed on Paramuricea clavata (Risso, 1827) off the coast of Corsica. These specimens had dark blue gills and light blue edge of the sheath of the rhinophores. To unravel whether this blue morphotype could mask hidden diversity, five specimens were sampled for molecular comparison.

Methods Six blue and two orange specimens, collected respectively on P. clavata and C. verticillata at 96 and 117 m depth, were photographed and stored. DNA was extracted and DNA-Barcoding, Bayesian Inference and Maximum Likelihood analyses were performed using Cytochrome C Oxidase subunit I, 16S rRNA and histone H3 molecular markers. A broader sequence dataset, including T. callogorgiae topotypical material and other Tritoniidae species, was considered.

Results DNA-Barcoding confirmed the blue specimens were indeed a morphotype of T. callogorgiae, despite the striking colour difference. Besides, phylogenetic analyses revealed a complex evolutionary history within this species, which is provisionally kept in Tritonia even though morphological features suggest it may possibly belong to a separate genus. Finally, a new trophic association with P. clavata, is reported.

Main conclusion Results showed that Tritonia callogorgiae has an extremely variable body colour pattern and diet. Furthermore, a complex phylogeny was revealed among this species and other Tritoniidae that should be elucidated in the future. Finally, evidence suggest the possible correlation between prey and colour variability that deserves future investigations.

Thematic Areas: Macroevolution, Biodiversity

ABSTRACT. Abstract Aim A recent work uncovered an unexpected diversity inside the genus Myxicola Koch in Renier in Meneghini, 1847 (Sabellidae, Annelida) in the Mediterranean Sea. The analysis of new samples from deep coralligenous and mesophotic assemblages highlighted the occurrence of potential new lineages within this genus. The increase in the number of species belonging to this genus calls for the need of a better understanding of the phylogenetic relationships occurring among them.

Methods Morphological analysis employed more than 40 different characters, to differentiate between taxa; in comparison with previous studies, the possibility to work on fresh material allowed us to detect characters appreciable only on live specimens. In addition, two mitochondrial markers (16S rDNA and COI) were used for molecular characterization and phylogenetic reconstruction.

Results Eight different lineages were recovered within the genus Myxicola in the Mediterranean Sea alone, while 4 additional lineages were obtained from public datasets (GenBank and BOLD). Two Mediterranean lineages resulted clearly distant from the remaining ones, allowing to retrieve a closer relationship with the genus Acromegalomma and suggesting that they should be considered as a different, new genus. Morphological results were consistent with molecular one, concerning the shape of abdominal uncini, radiolar skeleton and structure of lips, as well as ecological differences.

Main conclusion The possibility to work on environments scarcely known allowed the detection of an unforeseen biodiversity, falling into a genus previously characterized by few, cosmopolitan species. Although polychaetes have been recently studied for their abundant cryptic complexes, a vastly overlooked diversity still lies within this group, suggesting a largely underrated species diversity.

ABSTRACT. Aim Attention for rapid assessment of biodiversity and ecosystem integrity has been greatly revived by the advent of environmental DNA (eDNA) metabarcoding, a quick, cost-effective, and non-invasive molecular methodology. Traditionally targeted by standard parameters and/or indexes foreseen by the EU Water Directive, so far, the biological quality of the Apulian waters has never been explored using eDNA. Thus, a case study has been carried out, focused on a coastal zone subject to different levels and types of anthropic impacts.

Methods 18S and COI markers were used to screen for differences in the structure and organization of benthic macroinvertebrate communities at three stations near Brindisi (Apulia) in the Southern Adriatic coast: i) the Torre Guaceto Marine Protected Area as control site, and two impacted sites namely ii) the nearshore zone adjacent to the Brindisi main harbour, and iii) the proximate coastal zone offshore the ENEL thermoelectric power plant Federico II (Cerano). A user-friendly standardized protocol was implemented to obtain replicable and statistically reliable results. At each site, three sampling stations were located along a linear transect at 100, 500 and 1000m distance from the shoreline. Water and sediment samples were collected by SCUBA diving, in eight points at each station and sediment samples were sub-sampled three times. A total of 576 eDNA were extracted and a two step-PCR were performed to build the libraries for Illumina NovaSeqTM sequencing.

Results Bioinformatic analysis of reads and statistical study of alpha- and beta-diversity produced robust data of the community structures occurring across the investigated marine area, seeking for changes in benthic assemblages due to distance from the coast, anthropogenic impacts, and seasonality.

Main conclusion Our preliminary results will be reported here, witnessing the eDNA approach can be powerful to providing information on the welfare of natural marine environments, and calling attention for future applications of the same technique across multiple areas.

Thematic Areas Biodiversity, Conservation

ABSTRACT. Aim Cnidaria is one of the most ancient lineages of Metazoa and is characterized by the production of venom in specialized cells. The acquisition and diversification of venom plays a key role in the evolutionary success of the entire phylum. However, research on Cnidaria venom has been mostly focused on sea anemones, Anthozoans belonging to the subclass Hexacorallia. Notably, scarce information is available on the venom produced by the sister subclass of Octocorallia, or soft corals, and it mostly pertains to their small molecules content. Polyps of soft coral colonies are equipped with nematocyst-rich tentacles used to feed on zooplankton and to deter potential predators. The lack of information regarding polypeptide toxins of soft corals is a significant bias towards the understanding of the evolutionary history of Anthozoan venom toxins. Methods We used a combined transcriptomic-proteomic approach to investigate the venom of the white seafan Eunicella singularis, a Mediterranean soft coral inhabiting rocky bottoms at 20-30 m depth. We isolated venom polypeptides through selective chemical extraction of the nematocysts and we matched MS/MS proteomic data against a whole body assembled transcriptome. Finally, we performed comparative genomic and phylogenetic analyses of relevant proteins, to gain insights on their evolutionary history. Results We identified potential cytolytic and neurotoxic proteins in the venom of E. singularis, finding similarities with well characterized families of cnidarian toxins, such as the small cysteine rich peptides (SCRIPs). In addition, we detected uncommon polypeptides with novel cysteine frameworks. While some of these toxins seem to be more widespread across soft corals or either within all Anthozoa, other are restricted to specific Octocorallia clades. Homology models and surface electrostatic potentials were analyzed for the previously uncharacterized toxins, to understand their possible mode of action in comparison to well characterized proteins. Main Conclusions Our results provide the first description of the toxins from a soft coral’s venom, highlighting their potential activity and expanding our understanding of the evolutionary history of venom protein families in Anthozoa

ABSTRACT. Aim The great scallop (Pecten maximus) and the Mediterranean scallop (P. jacobaeus) are two European scallop species whose distribution abut at the Almeria-Oran front. These species were initially believed to have diverged shortly after the Messinian salinity crisis, around 5 million years ago (mya), but a number of genetic-based studies challenged this view by providing measures of genetic divergence indicative of a more recent split. Here, we use a genomic approach to reconstruct the divergence history of these two species and uncover the potential determinants that led to their differentiation.

Methods Our analyses were based on RAD sequencing data obtained from both scallop species and a chromosome-level reference genome. Firstly, we implemented demographic modelling to compare different speciation scenarios. Secondly, we calculated absolute and relative measures of genetic divergence between the two species to characterize levels of differentiation along the Pecten genome. Finally, we characterized picks of divergence to uncover the genomic context where they were located.

Results The two scallop lineages appeared to have diverged in allopatry around one mya. This was followed by secondary contact, which occurred at the end of the last glacial period. The Pecten genome was non-uniformly affected by gene flow during secondary contact and picks of divergence were found in genomic regions showing low recombination rates. Moreover, contrasting patterns of LD decay were detected for the two species, hinting at the presence of chromosomal inversions. Highly differentiated genomic regions were enriched for immune-related processes, mRNA modification and molybdenum metabolism and thus appeared to be adaptively important.

Main conclusion The great and the Mediterranean scallops diverged more recently than initially thought. Divergence accumulated over an initial period of allopatry and peaks of adaptively important differentiation were maintained despite secondary contact in genomic regions that are reproductively isolated.

Thematic Areas Populations and Species, Macroevolution.

ABSTRACT. Aim The occurrence of cryptic and pseudocryptic species is widespread in marine annelids; overlooked diversity can be expected in particular in iconic species that are considered very easy to identify. One of these species is Polyophthalmus pictus (Dujardin, 1839), an allegedly cosmopolitan species widespread in shallow, vegetated environments in European waters.

Methods Polyophthalmus specimens were sampled in several localities in the Mediterranean Sea, mainland European Atlantic coast, and Atlantic Islands, from both photophilous algae (0-15 m) and coralligenous assemblages (50-120 m). The morphological characterisation took into consideration characters traditionally considered informative for the taxonomy of this genus, while the molecular characterisation employed one mitochondrial marker (cytochrome c oxidase subunit I - COI) and one nuclear marker (ITS).

Results Shallow-water Polyophthalmus pictus belonged to four mitochondrial lineages, usually not occurring in syntopy. One lineage, retrieved only in Lebanon, was close to Indo-Pacific Polyophthalmus lineages, suggesting a non-indigenous origin in the Mediterranean. Canary and Azores Islands host a distinct mitochondrial lineage from the European mainland, but this distinction cannot be retrieved on the nuclear marker, suggesting a relatively recent diversification of the two lineages. The amplification of COI was not obtained for the majority of the coralligenous Polyophthalmus, but based on nuclear data, they belong to different, sometimes very distant lineages. Morphological data traditionally used do not show relevant variation among lineages, with the exception of the number and pattern of lateral ocelli.

Main conclusion As demonstrated for several marine annelids, Polyophthalmus pictus is a species complex. The diversity patterns detected seem to be associated mainly to ecological factors, while biogeography plays a less pronounced role, mostly due to the presence of pelagic dispersal stages in the life cycle of the species.

ABSTRACT. Abstract

Our knowledge of the role of fish microbiome in their health has been steadily increasing in the last decade, especially for species of commercial interest. Conversely, relatively few studies focus on the microbiome of wild fishes, especially apex predators like sharks, due to lower economic interest and to greater difficulty in obtaining samples. Studies investigating microbiome differences between diverse anatomical locations of sharks are limited, and the majority of available studies are focused on the microbial diversity present on the shark teeth, with the aim of preventing infections due to the bite of these animals or evaluating the presence of certain pathogens in healthy or diseased specimens.

Aim Here, we investigated the skin, mouth, gills, and cloaca microbiome of five individuals of two phylogenetically distant shark species (Prionace glauca and Somniosus rostratus), to get a better understanding of the diversity of the microbiome of these animals, how it changes throughout different anatomical locations and how much it is influenced and determined by the ecology and evolutionary relationship host-microbiome.

Methods To confirm the taxonomy of the sharks under study, we barcoded the specimens by sequencing the mtDNA COI from a biopsy of their skin. We also used 16S amplicon sequencing to investigate microbiome differences across the different anatomical locations and between the two shark species.

Results Microbial diversity analysis reveals that partially overlapping microbiomes inhabit different body parts of each shark species, while the communities are distinct between the two species.

Main conclusion Our results suggest that sharks’ microbiome species-specific differences are controlled by the ecology of the shark species. This is the first study comparatively analyzing the microbiome diversity of different anatomical locations in two shark species of the Mediterranean Sea.

ABSTRACT. Introduction Albeit sessile, cnidarian polyps actively capture large motile prey. Such predatory behavior is not limited to large polyps since small-sized polyps may access large prey by collective protocooperative predation. We investigated the benefits, the behavior, the cues inducing protocooperation, and the pattern of formation of groups by protocooperating polyps.

Methods First, we provided three diets (large and small, preys and a mixture of both) to Aurelia coerulea polyps for 36 days. Periodically, asexual reproduction, growth and interindividual distances were measured to evaluate polyps’ fitness and aggregation level. Secondarily, we described the behavior for small and large prey predation, and then provided prey homogenates to serve as cues for protocooperation. Direct observations were carried out for 10 minutes. The number of tentacles contractions were counted as a proxy for feeding excitement.

Results We reported higher reproduction (+37.5%) and growth (+0.15 mm) in polyps fed with both preys. The optimal group size for protocooperation was 3.05 ± 0.11 individuals. Protocooperative polyps appeared closer compared to polyps eating small prey (–1.71 ± 2.42 mm). The behavior “Active feeding” was significantly related to large prey predation, and it was mainly performed (occurrence >90%) when starved polyps previously fed with large prey were later stimulated with the large prey homogenate. This response decreased after 6 stimulation sessions, indicating habituation.

Main Conclusion Protocooperation is beneficial for the population and encourages polyps to create groups that may turn into stable aggregations. Predatory behavior could be learnt and mimicked, possibly inducing coordination. Cnidarians are extensively studied for the evolution of physiological and cognitive processes, as sister group of Bilateria. We suggest that cnidarians are the most primitive animals performing group-foraging, this relationship is supposed to involve learning and coordination. These findings are crucial for understanding sociality in the animal kingdom starting from brainless animals.

ABSTRACT. Aim Recent studies have evidenced that several bivalve species display an unusual pangenomic architecture, characterized by widespread hemizygosity and remarkable presence/absence variation (PAV) phenomena, which can affect several thousand protein-coding genes. Although these features might have a critical impact on phenotype, with important implications for local adaptation, this theme has been only marginally investigated in gastropods. Here, we preliminarily investigated the occurrence of gene PAV in the periwinkle Littorina, a well-established model for evolutionary biology studies in marine environments. Methods Following the refinement and annotation of the draft genome of Littorina fabalis, we analyzed the whole-genome resequencing data of 12 individuals belonging to two distinct ecotypes (“large” and “dwarf”), looking for signatures of PAV that might help to define the “openness” of the periwinkle pangenome and investigate the involvement of dispensable genes in specific biological pathways. In silico predictions were further validated through PCR. Results Gene PAV affected a non-negligible fraction of protein-coding genes (about 2000, ~6% of the total) in L. fabalis. Although structural variation was less prevalent than in bivalves, these findings indicate the presence of a pangenomic architecture that likely includes several hundred dispensable genes not represented in the reference genome. Interestingly, the accessory fraction of the periwinkle pangenome was significantly enriched in genes belonging to the AIG1 family of small GTPases, which have been previously implicated in stress adaptation and resistance towards infection in other mollusks. Moreover, PAV patterns correlated with observed phenotype, suggesting that dispensable genes may contribute to local adaptation. Main conclusion These findings confirm the occurrence of gene PAV in non-bivalve mollusks and highlight the need to adopt a paradigm shift towards the use of a pangenome instead of a monoploid assembly as a reference for genomic studies in this species.

ABSTRACT. Bruna Oršanić1, Andrea Toso2, Michele Solca3, Paolo Mariottini4, Chiara Papetti1, Giulia Furfaro2,5

1 Department of Biology, University of Padova, Padova, Italy. bruna.orsanic@studenti.unipd.it; chiara.papetti@unipd.it 2 Department of Biological and Environmental Sciences and Technologies – DiSTeBA, University of Salento, I-73100 Lecce, Italy. giulia.furfaro@unisalento.it 3 Museo di Biologia Marina ʺPietro Parenzanʺ, Via Vespucci 13/17, 73010 Porto Cesareo, Lecce, Italy. michelesolca@gmail.com 4 Department of Science, University of “Roma Tre”, Viale G. Marconi 446, 00146 Rome, Italy. paolo.mariottini@uniroma3.it 5 National Biodiversity Future Center (NBFC), 90133 Palermo, Italy.

Aim Nudibranchs (Gastropoda) are known for their extraordinary ability to obtain structures, cells or chemical compounds from their preys and include them in their own tissues or cells. These unique strategies co-evolved with the loss of the shell in the adult stage. In this framework, symbiotic relationships were already reported between some nudibranchs and unicellular algae but currently the pattern of distribution inside the body of the host is still lacking. To fulfil this gap of knowledge, Spurilla neapolitana (Delle Chiaje, 1841), a Mediterranean facelinid preying mainly on Anemonia Risso, 1827 anemones known to host symbiotic algae, was investigated using fluorescence to confirm the presence of the symbiont and describe its distribution. Furthermore, molecular techniques were used to confirm this symbiotic association and investigate on the systematic of the zooxanthellae involved.

Methods Seven S. neapolitana and two A. sulcata were collected from Taranto (Ionian Sea), observed and photographed alive using the fluorescence microscope before have being store in EtOH 96% for further molecular analyses. DNA was extracted from both nudibranchs and anemone and four molecular markers (COI, 16S, H3, ITS2) used for barcoding analysis.

Results Fluorescence clearly confirmed the presence of the symbiont in S. neapolitana and revealed a widespread distribution throughout almost all its dorsal surface. Furthermore, while the common markers confirmed the species identity, the nuclear ITS2 showed a double band within the nudibranch sample. This result is promising since confirm the presence, in the same tissues, of both host and symbiont thanks to the different length of ITS2 from different organisms.

Main conclusion This is the first study describing the distribution pattern of symbiotic algae inside the body of its nudibranch host. Furthermore, firstly in a nudibranch, this symbiotic relationship was investigated using the ITS2 marker to reveal simultaneously the presence of the host and its symbiont.