Integrative taxonomy of metazoan parasites of the bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) in the Mediterranean Sea, with the resurrection of Grillotia acanthoscolex Rees, 1944 (Cestoda: Trypanorhyncha).

Appropriate diagnoses of parasites of apex marine predators are crucial to understand their biodiversity, host specificity, biogeography, and life cycles. Such diagnoses are also informative of ecological and biological characteristics of both host and environment in which the hosts and their parasites live. We here (i) investigate the parasite fauna of a bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) obtained from the Gulf of Naples (Tyrrhenian Sea), (ii) characterize molecularly all its metazoan parasites, and (iii) resurrect and report the main morphological features and phylogenetic position of Grillotia acanthoscolex, a cestode species previously synonymized with Grillotia adenoplusia. A rich parasite fauna represented by eight different taxa was found, including two monogeneans (Protocotyle grisea and Protocotyle taschenbergi), one digenean (Otodistomum veliporum), four cestodes (Crossobothrium dohrnii, Clistobothrium sp., G. acanthoscolex, and G. adenoplusia), and one copepod (Protodactylina pamelae). Sequencing of these samples accounts for an important molecular baseline to widen the knowledge on the parasitic fauna of bluntnose sixgill sharks worldwide and to reconstruct their correct food chains. The bluntnose sixgill shark was found to be a definitive host for all endoparasites found here, confirming that it occupies an apex trophic level in the Mediterranean Sea. The taxa composition of the trophic parasite fauna confirms that the bluntnose sixgill shark mostly feeds on teleost fish species. However, the occurrence of two phillobothrid cestodes (C. dohrnii and Clistobothrium sp.) suggests that it also feeds on squids. Finally, we emphasize the importance of using integrative taxonomic approaches in the study of parasites from definitive and intermediate hosts to elucidate biology and ecology of taxa generally understudied in the Mediterranean Sea.

The genome of the rayed Mediterranean limpet Patella caerulea (Linnaeus, 1758).

Patella caerulea (Linnaeus, 1758) is a mollusc limpet species of the class Gastropoda. Endemic to the Mediterranean Sea, it is considered a keystone species due to its primary role in structuring and regulating the ecological balance of tidal and subtidal habitats. It is currently being used as a bioindicator to assess the environmental quality of coastal marine waters and as a model species to understand adaptation to ocean acidification. Here, we provide a high-quality reference genome assembly and annotation for P. caerulea. We generated ∼30 Gb of Pacific Biosciences high-fidelity data from a single individual and provide a final 749.8 Mb assembly containing 62 contigs, including the mitochondrial genome (14,938 bp). With an N50 of 48.8 Mb and 98% of the assembly contained in the 18 largest contigs, this assembly is near chromosome-scale. Benchmarking Universal Single-Copy Orthologs scores were high (Mollusca, 87.8% complete; Metazoa, 97.2% complete) and similar to metrics observed for other chromosome-level Patella genomes, highlighting a possible bias in the Mollusca database for Patellids. We generated transcriptomic Illumina data from a second individual collected at the same locality and used it together with protein evidence to annotate the genome. A total of 23,938 protein-coding gene models were found. By comparing this annotation with other published Patella annotations, we found that the distribution and median values of exon and gene lengths was comparable with other Patella species despite different annotation approaches. The present high-quality P. caerulea reference genome, available on GenBank (BioProject: PRJNA1045377; assembly: GCA_036850965.1), is an important resource for future ecological and evolutionary studies.

Chemical and Pharmacological Prospection of the Ascidian Cystodytes dellechiajei.

Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.

Hidden species diversity and mito-nuclear discordance within the Mediterranean cone snail, Lautoconus ventricosus.

The Mediterranean cone snail, Lautoconus ventricosus, is currently considered a single species inhabiting the whole Mediterranean basin and the adjacent Atlantic coasts. Yet, no population genetic study has assessed its taxonomic status. Here, we collected 245 individuals from 75 localities throughout the Mediterranean Sea and used cox1 barcodes, complete mitochondrial genomes, and genome skims to test whether L. ventricosus represents a complex of cryptic species. The maximum likelihood phylogeny based on complete mitochondrial genomes recovered six main clades (hereby named blue, brown, green, orange, red, and violet) with sufficient sequence divergence to be considered putative species. On the other hand, phylogenomic analyses based on 437 nuclear genes only recovered four out of the six clades: blue and orange clades were thoroughly mixed and the brown one was not recovered. This mito-nuclear discordance revealed instances of incomplete lineage sorting and introgression, and may have caused important differences in the dating of main cladogenetic events. Species delimitation tests proposed the existence of at least three species: green, violet, and red + blue + orange (i.e., cyan). Green plus cyan (with sympatric distributions) and violet, had West and East Mediterranean distributions, respectively, mostly separated by the Siculo-Tunisian biogeographical barrier. Morphometric analyses of the shell using species hypotheses as factor and shell length as covariate showed that the discrimination power of the studied parameters was only 70.2%, reinforcing the cryptic nature of the uncovered species, and the importance of integrative taxonomic approaches considering morphology, ecology, biogeography, and mitochondrial and nuclear population genetic variation.

Duplication and Losses of Opsin Genes in Lophotrochozoan Evolution.

Opsins are G-coupled receptors playing a key role in metazoan visual processes. While many studies enriched our understanding of opsin diversity in several animal clades, the opsin evolution in Lophotrochozoa, one of the major metazoan groups, remains poorly understood. Using recently developed phylogenetic approaches, we investigated the opsin evolution in 74 lophotrochozoan genomes. We found that the common ancestor of Lophotrochozoa possessed at least seven opsin paralog groups that underwent divergent evolutionary history in the different phyla. Furthermore, we showed for the first time opsin-related molecules in Bilateria that we named pseudopsins, which may prove critical in uncovering opsin evolution.

Parasites and prey of the nursehound shark Scyliorhinus stellaris (Linnaeus, 1758): Insights into hidden trophic web interactions in the Mediterranean Sea.

The metazoan parasite community and the stomach contents of the nursehound shark Scyliorhinus stellaris from the Gulf of Naples (central Mediterranean Sea) were studied. The nursehound shark harboured a poor parasite community composed of a species of gill monogenean (Hexabothrium appendiculatum) and three intestinal cestode taxa (Acanthobothrium coronatum and two unidentified species of the genera Yamaguticestus and Scyphophyllidium), all represented by adult stages. Hosts were mostly parasitized by individuals of A. coronatum, which was the most abundant species and contributed to almost 80% of the total number of parasites found. Conversely, other trophically transmitted parasites (i.e., Yamaguticestus sp. and Scyphophyllidium sp.) showed low prevalence and abundance. The parasite infracommunity was poor, showing low values of species richness, total mean abundance, and diversity indices. Overall, 52 prey items belonging to 13 taxa were identified in the stomach contents. Cephalopods were the most important prey items (represented by nine taxa) and the most diverse and abundant group. In the multivariate space provided by a principal component of mixed data (PCAmix), nursehound sharks distributed along two main axes, related to individual traits (first axis) and stomach contents (including empty ones, second axis). A logistic regression based on the first two axes of the PCAmix showed a significant influence of host individual traits and, to a lesser extent, of stomach contents, regarding the probability of being infected by A. coronatum. Alongside specific traits already associated with parasites transmission, our results highlight the importance of cephalopods in transferring cestode infections through trophic web interactions in the top-predator nursehound shark.

The complete mitochondrial genome of the zebra seabream Diplodus cervinus (Perciformes, Sparidae) from the Mediterranean Sea.

The complete nucleotide sequence of the mitochondrial (mt) genome of the demersal zebra seabream Diplodus cervinus (Lowe, 1838) was determined for the first time. The double stranded circular molecule is 16,559 base pairs (bp) in length and encodes for the typical 37 metazoan mitochondrial genes, and 2 non-coding regions (D-loop and L-origin). The gene arrangement of the D. cervinus mt genome follows the usual one for fishes. The nucleotide sequences of the mt protein coding and ribosomal genes of D. cervinus mt genome were aligned with orthologous sequences from representatives of the Sparidae family and phylogenetic relationships were inferred. Maximum likelihood analyses placed D. cervinus as a sister species of Diplodus sargus (Linnaeus, 1758).

A Comparative Analysis of Mucus Immunomodulatory Properties from Seven Marine Gastropods from the Mediterranean Sea.

The treatment of inflammatory and immune-related diseases due to dysfunctioning of the immune system necessitates modulation of the immune response through immunomodulatory compounds. Marine environments are considered as a new frontier for health benefit product implementations. Marine biodiversity is still a low explored resource, despite it is expected to represent an important platform for chemical bioactive compounds. Within the phylum Mollusca, gastropods are known to synthetize mucus, the latter presenting relevant bioactive properties, e.g., related to immunomodulant molecules able to activate the innate and acquired immune system. This study proposes a bioprospecting of the immunomodulant activity of mucus isolated from seven common gastropod species from the Gulf of Naples (Mediterranean Sea). Results showed that not all mucus displayed a significant cytotoxic activity on the two human cancer cell lines A549 and A2058. On the other hand, the mucus from Bolinus brandaris was strongly bioactive and was therefore thoroughly investigated at cellular, molecular, and protein levels on the human monocytes U937 line. It can conclusively induce monocyte differentiation in vitro and significantly stimulate natural immunity response.

Benthic litter in the continental slope of the Gulf of Naples (central-western Mediterranean Sea) hosts limited fouling communities but facilitates molluscan spawning.

Seafloor pollution by benthic litter is an emerging phenomenon, although debris colonization by biota remains largely unexplored. We characterized the litter of the continental slope (~400-600 m) of the Gulf of Naples (Mediterranean) and investigated its fouling biota through integrative taxonomic approaches. Plastic pieces (82 %) with land-based origin (96 %) and limited sizes (10-20 cm) were the items most commonly encountered, suggesting a transfer to deep waters through floating and sinking. The majority of the items were not fouled, and the debris hosted an impoverished biota, leading to hypothesize that benthic litter supports wide communities only in shallow waters. Higher colonization rates were observed for gastropod and cephalopod eggs with no preference for materials and sizes, suggesting that even small pieces of soft plastic provide a spawning habitat for molluscs and affect species' connectivity in the deep-sea ecosystem. Holistic approaches are necessary to evaluate interactions between litter and biota.

Drivers of parasite communities in three sympatric benthic sharks in the Gulf of Naples (central Mediterranean Sea).

Sharks play a key role in the functioning of marine ecosystems and maintenance of trophic web balance, including life cycles of parasites co-occurring in their habitats. We investigated the structure of parasite communities of three sympatric shark species (Etmopterus spinax, Galeus melastomus, and Scyliorhinus canicula) and explored both the influence of host features in shaping the communities and their role as biological indicators of environment stability in the Gulf of Naples (central Mediterranean Sea), a geographical area characterized by strong anthropic pressure. Parasites found were all trophic transmitted helminths with a complex life cycle, except Lernaeopoda galei, that is a ecto-parasite copepod. Communities were all similarly impoverished with 4-5 component species and low values of species richness and diversity. Higher abundance of cestode larvae of the genus Grillotia was found in G. melastomus, although their dominance in all host species suggests that the three sharks have a similar role as intermediate/paratenic hosts in local food webs. Similarly, high abundance of Grillotia larvae could also suggest the occurrence of high abundance of largest top predators in the area. Host morphological (fork length in S. canicula and G. melastomus and body condition index in G. melastomus) and physiological (sex and gonadosomatic and hepatosomatic indices in S. canicula) variables were differently correlated to parasite community structures depending by host species. Potential reasons for the present impoverished parasite communities are discussed.

Identification of the Marine Alkaloid Lepadin A as Potential Inducer of Immunogenic Cell Death.

Natural products and their synthetic analogs and derivatives are a traditional source of bioactive molecules with potential development as drug candidates. In this context, Marine Natural Products (MNPs) represent a rich reservoir of diverse molecular skeletons with potential pharmacological activity that, so far, has been mostly explored in cancer and infectious diseases. Starting from the development of a novel bioassay-guided screening platform for immunomodulatory compounds from an in-house MNPs library, we report the identification of the alkaloid lepadin A as a new model compound for immune-based anticancer activity with characteristics that suggest a possible mechanism as Immunogenic Cell Death inducer. The work describes the molecular-based bioprospecting in the Gulf of Naples together with the bioassay-guided fractionation, the chemical characterization of the alkaloid, and the biological activity in mouse dendritic cells (D1).

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction.

The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays-two phenotypic and one enzymatic-for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.

A molecular and ecological study of Grillotia (Cestoda: Trypanorhyncha) larval infection in small to mid-sized benthonic sharks in the Gulf of Naples, Mediterranean Sea.

AIM: Trypanorhyncha cestodes comprise a wide range of heteroxenous parasites infecting elasmobranchs as definitive hosts. Limited data exist on the larval infection of these cestodes and the role of intermediate and paratenic hosts in the life cycle of these parasites. We investigated the factors that determine the occurrence and the level of infection of Grillotia plerocerci in the skeletal muscles of various benthonic sharks and analyzed the parasites through an integrative taxonomic approach. LOCATION: Mediterranean Sea. METHODS: Sharks obtained as bycatch of commercial trawling activities (i.e., Etmopterus spinax, Galeus melastomus, and Scyliorhinus canicula) were used in this study. Data from a limited number of Dalatias licha and Scyliorhinus stellaris were also included. Grillotia plerocerci were molecularly characterized using the partial 28S large subunit rDNA. Boosted regression trees were used to model the relationship between the abundance of infection with both morphological and physiological predictors in each host. RESULTS: Plerocerci of Grillotia were detected in all shark species except S. stellaris. Host species significantly differed in terms of parasite abundance, with the highest and lowest prevalence and abundance of infection detected in G. melastomus and E. spinax, respectively. The relative influence of the traits involved in explaining the parasite abundance was related to the host size in G. melastomus, while both morphology- and physiology-related traits explained the patterns observed in E. spinax and S. canicula. The 28S rDNA sequences shared an identity of ∼99.40% with a Grillotia species previously found in the Mediterranean Sea. At intraspecific level, two different genotypes were found. A first type was retrieved only from D. licha, whereas a second type was found in G. melastomus, E. spinax, and S. canicula. MAIN CONCLUSIONS: Present results suggest that the two genotypes could be involved in different consumer-resource systems and confirm most of the examined shark species as transport hosts of Grillotia species for unknown larger top predators.

Morphological updates, host-specificity, molecular data and phylogenetic analysis of Acanthobothrium coronatum (Cestoda: Onchoproteocephalidea), a neglected parasite of the nursehound Scyliorhinus stellaris.

Acanthobothrium is the most speciose genus of onchoproteocephalidean cestodes, whose adults parasitize the intestine of elasmobranch fishes. Acanthobothrium coronatum, the type species of the genus described from Mediterranean elasmobranchs, remains a little known parasite, with the most recent reports dating back to the fifties. We hereby investigate host-specificity and redescribe A. coronatum from the same locality of its original description by using light and scanning electron microscopy approaches. Moreover, molecular and phylogenetic data inferred from the analysis of the 28S rDNA gene are reported for the first time. Out of the nine elasmobranch species examined from Gulf of Naples, we only detected A. coronatum in the intestine of Scyliorhinus stellaris, with infection patterns that supports evident host-specificity for this shark species. The genetic characterization of 28S rDNA showed 99.8-100% similarity with larvae previously found in Octopus vulgaris from the same area investigated here. Conspecificity between the present material and the larvae found in the octopus was also confirmed by the tree topology. The host-parasite phylogeny is discussed, even if additional molecular data are needed to clarify potential host-parasite patterns. Notwithstanding this limitation, this is the first molecular study revealing conspecificity between an adult Acanthobothrium species from a shark and the larvae found in an intermediate/paratenic host, shedding light on the transmission pathway of A. coronatum in S. stellaris. Finally, the taxonomic, molecular, and phylogenetic data presented here allow a better characterization of a neglected parasite.

The complete mitochondrial genome of the white seabream Diplodus sargus (Perciformes: Sparidae) from the Tyrrhenian sea.

The white seabream Diplodus sargus (Linnaeus, 1758) is a species of interest for commercial fisheries throughout its range of distribution and it is also reared using aquaculture techniques. Herein, we present the first complete sequence and annotation of the mitochondrial genome of this species. The D. sargus mitogenome is 16,515 base pairs in length and contains 13 protein-coding genes, 2 rRNA, 22 tRNA, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.3% A, 28.9% C, 26.8% T, and 17.0% G. Maximum likelihood analyses placed D. sargus as a sister species of Diplodus puntazzo. This study provides valuable information for further studying identification methods and evolutionary relationships of Sparidae species.

Molecular taxonomy confirms that the northeastern Atlantic and Mediterranean Sea harbor a single lancelet, Branchiostoma lanceolatum (Pallas, 1774) (Cephalochordata: Leptocardii: Branchiostomatidae).

Branchiostomatidae (lancelets or amphioxus) comprises about 30 species, several of which are well-established models in evolutionary development. Our zoological and ecological knowledge of the family is nonetheless limited. Despite evident differences can be found among known populations, the taxonomy of Branchiostoma lanceolatum (type species of the genus Branchiostoma) has never been investigated with modern methods through its range in the northeastern Atlantic and Mediterranean Sea. We address this via a multilocus molecular approach and comparing specimens collected from different European populations. Results obtained here confirm the presence of a single species inhabiting the range between the topotypical localities of B. lanceolatum (Atlantic Ocean) and of its junior synonym B. lubricum (Mediterranean Sea), without evincing geographical structure between populations. This suggests that environment most likely drives the characteristics observed in different geographic areas. The long larval phase and the slow mutation rate in lancelets may have played a key role in the evolutionary history of this iconic species.

The role of social media in compensating for the lack of field studies: Five new fish species for Mediterranean Egypt.

In the Mediterranean Sea, where biological invasions constitute a serious threat, the combination of citizen science and social networks amplified the power of proper field studies, recording species that would have otherwise presumably passed unnoticed. Based on data collected on several Facebook groups, we hereby first report the presence of five fish taxa (Kyphosus sp., Heniochus intermedius, Pomacanthus imperator, Pomacanthus maculosus and Abudefduf sp.) new for the Mediterranean Egypt, revise their distribution in the Mediterranean Sea and discuss their possible introduction pathways. Finally, we provide some considerations on the potentiality of social media for citizen science projects.

Insights into the role of deep-sea squids of the genus Histioteuthis (Histioteuthidae) in the life cycle of ascaridoid parasites in the Central Mediterranean Sea waters.

Ascaridoid nematodes comprise a wide range of heteroxenous parasites infecting top fish predators and marine mammals as definitive hosts, with crustaceans, squids, and fishes acting as intermediate/paratenic hosts. Limited data exist on the species and role of several intermediate and paratenic hosts in the life cycle of these parasites. In the aim of adding knowledge on the role of squid species in their life cycle, we have here investigated the larval ascaridoid nematodes collected from the deep-sea umbrella squid Histioteuthis bonnelli and the reverse jewel squid Histioteuthis reversa captured in the Central Mediterranean Sea (Tyrrhenian Sea). Morphological study and sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase subunit 2 (mtDNA cox2) gene locus revealed the occurrence of Anisakis physeteris and of an unidentified species of the genus Lappetascaris. Sequence analysis revealed that specimens of Lappetascaris from both squid species matched at 100% sequences previously deposited in GenBank from larval ascaridoids collected in octopuses of the genus Eledone of the Mediterranean Sea. The Bayesian inference tree topology obtained from the analysis of the fragments amplified showed that Lappetascaris specimens were included in a major clade comprising Hysterothylacium species collected in fishes of the families Xiphiidae and Istiophoridae. As regards the site of infection in the squid host species, A. physeteris larvae predominated (60.7%) in the gonads, while those of Lappetascaris (76.3%) were found infecting the mantle musculature. The overall high values of parasitic load suggest both squid species as transmitting hosts of third stage larvae of Lappetascaris to top predator fishes, as well as the umbrella squid as an intermediate/paratenic host in the life cycle of A. physeteris in the Mediterranean Sea.

Cohabiting with litter: Fish and benthic assemblages in coastal habitats of a heavily urbanized area.

Anthropogenic litter negatively impacts the marine environment and threatens biodiversity. At the same time, it represents a suitable substrate for the settlement of sessile species, thus potentially altering composition and structure of soft bottom benthic assemblages. By using a Remotely Operated Vehicle (ROV), we hereby investigated patterns of abundance, distribution, and origin of benthic litter in three subtidal habitats of a heavily urbanized area and tested whether litter distribution related to patterns of fish and mega- and macro-benthic diversity. Litter accumulation mostly occurred on soft bottoms, while rocky substrata were the least affected, albeit being particularly threatened by sea-based pollution. As expected, the highest biodiversity was observed on rocky bottoms, hosting notable biogenic formations (Cladocora caespitosa, Leptogorgia sarmentosa) despite the area is historically affected by anthropogenic activities. No correlation was found between biota and marine litter, suggesting that litter does not apparently influence biodiversity and distribution of the investigated assemblages.