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The Pinna nobilis , a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae . Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species-specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis , allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex-PCR successfully identified P. nobilis , P. rudis , and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

The philopatric and sedentary nature of female S. canicula, its high abundance as a bycatch and resilience to regular exploitation by bottom trawl fisheries and its widespread distribution, makes it a potentially good candidate as a biomonitor species. To evaluate this potential, microwave-assisted extraction (MAE) and Graphite Furnace Atomic Absorption Spectrometry (GF-AAS) were used to analyse Pb, Cd, and Cu in muscle tissue of individuals captured in the Western Mediterranean (Alicante, Spain). A monthly assessment of the concentration of these three analytes was conducted from November 2019 to November 2020 with 300 individuals. Results showed the existence of slightly higher mean concentrations during warmer seasons for Pb and Cd with mean concentrations for Pb and Cu peaking in Autumn 2019 and during Spring 2020 in the case of Cd. Significant differences in analyte concentrations found between non-consecutive months suggested gradual variability in time. Although space-wise, time-persistent concentration hotspots were observed throughout the sample area, the magnitude of these appeared to be variable in time and should be evaluated in future studies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 10 4 vs 2-5 x 10 5  cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plastic pollution is widespread in oceans and the ingestion of plastic by marine organisms is causing concern about potential adverse effects. The purpose of this study was to analyze the different types of plastics in the digestive tract of female small-spotted catshark (Scyliorhinus canicula). An alkaline digestion method using 10 % potassium hydroxide (KOH), was used. The samples were filtered and visually observed to classify the plastics according to size, shape, and color. Raman spectroscopy was further employed to identify the polymer types. The study found the presence of plastics in 89.5 % of the 200 females analyzed, including 10 polymers, with polystyrene (PS), polyamide-6 (PA6), polyvinyl chloride (PVC), and silicone rubber (SR) being the most common. The polymers identified largely reflect the results of similar studies in the marine environment and were similar to global polymer diversity of microplastics, which highlights the potential of S. canicula females for biomonitoring microplastic pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: The widespread mass mortality of the noble pen shell ( Pinna nobilis ) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations., Methods: We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome., Results and Discussion: In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 10 4 -1.60 × 10 5 ml -1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml -1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales , family Marnaviridae . The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus -a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IG declared a past co-authorship with the authors FC, DP, GD, JT-M, and JG-M to the handling editor., (Copyright © 2023 Carella, Prado, De Vico, Palić, Villari, García-March, Tena-Medialdea, Cortés Melendreras, Giménez-Casalduero, Sigovini and Aceto.)

The fan mussel Pinna nobilis is currently on the brink of extinction due to a multifactorial disease mainly caused to the highly pathogenic parasite Haplosporidium pinnae , meaning that the selection pressure outweighs the adaptive potential of the species. Hopefully, rare individuals have been observed somehow resistant to the parasite, stretching the need to identify the traits underlying this better fitness. Among the candidate to explore at first intention are fast-evolving immune genes, of which toll-like receptor (TLR). In this study, we examined the genetic diversity at 14 TLR loci across P. nobilis , Pinna rudis and P. nobilis  ×  P. rudis hybrid genomes, collected at four physically distant regions, that were found to be either resistant or sensitive to the parasite H. pinnae . We report a high genetic diversity, mainly observed at cell surface TLRs compared with that of endosomal TLRs. However, the endosomal TLR-7 exhibited unexpected level of diversity and haplotype phylogeny. The lack of population structure, associated with a high genetic diversity and elevated dN/dS ratio, was interpreted as balancing selection, though both directional and purifying selection were detected. Interestingly, roughly 40% of the P. nobilis identified as resistant to H. pinnae were introgressed with P. rudis TLR. Specifically, they all carried a TLR-7 of P. rudis origin, whereas sensitive P. nobilis were not introgressed, at least at TLR loci. Small contributions of TLR-6 and TLR-4 single-nucleotide polymorphisms to the clustering of resistant and susceptible individuals could be detected, but their specific role in resistance remains highly speculative. This study provides new information on the diversity of TLR genes within the P. nobilis species after MME and additional insights into adaptation to H. pinnae that should contribute to the conservation of this Mediterranean endemic species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Biodegradable polymers offer a potential solution to marine pollution caused by plastic waste. The marine biofilms that formed on the surfaces of poly(lactide acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were studied. Bioplastics were exposed for 6 months to marine conditions in the Mediterranean Sea, and the biofilms that formed on their surfaces were assessed. The presence of specific PLA and PHBV degraders was also studied. PHBV showed extensive areas with microbial accumulations and this led to higher microbial surface densities than PLA (4.75 vs. 5.16 log CFU/cm 2 ). Both polymers' surfaces showed a wide variety of microbial structures, including bacteria, fungi, unicellular algae and choanoflagellates. A high bacterial diversity was observed, with differences between the two polymers, particularly at the phylum level, with over 70% of bacteria affiliated to three phyla. Differences in metagenome functions were also detected, revealing a higher presence of proteins involved in PHBV biodegradation in PHBV biofilms. Four bacterial isolates belonging to the Proteobacteria class were identified as PHBV degraders, demonstrating the presence of species involved in the biodegradation of this polymer in seawater. No PLA degraders were detected, confirming its low biodegradability in marine environments. This was a pilot study to establish a baseline for further studies aimed at comprehending the marine biodegradation of biopolymers.

In this study, we investigated the presence of the parasite Haplosporidium pinnae , which is a pathogen for the bivalve Pinna nobilis , in water samples from different environments. Fifteen mantle samples of P. nobilis infected by H. pinnae were used to characterize the ribosomal unit of this parasite. The obtained sequences were employed to develop a method for eDNA detection of H. pinnae . We collected 56 water samples (from aquaria, open sea and sanctuaries) for testing the methodology. In this work, we developed three different PCRs generating amplicons of different lengths to determine the level of degradation of the DNA, since the status of H. pinnae in water and, therefore, its infectious capacity are unknown. The results showed the ability of the method to detect H. pinnae in sea waters from different areas persistent in the environment but with different degrees of DNA fragmentation. This developed method offers a new tool for preventive analysis for monitoring areas and to better understand the life cycle and the spread of this parasite.

A non-invasive laser fiber-optic method based on infrared sensors for heart rate (Hr) recording was applied to assess the physiological condition of Pinna nobilis . During 2017, the specimens of P. nobilis were sampled at three sites within the Boka Kotorska Bay, Montenegro and used for ex situ experiments with short-term reduction/restoration of ambient salinity to evaluate their physiological adaptive capacity based on heart rate recovery time (T rec ). Mean T rec for specimens from Sv. Nedjelja (reference site), Dobrota and Sv. Stasije were 72 ± 3, 91 ± 7 and 117 ± 15 min, while the coefficients of variation (CV) were 0.12, 0.13 and 0.17, respectively. Resting heart rate (Hr rest ) and T rec showed statistically significant differences between the groups of mussels from Dobrota and Sv. Stasije in comparison to the reference site. Statistically significant correlations were observed between T rec and shell length/width, which was not the case in comparison between Hr rest and shell length/width. The lower adaptive capacity within the P. nobilis specimens from Dobrota and Sv. Stasije in comparison to the reference site could occur due to stress induced by deterioration of environmental conditions, which could have led to impairment of the physiological state of the mussels evaluated by Hr. All the specimens of P. nobilis survived the experimental treatments; afterwards, they were successfully transplanted at the Dobrota site. The experimental unit with sensor technology applied in this study can provide Hr recording in real time and could have an application in monitoring the physiological/health state of P. nobilis individuals maintained in aquaria.

Background: The measurement of the energy available for growth (scope of growth, SFG) can be used in bivalves to make a long-term prediction in a short-term experiment of the condition of the individual. In order to tackle the best conditions for captive maintenance of Mediterranean Pinnids, a SFG study was conducted using Pinna rudis as a model species. Three diets were examined to test the viability of live microalgae and commercial products: i) a control diet using 100% of live microalgae based on the species Isochrysis galbana (t-ISO), ii) a 100% of commercial microalgae diet based on the product Shellfish Diet 1800®, and iii) a 50/50% mix diet of I. galbana (t-ISO) and Shellfish Diet 1800®., Results: SFG results showed significant differences among diets in the physiological functions measured and suggested lower acceptability and digestibility of the commercial product. Negative SFG values were obtained for the commercial diet which indicates that it should be rejected for both Pinnid maintenance. The mixed diet showed improved physiological performance compared to the commercial diet, resulting in a higher SFG that had no significant differences with the control diet. However, in the long-term, the lower digestibility of the mixed diet compared to the control diet could lead to a deterioration of individuals' conditions and should be considered cautiously., Conclusions: This work represents the first case study of SFG in Pinna spp. and provides fundamental data on dietary needs for the critically endangered species, P. nobilis., (© 2022. The Author(s).)

The pen shell Pinna nobilis is critically endangered due to a disease that has affected all open water populations since late 2016. Collection of early spats is considered a fundamental step for pen shell conservation. However, the identification between P. nobilis and P. rudis juveniles by morphology is a very difficult task. Furthermore, due to the small size of juveniles and high sensitivity to handling, the sampling for this purpose must not damage individuals. As a consequence, the application of molecular techniques for conservation strategies to identify threatened and endangered bivalve species is every day more and more necessary. In this study, we present the development of a multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples. Using species-specific primers, designed in the rRNA16S and rRNA12S mitochondrial genes, identification of species was obtained by cellular or extracellular DNA dissolved in water and differentiated based on the size of the amplified DNA fragments. • Development of a molecular multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples • Using specie-specific primers, the different species can be differentiated basing on the size of the amplified DNA fragments • This technique removes many of the limitations commonly associated with sampling of threatened and endangered juvenile bivalves for conservation strategies (sampling does not damage individuals)., Competing Interests: The authors certify that the material presented here is original work which has not been previously published. All biological samples were collected during management operations as part of a study plan approved by the Generalitat Valenciana (Conselleria de Agricultura, Medio Ambiente, Cambio Climático y Desarrollo Rural) and the Spanish Ministry for the Ecological Transition and the Demographic Challenge. Appropriate ethics, permissions to manipulate animals and other approvals were obtained for the submitted research. X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Anemonia viridis is an abundant and widely distributed temperate sea anemone that can form dense congregations of individuals. Despite the potential severity of its sting, few detailed cases have been reported. We report a case of a severe toxic reaction following an A. viridis sting in a 35-year-old oceanographer. She developed severe pain, itching, redness, and burning sensation, which worsened one week after treatment with anti-inflammatories, antihistamines and corticosteroids. Prompted by this event, and due to the insufficient risk prevention, lack of training for marine-environment users, and lack of research into sting-specific first-aid protocols, we evaluated the cnidocyst response to five different compounds commonly recommended as rinse solutions in first-aid protocols (seawater, vinegar, ammonia, baking soda, and freshwater) by means of the Tentacle Solution Assay. Vinegar and ammonia triggered an immediate and massive cnidocyst discharge after their application and were classified as activator solutions. Baking soda and freshwater were also classified as activator solutions, although with a lower intensity of discharge. Only seawater was classified as a neutral solution and therefore recommended as a rinse solution after A. viridis sting, at least until an inhibitory solution is discovered.

We examined a disease outbreak of the fan mussel, Pinna nobilis (L.), in the Alfacs Bay (South Ebro Delta, Spain) during a period of two years in three zones exposed to a summer salinity gradient resulting from agricultural freshwater discharges and distance to the open sea. Long-term monitoring was also conducted in Fangar Bay (North Ebro Delta), featuring lower salinities and no evidence of disease. Results showed that the salinity gradient of Alfacs Bay (37.4-35.7) was associated to cumulative mortality (100% near the mouth, 43% in middle regions, and 13% in inner regions), thus hindering the spread of pathogens. Young specimens showed to be more tolerant to disease than large adults but become vulnerable over time. In Fangar Bay, lower salinities (30.5-33.5) prevented the disease but individuals were highly vulnerable to Storm Gloria which caused 60% mortality in 3 weeks, and ~100% in 6 weeks., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present work, which is the first comparative study of the growth of the fan mussel Pinna nobilis in the western Mediterranean, encompasses 12 populations of this species living in different environments in France and Spain. Two hundred nine shells were processed and used to obtain growth records from the posterior adductor muscle scar. Size-at-age data were fitted to the Von Bertalanffy growth model. Considerable variability in growth parameters and age was detected among the populations. The results show that the only two fan mussel populations remaining in Spain, which live in an estuary and a coastal lagoon, occupy habitats that are optimal for fast growth, but individuals show low longevity, complicating the long-term conservation of the species. Multivariate analyses groups the populations into three groups (SO, EO and LG), and a general model is proposed for each group; the model can be used as an approximation to calculate the ages of individuals living in similar environments., (Copyright © 2019. Published by Elsevier Ltd.)

A mass mortality event is devastating the populations of the endemic bivalve Pinna nobilis in the Mediterranean Sea from early autumn 2016. A newly described Haplosporidian endoparasite (Haplosporidium pinnae) is the most probable cause of this ecological catastrophe placing one of the largest bivalves of the world on the brink of extinction. As a pivotal step towards Pinna nobilis conservation, this contribution combines scientists and citizens' data to address the fast- and vast-dispersion and prevalence outbreaks of the pathogen. Therefore, the potential role of currents on parasite expansion was addressed by means of drift simulations of virtual particles in a high-resolution regional currents model. A generalized additive model was implemented to test if environmental factors could modulate the infection of Pinna nobilis populations. The results strongly suggest that the parasite has probably dispersed regionally by surface currents, and that the disease expression seems to be closely related to temperatures above 13.5 °C and to a salinity range between 36.5-39.7 psu. The most likely spread of the disease along the Mediterranean basin associated with scattered survival spots and very few survivors (potentially resistant individuals), point to a challenging scenario for conservation of the emblematic Pinna nobilis, which will require fast and strategic management measures and should make use of the essential role citizen science projects can play.

Noble pen shell or fan mussel, Pinna nobilis Linnaeus (1758), protected since 1992, was incorporated into the Spanish Catalogue of Threatened Species (Category: Vulnerable, Royal Decree 139/2011). The status is presently in the process of being catalogued as critically endangered, pending approval by Spanish Government (https://www.mapama.gob.es/es/biodiversidad/participacion-publica/Borrador_OM_situacion_critica.aspx). The International Union for the Conservation of Nature (IUCN) alerted the countries of the Mediterranean basin to the "emergent situation" due to serious mortality events suffered by the fan mussel, putting it in serious risk of extinction. Thus, emergency actions have been implemented by Spanish authorities in which several research institutes from all over the country are involved. The parasite, Haplosporidium pinnae, was recently characterized by histology, TEM, SEM and molecular biology techniques and it was considered responsible for the mass mortality of P. nobilis in the Mediterranean Sea. In this context, the aim of this study has been to develop species-specific quantitative PCR (qPCR) protocol carrying out a fast, specific and effective molecular diagnose of H. pinnae. In this sense, the detection limit for qPCR was equal to 30 copies of SSU rDNA / ng of DNA using plasmid alone and when 100ng DNA of non-infected oyster were added. The qPCR assay revealed that 94% of the 32 analysed mantle tissues of fan mussel were infected by H. pinnae, showing a high sensitivity and specificity for its detection (100% if we don't consider negative and too much degraded samples). This technique will allow us to make quicker follow-ups of the disease, allowing us to get a better understanding of its evolution in order to help in the rescue of P. nobilis populations., Competing Interests: The authors declare that no competing interests exist.

For marine meta-populations with source-sink dynamics knowledge about genetic connectivity is important to conserve biodiversity and design marine protected areas (MPAs). We evaluate connectivity of a Mediterranean sessile species, Pinna nobilis. To address a large geographical scale, partial sequences of cytochrome oxidase I (COI, 590 bp) were used to evaluate phylogeographical patterns in the Western Mediterranean, and in the whole basin using overlapping sequences from the literature (243 bp). Additionally, we combined (1) larval trajectories based on oceanographic currents and early life-history traits and (2) 10 highly polymorphic microsatellite loci collected in the Western Mediterranean. COI results provided evidence for high diversity and low inter-population differentiation. Microsatellite genotypes showed increasing genetic differentiation with oceanographic transport time (isolation by oceanographic distance (IBD) set by marine currents). Genetic differentiation was detected between Banyuls and Murcia and between Murcia and Mallorca. However, no genetic break was detected between the Balearic populations and the mainland. Migration rates together with numerical Lagrangian simulations showed that (i) the Ebro Delta is a larval source for the Balearic populations (ii) Alicante is a sink population, accumulating allelic diversity from nearby populations. The inferred connectivity can be applied in the development of MPA networks in the Western Mediterranean.

A long-term experimental approach was undertaken to assess viability and resilience of the endangered Mediterranean fan mussel Pinna nobilis. Artificial and natural recruitment, mortality, population traits and juvenile growth were assessed in seasonal and annual surveys. In the Columbretes Islands, P. nobilis thrives in differing substrate types, from coarse sand to boulders, in Cymodocea nodosa meadows and among rhodoliths, and is always found sharing habitat with the less abundant sibling species P. rudis. In artificial collectors larval settlement occurred over a several months period, concentrating its peak in September and resulting from two separated spawning events. Recruitment in the collectors showed high inter-annual variability and was independent of depth, but positively correlated with seasonal water temperature increase in June. Natural recruitment of P. nobilis was low and showed little variability, evidencing the existence of intense post-settlement processes. Adult mortality was also low, thus leading to slow population dynamics and to the species' vulnerability to catastrophic events. Population size structure suggests the existence of a refuge size above 45 cm shell length. The fast growth during the first years of life would help shortening this vulnerability period. Altogether, essential information and tools for the species' conservation are provided, which will be critical in the current context of mass mortalities affecting P. nobilis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The pen shell Pinna nobilis (also known as the fan mussel) is an endemic bivalve of the Mediterranean Sea. Threatened by human activities, it has been listed as an endangered and protected species under the European Council Directive 92/43/EEC since 1992. The ecological role of this species is of importance because it filters and retains large amounts of organic matter from suspended detritus contributing to water clarity. In addition, as a hard substrate in the soft-bottom seafloor, it provides a surface that can be colonized by other (floral and faunal) benthic species. Here, we provide an overview of all available published studies on the pen shell, compiling available data and summarizing current knowledge on the conservation status and viability of populations over the full range of the Mediterranean Basin. Additionally, we discuss the different practices in applied methodology and identify gaps and new research areas in order to render conservation programmes of the species more effective., (© 2015 Elsevier Ltd. All rights reserved.)