Measuring immunocompetence in the natural population and captive individuals of noble pen shell Pinna nobilis affected by Pinna nobilis Picornavirus (PnPV).

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 104 vs 2-5 x 105 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.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Pathology, epidemiology, and phylogeny of mussel egg disease due to the microsporidian Steinhausia mytilovum (Field, 1924) in the Mediterranean mussel (Mytilus galloprovincialis).

Microsporidia are well known fungal pathogens of aquatic animals. However, the taxonomy of microsporidia is generally poorly resolved, which has consequently constrained our understanding of their pathology and epidemiology in marine animals. To date, microsporidia have been reported in both bivalves and gastropods, and microsporidia from mollusks have been classified in different genera. Despite ongoing work to better describe these genera, including detailed microscopic and ultrastructural images, so far we lack information on microsporidian phylogeny and pathogenicity of species within these genera. Here we investigate the microsporidian parasite Steinhausia mytilovum associated with the mussel, Mytilus galloprovincialis, in natural beds and farms along coast of southern Italy. A survey of M. galloprovincialis was conducted in 13 mussel farms and one natural bed between 2009 and 2020. We found the presence of S. mytilovum in 10 of the investigated farms, with a prevalence ranging between 14 and 100% of female mussels, depending on the population and season in which they were sampled. The parasite developed in the oocytes within a sporophorous vesicle (SV) where it produced 1-3 spores per cell, both in the cytoplasm and in the nucleus. Stenhausia mytilovum elicited an infiltrative (24.8%) or a strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective tissue, in some cases accompanied by gonadal atresia (24.8%), leading to loss of gonadal architecture. In 7% of cases no reaction was observed. Ultrastructural observations revealed a mitochondrial re-organization to interact with all the phases of parasite development; the mitochondria were arranged outside the parasitophorous vesicle (PV) or directly interacting with the spore inside vesicle. There are five taxonomic clades of microsporidians as identified by SSU ribosomal gene sequence data. Maximum likelihood analysis assigned S. mytilovum within the Clade IV, defined as the Class Terresporidia, with closest genetic relationship (83.6% identity) to an undetermined invertebrate ovarian microsporidian. The constant presence, prevalence, and severity of S. mytilovum in coastline populations of M. galloprovincialis populations in southern Italy may indirectly reflect immunocompetence at both individual and population levels.

Vibrio splendidus clade associated with a disease affecting Paracentrotus lividus (Lamarck, 1816) in Sardinia (Western Mediterranean).

This work reports for the first time the so-called bald sea urchin disease of the populations of Paracentrotus lividus in Sardinia (Italy). Following the disease occurrence reported by local fishermen of about 35% on the collected specimens for commercial purpose, the phenomenon was evaluated in two areas. Animals displayed external lesions represented by loss of spines and greenish/brownish skeleton patches. Microscopy of exoskeleton and tube feet showed haemocytosis with associated bacteria; gut and intestine histopathology revealed inflammatory lesions of different type and intensity. Microbiological and molecular analysis revealed the presence of the strains belonging to the Vibrio splendidus clade. Due to the important ecological role of Paracentrotus lividus in the Mediterranean Sea, this report represents a wake-up call for the entire basin worth of further insights.

Calcium Phosphate Particles Coated with Humic Substances: A Potential Plant Biostimulant from Circular Economy.

Nowadays, the use of biostimulants to reduce agrochemical input is a major trend in agriculture. In this work, we report on calcium phosphate particles (CaP) recovered from the circular economy, combined with natural humic substances (HSs), to produce a plant biostimulant. CaPs were obtained by the thermal treatment of Salmo salar bones and were subsequently functionalized with HSs by soaking in a HS water solution. The obtained materials were characterized, showing that the functionalization with HS did not sort any effect on the bulk physicochemical properties of CaP, with the exception of the surface charge that was found to get more negative. Finally, the effect of the materials on nutrient uptake and translocation in the early stages of development (up to 20 days) of two model species of interest for horticulture, Valerianella locusta and Diplotaxis tenuifolia, was assessed. Both species exhibited a similar tendency to accumulate Ca and P in hypogeal tissues, but showed different reactions to the treatments in terms of translocation to the leaves. CaP and CaP-HS treatments lead to an increase of P accumulation in the leaves of D. tenuifolia, while the treatment with HS was found to increase only the concentration of Ca in V. locusta leaves. A low biostimulating effect on both plants' growth was observed, and was mainly scribed to the low concentration of HS in the tested materials. In the end, the obtained material showed promising results in virtue of its potential to elicit phosphorous uptake and foliar translocation by plants.

Nodular-like growth and axial thickening in gorgonians are a defensive response to endolithic cyanobacteria, involving amyloid deposition.

An accurate approach to coral disease study is critical for understanding the global decline of coral populations. Such an approach should involve the proper use of medical concepts and terminology to avoid confusion and promote clarity in the coral disease literature. Inflammatory and neoplastic disorders have been frequently confused in corals. They are both reported as 'growth anomalies' because of their possible gross similarity, but in fact they are very different types of lesions and pathologic phenomena. In this work, we assessed the distribution and prevalence of growth anomalies, externally visible as nodular-like lesions, in the soft corals Eunicella cavolinii and E. singularis in 2008-2009 in 3 different areas along the Campanian coastline of Italy. Histopathology revealed them as chronic inflammatory lesions, resembling chronic inflammatory lesions of vertebrates, encapsulating an unidentified pathogen. Congo red and Masson Fontana histochemistry highlighted an amoebocyte infiltration with the presence of new apposition of melanin coupled with amyloid sheets intended as part of the defensive response, as reported in other invertebrates. A parallel molecular analysis of 16S rRNA of the lesions suggested that the causative agent is an endolithic cyanobacterium belonging to the order Nostocales. This is the first study assessing the presence of amyloid fibrils in corals.

Mussel Shell-Derived Macroporous 3D Scaffold: Characterization and Optimization Study of a Bioceramic from the Circular Economy.

Fish industry by-products constitute an interesting platform for the extraction and recovery of valuable compounds in a circular economy approach. Among them, mussel shells could provide a calcium-rich source for the synthesis of hydroxyapatite (HA) bioceramics. In this work, HA nanoparticles have been successfully synthesized starting from mussel shells (Mytilus edulis) with a two steps process based on thermal treatment to convert CaCO3 in CaO and subsequent wet precipitation with a phosphorus source. Several parameters were studied, such as the temperature and gaseous atmosphere of the thermal treatment as well as the use of two different phosphorus-containing reagents in the wet precipitation. Data have revealed that the characteristics of the powders can be tailored, changing the conditions of the process. In particular, the use of (NH4)2HPO4 as the phosphorus source led to HA nanoparticles with a high crystallinity degree, while smaller nanoparticles with a higher surface area were obtained when H3PO4 was employed. Further, a selected HA sample was synthesized at the pilot scale; then, it was employed to fabricate porous 3D scaffolds using the direct foaming method. A highly porous scaffold with open and interconnected porosity associated with good mechanical properties (i.e., porosity in the range 87-89%, pore size in the range 50-300 µm, and a compressive strength σ = 0.51 ± 0.14 MPa) suitable for bone replacement was achieved. These results suggest that mussel shell by-products are effectively usable for the development of compounds of high added value in the biomedical field.

Ontogeny of lymphoid organs and mucosal associated lymphoid tissues in meagre (Argyrosomus regius).

This study investigates the development of lymphoid organs and mucosal tissues in larval and juvenile meagre, Argyrosomus regius. For this purpose, meagre larvae were reared from hatch to the juvenile stage, under mesocosm conditions at 18-19 °C, using standard feeding sequences with live prey and artificial food. The kidney was evident upon hatch and included a visible pronephros, with undifferentiated stem cells and excretory tubules at 1 dph (3.15 ±â€¯0.1 mm SL). The thymus was first detected 8 dph (4.49 ±â€¯0.39 mm SL) and was clearly visible 12 dph (5.69 ±â€¯0.76 mm SL), 33 dph (15.69 ±â€¯1.81 mm SL) an outer thymocytic zone and inner epithelial zone were visible. The spleen was present 12 dph, located between exocrine pancreas and intestine and by 26 dph (11.84 ±â€¯1.3 mm SL) consisted of a mass of sinusoids filled with red blood cells. Melanomacrophage centers were found 83 dph (66.25 ±â€¯4.35 mm SL) in the spleen. Between 14-15 dph (6.9 ±â€¯1.1 mm SL), goblet and rodlet cells appear in the gill and intestinal epithelium. The lymphoid organs, which appear in the order of pronephric kidney (1 dph), thymus (8 dph) and spleen (12 dph) remarkably increase in size during the post-flexion stage. While functional studies are needed to confirm the activity of the immune response, the morphology of the lymphoid organs suggest that meagre is not immuno-competent until 83 dph.

Optimisation of artemisinin and scopoletin extraction from Artemisia annua with a new modern pressurised cyclic solid-liquid (PCSL) extraction technique.

INTRODUCTION: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particular for its artemisinin content, an active ingredient at the base of most antimalarial treatments, used every year by over 300 million people. In the last years, owing to low artemisinin content, research of new ways to increase the yield of the plant matrix has led to the use of the total extract taking advantage from the synergic and stabilising effects of the other components. OBJECTIVE: In this work we evaluated and compared the content of artemisinin and scopoletin in extracts of A. annua collected in the Campania Region (southern Italy), by two different extraction processes. METHODOLOGY: Artemisia annua plants were extracted by traditional maceration (TM) in hydroalcoholic solution as a mother tincture prepared according to the European Pharmacopeia and by pressurised cyclic solid-liquid (PCSL) extraction, a new generation method using the Naviglio extractor. RESULTS: The results showed that the PCSL extraction technique is more effective than traditional methods in extracting both phytochemicals, up to 15 times more, reducing the extraction times, without using solvents or having risks for the operators, the environment and the users of the extracts. CONCLUSION: The Naviglio extractor provides extracts with an artemisinin and scopoletin content eight times higher than the daily therapeutic dose, which should be evaluated for its stability over time and biological properties for possible direct use for therapeutic purposes.

Calcium phosphate-based nanosystems for advanced targeted nanomedicine.

Synthetic calcium phosphates (CaPs) are the most widely accepted bioceramics for the repair and reconstruction of bone tissue defects. The recent advancements in materials science have prompted a rapid progress in the preparation of CaPs with nanometric dimensions, tailored surface characteristics, and colloidal stability opening new perspectives in their use for applications not strictly related to bone. In particular, the employment of CaPs nanoparticles as carriers of therapeutic and imaging agents has recently raised great interest in nanomedicine. CaPs nanoparticles, as well as other kinds of nanoparticles, can be engineered to specifically target the site of the disease (cells or organs), thus minimizing their dispersion in the body and undesired organism-nanoparticles interactions. The most promising and efficient approach to improve their specificity is the 'active targeting', where nanoparticles are conjugated with a targeting moiety able to recognize and bind with high efficacy and selectivity to receptors that are highly expressed only in the therapeutic site. The aim of this review is to give an overview on advanced targeted nanomedicine with a focus on the most recent reports on CaP nanoparticles-based systems, specifically designed for the active targeting. The distinctive characteristics of CaP nanoparticles with respect to the other kinds of nanomaterials used in nanomedicine are also discussed.

Early evidence for a virus-like agent infecting the pest snail Theba pisana (Gastropoda: Pulmonata) in Southern Italy.

The Mediterranean land snail Theba pisana (Mollusca: Helicidae) is an introduced agricultural pest in many countries around the world, including Australia, Israel, USA and South Africa. In addition, this snail is an intermediate host of parasites of importance in both human and veterinary medicine. In this study, a natural population of T. pisana snails on the Domitian coast of Italy was surveyed following a mass mortality event. By light microscopy, 30% of the collected individuals showed in the calcium cells of the digestive gland the presence of hypertrophied nuclei containing eosinophilic to weakly basophilic inclusion bodies. Ultrastructural examination by transmission electron microscopy (TEM) showed nuclear inclusions constituted by a reticulated stroma into which unenveloped, roundish virus-like particles (38±4nm in diameter) were present. To the best of our knowledge this could be the first evidence for a virus-like agent infecting the gastropod T. pisana, which may open new biocontrol perspectives of the this pest worldwide.

Proliferation, apoptosis, and fractal dimension analysis for the quantification of intestinal trophism in sole (Solea solea) fed mussel meal diets.

BACKGROUND: The evaluation of intestinal trophism, mainly the mucosal layer, is an important issue in various conditions associated with injury, atrophy, recovery, and healing of the gut. The aim of the present study was to evaluate the kinetics of the proliferation and apoptosis of enterocytes by immunohistochemistry and to assess the complexity of intestinal mucosa by fractal dimension (FD) analysis in Solea solea fed different experimental diets. RESULTS: Histomorphological evaluation of all intestinal segments did not show signs of degeneration or inflammation. Cell proliferation index and FD were significantly reduced with a diet high in mussel meal (MM; p = 0.0034 and p = 0.01063, respectively), while apoptotic index did not show any significant difference for the same comparison (p = 0.3859). Linear regression analysis between apoptotic index (independent variable) and FD (dependent variable) showed a statistically significant inverse relationship (p = 0.002528). Linear regression analysis between cell proliferation index (independent variable) and FD (dependent variable) did not show any significant correlation (p = 0.131582). CONCLUSIONS: The results demonstrated that diets containing increasing levels of mussel meal in substitution of fishmeal did not incite a hyperplastic response of the intestinal mucosa. The mussel meal, which is derived from molluscs, could mimic the characteristics of the sole's natural prey, being readily digestible, even without increasing the absorptive surface of intestinal mucosa. Interestingly, from this study emerged that FD could be used as a numeric indicator complementary to in situ quantification methods to measure intestinal trophism, in conjunction with functional parameters.

The gonadotropin releasing hormone (GnRH)-like molecule in prosobranch Patella caerulea: potential biomarker of endocrine-disrupting compounds in marine environments.

It has been reported that endocrine disrupter compounds (EDCs) interfere with the endocrine system, mimicking the action of sex steroid hormones in different species of mollusks. Prosobranchs are frequently used as a reliable bioindicator to evaluate EDC exposure. In this article, we evaluate the effects of the xenoestrogen 4-n-nonylphenol (NP) in the prosobranch gastropod Patella caerulea, which exhibits protandrous hermaphroditism as its reproductive strategy. We isolated a partial sequence of a GnRH-like molecule from the gonads of Patella caerulea. The deduced amino acid sequence is highly homologous to that reported for the Lottia gigantea GnRH. Patella caerulea GnRH (pGnRH) mRNA expression is widespread in both male and female germ lines during gametogenesis. We suggest pGnRH as a novel biomarker for the early assessment of presence of EDCs and monitoring short and long-term impacts on Patella caerulea community structure.

Nocardiosis in Mediterranean bivalves: first detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy).

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.

Cytomorphology and PCNA expression pattern in bivalves Mytilus galloprovincialis and Cerastoderma edule with haemic neoplasia.

Haemic neoplasia (HN) is a pathologic condition reported in several bivalve species in different geographic areas. In this study we describe the cytomorphological features and the proliferative behaviour, assessed by the proliferating cell nuclear antigen (PCNA), of HN in common cockle Cerastoderma edule and Mediterranean mussel Mytilus galloprovicialis. In mussels the presence of at least 5 types of atypical haemocytes was detected, including A- and B-type cells, previously described in M. edulis and Mytilus sp., with predominance of A-type cells in early phases of the disease and B-type cells in more advanced stages. PCNA immunostaining was positive for 97 to 100% of the neoplastic cells, with both cytoplasmic (A cells) and nuclear patterns (B cells). Conversely, in C. edule there was no distinctive morphological cell sub-population, and staining atypical haemocytes with PCNA (range 93 to 100%) showed nuclear expression in early phases of disease and cytoplasmic expression in more advanced stages. The above findings suggest distinct histo-pathogenetic pathways for HN in mussels and common cockles.

Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Calcium phosphates from fish bones in sunscreen: An LCA and toxicity study of an emerging material for circular economy.

The use of sustainable and natural materials is an ever-increasing trend in cosmetic. Natural calcium phosphate (CaP-N) from food by-products and especially from fisheries (i.e., bones), has been suggested as a sustainable option to chemicals commonly used in cosmetic products, in particular to UV-filters in sunscreens. However, the environmental benefits and impacts of its production and use are still uncertain as they have never been quantified. In this paper, we report on toxicological characterization of CaP-N produced from incineration of fish meal in a pilot scale plant. Furthermore, we quantified the environmental burdens linked to the partial substitution of UV-filters by CaP-N through the life cycle assessment (LCA) comparing CaP-N with zinc oxide nanoparticles (ZnO NPs) as alternative option. CaP-N consists in a biphasic mixture 53:47 of hydroxyapatite:ß-tricalcium phosphate, and is made of round particles with a diameter in the range of a few microns. Toxicity tests on 4 aquatic species (Dunaliella tertiolecta, Tigriopus fulvus, Corophium insidiosum and Gammarus aequicauda) revealed that CaP-N does not produce any adverse effect, all the species showing EC/LC50 values higher than 100 mg L-1. Moreover, during the 96 h acute toxicity test on C. insidiosum, which is a tube-building species, the specimens built their tubes with the available CaP-N, further attesting the non-toxicity of the material. The LCA study showed that the environmental performance of CaP-N is better than that of ZnO NPs for 11 out of 16 impact categories analysed in this study, especially for the categories Ecotoxicity and Eutrophication of freshwaters (an order of magnitude lower), and with the exception of fossil resources for which CaP-N has a significantly higher impact than ZnO NPs (+140 %). Concluding, our study demonstrates that the replacement of ZnO NPs with CaP-N thermally extracted from fish bones in cosmetic products can increase their safety and sustainability.

Investigation of the highly endangered Pinna nobilis' mass mortalities: Seasonal and temperature patterns of health status, antioxidant and heat stress responses.

Recently, P. nobilis populations have suffered a tremendous reduction, with pathogens potentially playing a crucial role. Considering its highly endangered status, mechanisms leading to mass mortalities were examined in one or multiple pathogens infected populations. Thus, seasonal antioxidant enzymatic activities, hsp70 and catalase mRNA levels, were investigated in two different Greek populations, during mass mortality events in summer of 2020. Samples were collected from Fthiotis and Lesvos during February (ToC 14 ± 1.2 and 15 ± 1 respectively), April (ToC 18 ± 1.2 and 17 ± 1.3 respectively), and June (ToC 24.5 ± 1.5 and 21.5 ± 1.5 respectively) 2020. In July of the same year (ToC 26.5 ± 1.7 in Fthiotis and 24.5 ± 1.7 in Lesvos), no live specimens were found. All biochemical parameters and phylogenetic analysis suggest that pathogen infection increases P. nobilis sensitivity to water temperature, subsequently leading to mass mortality. The latter was obvious in Fthiotis individuals, in which Haplosporidium pinnae was also observed with Mycobacterium spp., compared to Lesvos individuals.

A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression.

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 × 104-1.60 × 105 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.

Calcium Phosphates from Fishery Byproducts as a Booster of the Sun Protection Factor in Sunscreens.

Calcium phosphates (CaPs) have been recently proposed as a bio- and eco-compatible alternative to UV filters in sunscreens, which are in the spotlight for being associated with health risks for both people and the environment. Here, natural CaPs extracted from fish bones have been tested as a booster of the sun protection factor (SPF), that is, as material working in synergy with UV filters to increase sunscreen UV-shielding efficiency, in combination with three of the most used UV filters, namely, octocrylene (OCR), octinoxate, and padimate-O, at different concentrations (10.0 and 20.0 wt %). The material obtained by calcination at 800 °C (CaP-N) was also enriched with Zn (CaP-Zn) or Mn (CaP-Mn) in an attempt to increase its SPF-boosting abilities. CaP-N and CaP-Zn consisted of a biphasic mixture of hydroxyapatite and beta tricalcium phosphate, while CaP-Mn presented a small quantity of Mn oxides. CaP-N was the most effective at increasing the SPF of the final emulsions, doubling the SPF of the formulation containing 20.0 wt % of OCR from 40.6 to 80.8. The results show that these CaPs, produced according to a circular economy approach, can be used as effective SPF boosters to decrease the concentration of UV filters used in sunscreen, while retaining high SPF values.