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.

Preliminary study of the population and reproductive dynamics of Echinaster sepositus (Spinulosida: Echinasteridae) in Cala del Racó / Estudio preliminar de la población y dinámica reproductiva de Echinaster sepositus (Spinulosida: Echinasteridae) en Cala del Racó

Abstract Introduction: The red starfish (Echinaster sepositus) is one of the most common asteroid species in the Mediterranean Sea. However, information about their biology or their role in benthic communities is scarce. Objective: This study aims to provide new information on the ecology of this species through the temporal characterization of the population of E. sepositus in Cala del Racó (Alicante, Spain) and the in situ monitoring of its reproductive cycle. Methods: For this purpose, three study areas were established at different depths. For each of the recorded starfish, data about the size, the substrate on which it was found, the area, the depth and the sex in the case of observing the reproduction were collected. Results: A total of 19 samplings have been carried out throughout a year of study. In this way, it has been possible to observe that the density of individuals increases in the shallower zone during autumn and winter, when the temperature drops to 14.13 ºC, while it decreases in spring and summer when the temperature rises to 27.17 ºC. Those results are reversed in the deepest part of the study. The highest density of individuals (0.51 ind/m2) occurred in October. Arborescent photophilic algae and crustose coralline algae were the substrates with the highest number of E. sepositus recorded. Medium to large specimens are located preferably on crustose coralline algae or arborescent photophilic algae, while smaller individuals were mostly located on Posidonia oceanica. No specimens of E. sepositus were observed spawning. Conclusions: Data leads to assume that there is a migration of starfishes towards more superficial areas when the water is at colder temperature and towards deeper areas when the temperature increases. It is valued the possibility that there is a change in the nutritional needs of E. sepositus throughout its development. According to our observations, the future reproduction studies should be concentrated between late-summer and early-autumn.

Resumen Introducción: La estrella de mar roja (Echinaster sepositus) es una de las especies de asteroideos más comunes del mar Mediterráneo. Sin embargo, la información sobre su biología o su papel en las comunidades bentónicas es escasa. Objetivo: Este estudio pretende aportar nueva información sobre la ecología de esta especie mediante la caracterización temporal de la población de E. sepositus en la Cala del Racó (Alicante, España) y el monitoreo in situ de su ciclo reproductivo. Métodos: Con este fin se establecieron tres zonas de estudio a distintas profundidades. Para cada una de las estrellas registradas se tomaron datos de tamaño, el sustrato sobre el que se encuentra, la zona, la profundidad y el sexo en caso de observar la reproducción. Resultados: A lo largo de un año de estudio se han realizado un total de 19 muestreos. De esta forma se ha podido observar que la densidad de individuos aumenta en la zona menos profunda durante otoño e invierno, cuando la temperatura del agua baja hasta los 14.13 ºC, mientras que se reduce en primavera y verano, cuando la temperatura se eleva hasta los 27.17 ºC. Este resultado se invierte en la zona más profunda del estudio. La mayor densidad de individuos ha sido observada en octubre (0.51 ind/m2). Las algas fotófilas arborescentes y las algas coralinales costrosas han sido los sustratos con un mayor número de E. sepositus registrados. Los ejemplares de tamaños medianos a grandes se localizan preferentemente sobre algas coralinales costrosas o algas fotófilas arborescentes, mientras que los individuos de menor tamaño se sitúan mayormente sobre Posidonia oceanica. No se observaron ejemplares de E. sepositus reproduciéndose. Conclusiones: Los datos permiten presuponer que existe una migración entre las zonas más superficiales, cuando la temperatura del agua es menor, y zonas más profundas cuando la temperatura aumenta. Se valora la posibilidad de la existencia de un cambio en los requerimientos nutricionales de E. sepositus a lo largo de su desarrollo. De acuerdo con nuestras observaciones, los estudios futuros sobre la reproducción de esta especie deben concentrarse entre finales de verano y principios de otoño.

The characterization of toll-like receptor repertoire in Pinna nobilis after mass mortality events suggests adaptive introgression.

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.

Microbiological Characterization of the Biofilms Colonizing Bioplastics in Natural Marine Conditions: A Comparison between PHBV and PLA.

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/cm2). 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.

Effect of Rinse Solutions on Rhizostoma pulmo (Cnidaria: Scyphozoa) Stings and the Ineffective Role of Vinegar in Scyphozoan Jellyfish Species.

Rhizostoma pulmo is a widely distributed scyphozoan in the Mediterranean Sea. Their stings result mainly in erythema, small vesicles, or/and pain, and cause a high number of bathers to seek assistance from first-aid services during the summer season. Despite the threat that jellyfish stings represent to public health, there is disagreement in the scientific community on first-aid protocols, with the dispute largely centered around the effectiveness of vinegar. In the present research, we investigated the effect of commonly used rinse solutions on nematocyst discharge in R. pulmo and the effect of vinegar on three more scyphozoans (Aurelia sp., Cassiopea sp., and Rhizostoma luteum). Scented ammonia, vinegar, and acetic acid triggered nematocyst discharge in R. pulmo. Vinegar also caused nematocyst discharge in Aurelia sp., Cassiopea sp., and R. luteum. In contrast, seawater, baking soda, freshwater, urine, and hydrogen peroxide were considered neutral solutions that did not induce nematocyst discharge. These results indicate that the use of vinegar, acetic acid, or commercial products based on these compounds is counterproductive. Their use can worsen pain and discomfort caused not only by R. pulmo stings but also by those of any scyphozoan. The use of seawater is recommended for cleaning the R. pulmo sting site until an inhibitor solution that irreversibly prevents nematocyst discharge is discovered.

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.

An incubation water eDNA method for a non-destructive rapid molecular identification of Pinna nobilis and Pinna rudis bivalve juveniles.

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).

Tracking a mass mortality outbreak of pen shell Pinna nobilis populations: A collaborative effort of scientists and citizens.

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.