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.

Haplosporidium pinnae Parasite Detection in Seawater Samples.

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 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.

Evaluation of Physiological State of Pen Shell Pinna nobilis (Linnaeus, 1758) by a Non-Invasive Heart Rate Recording under Short-Term Hyposalinity Test.

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 (Trec). Mean Trec 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 (Hrrest) and Trec 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 Trec and shell length/width, which was not the case in comparison between Hrrest 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.

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).

Trial Assay for Safe First-Aid Protocol for the Stinging Sea Anemone Anemonia viridis (Cnidaria: Anthozoa) and a Severe Toxic Reaction.

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.

Pinna nobilis in suboptimal environments are more tolerant to disease but more vulnerable to severe weather phenomena.

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.

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.

Haplosporidium pinnae sp. nov., a haplosporidan parasite associated with mass mortalities of the fan mussel, Pinna nobilis, in the Western Mediterranean Sea.

This study provides morphological and molecular characterization of a new species, Haplosporidium pinnae), very likely responsible for mass mortality of fan mussels, Pinna nobilis, in the Western Mediterranean Sea. The parasite was found in dead or moribund P. nobilis but did not occur in healthy fan mussels from locations that were not affected by abnormal mortality. Histological examination of infected fan mussels showed uninucleate cells of a haplosporidan parasite throughout the connective tissue and hemolymph sinuses of the visceral mass and binucleate cells and, rarely, multinucleate plasmodia were also detected in the connective tissue. Additionally, stages of sporulation occurred in the epithelium of the host digestive gland tubules. Spores were slightly ellipsoidal with a hinged operculum in one pole. Typical haplosporosomes were not found with TEM but vesicles with two concentric membranes resembling haplosporosomes were abundant in the cytoplasm of the multinucleate plasmodia occurring in host digestive gland tubules. SEM analysis showed multiple structures on the spore surface; some spores had two or four long tape-like filaments attached to the spore wall. Phylogenetic analysis based on the SSU rDNA sequence placed this parasite within a large clade including species of the order Haplosporida, not in the Bonamia/Minchinia subclade or the subclade containing most Haplosporidium species, but within a subclade of Haplosporidium sp. from Penaeus vannamei. Our results suggested that H. pinnae and the parasite of P. vannamei may represent a distinct new genus within the order Haplosporida.