Search Results (14)

The fan mussel Pinna nobilis is the largest bivalve species in the Mediterranean Sea and provides numerous ecosystem services. It is classified as critically endangered by IUCN (International Union for Conservation of Nature) due to severe mass mortality events throughout the Mediterranean. The aims of this work are as follows: (i) to assess the current recruitment potential of the species, (ii) to enhance recruitment by keeping juveniles in controlled conditions before releasing them back into the sea, and (iii) to assess the health status of recruits. In the period 2022–2023, larval collectors were set up in the Gulf of Trieste as part of the LIFE Pinna project. The collected individuals were kept in aquaria in two different facilities under different conditions: (a) a closed system with constant water temperature, live phytoplankton, and commercial food and (b) an open system with ambient seawater temperature and commercial food. A clear temporal and spatial variability in recruitment was observed: 13 recruits were found in 2022 and 50 recruits in 2023. The live specimens were between 0.5 and 8 cm in size upon collection and larger in 2023. The growth and survival rate did not differ significantly between the two systems, but the average monthly growth and survival rate were related to the initial size of the juveniles. Full article

This review addresses the critical conservation challenges faced by Pinna nobilis, the noble pen shell, a keystone umbrella species in Mediterranean marine ecosystems. Since 2016, the species has experienced catastrophic population declines due to mass mortality events likely driven by protozoan, bacterial, and viral infections. Despite these severe circumstances, small resilient populations persist in select estuaries and coastal lagoons across the Mediterranean, offering potential for recovery. We provide a comprehensive overview on research dedicated to Pinna nobilis’ biology, genetic variation, disease dynamics, and environmental factors influencing its survival, with a focus on refugia where populations still endure. Remarkably, recent studies have revealed signs of resistance in certain individuals and the potential for hybridisation with Pinna rudis. In this context, the possible impact of the increasing occurrence of hybridisation between Pinna nobilis and Pinna rudis on the conservation of their genetic diversity should be carefully considered. This review highlights the importance of ongoing conservation efforts including habitat restoration, protection of remaining populations, assessment of past and present genetic variability, and the development of captive breeding programmes. We aim to elucidate the need for continued studies on Pinna nobilis’ biodiversity, particularly its evolutionary dynamics, genetic makeup, and the interplay of environmental variables influencing its survival and persistence. Full article

Pinna nobilis, an ecologically significant and critically endangered bivalve endemic to the Mediterranean Sea, has been classified as “Critically Endangered” by IUCN due to habitat degradation, climate change, and mass mortality events caused by the protozoan parasite Haplosporidium pinnae. Effective conservation efforts require robust molecular tools for species identification and genetic monitoring, necessitating the development of optimized DNA extraction and amplification protocols for a non-invasive sampling protocol. In this study, we evaluated multiple DNA extraction methods—Chelex-100, the sodium chloride (NaCl) method, a modified CTAB protocol, and a commercial kit, NucleoSpin Tissue Kit—using minute shell fragments from both ethanol-preserved and air-dried (dead) samples. We optimized key parameters, including incubation times, temperatures, and sample preparation, to determine the most effective protocol for obtaining high-quality DNA suitable for downstream applications. Additionally, we assessed different PCR strategies, including nested and semi-nested approaches targeting the COI gene marker, to enhance species identification. To further refine the methodology, we evaluated novel specific primers for nested PCR, improving sensitivity and specificity in detecting P. nobilis DNA from minute and degraded samples. Our results provide an optimized, cost-effective, and time-efficient workflow for non-invasive molecular identification of P. nobilis, with broad implications for conservation genetics, biodiversity monitoring, and species recovery programs. Full article

The noble pen shell (Pinna nobilis) is a key bivalve species found in the Mediterranean that has suffered dramatic declines due to mass mortality events (MMEs) caused by pathogens like Haplosporidium pinnae. This study looks at the historical population structure of P. nobilis in Malo jezero, a coastal lagoon in Mljet National Park, Croatia, using data collected before the MME. During a field survey in 2018, data on the population density, size, and age of 3800 individuals, using a grid-based transect method, were collected. The population density ranged from 7.50 to 55.83 individuals per 100 m², with an average of 25.42 individuals per 100 m², over 11 520 m², reflecting a high abundance compared to other populations. All individuals were mature, with no juveniles or signs of recent recruitment. The estimated ages ranged from 8.00 to 44.34 years, with 20 individuals exceeding the expected maximum size. The population was comprised of older individuals, making it vulnerable to sudden events, due to a lack of younger individuals. The isolation of Malo jezero may limit larval exchange with other populations, contributing to recruitment challenges. This study provides important information for understanding P. nobilis populations and supports the potential of Malo jezero for future conservation and reintroduction efforts. Full article

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. Full article

Mortalities of Pinna nobilis populations set at risk the survival of the species from many Mediterranean coastline habitats. In many cases, both Haplosporidium pinnae and Mycobacterium spp. are implicated in mass mortalities of P. nobilis populations, leading the species into extinction. In the context of the importance of these pathogens’ role in P. nobilis mortalities, the present study investigated two Greek populations of the species hosting different microbial loads (one only H. pinnae and the second both pathogens) by the means of pathophysiological markers. More specifically, the populations from Kalloni Gulf (Lesvos Island) and from Maliakos Gulf (Fthiotis), seasonally sampled, were chosen based on the host pathogens in order to investigate physiological and immunological biomarkers to assess those pathogens’ roles. In order to determine if the haplosporidian parasite possesses a major role in the mortalities or if both pathogens are involved in these phenomena, a variety of biomarkers, including apoptosis, autophagy, inflammation and heat shock response were applied. The results indicated a decreased physiological performance of individuals hosting both pathogens in comparison with those hosting only H. pinnae. Our findings provide evidence for the synergistic role of those pathogens in the mortality events, which is also enhanced by the influence of seasonality. Full article

Due to the increasing mass mortality of Pinna nobilis, mainly caused by the protozoan Haplosporidium pinnae along the Mediterranean Sea, it is necessary to develop rapid and effective methods to detect the pathogen. The present study describes the development and validation of a species-specific assay based on hydrolysis probe chemistry to detect H. pinnae DNA from faeces and pseudofaeces of P. nobilis. During a study campaign in the Gulf of Trieste (Italy) in the spring and summer of 2022, 18 samples (10 faeces and 8 pseudofaeces) were collected. DNA was isolated from all samples and the presence of H. pinnae was tested by amplifying a small portion of 18S rDNA using qPCR. The newly developed assay detected positive H. pinnae in the faeces of the fan mussel in the spring, while no evidence of an outbreak of H. pinnae was found in the summer. In addition, the method proved to be noninvasive and can be used to monitor suspected H. pinnae infections in the early stages when bivalves are still vital. Furthermore, fecal analysis allows the monitoring of P. nobilis without dissecting tissues. The presented assay can also be used to routinely monitor the progress of mass mortalities caused by H. pinnae and to screen for the pathogen in live fan mussels and other environmental matrices, such as water, sediment, and faeces from other species that can host the protozoan. Full article

Pinna nobilis (Linnaeus, 1758) is the largest bivalve endemic to the Mediterranean. It is distributed in a wide range of coastal environments, including estuaries. Pinna nobilis has recently become a critically endangered species (with almost 100% mortality) along the entire Spanish Mediterranean coast. This may be due to coinfections caused by Haplosporidium pinnae and bacterial pathogens such as Mycobacterium spp. We extensively sampled P. nobilis from Mar Menor lagoon (SE Spain), a site where individuals still survive. Using conventional PCR, we found Haplosporidium spp. in 7.1% of mantle and faecal DNA samples in different individuals of P. nobilis. We identified and quantified Haplosporidium pinnae in P. nobilis using Sanger sequencing and qPCR. Faecal H. pinnae detection is non-invasive, unlike biopsies. Therefore, this non-lethal and non-invasive sampling method could contribute to the welfare of living populations, particularly in eutrophic environments, where they are prone to septicaemia. The use of faecal DNA analysis could be a major advance in epidemiology and recovery assessment studies of P. nobilis. Full article

Due to the rapid decrease of Pinna nobilis populations during the previous decades, this bivalve species, endemic in the Mediterranean Sea, is characterized as ‘critically endangered’. In addition to human pressures, various pathogen infections have resulted in extended reduction, even population extinction. While Haplosporidium pinnae is characterized as one of the major causative agents, mass mortalities have also been attributed to Mycobacterium sp. and Vibrio spp. Due to limited knowledge concerning the physiological response of infected P. nobilis specimens against various pathogens, this study’s aim was to investigate to pathophysiological response of P. nobilis individuals, originating from mortality events in the Thermaikos Gulf and Lesvos and Limnos islands (Greece), and their correlation to different potential pathogens detected in the diseased animals. In isolated tissues, several cellular stress indicators of the heat shock and immune response, apoptosis and autophagy, were examined. Despite the complexity and limitations in the study of P. nobilis mortality events, the present investigation demonstrates the cumulative negative effect of co-infection additionally with H. pinnae in comparison to the non-presence of haplosporidian parasite. In addition, impacts of global climate change affecting physiological performance and immune responses result in more vulnerable populations in infectious diseases, a phenomenon which may intensify in the future. Full article

Noble pen shells (Pinna nobilis) along the Eastern Adriatic coast were affected by mass mortalities similarly to the populations across the Mediterranean basin. Samples of live animals and organs originating from sites on Mljet Island on the south and the Istrian peninsula on the north of the Croatian Adriatic coast were analyzed using histology and molecular techniques to detect the presence of the previously described Haplosporidium pinnae and Mycobacterium spp. as possible causes of these mortalities. To obtain more information on the pattern of the spread of the mortalities, a study was undertaken in Mljet National Park, an area with a dense population of noble pen shells. The results of the diagnostic analysis and the velocity of the spread of the mortalities showed a significant correlation between increases in water temperature and the onset of mortality. Moderate to heavy lesions of the digestive glands were observed in specimens infected with H. pinnae. A phylogenetic analysis of the detected Haplosporidium pinnae showed an identity of 99.7 to 99.8% with isolates from other Mediterranean areas, while isolated Mycobacterium spp. showed a higher heterogeneity among isolates across the Mediterranean. The presence of Mycobacterium spp. in clinically healthy animals a few months before the onset of mortality imposes the need for further clarification of its role in mortality events. Full article

Pinna nobilis populations, constituting the largest bivalve mollusk endemic to the Mediterranean, is characterized as critically endangered, threatened by extinction. Among the various factors proposed as etiological agents are the Haplosporidium pinnae and Mycobacterium sp. parasites. Nevertheless, devastation of the fan mussel populations is still far from clear. The current work is undertaken under a broader study aiming to evaluate the health status of Pinna nobilis population in Aegean Sea, after the mass mortalities that occurred in 2019. A significant objective was also (a) the investigation of the etiological agents of small-scale winter mortalities in the remaining populations after the devastating results of Haplosporidium pinnae and Mycobacterium sp. infections, as well as (b) the examination of the susceptibility of the identified bacterial strains in antibiotics for future laboratory experiments. Microbiological assays were used in order to detect the presence of potential bacterial pathogens in moribund animals in combination with molecular tools for their identification. Our results provide evidence that Vibrio bacterial species are directly implicated in the winter mortalities, particularly in cases where the haplosporidian parasite was absent. Additionally, this is the first report of Vibrio mediterranei and V. splendidus hosted by any bivalve on the Greek coastline. Full article

The endemic fan mussel (Pinna nobilis) in the Mediterranean Sea is at high risk of disappearance due to massive mortality events. The aim of the study was to evaluate the antioxidant response of P. nobilis collected in the Balearic Islands (Western Mediterranean) before and after the mass mortality event. Individuals collected before (between 2011 and 2012) and after (between 2016 and 2017) the event were analyzed by histological, molecular, and biochemical methods to compare pathogenic loads and biochemical responses. All the individuals collected during 2016–2017 presented symptoms of the disease and were positive for Haplosporidium pinnae, while acid-fast bacteria or/and Gram-negative bacteria were detected in some individuals of both sampling periods. The activities of the antioxidant enzymes catalase and superoxide dismutase in the gills were significantly lower in P. nobilis affected with the parasite compared to those in the asymptomatic ones, while levels of malondialdehyde, as an indicator of lipid peroxidation, were higher in infected individuals. When analyzing the differential effects of H. pinnae and Mycobacterium sp. on P. nobilis, it was observed that significant effects on biomarkers were only observed in the presence of H. pinnae. Co-infection of P. nobilis by H. pinnae with other pathogens such as Mycobacterium sp. constitutes a serious problem due to its high mortality rate in the Balearic Island waters. This concerning situation for P. nobilis is favored by a reduction in antioxidant defenses related to H. pinnae infection that induces oxidative stress and cell damage. Full article

The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood. Full article

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such “lower risk” areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions. Full article