Haplosporidium Pinnae Research Articles

Pinna nobilis in the Mar Menor coastal lagoon: a story of colonization and uncertainty

Populations of the Mediterranean fan musselPinna nobilishave progressively decreased over the last decades as a result of anthropogenic activities. The rate of decline has strongly increased since 2016, when a mass mortality event triggered by the parasiteHaplosporidium pinnaeoccurred, and evidence exists thatMycobacteriumspecies may also have played a major role in the event. Indeed, the epidemic has spread throughout the Mediterranean, although coastal lagoons seem to offer a degree of ‘resistance’ against the parasite. In the early 1980s,P. nobilisappeared in the Mar Menor lagoon and rapidly became an important component of the benthos. However, colonization of the lagoon by the fan mussel was cut short in 2016 when a massive mortality event occurred, possibly as a consequence of the environmental collapse that occurred in the lagoon, parallel to the mortality that the species suffered in the Mediterranean that same year. In this study, we estimated the spatial distribution ofP. nobilisin the Mar Menor for 3 periods: 2003-2004, 2013 and 2016. The first 2 periods use published data, and the last period uses data collected in a new campaign. The probability of occurrence for the 3 periods was estimated using random forest and random forest regression-kriging models. The main environmental variables that determined the dispersion and colonization of the bivalve in the lagoon before 2016 are also identified.

Presence of Vibrio mediterranei associated to major mortality in stabled individuals of Pinna nobilis L.

A major epizootic event attributed to Haplosporidium pinnae leading to 100% mortality of Pinna nobilis L. populations along Mediterranean coastlines started in the fall of 2016. As a result, a project to rescue 221 adult individuals of the endangered pen shell, Pinna nobilis was conducted in November 2017 in the two areas of the Spanish coast where the species was still abundant and apparently free from infection by H. pinnae: Port Lligat in the Costa Brava, and the Alfacs Bay in the Ebro Delta. For biosecurity reasons, the 106 individuals from the Ebro Delta were stabled at the IRTA facilities located next to Alfacs Bay, whereas the 115 individuals from Port Lligat were stabled in different institutions throughout the Spanish territory. Initial biopsies showed that individuals from the Ebro Delta were free of the parasite, whereas most individuals from Port Lligat were already parasitized and died in the following months. Individuals at IRTA were hold in five tanks and fed ca. 4% of their dry weight with a mix of three species of phytoplankton and fine riverine sediments (13% OM). Seawater was filtered through 10, 5 and 1 μm to ensure the absence of the parasite and disinfected with UV light. No individuals died during the 4 initial months of captivity, but two died in April–May at temperatures from 17 to 19 °C. A peak of mortalities occurred during the summer months and early fall (53%) with maximums coinciding with temperatures above 25 °C. Individuals were again analyzed by PCR and histology for the presence of H. pinnae, Mycobacteria sp., and other locally important pathogens of commercial bivalves (Vibrio splendidus, V. aestuarianus and Herpesvirus OsHV-1 microVar), and therefore considered as potential pathogens of pen shells. However, with the exception of 3 individuals that were positive for Mycobacteria sp., results were all negative for the studied pathogens. Microbiological culture and isolation of bacteria from three moribund individuals, sacrificed for study purposes, showed V. mediterranei as the dominant species, and further PCR analyses confirmed the presence of the bacterium in ten deceased individuals. Overall, our results suggest the V. mediterranei is an opportunistic pathogen of stabled individuals possibly subjected to stress from captivity, and that antibiotic treatment (Florfenicol) combined with vitamins and mineral supplementation and reduction of water temperature (15 to 18 °C), can be used to mitigate (not to eradicate) the disease. Further research is needed to determine diets and stabling conditions that minimize captivity stress and prevent the emergence of the disease.

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 associated with mass mortality in endangered Pinna nobilis (Linnaeus 1758) fan mussels.

The fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

Real-Time PCR based test for the early diagnosis of Haplosporidium pinnae affecting fan mussel Pinna nobilis

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

Presence of Vibrio mediterranei associated to major mortality in stabled individuals of Pinna nobilis L.

Presence of Vibrio mediterranei associated to major mortality in stabled individuals of Pinna nobilis L.

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.