Haplosporidium Pinnae Research Articles

Internal transcribed spacer as effective molecular marker for the detection of natural hybridization between the bivalves Pinna nobilis and Pinna rudis.

The Pinna nobilis, a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae. Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species-specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis, allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex-PCR successfully identified P. nobilis, P. rudis, and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies.

Testing Bivalve Species as Potential Hosts for Haplosporidium pinnae, The Parasite Responsible for the Highly Endangered Status of the Fan Mussel Pinna nobilis

Testing Bivalve Species as Potential Hosts for Haplosporidium pinnae, The Parasite Responsible for the Highly Endangered Status of the Fan Mussel Pinna nobilis

Assessing habitat microscale conditions underlying with mortality and survival of Pinna nobilis in the Mar Menor coastal lagoon (SE Spain)

Pinna nobilis Linnaeus, 1758 is a Mediterranean endemic bivalve critically endangered due to a severe epidemic caused mainly by the parasite Haplosporidium pinnae. Presently, live populations in Spanish waters are restricted to the Ebro Delta (Catalonia) and the Mar Menor (Murcia). The coastal lagoon of the Mar Menor has faced several eutrophication crises since 2016, which pose a threat to the survival of P. nobilis. Since 2016, >95% of the specimens of this species have died due to recurrent episodes of eutrophication in the lagoon. An analysis of the mortality within the relict population of P. nobilis in the Mar Menor lagoon after the last population survey in 2019 has been carried out, together with a characterisation of some microscale factors that describe its current habitat in the lagoon. For this purpose, three sampling localities were selected along the east coast of the lagoon, where a study of both live and dead individuals was conducted. Additionally, a characterisation of the macrophyte beds and the sediment conditions surrounding these individuals was carried out, comparing the sediment near the recently dead and live individuals. All studied localities exhibit similar mixed macrophyte beds of Cymodocea nodosa (Ucria) Asch., 1870 and Caulerpa prolifera (Forsskål) J.V. Lamouroux, 1809. At the location where significantly less C. nodosa cover was observed, the mortality of P. nobilis individuals was higher, and the sediment near the dead individuals was also more disturbed (with a more negative Eh and higher percentage of organic matter). These results contribute to a better understanding of the dynamics of microscale factors following P. nobilis mortality events, facilitating the identification of potential survival areas.

Looking for the surviving optimal areas for the threatened species Pinna nobilis in a highly anthropized coastal lagoon (Mar Menor, SE Spain)

Pinna nobilis, an endemic bivalve species in the Mediterranean Sea, is critically endangered mainly due to the devastating impact of the protozoan parasite Haplosporidium pinnae. The population within the Mar Menor lagoon (Murcia, Spain) represents one of only two surviving populations along the Spanish coast. This relict population has witnessed a significant decline since 2016, primarily due to eutrophication episodes and other anthropogenic impacts affecting the lagoon. It is imperative to identify the most suitable zones for the survival of this species, where adult individuals could be translocated from affected areas to enhance their chances of survival. In the present study, we identify these zones through a Multi-Criteria Evaluation considering several factors that pose a threat to the species. This evaluation was complemented using Geographic Information Systems based multi-criteria decision analysis (GIS-MCDA). A total of 18 factors were weighted and combined, resulting in a surface area of 307.92 hectares (2.28% of the lagoon) with over 70% high suitability for species survival.

Detection of Minchinia mytili DNA in mussels (Mytilus galloprovincialis) after a mass mortality event in the Adriatic Sea

We investigated a mass mortality event of Mediterranean mussels (Mytilus galloprovincialis) in the Novigrad Sea, Croatia during winter 2022/2023. The mortality rate was up to 70% and was initially linked to an influx of freshwater from the Zrmanja River. Despite mitigation measures that included placing mussels deeper to avoid the influence of freshwater, mortalities continued. Subsequent analysis of 64 samples using cytology, histolology and real-time PCR to exclude listed diseases, yielded negative results. PCR testing for Haplosporidium pinnae revealed the presence of a haplosporidian-like DNA resembling Minchinia mytili. In the study of predominant bacteria, Psychrobacter sp., Colwellia sp., and Vibrio splendidus were detected. Histological examination showed no haplosporidium structures, and in situ hybridisation with probes for detection of Haplosporiidae did not confirmed the presence of M. mytili in mussel tissue. However, our study reports the first detection of M. mytili DNA in the Adriatic Sea, emphasising the need for extensive research and further analysis to determine the exact cause of these mass mortality events and the origin of the haplosporidium DNA.

Sanctuaries are not inviolable: Haplosporidium pinnae as responsible for the collapse of the Pinna nobilis population in Lake Faro (central Mediterranean)

The rapid spread of the protozoan Haplosporidium pinnae is having a strong negative effect on Pinna nobilis populations. A case study on a residual population in Lake Faro (Sicily, Central Mediterranean), whose long-term monitoring has revealed a dramatic decline following the 2018–2020 mass mortality event, is presented. In the framework of such monitoring, we performed tissue sampling on nine living P. nobilis, detecting the pathogen in seven of them. In contrast, other pathogens associated with P. nobilis disease in other areas, i.e., Mycobacterium spp. and Vibrio mediterranei, were not recorded. The surviving individuals (approximately twenty) showed that brackish areas only weakly mitigate the effects of H. pinnae disease and might not be resolutive. Nevertheless, the results show that Lake Faro may constitute one of the last Mediterranean P. nobilis sanctuaries.

First detection of Pinna nobilis infection by Haplosporidium pinnae in the sanctuary area of Thau lagoon, France

The noble pen shell Pinna nobilis is an endemic and emblematic giant bivalve whose populations have been recently affected by a mass mortality event (MME), primarily due to the spread of the pathogen Haplosporidium pinnae. Since the beginning of the MME in Spain in 2016, nearly one hundred percent of monitored open sea water populations have been decimated around the Mediterranean Sea. The only refuge areas with living P. nobilis populations were found in coastal lagoons. Today, the Thau lagoon in France is home to a vitally important population of P. nobilis and was thought to be safe from the parasite. Here, we report the first molecular detection of H. pinnae (isolate PN1) in tissue samples of moribund individuals from Thau lagoon.

First detection of the invasive protozoan Haplosporidium pinnae in the critically endangered bivalve Pinna nobilis in the Southern Mediterranean Sea (Bizerte Lagoon, Tunis) and update of its current status

Pinna nobilis (Linnaeus, 1758) populations have been severely damaged in the last few decades, and since early autumn 2016, a mass mortality event (MME) has drastically impacted populations in the Mediterranean Sea. Accordingly, the aim of the present study was to improve the knowledge on the status of P. nobilis populations in the Bizerte Lagoon (Tunisia) between 2016 and 2022. Before the MME, P. nobilis was found in the lagoon at depths from 1.5 to 6 m, with a density ranging from 2 to 30 ind/100 m². After the MME, mortality reached 100% in the monitored area, except in the eastern part of the Bizerte Lagoon near the Menzel Jemil site, where some living specimens were detected. Moreover, in 2022, successful recruitment was observed in the lagoon. Additionally, our results revealed the presence of Haplosporidium pinnae and Mycobacterium sp. in some living specimens sampled in the Bizerte Lagoon after the MME. This finding constitutes the first detection of both pathogens in Tunisia and in the entire Southern Mediterranean Sea.

Reproduction of the endangered fan mussel Pinna nobilis under natural and captivity conditions

Abstract A mass mortality event that started in 2016 has put the fan mussel Pinna nobilis close to the brink of extinction, leading to the species being classified as Critically Endangered in the Red List of the International Union for Conservation of Nature. Under current circumstances, with the persistence of the disease caused by Haplosporidium pinnae in the open sea, isolated populations in coastal lagoons and estuarine bays appear to be the main chance for the survival of the species. The low resilience of those populations highlights the importance of implementing additional measures to avoid the extinction of the fan mussel. One of these measures relies on completing its reproductive cycle in captivity. The reproductive window of the remaining fan mussel population in Alfacs Bay was explored to obtain gametes and to understand possible limitations in the reproductive activity in wild populations. For this purpose, during the reproductive season, several groups of individuals were relocated for short periods (~2 weeks) into the vivarium to obtain gametes under controlled conditions. Simultaneously, controlled temperature treatments and adapted food dosage for conditioning adult fan mussels out of their reproductive season in the wild were conducted in a recirculating aquaculture system. The reproduction of natural populations was mostly restricted to May, featuring temperatures around 20°C. A low success of spawning was obtained in the vivarium, with a 0% success in 11 out of 20 of the trials (six to nine individuals each) and an overall success of 16% (25 out of 156 individuals). A 100% success (14 out of 14) was obtained with individuals conditioned for ~2 months in the recirculating aquaculture system. A second conditioning of six of those individuals was attempted after a ~3‐month period, with a 33.3% success of spawning.

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.

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.

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.

Seasonality in Synergism with Multi-Pathogen Presence Leads to Mass Mortalities of the Highly Endangered Pinna nobilis in Greek Coastlines: A Pathophysiological Approach.

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.

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.

Haplosporidium pinnae Detection from the Faeces of Pinna nobilis: A Quick and Noninvasive Tool to Monitor the Presence of Pathogen in Early-Stage or during Fan Mussel Mass Mortalities

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.

RNA-Seq comparative study reveals molecular effectors linked to the resistance of Pinna nobilis to Haplosporidium pinnae parasite.

With the intensification of maritime traffic, recently emerged infectious diseases have become major drivers in the decline and extinction of species. Since 2016, mass mortality events have decimated the endemic Mediterranean Sea bivalve Pinna nobilis, affecting ca. 100% of individuals. These events have largely been driven by Haplosporidium pinnae's infection, an invasive species which was likely introduced by shipping. While monitoring wild populations of P. nobilis, we observed individuals that survived such a mass mortality event during the summer of 2018 (France). We considered these individuals resistant, as they did not show any symptoms of the disease, while the rest of the population in the area was devastated. Furthermore, the parasite was not detected when we conducted a PCR amplification of a species-specific fragment of the small subunit ribosomal DNA. In parallel, the transcriptomic analysis showed evidence of some parasite RNA indicating that theresistant individuals had been exposed to the parasite without proliferating. To understand the underlying mechanisms of resistance in these individuals, we compared their gene expression with that of susceptible individuals. We performed de novo transcriptome assembly and annotated the expressed genes. A comparison of the transcriptomes in resistant and susceptible individuals highlighted a gene expression signature of the resistant phenotype. We found significant differential expressions of genes involved in immunity and cell architecture. This data provides the first insights into how individuals escape the pathogenicity associated with infection.

Wide-Geographic and Long-Term Analysis of the Role of Pathogens in the Decline of Pinna nobilis to Critically Endangered Species

A mass mortality event (MME) affecting the fan mussel Pinna nobilis was first detected in Spain in autumn 2016 and spread north- and eastward through the Mediterranean Sea. Various pathogens have been blamed for contributing to the MME, with emphasis in Haplosporidium pinnae, Mycobacterium sp. and Vibrio spp. In this study, samples from 762 fan mussels (necropsies from 263 individuals, mantle biopsies from 499) of various health conditions, with wide geographic and age range, taken before and during the MME spread from various environments along Mediterranean Sea, were used to assess the role of pathogens in the MME. The number of samples processed by both histological and molecular methods was 83. The most important factor playing a main role on the onset of the mass mortality of P. nobilis throughout the Mediterranean Sea was the infection by H. pinnae. It was the only non-detected pathogen before the MME while, during MME spreading, its prevalence was higher in sick and dead individuals than in asymptomatic ones, in MME-affected areas than in non-affected sites, and it was not associated with host size, infecting both juveniles and adults. Conversely, infection with mycobacteria was independent from the period (before or during MME), from the affection of the area by MME and from the host health condition, and it was associated with host size. Gram (-) bacteria neither appeared associated with MME.

New insights about Haplosporidium pinnae and the pen shell Pinna nobilis mass mortality events

Since early autumn 2016, Mass Mortality Events (MME) have drastically impacted the population of the fan mussel Pinna nobilis in the Mediterranean Sea. Haplosporidium pinnae, a newly described Haplosporidian species, has been considered the causative agent of the mortality outbreaks in association to opportunistic bacterial pathogens. In the present study, we first reported a cytological description of H. pinnae in moribund specimens of P. nobilis which were collected in the Gulf of Taranto (Ionian Sea, Italy) during summer 2018. Different life-cycle stages of the parasite, including uni- and binucleate cells, small plasmodia, big multinucleate plasmodia and sporocysts with spores, were detected in all the examined animals and most of the parasite cells were present in gills, mantle and digestive gland, while the spores were found only in the latter organ. Histology and molecular biology were also performed, confirming the nature of the infectious agent, as already reported in the area. Additionally, molecular study revealed the presence of bacteria from the Mycobacterium ulcerans - M. marinum complex but no evident macroscopical or microscopical lesions, just as no bacteria referred to Mycobacterium were observed by histology. In conclusion, the present study aimed to provide further contributions to the understanding of the mortality of P. nobilis, pointing to the role of the cytological method of investigation both for diagnostic and epidemiological purposes, and discussing the current epidemic situation in the Adriatic sea.

Detection of Haplosporidium pinnae from Pinna nobilis Faeces

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.

Investigating population dynamics from parentage analysis in the highly endangered fan mussel Pinna nobilis.

Understanding dispersal patterns is a major focus for conservation biology as it influences local survival and resilience in case of local disturbance, particularly for sessile species. Dispersal can be assessed through parentage analyses by estimating family structure and self‐recruitment. This study documents the family structure of a pelagic spawner, Pinna nobilis, which is facing a major crisis that threatens its survival as most of its populations have been decimated by a parasite, Haplosporidium pinnae. In this context, we focused on a single population (Peyrefite, Banyuls‐sur‐mer, France) where 640 individuals were sampled in 2011, 2015, and 2018 and genotyped for 22 microsatellite markers. Genetic diversity was high and homogeneous among years, with mean allele numbers ranging between 13.6 and 14.8 and observed heterozygosities (H o) between 0.7121 and 0.7331. Low, but significant, genetic differentiations were found between 2011–2015 and 2015–2018. A parentage analysis described 11 clusters, including one prevailing, and revealed that 46.9% of individuals were involved in half‐sib relationships, even between years, suggesting that source populations were recurrent year after year. There were few individuals resampled between years (30 in 2015 and 14 in 2018), indicating a rapid turnover. Considering the large number of half‐sib relationships but the low number of relations per individual, we conclude that P. nobilis exhibit homogeneous reproductive success. Self‐recruitment was not detected, making this population highly vulnerable as replenishment only relies on connectivity from neighboring populations. In the context of the pandemic caused by H. pinnae, these results will have to be considered when choosing a location to reintroduce individuals in potential future rescue plans.