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.info:eu-repo/semantics/publishedVersio

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

All sequence files are available from the Genbank database (accession numbers MK142774-MK142779). The protocol is available from protocols.io (DOI: dx.doi.org/10. 17504/protocols.io.xmyfk7w).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 populationsProject MAPAMA ref. 28-5310LIFE IPE INTERMARES(LIFE15 IPE ES 012)IFAPA-UCV 014/2018 and co-funded by PHENOFISH ProjectState Plan for Scientific and Technical Research and Innovation 2013-2016, MINECO, ref. PTA2015-14709-IMinisterio de Economía, Industria y Competitividad, Gobierno de España, INIA CCAA (DOC INIA 8-2013)Project BF/HEM 15-1662Ciencias del Ma

Reduced Antioxidant Response of the Fan Mussel Pinna nobilis Related to the Presence of Haplosporidium pinnae

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 a ected 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 di erential e ects of H. pinnae and Mycobacterium sp. on P. nobilis, it was observed that significant e ects 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.En prens

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.info:eu-repo/semantics/acceptedVersio

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.En prens

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

20 Pág.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.This work was funded by: DG Pesca i Medi Mari (GOIB),EsMarEs (order IEO by MITECO, Spanish government), Life UFE IP-PAF INTEMARES (LIFE15 IPE ES 012) “Gestión integrada, innovadora y participativa de la Red Natura 2000 en el medio marino español,” the research project “Estado de conservación del bivalvo amenazado Pinna nobilis en el PNAC” (OAPN 024/2010), the project RECONNECT (MIS 5017160) of the Programme Interreg V-B “Balkan-Mediterranean 2014–2020.” MTES (French Government), DREAL (Direction Régionale Environnement Aménagement Logement) and Région Occitanie (France) for funding research and monitoring of Pinna.GC and PP were contracted under the INIA-CCAA cooperative research programme for postdoctoral incorporation from the Spanish National Institute for Agricultural and Food Research and Technology (INIA) (DOC INIA 8/2013 and 15/2015). MV-L was supported by a Juan de la Cierva-Incorporación postdoctoral contract (ICJI-2016-29329, MICIU Programme). ML-S and EÁ were supported by a Personal Técnico de Apoyo contract MINECO programme (PTA2015-11709-I and PTA2015-10829- I, respectively). CP and GS were supported by the project RECONNECT (MIS 5017160) financed by the Transnational Cooperation Programme Interreg V-B “Balkan-Mediterranean 2014–2020” and co-funded by the European Union and national funds of the participating countries. CP was supported by Sorbonne University.Peer reviewe

First Detection of Photobacterium spp. in Acute Hemorrhagic Septicemia from the Nursehound Shark Scyliorhinus stellaris

The nursehound Scyliorhinus stellaris is a threatened shark species and its population in the Mediterranean Sea is declining. Programs for captive breeding and repopulation in marine protected areas (MPA) are being carried out. Unfortunately, pathogens may hinder conservation plans for this species. An impactful disease of marine animals, caused by the bacteria Photobacterium damselae, has been detected with increased frequency in recent decades in both farmed and marine animals. The aim of this work was to determine the cause of a disease outbreak in eight captive nursehounds that died after 18 months of captivity. Gross necropsy observations were indicative of a presumptive diagnosis of hemorrhagic septicemia. Histological and molecular techniques were performed, to diagnose the etiological agents that could be involved in their mortality. Phylogenetic analysis indicated the presence of P. damselae, identified as subsp. damselae by PCR-duplex, and Photobacterium swingsii in the analyzed captive nursehound Scyliorhinus stellaris

The Nursehound Scyliorhinus stellaris Mitochondrial Genome—Phylogeny, Relationships among Scyliorhinidae and Variability in Waters of the Balearic Islands

The complete mitochondrial DNA sequence of the Nursehound Scyliorhinus stellaris has been determined for the first time and compared with congeneric species. The mitogenome sequence was 16,684 bp in length. The mitogenome is composed of 13 PCGs, 2 rRNAs, 22 transfer RNA genes and non-coding regions. The gene order of the newly sequenced mitogenome is analogous to the organization described in other vertebrate genomes. The typical conservative blocks in the control region were indicated. The phylogenetic analysis revealed a monophyletic origin of the Scyliorhininae subfamily, and within it, two subclades were identified. A significant divergence of Scyliorhinus spp. together with Poroderna patherinum in relation to the group of Cephaloscyllium spp. was observed, except for Scyliorhinus torazame, more related to this last cited clade. A hypothesis of a divergent evolution consequent to a selective pressure in different geographic areas, which lead to a global latitudinal diversity gradient, has been suggested to explain this phylogenetic reconstruction. However, convergent evolution on mitochondrial genes could also involve different species in some areas of the world

The Nursehound <i>Scyliorhinus stellaris</i> Mitochondrial Genome—Phylogeny, Relationships among Scyliorhinidae and Variability in Waters of the Balearic Islands

The complete mitochondrial DNA sequence of the Nursehound Scyliorhinus stellaris has been determined for the first time and compared with congeneric species. The mitogenome sequence was 16,684 bp in length. The mitogenome is composed of 13 PCGs, 2 rRNAs, 22 transfer RNA genes and non-coding regions. The gene order of the newly sequenced mitogenome is analogous to the organization described in other vertebrate genomes. The typical conservative blocks in the control region were indicated. The phylogenetic analysis revealed a monophyletic origin of the Scyliorhininae subfamily, and within it, two subclades were identified. A significant divergence of Scyliorhinus spp. together with Poroderna patherinum in relation to the group of Cephaloscyllium spp. was observed, except for Scyliorhinus torazame, more related to this last cited clade. A hypothesis of a divergent evolution consequent to a selective pressure in different geographic areas, which lead to a global latitudinal diversity gradient, has been suggested to explain this phylogenetic reconstruction. However, convergent evolution on mitochondrial genes could also involve different species in some areas of the world