Project description: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.
Project description: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.
Project description: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.
Project description: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 the resistant 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.
Project description:There is not information regards the role of miRNAs in the development of the external ear in mammals. The aim of this study was to determine the stage-specific expression of miRNAs during external ear development in order to identify miRNAs and their possible targets. GeneChip miRNA 3.0 arrays by Affymetrix were used to obtain miRNA expression profiles from mice fetal pinnae and skin back tissues at 13.5 dpc and 14.5 dpc, biological triplicates for each tissue were analyzed; one litter represents one biological replica, each litter with 16 fetuses in average.
Project description:Background: Anti-type 2 cytokine therapies represent promising interventions for chronic itch; however, their precise mechanisms in restoring nerve architecture and mitigating inflammation and pruritus remain incompletely understood. This study aimed to elucidate the mechanistic roles of IL-4, IL-13, and IL-31 in the pathophysiology of itch associated with type 2 inflammatory skin diseases. Methods: The effect of IL-4, IL-13, and/or IL-31 on neurite outgrowth and/or transcriptomic changes were analyzed in human and mouse dorsal root ganglion (DRG) neuronal cultures. Mouse ear pinnae were processed for histologic, transcriptomic, and proteomic analyses 4 days after intradermal injection of IL-4, IL-13, and/or IL-31. To evaluate functional correlations with neuronal responses, mice were subcutaneously challenged with IL-4, IL-13, and/or IL-31, and scratching behavior was monitored. Association between IL-4/IL-13–IL-4Rα axis and severity of atopic dermatitis (AD) was evaluated through correlative analyses of human DRG transcriptomic changes and AD transcriptomic datasets (GSE130588 and BioMap consortium). Results: IL-4 and IL-13 promote mouse and human DRG sensory neuron growth, with effects similar to or greater than IL-31. In mice, intradermal IL-4, IL-13, and IL-31 increased epidermal nerve growth; however, only IL-4 and IL-13 induced hyperplasia and immune cell recruitment. Multi-omic analyses revealed that IL-4 and IL-13 have a broader impact on neuroimmune interactions than IL-31. In a murine DRG neuron-eosinophil co-culture, IL-4Rα blockade reduced neurite growth. IL-13 and IL-31 elicited acute scratching, demonstrating their roles as direct pruritogens; IL-4 synergistically enhanced IL-13-induced itch, resulting in greater pruritic responses than IL-31. Additionally, a set of itch-associated genes upregulated by IL-4 and IL-13 and downregulated by dupilumab-mediated IL-4Rα blockade in human DRG neuronal cultures showed positive correlation with AD severity. Conclusions: These findings establish the IL-4/IL-13–IL-4Rα axis as a key regulator of inflammatory skin nerve innervation, neuroimmune interactions, barrier integrity, and itch response, highlighting its mechanistic role in modulating sensory neuronal function and shaping the inflammatory microenvironment that drives itch pathophysiology.