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The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL -1 , the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL -1 , and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL -1 . Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray's Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases., (Copyright © 2021. Published by Elsevier Inc.)

Bivalve stocks have been decreasing in the last decades largely due to emergent diseases and consequent mass mortality episodes. Cerastoderma edule (the edible cockle) is one of the most exploited bivalves in Europe and is among the most common hosts for trematodes, the most prevalent macroparasites in coastal waters but yet poorly studied. Therefore, in the present study, this bivalve species was used as host model to determine if trematode infection exerts a negative effect on bivalve energy metabolism and balance and if the tissues targeted by different trematodes influence the metabolic cost, with physiological and biochemical consequences. Cockles were experimentally infected with two trematode species, Himasthla elongata and Renicola roscovitus, that infect the foot and palps, respectively. Trematode infection exerted a negative effect on the metabolism of C. edule, the second intermediate host, by reduction of oxygen consumption. A different host biochemical response was found depending on trematode species, especially in regard to the level of oxygen consumption decrease and the preferential accumulation of lipids and glycogen. This study represents a step towards the understanding of host-trematode relationships that can be used to better predict potential conservation threats to bivalve populations and to maximize the success of stock and disease management., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently, we reported an emerging pathology named Brown Muscle Disease (BMD) affecting Asari clams inhabiting the most productive area for this species in France, the Arcachon Bay. The main macroscopic feature of the pathology relies on the atrophy of the posterior adductor muscle, affecting the ability of clams to burry. The research of the etiological agent of BMD privileged a viral infection. Contrary to healthy clams, infected animals are always found at the surface of the sediment and exhibit 30 nm virus-like particles in muscle, granulocytic and rectal cells. In order to get more insights on the etiology and impacts of the BMD on clams, we took advantage in the present study of next generation sequencing technologies. An RNA-Seq approach was used (i) to test whether viral RNA sequences can be specifically found in the transcriptome of diseased animals and (ii) to identify the genes that are differentially regulated between diseased and healthy clams. Contrary to healthy buried animals, in diseased clams one sequence showing extensive homologies with retroviridae-related genes was detected. Among the biological processes that were affected in diseased clams, the synaptic transmission process was the most represented. To deepen this result, a new sampling was carried out and the transcription level of genes involved in synaptic transmission was determined in healthy and diseased clams but also in clams with no visible sign of pathology but located at the surface of the sediment. Our findings suggest that muscle atrophy is a latter sign of the pathology and that nervous system could be instead a primary target of the BMD agent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bivalve populations are controlled by several biotic and abiotic factors. Parasitism is among the biotic factors but is often neglected. In the present study, we focused on the transcriptomic and biochemical responses of Cerastoderma edule when parasitized as first intermediate host by the trematode Bucephalus minimus (sporocyst, the most damaging stage), and taking into account seasonal patterns. In order to test the hypothesis that the presence of B. minimus compromises cockle regular gene expression and biochemical performance and increases their vulnerability to other parasite species infection, cockles were sampled every other month during one year in Arcachon Bay (French Atlantic coast). Overall, results showed that B. minimus induced its first intermediate host defence mechanism against oxidative stress (mainly at gene level), increased host metabolism and energy demand especially in summer (revealed at both gene and biochemical level, although without significant differences) and was accompanied by a higher metacercariae abundance. Results allowed to accept the posted hypothesis and to conclude that transcriptomic and biochemical markers can provide additional and ecologically relevant information about parasite effects on their hosts, reflecting the invasion effects of pathogens but also the environmental conditions that animals experience., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared. Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD. In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production. Most of the studied parameters underlined a cause-effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The present report presents the first evidence of disseminated neoplasia (DN) in cockles Cerastoderma edule from Arcachon Bay (France). Aneuploidy of neoplastic cells allowed the use of flow cytometry (FCM) to diagnose and stage DN. A 1year survey (2007) of the prevalence and intensity (% of aneuploid circulating cells in neoplastic cockles) was conducted. Prevalences ranged from 2.2% (June) to 13.6% (May), and disease intensity ranged from 18.7% (June) to 95.5% (September). These percentages were not correlated with seawater temperature, but rather showed unexplained oscillations over the year. Prevalence and intensity of DN were higher in cockles found at the surface of sediment compared to those buried normally (11.8% vs. 6.7% and 53.0% vs. 40.6%, respectively, p<0.05). DN could thus be one mechanism leading to unexplained presence of cockles at the surface of the sediment in Arcachon Bay. Ploidy characteristics of neoplastic cells were also investigated using FCM, revealing an unusual, broad continuum of ploidy distribution from 1.6 to 9.6n. Ploidy values were not in whole numbers in contrast to the rounded values reported in other studies. Ploidy varied according to DN intensity, with the ploidy distribution of neoplastic cells from lightly-diseased cockles being unimodal (3.7n median). In contrast, highly-diseased cockles showed a bimodal ploidy distribution (3.0n and 4.7n medians). This suggests that, in cockles from Arcachon Bay, mechanisms leading to aneuploidy are complex, developing during disease progression.