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Transcriptome-wide analysis of wild Asari (=Manila) clams affected by the Brown Muscle Disease: etiology and impacts of the disease

ABSTRACT: 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.

ORGANISM(S): Ruditapes philippinarum

PROVIDER: GSE119518 | GEO | 2018/11/28

REPOSITORIES: GEO

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Project description:Parasites of the genus Perkinsus spp. cause high mortalities and economic losses to the most noticeable bivalves produced in the worldwide aquaculture. In this study, we analyze how P. olseni influences the gene expression profiles of hemocytes from Manila clam (Venerupis philippinarum) using experimental infections along a temporal series and a Manila clam immune-enriched DNA microarray. Healthy and Perkinsus-infected clams (V. philippinarum) were obtained from Carril and Pontevedra shellfish farms, respectively (Galicia, NW Spain). The presence-absence of P. olseni was confirmed using the Ray`s fluid thioglycollate medium assay (RFTM) (Ray, 1966). Healthy clams were maintained in an open circuit filtered sea water tanks at 15°C with aeration. Natural infected animals were maintained in the same conditions using closed circuit sea water. All animals were fed daily with a mixture of microalgae containing Phaeodactylum tricornutum, Isochrysis galbana and Rhodomonas lens. Clams were acclimatized to the aquarium conditions for one week before the experiments were conducted. Perkinsus trophozoites were isolated from naturally infected animals following the protocol established by Ford et al., (2002). The concentration was adjusted to 5x104 trophozoites /ml in filtered sea water (FSW). Healthy clams (P. olseni free animals) (n=100) with a weight of 2.25 ± 0.64 g soft tissue, were notched in the shell and intramuscularly injected with 100 µl of the trophozoites suspension. Control animals (n=100) were injected with 100 µl of FSW. After infection, clams were maintained in 50 l tanks with aeration.Twenty animals from each experimental group and time point were sampled at 5, 10, 14, and 31 days post infection (pi).Hemolymph were extracted to perform microarrays experiments. In each condition hemolymph from three five individuals was pooled. Total RNA isolation was conducted following the manufacturer’s specifications. Isolated RNAs were treated with DNase I and purified again using the RNeasy Mini kit (Qiagen). A 8x15K Agilent 60-mer oligo-microarray (GPL16450) was used to compare gene expression profiles of clams after P. olseni infection with uninfected animals. The Agilent Feature Extraction Software (version 9.5.1) was used for the data extraction and background subtraction following standard procedures. The GeneSpring software (Agilent) was used to normalize and analyze the microarray fluorescence data.

Project description:A C. gallina microarray platform was developed to assess variations on transcritpomic profiles between different seasons and sampling sites A comparative analysis of gene expression was conducted in Chamelea gallina collected in two different areas along Abruzzi coasts subjected to different mortality events. In particular sampling activities have been performed in collaboration with fishing cooperatives along Abruzzi coasts, Chamelea gallina clams have been collected from two sites (T4 and T7) at 0.25 mi (ca. 0.4 km) at commercial size (25 mm) and harvested by hydraulic dredge. The harvesting has been conducted in three periods of 2014 (July, August and September). The time of sampling has been obliged by a dramatic reduction of harvesting in T4 that has been reported by fishing cooperatives during spring. After sampling, clams have been kept with ice till their arrival in laboratory, within 6 hours. From each sampling site and each period, the digestive gland of 30 clams has been dissected and frozen at -21Â°C, for a total of 150 clams divided in 5 pools for each period and each site of sampling. During the clam sampling, from the same sites, the upper layer of bed sediments (0â10 cm) was collected by a small grab sampler in July and September. Composite sediment samples have been refrigerated during transport and stored at â 20 Â°C within a few hours from sampling, until analysis. Briefly, about 400 g of sediment have been suitably homogenized and dried in an oven at 40 Â° C for 24 hours, then crushed, ground and sieved to 2 mm. A portion of about 100 g for each sample has been further ground and screened to 0.2 mm for heavy metal analysis. The content of cadmium (Cd), lead (Pb) and arsenic (As) have been performed, after extraction by aqua regia, by Inductively Coupled Plasma-Optical Emission Spectrometer ( ICP-OESâ¦..) the analysis have been recorded as means triplicate measurements. The determination of Hg have been done in Atomic Adsorption with Hydride System. Ten grams (accuracy Â± 0.0001 g) of dry and homogenized sediment of 0.2 mm has been put into a clean centrifuge tube, and a 1:1 (v/v) acetone/n-hexane (5 mL), and surrogate standard mixture (2-fluorobiphenyl and 4-terphenyl-d14) solutions were then added, after extraction and clean-up processes, the samples were analyzed with a gas chromatographerâelectron capture detector (GM-ECD) to evaluate concentrations of Polycyclic aromatic hydrocarbons (PAHs) and organophosphate pesticides (OPPs). Organochloride pesticides (OCPs) have been analysed, after purification, with static headspace GC Gene expression profiling was performed using an Chamelea gallina-specific oligo-DNA microarray of15,019 probes representing 12,064 transcripts based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

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Transcriptome-wide analysis of wild Asari (=Manila) clams affected by the Brown Muscle Disease: etiology and impacts of the disease

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