Project description:The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with high worldwide commercial importance. Nevertheless, diseases can cause high economical losses. For this reason, the study of immune genes in bivalve mollusks has increased in the last years. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application for the study of the gene transcription profiles of hemocytes from clams challenged with Vibrio alginolyticus through a time course. A comparative analysis of gene expression was conducted between R. philippinarum infected and non-infected by V. alginolyticus clam hemocytes. Clams (n=100) were notched in the shell next to the adductor muscles and injected with 100 Âµl of Vibrio alginolyticus, strain TA15, (10^8 UFC/ml in PBS) to mimic an intramuscular infection. Controls (n=100) were injected with 100 Âµl of PBS. After stimulation, clams were returned to the tanks and maintained at 15ÂºC until sampling at 3, 8, 24, and 72 hours after challenge Hemolymph (1 ml) was withdrawn from the adductor muscle of the clams with a 0.5mm diameter (25G) disposable needle. Hemolymph from four individuals was pooled and biological replicates were taken at each sampling point. Hemolymph was centrifuged at 4Â°C at 3000 g for 10 minutes. The pellet was resuspended in 250 Âµl of Trizol (Invitrogen). Total RNA isolation was conducted following the manufacturer's specifications in combination with the RNeasy mini kit (Qiagen) for RNA purification after DNase I treatment. Gene expression profiling was performed using an R. philippinarum oligo-DNA microarray of 13,671 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA 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.

Project description:Transcriptional profiling of different clam tissues (hemolymph, extrapallial fluid and mantle) in response to brown ring disease; Brown ring disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations.

Project description:Transcriptional profiling of different clam tissues (hemolymph, extrapallial fluid and mantle) in response to brown ring disease; Brown ring disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations. Two-condition experiment (healthy/diseased), 3 tissues, 6 biological replicates/tissue/condition Please note that mantle tissues from this study were labelled with Cy3 and hybridized against a set of reference samples that are not part of this study. Thus, the Cy5 data from the associated raw data files were excluded in the analysis. Please note that hemocytes are collected from hemolymph or extrapallial fluid and hybridized on the same array (i.e. technically as dual channel) but the results were processed as though they are single channel (Cy3 and Cy5 signals are calculated). The hemocytes from extrapallial fluid were labelled with Cy3, while hemocytes from hemolymph were labelled with Cy5. The raw data files that are associated with two sample records are linked as series supplementary files and are indicated in the sample description field.

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.

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Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

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