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Gene Expression Analysis of Ruditapes philippinarum Haemocytes after Experimental Perkinsus olseni Zoospore Challenge and Infection in the Wild

ABSTRACT: The production of Manila clam (Ruditapes philippinarum) is seriously threatened by the protistan parasite Perkinsus olseni. We characterized and compared gene expression of Manila clam haemocytes in response to P. olseni in a time-course (10 h, 24 h, 8 d) controlled laboratory challenge (LC), representing the first steps of infection, and in a more complex infection in the wild (WI) using a validated oligo-microarray containing 11,232 mostly annotated transcripts. Several immune-genes involved in NIK/NF-kappaB signalling, toll-like receptor signalling and apoptosis were activated at LC-10 h. However, down-regulation of others like lysozyme, histones, cathepsins and heat shock proteins indicated signals of immunodepression, which appeared exacerbated at LC-24 h, where only down-regulated genes were detected. A rebound of haemocytes activity occurred at LC-8 d as shown by up-regulation of genes involved in cytoskeleton organization and cell survival. WI showed a more complex picture and several immune-relevant processes, including cytoskeleton organization, cell survival, apoptosis, encapsulation, cell redox- and lipid- homeostasis were activated, involving the main mechanism of host response. Our results provide useful information, including potential biomarkers, to develop strategies for controlling Manila clam perkinsosis.

ORGANISM(S): Ruditapes philippinarum

PROVIDER: GSE99162 | GEO | 2019/05/13

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.

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Gene Expression Analysis of Ruditapes philippinarum Haemocytes after Experimental Perkinsus olseni Zoospore Challenge and Infection in the Wild

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