Project description:Bivalves are well known sentinel organism in the detection of environmental pollutants. Bioaccumulation of these contaminants in bivalves often manifests as specific alterations of their biological processes, which are used as biomarkers for environmental pollution. Tributyltin (TBT) is one such pollutant previously used as a biocide in marine antifouling paints, it now causes a number deleterious effects in bivalves leaching out of sediments in marine ecosystems. One effect extensively documented is shell abnormalities, including shell thickening and chambering. Changes in amino acid compositions of the shell matrix are associated with these deformations suggesting that TBT mode of action influences the biological control of shell biomineralization. This environmental toxicants effect on shell biomineralization was analyzed in this investigation at a transcriptional level in order to elucidate the normal shell biomineralization process. P. maxima animals were exposed to TBT in laboratory conditions and a concentration range for chronic and acute toxicity has been established. Animals exposed to chronic concentrations were analyzed for differential gene expression using PmaxArray 1.0 microarray platform and compared against control animals. Genes indentified as differentially expressed in association with TBT exposure included up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of novel transcripts were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This investigation has used a microarray to determine transcriptional effects of TBT on P. maxima and proposed the involvement of novel components in shell formation, aiding the elucidation of the process. Keywords: Expression profiling by array, stress response In order to determine to differential expression profiles for transcripts relevant to TBT exposure, 9 animals treated with TBT 50 ng1-1 were compared to 9 control animals untreated on a dual channel (Cy3 and Cy5) cDNA microarrays. The RNA for the 9 control animals was pooled together for a common reference while the RNA from the 9 treated animals was separated into 3 pooled replicates, each containing RNA from 3 individual animals. Each of the pooled treatment replicates were labeled (Cy3 or Cy5) as was the controls (opposing treatment label) and hybridized to a separate microarray chip, totaling 3 chips. Each chip had duplicate spot grids printed on the left and right of the chip providing technical replication. In total 6 microarrays were challenged and analyzed comprising 3 biological replicates each with 2 technical replicates.

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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Archivesica nanshaensis

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