Project description:Molluscan larval ontogeny is a highly conserved process typical of 3 principal developmental stages. A characteristic unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression on the microarray platform PmaxArray 1.0. A number of transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. Some of those isolated are known shell matrix genes while others are novel, these are discussed in relation to potential shell formation roles. This interdisciplinary investigation has married the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of shell biomineralization. Keywords: Temporal expression profiling by array Microarray is used to examine the temporal differential expression of transcripts from several bivalve larval development stages including 24hrs post fertilization, 3 days, 17 days, 20 days, 23 days, 26 days, 30 days, 35 days, 40 days. Differential expression profiles for transcripts of all the temporal samples was determined based on comparison to a common reference of unfertilized eggs. Each temporal larval sample included in the study has at least 3 replicate hybridizations. Dye flips have been incorporated in the replicates. A total of 46 microarray hybridizations were performed in this investigation for differential expression analysis.

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: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.

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Differential expression analysis of genes from the mantle tissue of pearl oyster: Pinctada maxima

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