Multi-transcript expression patterns in the gastrolith disk and the hypodermis of Cherax quadricarinatus at premolt

Variations in Gastrolith disk and hypodermis transcript expression paterrns related to induced molt cycle were identified in male Cherax quadricarinatus using a cDNA microarray estimated to contain 2180 unique sequences. Molt induction was performed by repeated alpha-ecdysone injections. Manipulated males were sacrificed at late premolt and reference populations included intact intermolt and sham-injected intermolt individuals. Isolated gastrolith disk and hypodermis cDNAs of premolted male crayfish were hybridized onto the microarray against the sham-injected male reference. A putative effect of the injection procedure was also tested by hybridizing gastrolith disks cDNAs of sham-injected males vs. intact intermolt males. Differentially-expressed genes were identified, sequenced, and annotated. The sham injections had almost no effect on the gene expression patterns. The late premolt stage was characterized by a dramatic up-regulation of the genes in the gastrolith disk and milder but definite down-regulation in the hypodermis. The induced gastrolith genes included structural genes of the gastrolith and genes related to protein synthesis, energy provision and cell proliferation. Many other genes revealed no resemblance to known protein or nucleotide sequences, and their functions are yet to be uncovered. It is hypothesized that some of these genes are related to the calcium carbonate transportation and gastrolith deposition machinery. The experiment submitted in this series includes male Cherax quadricarinatus in three experimental conditions. Individual variability was integrated into the hybridization design by using RNA populations of 4 (gastrolith disk) or 5 (hypodermis) individual crayfish males for each of three experimental conditions: injected-premolted, sham-injected and intact intermolted males. Four or five hybridization sets which are biological replicates, each composed of 3 slides, were carried out. In each set, one individual from each experimental group, the premolted, sham-injected and intact was hybridized vs. the other two cDNAs in a loop design, totaling 3 slides. The design was balanced with respect to the dye labeling of the RNA populations, such that each individual RNA population was labeled with one dye on one slide and with the other dye in two other slides (see graphic presentation of the hybridization design in the supplementary file GSE16866_Cq-Hybridization_design.pdf). Each of the 12 or 15 slide sets were analyzed as one integrative experiment, resulting in three binary comparisons among experimental conditions as follows: injected-premolted vs. sham-injected, sham-injected vs. intact intermolt reference, and injected-premolted vs. intact intermolt reference. Only the former two comparisons were deposited as samples. Data analysis was conducted generally according to: Yudkovski, Y., Shechter, A., Chalifa-Caspi, V., Auslander, M., Ophir, R., Dauphin-Villemant, C., Waterman, M., Sagi, A. & Tom, M. (2007). Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle. Insect Mol. Biol. 16, 661-674. Briefly, analysis was accomplished using the LIMMA package (Linear Models for Microarray data Analysis; Smyth, G.K. 2004. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3. http//www.bepress.com/sagmb/vol3/iss1/art3). The Cy5 and Cy3 intensities within each slide were normalized using global Loess method, and background correction was applied. LIMMA calculated for each spot on an individual slide an M-value [M=log2(Cy5/Cy3); Cy5/Cy3 calculated using the normalized emission intensities of the spot] and an emission intensity A-value [A=(log2(Cy5)*Cy3))/2]. Subsequently, LIMMA fitted a linear model to the expression data followed by Empirical Bayes smoothing. Inter-duplicate correlation method was used at the linear modeling step. A meanA value, which is the average spot emission intensity across the analyzed experiment, was calculated for each unique spot and only spots with meanA>9 were included in the analysis, avoiding faint emissions. The LIMMA summary statistics of each unique spot included an estimated average M-value across each binary comparison associated to the p-value, derived from a moderated t-statistic after multiple testing correction. All these parameters are found in the sample data table.