Absorbance was read spectrophotometrically using a microplate reader

d difference in expression. The distinctions between treatments, initially shown by PCA of Danoprevir differentially expressed genes, are borne out by differences in magnitude of expression, not by gene identity. Gene Ontology and Enrichment Tests Blast2GO was used for annotation of EST contigs and to test for enrichment of gene ontology terms between pairwise comparisons. Tests for enrichment of gene ontology terms found no significantly enrichment GO terms. As previously discussed, much of the differentiation between comparisons was in gene expression magnitude, not the presence or absence of different genes in the MAANOVA result. A test of GO enrichment is unable to elucidate this difference. Additionally, tests for enrichment are hampered by the lack of BLAST hits for 45% of the differentially expressed genes, precluding their inclusion in enrichment tests. Genes Involved in Thermal Injury After eight days of thermal challenge at 31uC, the nonpreconditioned corals bleached thoroughly. The differentially expressed genes between these non-preconditioned and control coral fragments illustrate the transcriptomic response of corals undergoing thermal injury. Our gene ontology analysis was informative for this comparison, with the 45 differentially expressed genes falling into GO IDs including response to oxidative stress, cellular homeostasis, and oxidation/reduction. Non-preconditioned corals are characterized by a more extreme modulation of many of the same genes differentially expressed in preconditioned corals. Notably, after eight days of thermal challenge these bleaching corals showed a marked increase in a heme-binding protein 2-like homolog, permease, glycine-rich RNA binding protein, chorion peroxidase, and a mannose-binding lectin. A decrease in transcripts was identified for homologs of a mannose-binding lectin, ricin b lectin, CD151, universal stress protein, NF-kB inhibitor, calumenin, group II decarboxylase, and prefoldin 2. It is important to distinguish that the up- and down-regulated mannose-binding lectins represent two distinct gene sequences. Spatial Ordination of Gene Expression PCA plots illustrate the spatial relationships of gene expression patterns amongst and between treatments. The first principal component separates preconditioned and nonpreconditioned treatments from controls after two, four, and eight days of thermal challenge. After eight days of thermal challenge, when non-preconditioned corals are undergoing bleaching, nonpreconditioned and preconditioned corals are distinctly different not just in their Symbiodinium density, but also in the ordination of their differential gene expression pattern, as illustrated by their separation on PC2. Differentially Expressed Genes Shared Across Treatments Following two days of 31uC thermal challenge, there is complete overlap in the identity of genes affected by non-preconditioned and preconditioned treatments during 31uC thermal challenge; all genes differentially expressed between S1 and Control are also differentially expressed between NPC and Control. However, the overlap of genes involved does not illustrate the full picture, as though the same genes are affected, the magnitude of expression varies considerable. There is a much more dramatic response from NPC/Control than from S1/Control both Gene Expression Co-occuring with Thermal Tolerance: Differential Expression between Preconditioned and Non-Preconditioned Treatments The comparison of NPC to S1 transcriptome respon