Heat maps of pathway enrichment analysis for temperature-regulated genes.

<p>(<b>A</b>) Up-regulated genes. (<b>B</b>) Down-regulated genes. Genes up or down-regulated by temperature stress at each time point were submitted to pathway enrichment analysis based on KEGG by ClueGO plugin of Cytoscape. Columns and rows in heat maps indicate treatments and enriched pathway terms, respectively. Sample names were displayed above the heat maps. Color scales represent <i>p</i> values of enrichment tests and gray cells indicate an empty value or a value >0.05.</p

Principle component analysis (PCA) of gene expression profiles.

<p>Normalized signal intensity values of all the probes called “present” in at least 2/3 (12) arrays were subjected to PCA using ArrayTrack. The x-, y- and z-axes represent PC1, PC2 and PC3, respectively. The colors and shapes of data points indicate temperature treatment and time of exposure: blue for cold (16°C), red for heat (34°C) and green for control (28°C), sphere for 2 h and square for 48 h after exposure. One of two overlapped heat-treated samples at 48 h is shown as yellow and sphere. The sample names were displayed within the figure.</p

Venn diagrams represent the number of differentially expressed genes.

<p>(A) Developmental processes-associated genes under 16, 28 and 34°C. Differentially expressed genes were identified by comparing their expression in 144 hpf samples (48 h after exposure) to that in 98 hpf samples (2 h after exposure) at the same temperature. (<b>B</b>) Temperature stress-regulated genes after 2 h of exposure. Differentially expressed genes were identified by comparing the expression of genes in 16°C-2 h and 34°C-2 h samples to that in 28°C-98 hpf samples. (<b>C</b> and <b>D</b>) Overlap between temperature-regulated genes and developmental processes-associated genes. Genes differentially expressed in 16°C-48 h and 34°C-48 h samples were compared to the development processes-associated genes identified from larvae maintained at 28°C?(28°C-144 hpf vs. 28°C-98 hpf). The official symbols of genes differentially expressed under different situations were displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037209#pone.0037209.s010" target="_blank">Table S6</a>.</p

Effects of temperature stress on the development of zebrafish larvae.

<p>(<b>A</b>) Representative images of zebrafish larvae before and after temperature stress. Zebrafish larvae at 96 hpf (maintained at 28°C from fertilization) were exposed to temperature stress for 48 h (cultured at 16, 28 or 34°C from 96 to 144 hpf). Images were taken under a stereomicroscope from Zeiss with a color CCD camera. Red and yellow dashed line indicates intestine lumen and yolk sac, respectively. SL: standard length (distance from the snout to the posterior tip of the notochord). ED: eye diameter. Scale bar: 500 µm. (<b>B</b>) Alcian blue staining of the cartilages of jaw and branchial arches. DMC: distance from the inner border of Meckel’s cartilage to the anterior end of ceratohyal. CL: ceratohyal length. Scale bar: 200 µm. (<b>C, D, E</b> and <b>F</b>) Bar charts demonstrate the effect of temperature stress on the SL, ED, DMC and CL, respectively. Horizontal axis indicates treatments described in (<b>A</b>) and (<b>B</b>). Error bars indicate standard deviations (n = 40–60). Different letters above the error bars indicate statistically significant differences determined by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (<i>p</i><0.05).</p

Expression profiles of genes in selected GO categories.

<p>(<b>A</b>) Regulation of gene expression<b>;</b> (<b>B</b>) Protein modification process; (<b>C</b>) Protein folding; (<b>D</b>) Response to stress; (<b>E</b>) Nucleic acid metabolic process; (<b>F</b>) Proteolysis; (<b>G</b>) Carbohydrate metabolic process; (<b>H</b>) Oxidation-reduction process; (<b>I</b>) Cellular component organization; (<b>J</b>) Transmembrane transport. All of the genes regulated by temperature stress were submitted to functional annotation using DAVID software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037209#pone.0037209-Dennis1" target="_blank">[63]</a> (<a href="http://david.abcc.ncifcrf.gov/tools.jsp" target="_blank">http://david.abcc.ncifcrf.gov/tools.jsp</a>) and 10 representative biological process GO categories were selected. Genes exhibiting a fold change ≥3.0 in at least one sample were displayed. Columns and rows in the heat maps represent samples and genes, respectively. Sample names were displayed above the heat maps. Color scale indicates fold changes of gene expression. Genes that belong to more than one functional category were displayed only once.</p

Validation of microarray data by qPCR.

<p>(<b>A</b>) Correlation between qPCR (x-axis) and microarray (y-axis) data. The numeric expression values of genes in selected samples were displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037209#pone.0037209.s007" target="_blank">Table S3</a>. The correlation between microarray and qPCR data were analyzed by Spearman’s rho test. Highly statistical significance [r (17) = 0.916, p = 0.00001] was observed. (<b>B</b>) Pair wise comparison of microarray and qPCR data for temperature stress-related marker genes. The expression of <i>cirbp</i> and <i>hmgb1</i> was detected in cold-treated samples (16°C-2 h and 16°C-48 h), and the expression of <i>hspb1</i> and <i>hsp47</i> were detected in heat-treated samples (34°C-2 h and 34°C-48 h), respectively. Error bars indicate standard deviations (n  = 3).</p

Experimental design.

<p>Zebrafish embryos were maintained at 28°C from fertilization to 96 hpf. Larvae at 96 hpf were exposed to 16, 28 or 34°C for 2 and 48 h, respectively. Samples exposed to different temperature were collected at 98 and 144 hpf and subjected to microarray analysis.</p

Body composition of zebrafish larvae before and after temperature stress exposure.

<p>Notes: 1) Zebrafish larvae at 96 hpf were exposed to 16, 28 or 34°C for 48 h. Samples exposed to different treatments were designated as 28°C -96 hpf, 16°C -48 h, 28°C -144 hpf and 34°C -48 h, respectively. 2) Data (µg/individual) are given as means ± standard deviations. The number of samples is displayed in parenthesis. 3) The numbers of individuals used for the measurement of wet mass, dry mass, protein, lipid and glycogen are 80, 80, 1, 20 and 20 respectively. 4) Different letters in the same row indicate statistically significant differences determined by ANOVA followed by Duncan’s multiple range test or Dunnett’s T3 test (<i>p</i><0.05).</p