After acclimation period, oysters were randomly assigned in 50-L tanks and incubated in the following acute and gradual treatment conditions. For acute temperature and acidity stress treatment, oysters were added into the tanks immediately with the maintaining conditions of temperature 25±1 °C and 31±1 °C, acidity pH7.8±0.05 and pH7.5±0.05, salinity 33±1‰ in recirculating seawater. The temperatures and pH values in the studies were near future levels and predicted for 2100 and 2300
Growth protocol
The adult pearl oysters, P.fucata, were purchased from the Marine Biology Research Station at Leizhou Bay, Guangdong Ocean University, Zhanjiang, China. Animals were pre-cultured one week in glass aquaria as performed by Fang et al (Sude Instant Sea Salt, 33±1‰ , pH8.1±0.05, at 19±1 °C) (2012).
Extracted molecule
total RNA
Extraction protocol
For microarray analysis, total RNA of mantle tissues from the two acute temperature treatments (25°C and 31°C), two acute acidity treatments (pH7.8 and pH7.5) samples at 72h and control (19°C and pH8.1) were extracted using TRIZOL Reagent (Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions.
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer's instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65 g Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer's instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5 m, PMT 100%, 10%, 16bit. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Description
Gene expression after 31°C, pH8.1,72hr in mantle
Data processing
Significant analysis of the microarray was employed to compare differentially expressed genes with an FDR <5%. Differentiallyexpressed genes between treatment groups and control group were also identified by both t-test and the fold-change criteria of the microarray and the genes with p<0.05 and fold change>2 were considered to be up- or down-regulated in compared with control. To determine the relationship between the different treatments, the total hierarchical clustering analysis was performed on the most significant genes (p<0.05; fold change>2) by cluster3.0 (de Hoon et al., 2004 [67]) and java treeview software (Saldanha, 2004[68]) using hierarchical and average linkage algorithm as long as one treatment representing significant difference. Expression trends analysis were carried out on all the most significant genes at different levels of temperature (Control, 25 C and 31 C) and acidity (Control, pH7.8 and pH7.5) treatment by used of MeV4.6 software (Saeed et al., 2006 [69]). Venn diagrams were produced using Venny program (Olivers, 2007 [70]) in order to compare and list the same and different expression trend genes in different treatment conditions. The differentially expressed genes between each treatment group and control were further analyzed by hierarchical clustering analysis and expression analysis respectively.
