2.10 Analysis of stress response genes
To reveal RNA expression changes in response to stress, we acclimated
100 adult shrimp for seven days at 28 °C in oxygenated water in
individual 120 L buckets. Shrimp were fed with clams at 5% of the total
shrimp weight daily. Every day, 50% of the water was changed. To
evaluate the transcriptional response of the shrimp to cold stress, 20
shrimp were maintained at 28 °C (CT, controls) and 20 shrimps were
maintained at 10 °C (LT) chiller, and the eyestalks from both groups
were sampled for RNA extraction at 12 h and 48 h, at which time the
shrimps began to die according a pilot experiment conducted under the
same conditions. Eyestalk mRNA was subjected to transcriptome sequencing
using the Illumina Hiseq X Ten system (carried out by Novogene
Biotechnology, Tianjin, China). We filtered the raw data to remove those
reads containing ambiguous bases (‘N’) or large numbers of low-quality
positions (> 10 positions with quality scores <
10). Hisat2 (v2.0.4) (Kim et al., 2015) was then used to map the high
quality reads to the M. japonicus genome. Read counts were
calculated using HTseq software (Anders et al., 2015) and the normalized
read counts (fragments per kilobase of transcript per million mapped
reads; FPKM) was used to estimate the expression level of the genes.
Differentially expressed genes (DEGs) were identified using DESeq2 (Love
et al., 2014) and genes with a log2 (fold-change (FC)) ≥1 and adjustedp -value <0.05 were defined as significant DEGs.