Transcriptome Analysis Revealed the Early Heat Stress Response in the Brain of Chinese Tongue Sole (Cynoglossus semilaevis)

Abstract

As a common influencing factor in the environment, temperature greatly influences the fish that live in the water all their life. The essential economic fish Chinese tongue sole (Cynoglossus semilaevis), a benthic fish, will experience both physiological and behavioral changes due to increases in temperature. The brain, as the central hub of fish and a crucial regulatory organ, is particularly sensitive to temperature changes and will be affected. However, previous research has mainly concentrated on the impact of temperature on the gonads of C. semilaevis. Instead, our study examines the brain using transcriptomics to investigate specific genes and pathways that can quickly respond to temperature changes. The fish were subjected to various periods of heat stress (1 h, 2 h, 3 h, and 5 h) before extracting the brain for transcriptome analysis. After conducting transcriptomic analyses, we identified distinct genes and pathways in males and females. The pathways were mainly related to cortisol synthesis and secretion, neuroactive ligand-receptor interactions, TGF beta signaling pathway, and JAK/STAT signaling pathway, while the genes included the HSP family, tshr, c-fos, c-jun, cxcr4, camk2b, and igf2. Our study offers valuable insights into the regulation mechanisms of the brain's response to temperature stress.

Keywords: Cynoglossus semilaevis; gene; heat shock; transcriptome.

Figure 1

Profiling of high-temperature treatment experiment and RNA-seq analysis. (A) Schematic illustration of C. semilaevis subjected to both control (CT—22 °C) and heat stress (HS—28 °C) treatments. Samples from the control group were collected before treatment, while samples from the heat stress group were collected at 1, 2, 3, and 5 h post-treatment. (B) Sequential methodologies for transcriptome analysis: 1. Genomic DNA extraction from caudal fin samples for genotypic sex determination. 2. Total RNA extraction from brain samples using Trizol reagent. 3. cDNA library construction. 4. Transcriptome sequencing. 5. Bioinformatic analysis including time series analysis, enriched KEGG pathways analysis, and differentially expressed gene analysis were conducted subsequently. Figure created using BioRender.com.

Figure 2

Differentially expressed gene analysis in females and males after heat shock. (A) Number of DEGs identified between control (CT) and heat stress (1 h, 2 h, 3 h, and 5 h) groups from females and males. Red color indicates up-regulated and blue color indicates down-regulated genes in HS vs. CT, respectively. (B) In females, distribution of DEGs depicted by a Venn diagram comparing the heat stress group (1 h, 2 h, 3 h, and 5 h) with the control group. (C) In males, the distribution of DEGs depicted by a Venn diagram comparing the heat stress group (1 h, 2 h, 3 h, and 5 h) with the control group.

Figure 3

Profiling of transcriptome time-series DEGs and enriched KEGG analysis of clusters. (A) Female time-series clustering analysis. (B) Male time-series clustering analysis. The graph color transitions from green to red to indicate gene expression trends from discrete to clustered. (C) KEGG enrichment analysis of DEGs contained in Cluster 1 and Cluster 2 (U shape) of females. (D) KEGG enrichment analysis of DEGs contained in Cluster 1 and Cluster 2 (U shape) of males. (E) KEGG enrichment analysis of DEGs contained in Cluster 3 and Cluster 4 (bell shape) of females. (F) KEGG enrichment analysis of DEGs contained in Cluster 3 and Cluster 4 (bell shape) of males.

Figure 4

Analyses of cluster 3 in females and males. (A,B). KEGG analysis results for cluster 3 in both females and males, respectively. (C,D). Gene relationship maps in females and males, highlighting the selected genes, respectively. Red indicates more important key genes compared with blue.

Figure 5

Heat map illustrating the relative expression of genes in females and males under heat stress. (A) Heat map of gene expression patterns in females derived from PPI analyses. (B) Heat map of gene expression patterns in males derived from PPI analyses. The rows represent the “expression levels of FPKM” and the columns represent treatment groups: control (CT1–3 at 22 °C) and heat stress (HS1h–HS5h at 28 °C). The higher expression values are displayed in red and lower expression values in blue.