Transcriptomic analyses on muscle tissues of Litopenaeus vannamei provide the first profile insight into the response to low temperature stress

Abstract

The Pacific white shrimp (Litopenaeus vannamei) is an important cultured crustacean species worldwide. However, little is known about the molecular mechanism of this species involved in the response to cold stress. In this study, four separate RNA-Seq libraries of L. vannamei were generated from 13°C stress and control temperature. Total 29,662 of Unigenes and overall of 19,619 annotated genes were obtained. Three comparisons were carried out among the four libraries, in which 72 of the top 20% of differentially-expressed genes were obtained, 15 GO and 5 KEGG temperature-sensitive pathways were fished out. Catalytic activity (GO: 0003824) and Metabolic pathways (ko01100) were the most annotated GO and KEGG pathways in response to cold stress, respectively. In addition, Calcium, MAPK cascade, Transcription factor and Serine/threonine-protein kinase signal pathway were picked out and clustered. Serine/threonine-protein kinase signal pathway might play more important roles in cold adaptation, while other three signal pathway were not widely transcribed. Our results had summarized the differentially-expressed genes and suggested the major important signaling pathways and related genes. These findings provide the first profile insight into the molecular basis of L. vannamei response to cold stress.

Fig 1. The cold challenge and survival rate of L. vannamei.

(A): Treatment processes of the shrimp. (B): Gradient temperature challenge and survival rates. Control: treated in control temperature of 28℃; T_2: treated in related temperature for 2 hours; T_48: treated in related temperature for 48 hours; Recover: treated in control temperature of 28℃ for 2 hours recovering. Red frame indicated the stress temperature.

Fig 2. Annotation of the transcriptome.

(A): The summary results with main annotation database; the X axis were labeled with various items; Y axis represented the proportion genes annotated by the functional database; 29,662 was the total numbers of Unigenes, only the percentage numbers were marked in other blue dots. (B): The Venn diagram between NR, KEGG, COG and Swissprot; the annotated gene numbers were exhibit in the related areas. (C): The pie chart of percentage of annotated species with NR database; the main species names were displayed under the pie chart, species (with the corresponding color) were displayed with descending order.

Fig 3. The main components and DEGs in RNA transport pathways.

(A): mRNA translation processes. (B): the annotated different expressed genes in EJC complex of T_2/CT. (C): the annotated different expressed genes in EJC complex of T_48/T_2. (D): the annotated different expressed genes in EJC complex of RC/T_48. The up-regulated genes were marked with red frame, down-regulated genes were marked with green frame.

Fig 4. GO and KEGG classification of the DEGs in each comparison.

(A): Comparison of T_2/CT. (B): Comparison of T_48/T_2. (C): Comparison of RC/T_48. The Y-axis represents the number of DEGs in each item.

Fig 5. Column and Volcano-plot of the DEGs in comparisons.

The columnar results of the DEGs are listed and the corresponding Volcano-plot figures are shown. Plots in the Volcano-plot figures represent the DEGs. The Y-axis in the columnar figures represents the DEG numbers in comparisons. In the Volcano-plot figures, the X-axis represents the comparison value of log₂^FoldChange, and the Y-axis represents the value of |-log₁₀^FDR|. The red color refers to up-regulated DEGs, blue color refers to down-regulated DEGs, and the black plot refers to no significantly different DEGs between the pairs of libraries (FDR value ≤ 0.001, |log₂^FoldChange| value ≥ 1).

Fig 6. Venn diagrams and heat maps of the major coexisted DEGs.

(A): The Venn diagrams of Total DEGs, GO annotated DEGs and KEGG annotated DEGs in the three comparisons; DEG numbers were labeled in the corresponding areas. (B): Hierarchical clustering analysis of the top 20% of Total coexisting DEGs in the three comparisons. (C): Hierarchical clustering analysis of the annotated Calcium signaling genes in cold stress. (D): Hierarchical clustering analysis of the annotated MAPK cascades genes in cold stress. (E): Hierarchical clustering analysis of the annotated Transcription factor genes in cold stress. (F): Hierarchical clustering analysis of the annotated Serine/threonine-protein kinase genes in cold stress.

Fig 7. Validation for the 15 transcriptomic DEGs in the comparison of T_2/CT.

(A): The Real-time PCR results of the 15 genes in treatments of CT and T_2; Y axis represented the Log2 value of the related expression. (B): Grouped results of the 15 genes; the blue columnar mean the Real-time PCR results, red columnar mean the RNA-Seq results; X axis indicated the gene names, Y axis represented the value of log₂^FoldChange value in the comparison of T_2/CT, the FoldChange value were obtained by2^–ΔΔCT method. Bars in this figure represented the standard deviation (SD).