Cold tolerance and acclimation of Songpu mirror carp and Barbless carp
Songpu mirror carp and Barbless carp are two closely related species whose larvae rearing at 28℃ exhibited similar developmental phenotypes at 15 dpf (Figure 1A). Larvae of Songpu mirror carp and Barbless carp at 9 dpf were used to compare their differences in tolerance to lethal cold stress at 10℃ or 8℃ for 24h (AS) and in cold acclimation to mild cold stress at 18℃ for 24h followed by exposure to lethal cold stress at 10℃ or 8℃ for 24h (CA) (Figure 1B). Then, larvae in AS and CA groups were recovered at 28℃ for another 24h to monitor their survival rates.
As shown in Figure 1C, the survival rate of Songpu mirror carp at 12 dpf was significantly higher than that of Barbless carp after exposure to lethal cold stress at 10℃, while no living larvae were detected in AS groups of Songpu mirror carp and Barbless carp exposed to lethal cold stress at 8℃. These data indicate that Songpu mirror carp has a stronger ability of cold tolerance to lethal cold stress than Barbless carp.
We further compared the difference of Songpu mirror carp and Barbless carp in cold acclimation. After pretreatment of 9 dpf larvae at 18℃ for 24h, 94.91% of Songpu mirror carp larvae and 96.84% of Barbless carp larvae in CA groups were able to survival at lethal cold stress at 10℃, while 90.61% of Songpu mirror larvae and 22.12% of Barbless carp larvae in CA group were able to survival at lethal cold stress at 8℃ (Figure 1D) (Additional file 1), indicating that both Songpu mirror carp and Barbless carp possess the ability of cold acclimation and Songpu mirror carp has a stronger ability of cold acclimation than Barbless carp.
RNA-seq and bioinformatics analysis
A total of twelve cDNA libraries for Songpu mirror carp and Barbless carp were constructed and subjected to high-throughput sequencing, followed by extensive bioinformatics analysis (Figure 2A). The total number of raw read pairs ranged from 11.93 to 15.26 million (M) and about 60% of the processed reads were mapped to the reference genome of common carp [46] after filtering out low quality reads (Table 1).
Principle component analysis (PCA) was used to examine the transcription profiles of genes in AS and CA groups. As shown in Figure 2B, there is an obvious consistency within the same group and a clear discrepancy among groups in PCA results, suggesting small variations within the same group and a high reliability of data. Moreover, the influence of cold stress at 18℃ led to 18% of the transcriptional variations (PC2) and 66% of variations (PC1) was due to the difference of two carp species (Figure 2B), indicating that Songpu mirror carp and Barbless carp are two closely related species of the carp family that possess different abilities of cold acclimation.
To validate the expression profiles from RNA-seq analysis, 13 genes including two transcripts of hmgb1 were measured with qPCR assays. Primers used for qPCR were listed in Additional file 2. The subunit S11 of the ribosomal gene 40S was used as the reference gene [47]. As shown in Figure 2C and Additional file 3, the Spearman bivariate correlation analysis revealed that data of RNA-seq and qPCR were significantly correlated (p < 0.00001, correlation coefficient = 0.9546), indicating the reliability of RNA-seq data.
Identification of differentially expressed genes for cold tolerance and acclimation between Songpu mirror carp and Barbless carp
After obtaining counts for annotated genes in two control groups (Ctrl) and cold acclimation groups (CA) of Songpu mirror carp and Barbless carp, we performed a comparative analysis of differentially expressed genes between two of Ctrl and/or CA groups (Figure 3A). Differentially expressed genes in group I represent the developmental and physiological differences between Songpu mirror carp and Barbless carp growing at normal temperature. Differentially expressed genes in group II represent the differences between Songpu mirror carp and Barbless carp during cold acclimation. Differentially expressed genes in group III represent cold-induced and -inhibited genes in Songpu mirror carp. Differentially expressed genes in group IV represent cold-induced and -inhibited genes in Barbless carp. The detail information of genes in each groups were listed in Additional file 4.
Next, we performed analysis of differentially genes with Venn's diagrams to identify potential genes that are specifically required for cold tolerance of Songpu mirror carp and for cold acclimation of Songpu mirror carp and Barbless carp. As shown in Figures 3B and 3C, differentially expressed genes in group I contain genes that are responsible for the difference of Songpu mirror carp and Barbless carp in cold tolerance to lethal stress (Figure 1C). Some of differentially expressed genes in groups II and III (a, b, c, a', b', and c') contain candidate genes that are responsible for the strong ability of cold acclimation in Songpu mirror carp. Some of differentially expressed genes in groups III and IV (d, e, d' and e') are involved in cold acclimation of both Songpu mirror carp and Barbless carp. Some of differentially expressed genes in group IV (f, g, f' and g') are likely required for cold acclimation of Barbless carp. The detail information and fold change of genes in each sets obtained by Venn's diagrams were displayed in Additional file 5. These sets of differentially expressed genes were further used for enrichment analysis to explore biological processes and signaling pathways for cold acclimation.
GO enrichment analysis to identify biological processes for the difference in cold acclimation between Songpu mirror carp and Barbless carp
Differentially expressed genes that are potentially associated with cold acclimation of Songpu mirror carp and Barbless carp were analyzed with GO enrichment and displayed in Additional file 6 and representatives of GO term belonged to the biological process obtained through REVIGO tool were displayed in Additional file 7. Candidate genes up-regulated in groups II and III (a, b, c) during the formation of a strong cold acclimation in Songpu mirror carp were overrepresented in biological processes including cellular component assembly involved in morphogenesis, secondary alcohol metabolism, drug transport, regulation of alternative mRNA splicing via spliceosome, cardiac muscle hypertrophy and cell-substrate adhesion (Figure 4A) and details of these processes can be visualized with the REVIGO Web server in Figure S1. Candidate genes down-regulated in groups II and III (a', b' and c') during the formation of strong cold acclimation in Songpu mirror carp were mainly enriched in biological processes including organonitrogen compound catabolism, fatty acid biosynthesis, membrane budding, striated muscle contraction, response to topologically incorrect protein and organic substance metabolism (Figures 4B and S2).
Candidate genes up-regulated in groups III and IV (d and e) during cold acclimation of both Songpu mirror carp and Barbless carp were mainly enriched in biological processes including lipid homeostasis, cellular biosynthesis, lipid transport, response to endogenous stimulus and other processes (Figures 4A and S3). Candidate genes down-regulated in groups III and IV (d' and e') during cold acclimation of both Songpu mirror carp and Barbless carp were overrepresented in biological processes such as protein catabolism, regulation of RNA splicing, striated muscle tissue development and primary metabolism (Figures 4B and S4).
Candidate genes up-regulated in groups III and IV (f and g) during cold acclimation of Barbless carp were mainly enriched in biological processes including monocarboxylic acid transport, rRNA processing, and monocarboxylic acid transport, cofactor metabolism and other processes (Figures 4A and S5). Candidate genes down-regulated in groups III and IV (f' and g') during cold acclimation of Barbless carp were overrepresented in biological processes including positive regulation of macroautophagy, organonitrogen compound catabolism, intracellular protein transport, protein deubiquitination and other processes (Figures 4B and S6).
KEGG enrichment analysis to identify signaling pathways for cold acclimation difference between Songpu mirror carp and Barbless carp
Differentially expressed genes that are potentially associated with cold acclimation of Songpu mirror carp and Barbless carp were analyzed with the KEGG pathway enrichment. Candidate genes up-regulated in groups II and III (a, b, c) during the formation of a strong cold acclimation in Songpu mirror carp were mostly enriched in signaling pathways such as leukocyte transendothelial migration, regulation of actin cytoskeleton, and FoxO signaling pathway.(Figure 5A and Additional file 8). Candidate genes down-regulated in groups II and III (a', b' and c') during the formation of strong cold acclimation in Songpu mirror carp were mainly overrepresented in signaling pathways such as protein processing in endoplasmic reticulum, antigen processing and presentation and amino sugar and nucleotide sugar metabolism (Figure 5B and Additional file 8). These signaling pathways are mainly classified into environmental information processing (Table 2).
Candidate genes up-regulated in groups III and IV (d and e) during cold acclimation of both Songpu mirror carp and Barbless carp were enriched in signaling pathways including cholesterol metabolism, PPAR signaling pathway and spliceosome (Figure 5A and Additional file 8). Candidate genes down-regulated in groups III and IV (d' and e') during cold acclimation of both Songpu mirror carp and Barbless carp were mainly overrepresented in the signaling pathway of proteasome (Figure 5B and Additional file 8). These signaling pathways are classified into organism systems (Table 2).
Candidate genes up-regulated in groups III and IV (f and g) during cold acclimation of Barbless carp were mainly enriched in signaling pathways such as insulin signaling pathway, PPAR signaling pathways, adipocytokine signaling pathway and ribosome biogenesis in eukaryotes (Figure 5A and Additional file 8). Candidate genes down-regulated in groups III and IV (f' and g') during cold acclimation of Barbless carp were overrepresented in signaling pathways of thermogenesis, cellular senescence and oxidative phosphorylation (Figure 5B and Additional file 8). These three signaling pathways are classified into organism systems, cellular processes, and metabolism (Table 3).
FoxO signaling pathway played a key role in the difference of cold acclimation between Songpu mirror carp and Barbless carp
FOXO is a transcription factor that plays important roles in transcriptional expression of genes involved in the control of cell survival [50]. The activity of FOXO was regulated by multiply mechanisms [50]. In this study, nine of up-regulated genes (a, b and c, in red) and two of down-regulated genes (a', b' and c', in green) in groups II and III during the cold acclimation of Songpu mirror carp were mapped to the FoxO signaling pathway and cellular events that were associated with FoxO signaling pathway include cell cycle regulation, regulation of autophagy, glycolysis/gluconeogenesis, muscle atrophy and cholesterol synthesis (Figure 6 and Table 4). Regulation of autophagy was down-regulated by cold-inhibited BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein) expression in groups III and IV (d' and e' in gray) of both Songpu mirror carp and Barbless carp (Figure 6). During the cold acclimation of Barbless carp, the AMPK signaling pathway was mainly activated by up-regulation of four genes in group IV (f and g in magenta) (Table 5) and oxidative stress resistance & DNA repair events downstream of FoxO signaling pathway were negatively controlled by down-regulation of two genes in group IV (f' and g' in aqua).
Next, inhibitors of FOXO (AS1842856), AMPK (Dorsomorphin 2HCl) and JNK (SP600125) and activator of Akt (SC79) were used to further verify the function of FoxO signaling in the formation of cold acclimation of Songpu mirror carp following a strategy in Figure 7A. Briefly, a final concentration of AS1842856 at 5 μM, Dorsomorphin 2HCl at 10 μM, SP600125 at 20 μM or SC79 at 4 μM in the embryo medium was used to treat twenty 9-dpf larvae of Songpu mirror carp in 50-mm dishes during cold acclimation at 18℃ for 24 hours. Then, the larvae were subjected to lethal cold stress at 8℃ for 24 hours in the pre-cooled embryo medium without inhibitor or activator. Lethal cold-treated larvae were recovered at 28℃ for 24 hours and the survival rates of larvae in different groups were calculated.
As shown in Figure 7B, the survival rate of Songpu mirror carp decreased to 76.62%, 70.9% and 16.72% after inhibition of FOXO, JNK and AMPK signaling pathways, respectively. Moreover, the survival rate of Songpu mirror carp was significantly reduced after activation of Akt signaling by SC79. We further detected the effects of inhibitors or activator on the phosphorylation levels of FOXO, AMPK and Akt using western blots (Figure S7). In comparison with those in the control at 28℃ (CK), levels of phosphorylated FOXO and AMPK significantly increased but phosphorylated Akt decreased during cold acclimation at 18℃ (CA); however, treatments with inhibitors (AS1842856 for FOXO and Dorsomorphin 2HCl for AMPK) abolished cold-induced phosphorylation of FOXO and AMPK (Figure 7C, left and middle panels). Additionally, the inhibition of Akt phosphorylation during cold acclimation (CA) was blocked by treatment with the SC79 activator (Figure 7C, right panel).
Taken together, these data suggest that FoxO signaling played a crucial role in cold acclimation of two carp species and distinctive signaling pathways upstream of FOXO activation contributed to the difference of cold acclimation in Songpu mirror carp and Barbless carp.