Figure 1.
WSSV induces the Warburg effect in the cellular proteome and metabolome of shrimp hemocytes at the replication stage (12 hpi) but not at the late stage (24 hpi).
Changes in the levels of enzymes and proteins (ellipses) and metabolites (rectangles) relative to PBS-injected controls are color-coded to represent up- (red) or down- (green) regulation. Yellow represents no change. Colorless boxes and ellipses indicate that no data was detected. Protein data were collected from 3–5 pooled samples of 5 shrimp using quantitative label-free proteomics and expressed on a logarithmic scale. Metabolomic data were collected from 5–6 pooled samples of 10 shrimp using LC-ESI/MS. Numeric values for the proteomic and metabolomic data are given in Tables S1 and S2, respectively.
Figure 2.
(A) WSSV infection increased the plasma lactate production at the WSSV genome replication stage (12 hpi), rather than at the late stage (24 hpi).
The levels of lactate production were profiled and measured by LC-ESI-MS. Each bar represents the mean ± SD from four or five independent samples, with each sample pooled from 10 shrimp. The asterisks indicates a statistically significant difference in lactate production (p<0.05). (B) WSSV-induced phosphorylation of 4E-BP1 was suppressed by treatment with the mTOR inhibitors Rapamycin and Torin 1. Two hours before WSSV injection, shrimp were treated with volume-matched solvent (PEG), Rapamycin (RAP; 0.02 µg/g shrimp) or Torin 1 (TR 1, 25 µg/g shrimp). At 24 h post WSSV injection, twelve shrimp were selected from each group and divided into four sets (A–D, E–H, I–L, M–P). From each set, four pooled samples (3 shrimp in each pool) were prepared by collecting gill samples and extracting the total proteins. Each pooled sample was then subjected to Western blotting with antibodies to phosphorylated 4E-BP1-PT37/46 and actin. (C) Shrimp LvRheb is induced after WSSV infection. At the indicated time points after WSSV injection, total protein was extracted from the gills of individual shrimp and subjected to Western blotting. The expression of LvRheb was significantly induced at 24 hpi. ICP11, which is a major WSSV very late protein, was used as a proxy to indicate the WSSV infection state. Actin was used as an internal control. (D) Time series showing that LvRheb dsRNA treatment successfully silenced LvRheb expression in the hemocytes of WSSV-infected shrimp. Three days after LvRheb dsRNA injection, shrimp were injected with WSSV. Hemocytes were collected after the indicated number of days and subjected to cDNA synthesis and real-time PCR. Injections of enhanced green fluorescent protein-dsRNA (EGFP dsRNA) and phosphate-buffered saline (PBS) were used as dsRNA controls. Each bar represents the mean ± SD from four pooled samples with 3 shrimp in each sample. An asterisk indicates a significant statistical difference between groups (p<0.05). (E) Gene silencing of shrimp LvRheb has no significant effect on the expression of the WSSV genes IE1 and VP28 during the first replication cycle (∼24 hpi). The mRNA expression of IE1 gene and VP28 were used as proxies to indicate the WSSV infection state. Each bar represents the mean ± SD from four pooled samples (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05). (F) Gene silencing of shrimp LvRheb also has no significant effect on the number of WSSV genome copies during the first replication cycle (∼24 hpi). Experimental conditions were as described above. The IQ Real WSSV Quantitative System was used to measure the number of copies of the WSSV genomic DNA. Each bar represents the mean ± SD from four pooled samples (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05).
Figure 3.
At 12-induced Warburg effect was suppressed by inhibiting mTOR, PI3K and Akt.
(A) At 12 h post WSSV injection, the accumulation of plasma lactate that was seen in the PEG-injected control group was suppressed by both Rapamycin (RAP) and Torin 1 (TR1). Each bar represents the mean ± SD from four pooled samples of hemolymph (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05). The effect of Rapamycin and Torin1 on (B) the expression of the WSSV genes IE1, DNA pol, VP28 and ICP11, and (C) WSSV genome copy number. Data were based on pooled samples of hemocytes (gene expression) or pleopods (genome copy number), with all samples being taken from the same sets of shrimp as those used in Figure 2B. Specifically, the PEG results were from sets E–H, the RAP results from I–L, and the TR1 results from N–P. (Please note that data from the set M was excluded because phosphorylation of 4E-BP1 was not successfully inhibited in this set.). (D) Each bar represents the mean ± SD of the relative glucose or lactate concentration of five pooled samples of hemolymph (3 shrimp in each pooled sample) under the indicated conditions. Statistically significant differences are shown by 1–3 asterisks, which respectively indicate p<0.05, p<0.005 and p<0.0005.
Figure 4.
WSSV replication was suppressed when the PI3K-Akt-mTOR pathway was inhibited by LY294002, MK2206 and Torin 1.
Shrimps were pretreated with LY294002 and samples were collected at 24(A–D) show the expression of WSSV (A) IE1, (B) VP28, and (C) ICP11 in shrimp hemocytes and (D) the viral copy number in pleopods from the same groups of LY294002-pretreated shrimp (n = 6–10 in each group). Graph (E) shows the mean WSSV copy number in five pooled samples of pleopods (3 shrimp in each pooled sample) collected from another batch of experimentally infected shrimp after pretreatment with the indicated inhibitor. Bars labeled with different letters indicate significantly different values (p<0.05).
Figure 5.
Pretreatment with Torin 1 inhibited the WSSV-induced Warburg effect at the genome replication stage of WSSV infection (12 hpi) in shrimp hemocytes.
Two hours after treatment with Torin 1, shrimp were injected with PBS or a WSSV inoculum. At 12(10 shrimp per pool) were collected from each group. Changes in the metabolomic levels of the WSSV-infected samples relative to the PBS controls are color-coded as described in Figure 1. Figure S3 shows changes in the metabolome at 24 hpi. Numerical data for 12 hpi and 24 hpi is given in Table S2. Changes in the metabolome for Torin-PBS versus PEG-PBS are shown in Fig. S3, with numerical data given in Table S2.
Figure 6.
Schematic representation of the WSSV-induced Warburg effect and the involvement of the PI3K-Akt-mTOR pathway at the viral genome replication stage (12 hpi) of the first WSSV replication cycle.
For the proteins investigated in this study, red indicates a positive involvement and gray indicates a partial involvement. Blue indicates important upregulated proteins and intermediates from previous studies: Glucose transporter (GLU1) is from Huang et al. [25], and G6PDH is from Chen et al. [9]. Elevated metabolic pathways are shown in pink. Pathway inhibitors are indicated in black boxes. Dashed lines indicate inferred regulatory mechanisms that were not investigated in the present study.