An Invertebrate Warburg Effect: A Shrimp Virus Achieves Successful Replication by Altering the Host Metabolome via the PI3K-Akt-mTOR Pathway
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).