Article Figures & Data
Figures
- Fig. 1.
Time course of MjVDAC expression in the shrimp stomach after specific RNA knockdown mediated by dsRNA injection. At each time point after dsRNA injection, total RNA was extracted from the stomach and reverse transcribed to cDNA. Injections of EGFP dsRNA and PBS were used as dsRNA controls. The EF1-α and MjANT genes were used as an internal control and a non-dsRNA target gene control, respectively.
- Fig. 2.
Gene silencing of shrimp MjVDAC decreases mortality after WSSV injection. Cumulative mortalities of shrimp challenged with PBS (control) or WSSV were observed after injection with PBS or double-stranded RNAs corresponding to MjVDAC or EGFP. Mortality was measured in each treatment group (n = 40) and was recorded every 24 h postchallenge. Differences in mortality levels between the MjVDAC dsRNA group and the EGFP dsRNA group were analyzed by Kaplan-Meier log rank χ2 tests. Significant differences in shrimp mortality are marked with asterisks and were found from 3 dpi to the end of the experiment (P < 0.05).
- Fig. 3.
Gene silencing of shrimp MjVDAC decreases WSSV DNA copy numbers after WSSV injection. Real-time qPCR was used to measure the number of copies of WSSV genomic DNA in WSSV-challenged shrimp that had been preinjected with PBS or double-stranded RNAs corresponding to MjVDAC or EGFP. An asterisk indicates a significant statistical difference between bracketed groups (P < 0.05).
- Fig. 4.
WSSV induces mitochondrial membrane permeabilization (MMP) in shrimp hemocytes. (A) After being injected with PBS or WSSV, shrimp hemocytes were collected at the indicated times and seeded into 24-well plates. JC-1 staining was used to indicate normal (red) versus low (green) mitochondrial membrane potential. DAPI (blue) staining was used to counterstain the nucleus. UV light treatment was used as the positive control. (B) Percentage of hemocytes showing mitochondrial membrane potential loss after WSSV infection. After seeding and staining, 300 to 1,500 hemocytes in each treatment were counted at different time points. Each bar represents the mean ± SD from 3 samples, where each sample was pooled from 3 shrimp. Asterisks indicate a significant difference between groups (P < 0.05).
- Fig. 5.
WSSV causes an increase in the ADP/ATP ratio, but not H2O2 concentration, in shrimp hemocytes. (A) Each bar represents the mean ± SD of the ADP/ATP ratios in 4 samples of pooled hemocyte lysate. Statistical significance is indicated by single (P < 0.05) or double (P < 0.005) asterisks. (B) Each bar represents the H2O2 concentrations (mean ± SD) in 4 pooled samples of shrimp hemocyte lysates. ND, no data. An asterisk indicates a significant statistical difference between groups (P < 0.05).
- Fig. 6.
WSSV infection alters the levels of glucose and lactate in shrimp plasma. Glucose (A) and lactate (B) concentrations in plasma were measured at the indicated time points in WSSV-challenged shrimp versus PBS-injected controls. Statistical significance is indicated by single (P < 0.05) or double (P < 0.005) asterisks.
- Fig. 7.
WSSV infection increases glucose-6-phosphate dehydrogenase (G6PDH) activity in shrimp hemocytes at 12 hpi. G6PDH activity was measured at the indicated time points in WSSV-challenged shrimp versus PBS-injected controls. Statistical significance is indicated by single (P < 0.05) or double (P < 0.005) asterisks.
- Fig. 8.
WSSV infection leads to a decrease in triglyceride levels in shrimp plasma. Plasma triglyceride concentrations were measured at the indicated time points in WSSV-challenged shrimp versus PBS-injected controls. Statistical significance is indicated by single (P < 0.05) or double (P < 0.005) asterisks.
- Fig. 9.
WSSV replication in shrimp. Histogram data represent the number of copies of WSSV genomic DNA (A) and WSSV VP28 mRNA (B) detected at the indicated time point, while the relative change from one time point (tn) to the next (tn + 1) is defined as (tn + 1)/(tn) and is shown by the dots.
- Fig. 10.
Schematic representation of the in vivo metabolic changes in shrimp hemocytes at the viral genome replication stage (12 h) (A) and the late stage (24 h) (B) of the first WSSV replication cycle. Measured concentrations or activities are shown in rounded boxes: red indicates an increase and green indicates a decrease relative to the respective PBS control. Yellow boxes showed no change. VDAC expression level data are from Wang et al., (36), Wang et al., (37), and our unpublished data. Hexokinase mRNA expression levels at 24 hpi are from unpublished microarray data. No HK data are presently available for 12 hpi. Increases in pathway use are indicated by the thicker solid arrows. Ψm represents membrane potential (no data are available for Ψm at 12 hpi). The shaded box at the top of the 12-h schematic encompasses concentration changes that are consistent with the Warburg effect. The shaded box at the bottom left of the 24-h schematic shows changes associated with MMP.
