GDH and ASAT are involved in WSSV replication.

<p>Three days after injection of the indicated dsRNA, shrimp were challenged with WSSV by injection, and 24h later, 4 hemocyte and pleopod samples (3 shrimp in each sample) were collected from each group. The hemocyte samples were used to measure mRNA expression while the pleopod samples were used to measure the viral copy number. <i>In vivo</i> gene silencing of (A) GDH and (B) ASAT by injection of corresponding dsRNA induced a significantly stronger decrease in the respective target mRNA expressions compared to the groups treated with non-specific EGFP dsRNA. <i>In vivo</i> gene silencing of GDH and ASAT decreased both (C) WSSV gene VP28 expression and (D) WSSV genome copy numbers at 24 h post WSSV injection. Asterisks indicate statistically significant differences between EGFP dsRNA control, GDH dsRNA and ASAT dsRNA groups (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

WSSV induces glutamate dehydrogenase (GDH) expression at the WSSV replication stage (12 hpi).

<p>(A) The mRNA level of SLC7A1 and SLC1A2 during WSSV infection. Gene expression in hemocyte samples (4 samples for each group at each time point; 4 shrimp in each sample) was analyzed by real-time PCR (qPCR). (B) The mRNA level of GDH, ASAT and GLS during WSSV infection. Gene expression in hemocyte samples (4 samples for each group at each time point; 4 shrimp in each sample) was analyzed by real-time PCR (qPCR). (C) Analysis of lysates from hemocytes collected at 12 and 24 hpi showed that the activities of GDH and ASAT, the key enzymes involved in the conversion of glutamate to α-ketoglutarate (α-KG), were upregulated at the WSSV genome replication stage (12 hpi). The values are shown as fold change compared to the PBS group. (D) Analysis of lysates from stomachs collected at 12 and 24 hpi showed that the level of α-KG was increased at 12 hpi. (Stomachs were used for this analysis because lysates from hemocytes did not contain detectable levels of α-KG.) Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV infection groups and the corresponding PBS control (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

Proposed model of how WSSV-induced glutamate-driven anaplerosis is regulated by the mTORC2 at 12 hpi.

<p>Gene expression and/or protein levels of enzymes (circle) and the mRNA levels of transporters (cylinders) were either increased (red), decreased (green) or remained unchanged (yellow). Expression and protein data are compiled from the present study and from Su <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146902#pone.0146902.ref005" target="_blank">5</a>]. The same color code is used for metabolites, which are shown in boxes. Except for α-KG, all metabolite data is from Su <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146902#pone.0146902.ref005" target="_blank">5</a>]. Details of the mTORC2 regulatory pathways are currently unknown. In addition to increasing energy levels (ATP), we speculate that replenishment of α-KG may also drive the production of lipid precursors (dashed red arrow).</p

WSSV-induced expression of GDH is regulated by mTORC2.

<p>(A) Schematic of the PI3K-Akt-mTOR pathway and the three inhibitors used in this experiment (black boxes). All of the results shown in this Figure are from WSSV-infected shrimp, i.e. the controls were not treated with the various inhibitors, but they were infected. (B) Real-time PCR (qPCR) analysis shows that the PI3K/mTORC1 inhibitor LY294002 had no significant effect on WSSV-induced GDH expression at 12 hpi. Gene expression was measured in 10 individual hemocyte samples for each group at each time point. (The number of data points shown may be less than 10 because any sample that failed to produce a sufficiently specific Bio-Rad amplification plot was rejected.) Bars represent the mean ± SD. Asterisks indicate statistically significant differences in WSSV-infected shrimp between the drug treatment groups and the corresponding vehicle-only control. The Mann-Whitney U test was used to test the non-normal LY294002 12 hpi GLS group data against the DMSO control. Student’s <i>t</i>-test was used for all other comparisons (** <i>p</i><0.01). (C) Inhibition of mTORC1 by Rapamycin significantly up-regulated the expression of GDH, ASAT and GLS mRNA at 12 hpi. Gene expression in hemocyte samples (4 samples for each group at each time point; 3 shrimp in each sample) was analyzed by real-time PCR (qPCR). Asterisks indicate statistically significant differences in WSSV-infected shrimp between the drug treatment groups and the corresponding vehicle-only control (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01). (D) Inhibition of mTORC1/C2 by Torin 1 significantly suppressed the expression of GDH, but no effect on ASAT and GLS at 12 hpi. Gene expression in pleopod samples (5–6 samples for each group at each time point; 10 shrimp in each sample) was analyzed by real-time PCR (qPCR). Each bar represents the mean ± SD. Asterisks indicate statistically significant differences in WSSV-infected shrimp between the drug treatment groups and the corresponding vehicle-only control (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01).</p

The glutaminolysis pathway is driven by the uptake of glutamate at 12 hpi.

<p>(A) Simplified schematic of the glutaminolysis metabolic pathway. Abbreviations: GLS, glutaminase; GLNA, glutamine synthetase; GDH, glutamate dehydrogenase; ASAT, aspartate aminotransferase; α-KG, α-ketoglutarate; OAA, oxaloacetate; SLC1A2: transporter involved in glutamate transport; SLC7A1: transporter involved in glutamine transport. Relative intensity of (B) glutamine and (C) glutamate was measured by LC-ESI-MS at the WSSV replication stage (12 hpi) and late stage (24 hpi). Samples of hemocytes (Hcy) and hemolymph (Hly) were collected at the indicated time points. Quantitative metabolomic LC-ESI-MS data were collected from a single replication of 5–6 samples of 10 shrimp each. (A summary of the hemocyte data appeared previously in Su <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146902#pone.0146902.ref005" target="_blank">5</a>]. Hemolymph data is from the same metabolomic study, but has not been previously published.) Asterisks indicate statistically significant differences between the PBS control and WSSV infection groups (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01).</p

Glutamate-driven anaplerosis produces α-KG, which is an essential anaplerotic TCA cycle metabolite during WSSV infection.

<p>(A) GDH dsRNA treatment significantly decreased GDH expression. Three days after treatment with EGFP dsRNA and GDH dsRNA, shrimp were challenged with WSSV and then subjected to α-KG replenishment (531 μg/g shrimp) 2h post WSSV injection. At 24 h post WSSV injection, 4 hemocyte and pleopod samples (3 shrimp in each sample) were collected from each group. The hemocyte samples were used to measure mRNA expression and ATP/ADP ratio, while the pleopod samples were used to measure the viral copy number. Asterisks indicate significant differences between each group (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001). (B) α-KG replenishment rescued the ATP/ADP ratio in the GDH dsRNA treated groups. Asterisks indicate significant differences between groups (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001). (C) α-KG replenishment rescued both WSSV mRNA expression and WSSV genome copy number in the GDH dsRNA treated groups. Asterisks indicate statistically significant differences between each group (Student’s t test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

16S_abundance_95-15

Summary of 16S rRNA based abundance calculation resul

Readme

Readm

Blastn results

Applying blastn to obtain the taxonomic information of each direct shotgun sequencing read that contain 16S rRNA gene sequenc

16S_GraPhlAn_20RA

Code for constructing phylogenetic tree in GraPhlA