Figure 2.

PGE2 signaling influences phenoloxidase (PO) activity and antimicrobial peptide (AMP) gene expression. Following treatment with PGE2 (500nM) (A) or when AgPGE2R was silenced (B), the expression of all nine mosquito prophenoloxidases (PPOs) were examined by qRT-PCR and compared to respective PBS or GFP controls. For both (A, B), mosquitoes were pooled (n=15) for analysis. Data were analyzed using an unpaired t test to determine differences in relative gene expression for each respective PPO gene between treatments. Bars represent mean ± SE of three independent biological replicates. (C) PO activity was measured from perfused hemolymph in mosquitoes primed with PGE2 (500 nM) and compared to PBS controls 24 h post-treatment (n=15 per treatment). (D) Additional experiments were performed measuring PO activity in which PGE2 (500nM) was injected into AgPGE2R- or GFP-silenced (control) mosquitoes (n=15 per treatment). For both (C, D), measurements (OD490) were taken for DOPA conversion assays at 5-min intervals from 0 to 30 min, as well as a final readout at 60 min. Data were analyzed using a two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons using GraphPad Prism 7.0. Bars represent mean ± SE of 3 independent experiments. In addition, PGE2 priming induced expression of antimicrobial peptide (AMP) genes (E), while AgPGE2R-silencing reduced AMP expression (F). For (E, F), data were analyzed using an unpaired t-test to determine differences in relative gene expression of each respective AMP gene between treatments. Bars represent mean ± SE of three independent replications. For all data, asterisks denote significance (**P < 0.01, ***P < 0.001, ****P < 0.0001). Genes that display significant differences in gene expression following PGE2 treatment are shaded in pink, while those genes differently regulated by AgPGE2R-silencing are displayed in blue.