Figure 3. Obp6 mediates the melanization cascade in adult tsetse.
(A) Survival following administration of clean wounds to the thoracic cuticle of siOBP6, siGFP and siOBP6R adults. Survival assays were performed in triplicate, using 25 flies per replicate. Red curve depicts a statistically significant difference in infection outcome (p<0.0001, log-rank test). (B) A representative micrograph of the cuticle of siRNA treated adults 3 hr post-wounding (hpw) with a clean needle. Melanin deposited at the wound site of siGFP and siOBP6R controls, and hemolymph exudate from a siOBP6 treatment individual, are identified by black and red arrowheads, respectively. Scale bar = 500 μm. Experiment was performed using four distinct flies per group (Figure 3—source data 1). (C) Quantitation of PPO1 and PPO2 in the hemolymph of siOBP6, siGFP and siOBP6R adults three hpw with a clean needle. Shown is a representative Western blot analysis using Drosophila anti-PPO1 and anti-PPO2 antibodies. 8 μl of pooled hemolymph was run per gel lane. Hemolymph was collected and pooled from four individuals from each group. Western blots were repeated in triplicate [Figure 3—source data 2 (for PPO1 westerns) and Figure 3—source data 3 (for PPO2 westerns)]. (D) PO activity in the hemolymph of siOBP6, siGFP and siOBP6R adults at 0 and 3 hpw with a clean needle. n = 5 biological replicates per group per time point for pre-wound readings, and n = 8 biological replicates per group per time point for post-wound readings. Data are presented as mean ± SEM. Bars with different letters indicate a statistically significant difference between pre- and post-wound values (specific p values are listed in the Figure 3—source data 4). Statistical test = 2 way ANOVA followed by Tukey’s HSD post-hoc analysis.
DOI: http://dx.doi.org/10.7554/eLife.19535.010
