Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. RNAi of Manduca proPSP decreases hemocyte spreading in vitro. Day 0 fifth-stage larvae were first injected with DMPC water (W) (a−c) or dsRNAs (d−i), 6 h later with PBS (c, f, i), 2×10⁷ cells of E. coli (EC) (a,d,g) or 1×10³ cells of P. luminescens (TT01) (b,e,h) and 18 h after the second injection were bled, hemocytes were collected and monolayers were prepared. Hemocytes were stained with FITC−phalloidin and visualized using fluorescence microscopy. Note the reduced hemocyte spreading in P. luminescens-infected insects compared with the E. coli-infected individuals and that this effect can further be reduced in the E. coli treatments by pre-injection of pro-PSP dsRNA (ds-proPSP), but not of dsRNA control (dsCON). Scale bar, 10 mm.

• Supplemental Figure S2 -

  Fig. S2. Appearance of hemocyte microaggregates formed in vitro. Uninfected day 0 fifth-stage Manduca caterpillars were injected with DMPC water (W) (a−c) or dsRNAs (d−i); 18 h later they were bled and hemocytes were collected. A suspension of 2×10⁷ E. coli (EC) (a,d,g) or 1×10³ P. luminescens (TT01) (b,e,h) cells ml⁻¹ was added to 10⁶ hemocytes in a 1:2 ratio and, following incubation for 15 min, the number of microaggregates was counted using an inverted microscope. PBS treatment was used as control (c,f,i). Note the reduction in microaggregates when caterpillars were injected with ds-proPSP prior to E. coli infection compared with the water- and dsCON-pre-treated insects. Scale bar, 20 mm.

• Supplemental Figure S3 -

  Fig. S3. Visual examination of melanotic nodules in tissues of day 0 fifth-stage Manduca larvae. Insects were pre-injected with proPSP dsRNA (ds-proPSP) (g−i), unrelated dsRNA control (dsCON) (d−f) or water (W) (a−c), and 6 h later injected with 2×10⁷ cells of E. coli (EC) (a,d,g), 1×10³ cells of P. luminescens (TT01) (b,e,h) or PBS (c,f,i). Caterpillars were dissected 18 h after the second injection. Note, first, the reduced nodulation in P. luminescens-infected insects (W+TT01) compared with the E. coli-infected individuals (W+EC) and, second, that this effect can further be suppressed by RNAi pre-treatment with ds-proPSP (ds-proPSP+EC, ds-proPSP+TT01) but not with dsCON (dsCON+EC, dsCON+TT01). Also note the lack of melanization in insects given no bacteria (W+PBS, dsCON+PBS, ds-proPSP+PBS).

• Supplemental Figure S4 -

  Fig. S4. Phagocytosed bacteria visualized in Manduca hemocytes. Green shows bacteria cells; red shows cell surface staining with the monoclonal antibody MS77, used here as a counterstain. Day 0 fifth-stage larvae were first injected with proPSP dsRNA (ds-proPSP) (g−i), dsRNA control (dsCON) (d−f) or water (W) (a−h), and 6 h later they were infected with labelled E. coli (EC) (a,d,g) or P. luminescens (TT01) (b,e,h) cells. PBS treatments were used as controls (c,f,i). Caterpillars were bled 18 h after the second injection and monolayers were prepared. The phagocytic competence of Manduca hemocytes for each treatment was examined using confocal microscopy. Note the decrease in the number of E. coli cells contained in phagocytic cells from insects injected with ds-proPSP prior to bacterial infection, compared with the W+EC and dsCON+EC treatments. Scale bar, 5 mm.