Choice assays

2 control solution and the other was painted with bacteria suspended in 10 mM MgCl 2 solution. Solution was applied to the leaf surface with a sterile cotton swab until the surface was visibly saturated but not dripping. Bacterial suspensions were made from overnight cultures, which were pelleted, washed and resuspended using 10 mM MgCl 2 then adjusted to an optical density of 1.0 (OD 600 ) using a WPA CO 8000 Cell Density Meter which measures optical density at 600 nm. Leaves were allowed to dry and then approximately 80 mixed age aphid nymphs (not yet reproductive) were introduced on the soil the base of the plant. Plants were caged individually and incubated at 21°C and 80% relative humidity under a light:dark 16:8 hr cycle with full spectrum cool blue fluorescent grow lights (54W T5 fluorescent with 6400K color temperature approximating daylight) positioned above the plants. Pea aphids feed on the undersides of leaves and on stems and we never observed aphids feeding on the upper surfaces of leaves in these experiments. Because broad bean stems would be difficult to paint with bacteria in a controlled manner we focused on aphids feeding on leaves. Our experimental set up mimicked natural conditions in which aphids would be viewing the undersides of leaves, in that they would be encountering the undersides of leaves that are illuminated from above. Placing aphids at the base of the plant forced them to approach each leaf pair via the stem and petiole, so they must choose one leaflet or the other (control or bacteria) from the petiole. The number of aphids feeding on control and bacterial leaves was observed after 4 hr and then every 7-17 hr for three days for a total of 4 time points. Aphids feeding on stems did not participate in the experiment and were not included in analyses. Eight plants were used in each replicate and assays were replicated three times for each strain. By 4 hr, most aphids had settled to feed and we found that plants had 30-70 aphids feeding on leaves. Additional time points were recorded as well, but we found that numbers of aphids on control versus bacterial leaves stayed consistent across the time frame of the experiment, so except when analyzing change over time ( Choice assays were performed on either whole plants, excised leaf pairs, or with artificial aphid diet. For assays using whole plants, two week old broad bean plants were used. The apical leaf pair of each plant was removed to encourage aphids to choose among fully expanded leaves. Each plant had three leaf pairs distributed in different orientations around the stem of the plant. Within each leaf pair one leaflet was painted with a 10 mM MgClcontrol solution and the other was painted with bacteria suspended in 10 mM MgClsolution. Solution was applied to the leaf surface with a sterile cotton swab until the surface was visibly saturated but not dripping. Bacterial suspensions were made from overnight cultures, which were pelleted, washed and resuspended using 10 mM MgClthen adjusted to an optical density of 1.0 (OD) using a WPA CO 8000 Cell Density Meter which measures optical density at 600 nm. Leaves were allowed to dry and then approximately 80 mixed age aphid nymphs (not yet reproductive) were introduced on the soil the base of the plant. Plants were caged individually and incubated at 21°C and 80% relative humidity under a light:dark 16:8 hr cycle with full spectrum cool blue fluorescent grow lights (54W T5 fluorescent with 6400K color temperature approximating daylight) positioned above the plants. Pea aphids feed on the undersides of leaves and on stems and we never observed aphids feeding on the upper surfaces of leaves in these experiments. Because broad bean stems would be difficult to paint with bacteria in a controlled manner we focused on aphids feeding on leaves. Our experimental set up mimicked natural conditions in which aphids would be viewing the undersides of leaves, in that they would be encountering the undersides of leaves that are illuminated from above. Placing aphids at the base of the plant forced them to approach each leaf pair via the stem and petiole, so they must choose one leaflet or the other (control or bacteria) from the petiole. The number of aphids feeding on control and bacterial leaves was observed after 4 hr and then every 7-17 hr for three days for a total of 4 time points. Aphids feeding on stems did not participate in the experiment and were not included in analyses. Eight plants were used in each replicate and assays were replicated three times for each strain. By 4 hr, most aphids had settled to feed and we found that plants had 30-70 aphids feeding on leaves. Additional time points were recorded as well, but we found that numbers of aphids on control versus bacterial leaves stayed consistent across the time frame of the experiment, so except when analyzing change over time ( Figure S1 ), we used data from 4 hr for analyses. At least 444 aphids were included in analyses for each strain.

Under dark conditions aphids were greatly delayed in settling to feed on whole plants. Therefore, to test aphid preference under dark and no UV light conditions choice assays were done using excised leaves. Methods for assays on excised leaves followed whole plant assays, except that a single leaf pair was removed from a plant and the petiole was embedded in a wedge of water agar in a Petri dish. Mixed age aphid nymphs were introduced into Petri dishes (not onto the leaf directly) and allowed to crawl onto the underside of the leaf. For assays testing preference in the absence of vision, two growth chambers with the same temperature and humidity were used, with lights turned off in one chamber and the other was illuminated with vertical fluorescent lights fixed in the door. For assays testing the importance of UV light on preference, plates were incubated in the same growth chamber but half of the plates were kept under a box constructed out of UV-filtering plexiglass (OP-3/UF-5), which filters out 98% of UV light. Each assay had five plates in each treatment and each treatment was replicated two (dark) or three (UV) times. Approximately 15-25 aphids per dish settled to feed on leaves during experiments in the dark and approximately 30-50 per dish settled during experiments under UV-filtering, for a total of at least n = 196 per dark/light treatment and n = 664 per UV/UV filtered treatment. The highly virulent and highly avoided strain Psy B728a was used for these experiments.