Zone of inhibition assay
Although immune-challenged individuals in this study were injected withE. coli , previous assays resulted in no measurable antibacterial activity on plates seeded with E. coli . Therefore, antimicrobial activity was assayed from zones of inhibition induced by samples on petri dishes containing agar seeded with Micrococcus luteus (ATCC 4698) (see Sadd and Schmid-Hempel, 2007 for methodological details). Briefly, M. luteus from a single colony on a streak plate were incubated in a shaking incubator overnight at 30°C in 7 mL of media (2.5 g peptone and 1.5 g meat extract in 500 mL of nanopure water, pH 7.0). From this culture, bacteria were added to liquid media containing 1% agar held at 40°C to achieve a final density of 1.5x105 bacterial cells/mL. Six milliliters of seeded medium were added to a 100 mm diameter petri dish to solidify. Sample wells were made using a Pasteur pipette (Volac D810) fitted with a ball pump, and 2 µL of sample hemolymph solution thawed on ice was added to each well. Negative (Grace’s insect medium, ThermoFisher Scientific, CAS: 11605094) control wells were also included on each plate. Plates were inverted, incubated for 48 h at 30°C, and then the diameter of inhibition zones was measured for each sample. Two diameter measurements of zones, perpendicular to one another, were measured for each sample using ImageJ (Schneider et al., 2012) and averaged (measurements were performed blind to treatment). Because zone of inhibition diameter does not increase linearly with antibacterial activity, measured zone diameters were converted, based on a standard curve, to units (mg/mL) of lysozyme (from hen egg white, MilliporeSigma, CAS: 12650-88-3). Each hemolymph sample was tested in duplicate, with the mean of the duplicates being used in analyses.