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.