Bt Spore Culture Preparation
To orally expose beetles to Bt, we created a spore culture supernatant
according to previously described protocols . Briefly, we inoculated 5
ml LB liquid media with Bacillus thuringiensis subsp. Morrisoni
bv. Tenebrionis (Btt; obtained from the Bacillus Genetic
Stock Center, Columbus, OH) from glycerol stock and incubated overnight
at 30°C. We streaked liquid Btt from the overnight culture onto
LB agar plates and incubated the plates overnight at 30°C. The next day,
18 individual colonies were picked and each inoculated into 5 ml of Bt
medium (w/V–0.75% bacto peptone (Sigma), 0.1% glucose, 0.34%
KH2PO4, 0.435%
K2HPO4) with the addition of 25 µL of
salt solution (w/V–2.46% MgSO4, 0.04%
MnSO4, 0.28% ZnSO4, and 0.40%
FeSO4) and 6.25 µL of 1M
CaCl2•2H2O; these cultures and a
negative control (Bt medium only) were allowed to grow overnight on a
bacterial shaker at 30°C, 200 rpm. The following day, 1 ml of the liquid
overnight culture was inoculated into 200 ml Bt medium with the addition
of 1 ml salt solution and 50 µl calcium chloride. These cultures and a
negative control were incubated for seven days in darkness on a
bacterial shaker at 30°C, 200 rpm; additional 1 ml salts solution and 50
µl calcium chloride were added after four days of incubation. Spore
cultures were centrifuged at 4,000 rpm for 15 minutes, washed once with
insect saline, and resuspended in Millipore H2O. We
calculated the final spore concentration of 8.62*109cells/ml by counting spores using a Hausser Scientific counting chamber.