Pesticide and Bt Spore Oral Diet Preparation
We prepared control diet by mixing 0.15 mg/ml standard diet (described above) in DI H2O. We prepared pesticide diets by combining approximate LC50 pesticide dilutions in H2O with 0.15 mg/ml standard diet (5.14 mg/ml OP and 0.188 mg/ml Pyr).Btt diet was prepared by mixing the previously described 8.62*109 cells/ml spore culture suspension with 0.15 mg/ml standard diet. Pesticide + Btt diet was prepared by diluting 9.75 µl/ml OP or 0.188 mg/ml Pyr in the spore culture suspension with 0.15 mg/ml standard diet. Oral diet plates were prepared by pipetting 50 µl of the appropriate treatment (Suppl. Fig. 1; control, control + Btt , OP, OP + Btt , Pyr, Pyr + Btt ) into 96-well cell culture plates. Plates were covered and allowed to dry overnight at 55°C.
We investigated the effects of diet disk preparation and pesticide treatments on Bt spore germination by preparing control, Btt, andBtt + pesticide diet disks as described above and counting the number of formed colonies from each treatment (Btt , Btt + OP or Btt + Pyr n=10; control n=6). We conducted the spore germination on two separate days. We stored dried diet disks for four or five days respectively at 30°C in the dark and then dissolved individual disks in 1 mL ultrapure water. To initiate germination and aid in the dissolving of the disks, we heated the samples to 50°C for ten minutes and then immediately plated the samples. For the bacteria culture plates, we spread 100µl of the final dilution onto LB agar, included a negative control with only water, incubated plates over night at 30°C and counted all Btt colonies after 15 hours. On the first day, we cultured bacteria from a 1:107 dilution in ultrapure water. On the second day, we used a 1:106 dilution, to ensure that sufficient colonies would grow. We analyzed the effect of treatment on the number of colony forming units (CFUs) using a generalized linear mixed model with day as a random factor and a negative binomial distribution.