Btt in vitro Growth Curve Assays
To measure in vitro effects of pesticides on Bt growth, we performed growth curve analyses for OP and Pyr exposure separately. For each analysis, Btt from a glycerol stock was freshly streaked onto Nutrient Broth (NB) agar plates and allowed to grow overnight at 30°C. We grew overnight cultures from single colony inoculates (n = 9) in 5 ml NB liquid media at 30°C, 200 rpm; cultures were incubated for only 16 hours to avoid entrance into sporulation phase. The following day, we grew log phase bacterial cultures by inoculating 100 µl of the overnight culture into 2.9 ml NB liquid media and incubated for 3.5 hours at 30°C, 200 rpm.
To replicate in vivo conditions, each growth curve analysis included control NB, 1:10 pesticide NB dilution, and 1:100 NB pesticide dilution treatments based on larval exposure OP and Pyr pesticide concentrations (described above). We added 200 µl of the appropriate treatment solution to a 96-well CYTO-One spectrophotometer plate. An aliquot of log phase bacterial culture (n = 9) was added to each well to achieve an initial OD600 = 0.4 (3 technical replicates/ culture). OD600 bacterial growth was measured every 15 minutes for 17 hours using a BioTek Epoch 2 spectrophotometer (30°C). Three or four independent growth curve replicates were performed for each pesticide.
We obtained growth curve metrics using the R package growthcurver . For OP and Pyr separately, we analyzed the individual effects of replicate and treatment on growth rate values (r) obtained from growthcurver using non-parametric Kruskal-Wallis tests. We measured the effect of pesticide treatment separately for each replicate if the effect of replicate was significant.