Impact of neonate pre-treatment conditions on insecticidal protein activity and laboratory bioassay variation

Variability in insecticidal bioassay has been observed among different laboratories, with different insecticidal molecules, and is present across different orders of insects. Efforts to standardize the laboratory methods used in bioassays of microbial products have been part of the overall attempt to minimize variation. The objective of this study was to determine if a particular pre-treatment condition can provide variation in a Spodoptera frugiperda laboratory population response when exposed to Vip3Aa19 insecticidal protein. Four different laboratory pre-treatment conditions were tested in fall armyworm neonates consisting of: 1) time after hatching; 2) prior feeding; 3) overnight storage; and 4) humidity. For all bioassays, neonates were subsequently exposed to a Vip3Aa19 dose approximating the LC_70. For the first condition, neonates were kept for four different times (0, 2, 6, 12 h) prior to Vip3Aa19 bioassay. For the feeding comparisons, neonates without prior feeding (0 h) were compared with 2, 6, 12 hours feeding on artificial diet before bioassays. The third condition involved storage of neonates overnight in a chamber at 14^oC as compared with larvae that were bioassayed within 4 h of hatching. The last condition involved pre-incubation at low humidity (~15%) or high humidity (>90%). Significant effects (P-value <0.05) of pre-treatment conditions were observed when neonates were fed prior to bioassay or when stored overnight at 14^oC. There was no significant difference observed in the other pre-treatment conditions. The results of this study will help standardize bioassay conditions for microbial products and identify possible sources of variation in bioassays.