Antifeedant Bioassay for Spodoptera littoralis

Spodoptera littoralis colonies are maintained at ICA-CSIC, reared on artificial diet, and kept at 22 ± 1 °C and >70% RH, with a photoperiod of 16:8 h (L:D) in a growth chamber. The bioassays were conducted as described [52 (link)]. The upper surface of Capsicum anuum (1.0 cm²) were treated with 10 μL of the test substance. The products were tested at an initial dose of 5 µg/µL (50 µg/cm²). Five Petri dishes with two sixth-instar S. littoralis larvae (>24 h after moulting) were allowed to feed in a growth chamber (until 75% larval consumption of control disks or tretament disks, environmental conditions as above). Each experiment was repeated 2 times. Feeding inhibition was calculated by measuring the disk surface consumption (digitalized with https://imagej.nih.gov/ij/, accessed on 23 March 2020) as % FI = [1 − (T/C) × 100], where T and C represent feeding on treated and control leaf disks, respectively. The antifeedant effects (% FI) were analyzed for significance by the nonparametric Wilcoxon signed-rank test. Extracts and compounds with an FI > 70% were further tested in a dose-response experiment (4–5 serial dilutions) to calculate their relative potency (EC₅₀, the effective dose to give a 50% settling reduction) from linear regression analysis (% FI on Log-dose).