# Examining the Effects of Induced Plant Defenses on _Spodoptera frugiperda_ Performance
## 2 Materials and Methods
### Experimental Procedure  
_S. frugigerda_ eggs were obtained from a commercially available colony (Benzon Research Inc., Carlisle, PA, USA) which originated from Mississippi, USA. These insects have been bred on a priority artificial diet and reared in a colony for multiple generations, and are used annually in field trials. For the subsequent experiments, newly hatched fall army-worm neonates (Benzon Research Inc., Carlisle, PA, USA) were fed on 1 of 5 diet treatments: a proprietary artificial diet (Southland Products Inc., Lake Village, AR, USA); uninduced _G. max_ c.v. Braxton; uninduced _G. max_ c.v. Gasoy; JA-induced _G. max_ c.v. Braxton; or JA-induced _G. max_ c.v. Gasoy. We initially set up \(\sim\)2000 insects and roughly divided them evenly across the treatments. Soybeans in the JA induction treatment were initially induced 24 h before being fed to larvae by spraying them until runoff with a 1 mM solution of JA (Tokyo Chemical Industry, Tokyo, Japan) dissolved in 0.5% ethanol. Non-induced plants were sprayed with a 0.5% ethanol solution as a control. Plants in the different induction treatments were also isolated in separate bioclimatic chambers (Conviron, Winnipeg, MB, Canada) on a 16L:8D cycle at 28 \({}^{\circ}\)C and 80% humidity to prevent volatile induction of the uninduced plants. Insects in the different diet treatments were also separated so that insects feeding on artificial diet, non-induced plants, and induced plants were in separate bioclimatic chambers (Conviron, Winnipeg, MB, Canada) each on a 16L:8D cycle at 28 \({}^{\circ}\)C and 80% humidity. Additionally, larvae were not directly placed on plants to ensure that feeding did not further induce the JA-sprayed plants or induce control plants. Larvae were instead fed leaf cuttings of plants ad libitum in 1 oz souffle cups with 30 mm Whatman filter paper discs applied with 50 mL of DI water to keep the leaves from drying out. Generally, across plant species, leaves not fully expanded are more well-defended and contain higher toxic secondary metabolites, so only fully expanded leaves were fed to the larvae, with leaves being removed mechanically at the pulvinus where the petiole meets the stem to prevent induction caused by wounding/injury, although cutting soybean leaves does not induce plant defenses [21; 26]. To maintain plant induction, the JA-induction treated plants were sprayed every other day for the duration of the experiment. Larvae were then assayed for mortality 7 days post-emergence from eggs. After this point, dead insects were removed, and the remaining live insects were arbitrarily culled by half, given the limited plant material for rearing insects to later instars. This left us with \(\sim\)800 caterpillars to rear and use for further experiments. The remaining individuals reared from the different diets were then fed until pupation, after which pupation was recorded, with pupae also being sexed and massed (Mettler Toledo, Greifensee, Switzerland). We used mortality (at seven days after insects hatched and were placed on diet treatments), development time (in days to pupation), and pupal mass (mg) as proxies for larval performance within a generation. Adult fecundity, taken as the total number of eggs laid per female, was also assessed for individuals on the artificial diet treatment in a separate experiment. Individuals used in this experiment were reared as larvae using the same setup/procedure as above. Upon pupation and then ecolsion, sex, pupal mass (mg), and adult mass (mg) were recorded for all individuals. Female moths that eclosed were then placed individually in large 3.7 L containers (Rubbermaid, Atlanta, GA, USA) with 2 male moths that also eclosed within 24 h of each other and provided a 20% sucrose-electrolyte solution (m/v; The Gatorade Company, Chicago, IL, USA). Cages were checked daily, with the number of eggs laid counted until the females died. A total of \(\sim\)1800 neonates were set up at the onset of this experiment which resulted in 18 breeding pairs (n = 18).