Caterpillar petri dish choice assay
To determine caterpillar preference for plant tissue with different
levels of salt stress, we conducted three two-choice assays and one
three-choice assay (Figure 1B ):
- No salt-treated plant leaf disc (0 mM) vs. High salt-treated plant
leaf disc (200 mM).
- Medium salt-treated plant leaf disc (100 mM) vs. High salt-treated
plant leaf disc (200 mM).
- No salt-treated plant leaf disc (0 mM) vs. Medium salt-treated plant
leaf disc (100 mM).
- No salt-treated plant leaf disc (0 mM) vs Medium salt-treated plant
leaf disc (100 mM) vs High salt-treated plant leaf disc (200 mM) (Panet al. , 2019).
1.5 cm diameter leaf discs were excised from the fourth fully expanded
leaf of each treatment plant (0 mM, 100 mM, and 200 mM). One leaf disc
from each treatment was placed at opposite ends of a petri dish (10 cm X
1.5 cm) lined with 3% agar. A single 2nd instarH. zea caterpillar was placed inside the petri dish, and the
petri dishes were sealed with parafilm to prevent the desiccation of
herbivores and plant tissue (Marsack and Connolly, 2022). Caterpillars
were given thirty minutes to make a choice in the petri dish, after
which the leaf disc the caterpillar had settled on was recorded. This
was referred to as the “first establish” (FE) parameter. Another
parameter called the “first finish” (F1) was reported as the first
leaf disc to be totally consumed by the caterpillar
(Supplementary Figure S1 ). In the three-choice test, a “second
finish” (F2) was also recorded, which was the second leaf disc to be
completely consumed by the caterpillar. First and second finishes were
recorded for a maximum of five days. After two days of feeding, leaf
area consumed or caterpillar consumption rate was determined by
photographing the remaining leaf disc area and analyzing images in
ImageJ (Supplementary Figure S2 )(Glozer, 2008; Connolly, Guiden
and Orrock, 2017; Orrock et al. , 2018; Marsack and Connolly,
2022).