# Chinese Cabbage Changes Its Release of Volatiles to Defend against _Spodoptera litura_
## 2 Materials and Methods
### Olfactory Preferences of M. similis (M. similis Olfactometer Experiments 1-4)  
We used a 4-arm olfactometer [30] with one glass sample bottle per arm to assess the olfactory preferences of 3-day-old mated females of _M. similis_. Each of the first three experiments included two empty sample bottles (blanks) and one bottle containing an undamaged, healthy plant. The fourth bottle in experiments 1-3 contained a mechanically damaged plant, a plant treated with larval regurgitate, or a plant infested with _S. litura_ for 12 h, respectively. In experiment 4, the sample bottles contained a healthy plant, a mechanically damaged plant, a plant treated with larval regurgitate, or a plant infested with _S. litura_ for 12 h. Each assay with four sample bottles was replicated 30 times. The trap ball was attached above the four selection arms; for each _M. similis_ individual, the selection was indicated by its climbing into the trap ball. An LED tube was hung 18.0 cm above an olfactometer, which was placed under a white canvas cover to diffuse the light source from interfering with the selection behavior of the wasps. In the experiment, a medical air compressor with low noise and relatively pure gas was selected as the gas delivery device, and a three-stage oil-water separator was used to filter out impurities in the gas, and then a clean and moist gas was produced after passing it into the filter bottle. By controlling the air compressor, the total gas flow rate is controlled at about 2.5 L min\({}^{-1}\), and the rotor flow device is adjusted to control the airflow rate through each gas source bottle at about 500 mL min\({}^{-1}\). Individual airflow was connected to each odor source and converged to a central glass piece where 4 _M. similis_ females were released. After thirty minutes following release, the location of each of the wasps was noted. Selection for an odor source was concluded when a wasp was present in one of the four ball traps. The experiment was replicated thirty times with 4 female wasps each time. Between replicates, glassware was cleaned by sequential rinsing in water, acetone, and pentane, after which the glassware was placed in an oven at 250 \({}^{\circ}\)C for 3 h.  
Behavioral Responses of S. litura Larvae to Chinese Cabbage and Volatile Compounds (S. litura Petri Dish Experiments 1 and 2, and S. litura Olfactometer Experiments 1 and 2)  
The preferences of _S. litura_ larvae for leaves treated in the indicated ways were compared in Petri dishes (20 cm diameter) containing a piece of filter paper with a drawn line that separated the dish into two equal sides. The experiments were conducted at 28 \(\pm\) 1 \({}^{\circ}\)C and in the dark. In Petri dish experiment 1, the following treatments were compared: CK (blank treatment without plants) vs. healthy leaves, and CK vs. leaves that had been infested by _S. litura_ for 12, 24 h, or 48 h. In Petri dish experiment 2, the following treatments were compared: healthy leaves vs. leaves that had been infested by _S. litura_ for 12, 24 h, or 48 h (the infesting larvae were removed from the leaves immediately before the experiment began). In each replicate of each treatment of the Petri dish experiments, one second- to third-instar larvae of _S. litura_ that had been starved for 24 h was placed in the center of the dish, and its feeding selection was recorded at 15 min. After a larva made a selection, it was removed, and the original leaf was immediately replaced with a new leaf for the next larva to choose. The Petri dish and filter paper were replaced every 3 larvae. The Petri dish experiments were conducted 3 times, each time with 30 replicates per treatment. A glass Y-tube olfactometer, consisting of a 12 cm long base tube and two 16 cm long arms at a 60\({}^{\circ}\) angle with an inner diameter of 2.0 cm, was used to observe the olfactoryresponse of _S. litura_ larvae to the volatiles of cabbage leaves. Before the start of each assay, we treated the system with compressed air for 10 min to ensure that the odors in the arms were equivalent.