FAW eggs and corn seeds were obtained from the National Plant Protection Scientific Observation and Experiment Station, Langfang. FAW were reared on an artificial diet until pupation in the artificial climate box (RXZ-500B, Ningbo Jiangnan Technology Co., Ltd., Ningbo, China) under controlled conditions (26 ± 1 °C, 65 ± 5% relative humidity, 16:8 h light/dark cycle). Adults and fourth to fifth instar larvae were used in oviposition, feeding, EAG, and behavioral assays. Both regular corn (RC: Zhengdan 958) and sweet corn (SC: Jinguan 597) were grown in pot culture in a greenhouse (26 ± 1 °C, 65 ± 5% relative humidity, 16:8 h light/dark cycle), and plants at the seedling stage (20 days) were used for behavioral choice experiments and volatiles collection.

All analytical standards, including hexanal (≥ 98% purity), trans-2-hexen-1-ol (≥ 97%), linalool (≥ 97%, mixture of isomers), β-caryophyllene (≥ 98%), cis-3-hexen-1-ol (≥ 98%), and mineral oil (light, suitable for GC), were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

A cage experiment simulated a field environment to assess the oviposition preferences of FAW on two different maize varieties. Ten seedling-stage RC and SC plants, each 10 plants, were placed on either side of the cage (1.5 × 1.0 × 1.5 m) (Figure 1A). A cotton ball soaked in a 10% honey solution was placed in the center of the cage for FAW to consume. Prior to testing, insects were sexed at the pupal stage. Upon emergence, males and females were paired and housed separately. Females exhibiting initial oviposition behavior were selected for the oviposition assays, with ten mated females used per replicate. Egg counts on leaves (excluding those on the cage walls) were counted 24 hours later. The oviposition preference experiment was conducted in a greenhouse (26 ± 1 °C, 65 ± 5% relative humidity, 16:8 h light/dark cycle) with 3 biological replicates.

The leaf disc test method established by Jermy (Jermy et al., 1968) was used to evaluate the feeding preference behavior of FAW larvae (Figure 2A). Before the test, the 4-5th instar FAW larvae used in the test were subjected to 12 hours of starvation, and the leaves of two types of maize at the seedling stage were trimmed to a length of about 5 cm. During the test, the prepared RC and SC leaves were placed in a 15 cm diameter culture dish, symmetrically arranged on both sides. During the test, a larva was placed in the center of the selection device, and the selection was recorded. 1 hour later, feeding preferences were assessed based on the area they ate. Leaves with larger feeding areas were considered to be preferred by FAW larvae, and this result was used to calculate the selection rate. The entire feeding choice test was completed in a greenhouse (26 ± 1 °C, 65 ± 5% relative humidity, 16:8 h light/dark cycle), and 80 biological replicates (n = 80 larvae) were performed.

Volatile organic compounds (VOCs) were collected from RC and SC seedlings using solid-phase microextraction (SPME) coupled with a 50/30 μm polydimethylsiloxane (PDMS) fiber assembly. Twenty minutes before collection, cover the stems and leaves of the corn plant above ground with a polyethylene terephthalate (PET) sampling bag to avoid mechanical damage to the plant during the operation. SPME technology was used for adsorption and collection for 6 hours. During adsorption, the SPME fiber was suspended and fixed above the sample using a support. 5 biological replicates were performed (n = 5 adsorption collections). Agilent Technologies (China) Co., Ltd., used gas chromatography-mass spectrometry to separate and identify volatile compounds.

Gas Chromatography (GC: 8890A) Conditions: Compound separation was performed on an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) with the following temperature program: initial temperature of 40 °C (hold 1 min), ramped at 5 °C/min to 150 °C (hold 2 min), then increased at 8 °C/min to 250 °C (hold 52 min). The carrier gas (He) flow rate was maintained at 1.0 mL/min with constant pressure mode. The injection port temperature was set at 250 °C with splitless mode, and the septum purge flow rate was 3.0 mL/min.

Mass Spectrometry (MS: 5977B) Parameters: The GC effluent was directly introduced into the mass spectrometer with the following operating conditions: electron impact (EI) ionization mode at 70 eV; ion source temperature 230 °C; quadrupole temperature 150 °C; mass range 40–550 amu at 1562 amu/s scanning speed. Compounds identification was achieved by retention time matching with standards and comparing mass spectra with NIST 17 library (match threshold >85%), and the relative content of each substance was calculated using area normalization.

Electroantennography (Tangshan Dinggan Technology Co., Ltd.) was used for recording the activity responses of the antennae of female FAW. Before the EAG recording, the odor source was prepared. The test substance was dissolved in mineral oil at various concentrations (0.1, 1, 10, and 100 mg/mL). The blank control (CK) consisted of mineral oil, and the positive control (CF) consisted of 100 mg/mL cis-3-hexen-1-ol. Before testing, 10 μL of each test substance was pipetted onto filter paper in a blue pipette tip (1 mL) and sealed with tin foil. During testing, both ends of the antenna were fixed to the electrodes using conductive adhesive, the test sequence for each group was CK, CF, test substance (0.1, 1, 10, and 100 mg/mL), and CF, CK. The test interval was 45 seconds, and each substance was repeated five times (n = 5 female antennae). The relative antennal response (%) induced by each test substance concentration was recorded.

To assess the behavioral responses of FAW larvae and adults to target volatile standards, tests were conducted using a Y-tube olfactometer and a three-chamber olfactometer, respectively. The Y-tube glass olfactometer (2.5 cm in diameter) comprises a 20 cm main tube and two 20 cm side tubes forming a 45-degree angle, and is housed in a steel chamber (1.0 × 0.8 × 0.8 m) equipped with a camera and a 40 W fluorescent lamp that delivers uniform illumination (~2000 lux) (Yi et al., 2023). The three-chamber olfactometer consists of two outer odor chambers and a central insect chamber. Compressed gas is introduced into the two outer chambers, and a common outlet at the top of the central chamber facilitates airflow. All experiments were conducted in a behavioral laboratory (26 ± 1 °C, 65 ± 5% relative humidity).

For larval behavioral testing (12-hour-starved 4-5th instar larvae), 20 μL of the volatile stimulus and 20 μL of mineral oil (CK) as a control were placed as the two odor sources in the Y-tube olfactometer. Individual larvae were placed in the olfactometer for behavioral choice testing. A choice was scored when the larva entered more than one-third of either arm and remained there for at least 30 seconds. 50 independent biological replicates (n = 50 larvae) were performed for each test substance. To mitigate positional bias, the left and right arms were swapped following every third test. The airflow was filtered through activated carbon and water before being delivered into the two side arms of the olfactometer at 300 mL/min, proceeding toward the central main arm.

For adult behavioral testing, 20 μL of each test substance and mineral oil (CK) were placed as odor sources in the olfactory chambers on either side of the three-chamber olfactometer (Figure 3A). 1 female moth were placed in the central compartment, 50 independent biological replicates (n = 50) were performed for each test substance. Purified air entered the two side chambers of the olfactometer simultaneously. An air pump then directed the odor from these chambers into the central compartment at a flow rate of 300 mL/min. Following each test, the chambers were thoroughly cleaned, and the left and right odor sources were swapped for the next trial to control for any positional effects. The experiment lasted from 9 pm to 5 am the following morning, which corresponds to the nocturnal activity period of FAW.

All data were analyzed using IBM SPSS Statistics v31 (SPSS Inc., Chicago, IL, USA). Student’s t-test was used to analyze significant differences in the oviposition preference test (P < 0.05). Chi-square tests were used to analyze significant differences in disk feeding and indoor olfactometer tests (P < 0.05). The significance of the difference in the relative content of the same volatile compound between the two corn varieties was analyzed using Student’s t-test (P < 0.05). The significance of the difference between the relative response of EAG was analyzed using one-way ANOVA and Tukey’s test (P < 0.05). EAG relative responses (%) = [(Treatment - CK)/(CF - CK)] × 100%. GraphPad Prism 9 software was used to plot these data.

Statistical analysis shows that in the oviposition preference test (Figure 1, Supplementary Table S1), mated FAW females preferred to lay eggs on SC maize plants, with the number of eggs on SC leaves being significantly higher than on RC leaves (t = 4.772, P < 0.001).

In this experiment, we recorded that 49 larvae chose to feed on sweet corn leaves, and 27 chose to feed on regular corn leaves. The results show that FAW larvae have a significant preference for feeding on sweet corn (χ² = 6.368, P = 0.01) (Figure 2).

Volatile compounds from the two corn varieties were collected by solid-phase microextraction (SPME) and separated and identified by gas chromatography-mass spectrometry (GC-MS). Qualitative analysis revealed no significant differences between SC and RC, with 21 compounds identified in both. However, quantitative analysis revealed significantly higher levels of four volatile compounds in SC compared to RC (Table 1). The specific compounds that differed were hexanal (t = -9.468, P < 0.001), trans-2-hexen-1-ol (t = -13.894, P < 0.001), linalool (t = -35.608, P < 0.001), and β-caryophyllene (t = -29.664, P < 0.001).

Mass spectrometry analysis revealed that four volatiles with significantly higher relative concentrations in SC than in RC were tested in mated and unmated female FAW insects. The results showed that the relative EAG responses of female corn borers to these four substances increased with increasing concentration. The EAG responses were lowest at 0.1 mg/mL and highest at 100 mg/mL (Figure 4). Specifically, at 100 mg/mL of hexanal and β-caryophyllene, mated females exhibited significantly higher EAG responses than unmated females. Furthermore, at 10 mg/mL of trans-2-hexen-1-ol, mated females exhibited significantly higher EAG responses than unmated females. At the same concentration of the other substances, no significant differences in EAG responses were observed between mated and unmated females (Table 2S).

Behavioral results obtained using a Y-tube olfactometer showed that hexanal, trans-2-hexen-1-ol, linalool, and β-caryophyllene all exhibited varying degrees of attraction to FAW larvae (Figure 5, Supplementary Table S3). Specifically, hexanal, trans-2-hexen-1-ol, and linalool were attractive to FAW larvae at all four concentrations. However, β-caryophyllene exhibited the most significant attraction only at 10 mg/mL and 100 mg/mL. In contrast, 0.1 mg/mL linalool (χ² = 37.098, P < 0.001) and 100 mg/mL hexanal (χ² = 39.093, P < 0.001) exhibited the most significant attraction.

Behavioral selection tests of FAW adults for volatile standards revealed that the four volatiles elicited behavioral responses that differed from those of larvae (Figure 3, Supplementary Table S4). All four volatiles exhibited significant attraction to adults at both 1 mg/mL and 10 mg/mL concentrations. At 0.1 mg/mL, trans-2-hexen-1-ol (χ² = 5.818, P = 0.016), and β-caryophyllene (χ² = 8.805, P = 0.003) exhibited weaker attraction than hexanal (χ² = 15.364, P < 0.001), linalool (χ² = 26.273, P < 0.001), but all significantly attracted FAW females. At a concentration of 100 mg/mL, hexanal (χ² = 32.818, P < 0.001), trans-2-hexen-1-ol (χ² = 17.780, P < 0.001), and β-caryophyllene (χ² = 39.093, P < 0.001) had a significant attraction effect on FAW females, while Linalool (χ² =3.571, P = 0.059) had no significant attraction effect.