Schematic of the experimental setup for studying magnetic orientation in fall armyworms.

Moths are tethered to a vertical shaft at the center of the virtual flight simulator, with an encoder recording their flight heading. A full-spectrum lamp illuminates the arena, while the computer controlling the experiment is positioned outside the light field to avoid interference. Moths are free to rotate in any direction during the assay. Full experimental details in Methods. The cylinder is illustrated as clear in the figure to reveal the internal setup, but it is opaque in the actual experiment.

The Earth’s magnetic field and visual cues guide migratory flight behavior in both field-captured and lab-raised fall armyworms.

(A) Flight orientation behavior of spring field-captured moths (“Spring Exp. Field”) in response to visual and geomagnetic cues. (B) Flight orientation behavior of fall field-captured moths (“Fall Exp. Field”) in response to visual and geomagnetic cues. (C) Flight orientation behavior of lab-raised fall-conditioned moths (“Fall Exp. Lab”) in response to visual and geomagnetic cues. (D) Flight orientation behavior of lab-raised control fall-conditioned moths (“Fall Control”), tested with consistent visual and geomagnetic alignment. Each group underwent five sequential 5-minute phases (I–V), with each subplot representing individual moths’ flight directions in the simulator. The length of each vector represents individual directedness (r), ranging from 0 to 1, where the outer edge of the plot corresponds to r = 1. The thick mean vector (MV) arrow represents the weighted average of individual orientations, calculated using Moore’s modified Rayleigh test (see Methods), and is red when there is significant group orientation but grey when it is not significant. The R* value quantifies the directedness of the MV. Dashed circles indicate thresholds for statistical significance, with radii corresponding to P < 0.05 and P < 0.01. Shaded sections of the outer diameters of the circles represent the 95% confidence limits of the group orientation. The outermost radius represents R* = 2.5. The black triangle denotes the position of the visual cue, while the red triangle indicates the direction of the expected migratory orientation in each season (north in spring, south in fall). The experimental setup included both the natural magnetic field (NMF, panels with light green background) and a changed magnetic field (CMF, panels with light blue background) where the horizontal magnetic field direction was reversed; further details of the magnetic field parameters in the NMF and CMF are shown in Fig. S1.

Fall armyworms exhibit a delayed response to changes in magnetic-visual cue alignment.

The behavioral data for (A) fall field-captured experimental (“Fall Exp. Field”, data from Fig. 2B), (B) fall lab-raised experimental (“Fall Exp. Lab”, data from Fig. 2C), and (C) fall lab-raised control (“Fall Control”, data from Fig. 2D) groups were analyzed in 30-second time bins, resulting in ten bins over each 5-minute phase of the experiment. For each group, Moore’s modified Rayleigh test was applied, and the obtained R* values were plotted against time. R* > 1 indicates a significant collective orientation within that 30-second interval, while R* < 1 indicates the absence of significant group-level orientation.

Visual information is essential for maintaining group flight orientation in fall armyworms.

(A) The lab-raised, fall-conditioned population lost significant group orientation under complete darkness (“Fall Exp. Dark”). (B) The lab-raised, fall-conditioned population exhibited a significant loss of group orientation under illuminated conditions where visual cues were reduced to the bare minimum (“Fall Exp. BMVC”), i.e., obvious visual cues such as the black triangle were not provided. (C) The distribution of Rayleigh test r-values for individual moth orientations across different treatment groups (Left), and the distribution of average directional change per second (Right), the latter reflecting flight stability. The box plots represent the interquartile range (IQR), with the horizontal line inside indicating the median. Whiskers extend to the most extreme data points within 1.5 times the IQR. Pairwise comparisons of the r-values were performed using the Mann-Whitney U test, with multiple comparisons corrected by the Benjamini-Hochberg method. Detailed statistical results are provided in Table S3. Comparisons of the average directional change per second were performed using one-way ANOVA followed by Tukey’s HSD post hoc test, with statistical results provided in Table S4. Groups labeled with different letters differ significantly (P < 0.05). We also analyzed the r-values and average directional change per second in the Fall Exp. Field, Fall Exp. Lab, Exp. Fall Control, and the experiment shown in this figure, with results consistent with those shown here (see Tables S3 and S4, Fig. S4).

The magnetic field conditions during experimental procedures in 2023 and 2024.

A Comparison of total magnetic field strength between the controlled magnetic field (CMF) and normal magnetic field (NMF) (t-test, ECMF = 42.21 μT, ENMF = 42.02 μT, P = 0.79), showing no significant difference. B There is no significant difference in the intensity of the horizontal component between CMF and NMF (t-test, HCMF = 31.18, HNMF = 31.35, P = 0.813). C There is no significant difference in magnetic inclination between CMF and NMF (t-test, αCMF = 38.22°, αNMF = 38.28°, P = 0.92). D Comparison of magnetic azimuth between CMF and NMF, demonstrating a highly significant difference (t-test, βCMF =175.62°, βNMF = 4.11°, P < 0.001). E The magnetic component B, measured in nanoteslas (nT), of the time-dependent electromagnetic field across a 10 kHz resolution bandwidth (RBW), was analyzed as a function of frequency f, expressed in megahertz (MHz). The data shown in the figure represents the maximum locked values observed over a 40-minute period at the experimental location (23.604°N, 101.977°E). Measurements were conducted using the NF5035 spectrum analyzer, MDF9400 magnetic field antenna, MDF960X preamplifier (with a gain of 25 dB), and a 10-meter Anoni RF cable. The spectrum in the frequency range of 1 MHz to 10 MHz was first measured using the antenna paired with the preamplifier. Subsequently, low-frequency noise within the range of 2 kHz to 1 MHz was detected using the internal probe of the spectrum analyzer. In the low-frequency range, significant peaks are observed, particularly in the0.2-1 MHz region, where the magnetic field intensity is notably higher. In contrast, within the frequency range of 1 MHz to 10 MHz, the noise intensity is relatively low, ranging between 0.01 nT and 0.001 nT. The overall trend in this range is fairly stable, with only minor fluctuations at specific frequency points.

Year-specific analysis of orientation behavior in fall field-captured fall armyworms (2023 and 2024).

A Orientation behavior of the field-captured population in fall 2023. B Orientation behavior of the field-captured population in fall 2024. Experiments were conducted using fall armyworms captured from the field, revealing consistent orientation behavior across both years. In both 2023 and 2024, moths oriented significantly toward the visual cue when visual and geomagnetic cues were aligned. However, after reversing the magnetic field, the strength of directional orientation gradually diminished. Due to limited sample sizes in each year, data from both years were pooled for a comprehensive analysis, as presented in Fig. 2B.

Spectral distribution of light provided by the full-spectrum lamp.

The spectral distribution of the full-spectrum lamp used in the experiment was measured using an ATP2000P spectrometer (Optosky Technology Co. Ltd., Xiamen, China). The sensor was positioned vertically upward at the location originally occupied by the moth to measure the spectral irradiance of the light after it passed through the softbox, diffusion paper, and transparent acrylic cover. The results indicate that light radiation was detectable across most wavelengths between 200 nm and 850 nm, confirming that: The experimental system provides a full-spectrum light environment spanning ultraviolet (UV) to near-infrared (NIR) wavelengths, covering the visual range relevant to moths. And the acrylic plate and diffusion paper did not significantly block light radiation at any specific wavelength, ensuring uniform spectral exposure within the flight arena. The ambient light level in the experimental environment was measured to be below 1 lux using a Testo 540 lux meter, manufactured by Testo SE & Co. KGaA (Titisee-Neustadt, Germany).

The lack of visual cues and other necessary visual information significantly affects moth orientation ability and flight stability.

A Box plots show the distribution of Rayleigh test r values for individual orientations of moths in different treatment groups. B Box plots show the distribution of average directional degree change per second, reflecting flight stability, across treatment groups. Boxes represent the interquartile range (IQR), with the horizontal line inside indicating the median. The “whiskers” extend to the most extreme data points within 1.5 times the IQR. Error bars indicate data variability, facilitating comparison between groups. Pairwise comparisons in A were conducted using the Mann-Whitney U test with multiple comparisons corrected by the Benjamini-Hochberg method. Detailed statistical results are provided in Supplementary Table S3. In B, comparisons were performed using one-way ANOVA followed by Tukey’s HSD post hoc test. Groups labeled with different letters differ significantly (P < 0.05). Detailed statistical results are provided in Supplementary Table S4.

Detailed data of flight orientation behavior assay.

Multiple comparison results of the mean Rayleigh’s r values of Exp. Spring across different experimental phases, using the Wilcoxon rank-sum test with multiple comparison correction performed by the Benjamini-Hochberg

Multiple comparisons of mean Rayleigh test r values across experimental conditions using the Wilcoxon rank-sum test (adjusted by the Benjamini-Hochberg method).

Tukey test for mean angular velocity (directional change per second) across experimental conditions.

Multiple comparisons of mean Rayleigh test r values across experimental conditions using the Wilcoxon rank-sum test adjusted by the Benjamini-Hochberg method.