eLife Assessment

This fundamental study presents experimental evidence on how geomagnetic and visual cues are integrated in a nocturnally migrating insect. The evidence supporting the conclusions is compelling. The work will be of broad interest to researchers studying animal migration and navigation.

Reviewer #1 (Public review):

Summary

The manuscript by Ma et al. provides robust and novel evidence that the noctuid moth Spodoptera frugiperda (Fall Armyworm) possesses a complex compass mechanism for seasonal migration that integrates visual horizon cues with Earth's magnetic field (likely its horizontal component). This is an important and timely study: apart from the Bogong moth, no other nocturnal Lepidoptera has yet been shown to rely on such a dual-compass system. The research therefore expands our understanding of magnetic orientation in insects with both theoretical (evolution and sensory biology) and applied (agricultural pest management, a new model of magnetoreception) significance.

The study uses state-of-the-art methods and presents convincing behavioural evidence for a multimodal compass. It also establishes the Fall Armyworm as a tractable new insect model for exploring the sensory mechanisms of magnetoreception, given the experimental challenges of working with migratory birds. Overall, the experiments are well designed, the analyses are appropriate, and the conclusions are generally well supported by the data.

Strengths

• Novelty and significance: First strong demonstration of a magnetic-visual compass in a globally relevant migratory moth species, extending previous findings from the Bogong moth and opening new research avenues in comparative magnetoreception.

• Methodological robustness: Use of validated and sophisticated behavioural paradigms and magnetic manipulations consistent with best practices in the field. The use of 5 min bins to study a dynamic nature of magnetic compass which is anchored to a visual cue but updated with latency of several minutes is an important finding and a new methodological aspect in insect orientation studies.

• Clarity of experimental logic: The cue-conflict and visual cue manipulations are conceptually sound and capable of addressing clear mechanistic questions.

• Ecological and applied relevance: Results have implications for understanding migration in an invasive agricultural pest with expanding global range.

• Potential model system: Provides a new, experimentally accessible species for dissecting the sensory and neural bases of magnetic orientation.

Weaknesses

Overall, this is a strong study, and the authors have completed an excellent major revision that has undoubtedly addressed most major and minor issues. The remaining points below are minor recommendations, and I acknowledge that differences in opinion are always possible:

(1) Structure and Presentation of Results

• I recommend reordering the visual-cue experiments to progress from simpler conditions (no cues) to more complex ones (cue-conflict). This would improve narrative logic and accessibility for non-specialist readers. The authors have chosen not to implement this suggestion, which I respect, but my recommendation stands.

(2) Ecological Interpretation

• The authors should expand their discussion on how the highly simplified, static cue setup translates to natural migratory conditions, where landmarks are dynamic, transient, or absent. Specifically, further consideration is needed on how the compass might function when landmarks shift position, become obscured, or are replaced by celestial cues. Additionally, the discussion would benefit from a more consolidated section with concrete suggestions for future experiments involving transient, multiple, or more naturalistic visual cues.

This point was addressed partially in one paragraph of the Discussion, which reads as follows:

"In nature, they are likely to encounter a range of luminance-gradient visual cues, including relatively stable celestial cues as well as transient or shifting local features encountered en route. Although such natural cues differ from our simplified laboratory stimulus, they may represent intermittently sampled visual inputs that can be optimally integrated with magnetic information, with the congruency between visual and magnetic cues likely playing a key role in maintaining a stable compass response. Whether the cues are static or changing, brief periods without them may still allow the subsequent recovery of a stable long-distance orientation strategy. Determining which types of natural visual cues support the magnetic-visual compass, and how they interact with magnetic information, including how their momentary alignment or angular relationship is integrated and how such visual cue-magnetic field interactions may require time to influence orientation, together with elucidating the genetic and ecological bases of multimodal orientation, will be important objectives for future research."

While this paragraph is informative, the wording remains lengthy, somewhat unclear, and vague. Shorter, clearer statements would improve readability and impact. For example:

• How could moths maintain direction during periods when only the magnetic field is present and visual landmarks are absent?

• Could celestial cues (e.g., stars) compensate, and what happens if these are also obscured?

• What role does saliency play when multiple visual landmarks are present simultaneously?

• How might a complex skyline without salient landmarks affect orientation?

Including simple, concise sentences that pose concrete open questions and suggest experimental designs would strengthen the discussion without creating space issues. In my view, a comprehensive discussion of how the simplified, static cue setup relates to natural migratory conditions-where landmarks are dynamic, transient, or absent-would add significant value to the paper.

(3) Methodological Details and Reproducibility

• The lack of luminance level measurements should be explicitly highlighted.

• The authors chose not to adjust figure legends by replacing "magnetic South" with "magnetic North." While I believe this would be more conventional and preferable, this is ultimately a minor stylistic issue.

(4) Conceptual Framing and Discussion

• Although the authors made a good attempt to explain the limitations of using an artificial visual cue, I believe there is room for a more explicit argument. For example, it could be stated clearly that this species is unlikely to encounter a situation in nature where a single, highly salient landmark coincides with its migratory direction. Therefore, how these findings translate to real migratory contexts remains an open question. A sentence or two making this point directly would strengthen the discussion.

(5) Technical and Open-Science Points

• Sharing the R code openly (e.g., via GitHub) should be seriously considered. The code does not need to be perfectly formatted, but making it available would be highly beneficial from an open-science perspective.

Reviewer #2 (Public review):

Summary:

The work titled "Geomagnetic and visual cues guide seasonal migratory orientation in the nocturnal fall armyworm, the world's most invasive insect" provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

Strengths:

It has been demonstrated that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long distance navigation. However, data are lacking for other insects. This study suggested that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects.

Weaknesses:

The visual cues used in the indoor experimental system designed by the authors may have some limitations in ecological relevance. The author may need more explanations on this experimental system.

In the revised manuscript, the authors have added explanations in the discussion section. I am fine with the revision.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary

The manuscript by Ma et al. provides robust and novel evidence that the noctuid moth <i>Spodoptera frugiperda</i> (Fall Armyworm) possesses a complex compass mechanism for seasonal migration that integrates visual horizon cues with Earth's magnetic field (likely its horizontal component). This is an important and timely study: apart from the Bogong moth, no other nocturnal Lepidoptera has yet been shown to rely on such a dual-compass system. The research therefore expands our understanding of magnetic orientation in insects with both theoretical (evolution and sensory biology) and applied (agricultural pest management, a new model of magnetoreception) significance.

The study uses state-of-the-art methods and presents convincing behavioural evidence for a multimodal compass. It also establishes the Fall Armyworm as a tractable new insect model for exploring the sensory mechanisms of magnetoreception, given the experimental challenges of working with migratory birds. Overall, the experiments are well-designed, the analyses are appropriate, and the conclusions are generally well supported by the data.

Strengths

(1) Novelty and significance: First strong demonstration of a magnetic-visual compass in a globally relevant migratory moth species, extending previous findings from the Bogong moth and opening new research avenues in comparative magnetoreception.

(2) Methodological robustness: Use of validated and sophisticated behavioural paradigms and magnetic manipulations consistent with best practices in the field. The use of 5-minute bins to study the dynamic nature of the magnetic compass which is anchored to a visual cue but updated with a latency of several minutes, is an important finding and a new methodological aspect in insect orientation studies.

(3) Clarity of experimental logic: The cue-conflict and visual cue manipulations are conceptually sound and capable of addressing clear mechanistic questions.

(4) Ecological and applied relevance: Results have implications for understanding migration in an invasive agricultural pest with an expanding global range.

(5) Potential model system: Provides a new, experimentally accessible species for dissecting the sensory and neural bases of magnetic orientation.

Weaknesses

While the study is strong overall, several recommendations should be addressed to improve clarity, contextualisation, and reproducibility:

We thank Reviewer #1 for the positive and encouraging evaluation of our study. We appreciate the recognition of our work’s strengths and are grateful for the constructive feedback on the remaining weaknesses, which will guide and strengthen our revisions.

Structure and presentation of results

Requires reordering the visual-cue experiments to move from simpler (no cues) to more complex (cue-conflict) conditions, improving narrative logic and accessibility for non-specialists.

Thank you for this thoughtful suggestion. While we appreciate the rationale for presenting results from simpler to more complex conditions, we kept the original sequence because it aligns with the logic of our study. Our initial aim was to determine whether fall armyworms use a magnetic compass integrated with visual cues, as shown in the Bogong moth. After establishing this phenotype, we then examined whether visual cues are required for maintaining magnetic orientation. We have also clarified in the Introduction that magnetic orientation in the Bogong moth relies on integration with visual cues, which provides readers with clearer context and improves the overall narrative flow.

Ecological interpretation

(a) The authors should discuss how their highly simplified, static cue setup translates to natural migratory conditions where landmarks are dynamic, transient or absent.

Thank you for raising this important point. We agree that natural migratory environments provide visual information that is often dynamic, transient, or intermittently absent, in contrast to the simplified and static cue used in our indoor experiments. Our intention in using a minimal, static cue was to isolate and test the fundamental presence of magnetic–visual integration in fall armyworms under fully controlled conditions.To address the reviewer’s concern, we have added a brief note in the Discussion indicating that fall armyworms may encounter both static and dynamic luminance-based visual cues in nature, such as light–dark gradients created by terrain features or more stable celestial patterns. Although these natural cues differ from our simplified laboratory stimulus, they may similarly provide asymmetric visual structure that can be integrated with magnetic information. We also note that determining which natural visual cues support the magnetic–visual compass will be an important direction for future work.

(b) Further consideration is required regarding how the compass might function when landmarks shift position, are obscured, or are replaced by celestial cues. Also, more consolidated (one section) and concrete suggestions for future experiments are needed, with transient, multiple, or more naturalistic visual cues to address this.

Thank you for this constructive suggestion. We appreciate the reviewer’s point that additional consideration of how the compass might function under shifting, obscured, or celestial visual cues would strengthen the manuscript. Given the limited evidence currently available for this species, we have incorporated a concise and appropriately cautious discussion addressing these possibilities.

Methodological details and reproducibility

(a) It would be better to move critical information (e.g., electromagnetic noise measurements) from the supplementary material into the main Methods.

Thank you for this helpful suggestion. In the revised manuscript, we have added the key electromagnetic noise measurements information to the main Methods section.

(b) Specifying luminance levels and spectral composition at the moth's eye is required for all visual treatments.

Thank you for this helpful comment. We have clarified in the Methods as well as the legend of Fig. S3 that both luminance levels and spectral composition were measured at the position corresponding to the moth’s head.

(c) Details are needed on the sex ratio/reproductive status of tested moths, and a map of the experimental site and migratory routes (spring vs. fall) should be included.

Thanks. We have added the reproductive status of the tested moths in the Methods, specifying that all individuals used were unmated 2-day-old adults.

(d) Expanding on activity-level analyses is required, replacing "fatigue" with "reduced flight activity," and clarifying if such analyses were performed.

Thank you for this comment. In this context, the term “fatigue” referred to the possibility that moths might gradually lose motivation or attention to orient when flying for an extended period in a simplified, artificial environment with limited sensory cues. Such a decrease in orientation motivation over time could, in theory, lead to a loss of individual orientation and consequently to the observed loss of group orientation. To test this possibility, we analyzed the orientation performance of each individual moth across different phases using the Rayleigh test. The <i>r</i>-value was used as a measure of individual directedness (higher <i>r</i>-values indicate stronger orientation). Our results showed that mean <i>r</i>-values did not differ significantly among the experimental phases (multiple comparisons, Table S2). This indicates that 25min measurement itself was not responsible for the loss of orientation. We did not perform a quantitative activity-level analysis in this study. However, as mentioned in Methods, flight activity was continuously monitored during the experiments by observing fluctuations in the pointer values on the experimental software, which corresponded to the moth’s rotational movements. If the pointer values remained unchanged for more than 10 seconds, the experimenter checked for wing vibrations by sound; if the moth had stopped flying, gentle tapping on the arena wall was used to stimulate renewed flight. Only individuals that maintained active flight throughout the experiment, with fewer than four instances of wingbeat cessation, were included in the analysis. We also mentioned that activity level analysis was not performed due to technical difficulties in the revised manuscript.

Figures and data presentation

(a) The font sizes on circular plots should be increased; compass labels (magnetic North), sample sizes, and p-values should be included.

Thank you for this helpful suggestion. Regarding the compass labels and statistical reporting, our analysis provides significance levels as ranges rather than exact <i>p</i>-values; therefore, we clarified in the figure legends that the two dashed circles correspond to thresholds for statistical significance <i>p</i> = 0.05 and <i>p</i> = 0.01, respectively. Sample sizes are already indicated within each panel. To avoid visual clutter caused by displaying both magnetic North and South, we show only the magnetic South direction (mS) consistently across panels, which can improve readability.

(b) More clarity is required on what "no visual cue" conditions entail, and schematics or photos should be provided.

Thank you for this comment. In our study, the “no visual cue” condition refers to the absence of the black triangular landmark inside the flight simulator. To improve clarity, we have updated the legend of Fig. 4 to explicitly state this and have referred readers to the schematic in Fig. 1, which illustrates the structure of the flight simulator. These additions clarify what the “no visual cue” condition entails without requiring additional schematics.

(c) The figure legends should be adjusted for readability and consistency (e.g., replace "magnetic South" with magnetic North, and for box plots better to use asterisks for significance, report confidence intervals).

Thank you. Regarding the choice of compass labeling, we intentionally used magnetic South (mS) rather than magnetic North (mN) because the main population tested in our experiments represents the autumn migratory generation. During autumn, fall armyworms orient southward when visual and magnetic cues are aligned. Using magnetic South in the plots therefore provides a clearer representation of cue alignment in this season and avoids potential confusion when interpreting the combined visual–magnetic information.

Conceptual framing and discussion

(a) Generalisations across species should be toned down, given the small number of systems tested by overlapping author groups.

Thank you for this valuable comment. In the revised manuscript, we have softened such statements in both abstract and maintext.

(b) It requires highlighting that, unlike some vertebrates, moths require both magnetic and visual cues for orientation.

Thank you for this helpful suggestion. We have added a sentence to the Discussion explicitly highlighting that, unlike some vertebrates capable of using magnetic information in the absence of visual cues, moths require the integration of both magnetic and visual cues for accurate orientation. This clarification emphasizes the distinct multimodal nature of compass use in migratory moths.

(c) It should be emphasised that this study addresses direction finding rather than full navigation.

Thank you for this important clarification. We have now made it explicit in the manuscript that our experiments address direction finding (i.e., orientation) rather than full navigation. This distinction is stated in both the Introduction and Discussion to clearly define the scope of the study.

(d) Future Directions should be integrated and consolidated into one coherent subsection proposing realistic next steps (e.g., more complex visual environments, temporal adaptation to cue-field relationships).

Thank you for this constructive suggestion. We agree that outlining realistic next steps is valuable. However, given the limited scope of the current data, we have only slightly expanded the existing forward-looking statements in the Discussion.

(e) The limitations should be better discussed, due to the artificiality of the visual cue earlier in the Discussion.

Thank you for this comment. We agree that the artificiality of the visual cue is an important limitation of the present study. Rather than extending speculative discussion, we have clarified this limitation in the revised Discussion and highlighted the key questions that future work must address.

Technical and open-science points

Appropriate circular statistics should be used instead of t-tests for angular data shown in the supplementary material.

Thank you for this comment. We have addressed this point (Fig. S1) in the revised supplementary material.

Details should be provided on light intensities, power supplies, and improvements to the apparatus.

Thank you. Light intensities are reported as spectral irradiance measurements in Supplementary Materials, which provide full wavelength-resolved information for the illumination used, although a separate measurement of total illuminance (lux) was not performed. We have also added the requested information on the power supplies.

The derivation of individual <i>r</i>-values should be clarified.

Thanks. We have clarified in the revised manuscript.

Share R code openly (e.g., GitHub).

Thanks. We are in the process of organizing the relevant R code, but have not been able to upload it to GitHub before the current revision deadline. The code is available from the corresponding author upon request.\

Some highly relevant - yet missing - recent and relevant citations should be added, and some less relevant ones removed..

Thanks. We added one recent relevant reference to the revised manuscript.

Reviewer #2 (Public review):

 

Summary:

 

This work provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

 

Strengths:

 

Although it has been demonstrated previously that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long-distance navigation, the study presented in this manuscript is still fundamentally important to the field of magnetoreception and sensory biology. It clearly shows that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects. I find the research very important, and the results are presented very well.

We thank Reviewer #2 for the positive and encouraging evaluation of our study. We appreciate the recognition of our work’s strengths.

Weaknesses:

The authors developed an indoor experimental system to study the influence of magnetic fields and visual cues on insect orientation, which is certainly a valuable approach for this field. However, the ecological relevance of the visual cue may be limited or unclear based on the current version. The visual cues were provided "by a black isosceles triangle (10 cm high, 10 cm 513 base) made from black wallpaper and fixed to the horizon at the bottom of the arena". It is difficult to conceive how such a stimulus (intended to represent a landmark like a mountain) could provide directional information for LONG-DISTANCE navigation in nocturnal fall armyworms, particularly given that these insects would have no prior memory of this specific landmark. It might be a good idea to make a more detailed explanation of this question.

We appreciate the constructive feedback on the weaknesses, which will guide and strengthen our revisions. To address the reviewer’s concern, we have added a brief note in the Discussion indicating that fall armyworms may encounter both static and dynamic luminance-based visual cues in nature, such as light–dark gradients created by terrain features or more stable celestial patterns. Although such natural cues differ from our simplified laboratory stimulus, they may represent intermittently sampled visual inputs that can be optimally integrated with magnetic information, whether the cues are static or changing, and brief periods without them may still allow the subsequent recovery of a stable long-distance orientation strategy.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major to Medium Suggestions

(a) Reordering of Visual Cue Tests

The manuscript currently presents cue-conflict experiments before the simpler "no visual cue" tests. For non-specialist readers, it would be more logical to start with the basic condition (no visual cues) and then move to progressively more complex ones. This provides a clearer and more logically sound narrative.

For example, the results could first demonstrate that without visual cues, the moths fail to orient (both in darkness and uniform light), and then show that introducing a single salient cue (a triangle on the horizon) restores directed behaviour. This would help readers understand the logic of the progression and should be better integrated throughout the Results and Discussion.

Thanks. We have responded this comment in Public Reviews.

(b) Translating Key Findings to Realistic Scenarios (LL 333-344 or where suitable in Discussion, and mentioning that we utilised a reductionist principle first in Intro, but clearly articulated that it is very simplified)

 

The main text (eg Discussion) should address how these findings translate to real-world conditions. The experimental design used a single, highly salient, and static cue, always aligned with the migratory direction. In nature, such a consistent landmark is unlikely-mountains or other features would shift position relative to the moth's trajectory as it flies.

Key questions arise which need to be addressed:

- How would the compass system adapt to changing landmark positions as the moth moves?

- What happens when no landmarks are visible (e.g. over flat plains or cloudy nights)?

- Would stellar or other cues take over in such cases? Your hypotheses, please.

Addressing these points - and proposing specific future experiments (e.g. with transient or multiple visual cues)-would strengthen the ecological relevance of the findings and show a clear way forward.

Thanks for your kind comments. We now explicitly state in the Introduction that our study employs a reductionist approach using a simplified visual environment to isolate magnetic-visual interactions. As the ecological questions raised by the reviewer cannot be addressed with the current dataset, we avoid extended speculation but have added brief clarification in the Discussion and addressed these points in the Public Reviews response. We also indicate that future work will need to examine the types of visual cues that can support magnetic orientation and how such cues couple with geomagnetic information.

Technical and Methodological Points

(a) Incomplete Methods Section

Critical technical information (e.g. electromagnetic noise measurements) currently appears only in supplementary figure legends. All such details should be included in the main Methods section if the word count allows (or include a short section in the main text with reference to more details in the supplementary material).

Thanks for your kind comments. We have addressed this as suggested in the Public Reviews.

(b) Lighting Conditions

Specify luminance levels (the amount of light emitted and passing through in quanta per unit of surface, eg m2) at the moth's eye and indicate whether spectral composition was consistent between treatments (with and without the visual cue).

Thanks for your comments. We have responded to this point in the Public Reviews.

(c) Figures

 

- Increase font sizes on circular histograms.

 

- Add compass labels (ideally magnetic North, mN, not south, etc, as it is usual in pertinent literature), sample sizes, and p-values on each panel.

 

- Replace "magnetic South" (mS) indicators with magnetic North (mN) to align with convention.

Thanks for your comments. We have responded to this point in the Public Reviews.

(d) Migratory Expectations

 

Include expected compass bearings for spring and autumn migrations (with citations) to relevant figures (Figure 2, 4, S2).

Thanks for your comments. We have added the information that “We recently found that fall armyworms from the year-round range in Southwest China (Yunnan) exhibit seasonally appropriate migratory headings when flown outdoors in virtual flight simulators, heading northward in the spring and southward in the fall, and this seasonal reversal is controlled by photoperiod (Chen et al., 2023).” in Introduction. Thus, we didn’t offer expected seasonal compass bearings in Results section.

(e) Add a map showing the experimental site and known migratory routes, clearly labelling spring vs fall routes. It would help justify expected headings.

Thank you for this suggestion. At present, there are no experimentally validated migratory routes (e.g., through mark-release-recapture or tracking approaches) for the specific fall armyworm population used in our study. Because these routes have not been biologically confirmed, we didn’t offer a presumed migratory map that may imply unwarranted certainty.

(f) Composition of Test Groups

Indicate sex ratios and reproductive status (mated/unmated) of tested moths, if known or comment if unknown, as both can affect migratory motivation and behaviour.

Thank you for this suggestion. We have responded to this point in the Public Reviews.

(g) Role and Nature of Visual Cues

While the results clearly show that orientation disappears without visual cues, the triangle cue is highly artificial. Well-studied Bogong moths are known to rely on views of Australian mountain ranges during their nocturnal migrations, but there is no evidence that armyworms use a similar strategy. Even for bogongs, it is not just one salient mountain always in front of them on migration. Discuss whether Fall Armyworm would encounter comparable natural cues in the field along their migratory route, or whether the triangle might simply provide a frame of reference rather than a true landmark.

Thank you for this comments. We have responded to this point in the Public Reviews.

(h) Future work could test:

- More naturalistic sky cues (moonlight, star fields).

- Varying the landmark's position relative to the magnetic field - slowly moving along - transient landmarks. Also, less salient landmarks and a more complex skyline, as it is usually more complex than just a single salient peak.

Thank you for this comments. We have responded to this point in the Public Reviews. Brief discussion as suggested has been added to the revised manuscript.

Minor Comments and Line-by-Line Suggestions

L70 - Check citation (possibly Mouritsen 2018). Missing in the list of references.

Thanks. This point has been addressed.

L75 - Consider citing the new and highly relevant preprint:

Pakhomov, A., Shapoval, A., Shapoval, N., & Kishkinev, D. (2025). Not All Butterflies Are Monarchs: Compass Systems in the Red Admiral (Vanessa atalanta). bioRxiv.

Thanks. We have cited this reference.

LL81-82 - Clarify vague phrasing; specify criteria for "good" vs "poor" orientation ability. Or reword/leave out.

Thanks for your comments.

L85 - "but one," not "bar one." 

Thanks. Corrected.

L124 - The 2 genetic citations are weakly linked to magnetoreception. We do not have a clear understanding of the insect magnetoreceptor and its underlying mechanism, so we simply cannot interpret genetic associations very well to underpin them to magnetoreception. For example, does noctuid's magnetic sense require a magnetised-based receptor and genes involved in biomineralization? Consider removing or softening claims. 

Thanks. Adressed.

LL123-126 - Define what for YOU constitutes "strong evidence" for magnetoreception (e.g. adaptive directional behaviour consistent with migratory orientation?). Is there such a thing as strong evidence at all?

Thanks for your comments. We agree that terms such as “confirmed” or “strong evidence” can overstate the certainty of magnetoreception findings, given the ongoing debates in the field. In the revised manuscript, we have toned down.

L153 - Indicate whether coils in NMF condition were powered or inactive.

Thanks for your comments. Addressed.

L163 - Justify use of multiple 5-min phases (e.g. temporal resolution of behaviour). It is confusing at the start, where first mentioned, and becomes clearer only towards the end, but it should be clearer at the start.

Thanks for your comments. The assay was divided into these 5-min segments to provide the temporal resolution needed to detect changes in flight orientation as the relative alignment of magnetic and visual cues was systematically altered. We now clarify this earlier in the Results.

LL167-171 - This is a good place where you can provide a map (main or supplementary with referencing) showing the study site and migration routes.

Thanks for your suggestion. We have responded to this point in the Public Reviews.

L174 - Avoid repetition of "expected."

Thanks. Addressed.

LL176-177 - Report 95% confidence intervals or equivalent and clarify which test (e.g. Moore's paired test) each p-value refers to.

Thanks for your suggestion.

LL189-191 - explain what fatigue means. I would remove fatigue and substitute it with "lowered flight activity". Also, the same statement comes later, so avoid repetitiveness and remove it in one place. The analysis of directedness is good throughout, but what about the analysis of activity level? Could you explain whether you did it or not, and if not, why, or if angular changes can serve as an activity proxy? Replace "fatigue" with "reduced flight activity." Avoid repetition. Clarify if activity level analysis was performed or if it was not, e.g. due to technical difficulties.

Thanks for your comments. We have responded to this point in the Public Reviews.

L196 - Note whether 95% CI overlaps with the expected direction. This is a crucial outcome.

Thanks for your comments.

LL203-205 - unclear, better to stick to "congruency", especially "initial congruency for the relationship between mN and visual cue" throughout.

Thanks for your suggestions.

L206 - Better to introduce a new subheading: "Laboratory-Reared Animals.".

Thanks for your suggestion. A new subheading has been added in the revised manuscript.

LL207-208 - Clarify which cues were available in Chen et al. (2023) and how they differ here.

Thanks for your comments. In Chen et al. (2023), the moths oriented under an artificial starry sky together with optic flow cues. In contrast, our experiments intentionally removed both the starry-sky pattern and optic flow to avoid introducing additional visual information when testing magnetic-visual integration for orientation. We have added further clarification regarding the conditions used in Chen et al. (2023) in the revised manuscript.

L228 - Use "lab-reared" consistently throughout the entire MS. Do not mix with lab-raised.

Thanks. Addressed by consistently using “lab-raised”.

Figure 2 - Confusing in parts, especially for people coming from birds and other vertebrates orientation background. At 12 o'clock, you usually expect either mN / gN (magnetic or geographic North) or the animal's own initial directional response used as control to compare the same animal's direction post-treatment. Here, your 6 o'clock is magnetic South in the first place - non-conventional. At 12 o'clock, better use mN or gN. Avoid using non-conventional references such as magnetic south. Remind readers of seasonally appropriate headings and refer to the map.

Thanks. We have responded to this point in the Public Reviews.

LL232-234 - Emphasize that cue-magnetic congruency is key. Highlight the most important point that the congruency between the seasonal migratory direction and visual cues is key, not that in spring/fall, visual cues must be towards or opposite to the migratory goal. But the visual cue could be in the migratory direction or opposite, or at an angle - this is for future direction.

Thanks. We have responded to this point in the Public Reviews.

Figure 2 and associated main text - highlight that you only tested the designs when in all seasons the salient and single visual cue was in the migratory direction (in spring it coincided with mN but in fall it was towards the magnetic south). Other directions of visual cues have not been tested, but for simplicity and consistency, you chose to do these ones as the first step, perhaps.

Thank you for this insightful comment. Yes, our experiments tested only the conditions in which the salient and single visual cue was aligned with the migratory direction. Other angular relationships between visual cues and the magnetic field were not examined in this study. For simplicity and consistency, we focused on this alignment as a first step toward understanding magnetic-visual cue integration in migratory orientation. We now highlight this in the Fig. 2 legend.

Figures captures/legends - hard to tell from the main text now, better to italicize figure caption text and visually space them from the main text.

Thanks for your suggestions.

LL 250-251 - mention to people more familiar with r - lowercase - what is the expected range for R uppercase. It is not bound 0-1 as r. Could it be negative? How large can it be?

Thanks. Thanks for the comment. After revisiting Moore (1980) we think that <i>R</i>* cannot take negative values. However, since <i>R</i>* = <i>R</i>*/N^ (3/2), it is not bounded between 0 and 1. We didn’t find any concept of an upper bound in the paper (https://doi.org/10.2307/2335330).

Figure 3 - Consider adding a horizontal line indicating the 5% significance threshold.

Thanks for your suggestions.

L 261 - need to have some narrative after the subheading before you insert Figure 3.

Thanks. Addreseed.

LL274-275 - highlight that the timeline of this congruency between mN and a landmark and the effect of this on directedness is not explored here, but worth doing in future. How long does a new congruency or a relationship between mN and a visual cue need to be exposed to the animal to regain its directional response? Clearly, it is just a question of time of exposure so that a new association is established. Suggest future work on time-dependent adaptation to new cue-field relationships.

Thanks for your suggestion. We have now included this point as a future direction in the revised Discussion.

Figure 4 & S4 - Replace letters with asterisks/brackets for significance. The use of the letter is confusing and unconventional.

Thanks for your suggestion.

Figure 4 caption - Clarify the main takeaway.

Thanks for your suggestion.

Figure 4 - bare minimum is confusing. I understand that you wanted to avoid "no visual cues" because, as long as the animal sees things, there are things to be used as visual cues, even if this is not the intention of the experimenter. However, it needs clarification and rewording. Better to be more specific, like "no black triangle and horizon were used, just the uniformly white cylinder", or something like that.

Thanks for your comments. In our setup it accurately describes the intentional removal of both the black triangle and the horizon, leaving only the uniformly white cylinder as the visual environment. This wording was chosen to reflect the practical limitations of producing a perfectly symmetrical flight simulator under laboratory conditions, and we therefore prefer to retain the original phrasing.

L328 - Remove Xu et al. (2021) citation (not relevant). This is an in vitro study with a protein which may not work exactly as it is claimed in the paper in vivo.

Thanks. Citation removed.

L349-350 - Clarify what "no visual cue" means (e.g., uniformly white cylinder, no horizon line). Include a photo or a schematic of the inner surface of the cylinder for this condition in the Supplementary Materials.

Thanks. We have responded to this point in the Public Reviews.

L380 & throughout - Replace "barely minimum visual cues" (BMVC) with "no visual cues", clarifying limitations in Methods, meaning that you can explain that absolutely no visual cues is practically impossible because, as long as there is light, animals can use some asymmetries as cues even if this is not the intention of the experimenter.

Thank you for this comment. We have decided to retain the term “barely minimum visual cues (BMVC)” because it accurately describes our experimental condition, which is distinct from a true “no visual cues” environment. In the revised Figure legend, we now clarify that BMVC refers to conditions in which obvious visual cues (i.e., features such as the black triangle in Fig. 1) were removed, while acknowledging that complete elimination of all visual information is not possible under illuminated conditions.

L396 - Be cautious when generalizing from two species tested by a research group that is not absolutely independent (some authors in bogong and armyworm works overlap). We saw examples in diurnal migratory butterflies (Monarchs), a more studied species than the armyworm, that the findings do not entirely translate to Red Admirals (Pakhomov et al. 2025 preprint mentioned). Suggestion to tone down any claims of broad generalisation throughout the manuscript.

Thank you for this comment. We have responded to this point in the Public Reviews.

LL402-407 - Note that, unlike birds (e.g. European robins), moths appear to require both magnetic and visual cues for orientation, whereas birds, mole rats and some other animals can use magnetic cues alone.

Thank you for this comment. We have responded to this point in the Public Reviews.

L410 - Specify that this is correct only in the Northern Hemisphere.

Thank you for this comment. Addressed.

LL415-416 - Acknowledge artificiality of single-cue setup (see the major comments above); integrate earlier in the Discussion.

Thank you for this comment. We have responded to this point in the Public Reviews.

LL420-425 - Consolidate Future Directions into a single subsection; include more concrete experimental ideas, for example, using more naturalistic, numerous transient landmarks (could be done in a virtual maze with LEDs on the wall of the cylinder with cues moving with time). Multiple visual cues. Manipulating with salience of cues - less simplistic, less salient.

Thank you for this comment. We have responded to this point in the Public Reviews.

L431 - Does this paper support this statement? I think it just tested the use of stellar cues in a zero magnetic field. It also dealt with direction finding, not navigation, which is a position-finding ability - a much more complex feat and might not be the ability of moths (requires further studies like with geographic and magnetic displacements, etc). Reword and check this. Show the distinction between direction finding and navigation.

Thank you for this comment. We have reworded the relevant sentence to use “orientation” instead of “navigation”.

L436-437 - Specify "global visual cues" (stellar, lunar, etc.) and merge all future directions into one coherent section.

Thank you for this comment. Addressed.

LL443-446 - A bit early to plan such studies because migratory direction could well be a complex multigenetic trait, so that you cannot approach it simply with the knock out of a single gene. The genetic basis of magnetic direction needs to be first demonstrated, which leads you to the Future Directions section.

Thank you for this helpful comment. We fully agree that migratory direction is likely a complex multigenic trait, and our intention was not to imply that knocking out a single gene would be sufficient to explain magnetic or migratory orientation. Our statement aimed only to highlight that identifying candidate genes is an important first step toward understanding the genetic basis of magnetic orientation.

Line 496 - Clarify whether optic flow was used (unlike previous studies).

Thank you for pointing this out. Clarified.

LL499-511 - Clarify the improvements done in Chen's system and their relevance.

Thank you for pointing this out. We reworded this sentence “The Flash flight simulator system was developed based on the early design of the Mouritsen-Frost flight simulator and adapted for our experiments in Yuanjiang”.

Line 531 - Report and compare light intensities between indoor and outdoor experiments.

Thanks for this comment. Unfortunately, due to the sensitivity limits of our current equipment, we were unable to reliably measure outdoor light intensities at night. However, we did not perform any open-top outdoor flight-simulator experiments; instead, we used field-captured moths but conducted all behavioral tests indoors.

L549 - Add make/model of power supplies.

Thanks. Addressed.

LL582-585 - Specify whether R code will be shared; recommend open access (e.g., GitHub, other open repositories). Reiterate the importance of open science and sharing all scripts. Also here, add citations to some studies where MMRT has been used recently.

Thank you for this comment. We have responded to this point in the Public Reviews.

Line 592 - Explain how individual r-values were derived from optical encoder data.

Thank you for this comment. Addressed.

L842-843 - t-tests are inappropriate for angular data; use circular tests (Watson-Williams, Mardia-Watson-Wheeler, etc.).

Thank you for this comment. Addressed.

L865 - Reword to avoid repetition of "fall." Example: "In field captured armyworms during fall migration".

Thank you for this comment. Addressed.

LL882-885 - Improve phrasing and language here. Confirming that - no colon after. "Both the acrylic plate and diffusion paper." Confirm relevance of spectra to moth visual sensitivity - add relevant citation to original studies showing that.

Thank you for this comment. Addressed.

L886 - Reword "uniform" - does not look uniform to me.

Thank you for this comment. Addressed.

Reviewer #2 (Recommendations for the authors):

 

The first two sentences of the abstract ("The navigational mechanisms employed by nocturnal insect migrants remain to be elucidated in most species. Nocturnal insect migrants are often considered to use the Earth's geomagnetic field for navigation, yet the underlying mechanisms of magnetoreception in insects remain elusive") are somewhat redundant. The authors may consider rewriting them.

Thank you for pointing this out. We have rewritten this opening to provide a more concise and non-repetitive introduction.