Female Moths Incorporate Plant Acoustic Emissions into Their Oviposition Decision-Making Process

Reviewer #1 (Public review):

Summary:

The authors demonstrate that female Spodoptera littoralis moths prefer to oviposit on well-watered tomato plants and avoid drought-stressed plants. The study then recorded the sounds produced by drought-stressed plants and found that they produce 30 ultrasonic clicks per minute. Thereafter, the authors tested the response of female S. littoralis moths to clicks with a frequency of 60 clicks per minute in an arena with and without plants and in an arena setting with two healthy plants of which one was associated with 60 clicks per minute. These experiments revealed that in the absence of a plant, the moths preferred to lay eggs on the side of the area in which the clicks could be heard, while in the presence of a plant the S. littoralis females preferred to oviposit on the plant where the clicks were not audible. In addition, the authors also tested the response of S. littoralis females in which the tympanic membrane had been pierced making the moths unable to detect the click sounds. As hypothesised, these females placed their eggs equally on both sites of the area. Finally, the authors explored whether the female oviposition choice might be influenced by the courtship calls of S. littoralis males which emit clicks in a range similar to a drought-stressed tomato plant. However, no effect was found of the clicks from ten males on the oviposition behaviour of the female moths, indicating that the females can distinguish between the two types of clicks. Besides these different experiments, the authors also investigated the distribution of egg clusters within a longer arena without a plant, but with a sugar-water feeder. Here it was found that the egg clusters were mostly aggregated around the feeder and the speaker producing 60 clicks per minute. Lastly, video tracking was used to observe the behaviour of the area without a plant, which demonstrated that the moths gradually spent more time at the arena side with the click sounds.

Strengths:

This manuscript is very interesting to read and the possibility that female moths might use sound as an additional sensory modality during host-searching is exciting and very relevant to the field of insect-plant interactions.

Weaknesses:

The study addresses a very interesting question by asking whether female moths incorporate plant acoustic signals into their oviposition choice, unfortunately, I find it very difficult to judge how big the influence of the sound on the female choice really is as the manuscript does not provide any graphs showing the real numbers of eggs laid on the different plants, but instead only provides graphs with the Bayesian model fittings for each of the experiments. In addition, the numbers given in the text seem to be relatively similar with large variations e.g. Figure 1B3: 1.8 {plus minus} 1.6 vs. 1.1 {plus minus} 1.0. Furthermore, the authors do not provide access to any of the raw data or scripts of this study, which also makes it difficult to assess the potential impact of this study. Hence, I would very much like to encourage the authors to provide figures showing the measured values as boxplots including the individual data points, especially in Figure 1, and to provide access to all the raw data underlying the figures.

Regarding the analysis of the results, I am also not entirely convinced that each night can be taken as an independent egg-laying event, as the amount of eggs and the place were the eggs are laid by a female moth surely depends on the previous oviposition events. While I must admit that I am not a statistician, I would suggest, from a biological point of view, that each group of moths should be treated as a replicate and not each night. I would therefore also suggest to rather analyse the sum of eggs laid over the different consecutive nights than taking the eggs laid in each night as an independent data point.

Furthermore, it did not become entirely clear to me why a click frequency of 60 clicks per minute was used for most experiments, while the plants only produce clicks at a range of 30 clicks per minute. Independent of the ecological relevance of these sound signals, it would be nice if the authors could provide a reason for using this frequency range. Besides this, I was also wondering about the argument that groups of plants might still produce clicks in the range of 60 clicks per minute and that the authors' tests might therefore still be reasonable. I would agree with this, but only in the case that a group of plants with these sounds would be tested. Offering the choice between two single plants while providing the sound from a group of plants is in my view not the most ecologically reasonable choice. It would be great if the authors could modify the argument in the discussion section accordingly and further explore the relevance of different frequencies and dB-levels.

Finally, I was wondering how transferable the findings are towards insects and Lepidopterans in general. Not all insects possess a tympanic organ and might therefore not be able to detect the plant clicks that were recorded. Moreover, I would imagine that generalist herbivorous like Spodoptera might be more inclined to use these clicks than specialists, which very much rely on certain chemical cues to find their host plants. It would be great if the authors would point more to the fact that your study only investigated a single moth species and that the results might therefore only hold true for S. littoralis and closely related species, but not necessary for other moth species such as Sphingidae or even butterflies.

Reviewer #2 (Public review):

This paper presents an interesting and fresh approach as it investigates whether female moths utilize plant-emitted ultrasounds, particularly those associated with dehydration stress, in their egg-laying decision-making process.

Female moths showed a preference for moist, fresh plants over dehydrated ones in experiments using actual plants. Additionally, when both plants were fresh but ultrasonic sounds specific to dehydrated plants were presented from one side, the moths chose the silent plant. However, in experiments without plants, contrary to the hypothesis derived from the above results, the moths preferred to oviposit near ultrasonic playback mimicking the sounds of dehydrated plants.

The results are intriguing, and I think the experiments are very well designed. However, if female moths use the sounds emitted by dehydrated plants as cues to decide where to oviposit, the hypothesis would predict that they would avoid such sounds. The discussion mentions the possibility of a multi-modal moth decision-making process to explain these contradictory results, and I also believe this is a strong possibility. However, since this remains speculative, careful consideration is needed regarding how to interpret the findings based solely on the direct results presented in the results section.

Additionally, the final results describing differences in olfactory responses to drying and hydrated plants are included, but the corresponding figures are placed in the supplementary materials. Given this, I would suggest reconsidering how to best present the hypotheses and clarify the overarching message of the results. This might involve reordering the results or re-evaluating which data should appear in the main text versus the supplementary materials.

There were also areas where more detailed explanations of the experimental methods would be beneficial.