Author response:
eLife Assessment
This valuable study raises the intriguing possibility that crickets use bat-associated odors as cues of predation risk, extending the classic bat-insect arms race beyond its usual acoustic framework. The authors combine fecal metabarcoding, behavioral assays, electrophysiology, chemical analyses, and field observations to show that Loxoblemmus equestris avoids the odor of the insectivorous bat Scotophilus kuhlii, and that synthetic (-)-limonene can elicit antennal responses, avoidance in the laboratory, and reduced calling activity in the field. However, the evidence is currently incomplete because the identity, biological source, natural concentration, and ecological specificity of limonene as a bat-derived predator cue require stronger support, including clearer quantification, contamination controls, individual-level odor data, and evidence that crickets can distinguish bat-associated limonene from common environmental sources. The work will be of interest to researchers in sensory ecology, chemical ecology, predator-prey interactions, and bat-insect coevolution.
We thank the editors for recognizing the novelty and significance of our work.
The central aim and contribution of this study is to provide direct evidence that an insect can detect a phylogenetically distant vertebrate predator, an insectivorous bat, via olfaction and initiate avoidance behavior. Our dietary analysis of bats and survey of potential prey in foraging habitats established a predator–prey relationship between the Asiatic lesser yellow house bat (Scotophilus kuhlii) and the cricket Loxoblemmus equestris. In addition, behavioral assays showed that the crickets strongly avoid air carrying bat body odor, and electrophysiological recordings using GC-EAD confirmed that volatiles from S. kuhlii body odor are detected by L. equestris antennae. Together, these results provide strong evidence that this insect can perceive and avoid the body odor of its predator, S. kuhlii. We are grateful that the editors and reviewers acknowledged the main conclusions.
We also investigated the sources of bat body odor, its major volatile components, and the behavioral, physiological, and ecological responses of the crickets to limonene. The purpose of these studies was to test the hypothesis that elemental perception—detection of a single compound—could serve as a mechanism by which crickets perceive bat odor. We found that limonene was present in bat odor, elicited antennal responses in crickets, induced avoidance behavior in olfactometer assays, and reduced calling activity in the field. Together, these results support the idea that elemental perception is a plausible and efficient strategy for initiating anti-predator behavior against bats.
We appreciate the editors’ constructive comments. The editors and Reviewer #1 suggested that limonene, as a bat-derived predator cue, requires more evidence, mainly for two reasons:
(1) Limonene is common in plants but rare in mammals; the reviewer raised the possibility that the limonene identified in our study may have been introduced as exogenous contamination during handling.
(2) The ability of crickets to distinguish bat-associated limonene from limonene originating from common environmental sources (e.g., plants) remains unclear.
Below we address these points and describe the revisions we will make to strengthen the manuscript.
On the potential contamination origin of limonene
We agree that limonene is common in plants and human-associated products, and we carefully considered this possibility. However, several lines of evidence suggest that contamination is highly unlikely. First, we followed strict experimental protocols: all instruments were cleaned with ethanol and oven-dried before each use; bats were held in stainless-steel cages and cloth bags made of degreased bleached cotton washed with purified water. Second, limonene was not detected in any blank controls (empty-chamber air samples for whole-body odor collection, nor blank cotton swabs for secretion analysis), whereas it was consistently identified in multiple bat snout-secretion samples. Third, previous studies have independently reported limonene in the secretions of several bat species (Faulkes et al., 2019, PeerJ; Zhang et al., 2022, Ann. N.Y. Acad. Sci.). Moreover, recent work suggests that skin-associated microorganisms can contribute to bat volatile profiles (Sun et al., 2026, BMC Biology), and some microbes possess enzymes involved in limonene biosynthesis. Therefore, we are confident that the limonene we detected originates from the bats themselves (either endogenously or via their microbiota), not from exogenous contamination.
On how crickets might distinguish bat-derived limonene from environmental sources
This is an insightful question. As discussed in our original manuscript (Discussion section), crickets may not rely exclusively on limonene as a standalone cue. First, our GC-EAD analyses showed that cricket antennae respond to multiple bat volatiles beyond limonene, suggesting that additional compounds, either alone or in synergistic blends, may contribute to predator recognition. Elemental perception via (–)-limonene therefore likely represents one effective strategy within a broader olfactory toolkit, rather than the sole mechanism. Second, under natural conditions, crickets could also integrate olfactory information with non-chemical ecological signals, such as temporal patterning (bats are active at night) and spatial patterning (specific foraging habitats), to further reduce false alarms.
However, fully testing these hypotheses would require substantial additional work. It would be necessary to quantify natural limonene concentrations in bat odors versus various plant sources, conduct choice experiments with ecologically relevant concentrations and blends, and perhaps manipulate the olfactory landscape in the field. It would also be necessary to examine how other volatile compounds in bat body odor interact with limonene, alone or together, to shape cricket recognition. After all, bat body odor contains dozens of compounds, and it is challenging to determine the necessity and sufficiency of each. These kinds of difficulties are not unique to our study; they are widespread in chemical ecology. Problems like how animals distinguish identical compounds from different biological sources are common in chemical ecology, and they are rarely solved in a single study. These lines of investigation, from quantifying natural concentrations to examining compound interactions, are important, but they are not the focus of the present study. So we have put this forward as an important direction for future research.
Revisions we will make：
(1) In the Methods section, we will add detailed descriptions of contamination controls and report blank-control results to demonstrate that exogenous contamination is very unlikely.
(2) In the Discussion section, we will expand the discussion of the possible biological sources of limonene (including microbiota) in light of our results and the literature.
(3) In the Discussion and Conclusion, we will state more cautiously the role of limonene as a bat-derived cue, acknowledging that while it is sufficient to trigger avoidance, additional work is needed to establish its ecological specificity.
We believe these revisions will address the editors’ and the reviewers’ concerns while preserving the main conclusion that olfaction can mediate bat detection by crickets.
Reviewer #1 (Public review):
The manuscript examines whether insects can use bat odor as a cue of predation risk. The authors focus on the insectivorous bat Scotophilus kuhlii and the cricket Loxoblemmus equestris. They first use fecal DNA metabarcoding to show that crickets are part of the bat's diet, and field surveys to show that L. equestris is abundant at local foraging sites. In laboratory Y-tube assays, the authors show that crickets strongly avoid air carrying bat body odor. Gas chromatography coupled with electroantennographic detection showed that cricket antennae respond to components of bat odor. Chemical analyses identified several volatile compounds, with 2,2-dimethylheptane and (−)-limonene associated with antennal responses. Further analyses suggested that snout secretions are likely to contribute to the bat's body odor. The authors then tested individual compounds. Among the commercially available candidates, (−)-limonene elicited a strong antennal response and was sufficient to cause avoidance in the olfactometer. In field plots, spraying (−)-limonene reduced cricket calling activity relative to pre-exposure levels, whereas calling increased in control plots treated with hexane. Overall, the study argues that crickets can detect a vertebrate predator through olfactory cues and that a single bat-associated volatile can trigger antipredator behavior.
This is an interesting and enjoyable study that addresses an understudied aspect of predator-prey interactions. The manuscript is clearly written, the experiments are presented in a logical sequence, and the figures are crisp and easy to follow. I really appreciated the combination of behavioral assays, electrophysiology, chemical analysis, and field observations.
My main issue concerns the identity and biological origin of the proposed bat odor cue, (−)-limonene. Limonene seems like an unusual compound to be emitted endogenously by a mammal, particularly by an insectivorous bat. It would be helpful if the authors could clarify whether mammals are known to synthesize this compound de novo, and, if not, what the likely source of this plant-associated terpene would be in S. kuhlii. Possible sources could include environmental exposure, diet, roosting material, handling, or temporary housing conditions.
I do not doubt that crickets avoid synthetic (−)-limonene. Indeed, this result is quite plausible given that limonene is widely used in insect repellent or repellent-associated fragrance products. However, this also makes contamination an important issue to address explicitly. How did the authors exclude the possibility that limonene entered the samples from human-associated sources, such as insect repellents, soaps, cleaning products, field equipment, cloth bags, cages, gloves, or other materials used while handling wild-caught bats? It would strengthen the manuscript to report limonene levels for individual bat odor collections, all relevant blanks, and any handling or housing controls.
More broadly, given the common occurrence of limonene in plants and human-associated products, I am not yet convinced that it would function as a reliable "keystone kairomone" as suggested around line 253. How would crickets distinguish bat-associated limonene from limonene emitted by a mint leaf, citrus peel, pine material, or other non-threatening environmental sources? The authors may wish to soften this interpretation or provide additional evidence that crickets respond to limonene in a bat-specific context, perhaps through concentration, temporal patterning, co-occurring volatiles, or enantiomeric composition.
We thank Reviewer #1 for the positive evaluation and for recognizing the study as “interesting and enjoyable.” We greatly appreciate the endorsement of our integrative approach combining behavioral assays, electrophysiology, chemical analysis, and field observations. The core conclusion that crickets can detect and avoid bats via olfaction is well supported by our data, and we are pleased that the reviewer has recognized this central finding.
We are grateful for the reviewer’s constructive comments on the biological source and ecological specificity of limonene. In our response to the editor above, we have already responded to both aspects in detail; here we will briefly restate the key points.
On the biological origin of limonene and potential contamination
We agree that limonene is common in plants and human-made products, but relatively unusual for a mammal to emit endogenously. We have carefully examined the possibility of contamination and believe it is highly unlikely for the following reasons:
(1) Strict experimental protocols: All experiments were conducted in a dedicated space. Instruments were cleaned with ethanol and oven-dried before and after each use. Cloth bags used to hold bats were made of degreased bleached cotton and washed with purified water; holding cages were stainless steel.
(2) Blank controls: Limonene was not detected in any blank control samples, neither in the empty-chamber air controls for whole-body odor collection nor in the blank cotton swabs used for secretion analysis. In contrast, limonene was consistently identified in multiple bat snout-secretion samples.
(3) Independent reports: Limonene has been previously identified in the secretions of several bat species (Faulkes et al., 2019, PeerJ; Zhang et al., 2022, Ann. N.Y. Acad. Sci.), indicating that its presence is not unique to our study or handling conditions.
(4) Potential microbial origin: Even if bats do not synthesize limonene de novo (a capacity for which there is currently no evidence), recent work shows that skin-associated microorganisms can substantially shape bat volatile odors (Sun et al., 2026, BMC Biology). Some of these microbes possess enzymes involved in limonene biosynthesis, making bat-associated microbiota a plausible biological source of this compound.
(5) Thus, the limonene we detected is highly likely to originate from the bats themselves (directly or via their microbes) rather than from contamination.
On how crickets distinguish bat-associated limonene from environmental sources
This is an excellent and important question. As we briefly discussed in the original manuscript, crickets may not rely exclusively on limonene as a bat-specific cue. Under natural field conditions, they could integrate olfactory information with other ecological cues, for example, temporal and spatial patterning (bats are active at night in specific foraging habitats), co-occurrence with other bat-specific volatiles (the full odor blend contains many compounds), or even concentration thresholds that differ between bat emissions and plant sources. We hypothesize that such context-specific integration could minimize false alarms.
However, we also recognize that fully testing these hypotheses would require substantial additional work: quantify natural limonene concentrations in bat odors versus various plant sources, conduct choice experiments with ecologically relevant concentrations and blends, and perhaps manipulate the olfactory landscape in the field. These are important questions, but they are not the central focus of the present study, whose primary aim is to provide evidence that olfaction—and elemental perception of a single compound—can function in this predator-prey system. We have therefore framed this as an important direction for future research.
Revisions we will make：
(1) In the Methods section, we will add detailed descriptions of contamination controls and present blank-control results to demonstrate that exogenous contamination is very unlikely.
(2) In the Discussion section, we will expand the discussion of limonene’s biological sources (including microbial contributions) and explicitly acknowledge the need for future work on how crickets might discriminate bat-derived from plant-derived limonene.
(3) In the Conclusion, we will more cautiously characterize limonene’s ecological role, emphasizing that it is sufficient to trigger avoidance but that its natural specificity requires further investigation.
We thank the reviewer again for these insightful comments, which will help us improve the manuscript.
Reviewer #2 (Public review):
Summary:
Many insects possess extremely sensitive olfactory systems that can detect chemical signals from distances of several kilometers. For decades, the arms race between bats and insects has served as a prime example of acoustic co-evolution. The auditory adaptations of insects to echolocation have been well documented. Cricket has a multi-sensory predator recognition system with keen olfactory, tactile, and auditory senses. However, whether crickets can use the scent of bats to avoid them remains unknown at present. The authors hypothesized that cricket prey (Loxoblemmus equestris) might eavesdrop on predator bat (Scotophilus kuhlii) VOCs as an early warning. L. equestris is one of the prey species of S. kuhlii, and the authors demonstrated that the body odor of the insectivorous bat S. kuhlii triggers robust avoidance and electrophysiological responses in the cricket L. equestris, and that a single compound, (-)-limonene, is sufficient to elicit this avoidance in the laboratory and suppress calling in the field. Overall, this paper has a complete chain of evidence and should be a highly praised study.
Comments:
(1) Olfactory eavesdropping can transcend the evolutionary divide between vertebrate predators and invertebrate prey, enabling invertebrates to trigger defensive avoidance behaviors in response to predator-derived volatile odors. This phenomenon is empirically well-documented and requires no excessive emphasis.
(2) Without quantitative analysis and without knowing the relative content of this key substance limonene, I don't quite understand how to determine the concentration of limonene standard for EAD, as well as the concentration in field experiments. How is the concentration of limonene determined in field spraying, and is this actually the case in the wild environment?
(3) Figures 1C and D should compare the GC-EAD response of L. equestris to the odor of bat body and the odor of bat nasal secretions. It should not be compared with the air control group. Figure 1D has the same problem.
We sincerely thank Reviewer #2 for the high praise (“complete chain of evidence,” “highly praised study”) and for the constructive suggestions to further improve the manuscript.
On the novelty of olfactory eavesdropping across the vertebrate–invertebrate divide
We agree with the reviewer that “olfactory eavesdropping can transcend the evolutionary gap between vertebrate predators and invertebrate prey” and that such phenomena have been documented. However, we would like to note that empirical examples remain relatively scarce, especially those that combine chemical identification, electrophysiology, behavioral assays, and field validation within a confirmed predator–prey relationship. We will adjust the wording in the Introduction and Discussion to more accurately reflect this current state of knowledge, acknowledging prior work while clarifying the added value of our study.
On quantitative analysis and concentration choices for limonene in EAG and field experiments.
EAG concentration gradients: The concentrations used in our EAG experiments (including the 1% and 10% v/v dilutions of (−)-limonene) were selected based on standard practices in insect chemical ecology and on previous studies investigating dose-dependent antennal responses to volatile compounds (e.g., Tang et al., 2024, Int. J. Biol. Macromol.). The goal was to determine whether L. equestris antennae are capable of detecting limonene across a range of concentrations, not to precisely match natural emission levels or to determine behavioral thresholds. Our data clearly show concentration-dependent antennal responses, establishing physiological sensitivity.
Field spray concentration: We acknowledge that the concentration used in the field experiment (10% v/v limonene sprayed over 25 m²) does not represent the exact amount of limonene naturally emitted by bats. Natural odor plumes are highly complex; the diffusion, dilution, and persistence of volatiles depend on multiple factors (airflow, turbulence, temperature, humidity, vegetation structure, etc.). Accurately reconstructing such dynamics would require detailed quantitative measurements and possibly fluid-dynamic modeling, which were beyond the scope of this study. The aim of the field experiment was functional: to test whether limonene, as a single bat-associated volatile, could alter cricket calling behavior under semi-natural conditions, not to establish the concentration threshold for this effect. Therefore, we did not design experiments to determine the exact concentration at which crickets begin to respond. The positive result supports the ecological relevance of limonene as an avoidance cue, but we do not claim that the applied concentration matches natural levels. We will clarify this point in the revised Methods and Discussion sections and acknowledge that quantitative characterization of natural bat-odor compositions and their diffusion dynamics is an important direction for future research.
On Figures 1C and 1D comparing bat body odor with air control rather than with snout secretions.
We thank the reviewer for this suggestion. The comparison between bat body odor and snout secretions is indeed novel and informative, and we agree that it could help identify anatomical sources of active volatiles. However, the purpose of Figures 1C and 1D in the current manuscript is to answer a more fundamental question: whether bat body odor (as a whole) contains volatile components that elicit antennal responses in crickets, compared to an odor-free control. This establishes the basic phenomenon of olfactory detection. The identification of snout secretions as the primary source of body odor is addressed separately in Figure 2, using HS-SPME-GC-MS and PCA. In the revised manuscript, we will clarify this rationale in the Methods and Results sections to avoid confusion. We also note that the reviewer’s idea, directly comparing GC-EAD responses to snout secretions versus whole-body odor, is an excellent suggestion for future experiments and would further strengthen the source attribution.
Revisions we will make：
(1) In the Introduction and Discussion, we will adjust the wording to more accurately reflect the current state of knowledge on olfactory eavesdropping across the vertebrate-invertebrate divide, acknowledging prior work while clarifying the added value of our study.
(2) In the Methods and Discussion, we will clarify the rationale for our concentration choices in the EAG and field experiments, acknowledging that our aim was functional (testing sufficiency) rather than determining quantitative thresholds.
(3) In the Methods and Results, we will clarify the rationale for comparing bat body odor with air controls in Figures 1C and 1D, and note that the reviewer’s suggestion of comparing with snout secretions is an excellent direction for future work.
We thank Reviewer #2 again for the thoughtful comments, which have helped us improve the manuscript.
