Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honeybee (Apis cerana) colonies

Asian honeybees use an impressive array of strategies to protect nests from hornet attacks, although little is understood about how antipredator signals coordinate defences. We compared vibroacoustic signalling and defensive responses of Apis cerana colonies that were attacked by either the group-hunting giant hornet Vespa soror or the smaller, solitary-hunting hornet Vespa velutina. Apis cerana colonies produced hisses, brief stop signals and longer pipes under hornet-free conditions. However, hornet-attack stimuli—and V. soror workers in particular—triggered dramatic increases in signalling rates within colonies. Soundscapes were cacophonous when V. soror predators were directly outside of nests, in part because of frenetic production of antipredator pipes, a previously undescribed signal. Antipredator pipes share acoustic traits with alarm shrieks, fear screams and panic calls of primates, birds and meerkats. Workers making antipredator pipes exposed their Nasonov gland, suggesting the potential for multimodal alarm signalling that warns nestmates about the presence of dangerous hornets and assembles workers for defence. Concurrent observations of nest entrances showed an increase in worker activities that support effective defences against giant hornets. Apis cerana workers flexibly employ a diverse alarm repertoire in response to attack attributes, mirroring features of sophisticated alarm calling in socially complex vertebrates.


Comments to the Author(s)
Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honey bee (Apis cerana) colonies, appears to be an interesting scientific manuscript, of original research on the honeybees' acoustical communication during pray-predator interaction. It can advance scientific knowledge on this field. It includes a relatively extensive load of data and conclusions are supported by results and analysis. In general, the manuscript is well written; however, there are few areas that require some improvement.
More specific: -The length of the manuscript is unnecessarily extensive and should be reduced. Especially at the sections of Introduction and Discussion. You must reduce the length and keep it focused just on the subject as described by its title.
-Lines 152-153. Overheating and asphyxiating are two different ways of bees killing the hornets without stinging. It's better if you separate the cases and the references at this sentence -Line 215. Why have you decided to use this range of frequencies covered by this type of microphone? In literature there are experiments applied with microphones covering a wider range of frequencies when recording acoustical communication of honeybees (and also during honeybees-hornets interactions) -Lines 223-225. This sentence is unclear though it seems important. I am not sure what do the authors mean. Recording other sounds outside the hive does not mean that they record all the bees' sound inside the hive (many differences especially in high frequencies between recorded bees and a variety of sounds that might be recorded from the background).
-General question regarding methodology: Have you applied any "control" recordings (i.e. recordings in hives without bees) to check the spectrum of background sounds (and noise) and "mask" your recording by removing them? If yes you must provide them in the manuscript.
-Line 610. Indeed, the function of antipredator pipes require confirmation. Have you tried to reproduce the sounds artificially, at the absence of hornets' attacks, and observe if they can result in any behavioural response by the nest mates? I understand that this is not the objective of the study but I am just wondering since you mention the need of playback experiments at line 661 -Lines 621-623. To my knowledge, Cyprian honeybees engulf Vespa orientalis in order to induce asphyxia (not the case of heat-balling). Please, have a look at Current Biology 17, R795-796 Decision letter (RSOS-211215.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Mattila
On behalf of the Editors, we are pleased to inform you that your Manuscript RSOS-211215 "Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honey bee (Apis cerana) colonies" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referees' reports. Please find the referees' comments along with any feedback from the Editors below my signature.
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Please submit your revised manuscript and required files (see below) no later than 7 days from today's (ie 29-Sep-2021) date. Note: the ScholarOne system will 'lock' if submission of the revision is attempted 7 or more days after the deadline. If you do not think you will be able to meet this deadline please contact the editorial office immediately.
Please note article processing charges apply to papers accepted for publication in Royal Society Open Science (https://royalsocietypublishing.org/rsos/charges). Charges will also apply to papers transferred to the journal from other Royal Society Publishing journals, as well as papers submitted as part of our collaboration with the Royal Society of Chemistry (https://royalsocietypublishing.org/rsos/chemistry). Fee waivers are available but must be requested when you submit your revision (https://royalsocietypublishing.org/rsos/waivers). The study describes vibroacoustic signalling behaviour of Asian honeybees during natural encounters with (1) dangerous giant hornets that threaten the entire colony and (2) with lessdangerous smaller hornets that threaten individuals. A third treatment exposes bees to van Der Vecht gland excretions of giant hornets, and two control treatments document signalling behaviour in the absence of predator cues. Results show that bees produce multiple types of vibroacoustic signals, possibly in conjunction with chemical signals, and that the rate of signalling increases with increasing threat. Perhaps most significant is the discovery of a previously undocumented signal that is structurally distinct from other signals and which is produced specifically and immediately in response to the presence of giant hornets. The study makes a strong argument that these eusocial insects have evolved a complex referential alarm communication system, though it correctly acknowledges that playback studies documenting receiver responses in the absence of predator cues will be needed to fully understand signal function.
Both reviewers believe the manuscript is interesting and significant, and both make several suggestions for minor revisions that will make it even stronger. The writing and presentation of the manuscript are excellent, but I agree with reviewer 2 that it is long. Where possible, please reduce the length, especially in the introduction and discussion. The first half of the third paragraph of the discussion, for example, is largely redundant with the results and could be removed. Both reviewers and I would like some clarification about defensive behaviours: reviewer 2 -differentiating between overheating and asphyxiating, reviewer 1 -describing 'bee carpet' behaviour, myself -explaining how body shaking deters predators. Both reviewers would like some clarification about the acoustical methods; I would also like to see the microphone model, recorder settings (sampling rate, bit rate, and file format), spectrogram settings (% overlap and windowing function -e.g., Hamming), a definition of 'frequency modulation' (it varies among researchers), and clarification of how events on the audio recordings were synchronized with events on the video given that they were recorded on separate devices with different (possibly drifting) clocks (L325). Reviewer 1 would like some clarification about the statistical methods, especially how mean signalling rates were calculated and whether the data met the assumptions of the parametric statistical models (I note that some variables are strongly skewedsee fig. 3); I am also curious whether colony size (e.g., number of bees) was known and whether that should be incorporated as a covariate in the models (since more bees presumably mean more signals) to better account for variance.
Minor edits: L93 -change to 'a class of'; L110 -missing period; L207 -change to 'in a vial'; L406there is no section '2b'; Table 1 -should "B" be "b" in rows 3 and 4?; Fig 1 caption -change 'strength' to 'significance', as 'strength' implies effect size Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) As per my knowledge, there are multiple things required to revise in your manuscript. 1. Addition of citations 2. Addition of missed information about defensive behavior in honeybees like 'bee carpet' and some others. 3. Clarification regarding microphone positioning and prevention in the colony, and recognition of acoustic signals. 4. Correct reference writing. 5. Calculation methods of mean signaling rate. 6. About statistical models-did you checked the distribution of data? 7. Filter or clean-up extraneous or noisy audio recordings, and generate spectrogram to compare bees' acoustic signatures under hornet attack and control. I would suggest to add a figure of spectrograms for this comparison. 8. Did you examine the link between the number of predators [hornets] and production of antipredator pipes? 9. Include the role of soundscape indices features in recognizing the acoustic patterns of bees that emit volatiles/chemical compounds on exposure of nasonov glands. 10. Clarification on some questions like; a] Does the predator also produce sound before or during the fight against the bees? In what frequency? b]-Does the strength of the colony effect which acoustic frequencies are produced? Or the number of attacking organisms? 11. Authors should add significant marks in all figures [2,4,5,6]  Reviewer: 2 Comments to the Author(s) Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honey bee (Apis cerana) colonies, appears to be an interesting scientific manuscript, of original research on the honeybees' acoustical communication during pray-predator interaction. It can advance scientific knowledge on this field. It includes a relatively extensive load of data and conclusions are supported by results and analysis. In general, the manuscript is well written; however, there are few areas that require some improvement.
More specific: -The length of the manuscript is unnecessarily extensive and should be reduced. Especially at the sections of Introduction and Discussion. You must reduce the length and keep it focused just on the subject as described by its title.
-Lines 152-153. Overheating and asphyxiating are two different ways of bees killing the hornets without stinging. It's better if you separate the cases and the references at this sentence -Line 215. Why have you decided to use this range of frequencies covered by this type of microphone? In literature there are experiments applied with microphones covering a wider range of frequencies when recording acoustical communication of honeybees (and also during honeybees-hornets interactions) -Lines 223-225. This sentence is unclear though it seems important. I am not sure what do the authors mean. Recording other sounds outside the hive does not mean that they record all the bees' sound inside the hive (many differences especially in high frequencies between recorded bees and a variety of sounds that might be recorded from the background).
-General question regarding methodology: Have you applied any "control" recordings (i.e. recordings in hives without bees) to check the spectrum of background sounds (and noise) and "mask" your recording by removing them? If yes you must provide them in the manuscript.
-Line 610. Indeed, the function of antipredator pipes require confirmation. Have you tried to reproduce the sounds artificially, at the absence of hornets' attacks, and observe if they can result in any behavioural response by the nest mates? I understand that this is not the objective of the study but I am just wondering since you mention the need of playback experiments at line 661 -Lines 621-623. To my knowledge, Cyprian honeybees engulf Vespa orientalis in order to induce asphyxia (not the case of heat-balling). Please, have a look at Current Biology 17, R795-796 ===PREPARING YOUR MANUSCRIPT=== Your revised paper should include the changes requested by the referees and Editors of your manuscript. You should provide two versions of this manuscript and both versions must be provided in an editable format: one version identifying all the changes that have been made (for instance, in coloured highlight, in bold text, or tracked changes); a 'clean' version of the new manuscript that incorporates the changes made, but does not highlight them. This version will be used for typesetting.
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Decision letter (RSOS-211215.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Mattila, I am pleased to inform you that your manuscript entitled "Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honey bee (Apis cerana) colonies" is now accepted for publication in Royal Society Open Science.
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We thank the Editor and both Reviewers for their thoughtful comments about our manuscript.
We have tried in good faith to address all of their ideas, concerns, and suggestions. As the Editor points out, many of the comments were similar, which provided strong guidance for revisions that have improved the clarity of our paper. Our specific responses are outlined below, point by point.

Associate Editor Comments to Author (Dr. David Wilson):
The study describes vibroacoustic signalling behaviour of Asian honeybees during natural encounters with (1) dangerous giant hornets that threaten the entire colony and (2) with lessdangerous smaller hornets that threaten individuals. A third treatment exposes bees to van Der Vecht gland excretions of giant hornets, and two control treatments document signalling behaviour in the absence of predator cues. Results show that bees produce multiple types of vibroacoustic signals, possibly in conjunction with chemical signals, and that the rate of signalling increases with increasing threat. Perhaps most significant is the discovery of a previously undocumented signal that is structurally distinct from other signals and which is produced specifically and immediately in response to the presence of giant hornets. The study makes a strong argument that these eusocial insects have evolved a complex referential alarm communication system, though it correctly acknowledges that playback studies documenting receiver responses in the absence of predator cues will be needed to fully understand signal function.
Both reviewers believe the manuscript is interesting and significant, and both make several suggestions for minor revisions that will make it even stronger. The writing and presentation of the manuscript are excellent, but I agree with reviewer 2 that it is long. Where possible, please reduce the length, especially in the introduction and discussion. The first half of the third paragraph of the discussion, for example, is largely redundant with the results and could be removed.
We strongly edited the Introduction and Discussion to keep the direction of those sections focused. In the Introduction, we removed a half page of writing (~200 words), including several references, although we had to add 1.5 sentences about specific defenses in response to questions from the Editor and both Reviewers (see next response). The Results section was trimmed by ~150 words. Major cuts were made to the Discussion, which was reduced in length by about one third (~1200 words).
Both reviewers and I would like some clarification about defensive behaviours: reviewer 2differentiating between overheating and asphyxiating, reviewer 1 -describing 'bee carpet' behaviour, myself -explaining how body shaking deters predators.
To address these concerns, we elaborated on our explanation of A. cerana defenses against hornets in the Introduction in the following ways: Appendix B The combination of papers we cite about bee balling (refs 89,94-96  To avoid overemphasizing heating, we edited the term "heat ball" to "bee ball" or simply "ball" throughout the manuscript. "Bee carpets" are a term used in the literature to describe the aggregations that A. mellifera workers make at entrances prior to bee balling. While the term "bee carpet" has not been applied to Asian bees (that we are aware), we fully agree that we need to mention these entrance aggregations in the Introduction, especially because we documented changes in bee numbers on hive fronts in our study. We added this statement about A. cerana: ""They often aggregate at the nest entrance as a first step [70,88,89], referred to a "bee carpets" in A. mellifera [90][91][92][93]" before describing the defenses that follow these entrance aggregations (ie. bee balling, dung spotting, body shaking).
Finally, we elaborated on the means by which body shaking is thought to work against predators: "Groups of workers also perform coordinated body shaking in response to hornets, a visually intimidating display that deters attackers from approaching the nest [77,[94][95][96][97]." Both reviewers would like some clarification about the acoustical methods; I would also like to see the microphone model, recorder settings (sampling rate, bit rate, and file format), spectrogram settings (% overlap and windowing function -e.g., Hamming), a definition of 'frequency modulation' (it varies among researchers), and clarification of how events on the audio recordings were synchronized with events on the video given that they were recorded on separate devices with different (possibly drifting) clocks (L325). We defined "frequency modulation" according to Schlegel et al. 2012 in the text as "change in frequency over the duration of a signal [64]".
We also described our method for synchronizing the audio and video recordings: "Clocks for videos were synchronized with corresponding audio recordings using a verbal mark that was announced at the start of both recordings." Reviewer 1 would like some clarification about the statistical methods, especially how mean signalling rates were calculated and whether the data met the assumptions of the parametric statistical models (I note that some variables are strongly skewed-see fig. 3); We expanded our explanation in the Methods about this calculation: "Using this final dataset of categorized signals, we calculated the number of each signal type that was produced per minute in each colony replicate. These values were averaged across minutes to determine mean rates of signalling per minute for each colony (for each signal type separately and for all signal types combined)." We think the request for a citation was given with the thought that the calculation was complicated, but it is straightforward and doesn't require a citation.
We tested the normality and homogeneity of variances of datasets that were subjected to parametric tests (ANOVAs and t-tests) and applied log transformations as necessary. The transformations either met the assumption of normality or improved normality; variance homogeneity was the norm. When homogeneity of variances is maintained, recent modeling shows that ANOVAs are highly robust to deviations from normality and maintain acceptable Type I error rates, according to Blanca et al. (2017), a statistical study that is highly cited by researchers (318 other studies to date). We cite it as well. We appreciate the push to more closely examine the distribution of our data. This revision required minor adjustments to reported ANOVA values whenever transformations were applied, but it did not change any of our conclusions. Original and transformed data are provided in our data file.
I am also curious whether colony size (e.g., number of bees) was known and whether that should be incorporated as a covariate in the models (since more bees presumably mean more signals) to better account for variance.
Unfortunately, we did not collect data on colony size, so we can't incorporate it into our statistical approach. We have to interpret our data with the experimental unit as the "colony", without knowing how it varied in size across replicates.
Minor edits: L93 -change to 'a class of' Fixed it, thanks!

L207 -change to 'in a vial'
Fixed it, thank you! L406 -there is no section '2b' Thank you, corrected to "section 3.2.4." Table 1 -should "B" be "b" in rows 3 and 4 Yes, thank you! We fixed it.

Reviewer 1
As per my knowledge, there are multiple things required to revise in your manuscript.

Addition of citations:
Line 107 Here, also describe acoustic emissions of 6 kHz frequency produced by Apis mellifera while confronting with V. orientalis [Papachristoforou et al., 2008]. The reviewer brings up an interesting idea, but one that is beyond the scope of what we can speculate about in our Discussion. We measured sounds, but we did not measure chemical emissions by alarmed workers. We noted that they exposed their Nasonov glands at entrances, and we strongly believe that future work should explore the potential multimodal nature of the vibroacoustic and chemical alarm signals released by workers when hornets attack their nest. Presently, we don't know how these chemical signals travel within colonies or which colony members they might affect. Thus, we feel it stretches the limits of our study to make guesses about how chemical signals alone could influence colony-level signalling (in the absence of hornet-related stimuli 2. Addition of missed information about defensive behavior in honeybees like 'bee carpet' and some others. Line157 Authors have missed one of the important defensive behaviors against predators in honeybees, and that behavior is designated as ''Bee-carpet". I would suggest adding a couple of sentences about this important behavior. For detailed defensive behavior in honeybees read a paper by Sharif et al. [2020] with title "Insect pests, parasitoids, and predators; Can they degrade the sociality of a honeybee colony, and be assessed via acoustically monitored systems?'' As described above, we added information about aggregations of workers at colony entrances (called bee carpets in A. mellifera), which occurs as a first step in bee balling, dung spotting, or body shaking.
3. Clarification regarding microphone positioning and prevention in the colony, and recognition of acoustic signals. Line 189 From the methodology section [2.2], I have the following questions to ask; 1-As you put the microphone close to the cluster of bees then how did you prevent the microphone from propolization?