Acoustic Signals and Behavior of the Invasive Freshwater Crayfish Cherax destructor (Clark, 1936)

Round 1

Reviewer 1 Report

Add more reference towards the behavioral changes and add more water quality changes

Author Response

Add more reference towards the behavioral changes and add more water quality changes

 

Thank you for the review, done. See line 75-80 (of the manuscript reviewed, accepted revisions).

Reviewer 2 Report

Dear Authors,

your study presents an interesting research with interesting results that you have managed to present well and put them in the context of known facts. Still, there are a few suggestions that I would like you to take into consideration before your article could be published. The discussion part should be particularly revised. Not all the references are relevant to your research, and a few important literature sources are missing. There is a feeling that you are uncertain whether your research is practical or not. You should be more certain when presenting your study.

Comments for author File: Comments.pdf

Author Response

Thank you for the review, Discussion was revised in agreement with reviewers’ suggestions. It was included some other references and some others not very fundamental were excluded. I replied to your precise requests on the pdf file here attached.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors reported acoustic signals and behavior in the freshwater crayfish Cherax destructor in the current study.

The main results showed that males and females produced two acoustic signals, high and low-frequency sounds. In grouped layout, animals at < 6 cm recorded more sound, suggesting that crayfish could use sounds for intraspecific communication.  

Main observations:

What are the mechanoreceptors and their neural pathway connections that participate in detecting and integrating the sounds in the nervous system of the crayfish?

However, in item 3.3, lines 223-224, the authors mentioned that "the analysis of synchronized acoustic and video data did not show any particular behavioral event/state associated with sound emission."

Thus, the conclusion is unclear (lines 314-315): "We could hypothesize that this species uses sounds to communicate with conspecifics, to court, to try to keep the group cohesive and to avoid fights."

Minor observations

The authors should be to indicate differences significative or not significative in Fig. 5.

Author Response

The authors reported acoustic signals and behavior in the freshwater crayfish Cherax destructor in the current study.

The main results showed that males and females produced two acoustic signals, high and low-frequency sounds. In grouped layout, animals at < 6 cm recorded more sound, suggesting that crayfish could use sounds for intraspecific communication.  

Main observations: 

What are the mechanoreceptors and their neural pathway connections that participate in detecting and integrating the sounds in the nervous system of the crayfish?

Thank you for the review.

Although many crustaceans produce sounds, their hearing skills are little known. A sound wave has two physical components, the pressure variations of the medium and the vibrations of water particles. To our knowledges, aquatic crustaceans are sensitive to the latter component. In crustaceans, sensory hairs on the body surface, internal chordal organs and statocysts can be stimulated by water vibrations. For Jézéquel et al 2021, the external cuticular hairs of the Homarus americanus are probably responsible for sound detection. In his study (Tautz & Sandeman 1980) using electrophysiological recordings of the sensory axons of chillipedes, he shows how the sensory hairs of Cherax destructorare sensitive to water vibration frequencies.

I added this part in the introduction (see line 52-58 of the manuscript reviewed, accepted revisions).

 

However, in item 3.3, lines 223-224, the authors mentioned that "the analysis of synchronized acoustic and video data did not show any particular behavioral event/state associated with sound emission."

Here we refer to a synchronisation that could help identify the mechanism of signal emission with a duration of milliseconds (the same order of acoustic signal).

 

Thus, the conclusion is unclear (lines 314-315): "We could hypothesize that this species uses sounds to communicate with conspecifics, to court, to try to keep the group cohesive and to avoid fights."

Here we mean in the behavioural context. However, following the suggestion of a other reviewer we change this conclusion.

 

Minor observations

The authors should be to indicate differences significative or not significative in Fig. 5.

Ok, we changed.

Reviewer 4 Report

The authors reported the acoustic signals and behaviour of the crayfish, and I am interested in this study. However, I think that some problems are in the experimental design. Firstly, the sample size is not enough, only 5 replicas in a single experiment, and only 3 replicas in the group, you should add the sample size up to less than 10-15, then the results will be more reliable. Secondly, the description of the experimental design and when the experiment was carried out are not described in detail. I don't know the size of the test tank, and I don't know what time of day or night the experiment was carried out. Because the daily rhythms of the animals could also affect the results of the experiment. Finally, the English language needs moderate editing.

 

Other comments are as follows:

1. Line 179-197: you should analyze the variation of individual acoustic signals rather than pooled data from 5 individuals in every single experiment.

2. If you like, I suggest you could add the representative videos of behavioral experiments as supplemental files, which allow the reader to visualize your experiments.

I encourage authors to ensure that their datasets are either deposited in publicly available repositories (where available and appropriate) or presented in the main manuscript or additional supporting files, in machine-readable format (such as spreadsheets rather than PDFs) whenever possible.”

Some of the English words in this paper are colloquial.

Author Response

The authors reported the acoustic signals and behaviour of the crayfish, and I am interested in this study. However, I think that some problems are in the experimental design. Firstly, the sample size is not enough, only 5 replicas in a single experiment, and only 3 replicas in the group, you should add the sample size up to less than 10-15, then the results will be more reliable.

 

We understand the points raised by the reviewer and we agree partially. In this study the number of replicas changes in agree with the specific hypothesis tested. A large part of the results of this study are not based on the analysis of dataset obtained on few replicas. We studied sound emissions of 46 specimens (23 males and 23 females); moreover, if we compare sounds emission rate in singled and in grouped animals (see line 236 of the manuscript reviewed, accepted revisions) we based our results on 10 replicas in singles specimens against 15 replicas in group (46 specimens). Otherwise, the results on behavioral states and events analysis (the most critical point raised by the reviewer) are based on three replicas of 3 or 2 specimens. This means that each replica contains the analysis of behavioural states/events of three/two specimens (9 specimens), making statistically stronger the value of each replica.

Finally, experimental tests, as is well known, take long time and require many logistic efforts. The addition of other replicas in our experimental tests would cause other issues concerning the physiological changes due to temporal pattern (daily, seasonal, breeding seasons) on the behaviors analyzed.

 

 

Secondly, the description of the experimental design and when the experiment was carried out are not described in detail. I don't know the size of the test tank, and I don't know what time of day or night the experiment was carried out. Because the daily rhythms of the animals could also affect the results of the experiment. 

We insert in the description of the experimental design the times when the experiments were carried out (see line 124). the dimensions of the tank were already present (see line 124 and line 125 of the manuscript reviewed, accepted revisions).

 

 

Finally, the English language needs moderate editing.

 

We checked the English with a native speaker.

 

 

Other comments are as follows:

1. Line 179-197: you should analyze the variation of individual acoustic signals rather than pooled data from 5 individuals in every single experiment.

The specific variations and the detailed description of acoustic signals will be object of another study (Buscaino et al, in preparation). Here, we evaluated the main behavioral contexts of the sound emissions because this can suggest the ecological role of sounds in this species.

If you like, I suggest you could add the representative videos of behavioral experiments as supplemental files, which allow the reader to visualize your experiments.  I encourage authors to ensure that their datasets are either deposited in publicly available repositories (where available and appropriate) or presented in the main manuscript or additional supporting files, in machine-readable format (such as spreadsheets rather than PDFs) whenever possible.”

 

Thanks for the suggestion, we included some videos as examples of behavioral events/states.

Reviewer 5 Report

The proposed manuscript sought to determine whether males and females of Cherax destructor generate acoustic signals and, if so, whether these signals are related with specific behavioural states or events. Since no previous research on the sound-generating capabilities of C. destructor was accessible, this manuscript of C. destructor acoustics and behaviour contributes to the growing number of studies on bioacoustics of invasive species. As stated in the introduction, this article on sound emission and the context of emissions could provide the baseline information for implementing a passive acoustic monitoring system in the natural environment. As a result, research into this highly invasive species of crayfish is both intriguing and vital from the standpoints of management and nature conservation. The substantial number of individuals tested, the behavioural component of the tests, and, as previously stated, the fact that the authors investigated the acoustic capacities of invasive species in laboratory conditions are the manuscript's strengths. However, the work includes several major imperfections and shortcomings that prevent me from gaining a better knowledge of the subject. The "Results" subtitle, for example, lacks a detailed acoustic analysis and the following data regarding the sound structure, acoustic variables, and intraspecific variability. Did the authors, for example, measure the duration of the sounds, their peak or fundamental frequency, and the number of pulses if any were present? In addition, according to the available literature on crayfish bioacoustics, the structure of the acoustic signal should be characterized. Later in the "Discussion," the authors reach a doubtful and overstated conclusion regarding their study and its relationship to other research. I reviewed the manuscript critically. As a result, for each line number, the following constructive, and critical remarks and recommendations are provided:

Line 15

“No of fight/no of encounter” replace with number of fight/number of encounter, if the authors consider “no” as number. Otherwise, it is not understandable what does “no of fight” means.

Line 21

“The number of signals emitted in FF group and in single M were significantly higher” add on the end compared to other layouts.

Line 41

Delete Moreover and start the sentence with “Recently,... “

Line 42

Delete also

Line 76

“as in inter as in intraspecific interactions” delete as in, add in both inter- and intraspecific interactions.

Line 80

The sentence is highly hypothetical. “The study of sound emission and the emissions context could furnish the baseline information to implement passive acoustic monitoring system in the natural environment.” - theoretically yes, but in this stage the sound emission should be strongly verified in laboratory conditions, then tested in natural habitat, and finally, passive acoustics can be applied. In this context, the sentence seems a bit too premature. Consider to modify (with less exaggeration) or to delete.

Line 91

Do not start the sentence with the number, put in the parentheses or modify. In addition, please indicate how did you determine the sex of each individual.

Line 94

Describe in more details the housing conditions during experiments and during holding situations.

Line 97

Subtitle 2.2. Experimental setup - did you consider to include the substrate, sand or fine gravel (less than 5 cm), into the tank? This way, you would insure that the high-frequency sounds, usually produced by the crayfish (or other animal) during moving or walking on the glass surface, are not produced during audio-video experiments. I am aware that this would also include some additional noise, produced during walking, but at least high-frequency component of the noise would be eliminated. In addition, how did you minimize the conduction of external noise to the tanks and therefore improved the quality of fish sound recording? Usually, two marble layers interspaced with two levels of rubber foam shock absorbers are used in these purposes.

Line 103

How did you address the effects of reverberation, resonance, and tank size on the characteristics of sound recorded inside small tanks, such as yours of capacity around 160 litters? The paper written by Akamatsu et al., 2002 (“Empirical refinements applicable to the recording of fish sounds in small tanks”) should be used as a guide when designing and conducting these types of the bioacoustics studies for laboratory animals. This is important, since high frequency sounds are strongly impacted by too small tanks, and the distance between animal and hydrophone during recordings. From the spectrogram/oscillogram of your sounds, it is obvious that both low and high frequency sounds are strongly affected by the tanks size (and possible distance between animal and hydrophone).

Line 153

Please indicate the AVISOFT parameters used to analyse the sounds, such as FFT, window type and % of frame, together with the version of the software used.

Line 183

“Grouped animals produced fewer low frequency sounds than single animals”

When summed, it seems that group individuals (all layouts) produce more LF sound than single, at least when looking at the total numbers from Table 3.? Or, do you mean, per each individual, single vs. grouped, if we put aside the layouts (maybe the same individual was used in different layouts, but produced sounds which are then summed as a "group" category)? Any ways, please include the mean number of the sounds per category single vs grouped.

Line 194 - 196

“Considering the high frequency sounds, the number of signals emitted in FFM group were significantly higher than the ones emitted in single layouts and FF group, but comparable to the other layouts”

Would not agree, the n. of sounds in FFM is highly different from other layouts according to its mean values (figure) and total number (Table 3). Therefore, delete “but comparable to the other layouts”.

Line 199 - 200

Why do you choose to present only the male acoustic signals in the figure? Are they complete the same as to the female sounds? In addition, please indicate the parameters from AVISOFT which were used to produce the figures (frame size, FFT, resolution, window type, etc..).

Line 201 (Table 3)

Not clear if the sound-producing individuals intertwine between categories In addition, not clear are those total numbers fixed per category and layout, or they intertwine with sex and group, e.g. is one individual (from “single” category) producing 13 sounds in F layout, and then after being used again in “group” category (let’s say FF layout), also producing 20 sounds. When summing, that would give 33 sounds for that individual, but in different context (single vs group). Please specify how did you separate individuals and how did they were used during the experiments, i.e. how many time did you used the same individual in the experiment.

Line 232

No clear is the figure 5 related to the “single” or “group “experiment? Maybe the comparison for each category (single and group with LF and HF sounds, so in total, four figure parts) should be best in order to facilitate the understanding.

Line 236

Replace showed with suggest

Line 243 - 246

“Moreover, C. destructor emitting more low frequency sounds compared to the high frequency sound, could make more effective the transmission of any information in the aquatic environment, where sounds at lower frequency can travel for longer distance than the higher frequency sounds [58].”

This sentence would make sense if we had the quantitative and qualitative data about the sound structure of the acoustic signals produced by the crayfish. Usually, in the marine environment, low frequency sounds are attributed to the low frequency call of the whales (infrasound), measuring couple of Hz in frequency, which (probably) propagate long distances due to their long wavelengths. In your case, you have arbitrary chosen the distinction line (of 20 kHz) between two sound types, without any reasonable explanation. Therefore, I cannot accept the further discussion about the sounds, their applicability or usage by the animal, since we know practically nothing about their spectral and temporal features.

Line 250

“We found that single male emitted more sounds than both single females and groups, 250 with exception of FF”

This sentence is very misleading. “Single male” would indicate that one, specific male emitted more sounds. Maybe the better fitting would be “the males from single M experiment” or something similar. In addition, when looking at Fig. 3, this is only true for LF sounds, therefore please be specific. Do you mean for LF sounds?

Line 251 - 252

Which sounds? This sentence is very speculative and has no direct links from your study, only if you include the additional results from your experiments (distance from the individuals, etc.). Even then, this is more the hypothesis, especially considering that single M emitted more sounds, so, to whome the group cohesion would be applied?!? If single males emitted more sounds, how could that be connected to group cohesion?

Line 252 - 253

Keep in mind that in the case of lobster, if the stridulating sounds are real commutative acoustic signals (maybe they really could be, due to their active and intentional nature, as in case with most stridulating sounds), they differ greatly from your acoustic signals. In your case, the signals presented in the text could be the by-product of some activity, such as moving, walking, etc.

Line 264 - 266

“The emission of acoustic signals in C. destructor could be used by females as an indirect way of establishing dominance avoiding injuring interaction.”

 How does this apply to your conducted experiments of states and events, regarding velocity, distance etc?

Line 268 - 269

“Increased emission of low-frequency sounds by the two females could also be explained as a possible call for male.”

You mean in FF experiments? This is highly speculative and it was no directly tested under your experiments, so it should be removed. Usually, the sexual selection works by means of male - male competition and female choice; in this case (usually) the male is the calling individuals. Maybe in your case the situation is different, but for now, I would remove this part.

Line 275

“These signals 274 were fewer compared to the low frequency sound and could be emitted accidently..”

Agree, therefore, you need to justify in more details the structure of these sounds as an active and intentional signal. In other words, are your sounds really produced intentionally and are not by-product of other activities? For HF sound, when looking at the spectrogram, this could be the case…

Line 277 - 278

“Considering the other behaviours, the behavioural events revealed no significant differences between the layouts”

How come, when the FFM differentiated from other layout according to your diagrams in encounters and fights? In addition, there were no tail flips in FFM. Please rewrite and specify.

Line 280 - 281

“Moreover, in the FFM groups the number of encounters and fights, although there is no statistical significance (which could emerge by increasing replicas), was higher…”

Higher compared to other layout or FF? Please indicate and be specific.

Line 281 - 282

“In this arrangement the animals also emitted more high-frequency sounds.”

Suggestion: This could be better visualized in the diagrams (such as Fig. 4.) where on the top of each layout, the total number of LF and HF sounds would be indicated.

Line 308 - 309

“In this work, any correlation was found between tail flip events and acoustic emission supporting the hypothesis that sounds probably are not involved in an antipredator strategy.”

I do not understand this part of sentence “any correlation was found”. Please, rewrite this in a more understandable sense. Do you mean, “the correlation”?

Line 312

This sounds as an exaggeration. The crayfish would be soniferous if it were really a matter of production of communicative sounds, which is questionable in your case. Second, “numerous sounds” would be applicable if the animal produce much more than two sounds per min.

Line 313 - 316

Highly speculative, should not be included in the conclusion. From sentence written like this, it appears (if I haven't read the manuscript) that you are 100% sure these are communicative sounds, for which we are not sure at the moment (at least before you provide more info on acoustic structure and other relevant parameters).

Line 316 - 319

Firstly, you should confirm the sounds in the lab, then investigate the sounds in the field in order to justify their spectro-temporal overlap (together with other acoustic similarities) and then you could indicate the usefulness of the sounds for PAM.

Minor corrections needed.

Author Response

The proposed manuscript sought to determine whether males and females of Cherax destructor generate acoustic signals and, if so, whether these signals are related with specific behavioural states or events. Since no previous research on the sound-generating capabilities of C. destructor was accessible, this manuscript of C. destructor acoustics and behaviour contributes to the growing number of studies on bioacoustics of invasive species. As stated in the introduction, this article on sound emission and the context of emissions could provide the baseline information for implementing a passive acoustic monitoring system in the natural environment. As a result, research into this highly invasive species of crayfish is both intriguing and vital from the standpoints of management and nature conservation. The substantial number of individuals tested, the behavioural component of the tests, and, as previously stated, the fact that the authors investigated the acoustic capacities of invasive species in laboratory conditions are the manuscript's strengths. However, the work includes several major imperfections and shortcomings that prevent me from gaining a better knowledge of the subject. The "Results" subtitle, for example, lacks a detailed acoustic analysis and the following data regarding the sound structure, acoustic variables, and intraspecific variability. Did the authors, for example, measure the duration of the sounds, their peak or fundamental frequency, and the number of pulses if any were present? In addition, according to the available literature on crayfish bioacoustics, the structure of the acoustic signal should be characterized. Later in the "Discussion," the authors reach a doubtful and overstated conclusion regarding their study and its relationship to other research. I reviewed the manuscript critically. As a result, for each line number, the following constructive, and critical remarks and recommendations are provided: 

We agree with the reviewer that sound should be described in more details. However, in this manuscript we describe the behavioural context of sound emissions because this could improve our knowledges on the ecological role of these sounds. The present study provides a consistent dataset and results and the specific description of the sounds will make the manuscript too long and difficult to read. In fact, the acoustic descriptions require many technical details in the results section, in the methods and in the discussions too. We are preparing a manuscript on only sound description of Cherax quadricarinatus/destructor sounds.

Line 15

“No of fight/no of encounter” replace with number of fight/number of encounter, if the authors consider “no” as number. Otherwise, it is not understandable what does “no of fight” means. 

Ok we replaced.

Line 21

“The number of signals emitted in FF group and in single M were significantly higher” add on the end compared to other layouts. 

Ok we added.

Line 41

Delete Moreover and start the sentence with “Recently,... “

We rewrited this part

Delete also

Ok, done

Line 76 

“as in inter as in intraspecific interactions” delete as in, add in both inter- and intraspecific interactions. 

Ok, done.

 

Line 80

The sentence is highly hypothetical. “The study of sound emission and the emissions context could furnish the baseline information to implement passive acoustic monitoring system in the natural environment.” - theoretically yes, but in this stage the sound emission should be strongly verified in laboratory conditions, then tested in natural habitat, and finally, passive acoustics can be applied. In this context, the sentence seems a bit too premature. Consider to modify (with less exaggeration) or to delete. 

Ok, deleted

Line 91

Do not start the sentence with the number, put in the parentheses or modify. In addition, please indicate how did you determine the sex of each individual. 

Done, see line 109-111 of the manuscript reviewed, accepted revisions

Line 94

Describe in more details the housing conditions during experiments and during holding situations. 

Done, see line 113-116 of the manuscript reviewed, accepted revisions

Line 97

Subtitle 2.2. Experimental setup - did you consider to include the substrate, sand or fine gravel (less than 5 cm), into the tank? This way, you would insure that the high-frequency sounds, usually produced by the crayfish (or other animal) during moving or walking on the glass surface, are not produced during audio-video experiments. I am aware that this would also include some additional noise, produced during walking, but at least high-frequency component of the noise would be eliminated. In addition, how did you minimize the conduction of external noise to the tanks and therefore improved the quality of fish sound recording? Usually, two marble layers interspaced with two levels of rubber foam shock absorbers are used in these purposes. 

We did not use sand because it fouled the water and compromised the quality of the videos for long time.

 

Line 103 

How did you address the effects of reverberation, resonance, and tank size on the characteristics of sound recorded inside small tanks, such as yours of capacity around 160 litters? The paper written by Akamatsu et al., 2002 (“Empirical refinements applicable to the recording of fish sounds in small tanks”) should be used as a guide when designing and conducting these types of the bioacoustics studies for laboratory animals. This is important, since high frequency sounds are strongly impacted by too small tanks, and the distance between animal and hydrophone during recordings. From the spectrogram/oscillogram of your sounds, it is obvious that both low and high frequency sounds are strongly affected by the tanks size (and possible distance between animal and hydrophone). 

We will consider this in the signal characterisation paper we are writing (Buscaino at al, in preparation)

 

Line 153

Please indicate the AVISOFT parameters used to analyse the sounds, such as FFT, window type and % of frame, together with the version of the software used. 

Done

Line 183

“Grouped animals produced fewer low frequency sounds than single animals” 

When summed, it seems that group individuals (all layouts) produce more LF sound than single, at least when looking at the total numbers from Table 3.? Or, do you mean, per each individual, single vs. grouped, if we put aside the layouts (maybe the same individual was used in different layouts, but produced sounds which are then summed as a "group" category)? Any ways, please include the mean number of the sounds per category single vs grouped.

We mean the number of sounds per specimen in single and group category. We added the averages in the results section.

 

Line 194 - 196

“Considering the high frequency sounds, the number of signals emitted in FFM group were significantly higher than the ones emitted in single layouts and FF group, but comparable to the other layouts”

Would not agree, the n. of sounds in FFM is highly different from other layouts according to its mean values (figure) and total number (Table 3). Therefore, delete “but comparable to the other layouts”.

Ok, done

Line 199 - 200

Why do you choose to present only the male acoustic signals in the figure? Are they complete the same as to the female sounds? In addition, please indicate the parameters from AVISOFT which were used to produce the figures (frame size, FFT, resolution, window type, etc..).

In the spectrogram male and female sounds do not present any evident differences, which is why I have not included both.

 

Line 201 (Table 3)

Not clear if the sound-producing individuals intertwine between categories. In addition, not clear are those total numbers fixed per category and layout, or they intertwine with sex and group, e.g.is one individual (from “single” category) producing 13 sounds in F layout, and then after being used again in “group” category (let’s say FF layout), also producing 20 sounds. When summing, that would give 33 sounds for that individual, but in different context (single vs group). Please specify how did you separate individuals and how did they were used during the experiments, i.e. how many time did you used the same individual in the experiment. 

The individual animals were only used once, I will add this information in the materials and methodsin the section “Experimental set-up” (see line 131 of the manuscript reviewed, accepted revisions).

 

Line 232 

No clear is the figure 5 related to the “single” or “group “experiment? Maybe the comparison for each category (single and group with LF and HF sounds, so in total, four figure parts) should be best in order to facilitate the understanding. 

The figure 5 is related to the group as proximity can only be calculated in grouped animals, individuals are alone in the tank. We consider all the group trials (FFM, MMF, FF, MM, MF) see line 317 of the manuscript reviewed, accepted revisions.

 

Line 236

Replace showed with suggest

The verb “suggest” indicate a no clear result of the study (is it?). On the contrary, there is no doubt that Cherax destructor produce acoustic signals. These signals are not caused by the movements of the water interacting with body parts and are unequivocally sound.

Line 243 - 246

“Moreover, C. destructor emitting more low frequency sounds compared to the high frequency sound, could make more effective the transmission of any information in the aquatic environment, where sounds at lower frequency can travel for longer distance than the higher frequency sounds [58].”

This sentence would make sense if we had the quantitative and qualitative data about the sound structure of the acoustic signals produced by the crayfish. Usually, in the marine environment, low frequency sounds are attributed to the low frequency call of the whales (infrasound), measuring couple of Hz in frequency, which (probably) propagate long distances due to their long wavelengths. In your case, you have arbitrary chosen the distinction line (of 20 kHz) between two sound types, without any reasonable explanation. Therefore, I cannot accept the further discussion about the sounds, their applicability or usage by the animal, since we know practically nothing about their spectral and temporal features. 

We agree partially with the reviewer. Sounds were subdivided in these two big categories because there is a clear distinction in the frequency peak (Buscaino at al, in preparation). There is no doubt that lower frequencies can travel for longer distance than higher frequencies and we cited this physical property in the discussion assuming that lower frequency sounds of Cherax d. can travel for longer distances. 

 

Line 250 

“We found that single male emitted more sounds than both single females and groups, 250 with exception of FF”

This sentence is very misleading. “Single male” would indicate that one, specific male emitted more sounds. Maybe the better fitting would be “the males from single M experiment” or something similar. In addition, when looking at Fig. 3, this is only true for LF sounds, therefore please be specific. Do you mean for LF sounds?

Thanks, we changed as suggested.

 

Line 251 - 252

Which sounds? This sentence is very speculative and has no direct links from your study, only if you include the additional results from your experiments (distance from the individuals, etc.). Even then, this is more the hypothesis, especially considering that single M emitted more sounds, so, to whome the group cohesion would be applied?!? If single males emitted more sounds, how could that be connected to group cohesion?

We agree with the reviewer and we modify this sentence point out that a communicative role of sounds must yet be proved and making clearer our logic line. 

 

Line 252 – 253

Keep in mind that in the case of lobster, if the stridulating sounds are real commutative acoustic signals (maybe they really could be, due to their active and intentional nature, as in case with most stridulating sounds), they differ greatly from your acoustic signals. In your case, the signals presented in the text could be the by-product of some activity, such as moving, walking, etc.

Yes in Cherax d. sounds are emitted both when the animal is in resting and in walking and we did not individuate a specific mechanism of production. We agree the sounds in lobster differ compared to sound of Cherax d., but here we just discussed an hypothetical role without any declaration.

Line 264 - 266

“The emission of acoustic signals in C. destructor could be used by females as an indirect way of establishing dominance avoiding injuring interaction.”

 How does this apply to your conducted experiments of states and events, regarding velocity, distance etc?

The variables that give strength to my hypothesis are the number of encounters and fights and there is no correlation with velocity/distances etc.

 

Line 268 - 269

“Increased emission of low-frequency sounds by the two females could also be explained as a possible call for male.”

You mean in FF experiments? This is highly speculative and it was no directly tested under your experiments, so it should be removed. Usually, the sexual selection works by means of male - male competition and female choice; in this case (usually) the male is the calling individuals. Maybe in your case the situation is different, but for now, I would remove this part. 

Ok, we agree and we deleted.

Line 275

“These signals 274 were fewer compared to the low frequency sound and could be emitted accidently..”

Agree, therefore, you need to justify in more details the structure of these sounds as an active and intentional signal. In other words, are your sounds really produced intentionally and are not by-product of other activities? For HF sound, when looking at the spectrogram, this could be the case…

Please see our preview answer on the possibility to add a full description of these sounds.

 

Line 277 - 278

“Considering the other behaviours, the behavioural events revealed no significant differences between the layouts”

How come, when the FFM differentiated from other layout according to your diagrams in encounters and fights? In addition, there were no tail flips in FFM. Please rewrite and specify.

We rewrited this part (see line 367-374 of the manuscript reviewed, accepted revisions ). 

 

Line 280 - 281 

“Moreover, in the FFM groups the number of encounters and fights, although there is no statistical significance (which could emerge by increasing replicas), was higher…”

Higher compared to other layout or FF? Please indicate and be specific.

Ok, we rewrited this part (see line 367 of the manuscript reviewed, accepted revisions)

Line 281 - 282

“In this arrangement the animals also emitted more high-frequency sounds.”

Suggestion: This could be better visualized in the diagrams (such as Fig. 4.) where on the top of each layout, the total number of LF and HF sounds would be indicated.

We already tried to insert the number of sounds into Fig. 4 but resulted more complex and harder to read jointly to letters of significances. We have reported all the acoustic data in Table 3.

 

Line 308 - 309

“In this work, any correlation was found between tail flip events and acoustic emission supporting the hypothesis that sounds probably are not involved in an antipredator strategy.”

I do not understand this part of sentence “any correlation was found”. Please, rewrite this in a more understandable sense. Do you mean, “the correlation”?

We have corrected the sentence.

Line 312

This sounds as an exaggeration. The crayfish would be soniferous if it were really a matter of production of communicative sounds, which is questionable in your case. Second, “numerous sounds” would be applicable if the animal produce much more than two sounds per min.

we changed sentence and the conclusion paragraph.

 

Line 313 - 316

Highly speculative, should not be included in the conclusion. From sentence written like this, it appears (if I haven't read the manuscript) that you are 100% sure these are communicative sounds, for which we are not sure at the moment (at least before you provide more info on acoustic structure and other relevant parameters).

We agree with reviewer, we changed sentence in a less speculative way.

 

Line 316 - 319

Firstly, you should confirm the sounds in the lab, then investigate the sounds in the field in order to justify their spectro-temporal overlap (together with other acoustic similarities) and then you could indicate the usefulness of the sounds for PAM.

We agree with reviewer, we changed sentence in a less speculative way.

 

 

 

 

 

 

 

 

Round 2

Reviewer 3 Report

In the current version, the authors had been include the main observations. 

Reviewer 4 Report

accepted

none