Reproduction under light pollution: maladaptive response to spatial variation in artificial light in a glow-worm

The amount of artificial light at night is growing worldwide, impacting the behaviour of nocturnal organisms. Yet, we know little about the consequences of these behavioural responses for individual fitness and population viability. We investigated if females of the common glow-worm Lampyris noctiluca—which glow in the night to attract males—mitigate negative effects of artificial light on mate attraction by adjusting the timing and location of glowing to spatial variation in light conditions. We found females do not move away from light when exposed to a gradient of artificial light, but delay or even refrain from glowing. Further, we demonstrate that this response is maladaptive, as our field study showed that staying still when exposed to artificial light from a simulated streetlight decreases mate attraction success, while moving only a short distance from the light source can markedly improve mate attraction. These results indicate that glow-worms are unable to respond to spatial variation in artificial light, which may be a factor in their global decline. Consequently, our results support the hypothesis that animals often lack adaptive behavioural responses to anthropogenic environmental changes and underlines the importance of considering behavioural responses when investigating the effects of human activities on wildlife.

I love the expermiment and the story, however I find the paper is not written very professional. Reading and commenting it felt like reading a student assignment, of a very good student but inexperienced paper writer (e.g. consistency in wording, finding functional terminology for the treatments, the function of a figure legend, the scope of the discussion). To improve this would have rather been the job of the senior authors before submission, not the job of a Proceedings reviewer. Still I kept reading and commenting below, since the experiments on glow worms are beautiful and light pollution is an important topic on the context of insect decline. 107 the width of the pronotum of the female (the structure that covers the dorsal surface of the thorax) at its widest part -change to … the maximum width of the pronotum (the structure that covers the dorsal surface of the thorax) of the female -107 measured it for what? … as a proxy of female body size (19) -Would latency to start glowing have been a variable to analyse? -108 why are treatment and control so differently in numbers? Give a good reason since this is suspicious -109-116 state clearly at the start of the paragraph which are the predictor variables included to logistic regression -160 terms that were part of your set-up and hypothesis should not be removed from the model, even if non-significant (this is the statistic answer to your question!). You wanted to test the importance of the Light intensity. It was not important but this is a result! Keep in in your models and discuss it in your discussion! (alternatively: show in the methods that it is not important, and shift the focus of the paper in hypotheses and analysis) -Do not name one condition of the light treatment "light treatment", otherwise the reader cannot distinguish factor name and factor levels.
- Figure 2: state purpose of this set up (to attract glow worms). Figures with their legends should be extractable and useful outside the paper -Complicated explanations in Lines 185-190 illustrate the problem with the name of the factor "distance", a confusing term for the location. Here it codes for a two-factor level categorical factor: illuminated or neighbouring, dark location. Meanwhile "distance" implies an intervalscaled gradient (something that increases or decreases with distance, make sure R really used as it "as factor"). In the methods you had named the same factor "locality", in Figure 2 distances are given in meters, in Figure 3 two levels "near" and "far". Be consistent. You should probably coin a better term, e.g. inside/outside light cone, factor "cone", or something similar, so everybody can understand your setup and analysis.
- Table 2: also provide information on the importance of your random factors date and locality (R marginal and R conditional) - Fig. 3: the survival analysis <for proportion females not yet glowing> is somehow expressed around the corner. How about analyzing <nrs of females glowing>, or analyzing the <latency until female glows> (should yield similar results). short term (this experiment) vs long term responses to illumination, -background knowledge on female and larval glow worm mobility -affecting their ability to leave or avoid lit areas as suggested -Sensory considerations, which light conditions allow male insects to pick up the signal, how sensory channels are affected by different ALAN techniques. Results of the mate attraction experiment should be picked up in the discussion -Importance of glow intensity of the dummy females: this variable was not significant but this needs to be discussed!! Line 241; direct -Authors contribution statement in the beginning and end of the draft (line 252) are contradictory. Consider contribution of AK Lastly, and I would not suggest this as this would be the most time consuming, the authors could investigate actual fitness of these glowworms looking at mating rates and/or offspring production. I hope my above suggestions are not overly daunting, but I do believe that this paper could take behavioral studies of light pollution to the next level and it is needed now in the field.
One minor detail that needs to be addressed -you use lux for ambient lighting but you use uW for radiance of glow worm lures. You need to either stick with photometric units (lux and lumens) or radiometric units (uWs or better yet, photons). This is very important.

Comments to the Author
The authors examine the movement patterns of female glowworms exposed to light pollution in experimental arenas in a laboratory.. They also examine how light pollution impacts the likelihood that a female glowworm will mate under artificial lights in the field. The combination of complimentary field and laboratory experiments is one of this paper's assets.
This paper focuses on a very specific study organism---glowworms. Many readers may find these organisms to be inherently interesting (insects that glow!). The addition of a few paragraphs in the introduction and discussion could give readers a broader perspective on how this study fits in with other research on how anthropogenic disturbances such as light pollution impact animal mating, movement, and courtship behaviors.
As I read through the paper, there were a few instances where I found myself wishing for more information. A few terms, such as "capital breeder" and "lantern," should be defined within the text for the benefit of readers who are unfamiliar with this study system. Further, the addition of more information about glowworm biology and natural history could help readers contextualize and interpret the results of this study. For example, readers may wish to know how far female glowworms typically travel at night, if this information is known.
Additional comments: Line 30,68-The term "capital breeders" is used throughout this manuscript (e.g., lines 30, 68), but is not defined. This term may be unfamiliar to readers.
Line 37-Spell out "advertisements" instead of "adverts." Line 89-What does the character immediately preceding "8 cm" in "(Ø 8 cm)" mean? Does this refer to one of the dimensions of the vials?
Line 93-Are light emission spectra available for the LED lights used in this experiment?
Line 95-Interesting! I'm trying to picture what the arenas might have looked like. Were the females most immediately in contact with the soil with the cardboard?
Line 99-Capitalize "pm." Line 101-I'd like to know more about how the position of the female was recorded. Was the females' location marked within the area? Was the distance from the last known location measured with a ruler? Line 103-"If a female settled multiple times, we used the last one as the settling location and time." How was "settling" defined? Additionally, I wonder if this approach would tend to underestimate the total distance moved be the female? For example, if a female moved 2 cm to the left, then 3 cm to the right along a straight line, would that be recorded as 5 cm or 1 cm traveled?
Line 105-Please define "lantern." Additionally, are there published data on the relationship between lantern size and female size? If so, please include a citation immediately after this statement: "To investigate if female size, which roughly correlates with lantern size and glow intensity," Line 123-Citation for this statistical analysis.
Line 148-How long do males stay with females after initial contact? Is it possible that some males might have left females prior to 1-2 AM, or is it more likely that males would stay with females all night?
Lines 205, 209-What is known about the distances female glowworms typically travel in the field at night? How does 1.2 m relate to the distances females typically travel?
Decision letter (RSPB-2019-2682.R0) 15-Jan-2020 Dear Mrs Elgert: I am writing to inform you that your manuscript RSPB-2019-2682 entitled "Reproduction under light pollution: maladaptive response to spatial variation in artificial light in a glow-worm" has, in its current form, been rejected for publication in Proceedings B.
This action has been taken on the advice of referees, who have recommended that very substantial revisions are necessary. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance.
The resubmission will be treated as a new manuscript. However, we will approach the same reviewers if they are available and it is deemed appropriate to do so by the Editor. Please note that resubmissions must be submitted within six months of the date of this email. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office. Manuscripts submitted after this date will be automatically rejected.
Please find below the comments made by the referees, not including confidential reports to the Editor, which I hope you will find useful. If you do choose to resubmit your manuscript, please upload the following: 1) A 'response to referees' document including details of how you have responded to the comments, and the adjustments you have made. 2) A clean copy of the manuscript and one with 'tracked changes' indicating your 'response to referees' comments document.
3) Line numbers in your main document.
To upload a resubmitted manuscript, log into http://mc.manuscriptcentral.com/prsb and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Resubmission." Please be sure to indicate in your cover letter that it is a resubmission, and supply the previous reference number. reviewers see value in the topic they have valid and substantial concerns with the current version of the manuscript. These need to be specifically addressed if this paper is to compete for space in Proceedings B.
Namely, two reviewers mention issues with the language, description, and scope of the paper as written. This could be part of the reason why the strength in experimental design or findings of the paper are getting lost behind what could be vast improved writing.
Additionally, given that Proceedings is a general topic journal the authors must bring their relevance and findings more clearly into their broader implications in both the Abstract and Discussion; at least better than is done here.
It is my hope that these comments will help the authors in revising their manuscript for publication here. However, if the authors cannot address these issues and the others raised I think this manuscript would be best served somewhere else.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) Review for RSPB-2019-2682, Jana Eccard, Animal Ecology, University of Potsdam Paper: Reproduction under light pollution: maladaptive response to spatial variation in artificial light in a glow-worm I love the expermiment and the story, however I find the paper is not written very professional. Reading and commenting it felt like reading a student assignment, of a very good student but inexperienced paper writer (e.g. consistency in wording, finding functional terminology for the treatments, the function of a figure legend, the scope of the discussion). To improve this would have rather been the job of the senior authors before submission, not the job of a Proceedings reviewer. Still I kept reading and commenting below, since the experiments on glow worms are beautiful and light pollution is an important topic on the context of insect decline. 109-116 state clearly at the start of the paragraph which are the predictor variables included to logistic regression -160 terms that were part of your set-up and hypothesis should not be removed from the model, even if non-significant (this is the statistic answer to your question!). You wanted to test the importance of the Light intensity. It was not important but this is a result! Keep in in your models and discuss it in your discussion! (alternatively: show in the methods that it is not important, and shift the focus of the paper in hypotheses and analysis) -Do not name one condition of the light treatment "light treatment", otherwise the reader cannot distinguish factor name and factor levels.
- Figure 2: state purpose of this set up (to attract glow worms). Figures with their legends should be extractable and useful outside the paper -Complicated explanations in Lines 185-190 illustrate the problem with the name of the factor "distance", a confusing term for the location. Here it codes for a two-factor level categorical factor: illuminated or neighbouring, dark location. Meanwhile "distance" implies an intervalscaled gradient (something that increases or decreases with distance, make sure R really used as it "as factor"). In the methods you had named the same factor "locality", in Figure 2 distances are given in meters, in Figure 3 two levels "near" and "far". Be consistent. You should probably coin a better term, e.g. inside/outside light cone, factor "cone", or something similar, so everybody can understand your setup and analysis.
- Comments to the Author(s) The authors present a study investigating the effects of direct light sources on mate attraction behaviors in the glow worm. They found that females under higher ALAN have decreased glowing onset or don't glow, that the females don't move to get away from the light pollution, and that males are less attracted to glowing females that are under direct light sources. The study is well done and could be published as is at a more specific journal, however, I feel that this study does not meet the high standards of novelty for Proc B as is currently written. However, I do think that the authors could add to this manuscript and make it very attractive to readers of Proc B.
As I was reading through the manuscript I was very curious as to what the difference in achromatic contrast was between the female glow and the ambient lighting. This would not be hard to measure and then model using a non-specific visual system and it would greatly add to the story. I suggest measuring the radiance of the glow worm lures at the angle that a male would be seeing them and also measuring the radiance of the background substrate (street, grass, etc?) that a male would be viewing the glow against. Do this for each of the different distances of the glow worm to see what the contrast is. This would predict if males can even see the glow of the glow worm under the different light treatments. If they easily can, then one could discuss how it isn't a visual detection issue but instead is likely a photophobic situation for males. I am very excited to see what you find.
Another avenue you could take it to set this paper apart from the previous work on Lampyridae, would be to look into the mechanism of the visual abilities of the female. What are they using to detect light levels to determine glowing or not? Are they using compound eyes or ocelli? If they are using only ocelli then it makes sense that they would not be able to determine where to move to for better lighting conditions. If they are using compound eyes, then what is there sensitivity to light levels and what is their acuity? Would they be able to see that there is an actual light gradient? I believe the authors are giving these beetles a lot of visual credit for being able to detect a light gradient of only one order of magnitude. This would be a very informative angle as well.
Lastly, and I would not suggest this as this would be the most time consuming, the authors could investigate actual fitness of these glowworms looking at mating rates and/or offspring production. I hope my above suggestions are not overly daunting, but I do believe that this paper could take behavioral studies of light pollution to the next level and it is needed now in the field.
One minor detail that needs to be addressed -you use lux for ambient lighting but you use uW for radiance of glow worm lures. You need to either stick with photometric units (lux and lumens) or radiometric units (uWs or better yet, photons). This is very important.

Referee: 3
Comments to the Author(s) The authors examine the movement patterns of female glowworms exposed to light pollution in experimental arenas in a laboratory.. They also examine how light pollution impacts the likelihood that a female glowworm will mate under artificial lights in the field. The combination of complimentary field and laboratory experiments is one of this paper's assets.
This paper focuses on a very specific study organism---glowworms. Many readers may find these organisms to be inherently interesting (insects that glow!). The addition of a few paragraphs in the introduction and discussion could give readers a broader perspective on how this study fits in with other research on how anthropogenic disturbances such as light pollution impact animal mating, movement, and courtship behaviors.
As I read through the paper, there were a few instances where I found myself wishing for more information. A few terms, such as "capital breeder" and "lantern," should be defined within the text for the benefit of readers who are unfamiliar with this study system. Further, the addition of more information about glowworm biology and natural history could help readers contextualize and interpret the results of this study. For example, readers may wish to know how far female glowworms typically travel at night, if this information is known.
Additional comments: Line 30,68-The term "capital breeders" is used throughout this manuscript (e.g., lines 30, 68), but is not defined. This term may be unfamiliar to readers.
Line 37-Spell out "advertisements" instead of "adverts." Line 89-What does the character immediately preceding "8 cm" in "(Ø 8 cm)" mean? Does this refer to one of the dimensions of the vials? Line 103-"If a female settled multiple times, we used the last one as the settling location and time." How was "settling" defined? Additionally, I wonder if this approach would tend to underestimate the total distance moved be the female? For example, if a female moved 2 cm to the left, then 3 cm to the right along a straight line, would that be recorded as 5 cm or 1 cm traveled?
Line 105-Please define "lantern." Additionally, are there published data on the relationship between lantern size and female size? If so, please include a citation immediately after this statement: "To investigate if female size, which roughly correlates with lantern size and glow intensity," Line 123-Citation for this statistical analysis.
Line 148-How long do males stay with females after initial contact? Is it possible that some males might have left females prior to 1-2 AM, or is it more likely that males would stay with females all night?
Lines 205, 209-What is known about the distances female glowworms typically travel in the field at night? How does 1.2 m relate to the distances females typically travel?
General interest: Is the paper of sufficient general interest? Marginal Quality of the paper: Is the overall quality of the paper suitable? Excellent Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? No Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author I understand that the authors don't want to add more analyses to the current study. I do hope that their future work does address my suggestions and questions.
I do have one more question: on lines 157-158 the authors claim that 1 to 2 lux is close to natural light levels at night. That is not true, that is not remotely true. Perhaps under a full harvest moon with good albedo (snow covered for instance), it may get to be between 1 and 10 lux. Most full moon conditions are between .1 and 1 lux. New moon conditions (which is more than 50 percent of the night) is less than .001 lux. So what do you mean? You even later say on line 248 that moonlight is around .05 to .1 lx, and again this actually ranges from .001 to 1 lux depending on lunar phase as well as lunar altitude and environmental conditions. So please rewrite lines 157 to 158 to be accurate and consistent with your discussion. I also think you need to caveat that your study's gradient is still quite bright.

Quality of the paper: Is the overall quality of the paper suitable? Excellent
Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? No

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author Please see attached comments (Tyler 2013 also attached)
Decision letter (RSPB-2020-0806.R0)

18-May-2020
Dear Mrs Elgert: Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. The reviewers' comments (not including confidential comments to the Editor) and the comments from the Associate Editor are included at the end of this email for your reference. As you will see, the reviewers and the Editors have raised some concerns with your manuscript and we would like to invite you to revise your manuscript to address them.
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Best wishes, Professor Hans Heesterbeek mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Dear Authors, Thank you for the resubmission of your manuscript. I find it to be improved. The updated manuscript has now been seen by two established researchers in the field (one old and one new reviewer). Both reviewers see much merit in the manuscript and its findings, but have concerns. Notably, Reviewer 1 is concerned with the lux ratings described in the paper and also with the fact that many of their suggestions were not addressed in the revised document. In fact, many instances in the Response to Reviewer comments letter the authors used the excuse of the Proceedings B page limit to avoid addressing critical reviewer concerns, such as better detailing the disturbing effects of light pollution on organisms other than glow-worms. This is not acceptable. Yes, there are limits to what you can write given space concerns, but the important issues should still be addressed rather than ignored. Reviewer 2 has some concerns about terminology and other small comments.
In addition to the Reviewer concerns I have some of my own to this revised version of the manuscript. First, I think the abstract still needs work to better highlight the broader implications of the study for the readers of Proc B, and to highlight the specific novelty of this study. Second, unlike the reviewers of this round I still find that the introduction is a hard read. As said previously, a better delve into past literature on the effect of light pollution across a broader range of organisms is relatively inexistent and could be much better addressed. Please do so. Now the Introduction just goes straight into the glow-worm system, disregarding a more detailed and broad review of the literature. Third, I find that this paper as written, however lovely the experiment, is too specific for a general audience, and does not need to be this way given the relative lack of clear studies showing the behavioral effect of light pollution in nature. I only see broader implications in the very last paragraph. This needs to be better addressed. The manuscript is also still slightly choppy in places and the authors could benefit by having it reread by colleagues outside their subject, before they turn in another revised version here.
Smaller concerns: Line 41 The sentence needs reworking. Line 47-50 Sentence grammatically incorrect. Also, if this sentence is supposed to be added to highlight the novelty of the current study then it is not clear that this is what you do here without expanding it. Line 265 remove 's' from movements Again we thank the authors for their manuscript, and we look forward to a revised paper that addresses all the current concerns.

Reviewer(s)' Comments to Author:
Referee: 2 Comments to the Author(s). I understand that the authors don't want to add more analyses to the current study. I do hope that their future work does address my suggestions and questions.
I do have one more question: on lines 157-158 the authors claim that 1 to 2 lux is close to natural light levels at night. That is not true, that is not remotely true. Perhaps under a full harvest moon with good albedo (snow covered for instance), it may get to be between 1 and 10 lux. Most full moon conditions are between .1 and 1 lux. New moon conditions (which is more than 50 percent of the night) is less than .001 lux. So what do you mean? You even later say on line 248 that moonlight is around .05 to .1 lx, and again this actually ranges from .001 to 1 lux depending on lunar phase as well as lunar altitude and environmental conditions. So please rewrite lines 157 to 158 to be accurate and consistent with your discussion. I also think you need to caveat that your study's gradient is still quite bright. Decision letter (RSPB-2020-0806.R1)

22-Jun-2020
Dear Mrs Elgert I am pleased to inform you that your manuscript entitled "Reproduction under light pollution: maladaptive response to spatial variation in artificial light in a glow-worm" has been accepted for publication in Proceedings B.
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109-116 state clearly at the start of the paragraph which are the predictor variables included to logistic regression RESPONSE: Clarified at the start of the paragraph: "To analyse the impact of the presence of artificial light and the glow intensity of the female (measured as pronotum size) on behaviours that were binary, we used logistic regression,…", lines 126 -128 160 terms that were part of your set-up and hypothesis should not be removed from the model, even if nonsignificant (this is the statistic answer to your question!). You wanted to test the importance of the Light intensity. It was not important but this is a result! Keep in in your models and discuss it in your discussion! (alternatively: show in the methods that it is not important, and shift the focus of the paper in hypotheses and analysis) RESPONSE: Variables that do not influence the response variables and do not contribute to the model outcome can be removed from the final model when the reasons are clearly explained, which we do on lines 179 -181. We are not in favour of altering hypotheses after an experiment has been carried out.
Do not name one condition of the light treatment "light treatment", otherwise the reader cannot distinguish factor name and factor levels.

RESPONSE:
We suspect the reviewer is referring to the three glow intensities (low, medium, high) within the light treatment. This has now been clarified, lines 169 -172. Complicated explanations in Lines 185-190 illustrate the problem with the name of the factor "distance", a confusing term for the location. Here it codes for a two-factor level categorical factor: illuminated or neighbouring, dark location. Meanwhile "distance" implies an interval-scaled gradient (something that increases or decreases with distance, make sure R really used as it "as factor"). In the methods you had named the same factor "locality", in Figure 2 distances are given in meters, in Figure 3 two levels "near" and "far". Be consistent. You should probably coin a better term, e.g. inside/outside light cone, factor "cone", or something similar, so everybody can understand your setup and analysis.

RESPONSE:
We agree that the choice of the term 'distance' was less suitable. We have changed the term as suggested to 'within or outside the cone of light'. We thank the reviewer for pointing out the problem with the use of these terms. Table 2: also provide information on the importance of your random factors date and locality (R marginal and R conditional) RESPONSE: We assume the reviewer is referring to r2 marginal (fixed factors only) and r2 conditional (fixed + random factors). However, definitions for r-square are problematic in models with multiple error terms, and there is not yet a consensus on how to calculate them -that is why we are not reporting them. They are not the same 'variance explained' as in linear models. However, our data will be freely available so anybody interested can calculate them if needed.

Discussion
The discussion is very limited.
RESPONSE: Because of the page limit of Proceedings B, we have to limit what we discuss. We have decided to not go in too many directions and keep speculations at a minimum, to avoid the paper becoming fragmented and too speculative. Thus, we have been selective in what we incorporate. Yet, we hope we have found a good balance in what we include in relation to the length of the paper. Sensory considerations, which light conditions allow male insects to pick up the signal, how sensory channels are affected by different ALAN techniques. Results of the mate attraction experiment should be picked up in the discussion RESPONSE: This is a big topic, which could fill a whole paper. We are currently focussing on part of these questions (current experiments) and before we have reliable results, we would rather not speculate on the matter (but stay tuned for coming papers).
Importance of glow intensity of the dummy females: this variable was not significant but this needs to be discussed!! RESPONSE: This is discussed on lines 267 -273: "Interestingly, the response of females to artificial light did not depend on their supposed glow intensity…" Line 241; direct

RESPONSE: The section has been rewritten
Authors contribution statement in the beginning and end of the draft (line 252) are contradictory. Consider contribution of AK RESPONSE: We apologize for the contradiction. AK contributed to the design of the experiment.

Referee: 2
The authors present a study investigating the effects of direct light sources on mate attraction behaviors in the glow worm. They found that females under higher ALAN have decreased glowing onset or don't glow, that the females don't move to get away from the light pollution, and that males are less attracted to glowing females that are under direct light sources. The study is well done and could be published as is at a more specific journal, however, I feel that this study does not meet the high standards of novelty for Proc B as is currently written. However, I do think that the authors could add to this manuscript and make it very attractive to readers of Proc B.
As I was reading through the manuscript I was very curious as to what the difference in achromatic contrast was between the female glow and the ambient lighting. This would not be hard to measure and then model using a non-specific visual system and it would greatly add to the story. I suggest measuring the radiance of the glow worm lures at the angle that a male would be seeing them and also measuring the radiance of the background substrate (street, grass, etc?) that a male would be viewing the glow against. Do this for each of the different distances of the glow worm to see what the contrast is. This would predict if males can even see the glow of the glow worm under the different light treatments. If they easily can, then one could discuss how it isn't a visual detection issue but instead is likely a photophobic situation for males. I am very excited to see what you find. RESPONSE: These are very interesting ideas, and certainly worth doing, but we believe they require additional work and would be the topic of another paper. It would then be worth manipulating also the artificial light conditions and not only light or not light, as we do in the present experiments. We are currently planning such an experiment, where we manipulate the quality and spectrum of the artificial light, and we will discuss these issues in more detail in that paper -the page limit for Proceedings B papers would not allow us to include and discuss the results from the coming experiments in this paper.
Another avenue you could take it to set this paper apart from the previous work on Lampyridae, would be to look into the mechanism of the visual abilities of the female. What are they using to detect light levels to determine glowing or not? Are they using compound eyes or ocelli? If they are using only ocelli then it makes sense that they would not be able to determine where to move to for better lighting conditions. If they are using compound eyes, then what is there sensitivity to light levels and what is their acuity? Would they be able to see that there is an actual light gradient? I believe the authors are giving these beetles a lot of visual credit for being able to detect a light gradient of only one order of magnitude. This would be a very informative angle as well.

RESPONSE:
The females are known to be able to move in response to light, as they respond to green LED lights (lines 237 -244). Moreover, even if the females would not be able to see the light gradient, they should still be able to discern between the bright and the dark end of the arena. Investigating the visual ability of females is another interesting topic, but goes beyond the purpose of this paper, which is ecologically oriented. It would be interesting to collaborate on the topic with researchers more familiar with the field, but it would be the topic of another paper.
Lastly, and I would not suggest this as this would be the most time consuming, the authors could investigate actual fitness of these glowworms looking at mating rates and/or offspring production.

RESPONSE: Yes, this would be really interesting to investigate, but as it turns out, raising glow-worms is not the easiest as it also demands the raising of snails, and the mortality commonly is high. Yet, this is included in future our plans.
I hope my above suggestions are not overly daunting, but I do believe that this paper could take behavioral studies of light pollution to the next level and it is needed now in the field.
One minor detail that needs to be addressed -you use lux for ambient lighting but you use uW for radiance of glow worm lures. You need to either stick with photometric units (lux and lumens) or radiometric units (uWs or better yet, photons). This is very important.

RESPONSE: Lux was chosen for the ambient light based on existing reference data for streetlights (lux is
usually used in connection with light pollution) and our existing equipment. Unfortunately, our LED lures were too dim for accurate measurements with our lux-meter. However, we were able to measure the LED lures with a spectrophotometer and, thus, report their values in uW. The conversion from lux to uW for ambient light is unreliable, and we have therefore chosen not to do the conversion.

Referee: 3
Comments to the Author(s) The authors examine the movement patterns of female glowworms exposed to light pollution in experimental arenas in a laboratory. They also examine how light pollution impacts the likelihood that a female glowworm will mate under artificial lights in the field. The combination of complimentary field and laboratory experiments is one of this paper's assets.
This paper focuses on a very specific study organism---glowworms. Many readers may find these organisms to be inherently interesting (insects that glow!). The addition of a few paragraphs in the introduction and discussion could give readers a broader perspective on how this study fits in with other research on how anthropogenic disturbances such as light pollution impact animal mating, movement, and courtship behaviors. We are pleased to submit a revised version of our manuscript RSPB-2020-0806 for your consideration. Below is a detailed point-by-point response of how we have attended to each of the reviewers' comments, and a copy of the manuscript with revisions made since the previous version marked as 'tracked changes'. The input provided by the reviewers has greatly strengthened the manuscript and we hope you will find the revision satisfactory. We look forward to a decision in due course.
We take the opportunity to thank the reviewers for their valuable input. Thank you for the resubmission of your manuscript. I find it to be improved. The updated manuscript has now been seen by two established researchers in the field (one old and one new reviewer). Both reviewers see much merit in the manuscript and its findings, but have concerns. Notably, Reviewer 1 is concerned with the lux ratings described in the paper and also with the fact that many of their suggestions were not addressed in the revised document. In fact, many instances in the Response to Reviewer comments letter the authors used the excuse of the Proceedings B page limit to avoid addressing critical reviewer concerns, such as better detailing the disturbing effects of light pollution on organisms other than glow-worms. This is not acceptable. Yes, there are limits to what you can write given space concerns, but the important issues should still be addressed rather than ignored. Reviewer 2 has some concerns about terminology and other small comments.
In addition to the Reviewer concerns I have some of my own to this revised version of the manuscript. First, I think the abstract still needs work to better highlight the broader implications of the study for the readers of Proc B, and to highlight the specific novelty of this study. Second, unlike the reviewers of this round I still find that the introduction is a hard read. As said previously, a better delve into past literature on the effect of light pollution across a broader range of organisms is relatively inexistent and could be much better addressed a better delve into past literature on the effect of light pollution across a broader range of organisms is relatively inexistent and could be much better addressed. Please do so. Now the Introduction just goes straight into the glow-worm system, disregarding a more detailed and broad review of the literature. Third, I find that this paper as written, however lovely the experiment, is too specific for a general audience, and does not need to be this way given the relative lack of clear studies showing the behavioral effect of light pollution in nature. I only see broader implications in the very last paragraph. This needs to be better addressed. The manuscript is also still slightly choppy in places and the authors could benefit by having it re-read by colleagues outside their subject, before they turn in another revised version here. RESPONSE: First, We have altered the focus of the abstract to more strongly focus on maladaptive responses to anthropogenic environmental changes and the importance of unravelling the underlying mechanism, with our contribution revealing a behavioral mechanism behind the effect of artificial light, and offering support for the hypothesis that maladaptive responses are common.

Appendix B
ii ii Third, the general interest of the paper has been increased by more strongly linking our work to the hypothesis that maladaptive responses are common when animals encounter conditions that they have not encountered in their recent evolutionary past (lines 43 -47, 50 -53). We have also extended the end part of the discussion by more broadly linking our results to the wider literature and stress the importance of investigating the behavioral mechanisms behind responses to disturbances. The text has in places been streamlined to be less choppy.
Smaller concerns: Line 41 The sentence needs reworking.

RESPONSE: We have reworked the sentence: "Vast areas of the earth are currently lit up at night by direct light emission from various sources, such as streetlights, advertisements, buildings, and cars, and by indirect skyglow." (lines 41 -43).
Line 47-50 Sentence grammatically incorrect. Also, if this sentence is supposed to be added to highlight the novelty of the current study then it is not clear that this is what you do here without expanding it.

Line 265 remove 's' from movements RESPONSE: We assume this refers to line 200 or 228 (no 'movements' on line 265). We have exchanged all 'movements' to 'movement' where suitable.
Again we thank the authors for their manuscript, and we look forward to a revised paper that addresses all the current concerns.

Reviewer(s)' Comments to Author:
Referee: 2 Comments to the Author(s). I understand that the authors don't want to add more analyses to the current study. I do hope that their future work does address my suggestions and questions.
I do have one more question: on lines 157-158 the authors claim that 1 to 2 lux is close to natural light levels at night. That is not true, that is not remotely true. Perhaps under a full harvest moon with good albedo (snow covered for instance), it may get to be between 1 and 10 lux. Most full moon conditions are between .1 and 1 lux. New moon conditions (which is more than 50 percent of the night) is less than .001 lux. So what do you mean? You even later say on line 248 that moonlight is around .05 to .1 lx, and again this actually ranges from .001 to 1 lux depending on lunar phase as well as lunar altitude and environmental conditions. So please rewrite lines 157 to 158 to be accurate and consistent with your discussion. I also think you need to caveat that your study's gradient is still quite bright.  How bright is moonlight. Astron. Geophys, 58, 31-32 that states that in temperate latitudes, in the summer, typical moonlight lies between 0.05 to 0.1 lx, with a maximum of ~0.3 lx.

Referee: 4 Review of RSPB---2020---0806
This study examines the timely and important topic of how spatial variation in artificial light affects female glow behavior and male mate attraction in the glow---worm Lampyris noctiluca.
This manuscript was a pleasure to read -it is well written, the experiments thoughtfully designed, the statistical analysis appropriate, and the results are presented clearly and concisely. Below we offer a few suggestions that we hope may further improve the paper.

Major suggestions:
1. Minorly confusing terminology: a)The variable called "Exposure to light" (i.e. in or out of the light cone) in the mate attraction experiment is a bit misleading because glowworms in the control treatment were also not exposed to light. Perhaps this variable could be called something like "position with respect to light fixture"? RESPONSE: We agree and have changed the variable name to "position with respect to light fixture", using the levels "inner position" and "outer position". b) Females' pronotal width was measured as a proxy for their glow intensity, and based on previous work this seems reasonable (as explained in lines 120---123). However, the manuscript switches between these two variable names (lines 126 vs. 133). It seems good practice to use what was actually measured as the variable name, so perhaps this could be changed to "pronotum width (glow intensity)". RESPONSE: We have followed your advice and changed the variable name as suggested.
2. Additional citations: Although anecdotal, the attached 2013 paper by John Tyler (What do glow---worms on their day off?) provides much relevant information on female glow---worm movement. Also relevant is iv iv experimental work by Booth et al. (2004J. Exp. Biol. 207, 2373---2378, which found that higher intensity lures were more attractive to L. noctiluca males.

RESPONSE: Thank you for these suggestions, we have added citations to these relevant sources (lines 262, 227 -229).
3.Show data for male response to glow intensity: Although dummy females' glow intensity did not turn out to have a significant effect in the male attraction experiment, it seems important to show these results for both experimental treatments (in a supplementary figure if need be). RESPONSE: Data from the 3 glow intensities has been added as a supplementary figure.

Minor suggestions:
Line 23 -Modify to read "A species potentially affected by light pollution…."? RESPONSE: The sentence has been rewritten ' We investigated if females of the common glow-worm Lampyris noctiluca -which glow in the night to attract males -mitigate negative effects of artificial light on mate attraction by adjusting the…', lines 23 -26.
Line 100 -Might be worth mentioning in the Introduction that glow---worm courtship is restricted to only 4 hours of darkness at your latitude -thus ALAN could have a more dramatic impact.

RESPONSE: Thank you, we have added the information to the method section, lines 107 -109.
Line 103 ---perhaps this should read "[peak] intensity ~940 microWatts [per cm2 per second]"? RESPONSE: Thank you for pointing this out, we have exchanged "u" for "µ". We used a spectrophotometer together with an integrating sphere for measuring the dummies, for the added measurements of ambient light we used both a lux-meter and a spectrophotometer together with cosine corrector. From the information on peak intensity (µW/nm, microwatts/nanometer) given by the spectrophotometer, we were able to calculate the total irradiance (µW). For clarity, we now have exchanged the total irradiance (µW) for the peak intensity (µW/nm) and added mention of the use of the integrating sphere. Lines 111 -114.
Lines 161---62 ---this range of dummy female intensities may not have been great enough to see a difference (Steven's power law) RESPONSE: This seems unlikely, as the range was chosen to reflect natural variation at our study site and the difference in brightness was easy to see with human eye.
Line 171-It may be worth mentioning that small n (low statistical power) in the control treatment of the mate attraction experiment might have made it difficult to detect an effect of glow intensity RESPONSE: This is now mentioned on lines 262 -264 in the Discussion: 'A larger sample size may be needed to detect the weaker effect of glow intensity on mate attraction as no significant effect of glow intensity on mate attraction was detected in the controls.' Lines 212---14 -As mentioned above, it would be good to show results for the 3 glow intensities from both control and + light treatments.

RESPONSE: Results from the 3 glow intensities has been added as a supplementary figure.
v v Lines 255---257-add "potentially" to clarify that we don't know if larvae actually do avoid light polluted areas RESPONSE: The sentence has been rewritten: 'Larvae have the potential to avoid light polluted areas by moving towards more undisturbed environments before they pupate to adults, as they can move up to 5 meters per hour [18], and do not pupate before they are 2-3 years old [18, 33, 42]. However, whether larvae can detect and respond to light pollution is currently unknown.' Lines 241 -246.
Lines 260---262-North American fireflies vary in emission wavelength ---"The degree to which this is an evolutionary change is unknown" -What does this mean? Clarify how this is relevant to adaptation to light pollution. RESPONSE: We have clarified the sentence: 'Fireflies of North-America differ in the wavelength of their glow depending on the local background, but the degree to which the differences have a genetic basis and indicate a potential for genetic adaptation is undetermined [44].' (lines 248 -251).
Lines 270---273 -Previous studies that have found an effect of glow intensity on male attraction should be cited again here. Also, the explanation given here does not seem consistent with the fact that glow intensity also did not influence male attraction in the dark controls (again, please show these data).

RESPONSE: Thank you for highlighting this. We have added citations to previous studies that have found an effect of brightness and an explanation for the lack of effect in the dark control: ' In areas with low intensity artificial light, differences among females in glow intensity may still influence mating success, as males are known to prefer brighter females [29, 45, 46]. A larger sample size may be needed to detect the weaker effect of glow intensity on mate attraction as no significant effect of glow intensity on mate attraction was detected in the controls.', (lines 261 -264).
Abstract 20 The amount of artificial light at night is growing worldwide, impacting the behaviour of nocturnal 21 organisms. Yet, we know little about the consequences of these behavioural responses for individual 22 fitness and population viability. We investigated if females of the common glow-worm Lampyris 23 noctilucawhich glow in the night to attract malesmitigate negative effects of artificial light on 24 mate attraction by adjusting the timing and location of glowing to spatial variation in light conditions. 25 We found females do not move away from light when exposed to a gradient of artificial light, but delay 26 or even refrain from glowing. Further, we demonstrate that this response is maladaptive, as our field 27 study showed that staying still when exposed to artificial light from a simulated streetlight decreases 28 mate attraction success, while moving only a short distance from the light source can markedly 29 improve mate attraction. These results indicate that glow-worms are unable to respond to spatial

171
We started each trial when dusk began to fall (approximately 10 PM), by turning on the glow of the 172 two dummy females, as well as the light from the pole in the artificial light treatment, while leaving 173 poles unlit in the control. We checked the dummy females 3-4 h later (at 1-2 AM) for the presence of 174 males and turned off all lights. Males are unlikely to escape from the traps (Elgert, personal 175 observation). 176 We conducted 38 replicates of the artificial light treatment, with three dummy glow intensities (low: n 177 = 13; medium: n = 14; high: n = 11, with two dummy females in each replicate) and 19 replicates of the 178 control, with three dummy glow intensities (low: n = 6; medium: n = 5; high: n = 8, with two dummy 179 females in each replicate). We distributed the treatments (presence of artificial light and glow 180 intensities) equally among the six sites.

181
In the analyses, we used the presence or absence of males in each dummy female trap as the response 182 variable, rather than the number of males caught, to rule out the possibility that the presence of one 183 male had attracted additional males. We analysed the data using a GLMM with binomial error and glow intensity as fixed factors, and date and site as random factors, with site nested within date. 188 We started with a full model and deleted non-significant interaction terms and fixed terms when this 189 was supported by the Akaike Information Criterion (AIC) and did not reduce the significance of other 190 terms [37]. All analyses were performed using SPSS 25. Significance was designated as P < 0.05.

193
Behavioural responses to light pollution 194 Artificial light in the experimental arena reduced the probability that females glowed (Table 1), 195 increased the latency to glowing for those that glowed (Cox Proportional Hazard model,Wald = 196 30.599, p < 0.001, Figure 3), and increased the probability that females went into hiding (Table 1).

197
Females that glowed continued to do so until the end of the trial, except for one female that glowed for 198 73 minutes under light and then ceased glowing. Artificial light did not influence the direction of 199 movements, the distance moved, whether they settled down, or the distance from the light when they 200 settled down (for those that settled) ( Table 1).    Interestingly, the response of females to artificial light did not depend on their supposed glow intensity, 256 measured as pronotum body sizewidth (which correlates with body and lantern size and, thus, with 257 glow intensity [29]). Moreover, the glow intensities of the dummies did not influence mate attraction 258 under artificial light, although the selected intensities reflected those of females in nature. These results 259 suggest that the strong intensity of the artificial light overrode differences in the weaker glow intensity 260 of females. In areas with low intensity artificial light, differences among females in glow intensity may 261 still influence mating success, as males are known to prefer brighter females [29,45,46]. A larger 262 sample size may be needed to detect the weaker effect of glow intensity on mate attraction as no 263 significant effect of glow intensity on mate attraction was detected in the controls.

264
In conclusion, our results support the suggestion that light pollution is one cause of the global decline         Tables   445   446   Table 1. Behavioural responses of glow-worm females exposed to a gradient of artificial light in an elongated arena.

Response
Light Control Logistic regression was used to analyse differences in proportions, and ANOVA to analyse differences in continuous variables.
447 Table 2. The influence of the presence of artificial light, and the position relative to the light fixtureinner or outeron the probability that a dummy female attracted one or more males.