Review of Open Biology manuscript: RSOB-22-0233
Ueda, A. et al. (2022)

Intense light unleashes male courtship behavior in wild-type Drosophila

Summary:
In this manuscript, Ueda et al. report the phenomenon that courtship behavior in wild-type male fruit flies is strongly induced by a sudden transition from dim to very bright light. In male-only chambers, male-male courtship was also initiated. Studies with mutant fly strains suggested that blue/UV light detection was required for this phenomenon to take place, and that olfactory cues might act to restrain male courtship behavior.

Strengths:

1. The study shows a robust and repeatable effect.
2. The study is concise and generally very well-written.
3. The graphs are effective at representing the data.
4. The finding is important, in that it points out the importance of controlling environmental conditions (especially light) when performing courtship and mating experiments, and could potentially explain differences in courtship observed in previously published studies.

Major Comments:

1. The results shown are interesting, but the study feels a bit incomplete because the authors have not yet examined how visual and olfactory information might interact mechanistically to influence male courtship behavior. If the editors feel that the current results have a strong enough impact for publication in this journal, then I would only ask that the authors include a more in-depth discussion of the known neural pathways involved in courtship and how visual and olfactory inputs might intersect with those pathways. However, the editors might choose to request that the authors delve deeper into the cellular/molecular mechanisms behind this phenomenon.
2. A statistics issue: From the Methods section, it seems that the authors ran ‘paired t-tests comparing the “low” vs. “high” light conditions’. However, in panel D, there were “baseline low”, “high”, and then “recovery low” conditions. Since there were 3 conditions, it would be more appropriate to run a 1-factor within-subject ANOVA, followed by post-hoc tests if the ANOVA shows significance.
3. A ceiling issue: In the studies of olfactory mutants, orco males have very high levels of “Chase” and “Wing Ext” behaviors even in the “low” condition, which cannot increase further in the “high” condition. Therefore, the lack of induction of courtship behavior is confounded by there being a ceiling effect in this strain of flies. Could the authors use a larger arena or perhaps pre-mate the males or use some other manipulation to lower their original courtship behavior in the “low condition”, ideally so that it resembles the levels observed in other strains?
4. In Figure 1, Panel B – it was unclear why the authors were showing the differences between Pulse and Sine song, since there didn’t appear to be data that analyzed each type of song separately. In fact, there are no data regarding courtship song, other than one statement that courtship songs “correlated with courtship-related wing extensions”. The authors should adjust how this material is covered in the text and in the figure, to either remove mention of courtship song or to add data showing the correlation between recorded song and visually observed mating behaviors. They could alternatively show in a graph that recorded song of Pulse and Sine types each was induced by high-intensity light.

Minor Comments:

1. The Introduction could use more of a broad starting paragraph or two instead of diving directly into what the authors did in this study.
2. When the authors mention the intensities of the “low” and “high” light conditions, it would be helpful to provide more context here. For example, please compare the 0.4klx and 18klx values with measurements that would be taken at different points during the day. It would also be helpful to provide context about the intensity of light that has been used in various studies of fruit fly behavior (especially studies of courtship). Along these lines, 0.4klx is referred to as “relatively low intensity light”, but this is a fairly high intensity for many types of behavioral studies (e.g., for sleep/rhythms studies). This could use more discussion.
3. Because the whole study is about light’s effects, it is important to provide as much info about the quality of the light used as possible. Could the authors please show spectrograms of the light emitted by their LED strips during both the low intensity and high intensity light conditions? This is important, so as to show the relative balance of blue/green/red from their LEDs and that the quality of light does not change when the intensity changes in their experiments.
4. When the authors say that they observed courtship increases “across WT strains”, please change this to read “Canton-S and white-Berlin wild-type strains”, since these are the only 2 wild-type strains that the authors are reporting data for.
5. Please provide N’s for all groups of animals used in the study. It was only mentioned that 8 males were used for panel C in Figure 1. I will also note that an N of 8 is relatively low – especially when reporting a new phenomenon, it might be worth repeating this experiment with another group of animals to make sure the effect is consistent. However, because the observed effects were so robust, I don’t feel that this is an absolute requirement for publication.
6. In the Figure 1 legend, please add “compared with Baseline Low” when describing the meaning of the asterisks for paired t-test comparisons.
7. Could use more discussion of why different responses might have been observed in the orco and smellblind olfactory mutants. In general, the discussion section could be expanded substantially. See for example Major Comment 1 above.
8. In the Methods section, please clarify what is meant by the “Wu Lab collection”. Also please clarify how the light intensity was adjusted using the LED strips. If any software was used to switch between low and high intensity, please mention this and make the code and wiring diagram available for future replication.
9. In the supplemental figure legend, for panel G, please change “intractograms” to “interactograms”.
10. Please check for other small typos/grammar issues.

Review of Open Biology manuscript: RSOB-22-0233.R1
Ueda, A. et al. (2023)

Intense light unleashes male courtship behavior in wild-type Drosophila

Major Comments:

1. Could use more discussion about why mutants to the white gene would not have a courtship response to the transition from low to high intensity light. If the mutants do in fact have increased sensitivity to light, would it actually be expected that they would show increased courtship even at relatively low light intensities? Could this effect be more due to the reduced visual acuity in white mutants, instead of their sensitivity to light?
2. Discussion, page 11, lines 7-8: The authors discuss here the results from olfactory mutants in terms of male-male courtship. It would be very helpful to know how these olfactory mutants affect M-F courtship in low vs. high light intensity conditions. If those data are available, it would be advised to add them to the study. But in their absence, the authors should discuss what they would expect to observe in those experiments.
3. I appreciate that the authors have included Figure S5 about the manufacturer’s reported spectrum for the LED strip that they used. But I really think it is important to include actual spectra recorded in the lab, for the LEDs at low and high intensities, but also for the incandescent light, for sunlight, and for the blue light source that was used in some experiments. This is important so that the reader can see the qualities of the light as opposed to just being provided with the intensities.
4. I appreciate that the authors added a single example of M-F song as a comparison with the song recorded in high light intensity in M-M conditions. But I am still not seeing any quantitative analysis of the song. For example, the authors could show what % of time that courtship song could be recorded in low-intensity light conditions vs. high-intensity light conditions. The authors state in the Results, page 5, lines 15-17 that “pulse and singe courtship songs… correlated with courtship-related wing extensions in the arena”. But there are no data about either courtship songs or wing extensions shown in the figures/tables. Along the lines of what I said in my first review, if the authors want to make statements about correlations, etc., they need to show the data and analyze it to support those statements.
5. Results, page 6, line 16: In a few earlier situations in the Results section, the authors report p-values, but after this point, they never do again. Please be consistent about reporting these statistical results. In addition, if these are results from ANOVAs, the authors should be reporting the F-values and degrees of freedom as well, not just the p-values.
6. Figure 3: I found it difficult to read the interactogram graphs. Would it be possible to edit the layout to make those bigger and therefore more legible? Also, is there any way to quantify these data in a more numerical way? In the original version of the article, the authors had included data from the tracker about the speed of the flies, etc., but that table has now been removed. I actually think that those analyses of the videos were helpful. In general, I would recommend moving Figures S1 and S2 to the main text, since those findings are more central to the main conclusions from the manuscript. In contrast, I’m not sure what the current Figure 3 adds to the manuscript that isn’t already captured by the Wing Extension, Chase, and Chaining data in Figures 1 and 2.

Minor Comments:

1. Intro, page 4, lines 13-17: It seems a bit strange that both disruption of the FruM gene and activation of FruM-expressing neurons cause M-M courting. It might be worth including more discussion of the possible reasons for this.
2. Intro, page 4, line 23: Edit to read “…WT males, using illumination with LEDs, incandescent lights, and sunlight. We examined this phenomenon…”
3. Results, page 5, line 14: Change “to” to “of”.
4. Results, page 6, lines 6-7: “…courtship behaviors in a second melanogaster strain, Berlin (Figure 2A).”
5. Results, page 6, line 8: Add “in” before “CS flies”.
6. Results, page 7, line 6: Add “that” after “indicate”.
7. Results, page 7, line 14: Add “we carried out” after “patterns among the male flies,”.
8. Results, page 7, line 16: Add “the” before “IowaFLI Tracker”.
9. Results, page 8, line 3: Change “is” to “are”.
10. Results, page 8, lines 10-12: Please add references for the statements made here about how mutations to the white gene affect visual processing, and that they result in a greater sensitivity to light.
11. Results, page 8, line 21: Edit to read “…demonstrates that the auditory cues generated…”
12. Results, page 9, line 6: Edit to read “…maximum score of 12, that a further increase in courtship…”
13. Discussion, page 9, lines 18-19: The authors mention studies here that examined the effects of blue and UV light on inhibition of male-male courtship. Since the intensity of light used is a central issue to the current study, it would be helpful to report what intensities of light were used in those cited reports.
14. Discussion, page 9, line 23: Add a comma after “normal”.
15. Discussion, page 10, line 5: Edit “oroco” to “orco”.
16. Discussion, page 10, line 9: Add “the” before “orco1/+ phenotype”.
17. Discussion, page 10, line 17: Change “folds” to “fold”.
18. Methods, page 12, line 7: Add “the” before “WT strain used”. Also, just to clarify, the authors refer to the second WT strain as WT-Berlin. Is this white-Berlin? That is a fairly common strain that I have seen used, but the authors seem to be using one that has wild-type red eyes. Can you make it clear in the manuscript which version of Berlin you were using, and what color eyes they have?
19. Methods, page 12, lines 10-12: Please add descriptions and references here for w1118 and wG. For example, it is important to know whether both of these mutations cause complete loss of the protein or more specific disruptions.
20. Methods, page 13, line 17: Change “define” to “defined”.
21. Figure 2: Since the incandescent and sunlight experiments used different light intensities from all the other graphs, I would recommend directly writing the light intensities into the figure itself for full clarity for the reader.
22. Table S1 legend: Change “blight” to “bright”.
23. Table S1 and legend: It was unclear to me how this experiment was run. Could the authors include more detail about the protocol that led to these data?
24. Table S1: Overall, I find it interesting that bright blue light seems to have a very different effect from bright white light. The article could use more discussion of why this might be. E.g., does this suggest that it is actually the longer wavelengths of light present in the intense white light that are causing the increase in courtship? Could the authors test that by filtering out the blue light from their white-light LEDs and see if this still causes an increase in courtship?
25. Table S2 and legend: Since the authors point out in the manuscript that the presence of a mini-w+ and the overall eye color of the fly seemed to correlate with courtship behavior, it would be helpful to mention in the figure legend which of these fly lines contained mini-w+ and also how many copies.

Review of Open Biology manuscript: RSOB-22-0233.R2
Ueda, A. et al. (2023)

Intense light unleashes male courtship behavior in wild-type Drosophila

Overall Review:

This version of the manuscript is much improved! The addition of the spectral plots, improved legibility of portions of the interactogram plots, inclusion of statistical results in a supplemental table, and expanded discussion of the effects of white gene mutations and mini-white transgenes on vision and courtship behavior were all important improvements. Although there are still a few confusing findings in the study, the authors have done a good job discussing them.

Minor Comments:

1. Even though it is only included in supplemental data, I would recommend including a spectrum for the blue light that was used to generate the data found in Table S2.
2. Results, page 8, line 19 – maybe specify here “(preliminary findings)” or “(unpublished observations)” to differentiate from the other published results that are mentioned in the same section about screening pigment effects.
3. Figure S1 – capitalize “Male-male courtship” label for consistency with “Male-female courtship” label below.