Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) Where the manuscript still has potential for improvement is the discussion, which in its current form does seem slightly self-contained and does not fully integrate the findings of previous studies on Drosophila rhodopsin7. As the introduction specifically points out that previous findings have been contradictory, this seems like a missed opportunity.
We thank the reviewer for this helpful suggestion and agree that the previous version of the Discussion did not sufficiently integrate earlier studies on Drosophila RH7. In the revised manuscript, we have expanded and restructured the discussion to explicitly incorporate key studies, including those by Ni et al. (2017), Senthilan et al. (2019), Lazopulo et al. (2019), and Meyerhof et al. (2024). In Discussion 5.4, we directly address similarities and discrepancies, such as differences in reported expression patterns, behavioral phenotypes, and experimental conditions. We use these inconsistencies to better position our findings and propose that RH7 acts as a modulatory photoreceptor with context dependent effects, which may explain the variability observed across studies. These changes provide a more integrated and coherent view of RH7 function.
(2) Similarly, the manuscript currently lacks a discussion of the possible relevance of rhodopsin7 (and other non-visual light receptors in other organisms) in the context of a species' environment and lifestyle, i.e., what is the relevance/benefit of having rhodopsin7 in the fly's everyday life?
We agree that the ecological relevance of RH7 deserved a more detailed discussion. According to the suggestion, we have now expanded the discussion to address the ecological and functional relevance of RH7 in the context of environmental light conditions and species-specific lifestyles (see Section 5.5). In particular, we now highlight that RH7 is frequently retained in species inhabiting sun-exposed environments and that its presence correlates with conditions of high light exposure, especially during early developmental stages. Together with our functional data in Drosophila, showing a role of RH7 in light dependent behavioral flexibility and circadian regulation, this supports the idea that RH7 contributes to the detection and integration of environmental light cues under natural conditions.
(3) An additional point concerns the title and abstract, which postulate rhodopsin7 roles in contrast vision as well as motion and brightness perception. Contrast remains poorly defined in the text, leaving it ambiguous whether it refers to bright/dark contrasts, e.g., along edges, or the temporal contrast that results from dark pulses (startle response). While the latter seems to apply here, the former is likely more intuitive. Thus, this aspect should be rephrased (also in the title) or properly clarified early on.
Thank you for this comment. We agree that the term “contrast” was ambiguous, as it can refer to spatial contrast (e.g., edge detection) or temporal contrast (changes in light intensity). Our study focuses on temporal contrast, such as light-dark transitions. Accordingly, we have revised the manuscript, replacing “contrast” with the more precise term “temporal changes in light intensity” throughout the relevant sections. In addition, to clarify this further, we conducted additional experiments with graded changes in illumination. These showed that Rh70 mutants have reduced responses compared to wild type in both abrupt (e.g. 30→0 µW/cm2) and gradual (e.g. 30→5 µW/cm2) transitions, indicating that RH7 contributes to detecting temporal changes in light intensity.
(4) Regarding motion detection, this is backed up by the optomotor response results, but the findings stand somewhat isolated from the other results, lacking a clear connection aside from general visual processing.
We have clarified the connection between motion detection and the other visual assays. In the Results section (4.7), motion detection is now described as “relying on the processing of spatiotemporal changes in luminance across the visual field,” explicitly linking it to general luminance-based visual processing. In the Discussion (5.3), we added the concise statement “These computations rely on changes in light intensity over space and time” to further emphasize this connection. We believe these changes make the relationship between the optomotor results and the other assays clearer and easier to follow.
(5) Lastly, brightness perception is mentioned in the abstract, but never again, possibly due to inconsistent phrasing throughout the manuscript.
We agree that the terminology was not used consistently throughout the manuscript. To improve clarity and consistency, we replaced the term "brightness perception" in the Abstract with "temporal changes in luminance", which more accurately reflects the focus of our study.
Reviewer #2 (Public review):
(1) It is not that clear to me what rhodopsin does in terms of circadian regulation (even though its function might be circadianly regulated). The clear role in the light/dark distribution of activity might not be circadian per se, but mostly light/dark- driven and there is no evidence here for a role in the entrainment of the clock.
We thank the reviewer for this important point. We agree that our current data does not provide sufficient evidence for a direct role of RH7 in circadian regulation or clock entrainment. In this revision, we have examined LD (12:12) behavior in more detail and report changes in several activity parameters (Figure 3A). While these differences could in principle involve circadian mechanisms, our experiments do not allow us to distinguish between circadian effects and acute light-driven modulation of locomotor activity. We therefore present these results without interpreting them as evidence for a circadian function of RH7, and instead focus on light-driven behavioral effects under LD conditions.
(2) The authors should cite Lazopulo, which nicely shows that Rh7 has an important role in peripheral neurons to allow flies to escape from blue light (see below).
We thank the reviewer for pointing this out. We have now cited Lazopulo et al. (2019) at multiple relevant points in the manuscript, specifically regarding atypical RH7 expression in peripheral neurons, the functional effects of RH7 outside the LNvs, and its protective role in light-dependent behavior (discussion 5.1, 5.4, and 5.5). We believe these additions ensure that the manuscript accurately reflects the existing literature and appropriately acknowledges the contribution of Lazopulo et al. to understanding RH7 function.
(3) The finding showing that Galphaz but not Galphaq can trigger signaling from light-excited Rh7 is a very intriguing finding to better understand Rh7 function. Since Galphaz is related to Gi/o, it would be interesting to test those, for example, by expressing RNAi with Rh7gal4 and testing the Light-dark or light-off response behavior.
We agree that testing additional Gi/o-type Gα subunits would be highly informative for further dissecting RH7 signaling. However, in our preliminary experiments, RNAi-mediated knockdown of Rh7 itself did not result in a robust or statistically significant behavioral phenotype under our conditions. This is consistent with previous reports that RNAi in Drosophila can show variable efficiency and often results in incomplete knockdown, leading to weak or undetectable phenotypes (Dietzl et al., 2007; Green et al., 2014; Perkins et al., 2015). Given these limitations, we did not further pursue RNAi-based approaches targeting additional Gα subunits in the current study. Instead, we complemented our experimental findings with additional comparative and in silico analyses using FlyPredictome, which support a potential association between RH7 and Gi/o-type signaling components. These results have now been incorporated into the revised manuscript (Discussion 5.2). We agree that further functional validation of Gi/o-type G protein involvement, for example using genetic mutants, interaction assays or alternative approaches, would be an important direction for future studies.
(4) The change in the locomotor activity distribution between light and dark in LD conditions provides a nice assay for Rh7 function. Since Lazopulo et al. (2019) have shown that wildtype but not Rh7 mutants do escape from blue light, it would be important to compare and discuss these LD behavior data with the Lazopulo results. Precisely, is this nighttime preference linked to blue light?
To address this point, we conducted additional experiments, which are now included in the supplementary materials. Instead of the usual white light–dark (LD) cycles, we tested blue light–dark and green light–dark conditions. However, we observed only minor differences and no significant shift in light preference, as reported by Lazopulo et al. (2019). A likely explanation for this discrepancy is the difference in experimental light intensity. In our assays, we used moderate light intensities, approximately 30 µW/cm² for white light and 5–6 µW/cm2 for color lights. Lazopulo et al. used substantially higher light intensities (2480 µW/cm2 for white light and approximately 600 µW/cm2 for colored lights). Even the higher intensities used in our arrhythmia experiments (~120 µW/cm2) remained well below those applied in their study.
Importantly, the behavioral paradigms differ: Lazopulo et al. assessed acute light preference/avoidance, whereas our experiments quantify locomotor activity distribution across the day under LD conditions. The latter is strongly shaped by circadian regulation and integrates multiple internal and external factors. Therefore, wavelength-dependent avoidance behavior observed under acute conditions may not directly translate into changes in daily activity patterns under moderate LD conditions. Although we cannot directly replicate the strong avoidance of blue light or preference for green light described by Lazopulo et al., our results do not contradict those of Lazopulo. The reduced activity during the midday phase in the wild-type flies, which is less pronounced in Rh70, may suggest a potential protective function or preventive behavior in the WT flies. Since the moderate light intensities used in our LD assays are much less aversive, no strong wavelength-dependent preference was observed.
(5) However, the paper would be strongly reinforced by correlating this with the LD behavior. The LD phenotype should be tested in flies with Rh7 expression rescued under Rh7gal4 control (as done for the startle response). This is important to show whether the expression pattern is likely responsible for the described Rh7 function in LD.
We agree that assessing LD behavior in flies with Rh7 expression rescued under Rh7-Gal4 control is important to link expression pattern to function. We have now performed these experiments. The Rh7-Gal4–driven rescue results in a substantial recovery of the LD locomotor phenotype, bringing it closer to control levels than the UAS-empty condition. While the rescue does not fully reach wild-type levels, the observed trend and significant differences across specific parameters support a functional contribution of Rh7-expressing cells. These data are now included in Figure 5C and described in section 4.6.
(6) If L5 and or M11 drivers are available, they should be used to rescue Rh7? Since expression in some clock neurons is shown, the rescue experiment should also be done with a clock neuron driver.
Thank you for this helpful suggestion. We agree that cell type-specific rescue using L5- and/or Mi1 drivers, as well as clock neuron drivers, would be highly informative.
However, implementing these experiments is currently not straightforward. Targeting these neuronal populations requires the use of split-Gal4 driver lines, which can differ in expression strength and are therefore not quantitatively comparable across combinations. In addition, the required genotype would be highly complex: the two split-Gal4 components will be located on separate chromosomes, the UAS-Rh7 transgene is on another chromosome, and the Rh70 mutation resides on the third chromosome. Combining all necessary elements in a single line would therefore require extensive genetic recombination. Importantly, we aim to avoid introducing mutations or markers on the X chromosome, as these are known to influence behavioral readouts. However, many of the available lines are in a white- background, and assembling the full rescue genotype while maintaining a controlled genetic background across all chromosomes is therefore particularly challenging. For these reasons, we have not pursued these rescue experiments in the current study but consider them an important direction for future work.
(7) In the same line, can the LD phenotype (or startle response phenotype of Figure 4) be restored by expressing Rh7 under ppk control, as shown for the blue light avoidance phenotype by Lazopulo et al?
We thank the reviewer for this suggestion. However, in our experiments, we did not observe a blue-light avoidance phenotype comparable to that described by Lazopulo et al. (2019). Since this behavioral effect did not occur in our assay, we did not perform rescue experiments with ppk-regulated Rh7 expression. As previously discussed, the absence of pronounced blue light avoidance in our experimental setup is likely due to the moderate light intensities we used, which are significantly lower than those applied in Lazopulo et al.
Therefore, a ppk-mediated rescue would not be meaningful in our context.
(8) Finally, the Rh7 "darkfly" rescued flies should be tested in LD.
We agree that testing Rh7Darkfly rescue flies under LD conditions provides important insight into Rh7 function. We have now performed these experiments. The Rh7Darkfly rescue leads to a marked shift of the LD locomotor phenotype toward the Darkfly phenotype, with a stronger effect than observed in the Rh7 rescue lines. While the rescue does not fully recapitulate either wild-type or Darkfly controls, the observed trend supports a contribution of RH7 to the modulation of LD behavior in this genetic background. These data are now included in Figure 5C.
Reviewer #3 (Public review):
(1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.
We thank the reviewer for this helpful comment. We have now clarified in the Materials and Methods exactly which tissues were collected and analyzed for the comparative qPCR experiments. Regarding Rh7 expression in the retina, in our hands, we did not detect signals in retinal support cells, likely due to low expression and masking by autofluorescence. However, low Rh7 mRNA levels detected by qPCR in retinal tissue, together with the faint staining reported by Kistenpfennig et al. (2017), suggest that expression in these cells is possible. We have added this clarification, along with the observation by Lazopulo et al. (2019), who reported Rh7 expression in multidendritic neurons, to the Discussion (section 5.1) to provide a more complete overview of RH7 localization.
(2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.
In the revised Figure 2A, we have included both mammalian melanopsins and lophotrochozoan opsins, which support our interpretation of RH7 as a primitive opsin. The color scheme has been updated to more clearly distinguish the different clades, and tardigrades are now represented with a red triangle rather than a separate color. These changes improve clarity and better highlight the intermediate phylogenetic position of RH7.
(3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.
We would like to thank the reviewer for this valid point. We agree that, due to the relatively simple design of our optomotor apparatus, the flies may have different viewing angles of the rotating grid. To illustrate this point, we have added representative video footage and further photographs of the experimental setup to the supplementary material. A more sophisticated optomotor apparatus that maintains a fixed viewing angle for the animals would certainly reduce experimental variability. Nevertheless, our current, simple setup yielded robust and statistically significant optomotor responses. To further strengthen the robustness of these results, we have increased the sample size from n = 20 to n = 30, thereby confirming the observed effects.
Recommendations for the authors:
Reviewing Editor Comments:
(1) Claimed impact of rh7 on circadian regulation.
As the data are presented at the moment, there is no evidence for a role in circadian regulation. At present, the data can be fully explained by the acute effects of light on behavior. Either remove the circadian from the text or consider specific entrainment and rescue experiments (see suggestions from Reviewer 2).
We thank the editor for this important comment. We agree that the original version of the manuscript overinterpreted the role of RH7 in circadian regulation, and that the observed phenotypes can be explained by acute effects of light on locomotor behavior under LD conditions. In the revised manuscript, we have substantially restructured and clarified the interpretation of our findings. We now explicitly frame our results as light-driven behavioral effects under LD (12:12) conditions and have removed claims suggesting a direct role of RH7 in circadian entrainment. Furthermore, we have also added new rescue experiments, including Rh7-Gal4–driven Rh7 and Rh7Darkfly rescue, which provide further functional insight. These data show partial restoration of the locomotor phenotype and genotype-dependent shifts in activity patterns, supporting a modulatory role of RH7 in light-dependent behavior, while not directly addressing circadian clock function.
We have further revised the text to ensure that conclusions remain strictly aligned with the presented data. In addition, we now emphasize that our data provide a basis for future studies exploring the potential role of RH7 in circadian entrainment.
(2) Improved discussion section
Both Reviewers 1 and 2 point out several aspects that should be better taken care of, especially embedding the new results into the context of the existing literature on rh7- Lazopulo et al 2019, Ni et al 2017, Meyerhof et al 2024 were particularly mentioned.
We have addressed the reviewers’ suggestions by revising the Discussion section. We now refer to Meyerhof et al. (2024), Lazopulo et al. (2019), and Ni et al. (2017) at multiple points throughout the Discussion to better place our findings in the context of the existing literature on RH7 function. These references are now included in Sections 5.1, 5.4, and 5.5 of the Discussion.
(3) The addition of vertebrate melanopsins, as pointed out by Reviewer 3 (they suggest mammalian, but consider adding other vertebrate and also lophotrochzoan proteins), in the phylogenetic tree will help to put the evolutionary ancestrality claim into a better perspective.
We performed new bioinformatic analyses using RefSeq proteomes and carefully selected representative species across relevant clades to construct a balanced phylogenetic tree. These additions support our interpretation of RH7 as a primitive opsin and help place its intermediate phylogenetic position into better perspective. The results are presented in the revised Figure 2A, which now also includes mammalian melanopsins and lophotrochozoan opsins.
(4) There were several questions raised on the qPCR and optomotor response experiments (Reviewer 3), as well as the definition of contrast (Reviewer 1).
We have addressed the reviewers’ comments by substantially improving the clarity and presentation of the qPCR and optomotor response experiments, as well as the definition of “contrast.” We now provide a more detailed description of the qPCR analysis in the Methods section. For the optomotor response, we have added representative images and videos of the experimental setup to the Supplementary Material to increase transparency. We further clarified that the term “contrast” was previously used ambiguously and may refer to different concepts. In the revised manuscript, we now consistently refer to “temporal changes in light intensity” to specify that our study focuses on light-dark transitions. In addition, we included additional experiments with graded changes in illumination, which show reduced responses in Rh7 mutants compared to wild type under both abrupt and gradual transitions.
(5) Supplementary Material needs sorting
Accession numbers are in Supplementary File 2, not Supplementary File 1. Supplementary Material S2B is in Supplementary File 3 - this is really confusing. Supplementary Materials S3 is probably Supplementary File 1, and the phylogenetic tree shown there actually looks identical to the tree shown in Figure 2A of the main manuscript.
We sincerely apologize for the confusion regarding the Supplementary Material numbering and file assignments. This issue likely arose during the upload process or transfer from the bioRxiv version of the manuscript. We have now carefully checked and corrected the Supplementary Files to ensure that all accession numbers, figures, and files are correctly assigned and consistently labeled throughout the revised submission.
Reviewer #1 (Recommendations for the authors):
(1) Here, I will address specific aspects of the manuscript, elaborating on the points mentioned in the public review and also listing detailed suggestions.
As mentioned in the public review, the discussion could do a better job of relating the obtained findings to previous publications. For example, the paper by Ni et al. (2017) investigates the role of Drosophila rhodopsin7 in circadian light entrainment and its interplay with dcry, which seems relevant in the context of rhodopsin7 apparently being only present in CRY-negative clock neurons (except DN1a).
We thank the reviewer for this helpful suggestion. We have expanded the manuscript to further explore the potential relationship between RH7 and DCRY. Specifically, we performed additional bioinformatic analyses, which are now presented in Results Section 4.1, and in discussion sections 5.4 and 5.5 addressing this aspect. While previous observations by Ni et al. (2017) and Kistenpfennig et al. (2017) have already pointed to a possible relationship between RH7 and DCRY, our phylogenetic analyses provide additional support for such a connection from an evolutionary perspective. Although our data do not yet allow us to infer the precise functional nature of this relationship, they further strengthen the evidence that Rh7 and DCRY are evolutionarily and functionally linked.
(2) Another relevant paper by Meyerhof et al 2024 (doi:10.1073/pnas.2400964121) investigates the role of rhodopsin7 in shade preference and startle responses to short light pulses. The lack of shade preference and the altered startle response in rh7 mutants could be related to the authors' own findings on altered behavior in darkness. Further, the paper shows rh7 mutants to more rapidly return to baseline activity after a light pulse, which is somewhat reminiscent of the abrupt activity decrease in rh7 mutants at the start of the dark phase (18h). Generally, I recommend using the contradictory findings mentioned in the introduction as a starting point to describe where the new findings expand previous knowledge and possibly resolve contradictions.
We thank the reviewer for this suggestion. We have revised the Discussion to integrate our findings with the available literature, including the recent study by Meyerhof et al. (2024), and explicitly compare similarities and differences between the reported phenotypes and our own observations.
These aspects are now addressed in the newly added Section 5.5, where we discuss the evolutionary retention of RH7 in sun-exposed species and relate our phylogenetic findings to the emerging evidence that RH7 may play a conserved role in mediating responses to sun-exposed and shaded environments.
(3) One specific aspect that does not fully convince me is the interpretation of the behavioral patterns concerning the short photoperiods (Figure 3C). The authors state that rh7 mutants lose the flexibility to shift their activity into the dark phase. However, drawing a vertical line through panels A, B, and C, it rather seems that activity peak times in wildtypes are fixed to 6-7h and 17-18h, while rh7 mutants do actually shift their peak activities with the decreasing photoperiod. A way of quantifying this would be to determine the phase differences between M- and E-peaks. It may also help to align plots in a way so that lightson of long and short photoperiod plots align. There is clearly a difference between WTs and mutants, but I would be more careful in calling this a reduction in flexibility.
We thank the reviewer for this comment. We also noticed that the behavioral patterns under short photoperiods can be interpreted in different ways. Based on this comment, as well as the comments from the other reviewers and the handling editor, we decided not to further discuss the potential circadian component in this manuscript. We agree that additional and more targeted experiments will be necessary to better separate circadian effects from light-induced behavioral responses. These experiments are beyond the scope of the present study but will be important to further investigate the potential role of RH7 in circadian regulation. Therefore, we revised the manuscript to focus on the light-induced behavioral effects of RH7 and avoided drawing conclusions about circadian flexibility that are not yet fully supported by our data. In this process, we removed the data showing actvity under short days from the manuscript.
Below, I list individual points according to their line number in the manuscript:
(1) Line 1: Consider rephrasing the title based on the above comment regarding contrast.
We have revised the title accordingly.
The new title is: RHODOPSIN 7: An ancestral non-canonical photoreceptor shaping light-responsive behavior
(2) Line 10: Vision is also used to adapt to the environment, so maybe change to "...also to otherwise/indirectly adapt behavior...".
We have revised the sentence as suggested to clarify the non-image-forming role of light in behavioral adaptation.
“Animals rely on light not only for vision but also to adapt behavior to their environment through non-image-forming pathways.”
(3) Line 15: Consider rephrasing the title based on the above comment regarding contrast, motion, and brightness perception.
We thank the reviewer for this suggestion. The title has been revised accordingly. The new title is: RHODOPSIN 7: An ancestral non-canonical photoreceptor shaping light-responsive behavior.
(4) Lines 23-25: This reads as if light perception and vision are the same. Please clearly distinguish to make clear that vision is only one way in which light can be perceived and influence behavior.
We have revised the text to clarify that vision represents only one modality of light perception and that light can influence behavior via both visual and non-visual pathways. “The ability to detect and respond to light is fundamental for many organisms and can influence behavior through both visual and non-visual pathways. Vision relies on….”
(5) Lines 26-27: Here and also later in the introduction, it is hard to follow how different groups of opsin light receptors relate to each other (e.g., opsins (in general,) rhodopsins, r-opsins, c-opsins, non-visual opsins), etc.) This is also related to the fact that opsins can be differentially grouped based on phylogeny/structure (e.g., r- vs. c-opsins) or role/localization (e.g., visual vs. non-visual). Please revisit the introduction with a non-expert reader in mind, and try to address the groups hierarchically. In fact, a small tree-like figure of the different groups may be a simple solution.
We thank the reviewer for this helpful suggestion. We have revised the Introduction to simplify the presentation of opsin classification. c-Opsins and r-opsins are now introduced only briefly to place RH7 within the rhabdomeric opsin family. Since Drosophila contains only rhabdomeric opsins and these are the focus of our study, we did not further expand the Introduction with a detailed discussion of c-opsins or add a classification figure.
(6) Line 33: Please expand to "...image formation in the eye, some..." to be more precise. We have revised the sentence to clarify that rhodopsin-mediated image formation refers to visual systems.
“While the primary function of rhodopsins is image formation in visual systems, some are also known to participate in non-image-forming functions.”
(7) Lines 46-55: This detailed information about the different Drosophila rhodopsins is not referred to anywhere else in the manuscript. I suggest shortening and focusing on rh7.
We thank the reviewer for the suggestion. We have shortened the section to focus on RH7, as the other rhodopsins are not discussed elsewhere in the manuscript. The revised text now highlights the debated expression and potential function of RH7.
“Drosophila melanogaster (Dmel) has seven rhodopsins (RH1-RH7), all belonging to the ropsin group. While RH1–RH6 are expressed in well-characterized photoreceptors, the expression pattern and function of RH7 remain less clear and are still debated.”
(8) Line 55: The statement that the expression of Rh7 remains debated also occurs later in the introduction (line 88). If in line 55, it is about the expression sites specifically, references should be added. Otherwise, it may be sufficient to only address this aspect in line 88.
The statement in line 55 specifically refers to the expression sites of RH7. References to Ni et al., 2017 and Senthilan et al., 2019 have been added to support this point.
(9) Line 61: Which of these features distinguishes rhodopsins from other opsin groups?
We thank the reviewer for this comment. The RH7 section has been thoroughly revised to clarify both the features shared with other rhodopsins and those that distinguish RH7 from other opsin groups. Key distinguishing features, including the extended N- and C-terminal tails and the absence of the QAKKMNV motif in ICL3, are now highlighted at the beginning of the paragraph, while additional Drosophila-specific features, such as the palindromic RCSI, are discussed subsequently.
“RH7 displays the canonical rhodopsin features, including seven transmembrane helices, a chromophore-binding lysine, and the LRTPXN motif located in the first intracellular loop (ICL1)….”
(10) Figure 1: For panel B (left), please increase label sizes. For panel B (right), the color scale makes it very hard to see low-level expression (e.g., in Mi15 and below). Please try adjusting the color scale and/or consider only showing the cell types also indicated in the left part of the panel. For ninaE, please add "aka Rh1" or similar for the less informed reader.
We have revised Figure 1 accordingly. Specifically, we increased the label sizes in panel B (left) to improve readability. For panel B (right), we adjusted the color scale to better visualize low-level expression and improve overall clarity. In addition, we updated the labeling of ninaE to “Rh1 (ninaE)” to aid accessibility for a broader audience.
(11) Lines 88-90: What exactly were the contradictions that emerged from previous studies? Also see the comment in the public review regarding the discussion of the results.
Thank you for pointing this out. We have clarified this in the revised manuscript. “Previous studies on RH7 have yielded conflicting results regarding its function and expression, including discrepancies in the identified neuronal populations and associated behavioral phenotypes”
(12) Line 91: Briefly explain the characteristics of white and yellow mutations so the reader can assess why these are relevant here.
We have clarified the role of the white and yellow genes in the genetic background. The revised text now reads:
“Both genes are expressed in the optic system (Davis et al., 2020; Supplementary Material) and are known to affect neuronal function and behavior (Bond et al., 2024; Drapeau et al., 2003; Rickle et al., 2025).”
In addition, we have visualized the expression data for yellow and white from Davis et al. in the Supplementary Material for more clarity.
(13) Lines 100-101: Circadian entrainment is not a typical visual function. Please rephrase.
The sentence has been revised for clarity and now reads:
“As an ancestral rhodopsin, RH7 appears to contribute to basic visual functions, such as contrast processing, as well as to non-visual functions like circadian synchronization, similar to melanopsins in mammals.”
(14) Line 105: The exact address is not necessary, mention BLASTP is enough.
Thank you. We have now removed the exact address and we now simply refer to BLASTP.
(15) Line 108-109: If default settings were used, the details are not needed.
We have omitted the details, as they are not necessary.
(16) Line 113: These settings seem to repeat line 108. Needed?
We thank the reviewer for pointing this out. In the original version, the settings in line 108 referred to the BLAST search, while those in line 113 pertained to the phylogenetic tree, where the parameters are essential, because phylogenetic inference depends on dataset- and method-specific choices. However, this entire section has now been revised, as the phylogenetic tree has been completely rebuilt with a much broader dataset, including mammalian melanopsins and lophotrochoan r-opsins.
(17) Line 122: Without further context, these abbreviations are non-informative and confusing to many readers. Provide details or rephrase. The same applies to DMEM, FBS, and Glo22F in the same paragraph. While many readers will understand instantly, keep the diverse audience of eLife in mind.
We have now added explanations for all abbreviations in the paragraph to improve clarity for a broad audience. Specifically, the Gα subunits (GNAS, GNAL, GNAQ, GNA11, GNA12, GNA13, GNAZ) are now clearly defined, and DMEM, FBS, and Glo22F are also explained as Dulbecco’s Modified Eagle Medium, fetal bovine serum, and a cAMP-sensitive luciferase reporter, respectively. These changes should make the methods clearer and more accessible to all readers.
(18) Line 144: "constant conditions" is not ideal as it could be misinterpreted as DD or LL. Please rephrase.
We agree with the reviewer and have removed the phrase “constant conditions” to avoid potential misinterpretation.
“All recordings were performed under 60% relative humidity and 25 °C…”
(19) Line 148: Stating Lux values for Drosophila is not informative as the unit is based on the spectral sensitivity of the human eye. Ideally, provide a spectrum of the used light source as a supplement.
We thank the reviewer for this suggestion. We agree that lux values alone are not informative for Drosophila, and we have now included the spectrum of the light source in Figure 3–figure supplement 1A.
“A standard day was defined as 12 h light (30 µW/cm2 ~ 100 lux, the spectrum of the light is shown in Figure 3–figure supplement 1A) and 12 h darkness”. In addition, to ensure complete consistency of the experimental conditions, we repeated some earlier experiments using the same light source as in the rest of the study, since the exact light source used in the initial experiments could no longer be verified. This ensures that all behavioral experiments presented in the revised manuscript were performed using the same light source.
(20) Lines 149-153: The startle response protocol is hard to understand when only reading it. It could help to state when (relative to lights-on) the first dark pulse was given and how many dark pulses were given per light phase.
We have clarified the timing and frequency of the dark pulses in the Methods section. We now specify that the first dark pulse was applied 1.5 h after lights-on and that six dark pulses were delivered during each 12 h light phase.
(21) Lines 159ff: The 4-hour startle response assay is not clearly distinguished from the previously mentioned assay (30 min D + 1.5h L). I could help to start with e.g., "In a second assay..." and to clearly state why this additional assay was performed. That is, what information can the 4-hour assay provide that the previous assay could not?
We have revised the text to clearly introduce the 4-hour startle response assay as a second, distinct paradigm (“In a second startle response assay…”).
We also clarified its purpose: in contrast to the initial assay, this paradigm was designed to measure the acute startle response independently of circadian influences. To this end, flies were rendered arrhythmic and exposed to repeated 4-hour light–dark cycles, preventing re-entrainment and isolating the startle response.
In addition, we now refer to a schematic of the light regime in Figure 3–figure supplement 1C.
(22) Lines 161-163: As flies were kept in LL aside from the dark pulses, it could be more intuitive to talk about 72h or three 24h intervals rather than 3 days. Otherwise, one might assume that the initial constant light was followed by 3 days of LD plus dark pulses.
We have revised the text to emphasize the total duration (“72 h”) to avoid confusion with a 24-hour LD regime. In addition, we now provide a schematic of the light regime in the Supplementary Material (Figure 3–figure supplement 1C).
(23) Line 172: Please write out CantonS once, as otherwise it only occurs in Table 1. Also, clarify that y+ and w+ refer to the (absence of the) yellow and white mutation, respectively.
We have revised the text to write out Canton-S (CS) upon first mention and clarified that y+ and w+ denote wild-type alleles of the yellow and white genes, respectively, meaning any mutations in these genes are absent.
(24) Table 1: Consider adding "(truncated RHSOPSIN7)" or similar to the "Darkfly" cell.
We have updated Table 1 to indicate that the Darkfly line carries a truncated RH7. The description now reads: “This line has been maintained for decades in constant darkness and carries numerous mutations, including a truncated RH7 lacking the last 21 amino acids.”
(25) Table 2: In the formatted paper, Table 2 should appear after section 3.5 so the reader can properly interpret the applications and names.
We have placed Table 2 immediately after section 3.5 as suggested.
(26) Table 3: Consider adding the product IDs of the used antibodies for convenience.
We have added the product IDs of all antibodies used to Table 3 for convenience.
(27) Lines 180ff: This section should initially state the purpose of establishing the rh7-Gal4 line, i.e., the expression of selected gene in rh7-expressing cells. Again, keep your (lee-informed) readers in mind.
We have revised the section to clarify the purpose and design of the Rh7-Gal4 line. It now states that the line was generated to identify Rh7-expressing cells and explains the use of a relatively large promoter region including exon 1 and 2.
(28) Line 190 w1118 flies do not occur in Table 1. While used by a commercial company here, please still include them for completeness.
We have added the w1118 line to Table 1 for completeness, as well as the BDSC Stock #8621 line, which was used as the background for the UAS-construct flies.
(29) Lines 192ff and 203ff: As for lines 180ff, start with a brief description of what the purpose of the respective works was.
We have revised the section to start with a brief description of the purpose of the rescue experiments. The text now clearly states that UAS constructs were generated to assess the ability of different Rh7 variants to rescue the phenotype, with UAS-Rh7 as the wild-type rescue, UAS-Rh7Darkfly carrying the Darkfly variant, and UAS-empty serving as a control. We have added an introductory sentence to the optomotor section in Materials & Methods to clarify the purpose of the experiments: “To investigate the contribution of Rh7 in the lamina and medulla to visual motion detection, male flies were individually tested for their optomotor walking behavior.” This makes clear why the assay was performed before describing the setup and measurements.
(30) Line 225: Just to be safe, make sure to not mix up Δ-CT and 2-ΔΔCT (see Livak & Schmittgen 2001, doi: 10.1006/meth.2001.1262).
We confirm that ΔCT was indeed used to calculate relative mRNA levels. To clarify this, we have now added the formula to the text: ΔCT = CT_α-tubulin − CT_Rh7.
(31) Line 229: Please state the light (cycle) conditions under which the flies were raised, as well as the sampling times at which they were fixed.
We have now specified the light cycle conditions under which the flies were raised (12 h light:12 h dark at 25 °C) in the Methods section. For qPCR experiments, tissues (retina, lamina, and brain) were carefully dissected from adult flies without prior fixation to preserve RNA integrity. Dissections were performed during the light phase and were not restricted to specific time points. These details have been added to the revised manuscript (section 3.10).
(33) Lines 296-297: This brief description could go at the start of section 3.2.
The sentence describing the GsX assay (“To test the functionality of RH7, we used a GsX assay designed to determine which Gα proteins can bind to a given GPCR, in this case RH7 (Ballister et al., 2018)”) has been moved to the Methods section (now 3.3) as a brief introduction to the approach.
(34) Line 305: Clarify that 2 different retinals were tested.
We clarified that two different retinals were tested. The text now reads: “We observed robust activation with the Gαz chimera, with both 9-cis-retinal and all-trans-retinal (Figure 2C).”
(35) Figure 2A, B: I suggest using a different color for the RH3,4,5, branch for better distinction from RH7.
We have generated a new tree and adjusted the branch colors to improve clarity between RH3,4,5, branch and RH7.
(36) See comment above regarding the shifting/flexibility of WTs vs. rh7 mutants.
Following the reviewers and editor's recommendations, we revised the manuscript to focus on the light-dependent effects of RH7 and no longer interpret our data in terms of shifting ability or circadian flexibility. The corresponding statements have been removed or revised accordingly.
(37) Line 348: Please change "occur" to "be triggered".
The wording has been updated as suggested, replacing “occur” with “be triggered” in the text.
(38) Line 355: I would mention the double mutants only after the results from the rh7 single mutants were described.
We have adjusted the order of the text accordingly and now introduce the double mutants only after describing the Rh7 single mutants.
(39) Line 362: See comment on line 159ff.
We have added a sentence at the beginning of the section to clearly introduce the 4-hour startle response assay as a second, distinct paradigm: “In a second startle response assay (4-hour assay), designed to measure the acute response independently of circadian influences…” This clarifies its purpose and distinguishes it from the initial assay.
(40) Line 368: Write "rh7 mRNA" to be precise.
We have updated the text to use “Rh7 mRNA” for precision, as suggested.
(41) Figure 5: If possible, please sort the panels in the order in which they are mentioned in the text. For panel E (right), the orientation of the lower schematic, relative to the upper one and the left panel, is unclear. Is it possible to add orientation indicators? Alternatively, could L5 and Mi1 neurons be indicated in the upper schematic instead?
We thank the reviewer’s helpful suggestion. Since reorganizing the figure panels was challenging and would have affected the overall layout, we instead revised the corresponding text to ensure that the order of panel descriptions matches the figure arrangement. To address the concern regarding panel E, we have added orientation indicators to the schematic to clarify the relative positioning.
(42) 419 Add "A)".
We have added an A).
(43) Line 432: Later on (line 598), it is mentioned that the other accumulated mutations in Darkfly may also have affected the observed patterns. Are there any specific mutations worth highlighting in this context aside from rh7?
We thank the reviewer for this comment. In response, we have expanded the Discussion to highlight additional mutations in the Darkfly genome that may contribute to the observed phenotype. Nonsense mutations in HisCl1 and Vha100-1 could affect visual processing and neuronal signaling, potentially altering sensory input and synaptic function. Missense mutations in DopR, bgm, and developmental regulators such as Sox21b may further modulate activity, sleep, and neural development. We have updated the Discussion accordingly to make these points clear.
(44) Lines 435-436: Unchanged mRNA levels do not necessarily equal the presence of the translated protein. Is there another way to properly confirm this? E.g., rh7 HCR in Darkfly?
We agree that unchanged mRNA levels alone do not confirm the presence of translated protein. While in situ HCR does not directly measure protein levels, it provides additional information on the cellular localization and presence of Rh7 transcripts beyond qPCR measurements. We have therefore performed Rh7 in situ in Darkfly and included this data in Figure 5—figure supplement 1B.
(45) Line 439: "activity" is too vague based on only the altered dark pulse response. I would rather suggest factors like increased protein concentrations/stability, increased sensitivity, constitutive activation, etc.
By “activity,” we refer to the molecular activity of RHODOPSIN 7 as reflected in the behavioral response. The Results have been revised to:
“When we exposed the Darkfly to dark pulses in a 12:12 light-dark cycle, they exhibited the opposite phenotype to Rh70 mutants, showing a stronger startle response (Figure 6A), possibly reflecting RHODOPSIN 7 adopting a constitutively active form in the Darkfly strain, driving the enhanced behavioral response.”
(46) Lines 442-446: The no-rescue control does not fully resemble WTs as they lack the gradual activity decrease after lights-off. The abrupt decrease (also in the 2 rescues) is more reminiscent of the rh7 mutants. This could be discussed in the context of what was mentioned at the start of this section (Meyerhof et al 2024 paper).
We agree that the rescue lines do not fully restore all aspects of the WT control locomotor pattern. One possible explanation is that the Rh7-Gal4 driver does not fully represent the endogenous Rh7 expression pattern. As described in the Results section, our in situ hybridization analysis identified additional Rh7-expressing cells, particularly in the optic system, that were not detected by the Rh7-Gal4 line (results, section 4.5). We have added this point to the Discussion to address how the broader Rh7 expression pattern may contribute to the observed behavioral effects (discussion 5.4).
(47) Line 472: "restored the reduced startle response" reads a bit convoluted. Better write e.g., "restored the enhanced startle response that was absent in the no-rescue mutants".
We have revised the text as suggested to clarify the phrasing.
(48) Lines 533-535: Please provide a reference for the statements about L5 and Mi1 functional roles. Also, this is the only part where there is some connection made between rh7 (expressed in Mi1) and contrast processing (which involves Mi1). As this is a rather weak connection, please consider the last paragraph of the public review section.
We have added references to support the functional roles of L5 and Mi1 neurons. L5 neurons relay signals from outer photoreceptors to the medulla and show strong contrast adaptation (Matulis et al., 2020). Mi1 neurons contribute to motion and contrast processing (Behnia et al., 2014; Matulis et al., 2020). To address the connection between Rh7 expression and contrast processing, we have carefully phrased the text to indicate a potential role of Rh7 in modulating contrast-related signals in Mi1, while noting that the functional link remains to be directly tested. We believe this wording, together with the added references, provides proper support for the statements and maintains a cautious interpretation.
If contrast perception shall indeed be investigated, this may be done by performing the optomotor assay with gray stripes of different contrast, with stripes where the edges are not sharp but show a gradual change from black to white.
We agree that contrast perception can be assessed in more detail using stimuli with defined contrast levels and gradual luminance transitions. However, our aim was not to directly measure contrast sensitivity, but rather to investigate behavioral responses to changes in luminance. We have clarified this distinction in the revised manuscript and now specify that the optomotor response reflects processing of spatial and temporal changes in luminance, while the light-off startle response measures responses to abrupt changes in luminance.”
(49) Lines 546-547: Are there references on how activity or behavior in general can affect external light cues?
We have added appropriate references to support this statement. In particular, we now cite Rieger et al. (2003), which demonstrates direct effects of light on locomotor activity (masking), and Helfrich-Förster (2017), which provides a comprehensive overview of circadian regulation and light-dependent behavioral modulation. These additions clarify how external light cues can directly influence behavior in addition to circadian clock control.
(50) Lines 557-560: See comment above regarding the shifting/flexibility of WTs vs. rh7 mutants.
We thank the reviewer for this comment. We agree that assessing shifting and behavioral flexibility would require dedicated experiments, such as jet lag or entrainment assays. We have therefore revised the focus of the manuscript and now primarily discuss RH7 function in light-dependent behavioral responses rather than broader flexibility of the circadian system. Future studies will further explore the potential contribution of RH7 to behavioral adaptation and flexibility using dedicated experimental approaches.
(47) Lines 564-567: How many species possess dcry but not rh7? This is an important distinction, as otherwise one could assume that both must always co-occur. Alone, this provides only limited support for a potential functional interplay. The findings by Ni et al. (2017) regarding dcry and rh7 in entrainment could, however, help strengthen this point.
We have clarified the evolutionary relationship between DCRY and RH7 in the revised manuscript. We now specify that, although DCRY is widespread across Bilateral and many non-panarthropod species possess DCRY without RH7, RH7 and DCRY show a strong evolutionary association within arthropods, with approximately 98% of DCRY-retaining arthropod species also retaining RH7. At the same time, the presence of RH7 in some lineages lacking DCRY suggests that RH7 may retain functional relevance in the absence of DCRY. Together with previous findings on the functional interaction between RH7 and DCRY in light adaptation (Ni et al., 2017), these evolutionary patterns support a conserved connection between the two light-sensing systems, while the exact nature of this relationship remains to be further investigated. These points are now shown and discussed in section 4.1 and 5.5.
(48) Lines 579-580: Here, one could explicitly mention that this is supported by the lack of diel startle response modulation in the per mutants (Figure 4A).
We have modified the text to explicitly highlight this point. The sentence now reads: “Dark-pulse experiments show that both wild-type controls and per0 mutants, which lack a functional circadian clock, exhibit a strong startle response, consistent with the absence of diel modulation of the response in per0 mutants (Figure 4A).”
(49) Lines 596-597: The increased night activity in Darkfly seems largely limited to the first half of the night (compare e.g. Figure 6A and Figure 3A). Again, the loss/alteration of rh7 seems to have its major effect after a transition from light to dark (see lines 442-446, start of the section).
We thank the reviewer for pointing out this issue. The wording in the previous version could have been misleading. By “dark phase,” we meant the 30-minute periods without lighting during the dark-stimulus experiments, and not the “night phase.” We have revised the text to clearly refer to the startle response.
We also clarify that the increased locomotor activity observed in the “Darkfly” strain outside of the startle response is likely caused by additional background mutations that accumulated during long-term rearing in constant darkness, and not exclusively by the RH7 deletion.
(50) Line 622: See comments regarding contrast and motion perception. Do the N- and Cterminal regions of RH7 and melanopsin share similarities in sequence or additional characteristics aside from phosphorylation sites? This could support a conserved role.
We thank the reviewer for this suggestion, which prompted us to perform a more detailed analysis of conserved features between RH7 and melanopsins. In addition to the extended and potentially highly phosphorylatable N- and C-terminal regions, we identified several conserved sequence motifs, including two in the C-terminal region and others within the transmembrane core. One of these motifs extends into the conserved DRY motif in the second intracellular loop (ICL2). These findings are now described in the Discussion (section 5.7) to highlight potential structural similarities beyond phosphorylation sites. The corresponding sequence alignments are provided in the supplementary material.
