Author response:
The following is the authors’ response to the original reviews.
Joint Public review:
Weaknesses:
(1) Controls for the genetic background are incomplete, leaving open the possibility that the observed oviposition timing defects may be due to targeted knockdown of the period (per) gene but from the GAL4, Gal80, and UAS transgenes themselves. To resolve this issue the authors should determine the egg-laying rhythms of the relevant controls (GAL4/+, UAS-RNAi/+, etc); this only needs to be done for those genotypes that produced an arrhythmic egg-laying rhythm.
(2) Reliance on a single genetic tool to generate targeted disruption of clock function leaves the study vulnerable to associated false positive and false negative effects: a) The per RNAi transgene used may only cause partial knockdown of gene function, as suggested by the persistent rhythmicity observed when per RNAi was targeted to all clock neurons. This could indicate that the results in Fig 2C-H underestimate the phenotypes of targeted disruption of clock function. b) Use of a single per RNAi transgene makes it difficult to rule out that off-target effects contributed significantly to the observed phenotypes. We suggest that the authors repeat the critical experiments using a separate UAS-RNAi line (for period or for a different clock gene), or, better yet, use the dominant negative UAS-cycle transgene produced by the Hardin lab (https://doi.org/10.1038/22566).
We have followed the referee advice,repeating the experiments with the dominant negative UAS-cycDN. They nicely confirm our conclusions: the abolition of the cellular clock in LNd neurons rule out the rhythmicity of oviposition. The results are presented in Fig. 3 of the new manuscript, panels H to N. We thank the reviewer for this suggestion that has definitely improved our paper, since it allows us to confirm our result using both a different driver and a different UAS sequence. In addition, we included the required GAL4 controls, which can be found in Panels E, L of the figure as well as average egglaying profiles for all genotypes involved (Panels B, D, F, I, K and M). Regarding the MB122Bsplit-Gal4>UAS-perRNAi experiment, we moved it to a supplementary figure (Figure 3S1). The paragraph where the new Figure 3 is discussed has been modified accordingly.
(3) The egg-laying profiles obtained show clear damping/decaying trends which necessitates careful trend removal from the data to make any sense of the rhythm. Further, the detrending approach used by the authors is not tested for artifacts introduced by the 24h moving average used.
The method used for the assessment of rhythmicity is now more fully explained and tested in the supplementary material. In particular, the issue of trend removal is treated in the second section of the SM, and the absence of "artifacts" (interpreted as the possibility of deciding that a signal is rhythmic when it is not, or vice versa) shown in figs. S3 to S5.
(4) According to the authors the oviposition device cannot sample at a resolution finer than 4 hours, which will compel any experimenter to record egg laying for longer durations to have a suitably long time series which could be useful for circadian analyses.
The choice of sampling every 4 hours is not due to a limitation imposed by the device used. In fact the device can be programmed to move at whatever times are desired. As mentioned in the Material and Methods section, "more frequent sampling gives rise to less consistent rhythmic patterns", because the number of eggs sampled at each time slot become too small. In particular, we have tested sampling at intervals of 2 hours, and we have observed that this doubles the work performed by the experimenter but does not lead to an improvement in the assessment of rhythmicity.
(5) Despite reducing the interference caused by manually measuring egg-laying, the rhythm does not improve the signal quality such that enough individual rhythmic flies could be included in the analysis methods used. The authors devise a workaround by combining both strongly and weakly rhythmic (LSpower > 0.2 but less than LSpower at p < 0.05) data series into an averaged time series, which is then tested for the presence of a 16-32h "circadian" rhythm. This approach loses valuable information about the phase and period present in the individual mated females, and instead assumes that all flies have a similar period and phase in their "signal" component while the distribution of the "noise" component varies amongst them. This assumption has not yet been tested rigorously and the evidence suggests a lot more variability in the inter-fly period for the egg-laying rhythm.
As stressed in the paper, and in the new Supplementary Material, the individual egg records are very noisy, which in general precludes the extraction of any information about the underlying period and phase. The workaround we (and others, e.g. Howlader et al. 2006) have used is analyzing average egg records for each genotype. Even though this implies assuming the same period and phase for all individuals, we have observed, using experiments with synthetic data, that small variations in individual periods (of the same amount as those present in real experiments where the period of some flies can be assessed individually) still allow us to use our method to decide if the genotype is rhythmic or not. This issue is discussed at length in the new Supplementary Material. There we also discuss an experiment with real flies, showing the individual records, and the corresponding periodograms, for each fly, for a rhythmic (Fig. S14) and an arrhythmic genotype (Fig. S17).
(6) This variability could also depend on the genotype being tested, as the authors themselves observe between their Canton-S and YW wild-type controls for which their egg-laying profiles show clearly different dynamics. Interestingly, the averaged records for these genotypes are not distinguishable but are reflected in the different proportions of rhythmic flies observed. Unfortunately, the authors also do not provide further data on these averaged profiles, as they did for the wild-type controls in Figure 1, when they discuss their clock circuit manipulations using perRNAi. These profiles could have been included in Supplementary figures, where they would have helped the reader decide for themselves what might have been the reason for the loss of power in the LS periodogram for some of these experimental lines.
We have added the individual periodograms of the arrhythmic lines to the Supplementary material (Figs. 3S2, 3S5 and panel G of Fig. 3S1), where they can be compared with their respective controls (Figs 3S3, 3S4, 3S6, 3S7 and panel F of Fig. 3S1).
(7) By selecting 'the best egg layers' for inclusion in the oviposition analyses an inadvertent bias may be introduced and the results of the assays may not be representative of the whole population.
We agree that the results may be biased for 'the best egg layers'. We remark however, that the flies that have been left out lay very few eggs, some of them even laying no eggs on a whole day. For these flies it is difficult to understand how one can even speak of egg laying rhythmicity (let alone how one can experimentally assess it). Thus, we think it might be misleading to speak of results as "representative of the whole population". Furthermore, it is even possible that the very concept of egg laying rhythmicity makes little sense if flies do not lay enough eggs.
(8) An approach that measures rhythmicity for groups of individual records rather than separate individual records is vulnerable to outliers in the data, such as the inclusion of a single anomalous individual record. Additionally, the number of individual records that are included in a group may become a somewhat arbitrary determinant for the observed level of rhythmicity. Therefore, the experimental data used to map the clock neurons responsible for oviposition rhythms would be more convincing if presented alongside individual fly statistics, in the same format as used for Figure 1.
In general, we have checked that there are no "outliers", in the sense of flies that lay many more eggs than the others in the experiment. But maybe the reviewer is referring to the possibility that a few rhythmic flies make the average rhythmic. This issue is addressed in the supplementary material, at the end of section "Example of rhythmicity assessment for a synthetic experiment". In short, we found that eliminating some of the most rhythmic flies from a rhythmic population makes the average a bit less rhythmic, but still significantly so. Conversely, if these flies are transferred to an arrhythmic population, the average is still non rhythmic.
Regarding "the number of individual records that are included in a group may become a somewhat arbitrary determinant for the observed level of rhythmicity", we stress that we have not performed a selection of flies for the averages. All of the flies tested are included in the average, independently of their individual rhythmicity, provided only that they lay enough eggs.
(9) The features in the experimental periodogram data in Figures 3B and D are consistent with weakened complex rhythmicity rather than arrhythmicity. The inclusion of more individual records in the groups might have provided the added statistical power to demonstrate this. Graphs similar to those in 1G and 1I, might have better illustrated qualitative and quantitative aspects of the oviposition rhythms upon per knockdown via MB122B and Mai179; Pdf-Gal80.
We are aware that in the studies of the rhythmicity of locomotor activity the presence of two significant peaks is usually interpreted as a “complex rhythm”, i.e. as evidence of the existence of two different mechanisms producing two different rhythms in the same individual. In our case, since the periodograms we show assess the rhythmicity of the average time series of several individuals, the two non-significant peaks could also correspond to the periods of two different subpopulations of individuals. However, a close examination of the individual periodograms, now provided as Supplementary Figures 3S2 to 3S9, does not show any convincing evidence of any of these two possibilities.
Another possibility could be that such peaks are simply an artifact of the method in the analysis of time series that consist of very few cycles and also few points per cycle. In the supplemenatry material we show that this can indeed happen. Consider, for example, periodograms 2 and 4 in Fig. S12 of the SM. Even though both of them display two non significant peaks, these periodograms correspond to two synthetic time series that are completely arrhythmic.
We have added to the manuscript a paragraph discussing the issue of possible bimodality (next to last paragraph in subsection "The molecular clock in Cry+ LNd neurons is necessary for rhythmic egg-laying").
Wider context:
The study of the neural basis of oviposition rhythms in Drosophila melanogaster can serve as a model for the analogous mechanisms in other animals. In particular, research in this area can have wider implications for the management of insects with societal impact such as pests, disease vectors, and pollinators. One key aspect of D. melanogaster oviposition that is not addressed here is its strong social modulation (see Bailly et al.. Curr Biol 33:2865-2877.e4. doi:10.1016/j.cub.2023.05.074). It is plausible that most natural oviposition events do not involve isolated individuals, but rather groups of flies. As oviposition is encouraged by aggregation pheromones (e.g., Dumenil et al., J Chem Ecol 2016 https://link.springer.com/article/10.1007/s10886-016-0681-3) its propensity changes upon the pre-conditioning of the oviposition substrates, which is a complication in assays of oviposition rhythms that periodically move the flies to fresh substrate.
We agree that social modulation can be important for oviposition, as has been shown in the paper cited by the reviewer. But we think that, in order to understand the contribution of social modulation to oviposition, it is important to know, as a reference for comparisons, what the flies do when they are isolated. Our aim in this work has been to provide such a reference.
Recommendations for the authors:
(1) The weaknesses identified in the Public review could be addressed as follows: etc.
We have followed the suggestions of the editor and addressed each of the weaknesses mentioned (see details above).
(2) Could the authors comment on their choice of using individual flies for their assay rather than (small) groups of flies? Is it possible that their assay would produce less noisy results with the latter?
First we want to emphasize that our aim here was to assess the presence of individual rhythmicity, free from any external influences, whether arising from environmental external cues (such as light or temperature changes) or by social interactions (with other females or males). However, we were also curious about the behavior when males were put in the same chamber with each female. We performed a few tests and the results were very similar to what we obtained with single females.
(3) Minor points:
(a) Line 57-58 - "around 24 h and a peak near night onset (Manjunatha et al., 2008). Egglaying rhythmicity is temperature-compensated and remains invariant despite the nutritional state": Rephrase to something simpler like temperature and nutrition compensated.
Corrected.
(b) Line 56-57 - "The circadian nature of this behavior was revealed by its persistence under DD with a period around 24 h and a peak near night onset (Manjunatha et al., 2008)." A better reference here would be to Sheeba et al, 2001 for preliminary investigations into the egg-laying rhythms of individual flies and McCabe and Birley, 1998 for groups of flies under LD12:12 and DD.
Suggestion accepted.
(c) Line 65-67 - "We determined..... molecular clock in the entire clock network reduced the LNv did not." This suggests that it was unknown until now that LNv does not have a role, whereas Howlader et al 2006 already suggested that. The reader becomes aware of this at a later part of the manuscript. Please revise.
This has been revised, and the citation to Howlader et al 2006 added to the new sentence.
(d) Line 67 - "impairing the molecular clock in the entire clock network reduced the circadian rhythm of.."; saying "Reduced the power of the circadian rhythm" might be better phrasing."
Suggestion accepted.
(e) Line 72 - using the Janelia hemibrain dataset.
Corrected
(f) Line 72 typo "ussing", should be 'using'.
Corrected.
(g) Line 94: why is the periodic signal the same for all on the first day of DD?
It is well known that in LD conditions activity is driven by the environmental light-dark cycle, which entrains the endogenous circadian clock of all flies. Even after the transition to DD, the effects of this entrainment persist for a few days, allowing the individual rhythmic patterns set by the light-dark cycle to remain synchronized for at least a few cycles. We are assuming that the same happens with oviposition. A sentence has been added explaining this (beginning of third paragraph of subsection "Egg-laying is rhythmic when registered with a semiautomated egg collection device").
(h) Figure 1A-D, Were all flies included or only rhythmic flies? Please make this clear. How do you distinguish rhythmic and arrhythmic flies in Figure 1E? Their representative individual plots of egg number graphs are required. Why was the number of flies under DD decreased from 20 to 18?
Throughout the paper, the analysis of average rhythmicity has been performed including all flies, since we postulate that even flies that individually can be classified as non rhythmic have a rhythm that is corrupted by noise, and that this noise can be partially subtracted by performing an average. The explanation of the characterization of rhythmic and arrhythmic individuals is in the Methods section, under the Data Analysis subsection. This is now fully developed in the Supplementary material, where the individual plots for some of the genotypes are included.
Regarding the question of the number of flies having "decreased from 20 to 18?", there is a misunderstanding here. The results depicted in Figure 1, and in particular in panel E, correspond to two different experiments: one performed only in LD (7 days, n=20), and a second one performed for 5 days in DD, with one previous day in LD (n=18).
(i) Figure E and K, Are n=20, 18, and n=30, 22 the total numbers of flies including both rhythmic and nonrhythmic? If so, it would be better to put them in the column, not in the rhythmic column.
The figure has been corrected.
(j) Line 107-108, please provide a citation for this statement.
We have added two references: Shindey et al. 2016, and Deppisch et al. 2022.
(k) Figure 1, 2, etc., please write a peak value inside the periodogram graph. This makes comparison easier.
The peak values have been added in all Figures.
(l) Line 184-185, Figure 2F, tau appears shorter in Clk4.1>perRNAi flies than in control, which suggests that DNp1 may play a role?
As explained in the Supplementary Material, the particularities of oviposition records (discrete values, noise, few samples per period, etc.) preclude an accurate determination of the period if the record is considered as rhythmic. In particular, Fig. S4 shows that differences of 1 hour between the real and the estimated periods are not unusual.
(m) Figure 4. Why are 2 controls shown? Please explain. Are they the same strains?
The two controls shown are the UAS control and the GAL4 control. This information has now been added to the figure.
(n) Line 314 'that' should be 'than'?
Corrected.
(o) Line 73-74 - Phrasing is not clear in: "LNds and oviposition neurons, consisting with, the essential role of LNds neurons in the control of this behavior.""
Corrected.
(p) Line 81-84 - "the experiments particularly demanding and labor-intensive. In this approach, eggs are typically collected every 4 hours (sometimes also every 2 hours), which usually implies transferring the fly to a new vial or extracting the food with the eggs and replacing it with fresh food in the same vial (McCabe and Birley, 1998; Menon et al., 2014)." McCabe and Birley had an automated egg collection device designed for groups of flies, which sampled eggs laid every hour for 6 days. Please remove this reference in this context
Reference removed.
(q) Line 91-92 - "The assessment of oviposition rhythmicity is challenging because the decision of laying an egg relies on many different internal and external factors making this behavior very noisy." This sentence makes it appear that 'assessment' is the limitation. Even locomotor activity is governed by many internal and external factors, yet we can obtain very robust rhythms. The sentence that follows is also not easy to digest. Can the authors frame the idea better?
We have rewritten the corresponding paragraph in order to make it more clear (second paragraph of the Results section). Additionally, the Supplementary Material contains now a more detailed explanation and analysis of the method used.
(r) Line 104-107 - rhythmic (with a period close to 24 h, Figure 1F) although the average egg record is strongly rhythmic with a period around 24 h (Figure 1B). Under DD condition, individual rhythmicity percentages are the same as in LD (Figure 1E) and their average record is also very rhythmic with a period of 24 h (Figure 1D). 'Strongly rhythmic' and 'very rhythmic' are less indicative of what is happening with the oviposition rhythm and can be phrased as robust instead, with a focus on their power measured.
We have accepted the suggestion.
(s) Line 108-110 - "Thus, egg-laying displays a much larger variability than locomotor activity, compounding the difficulty of observing the influence of the circadian clock on this behavior." The section discussed here does not illustrate the variability in egg-laying as much as the lack of robustness of the rhythm. The variation in rhythmicity going from CS flies (~70% rhythmic) to yw flies (~50% rhythmic) showcases the variability in this rhythm and how it is difficult to observe when compared to locomotor rhythms, which are usually consistently >90% rhythmic across multiple genotypes. These lines can be placed after the discussion about yw and perS flies. Moreover, previous studies using individual flies have reported that egg-laying rhythm is more variable than others Figure 1, Sheeba et al 2001.
We have accepted the suggestion, replacing "Thus, egg-laying displays a much larger variability than locomotor activity..." by "This shows that, at the individual level, egg-laying is much less robust than locomotor activity ..."
(t) Figure 1. Genotype notation within the figure panels is not consistent with the accepted / conventional notation or with the main text or legend notations throughout the manuscript.
We are sorry for this mistake. We have corrected the genotype names in Figures and text in order to make notation consistent across the paper.
(u) Supplementary Figure 1 Legend. Error in upper right corner? Not left corner? The photo does not clearly show the apparatus. The authors may wish to consider clearer images and more details about the apparatus including details of the 3D printing of the device and perhaps even include a short video where the motor moves the flies to a new chamber (This is only a suggestion to advertise the apparatus, not related to the review of the manuscript). They could also provide information about what fraction of females survived till the end of each trial when 21 flies were examined with 4-hour sampling across 4-5 cycles.
In general, more than 80% of the females are alive at the end of a one week oviposition experiment. We have added this information in the Methods section at the end of the corresponding subsection ("Automated egg collection device"). Regarding the eggcollection device, we have replaced the photographs in what is now Supplementary Figure 1S1, and a short supplementary movie showing its operation.
(v) The results depicted in Figure 2B are that of averaged time series. Hence the reader does not know 'the fact' that knocked-down animals are not completely rhythmic. Is the "not completely arrhythmic" in reference to flies with a power > 0.2 (weakly rhythmic) in their egg-laying rhythm or to the presence of ~40% of male flies (Supplementary Table 1) with a locomotor rhythm after perRNAi silencing of most of their clock neurons? This is confusing because no intermediate category of flies is discussed in Figure 2. Please edit for clarity.
We were referring to the rhythmicity of the genotype, not of the individuals. We have rewritten the corresponding paragraph in order to make it clearer (last paragraph of the first subsection of the Results section).
(w) Line 173 - ablation or electrically silencing all PDF+ neurons (Howlader et al., 2006). There were no experiments carried out using electrical silencing of PDF+ neurons in the referenced paper.
We are sorry for this mistake. This has been corrected (we have deleted the mention to electrical silencing).
(x) Line 173 - Shortening of period by nearly 3 hours cannot be considered minor.
We agree, and we have deleted the word "minor".
(y) Line 332-333 - "We also disrupted the molecular clock (or electrically silenced) in PDFexpressing neurons as well as in the DN1p group with no apparent effect on egg-laying rhythms". There was period shortening observed for pdf GAL4 > perRNAi manipulation so there was an effect on the egg-laying rhythm. Additionally, perRNAi based silencing does not electrically silence PDF neurons as the kir 2.1 was expressed only using Clk4.1 GAL4 in the Dn1ps. This line should be rewritten.
We have rewritten the paragraph mentioned (third paragraph of the Discussion) in order to make it more accurate.
(4) Page 22 - Data Analysis
Since the number of eggs laid by a mated female tend to show a downward trend, we proceeded as follows, in order to detrend the data (see the Supplementary Material for further details). First, a moving average of the data is performed, with a 6 point window, and a new time series T is obtained. In principle, T is a good approximation to the trend of the data. Then, a new, detrended, time series D is generated by pointwise dividing the two series (i.e. D(i)=E(i)/T(i), where i indexes the points of each series)." Can the authors provide a reference for this method of detrending? Smoothing can frequently introduce artifacts in the data and give incorrect period estimates. Additionally, the trend visible in the data, especially in Figure 1, suggests a linear decay that can be easily subtracted. Also, there is no discussion of detrending in the Supplementary material attached.
We are sorry for the confusion with the Supplementary materials. The method used for subtracting both noise and trend from the data is now fully explained in the new Supplementary Material. All the issues raised by the reviewer in this comment have been addressed there.
(5) Figure by figure
Page - Type (Figure or text) - Comment
(a) Page 6 Figure 1C There is remarkable phase coherence seen in the average egg laying time series for CS flies 5 days into DD and as the authors note in Lines 94-95 in the text "Under light-dark (LD) conditions, or in the first days of DD, it can be that the periodic signal is the same for all flies". Since this observation is crucial to constructing the figures seen later in the paper, a note should be made about why this rhythm could persist across flies, so deep into DD.
As mentioned above, we have added a couple of lines explaining why we think that the assumption of a synchronized periodic signal is reasonable, at least during the first cycles (second paragraph of the first subsection of section Results).
(b) Figure 1 G The effect of period/phase decoherence seems to be showing up here in the average profile for yw flies as they seem to completely dampen out after 2 days in DD and yet have a 24-hour rhythm in the averaged periodogram. The authors should make a note here if the LS periodogram is over-representing the periodicity of the first few days in DD or if comparing the first 3 vs. the last 3 days in DD gives different results.
The dampening observed in average oviposition records is a product of the dampening of the oviposition records, which is well known phenomenon, probably caused by the depletion of sperm in the female spermatheque. One of the aims of the method used in the paper was to avoid the bias introduced by this dampening, by means of a detrending procedure. This is explained in the Materials an Methods, and now full details are given in the new Supplementary Materials.
(c) Figure 1E, K Is this data pooled across 2-3 experiments, as discussed in lines 500-01 under 'Statistical Analysis'? Also, what test is being performed to check for differences between proportions here, seeing as there are no error bars to denote error around a mean value and no other viable tests mentioned in Statistical Analysis?
We are sorry for this omission. For the comparison of proportions we used the 'N-1' Chisquared test. We have added a sentence detailing this at the end of the Statistical analysis section.
(d) Figure 1 F, L Can the total number of weakly and strongly rhythmic values be indicated in the scatter plot?
Corrected.
(e) Figure 1F, L (legend) Is the Chi-squared test being performed on the proportion values of Figure 1(E, K) or for Figure 1(F, L)?"
The chi-squared test mentioned was used for Fig1 F-L. As explained above, for the comparison of proportions we used 'N-1' Chi-squared test. This has now been added to the legend of the figure
(f) Page 8 Figure 2B Seeing as individual flies with a LS periodogram power < 0.2 are considered weakly rhythmic in Figure 1 F, L can Clk856 > perRNAi flies on average also be considered weakly rhythmic, as the peak in the periodogram is above 0.3?
We prefer to use the weakly rhythmic class only for individual flies. Nevertheless, we agree that this periodogram shows that the genotype analyzed is not completely arrhythmic, and that this might be due to some remaining individual rhythmicity. As mentioned above, we have rewritten the last paragraph of the first subsection of section Results in order to discuss this.
(g) Figure 2D Can the authors comment on why there is a shorter period rhythm when PDF neurons have a dysfunctional clock, whereas previous evidence (Howlader et al., 2004) suggested that these neurons play no role in egg-laying rhythm? They should also refer to McCabe and Birley, 1998 to see if their results (where they observed a shorter period of ~19h with groups of per0 flies), might be of interest in their interpretations.
We have added a line commenting this in the corresponding subsection ("LNv and DN1 neurons are not necessary for egg-laying rhythmicity") of the Results, as well as a discussion of this in the third paragraph of the Discussion. In a nutshell, even though Howlader et al did not find a shortening when PDF neurons are ablated, they did find it in pdf01 flies.
(h) Figure 2 F, H As the authors mention in their Discussion on Page 16, lines 340-45, the manipulation of DN1p neurons might abolish the circadian rhythm in oogenesis as reported by Zhang et al, which is why they looked at this circuit driven by Clk4.1 neurons and comment that "The persistence of the rhythm of oviposition implies that it is not based on the availability of eggs but is instead an intrinsic property of the motor program". However, no change in fecundity is reported for either kir2.1 or perRNAi-based manipulations of these neurons, to help the reader understand if egg availability (at the level of egg formation) is playing any role in the downstream (and seemingly independent) act of egg laying. The authors should report if they see any change in total fecundity for either set of flies w.r.t their respective controls. Also, is the reduction in power seen with electrical silencing vs perRNAi expression of any relevance? Does the percentage of rhythmic flies change between these two manipulations?
In the line mentioned by the reviewer what we meant is that our results show that the rhythm of oviposition does not seem to be based in the rhythmic production of oocytes, which is not necessarily connected with the total number of eggs produced. We have modified the corresponding line in the paper, in order to avoid this misunderstanding. Regarding the "reduction in power" mentioned, it must be stressed that, in general, the height of the peak is correlated with the fraction of rhythmic individuals. The problem is that this fraction is a much more noisy output, and that is the reason why we have chosen to work with periodograms of averages.
(i) Figure 2 E and G, a loss of rhythmicity could also be due to a decrease in fecundity in the experimental lines. Since the number of eggs laid for each genotype is already known, can the authors show statistically relevant comparisons between the experimental lines and their respective controls? In this vein, can the averaged time series profiles also be provided for all the genotypes tested (as seen previously in Figure 1 A, C, G, I), perhaps in the supplementary?
We did not focus on fecundity in the present work. However, our observations do not seem to show any definite relationship with rhythmicity. We plan to address the issue of fecundity more systematically in a future work. The averaged time series profiles have now been added to the figure.
(j) Scatter plots showing the average period and SEM as seen in Figure 1 (F, L) would help in understanding if these manipulations have any effect on variation in the period of the egg-laying rhythm across flies. Particularly for pdf GAL4 > perRNAi flies which have a net shorter period, (but this might vary across the 34 flies tested).
We have added a Supplementary Figure (2S1) that shows that the shortening of oviposition period can be also observed at the individual level. We have also added a line commenting this in the corresponding subsection ("LNv and DN1 neurons are not necessary for egg-laying rhythmicity") of the Results, as well as a discussion of this in the third paragraph of the Discussion.
(k) Page 11 Figure 3B Does the presence of two peaks in the LS periodogram at a power > 0.2 indicate the presence of weakly rhythmic flies with both a short(20h) and a long(~27h) period component or either one? The short-period peak is nearly at p < 0.05 level of significance. So then, do most of the flies in MB122B GAL4 > perRNAi line show a weakly rhythmic shorter period?
(l) Figure 3D A similar peak is observed again at 20h (LS power > 0.2 and nearly at p < 0.05 significance level again) and a different longer one at (~30h) though this one is almost near 0.2 on the power scale. Given the consistency of this feature in both LNd manipulations, the authors should comment on whether this is driven by variation in periods detected or the presence of complex rhythms (splitting or change in period) in the oviposition time series for these lines.
(m) Figure 3 General scatter plots showing average period {plus minus} SEM could help explain the bimodality seen in the periodograms. Additionally indicating just how many flies are weakly rhythmic vs. strongly rhythmic can also help to illustrate how important the CRY+ LnDs are to the oviposition rhythm's stability.
For these three comments (k, l and m), we note that the issue of bimodality has been addressed above, in our response to Weakness 9.
(o) Figure 4B Same as comments under Figure 1, what is the statistical test done to compare the proportions for these three genotypes?
As mentioned above, for the comparison of proportions we used the 'N-1' Chi-squared test. We have added a sentence detailing this at the end of the Statistical analysis section.
(p) Figure 4C Are all flies significantly rhythmic? The authors should also provide an averaged LS periodogram measure for each genotype, to help illustrate the difference in power between activity-rest and egg-laying rhythms.
Yes, the points represent periods of (significantly) rhythmic flies. This has been added to the caption, to avoid misunderstandings. The differences that arise when assessing rhythmicity in activity records vs. egg-laying records is addressed at length in the Supplementary Material (see e.g. Fig S1).
(q) Page 15 Figure 5 - general As the authors discuss the possible contribution of DN1ps to evening activity and control over oogenesis rhythm, investigating the connections of the few that are characterized in the connectome (or lack thereof) with the Oviposition neurons, can help illustrate the distinct role they play in the female Drosophila's reproductive rhythm.
This information was in the text and the Supplementary Tables. Lines 273-275 of the old manuscript read: "The full results are displayed in Supplementary Tables 2 and Table 3, but in short, we found that whereas there are no connections between LNv or DN1 neurons and oviposition neurons..."
(r) Minor: The dark shading of the circles depicting some of the clusters makes it difficult to read. Consider changing the colors or moving the names outside the circles.
Figure corrected.
(s) Line 38: The estimated number of clock neurons has been revised recently (https://www.biorxiv.org/content/10.1101/2023.09.11.557222v2.article-info).
Thank you for the reference. We have corrected the number of clock neurons in the Introduction of the new manuscript.
