Circadian oscillation of perireceptor events influence olfactory sensitivity in diurnal and nocturnal mosquitoes

  1. Tanwee Das De
  2. Julien Pelletier
  3. Satyajeet Gupta
  4. Madhavinadha Prasad Kona
  5. Om P. Singh
  6. Rajnikant Dixit
  7. Rickard Ignell
  8. Krishanpal Karmodiya

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Abstract

Olfaction and circadian rhythm gate different behaviors in mosquitoes that are important for their capacity to transmit disease. However, the mechanisms of odor detection, and the circadian-guided changes in olfactory sensitivity across different mosquito species, remain largely unexplored. To this end, we performed a circadian-dependent RNA-sequencing study of the peripheral olfactory- and brain tissues of female Anopheles culicifacies and Aedes aegypti mosquitoes. Data analysis revealed a significant upregulation of genes encoding: (a) odorant binding proteins (OBPs), required for transportation of odorant molecules towards the olfactory receptors, and (b) xenobiotic-metabolizing enzymes (XMEs) during the day time in Aedes aegypti and during the dusk-transition phase in Anopheles culicifacies . While XMEs primarily function in the elimination of toxic xenobiotics, concurrent elevation of XMEs and OBPs are hypothesized to act cumulatively to regulate perireceptor events and odorant sensitivity. Electroantennographic analysis with both Anopheles gambiae and Aedes aegypti against diverse behaviorally relevant odorants, combined with XMEs inhibitors and RNA interference, establish the proof-of-concept that XMEs function in perireceptor events during odorant detection and influence the odorant sensitivity in mosquitoes. Additionally, the RNA-sequencing and RNAi-mediated knockdown data revealed that daily temporal modulation of neuronal serine proteases may facilitate the consolidation of the brain function, and influence the odor detection process in both diurnal and nocturnal mosquitoes. These findings provide the impetus to further explore the species-specific rhythmic expression pattern of the neuro-olfactory encoded molecular factors, which could pave the way to develop and implement successful mosquito control methods.

Graphical Abstract

Highlights

  • Circadian oscillation of perireceptor proteins possibly influences time-of-day dependent change olfactory sensitivity in diurnal and nocturnal mosquitoes

  • Diurnal and nocturnal mosquitoes depict distinct dynamic change in perireceptor proteins

  • Inhibition of cytochrome P450 gene minimizes antennal response to different odorants

  • Neuronal serine protease may consolidate brain function and odor detection

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In the present manuscript, the authors analyzed diel oscillations in the brain and olfactory organs' transcriptome of Aedes aegypti and Anopheles culicifacies. The analysis of their RNAseq results showed an effect of time of day on the expression of detoxification genes involved in oxidoreductase and monooxygenase activity. Next, they investigated the effect of time of day on the olfactory sensitivity of Ae. aegypti and An. gambiae and identified the role of CYP450 in odor detection in these species using RNAi. In the last part of the study, they used RNAi to knock down the expression of one of the serine protease genes and observed a reduction in olfactory sensitivity. Overall, the experiments are well-designed and mostly robust (see comment regarding the sample size and data analysis of the EAG experiments) but do not always support the claims of the authors. For example, since no experiments were conducted under constant conditions, the circadian (i.e., driven by the internal clocks) effects are not being quantified here. In addition, knocking down the expression of a gene showing daily variations in its expression and observing an effect on olfactory sensitivity is not sufficient to show its role in the daily olfactory rhythms. Knowledge gaps are not well supported by the literature, and overstatements are made throughout the manuscript. Our detailed comments are listed below.

    We sincerely thank the reviewer for their time and consideration, and appreciate the thorough review of our manuscript. Their insightful comments have greatly enriched our work. We also apologies for instances of overinterpreting the data. Your feedback has helped us recognize areas where clarity and caution are needed, and we are committed to addressing these concerns in our revisions. Thank you for your valuable input and guidance.

    Major comments

    Introduction

    1. Several statements made in the introduction are misleading and suggest that authors are trying to exaggerate the impact of their work. For example, "Furthermore, different species of mosquitoes exhibit plasticity and distinct rhythms in their daily activity pattern, including locomotion, feeding, mating, blood-feeding, and oviposition, facilitating their adaptation into separate time-niches (7, 8), but the underlying molecular mechanism for the heterogenous temporal activity remains to be explored." is not accurate since daily rhythms in mosquitoes' transcriptomes, behavior, and olfactory sensitivity have been the object of several publications. Even though some of them are listed later in the introduction, they contradict the claim made about the knowledge gap. See:

    Rund, S. S., Gentile, J. E., & Duffield, G. E. (2013). Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC genomics, 14(1), 1-19

    Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., & Duffield, G. E. (2011). Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences, 108(32), E421-E430

    Rund, S. S., Bonar, N. A., Champion, M. M., Ghazi, J. P., Houk, C. M., Leming, M. T., ... & Duffield, G. E. (2013). Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Scientific reports, 3(1), 2494

    Rund, S. S., Lee, S. J., Bush, B. R., & Duffield, G. E. (2012). Strain-and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. Journal of insect physiology, 58(12), 1609-1619

    Leming, M. T., Rund, S. S., Behura, S. K., Duffield, G. E., & O'Tousa, J. E. (2014). A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti. BMC genomics, 15(1), 1-9

    Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

    Rivas, G. B., Teles-de-Freitas, R., Pavan, M. G., Lima, J. B., Peixoto, A. A., & Bruno, R. V. (2018). Effects of light and temperature on daily activity and clock gene expression in two mosquito disease vectors. Journal of Biological Rhythms, 33(3), 272-288

    Response: We apologies for this oversight. In the revised manuscript, we have added these references and made changes to the text as suggested by the reviewer.

    The knowledge gap brought up in the next paragraph of the introduction doesn't reflect the questions asked by the experiments: "But, how the pacemaker differentially influences peripheral clock activity present in the olfactory system and modulates olfactory sensitivity has not been studied in detail." Specifically, the control of peripheral clocks by the central pacemaker has not been evaluated here.

    Response: This statement has been modified in the revised manuscript.

    "In vertebrates and invertebrates, it is well documented that circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" should also cite work on cockroaches and kissing bugs:

    Lubinski, A. J., & Page, T. L. (2016). The optic lobes regulate circadian rhythms of olfactory learning and memory in the cockroach. Journal of Biological Rhythms, 31(2), 161-169

    Page, T. L. (2009). Circadian regulation of olfaction and olfactory learning in the cockroach Leucophaea maderae. Sleep and Biological Rhythms, 7, 152-161

    Vinauger, C., & Lazzari, C. R. (2015). Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus. Journal of Experimental Biology, 218(19), 3110-3117

    Response: These references have been added in the revised manuscript as suggested by the reviewer.

    The sentence: "Previous studies showed that synaptic plasticity and memory are significantly influenced by the strength and number of synaptic connections (43, 44)." should be nuanced as the role of neuropeptides such as dopamine has also been showed to influence learning and memory in mosquitoes:

    Vinauger, C., Lahondère, C., Wolff, G. H., Locke, L. T., Liaw, J. E., Parrish, J. Z., ... & Riffell, J. A. (2018). Modulation of host learning in Aedes aegypti mosquitoes. Current Biology, 28(3), 333-344 Wolff, G. H., Lahondère, C., Vinauger, C., Rylance, E., & Riffell, J. A. (2023). Neuromodulation and differential learning across mosquito species. Proceedings of the Royal Society B, 290(1990), 20222118

    Response: We agree with the reviewer. We have modified this statement and added the references in the revised manuscript.

    Overall, the paragraph dealing with the idea that "circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" is very confusing. This paragraph discusses mechanisms of learning-induced plasticity but seems to ignore the simplest (most parsimonious) explanations for the circadian regulation of learning (e.g., time-dependent expression of genes involved in memory consolidation). In addition, the sentence quoted above is circumvoluted to simply say that training at different times of the day affects memory acquisition and consolidation. Although the authors did look at one gene involved in neural function, learning, memory, or circadian effects were not analysed in this study. Please reconsider the relevance of the paragraph.

    Response: We have modified this paragraph as per the suggestions of the reviewer in the revised manuscript.

    The sentence: "But, how the brain of mosquitoes entrains circadian inputs and modulates transcriptional responses that consequently contribute to remodel plastic memory, is unknown." should be rephrased. First, it should be "entrains TO circadian inputs", and second, it suggests that the study will be investigating circadian modulation of learning and memory, which is not the case. Furthermore, the term "remodel plastic memory" is unclear and doesn't seem to relate to any specific cellular or neural processes.

    Response: This statement has been removed from the revised manuscript.

    Given the differences in mosquito chronobiology observed even between strains, why perform the RNAi and EAGs on a different species of Anopheles than the one used for the RNAseq (or vice versa)?

    Response: We agree with the reviewer that there are differences in mosquito chronobiology between different strains and therefore species variation may be challenging for data interpretation. Considering the strict nocturnal behavioral pattern of An. culicifacies and dirurnal behavior of Aedes aegypti, we performed RNA-Seq study with these respective species. However, 1) due to unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available), 2) challenges in rearing and adaptation of An. culicifacies in a new environment/laboratory, 3) to validate the proof-of-concept of CYP450 function in odorant detection and olfactory sensitivity, we opt for the current collaborative study. We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (6–8)*. *For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates, therefore, the arguments for species difference can be overruled.

    S. S. C. Rund, J. E. Gentile, G. E. Duffield, Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC Genomics. 14 (2013), doi:10.1186/1471-2164-14-218. S. S. C. Rund, T. Y. Hou, S. M. Ward, F. H. Collins, G. E. Duffield, Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 108 (2011), doi:10.1073/pnas.1100584108. S. S. C. Rund, N. A. Bonar, M. M. Champion, J. P. Ghazi, C. M. Houk, M. T. Leming, Z. Syed, G. E. Duffield, Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Sci. Rep. 3, 2494 (2013).

    Results

    1. "As reported earlier, a significant upregulation of period and timeless during ZT12-ZT18 was observed in both species (Figure 1C)." Please provide effect size and summary statistics.

    Response: The statistics are provided in the Figure S2 in the revised manuscript.

    "Next, the distribution of peak transcriptional changes in both An. culicifacies and Ae. aegypti was assessed through differential gene-expression analysis. Noticeably, An. culicifacies showed a higher abundance of differentially expressed olfactory genes (Figure 1D)" Please provide effect size and summary statistics.

    Response: The statistics are provided in the Table 1 in the revised manuscript.

    "Taken together, the data suggests that the nocturnal An. culicifacies may possess a more stringent circadian molecular rhythm in peripheral olfactory and brain tissues." What do the authors mean by "stringent"? At this point, this should be stated as a working hypothesis, as the statement is not backed up by the data. It is possible that the fewer differentially expressed genes of Aedes aegypti are more central to regulatory networks and cascade into more "stringent" rhythmic control of activities and rhythms.

    Response: We thank the reviewer for this suggestion. We have modified this statement as suggested by the reviewer.

    The section title: "Circadian cycle differentially and predominantly expresses olfaction-associated detoxification genes in Anopheles and Aedes" doesn't make sense. The expression of genes can be modulated by circadian rhythms, but cycles don't express genes. Please rephrase. In addition, this whole section deals with "circadian rhythms" while no experiment has been conducted under constant conditions. The observed daily variations are therefore diel rhythms until their persistence under constant conditions is established.

    Response: We agree with the reviewer and changed the statement accordingly.

    "The downregulated genes of Ae. aegypti did not show any functional categories probably due to the limited transcriptional change." Could the authors explain if this is actually the phenomenon or due to a lack of temporal resolution in the study design (i.e., 4 time points)?

    Response: We do not agree with the reviewer’s comments about the lack of temporal resolution in the current study. The functional categories of differentially expressed genes are deduced by gene set enrichment analysis, which identify the classes of genes that are overrepresented in a large set of genes. The statistical significance value is dependent on the abundance of query and background genes. In our experiments, as the number of queries (i.e. number of downregulated genes) is limited, the enrichment tool, i.e. shinyGo didn’t able to show significant enrichment of downregulated genes with FDR cut-off 0.05 and top 10 pathways were selected. Though we have selected 4 time points, previous study by Rund et al. (BMC Genomics 2013) also showed that compared to Aed. aegypti, An. gambiae possess higher number of rhythmic genes (2.6 fold higher). Therefore, it can be stated that the data that we received is not due to the pitfalls of study design, but probably the physiological difference between Anopheles and Aedes mosquitoes.

    "a GO-enrichment analysis was unable to track any change in the response-to-stimulus or odorant binding category of genes (including OBPs, CSPs, and olfactory receptors)." This finding doesn't corroborate the statements made previously and doesn't align with previously published studies. Is it due to pitfalls in the study design?

    Response: The functional categories of differentially expressed genes are deduced by gene set enrichment analysis, which identify the classes of genes that are overrepresented in a large set of genes. The statistical significance value is dependent on the abundance of query and background genes. Though, differential expression analysis revealed a significant upregulation of a subset of CSPs (~ 5-fold) and OBP6 (~3.3-fold) transcripts in An. culicifacies mosquitoes during ZT12, as the number of queries (i.e. number of chemosensory genes) is limited (i.e. 3), the enrichment tool, i.e. shinyGo didn’t able to show significant enrichment of these categories of genes when FDR cut-off 0.05 and top 10 pathways were selected.

    Moreover, we do not agree with the reviewer regarding the comment on pitfalls of study design because our previous experiments with An. culicifacies according to diel rhythm, considering more extended time points, also revealed similar expression pattern of chemosensory genes (Das De et.al., 2018).

    "In contrast, three different clusters of OBP genes in Ae. aegypti showed a time-of-day dependent distinct peak in expression starting from ZT0-ZT12 (Figure 2F)." Please provide summary statistics.

    Response: Please find the table for summary statistics in the supplemental file 1.

    "In the case of An. gambiae, the amplitudes of odor-evoked responses were significantly influenced by the doses of all the odorants tested (repeated measure ANOVA, p {less than or equal to} 2e-16) (Figure S4B)." Did the authors use a positive control for the EAGs? How did the authors normalize the responses across the two species? Given the way the data is presented, how were the data normalized to allow inter-species comparisons? In addition, It is highly unlikely that all the mosquito preps used in the EAG assay responded to all the odors tested. If that was the case, then the dataset includes missing data for certain odors and time points. We believe the authors have ensured there are at least a certain number of responses per odor and time point combinations. If this is true, repeated measures ANOVA is not suited for analyzing this data because this statistical technique requires all repeated measures within and across preps without missing values. Also, the authors need to correct the summary statistics for multiple comparisons within this framework to avoid inflating type-I errors. Has this been done?

    Response: In our study involving An. gambiae, we observed significant influences of odorant doses on the amplitudes of odor-evoked responses (repeated measure ANOVA, p ≤ 2e-16) (Figure S4B). It's important to note that we did not employ a separate positive control for the electroantennogram (EAG) assays, as the compounds utilized in our research are already known to be EAG active in at least one of the mosquito species under investigation (mentioned in supplementary file 3).

    Our primary objective for performing EAG studies is to correlate the diel-rhythmic molecular data with the diel-rhythmic electroantennographic response in nocturnal and diurnal mosquitoes. To address the normalization of responses across the two species, we opted to control for dose and time rather than normalizing using one of the EAG active compounds. Further, the EAG responses were measured in relation to solvent control. In our experimental design, we utilized different batches of mosquitoes from the same cohort to test each odorant at various time points. EAG responses were acquired using the same mosquito across different dilutions for a single odor or volatile compound, rather than across time points. Hence, we didn’t end up with missing values.

    For individual species analysis, we performed repeated measures ANOVA for each compound's EAG response, considering dose and time as variables. This enabled not only enabled us select compounds which where ‘Time’ or its interaction terms were found to be significant. Subsequently, for compounds showing significance, we conducted a basic one-way ANOVA using only time as a variable, segregating the data by each individual dose. Post-hoc Tukey tests were then carried out to compare between time points. When comparing between species, we generated a dataset by combining both species and adding species as a variable as well. Repeated measures ANOVA for each compound's EAG response, considering species, dose, and time as variables, was applied. This enabled us select compounds which where ‘Time’ or its interaction terms were found to be significant. For significant compounds, a two-way ANOVA was performed using time and species as variables. Data were segregated by each individual dose, and post-hoc Tukey tests were employed to compare between time points. It's worth mentioning that our analysis aims to account for repeated measures within and across preparations. Additionally, we have implemented post-hoc Tukey tests to correct for multiple comparisons within this framework, ensuring that we avoid inflating type-I errors in our statistical interpretations.

    "Ae. aegypti was found to be most sensitive to all the odorants (4-methylphenol, β-ocimine, E2-nonenal, benzaldehyde, nonanal, and 3-octanol) during ZT18-20 except sulcatone (Figure 3C - 3H)." Although some of these chemicals are associated with plants and Ae. aegypti is suspected to sugar feed at night, how do the authors explain that the peak olfactory sensitivity occurs at night for compounds such as nonanal? It would be interesting to discuss how these results compare to previous studies such as:

    Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

    Response: The possible explanations have been added in the revised MS.

    "Additionally, our principal components analysis also illustrates that most loadings of relative EAG responses are higher towards the Anopheles observations (Figure S4C)." The meaning of this sentence is unclear? Please clarify.

    Response: Considering the limited clarity of the statement we have removed it from the revised manuscript.

    "Taken together these data indicate that An. gambiae may exhibit higher antennal sensitivity to at least five different odorants tested, as compared to Ae. aegypti." As mentioned above, how did the authors normalized across species to allow comparisons? If not normalized, how do you ensure that higher response magnitudes correlate with higher olfactory sensitivity, given potential differences in the morphology or size differences between the two species? Furthermore, An. gambiae has been exclusively used in the EAG assay. Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable. This, however, is a major caveat in the experiment design, given previous studies (indicated below) have reported species-specific variations in olfactory sensitivity. In its present form, the EAG data from An. gambiae is not a piece of appropriate evidence that the authors could use to complement or substantiate the findings from other aspects of this study on An. culicifacies.

    Wheelwright, M., Whittle, C. R., & Riabinina, O. (2021). Olfactory systems across mosquito species. Cell and Tissue Research, 383(1), 75-90. Wooding, M., Naudé, Y., Rohwer, E., & Bouwer, M. (2020). Controlling mosquitoes with semiochemicals: a review. Parasites & Vectors, 13, 1-20.

    iii. Gupta, A., Singh, S. S., Mittal, A. M., Singh, P., Goyal, S., Kannan, K. R., ... & Gupta, N. (2022). Mosquito Olfactory Response Ensemble enables pattern discovery by curating a behavioral and electrophysiological response database. Iscience, 25(3).

    Response: The data is normalized as described above in the point 15. Also, it is technical limitation that we had to use multiple species of the mosquito for this study (please refer to the point 7).

    The reviewer’s statement “Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable” is not true, as we never assume similar olfactory sensitivity between An. culicifacies and An. gambiae. We only consider nocturnal activity for both the mosquito species. Moreover, we are aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (no other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (6–8)*. *For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Furthermore, we would like to emphasize that the primary focus of the current manuscript is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validated this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

    "Similar to An. gambiae, a comparatively high amplitude response was also observed in An. stephensi (Figure S4D)." This is interesting but what would be even more relevant to the present study is to discuss how the time-dependent responses compare between the two Anopheles species.

    Response: We agree that it will be interesting to compare time-dependent response between the two Anopheles species. However, it is not our primary interest and objectives, and is beyond the scope of the current manuscript. Thus, we remove the data from the revised MS.

    The paragraph titled "Daily temporal modulation of neuronal serine protease impacts mosquito's olfactory sensitivity" is confusing because the authors move on to test the effect of knocking down a serine protease gene (found to be differentially expressed throughout the day) on olfactory sensitivity. While this is interesting in and of itself, the link between the role of this gene in learning-induced plasticity, the circadian modulation of "brain functions" and olfactory sensitivity is 1) unclear and 2) not explicitly tested. We agree with the authors that what has been tested is "the effect of neuronal serine protease on circadian-dependent olfactory responses," but the two paragraphs leading to it seem to be extrapolating functional links that have yet to be determined. In this context, their conclusions that "Our finding highlights that daily temporal modulation of neuronal serine-protease may have important functions in the maintenance of brain homeostasis and olfactory odor responses." is misleading because although they used the hypothetical "may", the link between the temporal modulation of one serine protease gene and the maintenance of brain homeostasis is not explicitly tested here.

    Response: Though, we strongly believe that neuronal serine protease are involved in remodelling of extracellular matrix and the maintenance of brain homeostasis, the limitation of experimental validation by neuroimaging (out of the scope of the current manuscript), restricting us to draw the conclusion. Therefore, we have modified our conclusions based on the available data as suggested by the reviewer.

    Discussion

    1. The first sentence of the discussion: "In this study, we provide initial evidence that the daily rhythmic change in the olfactory sensitivity of mosquitoes is tuned with the temporal modulation of molecular factors involved in the initial biochemical process of odor detection i.e., peri-receptor events" is not true since studies from Rund and Duffield previously revealed the daily modulation of OBP gene expression. It also contradicts the next sentence: "The findings of circadian-dependent elevation of xenobiotic metabolizing enzymes in the olfactory system of both Ae. aegypti and An. culicifacies are consistent with previous literature (26, 31), and we postulate that these proteins may contribute to the regulation of odorant detection in mosquitoes."

    Response: This statement is modified in the revised manuscript.

    The use of "circadian" in the discussion of the results is also misleading as only diel rhythms were evaluated in the present study.

    Response: This is changed in the revised manuscript.

    "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58)." This is not really what these references show.

    Response: The statement and the references have been changed in the revised manuscript.

    "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58), it can be hypothesized that detection of any specific signal in such a noisy environment, mosquitoes may have evolved a sophisticated mechanism for rapid (i) odor mobilization and (ii) odorant clearance, to prevent anosmia (24)." One could argue that this is a requirement for all insects, regardless of the size of their olfactory repertoire.

    Response: We agree with the reviewer and modified the text accordingly.

    "Taken together, we hypothesize that circadian-dependent activation of the peri-receptor events may modulate olfactory sensitivity and are key for the onset of peak navigation time in each mosquito species." This is not entirely accurate since spontaneous locomotor activity rhythms are also observed in the absence of olfactory stimulation. While "navigation" does imply olfactory-guided behaviors, "peak navigation time" appears to be driven by other processes. See, for example, all studies testing mosquito activity rhythms in locomotor activity monitors. Response: Considering the concern of the reviewer, we have modified the text.

    "Due to technical limitations, and considering the substantial data on the circadian-dependent molecular rhythmicity" please clarify what the technical limitations were. Is this something that prevented the authors specifically, or something tied to mosquito biology and would prevent anybody from doing it? Also, why couldn't the transcriptomic analysis be performed on An. gambiae?

    Response: As previously mentioned, primarily, unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available) is the major challenge for us to proof our hypothesis. Secondly, transportation of An. culicifacies was not possible due to Govt. regulations and also adaptation and establishment of the colony of An. culicifacies take long time as it is not easily adapted (Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. *Trans R Soc Trop Med Hyg. *1999;93:573–577) in a new environment/laboratory. Thirdly, An. culicifacies colony was not available at our collaborative laboratory. These are the major technical limitations.

    Therefore, to validate the hypothesis of CYP450 function in odorant detection and olfactory sensitivity, we opt for the current collaborative study. We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (6–8)*. *For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Performing another RNA-Seq study with An. gambiae would not be possible for the current MS. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

    "In contrast to An. gambiae, the time-dose interactions had a higher significant impact on the antennal sensitivity of Ae. aegypti. An. gambiae showed a conserved pattern in the daily rhythm of olfactory sensitivity, peaking at ZT1-3 and ZT18-20." These two sentences are very confusing. Doesn't it simply mean that the co-variation is not linear or not the same across odors? In addition, what does it mean for a pattern to be more conserved? How can one conclude about the "conserved" nature of a pattern by looking at time-dependent variations in dose-response curves?

    Response: This section of discussion is re-written in the revised version of the manuscript.

    "Together these data, we interpret that mosquito's olfactory sensitivity possibly does not follow a fixed temporal trait" is unclear and suggests that the authors are discussing global versus odor-specific rhythms. Please rephrase.

    Response: This section of discussion is re-written in the revised version of the manuscript.

    "Moreover, we hypothesize that under standard insectary conditions, mosquitoes may not need to exhibit foraging flight activity either for nectar or blood, and during the time course, it may minimize their olfactory rhythm, which is obligately required for wild mosquitoes." This hypothesis is not supported by the results of the study and contradicts work by others (Rund et al., Eilerts et al., Gentile et., etc).

    Response: This section of discussion is re-written in the revised version of the manuscript.

    The same comment applies to "Therefore, it is reasonable to think that the mosquitoes used for EAG studies may have adapted well under insectary settings and, hence carry weak olfactory rhythm." as this statement is not supported by results of the present study or comparisons of the results to previous studies based on field-caught mosquitoes. Although it is an interesting question to ask in the future, it should be stated as a future research avenue rather than a working hypothesis that results from the present study.

    This section of discussion is re-written in the revised version of the manuscript.

    "Aedes aegypti displayed a peak in antennal sensitivity at ZT18-20 to the higher concentrations of plant and vertebrate host-associated odorants tested. Given the time-of-day dependent multiple peaks (at ZT6-8 and ZT18-20 for benzaldehyde and at ZT12-14 and ZT18-20 for nonanal) in antennal sensitivity to different odorants, our data supports the previous observation of bimodal activity pattern of Ae. aegypti (50)." Rephrase by saying that results are "aligned with the previous observations of bimodal activity". Olfactory rhythms don't "support" the activity patterns because olfactory processes and spontaneous locomotor activity are independent processes.

    Response: We have made these changes in the revised manuscript as per the suggestions of the reviewer.

    "our preliminary data indicate that Anopheles spp. may possess comparatively higher olfactory sensitivity to a substantial number of odorants as compared to Aedes spp." Consider removing this sentence unless the way the data has been normalized to allow for comparisons between species is clarified.

    Response: This statement is removed from the revised manuscript.

    In "A significant decrease in odorant sensitivity for all the volatile odors tested in the CYP450-silenced Ae. aegypti," please change "silenced" to "reduced" because RNAi doesn't silence (i.e. knockout) gene expression.

    Response: It has been modified as per the suggestions of the reviewer.

    The title "Neuronal serine protease consolidates brain function and olfactory detection" is extremely misleading. Do the authors refer to memory consolidation, which has not been tested here? What is brain function consolidation??

    Response: We agree with the reviewer. The title has been modified in the revised manuscript.

    The reference used in "Despite their tiny brain size, mosquitoes, like other insects, have an incredible power to process and memorize circadian-guided olfactory information (7)." is not appropriate. Also, "circadian-guided" is unclear. Consider replacing it with "circadian-gated".

    Response: It has been modified as per the suggestions of the reviewer.

    What is the "the homeostatic process of the brain"?

    Response: The process of maintaining a stable state can be defined as homeostasis. Here, the statement "the homeostatic process of the brain" is used to convey that after the active host-seeking/olfaction phase of mosquitoes during which the co-ordinated and integrated functions of both olfactory and neuronal system is required for crucial decision-making events, brain may undergo a homeostatic process (comes down from excitatory state to stable state) during the resting period. However, in view of reviewer’s concern we have modified the statement.

    "the temporal oscillation of the sleep-wake cycle of any organism is managed by the encoding of experience during wake, and consolidation of synaptic change during inactive (sleep) phases, respectively (70)." By experience, do the authors refer to learning? This seems out of topic as this process has not been evaluated here.

    Response: It has been modified as per the suggestions of the reviewer.

    "We speculate that after the commencement of the active phase (ZT6-ZT12), the serine peptidase family of proteins in the brain of Ae. aegypti mosquitoes may play an important function in consolidating brain actions (after ZT12) and aid circadian-dependent memory formation." The value of this statement is unclear. Circadian-dependent memory formation is not being evaluated here, and the results from the present study do not directly support this speculation, also because other processes involved in memory formation are not evaluated here. This seems at odds with the literature on learning and memory.

    Response: We have modified these statements in the revised manuscript and mentioned it as future research hypothesis.

    "Subsequent work on electrophysiological and neuro-imaging studies are needed to demonstrate the role of neuronal-serine proteases in the reorganization of perisynaptic structure." Sure. But the link between "the role of neuronal-serine proteases in the reorganization of perisynaptic structure" and rhythms in olfactory sensitivity is unclear.

    Response: It has been modified as per the suggestions of the reviewer.

    As a general comment, EAGs seem inappropriate to evaluate the effect of the central-brain processing in the regulation of peripheral olfactory processes. This is a critical comment that needs to be considered by the authors and clarified in the manuscript. If rhythms of central brain processes are important for olfactory-guided behaviors, these should be evaluated at the level of the central brain or via behavioral metrics. The effect of the RNAi knockdowns on peripheral sensitivity is interesting, but its link with central processes is unclear and doesn't support the speculations made by the authors about learning and memory.

    Response: We agree with the reviewer that EAG study is not enough/appropriate to comment on the effect of central-brain processing in the regulation of olfactory processes. Further validation by either neuroimaging or behavioral studies are needed to make any conclusion. We clearly mention in the manuscript that our data indirectly indicating this function of serine protease and further confirmatory studies are needed to prove this hypothesis.

    Methods

    1. No explanations are provided for how the EAG data are normalized to allow comparisons between species.

    Response: Please refer to the response of the point no. 15 of the reviewer 1.

    Figures

    1. Figure 1: The daily rhythm depicted in A, are not representative of the actual profiles. See: Benoit, J. B., & Vinauger, C. (2022). Chapter 32: Chronobiology of blood-feeding arthropods: influences on their role as disease vectors. In Sensory ecology of disease vectors (pp. 815-849). Wageningen Academic Publishers. Or any other paper on mosquito activity rhythms.

    Response: Considering the reviewer’s concern we have revised the figure.

    Figure 3 and 4: The EAG results are plotted twice. This is redundant and misleading as it makes the reader think there is more data than actually presented.

    Response: Considering the reviewer’s comment we shifted figure 4 into the supplemental file.

    Figure 5: Please clarify the sample size for each panel. In C - F, what would be used as a reference? In other words, what is a Relative EAG Response of 1? And if it is "relative", are the units really mV? In E and F, it would be great to show how the Ethanol control compares to the no solvent condition. This could be placed in supplementary materials.

    Response: The sample size was mentioned in the figure legends. However, for the reviewer’s clarification, the odor response was tested with 40 individual mosquitoes of control and dsrRNA-treated groups. Therefore, sample size N=40 for Fig. 5C.

    Respective solvent control (hexane solvent) used as a reference to calculate the relative EAG response for both the dsrLacZ and dsrCYP450 group. As it is relative EAG amplitude we have removed the unit in the revised MS.

    Figures 5 and 6, given the dispersion in the EAG data, the treatments where N=40 appear robust, but the interpretation of results from treatments where N=6 may be limited due to the low sample size. This limitation is visible in Figure 5F, for example, where ABT-Aceto is different from Cont-Aceta but not PBO-Aceto because one individual shows a higher response.

    Response: We agree that probably, by increasing the sample size for inhibitor treatment experiment, may decrease these inter-individual differences and increase the overall significance value. However, our robust knock-down data showed significant results and simultaneously it complements the inhibitor study in Ae. aegypti, we do not think of any disparity in the data. Moreover, EAG response to human blend, nonanal and benzaldehyde showed similar significant results in both RNAi and inhibitor studies. Accounting, the different knock-down efficiency in dsRNA injected mosquitoes, the phenotypic assays (EAG recordings) were carried out with 40 control and 40 dsRNA-treated mosquitoes. And, we observed significant reduction in EAG response following inhibitor treatment in An. gambiae, when we tested for 6 ethanol and 6 inhibitor treated mosquitoes. Thus, we followed the similar protocol for Ae. aegypti also. However, inter-individual difference in response is affecting the significance value.

    Figure S6: how does this support that synaptic plasticity is influenced by "Time-of-day dependent modulation of serine protease genes in the brain"?

    Response: We agree with the reviewer’s concern that with only EAG data it is not possible to comment on synaptic plasticity. We apologize for it and revised the statement in the MS.

    Minor comments

    What do the authors mean by "consolidation of brain functions"? Memory consolidation? Please clarify.

    Response: The consolidation of brain function or memory consolidation means to the process of stabilizing the memory that an organism gains through the process of experience or training/learning phase. Memory consolidation initiates with rapid change in de-novo gene expression regulated by several transcription factors, effector genes and non-coding RNAs, known as molecular consolidation followed by cellular consolidation that involves cellular signal transmission within the neurons in the brain. The molecular and cellular consolidation are the basis for system level consolidation which is a slow process and involves communication among neurons located different regions of the brain. The system level consolidation is very important for the reorganization of the brain circuits to maintain long-term memory. The concept of system consolation is very much well evident in humans. Additionally, several studies in Drosophila also showed that fruit fly develop olfactory memories after classical conditioning or olfactory training through system consolidation process.

    Moreover, accumulating data from humans suggest that sleep helps in memory consolidation. Sleep is basic drive for all animals that help to build memories. There are two hypothesis and respective compelling evidences for that. First hypothesis and the supporting molecular and electrophysiological data convey that sleep facilitate the homeostatic processes of the brain involving loosening of synaptic connections between the overactive neurons, structural modification of synapse which consequently help in memory formation. The second hypothesis state the important contribution of sleep in system consolidation and long-term memory potentiation. Studying the electrical activity of the brain and the recent advancement of fMRI scan indicate reorganization of neural activity between brain regions during sleep-related memory consolidation.

    There are several experimental evidences in support of both the theory for humans as well as in fruit fry Drosophila melanogaster. In mosquitoes, the studies related to the function of brain are primarily restricted to the mechanism of odor coding and memory formation has been correlated with Dopamine neurotransmitter signalling. In view of the rapid adaptation potential, change in host-preference and evolution of temporal host-seeking behaviour, it can be hypothesized that mosquito brain also undergo the process of memory consolidation (either following any of the two hypothesized path or cumulatively apply the both) to learn new information in order to effectively shape future actions.

    Furthermore, according to the fundamental principle of modern neuroscience learning and memory are achieved either by the formation of new synaptic connections or changing in existing connections between neurons. The ability of synapses to either strengthen or weaken the communications is called plasticity which is influenced by learning and experience and facilitate organism’s adaptation and survival.

    Reference:

    1. Cervantes-Sandova, A. Martin-Peña, J. A. Berry, R. L. Davis, System-like consolidation of olfactory memories in Drosophila. J. Neurosci. 33, 9846–9854 (2013).
    2. In "Similar to previous studies (26), the expression of a limited number of rhythmic genes was visualized in Ae. aegypti" please replace "visualized" with "observed".
    3. Marshall, N. Cross, S. Binder, T. T. Dang-Vu, Brain rhythms during sleep and memory consolidation: Neurobiological insights. Physiology. 35, 4–15 (2020).
    4. Brendon O. Watson and György Buzsáki. Sleep, Memory & Brain Rhythms. Daedalus, 144(1): 67–82 (2015). doi:10.1162/DAED_a_00318

    Figure 2A, please clarify in the caption what FDR stands for.

    Response: FDR stands for “false discovery rate”. FDR is an adjusted p-value to trim false positive results.

    In "To further establish this proof-of-concept in An. gambiae, three potent CYP450 inhibitors, aminobenzotriazole(52), piperonyl butoxide(53), and schinandrin A (54), was applied topically on the head capsule of 5-6-day-old female mosquitoes" replace "was applied" with "were applied".

    Response: These changes are made in the revised manuscript.

    "Interestingly, our species-time interaction studies revealed that An. gambiae exhibits time-of-day dependent significantly high antennal sensitivity to at least four chemical odorants compared to Ae. aegypti, except phenol." is unclear. Please reword.

    Response: The statement has been revised in the MS.

    In "Similar observations were also noticed with An. stephensi." replace "noticed" with "made". Response: We have modified the statement in the revised version of the manuscript.

    Reviewer #1 (Significance (Required)):

    Such a study has the potential to be valuable for the field, but its value and significance are hindered by an accumulation of overstatements, the fact that prior work in the field has been minimized or omitted, and a lack of support for the stated conclusions.

    In this context, the advances are only slightly incremental compared to the work produced by Rund et al., and the mechanistic hypotheses emitted to link the genes selected for knockdown experiments and olfactory sensitivity are not clearly supported by the evidence presented here. The main strength of the paper is to show the role of CYP450 in olfactory sensitivity.

    The audience is fairly broad and includes insect neuro-ethologists, molecular biologists, and chronobiologists.

    Our field of expertise:

    • Mosquito chemosensation

    • Learning and memory

    • Chronobiology

    • Electrophysiology

    • Medical entomology

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This report combines an examination of peripheral transcriptomes and general olfactory sensitivity in an effort to underscore the importance of peri-receptor components in circadian-directed modulation of olfaction across both Aedine and Anopheline mosquitoes. While the authors do a nice job of raising the importance of the often-underappreciated spectrum of insect olfactory peri-receptor proteins, the impact of their study is undercut by technical concerns regarding methods and data presentation. That several of these concerns (detailed below) are explicitly acknowledged by the authors as limitations of this study does not mitigate their impact in eroding confidence in these data and this study.

    All in all, as a result of these concerns, I am unconvinced as to the overall merits of this somewhat interesting but generally uneven study.

    We sincerely thank the reviewer for their time and consideration, and appreciate the thorough review of our manuscript. Their insightful comments have greatly enriched our work. We also apologies for instances of overinterpreting the data. Your feedback has helped us recognize areas where clarity and caution are needed, and we are committed to addressing these concerns in our revisions. Thank you for your valuable input and guidance.

    Major concerns:

    1. That the authors use An. culicifacies for their transcriptome studies and An. gambiae (G3) for the olfactory physiology does not work. The 'technical limitations' (read studies done at two different locations) make this report an unwelcome melding of what should perhaps be two distinct studies. In order to maintain this forced marriage as a single report I would suggest the authors utilize An. culicifacies for both components. Alternatively, they can do both parts with An. gambiae but here I would strongly urge them to use any strain other than G3 which as a result of its now decades-long laboratory residence has long since lost its relevance to natural populations of Anopheline vectors. Response: We agree with the reviewer that there is significant species-specific variation in olfactory sensitivity of mosquitoes. Considering the strict nocturnal behavioral pattern of An. culicifacies and dirurnal behavior of Aedes aegypti, we performed RNA-Seq study with these respective species. However, 1) due to unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available), 2) challenges in rearing and adaptation of An. culicifacies in a new environment/laboratory (An. culicifacies take long time as it is not easily adapted, Ref: Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. *Trans R Soc Trop Med Hyg. *1999;93:573–577), 3) An. culicifacies colony was not available at our collaborative laboratory, 4) to validate our hypothesis of CYP450 function in odorant detection and olfactory sensitivity of mosquitoes, we opt for the current collaborative study.

    We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (6–8)*. *For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Performing another RNA-Seq study with An. gambiae would not be possible for the current MS. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

    The 70-80% alignment rate reported to the An. culicifacies reference genome significantly erodes this reader's confidence in the integrity of their analyses. That low level of alignment can have dramatic impacts on the estimation of transcript abundance has been repeated demonstrated (see, Srivastava, A., Malik, L., Sarkar, H. et al.. Genome Biol 21, 239, 2020, https://doi.org/10.1186/s13059-020-02151-8). This may (in part) explain why olfactory receptors have been largely absent from this data set.

    Response: We agree with the reviewer that alignment rate could have been better but this should not affect the quantitative information we are referring to in this manuscript. The alignment rates could have impacted the qualitative information which can vary due to multiple reasons including the quality of the reference genome. As it is evident from the analysis that in Ae. aegypti 90% of the reads are aligned to the reference genome, still we did not observe any difference in the abundancy of olfactory receptor genes. Previous microarray analysis in An. gambiae by Rund et.al. 2013, also did not show diel rhythmic expression of any OR genes.

    The issue of species choice is further complicated by questions regarding the An. culicifacies species complex which contains 5 cryptic species. How did the authors confirm they are indeed working with An. culicifacies species A -there is no mention regarding the molecular identification.

    Response: The An. culcifacies species A colony has been colonized at NIMR since 1999, with routine checks performed to verify its purity of species by analyzing inversion genotypes on chromosomes for the presence of sibling species (see the references). But at that time, we had three sibling species--A, B, C; subsequently, we lost B and C. Giving old references will not serve the purpose. Later we verified sibling species A by inversion genotype on chromosome and molecular tools. However, we do not have any published reference for that verified data.

    The species can be identified by performing 28S rDNA-based PCR (Singh et al, 2004) and cytochrome oxidase II-based PCR (Goswami et al 2006). Sequencing can also serve the purpose.

    Singh OP, Goswami G, Nanda N, Raghavendra K, Chandra D, Subbarao SK. An allele-specific polymerase chain reaction assay for the identification of members of Anopheles culicifacies complex. J Biosci. 2004; 29: 275—280 10.1007/bf02702609

    Goswami G, Singh OP, Nanda N, Raghavendra K, Gakhar SK, Subbarao SK. Identification of all members of the Anopheles culicifacies complex using allele-specific polymerase chain reaction assays. Am J Trop Med Hyg. 2006; 75: 454-460. doi: 10.4269/ajtmh.2006.75.454

    Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. *Trans R Soc Trop Med Hyg. *1999;93:573–577

    The switch from dsRNAi studies in Aedes to protease inhibitor studies in Anopheles adds to the interspecies confusion.

    Response: Our main goal in this study was to evaluate the function of CYP450 in mosquito’s odor detection and olfactory sensitivity. Our data as well as previous data (Rund et.al. 2011, Rund et.al. 2013) suggesting that the basic mechanism of odor detection and peri-receptor events are similar for both An. gambiae, An. culicifacies and Ae. aegypti, and the role of detoxification genes are very much evidenced from these data. Based on our RNA-Seq data on Ae. aegypti, we shortlisted one CYP450 gene for functional knockdown assays. However, for Anopheles we used An. gambiae for functional validation. Thus, it was not possible for us to select appropriate CYP450 gene from An. gambiae. That is why, we plan for using CYP450 protein inhibitors which block the function of all the CYP450 expressing in the olfactory system of mosquitoes. Expectedly, we also observed much more pronounced reduction of olfactory sensitivity when inhibitors were applied compared to dsRNAi mediated knock-down the function of only one CYP450 protein. These data indicate that Anopheles also possess similar mechanism of perireceptor events for odor detection and CYP450 plays an important role in it.

    The olfactory shifts presented in Fig 3 are somewhat underwhelming. In An. gambiae this mostly seen at very high (to my eyes, non-biologically relevant) 10-1 dilutions. In Aedes, while statistically significant, the EAG values (especially for 4MePhenol) are very low and therefore suspect and unconvincing. It is also unclear how 'Relative EAG Responses' were derived?? Does this mean relative to solvent alone controls??

    Response: Yes, relative EAG response means relative to respective solvent control. We also make necessary changes in the text as well as in the figures for better understanding and representation.

    The same data set seems to have been presented in Figures 3 and 4, with the latter's absence of salient details e.g. haphazard odor concentrations which are seen only when legend is examined). These factors make the inclusion of Figure 4 less obvious.

    Response: Depending on the reviewer’s concern we shifted the Figure 4 into the supplemental data and we are sorry for the miscommunication.

    I am concerned that the data in Figure 5B is derived from only those samples with altered EAGs. I believe that all injected mosquitoes should be assayed in order to better understand the actual efficacy of the treatment. The cherry picking of samples is troubling.

    Response: We pooled five heads for each replicate and we performed the assay with three replicates. That mean we have taken heads from 15 mosquitoes for each experimental setup (control vs knock-down). It is true that we did not consider all the 40 mosquitoes that we used for EAG-recordings. However, we believe that 15 mosquitoes will be a good representation of the population. And the error bars among replicates of the knock-down mosquitoes, compared to the dsLacZ group, clearly indicates the disparity in knock-down efficiency among individuals.

    As is true for earlier figures, Figure 5c-f is lacking critical information about concentration (also not presented in figure legend) and should be done within the context of a multi-point dose response study. The data in its current form is not acceptable.

    Response: We apologize for the mistake for not mentioning the concentration of the inhibitors. Now, we added this information in the revised manuscript.

    The same data concerns apply to Figure 6d-g.

    Response: We apologize for the mistake for not mentioning the concentration of the inhibitors. Now, we added this information in the revised manuscript.

    The inclusion of An. stephensi data Figure S4D seems thrown in as an after-thought and without good reason.

    Response: Our RNA-Seq data on An. culicifacies and Aedes aegypti revealed similar abundance and expression pattern of rhythmic transcripts specifically for peri-receptor transcripts, as reported before by Rund et. al. 2011 & 2013 for Aedes aegypti and Anopheles gambiae. Moreover, we observed significant difference in EAG response between Aedes *aegypti *and Anopheles gambiae, we hypothesized that higher abundance of rhythmic peri-receptor transcripts possibly has correlation with high EAG response in Anopheles. Therefore, to get an idea about the EAG response for other Anopheles sp. we used An. stephensi, and observed similar difference in EAG response. Though, it will be interesting to compare time-dependent response between the two Anopheles species, it is not our primary interest and objectives, and is beyond the scope of the current MS and the objective can be elaborated further in future.

    I am unsure how shifts in CNS levels of P450 or serine proteases impact peripheral EAG recordings? This is especially so given that any effects on synaptic plasticity/efficacy that might occur are expected to be downstream of the peripheral antennae being recorded in EAGs. The authors do not do a great job explaining away that paradox even though that section in the discussion seems overly speculative.

    Response: We agree with the reviewer that EAG study is not enough/appropriate to comment on the effect of central-brain processing in the regulation of olfactory processes. Further validation by either neuroimaging or beavioral studies are needed to make any conclusion. And we clearly mention in the MS that our data indirectly indicating this function of serine protease and further confirmatory studies are needed to proof this hypothesis. However, it is not possible for us to perform all the experiments now, due to technical and infrastructural limitations. Thus, we hypothesized it as future research endeavour. Moreover, considering the reviewer’s concern we have modified the text and removed the overstatements and speculations.

    The authors discussion on peri-receptor protein oscillation seems premature given the data that is presented (regardless of the caveats discussed above) center on transcript abundance. There is no data on protein abundance, which while related, is an entirely different question/issue.

    Response: Yes, we agree that our hypothesis of peri-receptor protein oscillation is based on our RNA-Seq data. However, later we validated our hypothesis by knock-down studies in mosquitoes as well as we used CYP450 protein inhibitors, where also we observed significant results of decrease in olfactory sensitivity. It is true that we do not have any data on protein abundance, but several previous studies along with our data showed the similar expression profiling of peri-receptor genes, which clearly indicates that the rhythmic expression pattern of these genes are conserved among mosquitoes. None of the previous studies address the hypothesis regarding the peri-receptor events and possible function of XMEs in odorant detection, which is the uniqueness of our study. Therefore, we believe that after functional validation by dsRNAi and inhibitor study, we are able to validate our hypothesis for scientific acceptance. While, CYP450 has been reported to have crucial role in xenobiotic detoxification, its role in odor detection has not been explored yet. We agree that further biochemical validation is required to see the interaction between CYP450 and odor molecules, and how CYP450 is modifying the odorant chemicals either for its detection or for its inactivation. But, such study is out of the scope of the MS and will be our future research endeavour. However, our current data and the MS will have large impact for designing of strategies for application of insecticides, as overlapping the timing of application of insecticide and rhythmic expression/natural upregulation of XMEs could accelerate the inactivation of insecticides and rapid generation of resistant mosquitoes. Thus, we believe that the current revised MS have potential data and would be valuable for publication.

    Minor concerns:

    The authors routinely confuse transcript abundance derived from their RNAseq data with gene expression. The former reflects the steady-state snapshot levels of transcripts encompassing\ synthesis, use and decay while the latter is limited to the rate of transcription requiring nuclear run on or single-nucleus RNAseq approaches. Response: Thank you for your insightful comment. We appreciate your clarification regarding the distinction between transcript abundance and gene expression. In the revised manuscript, we have included a clarification stating that 'transcript abundance is referred to as gene expression, unless explicitly stated otherwise”.

    There are numerous typos, spelling errors and other grammatical mistakes-a copy editor is needed.

    Response: In the revised manuscript, we have carefully corrected the spelling errors and other grammatical mistakes.

    Many of the supplemental figures are error filled, lacking sufficient details and otherwise difficult to parse/understand. I recommend revisiting/removing many of these/

    Response: We have improvised on the supplementary figures in the revised manuscript as suggested by the reviewer.

    __ Reviewer #2 (Significance (Required)):__

    In light of the serious concerns described above there is limited significance to this study. Similarly these concerns erode almost all of any advance to the field this study might have offered. The audience of interest would be highly specialized

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This report combines an examination of peripheral transcriptomes and general olfactory sensitivity in an effort to underscore the importance of peri-receptor components in circadian-directed modulation of olfaction across both Aedine and Anopheline mosquitoes. While the authors do a nice job of raising the importance of the often-underappreciated spectrum of insect olfactory peri-receptor proteins, the impact of their study is undercut by technical concerns regarding methods and data presentation. That several of these concerns (detailed below) are explicitly acknowledged by the authors as limitations of this study does not mitigate their impact in eroding confidence in these data and this study.

    All in all, as a result of these concerns, I am unconvinced as to the overall merits of this somewhat interesting but generally uneven study.

    Major concerns:

    1. That the authors use An. culicifacies for their transcriptome studies and An. gambiae (G3) for the olfactory physiology does not work. The 'technical limitations' (read studies done at two different locations) make this report an unwelcome melding of what should perhaps be two distinct studies. In order to maintain this forced marriage as a single report I would suggest the authors utilize An. culicifacies for both components. Alternatively, they can do both parts with An. gambiae but here I would strongly urge them to use any strain other than G3 which as a result of its now decades-long laboratory residence has long since lost its relevance to natural populations of Anopheline vectors.
    2. The 70-80% alignment rate reported to the An. culicifacies reference genome significantly erodes this reader's confidence in the integrity of their analyses. That low level of alignment can have dramatic impacts on the estimation of transcript abundance has been repeated demonstrated (see, Srivastava, A., Malik, L., Sarkar, H. et al.. Genome Biol 21, 239, 2020, https://doi.org/10.1186/s13059-020-02151-8). This may (in part) explain why olfactory receptors have been largely absent from this data set.
    3. The issue of species choice is further complicated by questions regarding the An. culicifacies species complex which contains 5 cryptic species. How did the authors confirm they are indeed working with An. culicifacies species A -there is no mention regarding the molecular identification.
    4. The switch from dsRNAi studies in Aedes to protease inhibitor studies in Anopheles adds to the interspecies confusion.
    5. The olfactory shifts presented in Fig 3 are somewhat underwhelming. In An. gambiae this mostly seen at very high (to my eyes, non-biologically relevant) 10-1 dilutions. In Aedes, while statistically significant, the EAG values (especially for 4MePhenol) are very low and therefore suspect and unconvincing. It is also unclear how 'Relative EAG Responses' were derived?? Does this mean relative to solvent alone controls??
    6. The same data set seems to have been presented in Figures 3 and 4, with the latter's absence of salient details e.g. haphazard odor concentrations which are seen only when legend is examined). These factors make the inclusion of Figure 4 less obvious.
    7. I am concerned that the data in Figure 5B is derived from only those samples with altered EAGs. I believe that all injected mosquitoes should be assayed in order to better understand the actual efficacy of the treatment. The cherry picking of samples is troubling.
    8. As is true for earlier figures, Figure 5c-f is lacking critical information about concentration (also not presented in figure legend) and should be done within the context of a multi-point dose response study. The data in its current form is not acceptable.
    9. The same data concerns apply to Figure 6d-g.
    10. The inclusion of An. stephensi data Figure S4D seems thrown in as an after-thought and without good reason.
    11. I am unsure how shifts in CNS levels of P450 or serine proteases impact peripheral EAG recordings? This is especially so given that any effects on synaptic plasticity/efficacy that might occur are expected to be downstream of the peripheral antennae being recorded in EAGs. The authors do not do a great job explaining away that paradox even though that section in the discussion seems overly speculative.
    12. The authors discussion on peri-receptor protein oscillation seems premature given the data that is presented (regardless of the caveats discussed above) center on transcript abundance. There is no data on protein abundance, which while related, is an entirely different question/issue.

    Minor concerns:

    1. The authors routinely confuse transcript abundance derived from their RNAseq data with gene expression. The former reflects the steady-state snapshot levels of transcripts encompassing\ synthesis, use and decay while the latter is limited to the rate of transcription requiring nuclear run on or single-nucleus RNAseq approaches.
    2. There are numerous typos, spelling errors and other grammatical mistakes-a copy editor is needed.
    3. Many of the supplemental figures are error filled, lacking sufficient details and otherwise difficult to parse/understand. I recommend revisiting/removing many of these/

    Significance

    In light of the serious concerns described above there is limited significance to this study. Similarly these concerns erode almost all of any advance to the field this study might have offered. The audience of interest would be highly specialized

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In the present manuscript, the authors analyzed diel oscillations in the brain and olfactory organs' transcriptome of Aedes aegypti and Anopheles culicifacies. The analysis of their RNAseq results showed an effect of time of day on the expression of detoxification genes involved in oxidoreductase and monooxygenase activity. Next, they investigated the effect of time of day on the olfactory sensitivity of Ae. aegypti and An. gambiae and identified the role of CYP450 in odor detection in these species using RNAi. In the last part of the study, they used RNAi to knock down the expression of one of the serine protease genes and observed a reduction in olfactory sensitivity. Overall, the experiments are well-designed and mostly robust (see comment regarding the sample size and data analysis of the EAG experiments) but do not always support the claims of the authors. For example, since no experiments were conducted under constant conditions, the circadian (i.e., driven by the internal clocks) effects are not being quantified here. In addition, knocking down the expression of a gene showing daily variations in its expression and observing an effect on olfactory sensitivity is not sufficient to show its role in the daily olfactory rhythms. Knowledge gaps are not well supported by the literature, and overstatements are made throughout the manuscript. Our detailed comments are listed below.

    Major comments

    Introduction

    Several statements made in the introduction are misleading and suggest that authors are trying to exaggerate the impact of their work. For example, "Furthermore, different species of mosquitoes exhibit plasticity and distinct rhythms in their daily activity pattern, including locomotion, feeding, mating, blood-feeding, and oviposition, facilitating their adaptation into separate time-niches (7, 8), but the underlying molecular mechanism for the heterogenous temporal activity remains to be explored." is not accurate since daily rhythms in mosquitoes' transcriptomes, behavior, and olfactory sensitivity have been the object of several publications. Even though some of them are listed later in the introduction, they contradict the claim made about the knowledge gap. See:

    Rund, S. S., Gentile, J. E., & Duffield, G. E. (2013). Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC genomics, 14(1), 1-19

    Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., & Duffield, G. E. (2011). Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences, 108(32), E421-E430

    Rund, S. S., Bonar, N. A., Champion, M. M., Ghazi, J. P., Houk, C. M., Leming, M. T., ... & Duffield, G. E. (2013). Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Scientific reports, 3(1), 2494

    Rund, S. S., Lee, S. J., Bush, B. R., & Duffield, G. E. (2012). Strain-and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. Journal of insect physiology, 58(12), 1609-1619

    Leming, M. T., Rund, S. S., Behura, S. K., Duffield, G. E., & O'Tousa, J. E. (2014). A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti. BMC genomics, 15(1), 1-9

    Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

    Rivas, G. B., Teles-de-Freitas, R., Pavan, M. G., Lima, J. B., Peixoto, A. A., & Bruno, R. V. (2018). Effects of light and temperature on daily activity and clock gene expression in two mosquito disease vectors. Journal of Biological Rhythms, 33(3), 272-288

    The knowledge gap brought up in the next paragraph of the introduction doesn't reflect the questions asked by the experiments: "But, how the pacemaker differentially influences peripheral clock activity present in the olfactory system and modulates olfactory sensitivity has not been studied in detail." Specifically, the control of peripheral clocks by the central pacemaker has not been evaluated here.

    "In vertebrates and invertebrates, it is well documented that circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" should also cite work on cockroaches and kissing bugs:

    Lubinski, A. J., & Page, T. L. (2016). The optic lobes regulate circadian rhythms of olfactory learning and memory in the cockroach. Journal of Biological Rhythms, 31(2), 161-169

    Page, T. L. (2009). Circadian regulation of olfaction and olfactory learning in the cockroach Leucophaea maderae. Sleep and Biological Rhythms, 7, 152-161

    Vinauger, C., & Lazzari, C. R. (2015). Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus. Journal of Experimental Biology, 218(19), 3110-3117

    The sentence: "Previous studies showed that synaptic plasticity and memory are significantly influenced by the strength and number of synaptic connections (43, 44)." should be nuanced as the role of neuropeptides such as dopamine has also been showed to influence learning and memory in mosquitoes:

    Vinauger, C., Lahondère, C., Wolff, G. H., Locke, L. T., Liaw, J. E., Parrish, J. Z., ... & Riffell, J. A. (2018). Modulation of host learning in Aedes aegypti mosquitoes. Current Biology, 28(3), 333-344

    Wolff, G. H., Lahondère, C., Vinauger, C., Rylance, E., & Riffell, J. A. (2023). Neuromodulation and differential learning across mosquito species. Proceedings of the Royal Society B, 290(1990), 20222118

    Overall, the paragraph dealing with the idea that "circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" is very confusing. This paragraph discusses mechanisms of learning-induced plasticity but seems to ignore the simplest (most parsimonious) explanations for the circadian regulation of learning (e.g., time-dependent expression of genes involved in memory consolidation). In addition, the sentence quoted above is circumvoluted to simply say that training at different times of the day affects memory acquisition and consolidation. Although the authors did look at one gene involved in neural function, learning, memory, or circadian effects were not analyzed in this study. Please reconsider the relevance of the paragraph.

    The sentence: "But, how the brain of mosquitoes entrains circadian inputs and modulates transcriptional responses that consequently contribute to remodel plastic memory, is unknown." should be rephrased. First, it should be "entrains TO circadian inputs", and second, it suggests that the study will be investigating circadian modulation of learning and memory, which is not the case. Furthermore, the term "remodel plastic memory" is unclear and doesn't seem to relate to any specific cellular or neural processes.

    Given the differences in mosquito chronobiology observed even between strains, why perform the RNAi and EAGs on a different species of Anopheles than the one used for the RNAseq (or vice versa)?

    Results

    "As reported earlier, a significant upregulation of period and timeless during ZT12-ZT18 was observed in both species (Figure 1C)." Please provide effect size and summary statistics.

    "Next, the distribution of peak transcriptional changes in both An. culicifacies and Ae. aegypti was assessed through differential gene-expression analysis. Noticeably, An. culicifacies showed a higher abundance of differentially expressed olfactory genes (Figure 1D)" Please provide effect size and summary statistics.

    "Taken together, the data suggests that the nocturnal An. culicifacies may possess a more stringent circadian molecular rhythm in peripheral olfactory and brain tissues." What do the authors mean by "stringent"? At this point, this should be stated as a working hypothesis, as the statement is not backed up by the data. It is possible that the fewer differentially expressed genes of Aedes aegypti are more central to regulatory networks and cascade into more "stringent" rhythmic control of activities and rhythms.

    The section title: "Circadian cycle differentially and predominantly expresses olfaction-associated detoxification genes in Anopheles and Aedes" doesn't make sense. The expression of genes can be modulated by circadian rhythms, but cycles don't express genes. Please rephrase. In addition, this whole section deals with "circadian rhythms" while no experiment has been conducted under constant conditions. The observed daily variations are therefore diel rhythms until their persistence under constant conditions is established.

    "The downregulated genes of Ae. aegypti did not show any functional categories probably due to the limited transcriptional change." Could the authors explain if this is actually the phenomenon or due to a lack of temporal resolution in the study design (i.e., 4 time points)?

    "a GO-enrichment analysis was unable to track any change in the response-to-stimulus or odorant binding category of genes (including OBPs, CSPs, and olfactory receptors)." This finding doesn't corroborate the statements made previously and doesn't align with previously published studies. Is it due to pitfalls in the study design?

    "In contrast, three different clusters of OBP genes in Ae. aegypti showed a time-of-day dependent distinct peak in expression starting from ZT0-ZT12 (Figure 2F)." Please provide summary statistics.

    "In the case of An. gambiae, the amplitudes of odor-evoked responses were significantly influenced by the doses of all the odorants tested (repeated measure ANOVA, p {less than or equal to} 2e-16) (Figure S4B)." Did the authors use a positive control for the EAGs? How did the authors normalize the responses across the two species? Given the way the data is presented, how were the data normalized to allow inter-species comparisons? In addition, It is highly unlikely that all the mosquito preps used in the EAG assay responded to all the odors tested. If that was the case, then the dataset includes missing data for certain odors and time points. We believe the authors have ensured there are at least a certain number of responses per odor and time point combinations. If this is true, repeated measures ANOVA is not suited for analyzing this data because this statistical technique requires all repeated measures within and across preps without missing values. Also, the authors need to correct the summary statistics for multiple comparisons within this framework to avoid inflating type-I errors. Has this been done?

    "Ae. aegypti was found to be most sensitive to all the odorants (4-methylphenol, β-ocimine, E2-nonenal, benzaldehyde, nonanal, and 3-octanol) during ZT18-20 except sulcatone (Figure 3C - 3H)." Although some of these chemicals are associated with plants and Ae. aegypti is suspected to sugar feed at night, how do the authors explain that the peak olfactory sensitivity occurs at night for compounds such as nonanal? It would be interesting to discuss how these results compare to previous studies such as:

    Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

    "Additionally, our principal components analysis also illustrates that most loadings of relative EAG responses are higher towards the Anopheles observations (Figure S4C)." The meaning of this sentence is unclear? Please clarify.

    "Taken together these data indicate that An. gambiae may exhibit higher antennal sensitivity to at least five different odorants tested, as compared to Ae. aegypti." As mentioned above, how did the authors normalized across species to allow comparisons? If not normalized, how do you ensure that higher response magnitudes correlate with higher olfactory sensitivity, given potential differences in the morphology or size differences between the two species? Furthermore, An. gambiae has been exclusively used in the EAG assay. Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable. This, however, is a major caveat in the experiment design, given previous studies (indicated below) have reported species-specific variations in olfactory sensitivity. In its present form, the EAG data from An. gambiae is not a piece of appropriate evidence that the authors could use to complement or substantiate the findings from other aspects of this study on An. culicifacies.

    i. Wheelwright, M., Whittle, C. R., & Riabinina, O. (2021). Olfactory systems across mosquito species. Cell and Tissue Research, 383(1), 75-90.

    ii. Wooding, M., Naudé, Y., Rohwer, E., & Bouwer, M. (2020). Controlling mosquitoes with semiochemicals: a review. Parasites & Vectors, 13, 1-20.

    iii. Gupta, A., Singh, S. S., Mittal, A. M., Singh, P., Goyal, S., Kannan, K. R., ... & Gupta, N. (2022). Mosquito Olfactory Response Ensemble enables pattern discovery by curating a behavioral and electrophysiological response database. Iscience, 25(3).

    "Similar to An. gambiae, a comparatively high amplitude response was also observed in An. stephensi (Figure S4D)." This is interesting but what would be even more relevant to the present study is to discuss how the time-dependent responses compare between the two Anopheles species.

    The paragraph titled "Daily temporal modulation of neuronal serine protease impacts mosquito's olfactory sensitivity" is confusing because the authors move on to test the effect of knocking down a serine protease gene (found to be differentially expressed throughout the day) on olfactory sensitivity. While this is interesting in and of itself, the link between the role of this gene in learning-induced plasticity, the circadian modulation of "brain functions" and olfactory sensitivity is 1) unclear and 2) not explicitly tested. We agree with the authors that what has been tested is "the effect of neuronal serine protease on circadian-dependent olfactory responses," but the two paragraphs leading to it seem to be extrapolating functional links that have yet to be determined. In this context, their conclusions that "Our finding highlights that daily temporal modulation of neuronal serine-protease may have important functions in the maintenance of brain homeostasis and olfactory odor responses." is misleading because although they used the hypothetical "may", the link between the temporal modulation of one serine protease gene and the maintenance of brain homeostasis is not explicitly tested here.

    Discussion

    The first sentence of the discussion: "In this study, we provide initial evidence that the daily rhythmic change in the olfactory sensitivity of mosquitoes is tuned with the temporal modulation of molecular factors involved in the initial biochemical process of odor detection i.e., peri-receptor events" is not true since studies from Rund and Duffield previously revealed the daily modulation of OBP gene expression. It also contradicts the next sentence: "The findings of circadian-dependent elevation of xenobiotic metabolizing enzymes in the olfactory system of both Ae. aegypti and An. culicifacies are consistent with previous literature (26, 31), and we postulate that these proteins may contribute to the regulation of odorant detection in mosquitoes."

    The use of "circadian" in the discussion of the results is also misleading as only diel rhythms were evaluated in the present study.

    "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58)." This is not really what these references show.

    "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58), it can be hypothesized that detection of any specific signal in such a noisy environment, mosquitoes may have evolved a sophisticated mechanism for rapid (i) odor mobilization and (ii) odorant clearance, to prevent anosmia (24)." One could argue that this is a requirement for all insects, regardless of the size of their olfactory repertoire.

    "Taken together, we hypothesize that circadian-dependent activation of the peri-receptor events may modulate olfactory sensitivity and are key for the onset of peak navigation time in each mosquito species." This is not entirely accurate since spontaneous locomotor activity rhythms are also observed in the absence of olfactory stimulation. While "navigation" does imply olfactory-guided behaviors, "peak navigation time" appears to be driven by other processes. See, for example, all studies testing mosquito activity rhythms in locomotor activity monitors.

    "Due to technical limitations, and considering the substantial data on the circadian-dependent molecular rhythmicity" please clarify what the technical limitations were. Is this something that prevented the authors specifically, or something tied to mosquito biology and would prevent anybody from doing it? Also, why couldn't the transcriptomic analysis be performed on An. gambiae?

    "In contrast to An. gambiae, the time-dose interactions had a higher significant impact on the antennal sensitivity of Ae. aegypti. An. gambiae showed a conserved pattern in the daily rhythm of olfactory sensitivity, peaking at ZT1-3 and ZT18-20." These two sentences are very confusing. Doesn't it simply mean that the co-variation is not linear or not the same across odors? In addition, what does it mean for a pattern to be more conserved? How can one conclude about the "conserved" nature of a pattern by looking at time-dependent variations in dose-response curves?

    "Together these data, we interpret that mosquito's olfactory sensitivity possibly does not follow a fixed temporal trait" is unclear and suggests that the authors are discussing global versus odor-specific rhythms. Please rephrase.

    "Moreover, we hypothesize that under standard insectary conditions, mosquitoes may not need to exhibit foraging flight activity either for nectar or blood, and during the time course, it may minimize their olfactory rhythm, which is obligately required for wild mosquitoes." This hypothesis is not supported by the results of the study and contradicts work by others (Rund et al., Eilerts et al., Gentile et., etc).

    The same comment applies to "Therefore, it is reasonable to think that the mosquitoes used for EAG studies may have adapted well under insectary settings and, hence carry weak olfactory rhythm." as this statement is not supported by results of the present study or comparisons of the results to previous studies based on field-caught mosquitoes. Although it is an interesting question to ask in the future, it should be stated as a future research avenue rather than a working hypothesis that results from the present study.

    "Aedes aegypti displayed a peak in antennal sensitivity at ZT18-20 to the higher concentrations of plant and vertebrate host-associated odorants tested. Given the time-of-day dependent multiple peaks (at ZT6-8 and ZT18-20 for benzaldehyde and at ZT12-14 and ZT18-20 for nonanal) in antennal sensitivity to different odorants, our data supports the previous observation of bimodal activity pattern of Ae. aegypti (50)." Rephrase by saying that results are "aligned with the previous observations of bimodal activity". Olfactory rhythms don't "support" the activity patterns because olfactory processes and spontaneous locomotor activity are independent processes.

    "our preliminary data indicate that Anopheles spp. may possess comparatively higher olfactory sensitivity to a substantial number of odorants as compared to Aedes spp." Consider removing this sentence unless the way the data has been normalized to allow for comparisons between species is clarified.

    In "A significant decrease in odorant sensitivity for all the volatile odors tested in the CYP450-silenced Ae. aegypti," please change "silenced" to "reduced" because RNAi doesn't silence (i.e. knockout) gene expression.

    The title "Neuronal serine protease consolidates brain function and olfactory detection" is extremely misleading. Do the authors refer to memory consolidation, which has not been tested here? What is brain function consolidation??

    The reference used in "Despite their tiny brain size, mosquitoes, like other insects, have an incredible power to process and memorize circadian-guided olfactory information (7)." is not appropriate. Also, "circadian-guided" is unclear. Consider replacing it with "circadian-gated".

    What is the "the homeostatic process of the brain"?

    "the temporal oscillation of the sleep-wake cycle of any organism is managed by the encoding of experience during wake, and consolidation of synaptic change during inactive (sleep) phases, respectively (70)." By experience, do the authors refer to learning? This seems out of topic as this process has not been evaluated here.

    "We speculate that after the commencement of the active phase (ZT6-ZT12), the serine peptidase family of proteins in the brain of Ae. aegypti mosquitoes may play an important function in consolidating brain actions (after ZT12) and aid circadian-dependent memory formation." The value of this statement is unclear. Circadian-dependent memory formation is not being evaluated here, and the results from the present study do not directly support this speculation, also because other processes involved in memory formation are not evaluated here. This seems at odds with the literature on learning and memory.

    "Subsequent work on electrophysiological and neuro-imaging studies are needed to demonstrate the role of neuronal-serine proteases in the reorganization of perisynaptic structure." Sure. But the link between "the role of neuronal-serine proteases in the reorganization of perisynaptic structure" and rhythms in olfactory sensitivity is unclear.

    As a general comment, EAGs seem inappropriate to evaluate the effect of the central-brain processing in the regulation of peripheral olfactory processes. This is a critical comment that needs to be considered by the authors and clarified in the manuscript. If rhythms of central brain processes are important for olfactory-guided behaviors, these should be evaluated at the level of the central brain or via behavioral metrics. The effect of the RNAi knockdowns on peripheral sensitivity is interesting, but its link with central processes is unclear and doesn't support the speculations made by the authors about learning and memory.

    Methods

    No explanations are provided for how the EAG data are normalized to allow comparisons between species.

    Figures

    Figure 1: The daily rhythm depicted in A, are not representative of the actual profiles. See: Benoit, J. B., & Vinauger, C. (2022). Chapter 32: Chronobiology of blood-feeding arthropods: influences on their role as disease vectors. In Sensory ecology of disease vectors (pp. 815-849). Wageningen Academic Publishers. Or any other paper on mosquito activity rhythms.

    Figure 3 and 4: The EAG results are plotted twice. This is redundant and misleading as it makes the reader think there is more data than actually presented.

    Figure 5: Please clarify the sample size for each panel. In C - F, what would be used as a reference? In other words, what is a Relative EAG Response of 1? And if it is "relative", are the units really mV? In E and F, it would be great to show how the Ethanol control compares to the no solvent condition. This could be placed in supplementary materials.

    Figures 5 and 6, given the dispersion in the EAG data, the treatments where N=40 appear robust, but the interpretation of results from treatments where N=6 may be limited due to the low sample size. This limitation is visible in Figure 5F, for example, where ABT-Aceto is different from Cont-Aceta but not PBO-Aceto because one individual shows a higher response.

    Figure S6: how does this support that synaptic plasticity is influenced by "Time-of-day dependent modulation of serine protease genes in the brain"?

    Minor comments

    What do the authors mean by "consolidation of brain functions"? Memory consolidation? Please clarify.

    In "Similar to previous studies (26), the expression of a limited number of rhythmic genes was visualized in Ae. aegypti" please replace "visualized" with "observed".

    Figure 2A, please clarify in the caption what FDR stands for.

    In "To further establish this proof-of-concept in An. gambiae, three potent CYP450 inhibitors, aminobenzotriazole(52), piperonyl butoxide(53), and schinandrin A (54), was applied topically on the head capsule of 5-6-day-old female mosquitoes" replace "was applied" with "were applied".

    "Interestingly, our species-time interaction studies revealed that An. gambiae exhibits time-of-day dependent significantly high antennal sensitivity to at least four chemical odorants compared to Ae. aegypti, except phenol." is unclear. Please reword.

    In "Similar observations were also noticed with An. stephensi." replace "noticed" with "made".

    Significance

    Such a study has the potential to be valuable for the field, but its value and significance are hindered by an accumulation of overstatements, the fact that prior work in the field has been minimized or omitted, and a lack of support for the stated conclusions.

    In this context, the advances are only slightly incremental compared to the work produced by Rund et al., and the mechanistic hypotheses emitted to link the genes selected for knockdown experiments and olfactory sensitivity are not clearly supported by the evidence presented here. The main strength of the paper is to show the role of CYP450 in olfactory sensitivity.

    The audience is fairly broad and includes insect neuro-ethologists, molecular biologists, and chronobiologists.

    Our field of expertise:

    • Mosquito chemosensation
    • Learning and memory
    • Chronobiology
    • Electrophysiology
    • Medical entomology
  4. Version published to 10.1101/2023.10.19.563057v1 on bioRxiv