Blood-derived dietary protein promotes sleep in the mosquito Aedes aegypti

Reviewer #1 (Public review):

Summary:

The presented investigation aims to expand the sleep definition and its relationship with blood meal and/or circadian clock in the mosquito, Aedes aegypti. The authors exhausted the established sleep analytical paradigm and three behaviour toolkits: LAM10, EthoVision, and DART. They also investigated the potential underlying molecular mechanism by using dsRNA injection (LkR) and KO mosquito (Cyc-/-).

Strengths:

The authors presented a very solid dataset showing posture changes and increase in the arousal threshold of mosquito after 10 minutes of immobility. This is major clarification and extension to our understanding in insect sleep beyond Drosophila. Inclusion of analytical parameters such as bout length, waking activity and pDoze/Wake provide critical reminder for other investigators of the steps needed for defining sleep in a new species. The investigation, with its technical span in behaviour assays, therefore, establish a good standard for mosquito sleep analysis to the same quality seen in the landmark studies (Shaw et al 2000 and Hendricks et al 2000) for Drosophila sleep. The pioneering data showing clear effect of blood meal and LkR reduction on locomotion and sleep provides an entry point for further investigations. The author has addressed previous concern on coincidence of sleep increase and locomotion reduction by using their two high-res. video tracking velocity or pDoze/Wake, showing that the "sleepy" mosquitos remain capable to reach high speed locomotion albeit less frequently. The authors also discuss the possibility of ATP and alternative explanation regarding sugar content in diet.

Reviewer #2 (Public review):

Summary:

Zhang et al. investigate how blood feeding and dietary protein influence sleep in the mosquito Aedes aegypti. The authors first establish a behavioural definition of sleep using postural analysis and arousal threshold measurements, then demonstrate that both blood meals and a bovine serum albumin (BSA)-based protein diet increase sleep for several days. They further show that RNAi-mediated knockdown of the leucokinin receptor (Lkr) enhances sleep, implicating neuropeptide signalling in the regulation of postprandial sleep.

Strengths:

The central question is well-motivated, and the experimental approach is systematic. The use of multiple independent methods to characterise sleep - postural analysis, infrared activity monitoring, videography, and arousal threshold - provides converging evidence. The 10-minute immobility criterion is grounded in the arousal threshold data, bouts exceeding 10 minutes corresponding to the first bin at which a significant effect emerges. The demonstration that the sleep increase is already detectable before oviposition establishes that the phenotype begins with feeding rather than with the completion of the reproductive cycle. The BSA feeding experiment is a particularly effective demonstration that dietary protein, rather than other blood components, is a key regulator of the sleep increase. The conservation of leucokinin signalling in sleep regulation between Drosophila and Ae. aegypti is a noteworthy finding that adds comparative depth. The "opportunistic versus determined" host-seeking distinction is appropriately framed as a hypothesis for future testing rather than as a conclusion drawn from the present data, and the limits of the design with respect to reproductive physiology are stated explicitly.

Weaknesses:

(1) Confound of reproduction and sleep. Blood and BSA both support egg development, so neither condition isolates nutrient sensing from reproductive physiology. The relative contributions of diet, egg development and post-reproductive recovery remain undetermined.

(2) Sleep versus reduced locomotion. The pDoze and pWake measures are defined here as proportions of time above or below a velocity threshold, rather than as the per-minute transition probabilities of the established definition (Wiggin et al. 2020, PNAS). So defined, they are equivalent to percent sleep and percent wake and cannot distinguish a sleep-like state from the mechanical consequences of engorgement.

(3) Data availability. Raw data are stated to be available on request rather than deposited in a public repository, which makes independent reanalysis less straightforward than it need be.

Author response:

The following is the authors’ response to the original reviews.

We thank both reviewers for their thoughtful and constructive evaluations of our manuscript. We are grateful that both reviewers found the study to provide a strong behavioral framework for defining sleep in Aedes aegypti and appreciated the breadth of the behavioral and genetic approaches used. We also appreciate the reviewers’ careful identification of several issues requiring clarification, particularly regarding the interpretation of post-blood-meal sleep, the support for the 10-min sleep threshold, possible nutritional confounds in the BSA experiments, the framing of the host-seeking model, and the description of statistical analyses. In the revised manuscript, we have addressed these concerns by clarifying our rationale, tempering several conclusions, revising the statistical reporting and methods, explicitly stating sample sizes, and expanding the Discussion to better acknowledge limitations and alternative interpretations. Where appropriate, we have also revised the text to distinguish more clearly between increased sleep and reduced locomotion, and to frame mechanistic conclusions more cautiously.

Public Reviews:

Reviewer #1 (Public review):

(1) Conventionally, a coincidence of sleep increase and locomotion reduction would weaken the certainty of a sleep increase assessment. The authors implied this concurrence observed after blood meal is derived from internal "drowsy" neural state instead of physical "cripple", but they did not use their two high-resolution video tracking velocity or pDoze/Wake to clarify this.

Thank you for addressing this point. We understand the need to validate locomotion when used as a readout of sleep. We note that analysis of waking activity is normalized to time spent awake, and therefore should be separate from the time spent inactive that is classified as sleep. Based on the reviewers’ suggestions we have reanalyzed some data and revised the relevant sections in include this analysis.: In brief we performed pDoze/pWake analyses on the two high-resolution tracking video from EthoVision XT system. A velocity threshold of 0.4 mm/s was used, with velocities above 0.4 mm/s defined as wake/activity and velocities below 0.4 mm/s defined as doze/sleep state. pWake and pDoze were defined as proportional time metrics of wake/active (velocity > 0.4 mm/s) and doze/sleep (velocity > 0.4 mm/s) within each LD cycle. The conclusion that sleep is increased following blood feeding is supported by these data. We also note (as described in response to Reviewer 2, that this paper represents a step towards describing sleep in mosquitoes. We hope that future application of approaches used in Drosophila, such as brain imaging and indirect calorimetry will further refine our understanding. Along these lines, we have also included a section in the Discussion about how additional measures, including systems like FlyVista might be applied in the future.

(2) The major molecular component underlying blood meal effect on sleep/locomotion is less certain, because the BSA solution used for feeding contains ATP, which itself is able to enter haemolymph and potentially exerts sleep/locomotion effect. Additionally, the basal or control sleep recording is done after sucrose feeding. It is, however, unclear from the method if this is 10% too? And if the observed sleep level increase after a blood meal is a result of sugar level reduction in the blood (~0.1%).

We thank the reviewer for raising this important issue. We think it is unlikely that the small amount of ATP used for feeding is driving the sleep phenotype. We have now included this point as a caveat within the discussion, and explained its inclusion.

(2) Sucrose concentration in controls

We apologize that this was not clearly stated. Yes, the control mosquitoes were maintained on 10% sucrose, and we have now clarified this explicitly in the Methods and figure legends where relevant.

(3) Could the effect reflect reduced sugar intake rather than blood/protein?

We think this is unlikely, however it cannot be ruled out based on the experiments we have run. We have added discussion of this point. However, we note in fruit flies, this has been studied extensively, and loss of sugar under certain contexts reduces sleep. The points above highlight the need for systematic analysis of the dietary components that contribute to sleep in mosquitoes. While we regret being unable to include them in this manuscript, we note that many of these experiments are challenging (with many controls) and have been ongoing for over a decade (with contributions from many labs) in Drosophila.

Reviewer #2 (Public review):

(1) The authors settle on a 10-minute immobility threshold, but their own data do not convincingly support this choice… A 15-minute threshold would be better supported by the data as presented.

We appreciate this evaluation of the sleep threshold. We chose 10 minutes because the first significance in arousal threshold is at the time-point of 10-15 minutes. Therefore, we believe that sleep bouts longer than 10 minutes should be qualified as sleep. We are particularly interested in why arousal threshold continues to increas at 15 minutes. This is either incomplete sleep between minutes 10 and 15 or the presence of multiple sleep states. We have established a new system in the lab using Zantiks that we believe will allow for simultaneous recording of posture and arousal threshold. We now explicitly comment on this in the discussion, and the need for further analysis of the timeframe for which sleep is defined. Nevertheless, we believe we have honed in on a period of 10-15 minutes that serves as a good proxy for sleep regulation. We hope that this initial description of sleep in mosquitoes provides an initial step towards defining sleep, and that future studies that include techniques applied in Drosophila including brain imaging, indirect calorimetry and additional videography will define more nuanced changes in sleep. We have written in limitations and future opportunities to better define sleep throughout the manuscript.

(2) The primary experimental paradigm measures sleep beginning at Day 4 post-blood feeding, immediately after oviposition... what is being measured as ‘sleep’ could reflect post-reproductive quiescence or recovery rather than diet-induced sleep per se. The BSA experiment partially addresses this, but since BSA also triggers vitellogenesis and egg production, the confound persists.

We agree this is an important concern. Our intent in measuring sleep after oviposition was to isolate prolonged post-feeding effects from the well-established transient suppression of host-seeking that occurs during the first ~72 h after blood feeding. However, as the reviewer notes, this design does not by itself distinguish post-feeding sleep from other physiological processes associated with reproduction, including vitellogenesis, oviposition, or post-reproductive recovery. To address this issue, we included the experiment measuring sleep immediately after blood feeding, before oviposition. We agree, however, that this rationale should have been stated more clearly and that the limitation remains relevant, particularly because BSA can also support egg development. In the revised manuscript, we have therefore: In the current version we have clarified more explicitly that the immediate post-blood-meal recording was included to show that the sleep increase begins before oviposition; We have also tempered our interpretation of the Day 4–5 phenotype to avoid implying that it is purely diet-driven and fully independent of reproductive state; and expanded the Discussion to acknowledge that blood feeding, protein feeding, and reproductive physiology are closely linked in female mosquitoes and that our current experiments do not fully disentangle these processes. These changes frame the data more cautiously: blood/protein feeding is sufficient to induce a sleep-promoting state that begins immediately after feeding and persists into the post-oviposition period, but the relative contributions of nutrient sensing, egg development, and reproductive recovery remain to be determined.

(3) The opportunistic vs. determined host-seeking hypothesis… requires actual measurement of host-seeking alongside sleep to be substantiated, or at least the caveats need to be discussed more explicitly.

We agree with the reviewer. Our intention was to present this as a conceptual model motivated by the temporal dissociation between published host-seeking recovery and the prolonged sleep phenotype observed here, not as a demonstrated behavioral framework directly tested in this study. In the revised manuscript, we have substantially softened this section by clarifying that we did not directly measure host-seeking behavior in the current study; adding explicit caveats that the proposed framework remains speculative until sleep and hostseeking are measured simultaneously in the same animals across the same post-feeding time course. We appreciate this comment and agree that the distinction should be presented as a model for future testing rather than as a central conclusion established by the current data.

(4) The methods describe ‘one-way ANOVA, followed by Mann-Whitney tests with Welch’s correction,’ which is an internally inconsistent combination…

We thank the reviewer for catching this lack of clarity. We apologize for this inconsistency. We have fixed this error. In the revised manuscript, we have carefully rewritten the statistical analysis section to specify: which datasets were analyzed using parametric tests (e.g., ANOVA, with appropriate post hoc comparisons where assumptions were met), which datasets were analyzed using non-parametric tests (e.g., Mann-Whitney), and where Welch’s correction was applied, specifically for unequal-variance t-tests, not Mann-Whitney tests. We have also revised Methods, Figure legends and reporting throughout to ensure that the statistical test named in the text matches the reported test statistics. The changes include statistical methods rewritten for consistency and accuracy, and updated figure legends that include exact sample sizes. In addition, one summary spreadsheet of statistical analysis throughout this study is provided and will be submitted as a supplementary file.