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 a KO mosquito (Cyc-/-).

Strengths:

The authors presented a very solid dataset showing posture changes and an increase in the arousal threshold of the mosquito after 10 minutes of immobility. This is a major clarification and extension to our understanding of 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 establishes 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 a clear effect of blood meal and LkR reduction on locomotion and sleep provides an entry point for further investigations.

Weaknesses:

Despite the versatility of the behaviour and transgenic methods in this manuscript, there are two logical gaps in the conclusion, which are related to the effect of blood meal/BSA/LkR KD on A. aegypti sleep:
(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.
(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%).

Reviewer #2 (Public review):

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. The authors propose that elevated sleep persists well beyond the restoration of host-seeking behaviour, suggesting the existence of distinct "opportunistic" versus "determined" host-seeking phases.

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 BSA feeding experiment is a particularly effective demonstration that dietary protein, rather than other blood components, is the 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.

Weaknesses

(1) Sleep definition.

The authors settle on a 10-minute immobility threshold, but their own data do not convincingly support this choice. The arousal threshold data (Figure 1G) show no significant difference between the 1-5 min and 6-10 min bins (P=0.246), with significance emerging only at the 11-15 min bin. The postural analysis likewise indicates that sleep-associated postures appear at ~20 min during the day and ~11 min at night. A 15-minute threshold would be better supported by the data as presented. The previous literature used 120 minutes for this species (Ajayi et al. 2022), making this a dramatic shift.

(2) Confound of reproduction and sleep.

The primary experimental paradigm measures sleep beginning at Day 4 post-blood feeding, immediately after oviposition. Animals have undergone gut distension, vitellogenesis, and oviposition, and 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 (as the authors note), the confound persists.

(3) Opportunistic vs. determined host-seeking hypothesis.

This framework is presented as a key conceptual contribution, but the paper contains no data on host-seeking behaviour. The authors infer two phases from the temporal mismatch between a 72-hour host-seeking suppression window (from prior studies) and elevated sleep through Day 5 (~120 hours). While this is an interesting hypothesis, it requires actual measurement of host-seeking alongside sleep to be substantiated, or at least the caveats need to be discussed more explicitly.

(4) Statistical approach.

The methods describe "one-way ANOVA, followed by Mann-Whitney tests with Welch's correction," which is an internally inconsistent combination: Mann-Whitney is non-parametric and does not use Welch's correction (which applies to t-tests). Throughout the figures, F-statistics (parametric) are reported alongside what appear to be non-parametric tests. The statistical framework needs to be clarified and made consistent. Exact sample sizes per group should also be stated explicitly in the methods for all experiments.