Characterization of sleep and arousal threshold in female Ae. aegypti.

(A-D) Representative images of adult female Ae. aegypti either in active state or during sleeping. E) Quantification of sleep onset latency in mosquitoes using postural recordings from an iPhone 16 Pro. Sleep onset latency was significantly longer during the day than at night (ANOVA with Mann-Whitney test, F1,12=7.792, P=0.0013). Individual data points are shown as gray (day) and black (night) dots. (F-I) A modified version of Drosophila Arousal Tracking (DART) system was used to assess sleep responsiveness in mosquitoes. The setup records mosquito movements while simultaneously delivering mechanical stimuli through a digital analog converter (DAC). All measurements were taken from sleeping mosquitos and recorded hourly, beginning at ZT0. (F) Schematic of the vibratory stimulus setup used to assess percent of sleep responsiveness. (G) Arousal threshold measurements in the DART system. The response probability was greater for animals that had been inactive for 1–9 minutes than that had been inactive for (≥10 minutes) One-way ANOVA F4,145=6.817, (1-5) vs. (6-10) P= 0.2460; (1-5) vs. (11-15) P= 0.0051, N=14. (H-I) Individual mosquito trajectories showing increased sleep duration (H, paired t test, P=0.0478) and sleep bout length (I, paired t test, P=0.0041). Error bars represent the standard error of the mean (±SEM). Here and in subsequent figures, asterisks indicate the level of significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Effects of blood feeding on sleep patterns in Ae. aegypti.

(A) Schematic representation of the experimental design. Female mosquitoes, aged 3-5 days post-eclosion (dpe), were either sugar-fed or blood-fed. Following a 72-hour period of oviposition, individual animals were loaded to the infrared-based activity monitors (LAM10, diameter=10mm) used for tracking sleep and activity. (B) Sleep profile (minutes per hour) across the 24-hour cycle on Day 4 post-oviposition. Blood-fed mosquitoes (red line) displayed a consistently elevated sleep profile compared to sugar-fed counterparts (gray line). Light-colored shadows indicate the ±SEM error bar, and white and black boxes indicate the daytime and nightime, respectively. (C) Comparison of total sleep of blood-fed mosquitoes (red cycles) on Day 4 post blood meal to sugar-fed controls (gray diamonds). Blood-fed mosquitoes showed a significant increase in total sleep compared to sugar-fed mosquitoes (F1, 30= 135.8, P<0.0001). (D) Total sleep duration across Day 4 to 9 post-blood meal. Blood-fed mosquitoes (red cycles) exhibited significantly increased sleep compared to sugar-fed controls (gray diamonds) on Days 4 and 5 (Day 4: F1, 30= 135.8, P<0.0001; Day 5: F1,30=79.00, P<0.0001). (E-G) Changes of sleep architecture of blood-fed mosquitoes (red cycles) on Day 4 post blood meal compared to sugar-fed controls (gray diamonds). Blood-fed mosquitoes demonstrated a significantly lower waking propensity, P(Wake), compared to sugar-fed mosquitoes (E, F1,30=29.79, P<0.0001). Blood-fed mosquitoes exhibited a significantly higher sleeping propensity, P(Doze), compared to sugar-fed controls on Day 4 post-blood feeding (F, F1,30=64.44, P<0.0001). Error bars represent the standard error of the mean (±SEM). Data points in the bar graphs represent individual animals. Waking activity (activity per waking minute) was reduced on Day 4 post blood meal compared to that of sugar-fed animals, indicating lower movement intensity after blood feeding (G, F1,30=19.98, P<0.0001).

Validation of blood feeding induced sleep in female Ae. aegypti using EthoVision.

(A) Schematic of the setup for behavioral recording via EthoVision XT. Female mosquitoes, at the age of 1-3 dpe, were either sugar-fed or blood-fed, and immediately transferred to a 6-well plate after meal for monitoring activities over one LD cycle using EthoVision XT, which allowed for track visualization and analysis of sleep patterns. One animal was housed in one well of the plate. (B) Total daily sleep duration in blood-fed mosquitoes (red) compared to sugar-fed controls (gray). A significant increase of daily sleep exhibited in blood-fed group compared to sugar-fed (F1,12=24.01, P<0.0001). (C) Sleep profiles across the 24-hour cycle for sugar-fed and blood-fed mosquitoes via EthoVision recording. Blood-fed mosquitoes (red) showed a distinct sleep pattern, with increased sleep duration observed during both day and night periods compared to sugar-fed counterparts (gray). Light-colored shadows indicate the ±SEM error bar, and white and black boxes indicate the light and dark, respectively. (D) Representative tracks of sugar-fed (left) and blood-fed (right) mosquitoes under EthoVision recording. The red lines indicate the movement paths of the mosquitoes, illustrating differences in activity levels between the two feeding groups. (E) Total traveled distance (meters per day) by sugar-fed and blood-fed mosquitoes. Blood-fed mosquitoes (red cycles) showed a significantly shorter distance compared to sugar-fed controls (gray diamonds, F1,12=13.06, P=0.0058), indicating reduced activity levels. Error bars represent the standard error of the mean (±SEM) and each dot on the bar graphs represents individual animal.

Dietary protein increases sleep in female Ae. aegypti via BSA feeding.

(A) Schematic overview of the experimental design. Female mosquitoes, aged 3-5 dpe, were either sugar-fed or fed Bovine Serum Albumin (BSA). Following a 72-hour period of oviposition, sleep was monitored using custom-built infrared activity monitors over a 7-day recording period. (B) Comparison of total sleep of BSA-fed mosquitoes (blue squares) on Day 4 post blood meal to sugar-fed controls (gray diamonds). Total sleep duration of BSA-fed mosquitoes showed a significant increase compared to sugar-fed mosquitoes (F1,30=26.68, P<0.0001). (C) Sleep distribution across one LD 15hr:9hr cycle on Day 4 post-BSA meal. BSA-fed mosquitoes (blue line) demonstrated elevated sleep levels during both day and night periods compared to sugar-fed mosquitoes (gray line). Light-colored shadows indicate the ±SEM error bar, and white and black boxes indicate the daytime and nighttime, respectively. (D) Total sleep duration across Days 4 to 9 post-BSA feeding. BSA-fed mosquitoes (blue) exhibited significantly increased sleep compared to sugar-fed controls (gray) on Days 4 and 5 post-BSA feeding (Day 4: F1,30=26.68, P<0.0001; Day 5: F1,30=15.17, P=0.0005). (E-G) Changes of sleep architecture of BSA-fed mosquitoes (blue) on Day 4 compared to sugar-fed mosquitoes (gray). Significantly lower P(Wake) (E, F1,30=22.90, P<0.0001) and higher P(Doze) (F, F1,30=12.91, P=0.0003) was showed in BSA-fed population compared to sugar-fed group on Day 4 post-BSA feeding. Data points in the bar graphs represent individual animals and error bars represent the standard error of the mean (±SEM). BSA-fed mosquitoes exhibited reduced waking activity compared to sugar-fed controls on Day 4 post-BSA feeding (G, F1,30=23.63, P<0.0001).

Role of the leucokinin receptor (lkr) in sleep regulation of female Ae. aegypti.

(A) Schematic overview of the experimental design for sleep monitoring in dsRNA-injected mosquitoes. (B) Daily sleep duration was significantly induced in dsLKR-injected mosquitoes (magenta cross markers) compared to dsEGFP controls (green diamonds) (F1,25=15.89, P=0.0012). (C) Sleep profiles of female mosquitoes injected with either dsLKR or dsEGFP across the 24-hour cycle of Day one post injection. Mosquitoes with LKR knockdown (magenta) displayed a distinct sleep pattern, with increased sleep observed during both day and night periods compared to dsEGFP group (green). Light-colored shadows indicate the ±SEM error bar, and white and black boxes indicate the light and dark periods, respectively. (D) Total sleep duration across Days 1 to 6 post-siRNA injection. Mosquitoes injected with dsLKR (magenta line with cross markers) exhibited significantly increased sleep compared to controls injected with dsEGFP (green line with diamond markers) on Days 1 and 2 (F1,25=15.89, P=0.0012 for Day 1 and F1,25=15.18, P=0.0015 for Day 2). (E-G) Comparisons of P(Wake), P(Doze) and waking activity of mosquitoes injected with dsLKR to dsEGFP controls on Day 1 post injection. After knockdown of LKR, female mosquitoes demonstrated a significantly lower probability of waking compared to dsEGFP controls (E, F1,25=13.19, P=0.0002). In contrast, the sleeping propensity, P(Doze), was significantly induced in dsLKR-injected mosquitoes compared to dsEGFP controls (F, F1,25=9.48, P=0.0012). dsLKR mosquitoes (magenta cross markers) exhibited significantly reduced waking activity compared to dsEGFP controls (green diamonds, G, F1,25=11.71, P=0.0053). Error bars represent the standard error of the mean (±SEM), and unpaired t-test was used for significance: **P<0.01, ***P<0.001, ****P<0.0001.

Effects of blood feeding on sleep and activity in Ae. aegypti females.

(A) Total sleep duration in sugar-fed Ae. aegypti versus blood-fed with analysis in 30-min intervals. Sleep of blood-fed mosquitoes (red cycles) was significantly increased compared to sugar-fed controls (gray diamonds, F1,30=111.5, P<0.0001) (B) Paired dot plot displayed the averaged sleep for either sugar-fed or blood-fed individual subjects during day and night periods. Blood-fed mosquitoes (red dots) showed significantly higher sleep levels during both day and night compared to sugar-fed controls (light gray diamonds, F1,30=36.00, P<0.0001 for day and F1,30=44.44, P<0.0001 for night). (C) Daily activity across Days 4 to 9 post-blood meal. Blood-fed mosquitoes (red line) exhibited significantly reduced activity compared to sugar-fed controls (dark gray line) on Days 4 and 5 (F1,30=10.75, P=0.0045 for Day 4 and F1,30=10.08, P=0.0061 for Day 5). (D) Total daily activity for sugar-fed (gray diamonds) and blood-fed (red cycles) mosquitoes on Day 4 post blood meal. Blood-fed mosquitoes displayed significantly lower daily activity levels compared to sugar-fed controls (F1,30=10.75, P=0.0045). (E) Hourly activity profiles across the 24-hour cycle for sugar-fed (gray line) and blood-fed (red line) mosquitoes. Blood-fed mosquitoes showed a distinct reduced activity during both day and night periods. Light-colored shadows indicate the ±SEM error bar, and white and black boxes indicate the daytime and nighttime, respectively. Error bars represent the standard error of the mean (±SEM), and asterisks indicate the level of significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.