Light manipulation of mosquito behaviour: acute and sustained photic suppression of biting activity in the Anopheles gambiae malaria mosquito

Abstract

Background: Host-seeking behaviours in anopheline mosquitoes are time-of-day specific, with a greater propensity for nocturnal biting. We investigated how a short exposure to light presented during the night or late day can inhibit biting activity and modulate flight activity behaviour.

Results: Anopheles gambiae (s.s.), maintained on a 12:12 LD cycle, were exposed transiently to white light for 10-min at the onset of night and the proportion taking a blood meal in a human biting assay was recorded every 2 h over an 8-h duration. The pulse significantly reduced biting propensity in mosquitoes 2 h following administration, in some trials for 4 h, and with no differences detected after 6 h. Conversely, biting levels were significantly elevated when mosquitoes were exposed to a dark treatment during the late day, suggesting that light suppresses biting behaviour even during the late daytime. These data reveal a potent effect of a discrete light pulse on biting behaviour that is both immediate and sustained. We expanded this approach to develop a method to reduce biting propensity throughout the night by exposing mosquitoes to a series of 6- or 10-min pulses presented every 2 h. We reveal both an immediate suppressive effect of light during the exposure period and 2 h after the pulse. This response was found to be effective during most times of the night: however, differential responses that were time-of-day specific suggest an underlying circadian property of the mosquito physiology that results in an altered treatment efficacy. Finally, we examined the immediate and sustained effects of light on mosquito flight activity behaviour following exposure to a 30-min pulse, and observed activity suppression during early night, and elevated activity during the late night.

Conclusions: As mosquitoes and malaria parasites are becoming increasingly resistant to insecticide and drug treatment respectively, there is a necessity for the development of innovative control strategies beyond insecticide-treated nets (ITNs) and residual spraying. These data reveal the potent inhibitory effects of light exposure and the utility of multiple photic pulses presented at intervals during the night/late daytime, may prove to be an effective tool that complements established control methods.

Keywords: Anopheles gambiae; Behaviour; Biting; Blood-feeding; Circadian rhythm; Flight activity; Light; Locomotor activity; Malaria; Mosquito; Photic; Physiology.

Fig. 1

Schematic representation of the biting behavioural assays. a Standard photoschedule that the An. gambiae (s.s.) mosquitoes were reared on and experimented under. Zeitgeber time (ZT) 12 is the time of lights off, and ZT24/0 occurs at the end of the 1 h dawn transition. b Method for testing the immediate (acute) and sustained (chronic) effects of a single light pulse presented during the onset of the night on biting activity. The dotted line represents a single, one-time light pulse for all experimental mosquitoes. c Method for testing the effects of dark acclimation on blood-feeding behaviour during the late daytime and the dusk transition. d Method for assaying the immediate acute inhibition of biting behaviour by a single light pulse tested at different times throughout the night. e Method for testing the sustained (chronic) inhibition of biting behaviour 2 h following light treatment and with multiple light pulses delivered throughout the night. Each batch of mosquitoes was assayed for blood-feeding in the dark immediately prior to the next time series batch being exposed to light. The dotted line represents all experimental mosquitoes were light-pulsed repeatedly. White-black bar represents the environmental LD cycle. A grey section (e) represents subjective day when the LD cycle ends in the experiment, and there is a transition to constant darkness. Arrows directly above the light-dark bar pointing down indicate the time of blood meal (Investigator’s arm) being offered (b, c, d, e); arrows directly below the light-dark bar pointing up to indicate the time specific presentation of white light (b, d, e). A black box below the light-dark bar indicates the administration of a pretreatment of darkness during the light phase of the LD cycle (c)

Fig. 2

Experiment 1: Single light pulse treatment revealed immediate (acute) and sustained (chronic) suppression of biting. Representative biting percentages of mosquitoes either after receiving a single 10 min, ~300 lux white light pulse at ZT12 or transitioning normally into the constant dark (control treatment). Light and control treated mosquitoes were simultaneously offered a blood meal for 6 min at ZT12.2 and then again at ZT14, 16, 18, and 20. The percentage of mosquitoes per container that blood-fed was recorded immediately after the blood meal was offered by observing blood in the abdomen. Two-way ANOVA (effect of treatment, F _(1,35) = 13.8, P < 0.001; effect of ZT, F _(4,35) = 11.6, P < 0.001; interaction, F _(4,35) = 3.7, P < 0.05) followed by Tukey post-hoc tests (*P < 0.05). Values shown are mean ± SEM

Fig. 3

Experiment 2: Mosquitoes exposed to darkness during the late daytime exhibit increased biting activity. Mosquito biting behaviour is increased after receiving a 15 min dark pretreatment when tested at two different times of the late day including during the dusk transition. Blood meals were offered at ZT9.5 and ZT11.5 in the dark following either a dark pretreatment or normal exposure to light of the LD cycle and dusk transition (controls). Dusk progression is indicated by the horizontal white/black bar occurring at ZT11-12. Two-way ANOVA (effect of treatment, F _(1,15) = 12.0, P = 0.003; effect of ZT, F _(1,15) = 2.6, P = 0.125; interaction, F _(1,15) = 0.02, P = 0.907), followed by post-hoc tests (*P < 0.05). Values shown are mean ± SEM

Fig. 4

Experiment 3: Immediate (acute) inhibition of biting behaviour by a single light pulse tested at different times of the diel night. Inhibition of blood-feeding in mosquitos that were blood-fed in the light versus fed in the dark. The inhibitory effect of light on blood-feeding declined as the dark period progressed. Blood meals were offered during the biological night at ZT12, 14, 16, 18, 20 and 22 under either light or dark conditions. Experimental mosquitoes were subject to a single light pulse while simultaneously being blood-fed. Two-way ANOVA (effect of treatment, F _(1,32) = 50.6, P < 0.001; effect of ZT, F _(5,32) = 2.2, P = 0.097; interaction, F _(5,32) = 1.8, P = 0.161), followed by post-hoc tests (*P <0.05, **P <0.01 and ***P <0.001). Values are mean ± SEM

Fig. 5

Experiment 4: Sustained (chronic) inhibition of biting behaviour by multiple light pulses delivered during the diel night. Inhibition of blood-feeding in mosquitos that were blood-fed one h 50 min after receiving a light pulse versus controls. Light pulses were administered every 2 h throughout the dark phase starting at ZT12. Excluding mosquitoes offered a blood meal at ZT14, experimental mosquitoes were subject to multiple light pulses. Blood meals were offered for 6 min during the biological night at ZT14, 16, 18, 20, 22, and CT24/0 under dark conditions. Two-way ANOVA (effect of treatment, F _(1,47) = 51.9, P < 0.001; effect of ZT, F _(5,47) = 1.6, P = 0.194; interaction, F _(5,47) = 3.4, P = 0.013) followed by post-hoc tests (*P < 0.05, **P < 0.01, and ***P < 0.001). Values are mean ± SEM

Fig. 6

Experiment 5: Light exposure during the night modulates locomotor/flight activity behaviour in a time-specific manner. a Representative locomotor/flight activity plots during a 4 h time window of four individual mosquitoes. Mosquitoes were entrained to 12 h/12 h LD conditions (with one h dawn and dusk transitions). Activity during control days (days 2 and 3) under this normal photoperiod are shown (upper panels), and on day 4 of the experiment, mosquitoes were exposed to a 30 min, 300 lux white light pulse administered at a precise circadian time (lower panels). Control Day 1 is not shown, which was the first 24 h after introduction to the LAM unit. Running from left to right are four individual mosquitoes exposed to light at ZT12, ZT16, ZT22 or CT24/0. The 4 h time window shown for each mosquito is centred on this pulse time. b Mean flight activity during a 30 min light pulse delivered at precise Zeitgeber time (ZT)/circadian time (CT) (dotted bars) compared to activity during the same time period on the two prior, non-pulsed days (black and striped bars). One-way repeated measures ANOVAs (ZT12, effect of day, F _(2,53) = 17.9, ***P < 0.001; ZT16, F _(2,164) = 9.3, ***P < 0.001; ZT22, F _(2,65) = 14.4, ***P < 0.001; and ZT24, F _(2,29) = 4.2,*P <0.05) followed by post-hoc tests (*P < 0.05, **P < 0.01, ***P < 0.001). Values shown are mean ± S.E.M. of individual mosquito activity (n = 16–38 mosquitoes per time point). Activity is scored as the average number of LAM beam crossings per minute during the 30 min pulse duration determined for each individual mosquito

Fig. 7

Sustained effects of a 30 min light pulse on An. gambiae mosquito flight activity. Mean flight activity measured during a 60 min period at various intervals over 8 h (1, 2, 3, 4 and 8 h), following exposure to a 30 min light pulse. Analysis was conducted at times of the circadian cycle that correspond with changes in activity that were detected during the 30 min exposure to light, i.e. at ZT12 (a), ZT16 (b), ZT22 (c) and CT24/0 (d) (see also Fig. 6 for mean flight activity measured during the light pulse administration). Mean flight activity is compared on the day of treatment (triangle symbols) to activity during the same period on the two prior, non-pulsed days (circle and square symbols). A pulse administered at ZT22 significantly inhibited activity during 1 h following the pulse (i.e. at ZT22.5–23.5; Table 1). No other significant differences between treatment and control days were observed during the 8 h following the time-specific light pulses Table 1). One-way RM-ANOVAs followed by post-hoc tests (***P < 0.001). Values shown are mean ± S.E.M. of individual mosquito activity. Activity is scored as the average number of LAM beam crossings per minute during the 60 min duration determined for each individual mosquito at the specified time intervals