In this study, we have explored how light can modify An. gambiae blood-feeding behaviour. We used timed white light treatments under controlled laboratory conditions presented singularly or as multiple pulses at various intervals throughout the night, to explore the efficacy of light to reliably suppress adult female mosquito biting and modify flight activity. The current work builds upon our earlier studies of time-of-day-specific blood-feeding activity in An. gambiae mosquitoes, and how temporal changes in behaviour correlate with physiological and molecular changes in olfactory modalities . Using a human arm blood-feeding assay, the Pimperana strain exhibits a 24 h rhythm in biting, peaking during the early night. This rhythm is preserved under constant conditions of darkness, thereby revealing that the rhythm is also driven, at least in part, by the endogenous circadian clock of the mosquito [19, 20]. The experiments in the current study also complement the work of Das & Dimopoulos , that explored biting suppression in an outbred strain during the late night using short pulses of bright light (2 min, 800–1000 lux) and with an artificial membrane feeder in the absence of a human .
The current study was designed first to explore the potential modulation of blood-feeding behaviour by light specifically during the early night and late daytime, times of the 24 h day that are proving to be critical in residual biting activity even when ITNs have been introduced to a malaria endemic area. Secondly, on establishing an efficient and effective level of suppression during the early night, we wished to determine if the immediate suppressive effect could be reproduced at all times of the night. Thirdly, on discovery of a long duration (2–4 h) chronic suppression of biting following a single pulse of light, and to develop an efficient method to suppress biting activity throughout the night, mosquitoes were exposed to multiple short, discrete pulses of light. To complement the biting studies, and since flight activity is a component of host-seeking behaviour , we tested for modulatory effects of a light pulse treatment presented at different times of the 24 h day upon a quantitative measure of general mosquito flight activity.
Anopheline mosquitoes are predominately nocturnal, concentrating their feeding and flight activity (swarming, migration and host-seeking) to the night [12, 13, 16, 20, 27]. We, therefore, hypothesised that a light pulse would inhibit or reduce the propensity to bite a human host when administered during the dark phase of the LD cycle. In Experiment 1 of our investigation, An. gambiae mosquitoes demonstrated an acute (tested at ZT12:10) and sustained (tested at ZT14) inhibition of biting activity after a receiving a single 10 min white light pulse presented at ZT12. Remarkably, the level of biting suppression assayed on the human arm immediately after cessation of the light pulse was at an average of 42% of biting control levels. This inhibition remained high at 32% for 2 h after delivery of the light pulse; and somewhat surprisingly, the photic effect upon biting behaviour in some trials was sustained for up to 4 h after the pulse.
The results of our second experiment in which we examined the potential for light exposure during the late daytime to suppress biting activity were as predicted from our previous work. In Rund et al.  using the same strain of mosquitoes, we had observed that the biting propensity of mosquitoes studied under diel conditions to be close to 0% during the day phase of the LD cycle . However, under constant dark (circadian) conditions, while a distinct 24-h rhythm persisted for several days, the level of biting during the ‘subjective’ daytime was elevated up to 70% of maximal night-time biting levels. We, therefore, hypothesised that the presence of light during the daytime has a direct suppressive effect on the biting behaviour of the mosquito. Furthermore, in a limited experiment, the effect of a 15 min pretreatment of darkness on biting activity was examined and revealed an elevation when tested at each of three different daytime phases of the diel cycle (ZT0, ZT4 and ZT8) . To examine this phenomenon in a more controlled and complete fashion, we tested the effect of a 15 min treatment of darkness presented during the late daytime (ZT9.25) or during dusk (ZT11.25) before undertaking the standard blood-feeding assay. The results of this procedure revealed a distinct elevation in biting activity as compared to time-matched control mosquitoes that had simply been exposed to light within the normal LD cycle. The effect was potent, as the dark-treatment during the late daytime (2 h prior to dusk or during actual dusk) resulted in a 58% elevation in biting propensity compared with control mosquitoes. Moreover, this effect was observed and equally effective at both times of the late daytime that were tested. This is an important finding since it suggests that while An. gambiae mosquitoes concentrate their blood-feeding during the night, and that this behaviour is driven by their endogenous circadian clock [19, 20], there is a residual negative masking effect of light that occurs during the daytime.
In the third experiment, we tested the immediate response of the mosquitoes to light while simultaneously offering a human blood meal. These tests were performed at one of six different times of the night to evaluate whether differential responses would occur that were time-of-day dependent. By testing at 2 h intervals, this immediate inhibition effect was found to occur during the entire night, i.e. at times tested during the early to late night (i.e. ZT12-ZT22), and resulting in a dramatic suppression of biting activity of 42–66% as compared to the time-matched controls. Interestingly, a clear incremental decline of suppression was observed as the night progressed. The differential responses that were time-of-day specific suggest an underlying circadian property of the mosquito physiology that results in the altered treatment efficacy.
Based on the observations of these experiments, we expanded this approach to photic manipulation of the mosquitoes to develop a method to reduce biting propensity throughout the night by exposing mosquitoes to a series of light pulses presented every 2 h. We hypothesised that this protocol would result in a sustained inhibition of blood-feeding behaviour throughout the duration of the night. In this fourth experiment, the protocol elicited a sustained suppression of biting activity that was observed during the early to middle of the night, as well as at the very end of the night/dawn (ZT14, 16, 18 and CT0/24). While statistically significant differences were not observed for ZT20 and ZT22, the means for the pulsed groups were lower than the time matched control groups. This was somewhat surprising as we had hypothesised that the sustained response would be equal at all times of the night. However, these data suggest in a manner similar to the immediate responses tested in experiment 3, that the sustained effects of light are influenced by an underlying circadian property of the system. Additionally, the change in biting propensity may reflect an increased homeostatic drive to blood feed as the night progresses that then competes with the light-suppressive mechanism. However, despite the reduced efficacy of light delivered during the middle of the night, the sustained response using the multiple pulse approach provided suppression of biting during the early to middle night and late night/dawn phases of the night. As these times of night are critical phases of the diel cycle when humans are most susceptible to biting events as they are unprotected when not sleeping under a bed net, the results of the experiment suggest that indeed this multi-pulse method might be effective in the control of mosquito biting events in the field.
These striking results on photic manipulation of An. gambiae blood-feeding behaviour generates several questions regarding the mode of action by which photic cues impact mosquito physiology and behaviour. It is likely that the mechanism within the brain circuitry for the acute behavioural response (suppression of biting) to light is mediated by a neuronal response (potentiation and integration) and/or phosphorylation of protein(s) due to its rapid response; whereas the sustained effect of photic stimulation is likely to result from the turnover of protein and/or de novo change in gene expression and subsequent change in protein synthesis [28, 29]. Such changes in gene expression have been observed in Drosophila head and An. gambiae head and whole body following photic stimulation [19, 30, 31]. However, it is unlikely that the behavioural effects observed in the current study are operated through changes in the circadian clock [14, 32]; yet in theory, a phase shift of the clock could change the temporal profile of the 24 h biting preference rhythm . The clock in other organisms has been shown to be reset by light by 2–3 h but not at 1 h after the start of a precisely-timed photic stimulus [33, 34]. Therefore, a shift of the circadian clock would not explain the acute suppression of biting that is observed immediately after the start of the light pulse (i.e. within 15 mins at ZT12.25), or during the actual exposure to the light, as observed at various times during the night. A shift of the clock might, however, contribute to the changes observed after two or more hours after photic treatment (e.g. at ZT14 when treated with light for 10 min at ZT12). As light presented during the early night (ZT12–14) would be predicted to produce a phase delay of the clock and light during the middle to late night (ZT15–24/0) would result in a phase advance [19, 35], these adjustments may well shift the position of the biting rhythm [19, 20]. These shifts of the clock, based on a circadian light phase response curve (PRC) for An. gambiae , might explain some but not all of the results of Experiment 4, where mosquitoes were exposed to multiple pulses of light at night. We would predict to generate relatively small shifts of the clock, and maximally a 2 h average shift when the light is presented at ZT14 (delay) or ZT15 (advance). However, since the average biting propensity of the control groups of mosquitoes in Experiment 4 was ≥ 80% when examined at different times across the night, and the biting activity of light-pulsed mosquitoes tested at ZT16 and ZT18 was ≤ 50%, factors other than a shift of the endogenous biting rhythm would have to account for this suppression of biting. The exception would be a discrete light pulse delivered at ZT22 and tested 2 h later at CT24/0 since a phase advance might shift the biting rhythm into the subjective day, and thus to a phase of the circadian cycle where we would expect reduced biting activity [19, 20].
While not explicitly tested in the current investigation, we hypothesise a dose dependency of photons per unit time, such that a higher light intensity would require a lower exposure duration to reach the desired level of suppression of feeding . For example, 1 min at 5000 lux may provide the equivalent inhibition of feeding as 10 min at 300 lux and 30 min at 30 lux. Therefore, it is plausible that an increased dose of light could unmask some of the subtle effects of light upon suppression, such as at ZT20–22 in the multi-pulse experiment, as well as simply increase the treatment efficacy at all phases of the night and late daytime. A similar prediction could be made for a dependency upon the specific wavelength(s) of light upon the efficacy of the pulse treatment [36, 37]. We predict that there will be a specific wavelength(s) of light that will have maximal efficacy in the immediate and sustained suppression of biting behaviour and related effects of suppression or elevation of flight activity. Selecting a monochromatic light source in this maximal range would allow for modulation of behaviour at lower intensities of light. Additionally, selecting a monochromatic light source that is outside of the blue colour of the spectrum, even if sub-optimal, might allow for manipulation of mosquito behaviour without disruption of human behaviour/physiology (e.g. human arousal, circadian phase shifting) [38, 39]. In the current study we purposefully used broad-spectrum white light, but having established a baseline level of suppression, testing different colours of light might allow for enhanced utility of light as a vector control method. Ultimately, testing this variable as well as different light intensities and durations of exposure is beyond the scope of the current study.
In an effort to understand the broader impact of light on An. gambiae behaviour, we examined mosquito flight activity during and several hours following treatment with white light at different phases of the circadian cycle including both the night and subjective daytime (tested while in constant darkness). Adult female mosquitoes exposed to the photic treatment demonstrated an acute flight inhibition during a 30-min light pulse when presented at the start of night at ZT12 (a 79% suppression of activity compared to the same Zeitgeber time of control days), and conversely an acute elevation of flight activity during an equivalent light pulse presented at ZT16, ZT22 or CT24/0 (with a dramatic ≥ 1000% increase of activity when the mosquito was treated at the end of night or dawn). There was also a sustained effect on flight activity during the 1 h immediately following cessation of the light when the administration of the light pulse was during the late night at ZT22; however, the activity was reduced (by 80%), not increased as observed during the time of pulse administration. In the time-specific light pulse treatment groups examined (namely ZT12, ZT16, ZT22 and CT24/0), this was the only significant change in flight activity detected over the course of the 10 h following the photic treatment.
We propose that the sudden inhibition of flight activity seen at ZT12, and elevation of activity at ZT22 and ZT24, represent ‘masked’ responses  and comparable to the immediate suppression of biting activity during exposure to the light pulse, as briefly discussed above. In this mechanism upon the area becoming illuminated the mosquito behaviour is acutely altered in a negative or positive manner, and that a shift in the circadian timing system is not directly responsible for the immediate behavioural response. Negative, positive and paradoxical masked responses to light have been documented in a similar fashion in laboratory rodents and Drosophila [40,41,42]. Furthermore, complementary to the data presented here, modifications to swarming behaviour of anophelines in response to changes in light exposure have been reported in the field and laboratory [36, 43]. For example, when located inside a dark outdoor barn, male An. maculipennis atroparvus demonstrated typical swarming behaviour; however, when the light intensity of the area increased by ~1.0 log lux, swarming abruptly stopped . Interestingly, when the light was prevented from entering the area swarming recommenced. This suggests that the acute inhibition of activity by light was, like the data we present here, a masked response to the stimulus and not an endogenous response mediated by the circadian clock. Masked responses may provide increased fitness benefits to the organism by allowing a rapid reaction to environmental change. For example, upon the sudden illumination of an occupied area, survivorship is increased (e.g. by avoiding potential predation or desiccation) . As clock-mediated adaptation takes considerably longer to manifest (> 1 h), it would be evolutionary detrimental for a clock mechanism to orchestrate such responses.
The result at ZT12 of photic suppression of flight activity is perhaps not surprising. The intense and short-lived elevation in general activity that normally corresponds with dusk/onset of night is likely related to behaviours such as the movement from resting sites, swarming, migration and sugar feeding [12, 16, 27]. An interaction of environmental variables such as light and endogenous rhythms can be seen in a modification to the profile of flight activity at dusk. Also studied using the LAM unit assay, both An. gambiae and An. coluzzii exhibit a different minute-to-minute pattern of activity depending on whether mosquitoes are exposed to a ‘natural’ dusk transition or maintained under conditions of constant darkness . As has been demonstrated in various mosquito species including anophelines, the light intensity is an important factor in the timing of swarming events [12, 36], such that intensities above certain thresholds can result in cessation of swarming or inhibition of its onset.
While we believe that the current study is the first to quantify changes in flight activity of An. gambiae mosquitoes over the 24-h circadian cycle, Jones et al.  qualitatively described a “lights on reaction” in An. gambiae flight activity when exposed to light under DD conditions and using an acoustic assay . Consistent with the current investigation, a decrease in activity was observed at ZT12, marked increases when the light was presented later in the night, and no responses observed during the subjective daytime. The consistency of observations between studies despite using different experimental approaches suggests that the acute responses to photic stimuli represent a highly reproducible behavioural response that is conserved across An. gambiae strains.
From the experimental design of the study used here, it is unclear if the observed increases in flight activity observed during the night and dawn were photophilic, photophobic or reflect an escape response. The modulation of locomotion/flight activity in our LAM assay could represent positive or negative phototaxis, such as an attraction to the exit of a shelter, or a shelter-seeking ‘escape’ response . Alternatively, it could be a neutral phototaxic response, i.e. no specific orientation, and simply represents an increase or decrease in generalised flight activity, e.g. the photic suppression of activity that we observe with a treatment at ZT12 could represent a suppression of swarming-related ‘excited’ activity. However, without further experimentation, using an arena apparatus, for example, it is unclear what the suppressed (at ZT12) or elevated flight activity (at ZT16, ZT22 and ZT24/0) represents in the natural behavioural repertoire of the mosquito.
The mechanism by which the photic cues are sensed by the mosquito, that in turn influence brain-behaviour pathways involved in the modulation of biting and flight activity, is likely to include the photoreception cascade of the compound eye [46, 47]. The use of broad spectrum white light does not favour or preclude one specific opsin photopigment or class of ommatidium involved in detecting blue, green or red wavelengths of light [14, 47, 48]. The immediate response to photic stimuli are indeed likely to be conveyed by the compound eyes and via their neuronal connections to the brain, but it is plausible that the sustained effects of the light treatment involve non-retinal photoreception. This might include the flavoprotein cryptochrome photopigment system (specifically CRY1 in An. gambiae), found expressed throughout the insect body [14, 49], and that is important in the photic-resetting mechanism of the insect circadian clock [49,50,51,52,53]. Obviously identifying the specific opsins, photoreceptors, and downstream neuronal and endocrine pathways associated with the behavioural responses reported herein generates interesting questions for future investigation.
We note this is a laboratory-based study, using one inbred mosquito line, with mosquitoes kept in small containers, and shielded from any low-level nocturnal light and from continuous exposure to host odours. To complement any field-trials, future laboratory work might include a determination of the optimal wavelength of light to produce a given behavioural response, generate a photic dose response curve, optimize the spacing and timing of light pulses, and explore further the effect of low-level nocturnal light, e.g. moonlight, on behaviour [54,55,56]. The suppressive nature of light on mosquito biting on successive and subsequent nights of treatment, and in the constant presence of host cues might also be aspects worthy of evaluation.