Highlights d Diurnal/nocturnal mosquitoes have distinct circadian neural circuit and PER cycling d Diurnal versus nocturnal mosquitoes have distinct clockmodulated light preferences d Light preference is dependent on mosquito sex and species, time of day, and color d Circadian clock modulates timing and valence of mosquito light responses

Drosophila CRYPTOCHROME (dCRY) mediates electrophysiological depolarization and circadian clock resetting in response to blue or ultraviolet (UV) light. These light-evoked biological responses operate at different timescales and possibly through different mechanisms. Whether electron transfer down a conserved chain of tryptophan residues underlies biological responses following dCRY light activation has been controversial. To examine these issues in in vivo and in ex vivo whole-brain preparations, we generated transgenic flies expressing tryptophan mutant dCRYs in the conserved electron transfer chain and then measured neuronal electrophysiological phototransduction and behavioral responses to light. Electrophysiological-evoked potential analysis shows that dCRY mediates UV and blue-light-evoked depolarizations that are long lasting, persisting for nearly a minute. Surprisingly, dCRY appears to mediate red-light-evoked depolarization in wild-type flies, absent in both cry-null flies, and following acute treatment with the flavin-specific inhibitor diphenyleneiodonium in wild-type flies. This suggests a previously unsuspected functional signaling role for a neutral semiquinone flavin state (FADH • ) for dCRY. The W420 tryptophan residue located closest to the FAD-dCRY interaction site is critical for blue-and UV-light-evoked electrophysiological responses, while other tryptophan residues within electron transfer distance to W420 do not appear to be required for light-evoked electrophysiological responses. Mutation of the dCRY tryptophan residue W342, more distant from the FAD interaction site, mimics the cry-null behavioral light response to constant light exposure. These data indicate that light-evoked dCRY electrical depolarization and clock resetting are mediated by distinct mechanisms. cryptochrome | flavoprotein | phototransduction | Drosophila | circadian clock C RYPTOCHROME (CRY) is a highly conserved and evolutionarily ancient flavoprotein expressed widely in prokaryotes and eukaryotes. Light-sensitive Drosophila CRYPTOCHROMEs (dCRYs) exhibit 2 absorbance peaks at 365 nm [ultraviolet (UV) light] and 450 nm (blue light) both in vitro and in cells (1-3). Light activation of dCRY initiates a relatively slow (∼1 h) irreversible process of ubiquitin-mediated degradation of the clock protein TIMELESS (TIM) and dCRY itself, thus resetting the circadian clock (4-8). More recently, dCRY was discovered to mediate rapid membrane depolarization (τ on ∼ 100 ms) and an increased spontaneous action potential firing rate in the lateral ventral neurons (LNvs) in response to blue and UV light (9-12). The mechanisms for light-activated dCRY leading to TIM degradation/clock resetting and membrane depolarization may differ based on timing of the response onset and reversibility/irreversibility following light exposure. For clock resetting, the light activation of dCRY leads to displacement of its short helical C-terminal tail (CTT) allowing TIM binding, which triggers the JETLAG (JET) ubiquitin-ligasemediated targeting of TIM ...

We developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adult Drosophila brains that captures circadian circuit-wide cycling under simulated day/night cycles. Light input analysis confirms that CRYPTOCHROME (CRY) is the primary circadian photoreceptor and mediates clock disruption by constant light (LL), and that eye light input is redundant to CRY; 3-h light phase delays (Friday) followed by 3-h light phase advances (Monday morning) simulate the common practice of staying up later at night on weekends, sleeping in later on weekend days then returning to standard schedule Monday morning [weekend light shift (WLS)]. PER and TIM oscillations are highly synchronous across all major circadian neuronal subgroups in unshifted light schedules for 11 d. In contrast, WLS significantly dampens PER oscillator synchrony and rhythmicity in most circadian neurons during and after exposure. Lateral ventral neuron (LNv) oscillations are the first to desynchronize in WLS and the last to resynchronize in WLS. Surprisingly, the dorsal neuron group-3 (DN3s) increase their within-group synchrony in response to WLS. In vivo , WLS induces transient defects in sleep stability, learning, and memory that temporally coincide with circuit desynchrony. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.

Billions of people subject themselves to phase-shifting light signals on a weekly basis by remaining active later at night and sleeping in later on weekends relative to weekday for up to a 3hr weekend light shift (WLS). Unnatural light signals disrupt circadian rhythms and physiology and behavior. Real-time light responses of mammalian suprachiasmatic nucleus are unmeasurable at single cell resolution. We compared Drosophila whole-circadian circuit responses between unshifted daytime/nighttime schedule and a 3hr WLS schedule at the single-cell resolution in cultured adult Drosophila brains using real-time bioluminescence imaging of the PERIOD protein for 11 days to determine how light shifts alter biological clock entrainment and stability. We find that circadian circuits show highly synchronous oscillations across all major circadian neuronal subgroups in unshifted light schedules. In contrast, circadian circuits exposed to a WLS schedule show significantly dampened oscillator synchrony and rhythmicity in most circadian neurons during, and after exposure. The WLS schedule first desynchronizes lateral ventral neuron (LNv) oscillations and the LNv are the last to resynchronize upon returning to a simulated weekday schedule. Surprisingly, one circadian subgroup, the dorsal neuron group-3 (DN3s), robustly increase their within-group synchrony in response to WLS exposure. Intact adult flies exposed to the WLS schedule show post-WLS transient defects in sleep stability, learning, and memory. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.Significance StatementThe circadian clock controls numerous aspects of daily animal physiology, metabolism and behavior. Shift work in humans is harmful. Our understanding of circadian circuit-level oscillations stem from ex vivo imaging of mammalian suprachiasmatic nucleus (SCN) brain slices. However, our knowledge is limited to investigations without direct interrogation of phase-shifting light signals. We measured circuit-level circadian responses to a WLS protocol in light sensitive ex vivo Drosophila whole-brain preparation and find robust sub-circuit-specific oscillator desynchrony/resynchrony responses to light. These circuit-level behaviors correspond to our observed functional defects in learning and memory, and sleep pattern disruption in vivo. Our results reflect that WLS cause circadian-circuit desynchronization and correlate with disrupted cognitive and sleep performance.

Mosquitoes pose widespread threats to humans and other animals as disease vectors. Day-vs. night-biting mosquitoes occupy distinct time-of-day niches and exhibit very different innate temporal attraction/avoidance behavioral responses to light, yet little is known about their circuit or molecular mechanisms. Day-biting diurnal mosquitoes Aedes aegypti are attracted to light during the day regardless of spectra. In contrast, night-biting nocturnal mosquitoes Anopheles coluzzii avoid short, but not long wavelength light. Attraction/avoidance behavioral responses to light in both species change with time-of-day and show distinct sex and circuit differences. The basis of diurnal versus nocturnal behavior is driven by clock timing, which cycle anti-phase between day-biting versus night-biting mosquito species. Disruption of the circadian molecular clock severely interferes with light-evoked attraction/avoidance behavior in mosquitoes. In summary, attraction/avoidance mosquito behaviors are circadian and light regulated, which may be applied towards species specific control of harmful mosquitoes.