Distinct mechanisms of Drosophila CRYPTOCHROME-mediated light-evoked membrane depolarization and in vivo clock resetting.

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

Investigating circadian disruption using real-time longitudinal imaging of the entire Drosophila circadian neural network

Circadian oscillators are capable of endogenous oscillations in free-running conditions. Many studies show that the coordination of the biological clock with environmental inputs, such as daily solar cycles, is critical for regulating physiological and behavioral activities. Conversely, billions of people worldwide are subjecting themselves to chronic misalignment and disruption of biological clocks with environmental inputs. In this study, we developed an entrainment protocol we call LD Strobe that permits us to obtain real-time, longitudinal bioluminescence imaging of the entire Drosophila circadian neural circuit in adult cultured brains, ex vivo. With the LD Strobe schedule, we can now examine circadian disruptions that could potentially lead to the progression of human-related diseases. In the following proposed projects we aim to (1) capture the Drosophila circuit-wide responses to Weekend Light Shifts, (2) determine Drosophila behavioral outputs generated by circadian-disrupted flies after undergoing a simulated weekend, (3) examine the circuit-wide response of flies in a genetic background with uncharacterized disruptions to circadian-modulated physiology and behavior, and (4) determine the circadian regulation light-evoked attraction and avoidance behaviors in daytime- and nighttime-biting mosquitos. Due to similarities between mammalian and insect circadian molecular and neural circuits, features of the circadian physiological and behavioral outputs from the proposed projects may be broadly observed and applicable to humans

Circadian Regulation of Light-Evoked Attraction and Avoidance Behaviors in Daytime- versus Nighttime-Biting Mosquitoes

Mosquitoes pose widespread threats to humans and other animals as disease vectors [1]. Day- versus night-biting mosquitoes occupy distinct time-of-day niches [2, 3]. Here, we explore day- versus night-biting female and male mosquitoes' innate temporal attraction/avoidance behavioral responses to light and their regulation by circadian circuit and molecular mechanisms. Day-biting mosquitoes Aedes aegypti, particularly females, are attracted to light during the day regardless of spectra. In contrast, night-biting mosquitoes, Anopheles coluzzii, specifically avoid ultraviolet (UV) and blue light during the day. Behavioral attraction to/avoidance of light in both species change with time of day and show distinct sex and circadian neural circuit differences. Males of both diurnal and nocturnal mosquito species show reduced UV light avoidance in anticipation of evening onset relative to females. The circadian neural circuits of diurnal/day- and nocturnal/night-biting mosquitoes based on PERIOD (PER) and pigment-dispersing factor (PDF) expression show similar but distinct circuit organizations between species. The basis of diurnal versus nocturnal behaviors is driven by molecular clock timing, which cycles in anti-phase between day- versus night-biting mosquitoes. Observed differences at the neural circuit and protein levels provide insight into the fundamental basis underlying diurnality versus nocturnality. Molecular disruption of the circadian clock severely interferes with light-evoked attraction/avoidance behaviors in mosquitoes. In summary, attraction/avoidance behaviors show marked differences between day- versus night-biting mosquitoes, but both classes of mosquitoes are circadian and light regulated, which may be applied toward species-specific control of harmful mosquitoes

Weekend light shifts evoke persistent Drosophila circadian neural network desynchrony

We developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adul