CRYPTOCHROME mediates behavioral executive choice in response to UV light

Significance

Many animals exhibit behavioral responses to UV light, including harmful insects. Recently, the explosive spread of diseases carried by mosquitoes has increased motivation to better understand insect UV phototransduction. CRYPTOCHROME (CRY) is a highly conserved nonopsin photoreceptor expressed in a small number of brain circadian and arousal neurons in Drosophila melanogaster that mediates cell-autonomous electrophysiological membrane excitability in response to UV light. CRY signaling modulates multiple fly behaviors evoked by UV light, including acute nighttime arousal responses to light flashes and phototaxis toward low-intensity UV light. Loss of CRY or the redox sensor HYPERKINETIC (HK) leads to the loss of ability to avoid high-intensity UV light; thus, CRY signaling exhibits novel features of behavioral executive choice.

Abstract

Drosophila melanogaster CRYPTOCHROME (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, we tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons.

For nearly a century, it has been assumed that insect behavioral responses to UV light are exclusively mediated by UV-sensitive opsins expressed in eyes and other external photoreceptors. However, organisms express other nonopsin photoreceptors, including the blue-light-sensitive flavoprotein CRYPTOCHROME (CRY). In Drosophila melanogaster, CRY mediates rapid membrane depolarization and increased spontaneous action potential firing rate in the lateral ventral neurons (LNvs), which are involved in arousal and circadian behavioral responses (1–7). Blue-light-activated CRY couples to membrane depolarization in Drosophila LNv neurons by a redox-based mechanism involving potassium channel heteromultimeric complexes, consisting of the downstream redox sensor cytoplasmic potassium beta (Kvβ), HYPERKINETIC (HK), and ion-conducting voltage-gated potassium alpha (Kvα) ether-a-go-go family subunits (8). Electrical activity in the LNvs contributes to circadian rhythms (9–11), and, reciprocally, LNv neuronal firing rate is circadian-regulated (1, 2, 12). Circadian regulation of firing rate is widely conserved in other invertebrate and vertebrate species (13, 14). For insects, CRY is characterized as the primary photoreceptor for blue–light-activated circadian entrainment (15–21). CRY-expressing large LNvs (l-LNvs) also mediate acute behavioral arousal responses to blue-light-containing spectra (4–7). Arousal and circadian functions are not strictly segregated between the LNv subsets because the small LNvs (s-LNvs) also contribute to arousal (5), and clock cycling is robustly altered in the l-LNv in response to light entrainment cues (22, 23).

Many insects, including Drosophila, display strong spectral sensitivity for short-wavelength light. The ability to sense and respond to UV light is important because it guides physiological and behavioral responses to sunlight that are crucial for survival. The absorbance spectra of purified CRY from Drosophila at the oxidized baseline state of the flavin dinucleotide chromophore show a strong UV peak near 365 nm, in addition to the 450-nm blue-light peak (24–26). Furthermore, UV light triggers CRY degradation in cultured cells that heterologously express fly CRY (27). To test the in vivo functional significance of these findings, we measured behavioral and electrophysiological responses to UV light near the CRY UV peak.

Results

CRY Mediates Opsin-Independent Electrophysiological Responses to UV Light in l-LNvs.

Light-modulated behaviors are driven by the modulation of membrane excitability in contributing neurons, such as the l-LNvs (5, 6, 8–11). We tested whether similar l-LNv electrophysiological response (firing frequency light on/light off) was observed in response to UV light. Control fly l-LNvs respond to UV light with varying degrees to different intensities (Fig. 1 A and E; 365 nm; low, 20 μW/cm²; intermediate, 150 μW/cm²; and high, 640 μW/cm²). The l-LNv response to UV light is significantly attenuated in cry-null mutant flies (cry^−/−; Fig. 1 B and E) and hk-null mutant flies (hk^−/−; Fig. 1 C and E) relative to control (Fig. 1 A and E).

Fig. 1.

l-LNv electrophysiological response to UV light is attenuated in flies lacking CRY-based phototransduction. (A–D) Representative trace for control l-LNv UV light response (A) (365 nm, 640 µW/cm², violet bar; lights off, <0.01 µW/cm², black bar; the gap in the x axis removes <1 s, wherein a noise transient is caused by manual opening of the shutter to expose the prep to light) vs. representative traces for cry^−/− (B), hk^−/− (C), and “cry rescue” (D) flies (pdf-GAL4–driven LNv UAS-CRY expression in a cry^−/− background). (E–G) Dose–response quantification of l-LNv firing frequency (FF) response (FF on/FF off) to UV light at low (20 µW/cm²), intermediate (150 µW/cm²), and high (640 µW/cm²) intensities. (E) Electrophysiological response of control flies increase with increasing intensities of UV light (1.19 ± 0.04, n = 17, low; 1.33 ± 0.07, n = 15, intermediate; 1.77 ± 0.12, n = 15, high intensity). The significantly attenuated UV light responses of cry^−/− (1.04 ± 0.02, n = 17, P = 0.01, low; 1.17 ± 0.06, n = 15, P = 0.129, intermediate; 1.35 ± 0.07, n = 15, P = 0.005 vs. control, high intensity) and hk^−/− (0.99 ± 0.04, n = 15, P = 0.002, low; 1.13 ± 0.03, n = 14, P = 0.049, intermediate; 1.37 ± 0.07, n = 26, P = 0.008 vs. control, high intensity) flies do not differ from each other (P = 0.622, low; P = 0.879, intermediate; P = 0.978, high intensity). (F) Dose–response quantification of FF for control vs. cry^−/− and cry rescue flies. Full rescue is achieved at low (1.18 ± 0.03, n = 15, P = 0.99 vs. control) and intermediate intensities (1.24 ± 0.03, n = 15; P = 0.14 vs. control), but is incomplete at high-intensity UV light (1.45 ± 0.05, n = 15, P = 0.03 vs. control and P = 0.68 vs. cry^−/−). (G) Dose–response quantification of FF for control vs. hk^−/− and pdf-GAL4–driven rescue of WT-HK (UAS-HK-WT) or of redox sensor-disabled point mutant HK-D260N (UAS-HK-D260N), both in hk^−/− genetic background. WT-HK rescue flies also achieve rescue at low (1.21 ± 0.03, n = 16, P = 0.97 vs. control) and intermediate (1.26 ± 0.03, n = 16, P = 0.732 vs. control) intensities, but not at high intensity (1.34 ± 0.04, n = 16, P = 0.001 vs. control, and P = 0.99 vs. hk^−/−). The redox sensor-disabled point mutant HK-D260N fails to rescue the light response at all UV light intensities (1.03 ± 0.04, n = 13, P = 0.033, low; 1.09 ±0.03, n = 15, P = 0.004, intermediate; 1.19 ± 0.05, n = 11, P ≤ 0.001 vs. control, high intensity; P ≥ 0.417 vs. hk^−/− all intensities). (H) Representative trace for glass^60j (gl^60j) mutant l-LNv UV light response. (I) Representative trace for gl^60j- cry^−/− double-mutant l-LNv UV light response. (J) Dose–response quantification FF for gl^60j and gl^60j- cry^−/− double-mutant flies. gl^60j flies response do not significantly differ from control (1.20 ± 0.06, n = 18, P = 0.87, low; 1.19 ± 0.05, n = 14, P = 0.15, intermediate; 1.54 ± 0.07, n = 16, P = 0.098 vs. control, high intensity). gl^60j- cry^−/− double mutant has significantly attenuated UV response compared with control (1.01 ± 0.03, n = 20, P = 0.002, low; 1.08 ± 0.04, n = 32, P = 0.003, intermediate; 1.26 ± 0.05, n = 28, P ≤ 0.001 vs. control, high intensity) and do not differ from cry^−/− response (P = 0.499, low; P = 0.252, intermediate; P = 0.157 vs. cry^−/−, high intensity). *P < 0.05; **P < 0.01; ***P < 0.001.

To determine whether CRY-mediated l-LNv UV light responses are cell-autonomous, we performed genetic rescue experiments. Genetic rescue of LNv-targeted expression of CRY in cry^−/− genetic background cell-autonomously rescues the l-LNv UV light response at low and intermediate intensities, but incompletely at high intensity (Fig. 1 D and F). Similarly, LNv-targeted expression of WT-HK in hk^−/− genetic background rescues l-LNv UV light response at low and intermediate intensities, but again not at high intensity (Fig. 1G). Expression of redox sensor-disabled HK point mutant, HK-D260N, does not rescue l-LNv response to UV light at all intensities (Fig. 1G). Thus, electrophysiological responses to UV light are specifically mediated by light-activated CRY coupled to the membrane via HK redox sensor, consistent with previous findings using blue light (8).

Because low levels of CRY are expressed in the R7 and R8 photoreceptors (28), we recorded from l-LNv of sevenless mutant flies, which lack all R7 photoreceptors (29). The l-LNv UV light responses of sevenless flies do not significantly differ from control flies (Fig. S1). To determine whether opsin-based photoreceptors also contribute to the l-LNv electrophysiological response to UV light, we recorded from the l-LNv neurons of glass^60j (gl^60j) flies, which lack eyes and other external photoreceptors (and DN1p circadian cells). The l-LNv UV-light responses of gl^60j flies are qualitatively lower, but do not significantly differ from control flies (Fig. 1 H and J). The l-LNv UV light response of glass^60j-cry^−/− (gl^60j-cry^−/−) double-mutant flies is indistinguishable from that of cry^−/− flies (Fig. 1 I and J). These results suggest that CRY mediates electrophysiological responses to UV in the l-LNvs in an opsin-independent manner. gl^60j-cry^−/− double-mutant flies show some residual electrophysiological UV response at higher intensities, indicating the presence of a yet-to-be identified third photoreceptor for the l-LNvs, consistent with earlier findings (17).

Fig. S1.

l-LNv electrophysiological UV light response is not attenuated in sevenless flies. Dose–response quantification of l-LNv firing frequency (FF) response (FF on/FF off) to UV light at low (20 µW/cm²), intermediate (150 µW/cm²), and high (640 µW/cm²) intensities. l-LNv electrophysiological UV response of sevenless flies do not significantly differ from that of control (1.23 ± 0.06, n = 9, P = 0.547, low; 1.37 ± 0.05, n = 9, P = 0.661, intermediate; 1.72 ± 0.14, n = 9, P = 0.816 vs. control, high intensity).

Acute Arousal Behavioral Response to UV Light Is CRY-Dependent.

CRY is expressed in circadian, arousal, and photoreceptor neurons (3, 11, 28, 30), including l-LNvs, which mediate acute arousal behavioral responses to blue light at physiological intensities that transmit the head and eye cuticles (8). We measured the proportion of UV light transmittance through eye and head cuticle tissue using a 365-nm LED light source using procedures described in ref. 3. Eye cuticles are >85% transparent, and head cuticles are nearly 50% transparent to 365-nm UV light (Fig. S2). We then measured acute behavioral responses to 5-min pulses of 365-nm UV (3 mW/cm²) or 595-nm orange (7 mW/cm²) LED light in the middle of the subjective night at zeitgeber time (ZT) 18, ZT19, and ZT20 for three consecutive nights in control and cry^−/− mutants, as well as no receptor potential A null mutant (norpA^P24) and gl^60j mutant flies, which, respectively, have defects in opsin phototransduction in the eyes and the ocelli and external photoreceptor development. A representative averaged behavioral actogram of control flies (n = 32) responding to UV light pulses is shown (Fig. 2A). We examined flies that were asleep immediately before the light pulse (4, 5). The percentage of flies that awaken in response to 365-nm UV light is significantly lower in cry^−/−, norpA^P24, and gl^60j mutant flies compared with control (Fig. 2B). In response to 595-nm orange light, the percentage of flies that awaken is comparable between control and cry^−/− flies. However, a significantly higher percentage of norpA^P24 flies awaken, whereas a significantly lower percentage of gl^60j flies awaken in response to orange light relative to controls, indicating that the norpA^P24 and gl^60j flies differ in their light responses (Fig. 2B). Recently, Drosophila transient receptor potential A1 (TRPA1) channel has been implicated as an H₂O₂-sensitive high-intensity UV (600–5,000 mW/cm²) sensor (31). We tested acute arousal responses of trpA1-null (trpA1¹) flies to UV light (3 mW/cm²) and orange light (7 mW/cm²). Acute arousal response of trpA1¹ flies to either UV or orange light pulses is indistinguishable from that of control flies (Fig. S3), indicating that acute arousal responses to UV light pulse is not mediated by TRPA1 at the light intensities tested.

Fig. S2.

Drosophila head and eye cuticles transmit UV light. The proportion of 365-nm UV light transmitted through cuticle tissues was calculated as the amount of UV light transmitted through either eye (n = 10) or head (n = 7) cuticle tissues divided by baseline measurements of transmittance through a droplet of PBS. ***P < 0.001.

Fig. 2.

Drosophila acute arousal response to UV light is CRY-dependent. (A) Representative averaged double-plotted actogram of n = 32 flies given three 5-min light pulses (365 nm, 3 mW/cm²) during three consecutive nights. Flies respond acutely to light pulses, but remain entrained to the LD 12:12 environmental cues. (B) Percentage of sleeping flies that awaken during the light pulse for UV (365 nm, 3 mW/cm²) and orange light (595 nm, 7 mW/cm²). Compared with control flies (0.72 ± 0.03, n = 384 flies for UV), a significantly lower percentage of cry^−/− flies (0.61 ± 0.04, n = 192 flies, P = 0.039 vs. control) awaken in response to UV light pulse. Percentage of sleeping cry^−/− flies that awaken during orange light pulse does not differ from percentage of sleeping control flies that awaken (0.32 ± 0.03, n = 224 flies for control; 0.38 ± 0.04, n = 128 flies, for cry^−/−; P = 0.264 cry^−/− vs. control). Both norpA^P24 and gl^60j flies have a significantly lower percentage of flies that awaken in response to UV light pulses (0.54 ± 0.03, n = 192 flies, P ≤ 0.001 for norpA^P24 vs. control; 0.09 ± 0.02, n = 64 flies, P ≤ 0.001 for gl^60j vs. control). A significantly higher percentage of norpA^P24 flies awaken (0.56 ± 0.03, n = 160 flies, P ≤ 0.001 for vs. control), whereas a significantly lower percentage of gl^60j flies awaken in response to orange light pulses (0.13 ± 0.02, n = 64 flies, P ≤ 0.001 vs. control). (C–F) Time course of activity of awake flies during and after UV (C and E) or orange (D and F) light pulse. Each point on the graph represents a bin of 5 min, with the first bin collected during the pulse. (C) During the UV light pulse, control flies show a dramatic increase in arousal activity (activity/baseline is 2.98 ± 0.23, n = 384 flies), whereas cry^−/− flies remain relatively inactive (1.35 ± 0.12, n = 192 flies, P ≤ 0.001 vs. control), only responding after the pulse (cry^−/− vs. control, P > 0.213 for all bins, after the light pulse). (D) cry^−/− and control fly activities do not differ during and after the orange light pulse (n = 128 flies, cry^−/− vs. control, n = 224 flies, P > 0.064 for all bins). (E) Activity of awake norpA^P24 flies does not differ from that of control flies (n = 192 flies, norpA^P24 vs. control, n = 384 flies, P > 0.174 for all bins). gl^60j flies show a significantly lower arousal response both during and after the UV light pulse (n = 64 flies, gl^60j vs. control, P < 0.010 for bins during and 5–20 min after the light pulse). (F) gl^60j flies show a significantly lower arousal response during and after orange light pulse (n = 64 flies, gl^60j vs. control, n = 224 flies, P < 0.018 for bins during and 5–25 min after the light pulse). norpA^P24 flies have significantly higher activity than control flies during the orange light pulse (n = 160 flies, norpA^P24 vs. control, P ≤ 0.001), but do not differ in activity after the orange light pulse (norpA^P24 vs. control, P < 0.332 for all bins, after the light pulse). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S3.

Drosophila CRY-mediated acute arousal response to UV light is not attenuated in mutants lacking trpA1. (A) Percentage of sleeping flies that awakened during the 5-min light pulse for UV (365 nm, 3 mW/cm²) and orange (595 nm, 7 mW/cm²) light. Percent of sleeping trpA1¹ flies that awaken in response to UV (0.72 ± 0.03, n = 192 flies, P = 0.928 vs. control) or orange (0.32 ± 0.02, n = 192 flies, P = 0.919 vs. control) light does not differ from that of control flies. (B) Time course of activity of awake control and trpA1¹ flies during and after UV light pulse does not differ from each other (all P > 0.481 vs. control). (C) Time course of activity of awake control and trpA1¹ flies during and after orange light pulse does not differ from each other (all P > 0.115 vs. control).

We also examined the behavioral responses of flies that were awake immediately before the light pulse. During the UV light pulse (just after 0 min), awake control flies show increased arousal activity, whereas awake cry^−/− flies remain relatively inactive during the UV light pulse, then show a delayed response minutes later after the UV light pulse (Fig. 2C). CRY-mediated acute arousal activity is specific to UV light, because the cry^−/− response to orange light does not differ from that of control flies (Fig. 2D). Acute arousal activity of awake norpA^P24 flies in response to UV light pulse is indistinguishable from that of control flies (Fig. 2E). norpA^P24 flies show increased acute arousal responses during the orange light pulse, but after the orange light pulse, their activity does not differ from that of control flies (Fig. 2F). gl^60j awake flies do not respond to either UV or orange light pulses (Fig. 2 E and F). This finding suggests that acute arousal behavioral response to UV may be modulated by DN1 cells and/or Hofbauer–Buchner (HB) eyelet, which is functionally defective in the gl^60j mutants, but not in norpA^P24 mutants (17, 32–35).

CRY Mediates Executive Choice Attributes of Positive Phototaxis and Avoidance Behaviors to Different Intensities of UV Light.

Light can serve as either a repellent or an attractive signal for an animal’s behavior, depending on intensity and spectra. Many insects exhibit an innate spectral attraction to low-intensity UV light, as shown by phototaxis behavioral assays (36–38). In contrast, high-intensity UV light induces avoidance behavior, particularly in larvae and egg-laying females (39, 40), and reduces mating activity in adult male Drosophila (41).

Positive phototaxis behavior of adult male flies in response to very low-intensity UV light (3 µW/cm² 365-nm LED, 5 min per exposure) was measured by using a retrofitted Trikinetics DAM5 Drosophila Activity Monitor attached to a light-tight chamber holding a population of 40 flies (Fig. 3A). Positive phototaxis is measured by increased activity levels (counts per min) from flies migrating to the light-transparent activity monitor in the front (Fig. 3A). WT control flies show robust attraction in response to 5-min pulses of very low-intensity UV light (Fig. 3 B and D). Positive phototaxis to UV light is significantly attenuated in cry^−/− flies compared with control flies (Fig. 3 B and D). Interestingly, control flies choose to linger in the previously light-exposed region after the light-pulse long after the light has been turned off, compared with cry^−/− flies, which leave the previously light-exposed region quickly (Fig. 3D). This finding suggests that CRY potentially mediates aspects of executive choice, specifically in choosing to linger in a previously light-exposed region, in addition to simple acute sensory function (Fig. 3 B and D). Both norpA^P24 and gl^60j flies show little attraction toward very low-intensity UV light (Fig. 3 B and F). Thus, external photoreceptors have a primary acute sensory role for UV phototaxis, whereas CRY modulates the magnitude and duration of the response. trpA1¹ mutant flies do not exhibit attenuated positive phototaxis in response to 5-min pulses of very–low-intensity UV light, but, surprisingly, show significantly higher positive phototaxis compared with control flies (Fig. S4 A and C). Orange light (3 µW/cm² 595-nm LED, 5 min per exposure) fails to evoke strong positive phototaxis (Fig. 3 C, E, and G and Fig. S4 B and D).

Fig. 3.

Drosophila positive phototaxis behavior toward UV-light is attenuated in mutants lacking CRY- and in mutants lacking external photoreceptors. (A) A DAM2 Drosophila Activity Monitor (32 channels with dual infrared beams; Trikinetics) was mounted to the front of the light-tight chamber holding a population of 40 flies and sealed with a glass cover on the outer face. (B) Average phototaxis activity counts per min toward a very low-intensity UV light pulse (365 nm, 3 µW/cm², five exposures of 5-min light; indicated by violet arrows) followed by 55 min of darkness starting at circadian time (CT) 21 to CT 3 for control (nine experimental repeats, n = 40 flies per experiment), cry^−/− (three experimental repeats, n = 40 flies per experiment), norpA^P24 (four experimental repeats, n = 40 flies per experiment), and gl^60j flies (four experimental repeats, n = 40 flies per experiment). (C) Average phototaxis activity counts per min toward five 5-min orange light pulses (595 nm, 3 µW/cm², indicated by orange arrows) followed by 55 min of darkness starting at CT 21 to CT 3 for control (four experimental repeats, n = 40 flies per experiment), cry^−/− (five experimental repeats, n = 40 flies per experiment), norpA^P24 (three experimental repeats, n = 40 flies per experiment), and gl^60j flies (six experimental repeats, n = 40 flies per experiment). (D–G) Average phototaxis activity in 5-min bins relative to the UV (D and F) or orange (E and G) light pulses averaged from B and C. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S4.

Drosophila-positive phototaxis behavior toward UV light is not attenuated in mutants lacking trpA1. (A) Average phototaxis activity counts per min toward a very low-intensity, five 5-min UV light pulses (365 nm, 3 µW/cm²; indicated by violet arrows), followed by 55 min of darkness starting at CT 21 to 3 for control (nine experimental repeats, n = 40 flies per experiment) and trpA1¹ flies (four experimental repeats, n = 40 flies per experiment). (B) Average phototaxis activity counts per min toward a very low, five 5-min orange light pulse (595 nm, 3 µW/cm²; indicated by orange arrows), followed by 55 min of darkness starting at CT 21 to 3 for control (four experimental repeats, n = 40 flies per experiment) and trpA1¹ flies (three experimental repeats, n = 40 flies per experiment). (C and D) Average phototaxis activity in 5-min bins relative to the UV (C) or orange (D) light pulses averaged from A and B. **P < 0.01.

CRY potentially contributes to executive choice evoked by UV light. To test this hypothesis directly, we measured behavioral avoidance responses to high-intensity UV light (400 μW/cm²). A modified Trikinetics Drosophila Activity Monitor system was fitted for long tubes with two infrared photobeams separated by 8.4 cm that measure locomotor activity at different zones for a single fly, with food and air holes placed equally on either side of the long tube to prevent food and air spatial preferences (Fig. 4A). Adult male flies were 12:12 light–dark (LD)-entrained in standard white light (3 d), followed by 12:12 LD entrainment in UV light (3 d). Then, an opaque screen was placed covering one side of each long tube, so that half the length of the tube was exposed to high-intensity UV light, and the other half of the tube was shaded, thus blocking all direct UV light (Fig. 4B). Each infrared photobeam on either side of the long tube allowed us to measure the fly’s choice of locomotor activity, either in the zone of the tube exposed to high-intensity UV light or in the zone of the tube shaded from direct UV light. To measure potential time-of-day effects, high-intensity UV light was on for 12 h, matching the entrained daytime (ZT0–12), followed by all lights off (ZT12–24). This schedule presented flies with a choice between activity in the high-intensity UV light-exposed environment vs. escape to the covered environment shaded from direct UV light at all times during daytime for 10 d (Fig. 4B). We refer to this as the “Mad Dogs and Englishmen” experiment [after Noel Coward, 1931 (42)].

Fig. 4.

CRY-based phototransduction contributes to UV light avoidance behavior in Drosophila. (A) Diagram of the “light choice” apparatus. Standard Trikinetics Drosophila activity monitors were modified to fit behavior tubes of 2× length, which have food and air hole on both sides of the tube. Flies are first entrained in standard 12:12 white light LD without any cover. The 12:12 white light LD is then replaced by 12:12 UV light LD. (B) Half of the monitor is then covered with cardboard to provide flies a choice between UV light-exposed (400 µW/cm²) and shaded environments. UV light is turned on only during the entrained daytime (ZT0-12). (C–E) Preference for UV-exposed vs. shaded environment is measured by percent of activity in each environment over total amount of activity for each ZT. Gray shade indicates shaded environment preference (light avoidance), and violet-shade indicates UV environment preference. (C) cry^−/− flies have a significant defect UV light avoidance behavior at all times of the day compared with control flies and prefer the UV environment over the shaded (cry^−/−, n = 78 vs. control, n = 76, all P < 0.05). (D) Similarly, hk^−/− flies also have a significant defect in UV light avoidance behavior at all times of the day compared with control flies and prefer the UV environment over the shaded (hk^−/−, n = 77 vs. control, all P < 0.05). (E) Mutant flies lacking all opsin-based external photoreceptors (gl^60j) show significantly less UV avoidance compared with control flies only during the midday, ZT1-6 (gl^60j, n = 76 vs. control, all P < 0.05). (F) Average percent activity in UV-exposed environment during the day vs. night. cry^−/− and hk^−/− flies have significantly higher activity in the UV-exposed environment during the day compared with control flies (P < 0.05). Daytime percent activity in UV-exposed environment of gl^60j flies does not significantly differ from control. Percent activities in UV-exposed environment for cry^−/−, hk^−/−, and gl^60j flies during the nighttime do not differ from control. *P < 0.05.

Control and gl^60j flies significantly avoid UV light and strongly prefer to be in the shaded environment, including during the midday (Fig. 4 C–E and Fig. S5 A and D). The gl^60j flies are not as effective as control flies for UV avoidance, but show the same pattern of avoidance. In contrast, cry^−/− and hk^−/− flies significantly prefer the high-intensity UV light environment over the shaded environment during the daytime, particularly during the early morning and all afternoon hours, and exhibit significantly attenuated avoidance behavior to high-intensity UV light compared with controls at all times of day (Fig. 4 C and D and Fig. S5 B and C). To control for potential olfactory cues deposited by flies during daytime activity, we analyzed for environmental preference for both sides of the monitor (ZT12–24) when the UV light is off. No differences in preferences are detected between all four genotypes during subjective nighttime (Fig. 4F). However, on an hour-by-hour basis, cry^−/− and hk^−/− flies show small, but significant, preferences for the covered side during all of the night (Fig. S5 B and C). Similarly, control and gl^60j flies show small, but significant, preferences for the covered side during half or nearly half of the night (Fig. S5 A and D). This nighttime activity might reflect residual olfactory cues left during daytime activity or differences in food quality on the covered side. Thus, results show clearly that CRY and its downstream redox sensor HK mediate choice in avoidance behavior in response to high-intensity UV light during day. This territorial preference does not extend in the absence of UV light.

Fig. S5.

CRY-based phototransduction mediates Drosophila choice of light environment. Average activity count over ZT time in the UV-exposed environment (365 nm, 400 µW/cm²; violet bars) vs. the shaded environment (black bars) is shown. The UV light was on during the daytime (ZT0–12) and off during the nighttime (ZT12–24; shaded gray on the graphs). (A) Control flies prefer the shaded environment over the UV-exposed one during the midday (n = 76). (B and C) cry^−/− flies (n = 78) (B) and hk^−/− flies (C) both lack the preference for shaded environment in the midday and prefer the UV-exposed environment at other times of the day (n = 77). (D) gl^60j flies choose the shaded environment over the UV-exposed one during the midday (n = 76), similar to control flies. *P < 0.05; **P < 0.01; ***P < 0.001.

Discussion

The results above show that both CRY- and opsin-based photoreceptors contribute to UV light-sensing and behaviors. The l-LNv electrophysiological UV light responses increase monotonically with increasing UV light intensity. The l-LNv electrophysiological response to UV light is severely attenuated in cry^−/− and hk^−/− null mutants, along with qualitative decreases seen in gl^60j mutants (Fig. 1). There is a small residual l-LNv electrophysiological light response even in gl^60j-cry^−/− double mutants (Fig. 1J), suggesting that there is another short-wavelength light photoreceptor that has yet to be identified. Subtleties in our data suggest potential circuit-level effects for encoding light. Gene-replacement rescue experiments in cry^−/− and hk^−/− null backgrounds show intensity-dependent degrees of rescue, for which rescue is complete for lower light intensities, but incomplete for higher light intensities (Fig. 1 F and G). This result may be due to the fact that the genetic rescue is limited to the LNv, not all neurons that ordinarily express CRY.

Mutants lacking CRY show significantly altered behavioral responses to UV light by three very different assays: (i) acute arousal response to high-intensity UV light flashes during the night; (ii) positive phototaxis for very low-intensity UV light; and (iii) avoidance of high-intensity UV light. The ability to discern the changes in intensity, spectral content, timing, and exposure length of light provides valuable environmental information crucial to an organism’s well-being and survival. UV light-avoidance behavior has been demonstrated in foraging larva and egg-laying activity in females (39, 40). We demonstrate that the CRY/HK signaling pathway mediates UV light avoidance behavior in adult male Drosophila. During peak UV light intensity (midday in most natural environments), flies (especially males) tend to take a “siesta” rest and thus avoid heat exposure and desiccation. UV light avoidance behavior is highest during the midday (Fig. 4), despite unvarying UV intensity for our experimental conditions during the daytime (ZT0-12). This finding suggests that CRY/HK-mediated UV light avoidance behavior may be under circadian control, comparable to larval avoidance behavior shown to be dependent on opsin-based photoreceptors and subsets of circadian pacemaker neurons and circadian genes (43, 44).

CRY dually mediates attraction and avoidance behaviors to UV light depending on UV light intensity. The differences in CRY-mediated behavioral response to varying intensities of UV light poses the interesting question of whether CRY may be important, not only for the acute sensory detection of the light, but also for modulating more complex aspects of behavior, such as executive choice. CRY-mediated behavioral responses likely depend on spectral composition, intensity, and duration of light exposure, as well as integration with other sensory cues, most notably temperature (45–49). CRY-mediated electrophysiological light responses vary monotonically depending on UV light intensity. Thus, the cell-autonomous neuronal CRY light sensor codes for graded responses to UV light intensity rather than gated on/off responses.

Opsin-based light sensing is clearly critical for behavioral light responses. The gl^60j mutant exhibits the developmental loss of all external opsin-based photoreceptors, HB eyelet, and the DN1p subset of circadian neurons (17, 32, 33). The DN1s have been implicated for light-evoked morning arousal activity (50, 51). HB eyelet cells project into the accessory medulla and to the LNvs (52, 53). The norpA^P24 mutant disrupts opsin-based phototransduction in eyes without disrupting phototransduction in the HB eyelet or development of the DN1p circadian neurons; thus, the gl^60j and norpA^P24 mutants are not functionally equivalent (17, 34, 35). In contrast to the dramatic loss of arousal response to UV light in gl^60j mutants, norpA^P24 mutants show UV light arousal responses that closely resemble those of controls (Fig. 2E), suggesting the DN1ps and/or the HB eyelet may also contribute to the UV light arousal response. CRY’s contribution is functionally distinct from that of opsins, as shown by both electrophysiological and behavioral results. In conclusion, CRY is a major modulator of a wide range of fly behavioral responses to UV light.

Materials and Methods

Locomotor activity was recorded by using the TriKinetics Drosophila Activity Monitor system (9). l-LNv recordings were performed on acutely dissected adult fly brains in whole-cell current clamp mode (1, 2). Extended information on materials and methods is described in SI Materials and Methods, including protocols for electrophysiology, optics, genetics, behavioral testing, and statistical analysis.

SI Materials and Methods

Generation of gl^60j-cry^−/− Double-Mutant Line by Homologous Recombination.

We generated the gl^60j-cry^−/− double-mutant line (same as gl^60j-cry⁰¹) by standard recombination genetic crosses of gl^60j (Bloomington Stock Center; BL 509) and the cry⁰¹ line provided by Jeff Hall, Brandeis University, Waltham, MA (54). We verified glass and cry loss in our gl^60j-cry⁰¹ double-mutant line by phenotype, genetic markers, and PCR.

Electrophysiology.

Patch-clamp measurements were performed on acutely dissected adult fly brains as described (1, 3). l-LNv recordings were made in whole-cell current clamp mode. After allowing membrane properties to stabilize after whole-cell break-in, 30–60 s of recording in current clamp configuration was obtained under dark conditions before lights on. The UV wavelength range was isolated by using a filter cube designed for DAPI imaging (Olympus). Light intensities were determined by a Newport 842-PE Power/Energy meter. The UV dose–response curve was performed by placing neutral density filters of varying degrees of opaqueness in the light path from the mercury bulb to the brain prep. Dose–response to light was quantified by firing frequency during lights on/firing frequency during lights off (which was averaged from dark period before and after the lights-on period). Most recordings lasted for the duration of the full dose–response curve, and all intensities for all genotypes include at least five different brains.

Behavioral Experiments.

Light pulse arousal.

Flies were entrained for a minimum of 3 d to a normal LD 12:12 cycle. During the dark phase, they were given three consecutive 5-min pulses of UV (365 nm, 3 mW/cm²) or orange (595 nm, 7 mW/cm²) at ZT 18, 19, and 20, for three nights. Activity during all of these pulses and for the subsequent 40 min was binned in 5-min intervals and averaged. Statistical tests between genotypes at each 5-min bin were conducted by t test. Sleeping flies, defined by inactivity for 5 min preceding the pulse, were assessed for the percentage that woke up during the pulse. Active flies were assessed for the change in their activity during and after the pulse; changes were expressed as activity of bin/activity during baseline.

Behavioral phototaxis.

An electrophoresis system photodocumentation hood (15 × 20 cm; Fisher Biotech) was converted into a light-tight chamber to hold a population of flies. A DAM2 Drosophila Activity Monitor (32 channels with dual infrared beams; Trikinetics) was mounted to the front of the chamber and sealed with a glass cover on the outer face. For each experiment, a population of 40 adult flies was directly placed into the chamber. w¹¹¹⁸, cry⁰¹, and gl^60j flies were independently tested. The entire apparatus was then placed in a light-sealed enclosure, where the flies were left to habituate for 30 min before starting the recording. To capture time-of-day effects for phototactic responses, behavioral activity was recorded from CT 21 to 3. An LED light source was turned on for five cycles of 5-min pulses, followed by 55 min of constant darkness. The LED light source was either (i) a Prizmatix UHP-Mic-LED-595 (595 nm) or (ii) a Prizmatix Mic‐LED‐365, High Power UV LED (365 nm). The light intensity for both LED sources was set to 3 µW/cm². Because of the narrow collimated light emission of these two light sources, they were reflected off an inner surface of the enclosure so that their light emissions covered all 32 channels of the activity monitor. Data were collected by the DAM2 Drosophila Activity Monitor to be analyzed in 1-min bins. Excel was used for quantitative analysis of average population activity in response to the light pulses relative to baseline measurements of activity in darkness. Statistical significance was determined by using the t test.

Light intensity: Phototaxis.

• Raw (directly in front of LED) = 6 mW/cm².

• Reflected (on behavior monitors) = 90 µW/cm².

• Through glass (within chamber) = 3 µW/cm².

Light choice.

Standard Trikinetics activity monitors were modified to remove the center barrier to accommodate 15-cm-long, 5-mm-diameter glass tubes (Trikinetics). Two air holes were drilled into the tube equidistant from the ends, which were both plugged with fly food on both sides, and sealed with paraffin wax after the fly was introduced to the tube. Flies were entrained in the tubes for 3 d in white light 12:12 h LD and 3 d in UV light (365 nm, 400 µW/cm²), uncovered. Continuing the LD schedule in UV light (UV light on during ZT0–12), one-half of the monitor was covered with cardboard, providing the flies with a choice of a shaded environment during the 12 h of UV light. Activity for each fly in the UV-exposed vs. shaded side of the tube was averaged over 10 d, and statistical preferences were determined by t test. Each genotype was tested over five separate behavioral runs with >76 flies total per genotype.

Transparency of Drosophila cuticles to UV light.

The proportion of UV light (365 nm) that is transmitted through head and eye cuticles was measured by using a modified version of a protocol described in ref. 3. Briefly, adult male flies were dissected in ice-cold PBS for removal of head or compound eye cuticle tissue within 3 d of eclosion. Three blue-black light strips were mounted facing downward in a light-tight enclosure to shine UV light on a Li-Cor LI-250A light meter with the Li-Cor Quantum sensor facing upward. The quantum sensor was wrapped in aluminum foil with a small pinhole made in the foil with a diameter smaller than the cuticle samples. The transparency of the cuticles was measured as the amount of 365 nm transmitted through the cuticle tissues divided by baseline measurements of the amount of UV light that passes through a small droplet of PBS covering the pinhole. The amount of light transmitted was measured for 10 compound eye and 7 head cuticle tissue samples. A statistical comparison of the percentage of UV light that passes through eye vs. head cuticles was determined by using the Student t test.