Drosophila melanogaster flies were raised on standard media (yeast, cornmeal, agar) at 25 ± 1°C and 40–60% relative humidity in 12:12 h Light:Dark cycles. All flies used in experiments were first isogenized (backcrossed) to the w1118 genetic background for a minimum of six generations. All behavioral experiments used 3–4-day post-eclosion adult male flies. We generated pJFRC7 vectors containing cryptochrome 1 from Drosophila melanogaster (Dm), An. gambiae (Ag), and Ae. aegypti (Ae) in frame with eGFP. Use of the pJFRC7 vector allows for a controlled site-specific PhiC31 genomic insertion site. DNA constructs were then sent to the vendor Bestgene for fly embryonic injection and screening for successful transgenesis. Experimental transgenic flies backcrossed to the common wild-type w1118 background for a minimum of 6 generations. Genotyping primers were designed with the following sequences: AeCRY1 Forward: CGA GAA AGT GCA GGC CAA CAA TC, AeCRY1 Reverse: GT TCT TCA ACT CCG GCA GAT ATC, AgCRY1 Forward: CAG CCA GTT CAA GTA TCC GG, and AgCRY1 Reverse: CGG TTC GTG CAC AAA CTG TG. Experimental transgenic flies were crossed with a cry-null background (obtained from Jeff Hall, Brandeis University), then with a crypGAL4-24 driver line for CRY-neuron specific expression of DmCRY or mosquito CRY1.

Adaptations to the behavioral assays from Nitabach et al. (2002), Chiu et al. (2010), and Nave et al. (2021) were made for testing constant dark conditions for circadian behavior following 12 h:12 h light:dark entrainment (LD:DD) tested under two light intensities of l lux and 400 lux white light. Adult male flies (2–4 days post-eclosion) were anesthetized over CO₂ and individually loaded into borosilicate activity tubes. The TriKinetics Locomotor Activity Monitoring System was used to track fly behavior over a protocol of: 12:12 h Light:Dark (LD) entrainment for 7 days, then 7 days of constant dark (DD) conditions. Actograms were generated using Clocklab software. Average activity education graphs and its statistics were measured using FaasX software, then graphed using Microsoft Excel. Within FaasX, the CycleP analysis toolkit was used to calculate % rhythmicity from periodogram analysis with the following scoring criteria for flies in DD: minimum power ≥ 20, minimum width (h) ≥ 2, Chi-square significance ≥ 0.05 and calculation of tau. Data are reported as averages ± standard error mean. Anticipation index measurements during LD were adapted for the entrainment duration from Harrisingh et al. (2007) and Sheeba et al. (2010) taking the average activity in the 3 h preceding lights on (morning anticipation) or lights off (afternoon/evening anticipation) as a ratio over the average activity in the 6 h preceding lights on or off for individual flies over 5 days of LD entrainment. The reported values for anticipation index are an average of all the flies over the 5 days of LD entrainment.

Experimental transgenic flies were dissected for ex vivo brain preparations after 3–5 days of 12:12 h LD entrainment. Dissections began approximately 1 h before each ZT time point measured (ZT5, 11, 17, 23). Immunocytochemistry (ICC) experiments were performed for all genotypes in a given experiment, then repeated a minimum of 3 times to optimize statistical analysis and minimize experimental error. Dissected brains were placed in chilled 1X PBS, fixed in 4% paraformaldehyde (PFA) for 30 min, washed 3 × 10 min in PBS-Triton-X 1%, incubated in blocking buffer (10% Horse Serum-PBS-Triton-X 0.5%) at room temperature before incubation with rabbit α-TIM, polyclonal (1:1,000) antibodies overnight in 4°C. 3 rinse steps were performed at 10 min intervals with PBS-Triton-X 0.5% then incubated in goat α-rabbit-Alexa- 594 (1:1,000) secondary antibodies in blocking buffer overnight in 4°C. Brains were then rinsed 5 times at 15 min intervals in PBS-Triton-X 0.5% before mounting in Vectashield mounting media (Vector Laboratories). Sample slides were imaged using a Leica SP8 confocal microscope. We reproduced the TIM and CRY-GFP experiments published in Au et al. (2022), Current Biology and pooled the data with the earlier data for the results and updated total n’s reported in Figures 1, 2. The n’s for the new data added to the earlier data are: for ZT 5: DmCRY: 7, AeCRY1: 4, AgCRY1: 9, cry-null: 7; for ZT 11: DmCRY: 10, AeCRY1: 4, AgCRY1: 7, cry-null: 7; for ZT 17: DmCRY: 4, AeCRY1: 16, AgCRY1: 15, cry-null: 12; for ZT 23: DmCRY: 10, AeCRY1: 14, AgCRY1: 22, cry-null: 18.

For the data in Figures 1, 2, brain samples were imaged with a Leica SP8 confocal microscope with 594 nm antibody fluorescence for TIM signal and 488 nm CRY-GFP signal. FIJI/ImageJ analysis software was utilized for quantification of ventral lateral neuronal. Maximum intensity projections were generated using the Z stack tool. Fluorescent quantification of TIM and CRY-GFP signal were obtained by marking regions-of-interest on LNv (small and large LNvs) soma identified by morphology and anatomical positioning within each brain sample. Fluorescent values for the total number of neurons in a brain are normalized to the background brain fluorescence, then measurements of all neurons from all brain samples are averaged together.

Previously established whole-cell current-clamp protocols from Baik et al. (2019a) were modified to run our light-evoked potential electrophysiology experiments. Adult male fly brains were dissected in external recording solution consisting of: 122 mM NaCl, 3 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mM glucose, 10 mM HEPES, 7.2 pH, and calibrated to an osmolarity of 250–255 mOsm. The internal recording solution consists of: 102 mM Kgluconate, 17 mM NaCl, 0.085 mM CaCl₂, 1.7 mM MgCl₂ (hexahydrate), 8.5 mM HEPES, 0.94 mM EGTA, 7.2 pH, and is calibrated to an osmolarity of 232–235 mOsm. Custom multichannel LED source (Prizmatix/Stanford Photonics, Palo Alto, CA, United States) fitted to the Olympus BX51 WI microscope was used as the primary light source for our electrophysiology experiments. LED peak wavelengths are as follows: UV (365 nm), violet (405 nm), blue (450 nm), and red (635 nm), and all exposures were set to an intensity of 200 μW/cm² by use of a Newport 842-PE Power/Energy meter. Each LED was triggered on and off for each sweep with TTL pulses programmed by pClamp (Molecular Dynamics) data acquisition software. The light-evoked potential protocol is as follows: 50 s of dark for baseline recording, 5 s of colored-light stimulation, then 95 s of inter-pulse darkness for recovery back to baseline. The protocol repeats five times per recording. For analysis, sweeps are averaged, and baseline adjusted to pre-pulse signal, then low-pass noise filtered using Gaussian and Butterworth filters in the ClampFit 10 software (Molecular Dynamics). Our light-evoked potential protocol captures averaged light-evoked changes in membrane potential (Fogle et al., 2011; Baik et al., 2019a; Au et al., 2022), thus providing a kinetically robust light-evoked potential.

Standard LD light choice assays were conducted using behavioral protocols developed in previous studies (Baik et al., 2017; Au et al., 2022). The locomotor activity of individual flies was measured using the TriKinetics Locomotor Activity Monitoring System via dual infrared beam-crossing, recording total crosses in 1-min bins. Individual flies housed on glass tubes have a choice of exposure to a lighted side or in a dark side blocked by aluminum foil of the two infrared sensor tube. Percentage activity and statistics were measured using Microsoft Excel. Custom LED fixtures were built using Waveform Lighting blue and red LEDs with a narrow peak wavelength of 450 and 405 nm, respectively, and intensity-tuned to 10 and 400 μW/cm² for low and high intensity light exposures, respectively.

All reported values are represented as mean ± SEM. Values of n refer to the total number of experimental flies tested over all replicates of an experiment (minimum of three replicates). Firing frequency values are calculated as a ratio of spikes during the 5 s of lights on/average baseline firing rate binned in 10 s increments. Statistical tests were performed using Minitab, Matlab, and Microsoft Excel software. Statistical analysis began with performing an Anderson-Darling normality tests to determine normality of data. Variance was determined using F-tests for normally distributed data, then significance was determined using two-sample, one-tailed T-tests with alpha values of 0.5 before pairwise correction. Significance for non-normal data was determined by Mann-Whitney U-tests. Spike firing and membrane potential quantifications were performed using custom Matlab scripts and Clampfit software. Multi-comparison tests leading to Type I error/false positives were mitigated by a more stringent test of p-value adjustment based on false discovery rate (FDR, Benjamini and Hochberg, 1995, see also Au et al., 2022). A standard FDR threshold of 0.1 was then implemented in order to indicate significance as an expected proportion of false positives that is no greater than 10%.

Diurnal Aedes aegypti (Ae. aegypti) and nocturnal Anopheles gambiae (An. gambiae) mosquitoes are anthropophilic mosquitoes that occupy opposite day/night temporal niches. To determine whether heterologous CRY1 expression might disrupt the circadian clock, we compared circadian behavior in constant darkness (DD) in UAS-flies on a cry-null background expressing either Drosophila CRY (DmCRY), AeCRY1, AgCRY1 under the crypGAL4-24 (drives expression in all cells that ordinarily express CRY, Zhao et al., 2003) vs. negative control cry-null flies using two white light intensities of 1 and 400 lux. The expression of AgCRY1 is not sufficient to confer nocturnal activity at either 1 or 400 lux white light (Figures 3, 4) in Drosophila, in contrast to the robust nocturnal behavior seen in Anopheles mosquitoes (Baik et al., 2020). For low-intensity 1 lux LD entrainment, there are no significant differences in % rhythmicity between DmCRY, AeCRY1, and AgCRY1 expressing flies (Figure 3). In contrast, cry-null flies show significantly less % rhythmicity relative to DmCRY, AeCRY1, and AgCRY1 expressing flies (Figure 3). Similarly, at the higher-intensity 400 lux LD entrainment, there are no significant differences in % rhythmicity between DmCRY, AeCRY1, and AgCRY1 expressing flies, while again cry-null flies show significantly less % rhythmicity relative to DmCRY, AeCRY1, and AgCRY1 expressing flies (Figure 4). Thus, AgCRY1 nor AeCRY1 expression disrupts the circadian clock in Drosophila. Further analysis shows that AgCRY1 expressing flies show significantly longer period length (tau, τ) in constant darkness compared with DmCRY, AeCRY1, and cry-null following 1 and 400 lux light entrainment (Supplementary Figure 1) and that cry-null flies show significantly shorter period length than DmCRY, AeCRY1, and AgCRY1 expressing flies following 1 and 400 lux light entrainment (Supplementary Figure 1). Further, AgCRY1 expressing flies show significantly less morning anticipatory behavior and significantly greater evening anticipatory behavior compared with DmCRY, AeCRY1, and cry-null during 1 and 400 lux light entrainment (Supplementary Figure 2). In an earlier paper, we also found that circadian clock function measured by free running behavior in constant darkness and morning anticipatory behavior are not well correlated (Sheeba et al., 2010). Previous work from the Helfrich-Forster group concluded that eye photoreceptor inputs are primarily responsible for modulating morning anticipation in the absence of a functional circadian clock (Schlichting et al., 2015). However, the present results suggest that Cryptochromes may also modulate morning and evening anticipation, and perhaps this is not surprising that Cryptochromes from opposing temporal niches for diurnal vs. nocturnal animals might drive differences in anticipatory behavior.

Upon photoactivation, DmCRY resets the circadian molecular clock by binding with the clock protein TIMELESS (TIM) and setting it for degradation (Emery et al., 1998; Stanewsky et al., 1998; Koh et al., 2006). The circadian clock cycles in anti-phase fashion comparing diurnal Aedes mosquitoes (PER levels in the s-LNv peak at ZT23) vs. nocturnal Anopheles mosquitoes (PER levels in the s-LNv peak at ZT11, Baik et al., 2020). To determine if diurnal AeCRY1 or nocturnal AgCRY1 is sufficient to set the circadian clock to its peak timing of TIM protein expression, transgenic flies were entrained for at least 3 days of 12:12 h Light: Dark (LD) and immunocytochemistry experiments were used to measure TIM levels at time points ZT5, ZT11, ZT17, and ZT23. Fluorescent TIM signals were quantified in the ventral lateral neuronal subgroup (LNvs) and showed peak signal at ZT23 and the lowest signals at ZT5 and ZT11 for control DmCRY, AeCRY1, and AgCRY1 expressing flies (Figures 1A–C). Negative control cry-null flies show a similar TIM expression pattern in the LNvs (Figure 1D). Fluorescent measurements of TIM signal during ZT17 are significantly different and are more than twofold greater in flies expressing AeCRY1 than AgCRY1, suggesting diurnal AeCRY1 is less light sensitive than nocturnal AgCRY1. However, TIM signal at ZT5, ZT11, and ZT23 does not differ between AeCRY1 and AgCRY1 flies (summary of TIM measurements, Figure 1E). Transgenic expression of mosquito CRY1 in flies also includes N-terminal fusion of eGFP for protein expression verification. DmCRY expression measured by eGFP signal shows low expression during ZT5 and ZT11 with peak expression during ZT23 (Figure 2A). AeCRY1 expression is markedly higher than DmCRY, but exhibits a similar cycling pattern with ZT5 and ZT11 showing the lowest protein levels, and ZT17 and ZT23 showing the highest protein levels (Figure 2B). AgCRY1 protein expression is consistently high during all time points (Figure 2C), but the levels are within an order of magnitude compared with DmCRY and AeCRY1 protein expression levels (summary of CRY-GFP measurements, Figure 2D). In summary, AeCRY1 and AgCRY1 expression in flies does not disrupt the circadian clock nor alter the timing of the TIM expression peak. Between genotype differences in absolute protein levels may be due to codon usage or differences in protein stability of different CRY proteins.

Drosophila ventral lateral neurons are circadian/arousal neurons that drive CRY-dependent acute electrophysiological light responses (Holmes et al., 2007; Sheeba et al., 2008b; Fogle et al., 2011, 2015; Giachello et al., 2016; Baik et al., 2017, 2019a; Hong et al., 2018; Au et al., 2022). We expressed AeCRY1, AgCRY1, and control DmCRY in cry-null genetic background flies with the UAS/GAL4 expression system, then measured the light on/light off ratio of action potential firing frequency in response to 200 μW/cm² 450 nm blue-light from whole-cell patch-clamp recordings of l-LNvs in transgenic flies. For these experiments, we used the crypGAL4-24 driver line that drives expression in all CRY neurons (Zhao et al., 2003).

Positive control DmCRY expression driven by the crypGAL4-24 line mediates robust and significant increases in action potential firing frequency (FF) in the l-LNvs in response to 200 μW/cm² blue-light (450 nm) relative to cry-null negative controls (Figure 5A, blue column vs. gray column) and mediates significant sustained increases in firing frequency in response to blue-light (Figure 5A). AeCRY1 driven by the crypGAL4-24 line also shows significant increases in FF in the l-LNvs in response to 200 μW/cm² blue-light relative to cry-null negative controls (Figure 5A, orange column vs. gray column). However, after adjusting for false discovery rate (FDR), there is no significance difference observed between these two groups. This is unlike AgCRY1 driven by the crypGAL4-24 line, which shows robust and significant increases in FF in the l-LNvs in response to 200 μW/cm² blue-light relative to cry-null negative controls (Figure 5, purple column vs. gray column) even after adjusting for FDR, suggesting a greater blue light response for AgCRY1 compared to AeCRY1. Further, the AgCRY1 blue-light FF response does not significantly differ from the DmCRY blue-light FF response (purple column vs. blue column, Figure 5A). Comparing the 200 μW/cm² blue-light-evoked FF ratio during stimulus and subsequent 10 s bins post-stimulus up to 40 s, AgCRY1 FF is significantly greater than AeCRY1 FF 30 s post-stimulus (Figure 5E), but again, does not show significance after FDR adjustment. The positive control DmCRY FF is significantly greater than the cry-null negative control FF during stimulus and at the 10 and 30 s bins (Figure 5B).

Previous work shows that light activated CRY mediates changes in membrane potential through the voltage gated potassium channel beta subunit and modulation of potassium channels (Fogle et al., 2011, 2015; Giachello et al., 2016; Baik et al., 2017, 2018, 2019a; Hong et al., 2018; Tabuchi et al., 2021). To determine whether mosquito CRY expression alters LNv basal electrophysiological processes, we plotted basal l-LNv firing rates, basal resting membrane potential values and firing mode (tonic vs. burst firing) across the time of day of the recordings (Figure 6). The range of l-LNv firing rates and the average resting membrane potentials from the present set of whole-cell patch-clamp recordings for DmCRY expressing neurons are similar to previously reported values around –40 mV (the mean is −37 mV, Figures 6B,D,F). Basal firing rates and resting membrane potentials for DmCRY expressing flies are significantly lower than cry-null, AeCRY1 and AgCRY1 expressing flies (Figures 6E,F). The majority of the l-LNv recordings are from neurons during the day between ZT6-ZT12 and include a few recordings for the first few hours of night up until ZT16. None of the genotypes shows clear time of day differences in basal firing rate or membrane resting potential. However, these experiments were not designed to test time of day distributions as the present data cluster during midday. There are relatively few nighttime recordings and recordings from early morning and late night are not represented. Previous publications designed to test this question, including several of our own, show firing rates trending high at the beginning of day that tend to decrease at night (Cao and Nitabach, 2008; Sheeba et al., 2008b,2010; Flourakis et al., 2015; Smith et al., 2019). Consistent with most earlier publications, we observe predominantly tonic action potential firing in l-LNv recordings (Holmes et al., 2007; Cao and Nitabach, 2008; Sheeba et al., 2008b,2010; McCarthy et al., 2011; Seluzicki et al., 2014; Flourakis and Allada, 2015; Flourakis et al., 2015; Fogle et al., 2015; Buhl et al., 2016, 2019; Baik et al., 2017, 2019a; Li et al., 2018; Smith et al., 2019; Au et al., 2022). Burst firing as the predominant firing mode in l-LNv has been reported by another group (Muraro and Ceriani, 2015; Fernandez-Chiappe et al., 2021), however, they do not systematically address firing mode as a function of time of day.

Light-evoked averaged potentials are more kinetically reliable than light onset and CRY mediated action potential firing (Fogle et al., 2011; Baik et al., 2019a; Au et al., 2022). The blue-light-evoked response of DmCRY relative to the cry-null negative control shows strong depolarization then a slowly tapering sustained response over the 10 s following light stimulus offset (Figure 7A) with a qualitatively similar response recorded from neurons expressing AeCRY1 relative to the cry-null negative control (Figure 7B). In contrast, the blue-light-evoked response of neurons expressing AgCRY1 relative to the cry-null negative control show sustained significant depolarization during lights on, followed by a very long sustained depolarization response that lasts tens of seconds (Figure 7C). The blue-light response of AgCRY1 relative to AeCRY1 exhibits a significantly longer and more sustained membrane depolarization event lasting for tens of seconds evoked by a 5 s pulse of 200 μW/cm² blue-light relative to the shorter-lasting AeCRY1 evoked blue-light potential (Figure 7D). The significantly higher AgCRY1 blue-light-evoked depolarization for most of the duration of the evoked potential occurs after approximately 15 s post-stimulus relative to AeCRY1 (Figure 7D). These results, particularly the similar duration of the evoked potential blue-light response between AeCRY1 and DmCRY, suggest no direct relationship between CRY expression levels (Figure 3) and the magnitude of the physiological light response (Figures 2, 5, 6), confirming earlier findings concerning this (Baik et al., 2017, 2019a,b; Au et al., 2022). The AgCRY1 blue-light-evoked potential is significantly greater than that for the cry-null negative control for almost the entire duration up to 40 s from the stimulus onset (Figure 7C), while the much weaker AeCRY1 evoked potential is only significantly higher than the cry-null negative control for the first few seconds following stimulus onset (Figure 7B), but after FDR adjustment, it does not show significant differences. AgCRY1 confers a more sustained light response than DmCRY (Figures 7A,C). Representative voltage traces showing light-evoked depolarization and increased action potential firing frequency in patch-clamp recordings of l-LNvs during the 5 s of blue-light stimuli and 60 s post-light stimulus for positive control DmCRY/cry24, AeCRY1/cry24, AgCRY1/cry24, and negative control cry-null flies are shown in Figure 8, where the blue bar indicates 5 s of 200 μW/cm² 450 nm blue-light stimulus.

As expected, there are no significant differences in light-evoked FF between all four CRY genotypes in response to 200 μW/cm² violet-light (405 nm) (Figure 9A), as there is a trough of the CRY action spectra around 405 nm and Rh7 and other opsin photoreceptors are activated in this range of the color spectra (Ni et al., 2017; Sakai et al., 2017; Baik et al., 2019b). The depolarization magnitude and duration of DmCRY, AeCRY1, AgCRY1, and negative control cry-null responses to violet-light are similar and indistinguishable from cry-null and are at a lower magnitude of FF ratio and depolarization magnitude and duration relative to intensity matched blue-light stimuli (compare Figure 9 vs. Figure 5). The violet-light-evoked increases in l-LNv firing frequency (Figures 9B–E) and light-evoked depolarization (Figure 10) during and after the violet-light stimulus are weak and do not differ systematically between the different CRY genotypes. These results are consistent with earlier findings that CRY is not activated by violet-light and is consistent with earlier findings that Rh7 is the primary non-image forming visual violet-light photoreceptor in LNvs (Ni et al., 2017; Sakai et al., 2017; Baik et al., 2019b). Representative voltage traces showing light-evoked depolarization and increased action potential firing frequency in patch-clamp recordings of l-LNvs during the 5 s of violet-light stimuli and 60 s post-light stimulus for positive control DmCRY/cry24, AeCRY1/cry24, AgCRY1/cry24, and negative control cry-null flies are shown in Figure 11, where the violet bar indicates 5 s of 200 μW/cm² 405 nm violet-light stimulus.

Diurnal mosquitoes are behaviorally attracted to short-wavelength light (UV, blue), while nocturnal mosquitoes behaviorally avoid short wavelength light (Baik et al., 2020). CRY1 is a strong photoreceptor candidate to drive these species-specific attraction/avoidance behavioral light responses. In our recent study Au et al. (2022) testing transgenic Drosophila that express diurnal AeCRY1 or nocturnal AgCRY1 in a cry-null genetic background, we find that AeCRY1 expressing flies show strong photo-attraction behavioral responses to a wide intensity range (1–400 μW/cm²) of UV (365 nm) light. In contrast, nocturnal AgCRY1 expressing flies show discernable photo-attraction behavioral responses to UV light at very low intensities (1 μW/cm²) but show significant photo-avoidance behavioral responses to higher UV light intensities (at 10 and 400 μW/cm² of UV light). Here, we examine the role for CRY1s for conferring day- vs. night-active mosquito species-specific light choice behaviors to other wavelengths by performing blue (450 nm) and violet (405 nm) light choice behavioral assays with flies expressing DmCRY, AgCRY1, AeCRY1 under the crypGAL4-24 promoter at low (10 μW/cm²) and high (400 μW/cm²) light intensities using an environmental light choice preference test. At low intensity (10 μW/cm²) 450 nm blue-light, cry-null flies show significantly greater attraction to blue-light relative to all CRY expressing fly groups (Figures 12A–C). Flies expressing DmCRY, AgCRY1, or AeCRY1 show weak or no behavioral attraction to low intensity blue-light (Figures 12A–D). The average% activity of flies in the blue lit environment over the first 30 min shows no significant differences between flies expressing DmCRY, AeCRY1, or AgCRY1 (Figure 12E).

In contrast, at higher intensity 400 μW/cm² 450 nm blue-light, the genotypes behavioral light responses diverge: DmCRY expressing flies exhibit relatively neutral responses to the blue lit environment, showing moderate photo-attraction for 15 min of blue-light exposure, then moderate photo-avoidance to 400 μW/cm² 450 nm blue-light for the next 15 min (Figure 13A). AeCRY1 expressing flies show significantly greater behavioral attraction to high intensity blue-light relative to cry-null and AgCRY1 expressing flies at many time points (Figures 13B,D). AgCRY1 expressing flies exhibit the greatest significant light avoidance to the high-intensity blue-light exposed environment relative to other genotypes (Figures 13A–E). This is confirmed by average% activity plots for each CRY expressing genotype showing that AeCRY1 expressing flies have significantly greater activity in higher intensity blue-light than either AgCRY1 or DmCRY, and that AgCRY1 have significantly the least amount of activity in high intensity blue-light relative to AeCRY1 or DmCRY (Figure 13E).

At low intensity 405 nm violet-light (10 μW/cm²), DmCRY and AgCRY1 expressing flies both show behavioral photo-attraction to the low intensity violet lit environment (Figures 14A,C,D), while cry-null and AeCRY1 expressing flies show less behavioral photo-attraction to the violet lit environment (Figures 14B,D). The average% activity plots for each CRY expressing genotype shows AeCRY1 expressing flies show significantly the least behavioral activity in low intensity violet-light while DmCRY expressing flies show significantly the most behavioral activity in low intensity violet-light (Figure 14E). Control cry-null and DmCRY expressing flies both behaviorally avoid high intensity violet-light (400 μW/cm², Figure 15A), except during the first 10 min of violet-light exposure for DmCRY expressing flies. The behavioral responses to high intensity violet-light are divergent between AgCRY1 and AeCRY1 expressing flies: AgCRY1 expressing flies behaviorally avoid high intensity violet-light while AeCRY1 expressing flies are behaviorally attracted to high intensity violet-light, consistent with the previously reported general attraction of Ae. aegypti mosquitoes to all visible light wavelengths (Figures 15A–E, see also Baik et al., 2020). The average% activity plots for each CRY expressing genotype shows that AeCRY1 expressing flies show significantly the greatest behavioral activity in high intensity violet-light while AgCRY1 expressing flies show significantly the least behavioral activity in high intensity violet-light (Figure 15E). Taken together for responses to varying intensities of violet-light, these complex behavioral effects may be due either to direct effects through mosquito CRY proteins or possibly due to unknown CRY interactions with the major violet-light sensor Rh7 that co-expresses in the LNv subgroups to mediate multi-photoreceptor inputs for light attraction/avoidance behavioral responses (Ni et al., 2017; Baik et al., 2018, 2019b), or image forming photoreception in the eyes Altogether, these results indicate the blue and violet-light intensity-dependent light attraction/avoidance behaviors significantly diverge between AeCRY1 and AgCRY1 expressing flies and that these behavioral results are consistent with the distinct diurnal and nocturnal mosquito attraction/avoidance responses to short-wavelength light. Taken together, the data provides further support to our conclusions that CRY photoreceptors mediate species-specific physiological and behavioral light responses (Baik et al., 2020; Au et al., 2022).