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Journal Pre-proof

Control of odor sensation by light and cryptochrome in the Drosophila antenna

Dhananjay Thakur, Sydney Hunt, Tiffany Tsou, Miles Petty, Jason M. Rodriguez,

Craig Montell

PII:

S2589-0042(25)00704-7

DOI:

https://doi.org/10.1016/j.isci.2025.112443

Reference:

ISCI 112443

To appear in:

ISCIENCE

Received Date: 12 December 2024
Revised Date: 14 February 2025
Accepted Date: 11 April 2025

Please cite this article as: Thakur, D., Hunt, S., Tsou, T., Petty, M., Rodriguez, J.M., Montell, C., Control

of odor sensation by light and cryptochrome in the Drosophila antenna,

ISCIENCE

(2025), doi:

https://

doi.org/10.1016/j.isci.2025.112443

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Control of odor sensation by light and

cryptochrome in the Drosophila antenna

Dhananjay Thakur, Sydney Hunt, Tiffany Tsou,

Miles Petty, Jason M. Rodriguez and Craig Montell*,**

Department of Molecular, Cellular, and Developmental Biology,

and the Neuroscience Research Institute,

University of California, Santa Barbara, Santa Barbara, CA 93106, USA

*Corresponding author: Craig Montell

**Lead contact: Craig Montell

Email:

cmontell@ucsb.edu

Keywords:

Olfaction, smell, light, ultraviolet, reactive oxygen species, ROS, hydrogen

peroxide, H

, cryptochrome, TRPA1, olfactory receptor neurons, ORNs, olfactory

sensory neurons, OSNs, support cells

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Abstract

Olfaction is employed by the fruit fly,

Drosophila melanogaster

, to differentiate

safe from harmful foods and for other behaviors. Here, we show that ultraviolet (UV) or

blue light reduces the fl

y’s behavioral aversion, and the responses of olfactory receptor

neurons (ORNs) to certain repellent odors, such as benzaldehyde. We demonstrate that

cryptochrome

(

cry

) is expressed in antennal support cells and is required for the light-

dependent reduction in aversion. Light activation of Cry creates reactive oxygen species

(ROS), and ROS activates the TRPA1 channel. We found that TRPA1 is required in

ORNs for benzaldehyde repulsion and is activated

in vitro

by benzaldehyde. We

propose that light-activation of Cry and creation of ROS persistently stimulates and then

desensitizes TRPA1, preventing activation by benzaldehyde. Since flies begin feeding

at dawn, we suggest that the light-induced reduction in odor avoidance serves to lower

the barrier to feeding following the transition from night to day.

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Introduction

Chemosensation includes the senses of taste and smell, and it is known that

taste is greatly influenced by multiple types of sensory stimuli. In humans, the

perception of sweetness and bitterness is affected by food temperature, food texture,

sound and external light.

1-8

The receptor mechanisms underlying how these sensory

features impact taste have been characterized using model organisms, ranging from

flies

9-13

to mice.

In the case of olfaction, some people associate certain odors with

specific colors.

15,16

In principle, environmental light could impact odor acuity or valence through

direct light reception by olfactory organs. To address this question, we elected to focus

on the fruit fly,

Drosophila melanogaster

, since one of the olfactory organs, the antenna,

responds to multiple types of sensory inputs.

While the 3

segment of the antenna

contains a high density of olfactory receptor neurons (ORNs), other parts of the

antennal

structure, namely the Johnston’s organ and arista, are sensitive to stimuli such

as humidity

18-20

, sound

wind

, gravity

, and changes in temperature.

The

possible integration of odor detection with sensation of other physical parameters is

under active investigation. Smell acuity, for example, is known to be sensitive to

ambient temperature,

26,27

and changes during the circadian rhythm.

Drosophila

antennae are situated on the dorsal surface of the fly and are known

to express photoreceptive proteins such as the ultraviolet (UV) and blue light sensitive

rhodopsins

, and cryptochrome (Cry).

However, it is not known if the antennae sense

and respond directly to ambient light, or if light sensation by olfactory organs has an

impact on the odor response. Nevertheless, it has been documented that sensation of

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blue light by the

Drosophila

compound eyes can reduce the activity of a particular class

of olfactory receptor neurons that detect the male pheromone, cis-vaccenyl acetate.

Here, we discovered that ambient, short-wavelength light reduces the aversion to

multiple odorants, such as the noxious odorant, benzaldehyde. The light is detected

directly by olfactory sensilla in the antenna, and this depends on Cry, which is

expressed in central pacemaker neurons in the brain, where it contributes to light-

entrainment of circadian rhythms.

32-35

Surprisingly, we found that Cry is required in

support cells rather than ORNs in the sensilla. Our work supports the model that light

activation of Cry results in induction of reactive oxygen species, which causes persistent

activation and subsequent refractoriness of the TRPA1 channel in ORNs to

responsiveness to benzaldehyde. This work highlights a role for light in modifying the

olfactory response, which does so through light reception in a new class of light

responsive cells present in an olfactory organ.

Results

DART2 assay for olfactory avoidance of repellents

To assay whether olfactory sensation in

Drosophila

is impacted by light, we

modified a two-way choice assay (direct airborne repellent test, DART)

consisting of a

tube capped at one end with a repellent and the solvent that was used to solubilize and

dilute the repellent at the other end with lights directed from above (Figure 1A). To

ensure that there was no contact between the flies and the chemicals during the assay,

the caps included meshed chambers, creating a separation of 8 mm between the flies

and the chemicals. We transferred flies to the tube, allowed them to acclimate for 20

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minutes without any odorants, added the odorants, and then recorded their positions

over the course of 60 minutes. To optimize the assay, we first focused on characterizing

olfactory behavior in the dark under near-infrared illumination with a video camera

(Figure 1A).

We tracked the responses of the flies by calculating the transverse component of

their center of mass (CoM) per frame and normalizing it to the length of the tube. We

found that the CoM provided the most precise quantitative alignment with the visually

observed distribution of flies in the assay tube. The most extreme avoidance or

attraction to a chemical, as seen when the flies cluster at one end or the other (Figure

S1A), produces a CoM of 1.0 or 0 (Figure S1B). If the flies distributed homogeneously

throughout the tube (Figure S1A), this would indicate neither aversion nor attraction to

the chemical and result in a CoM of 0.5 (Figure S1B). Other sample distributions

indicating less than the most severe repulsion (e.g. right bias, right cluster or slight right

bias; Figure S1A) produce CoM >0.5

– <1.0 (Figure S1B), while less than the most

severe attraction (e.g. left bias, left cluster or slight left bias; Figure S1A), result in CoM

– <0.5 (Figure S1B). In contrast to the CoM metric, measuring the position of the

flies in terms of avoidance index (Figure S1C) could not discriminate between subtle

differences in the distribution of the flies within the tubes. Therefore, we used CoM as

the measure of odor responses throughout this study.

Benzaldehyde (BA) is a naturally occurring compound synthesized in plants,

100

which is a repellent odorant to

Drosophila

However, it is not sensed exclusively

101

through olfaction since removal of the olfactory organs significantly reduces but does

102

not eliminate BA avoidance.

Using DART2 assays, we found that the flies were

103

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indifferent to 0.1% or 0.5% BA, while increasing the BA to just 1% elicited strong

104

avoidance (Figure S1D). Elevating the BA to 10% caused extreme avoidance, with the

105

flies clustering near the right side of the DART2 tube (Figure S1D).

106

In order to calibrate the variability of the response as a function of the number of

107

flies in the DART2 assay, we tested the avoidance to 1% BA with different numbers of

108

flies ranging from 1 to 64. We found that there was considerable variation in the

109

response over time if the assays included only 1, 2, or 4 flies (Figures 1B

–E). In close

110

concordance with previous work that shows that collective behavior produces more

111

robust responses to aversive cues,

we found that increasing the number of flies in the

112

assay (≥8) resulted in less variability in the BA response (Figures 1B and F–I). In

113

assays with 8 and 16 flies, the CoM moved increasingly away from BA over the first 20

114

minutes, reaching a steady-state of aversion that was maintained for the remainder of

115

the 60-minute-long assay (Figures 1F and G). Including greater numbers of flies in the

116

assay (32 or 64) caused slight decreases in the average CoM relative to the assays with

117

16 (Figures 1H and I) as some of the flies were forced into the side of the tube closer to

118

the repellent due to overcrowding. In order to obtain minimal variance and maximum

119

effect size (Figure 1B), we elected to use 8-16 flies per tube for the remaining analyses.

120

Next we tested whether there was sexual dimorphism in the response to BA. We found

121

that the responses were indistinguishable when we tested males only, females only, or

122

a mix of both sexes (Figure S1E). Therefore, we used a mix of males and females in

123

each tube for all further analyses.

124

125

UV and blue illumination reduce aversion to benzaldehyde

126

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To address whether exposure to light impacted the responses to BA, we

127

performed DART2 assays while exposing flies to ‘white light’ illumination, which

128

consisted of a combination of red, green, blue and UV LEDs each at an intensity of

129

~100 μW/cm

. We found that in the presence of white light, the avoidance of 1% BA

130

was significantly reduced (Figure 2A). We then tested the impact of different

131

wavelengths of light and found that ultraviolet light (UV; 365 nm) or blue light (450 nm),

132

but not green light (520 nm) or red light (625 nm), suppressed the repulsion to BA

133

relative to that they exhibited in the dark (Figure 2B). The aversion was decreased

134

significantly when the flies were exposed to ≥20 μW/cm

of UV or ≥18 μW/cm

blue

135

illumination (Figures 2C and D). We assayed the impact of 100 μW/cm

UV light on

136

repulsion by different concentrations of BA ranging from 0.1

–10%. While the response

137

to 1.0% benzaldehyde was suppressed by UV, there were no effects of UV on lower BA

138

levels (0.1% and 0.5%), and the highest concentration of BA (10%) was so aversive

139

(CoM = 0.93 ±0.01) that the UV light had no impact (Figure 2E).

140

To determine if UV light modified the responses to other odorants, we tested

141

AITC (allyl isothiocyanate), which is a potent noxious odorant to flies,

citronellal, which

142

is a previously described repellent,

isovaleric acid (a product of fermentation) and

143

lemongrass, a widely used insect repellent.

To conduct these experiments, for each

144

repellent, we first established a concentration of the odorant that produces a significant

145

effect in the dark, but does not cause a ceiling effect (CoM ~0.65-0.8).

As controls, we

146

also included propionic acid (a constituent of fly food), and limonene (a neutral odor).

147

The responses of the flies were not significantly different under UV and dark conditions

148

to propionic acid, limonene, AITC, and citronellal (Figure 2F). However, similar to

149

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benzaldehyde, the aversion was significantly reduced when flies were tested with

150

isovaleric acid and lemongrass (Figure 2F).

151

152

ORN responses are suppressed by light, via a rhodopsin independent mechanism

153

To test if light directly modulates the response of the ORNs, we performed

154

electroantennograms (EAGs), which are field recordings performed by placing a

155

recording electrode on the antenna. We exposed flies to 1% BA either in the dark or

156

during 5 minutes of blue light. Upon exposure to blue light, the response to BA was

157

attenuated to 41.6 ±6.2% of the response in the dark (Figures 3A and B), showing that

158

light affects ORN activity in the antenna. These results raise the possibility that there is

159

a light sensor in the antenna that serves to modulate the olfactory response.

160

The main light receptors are rhodopsins, which initiate phototransduction

161

cascades.

To address whether a rhodopsin is required for suppressing the repulsion

162

to BA, we tested the requirements for rhodopsins that are activated by UV light. Flies

163

encode seven rhodopsins, four of which are effectively activated by UV light (Rh1, Rh3,

164

Rh4, and Rh7).

42-45

Six of the rhodopsins (Rh1-Rh6) engage a trimeric G-protein that

165

couples to a phospholipase C

(PLC) encoded by the

norpA

gene,

while Rh7

166

signaling is mediated through PLC21C.

We found that flies lacking

norpA

showed

167

similar aversion to BA under UV light as control flies (Figure 3C), indicating that

168

rhodopsins that engage this G-protein-activated phospholipase C

are not the receptors

169

enabling the light-dependent suppression of BA repulsion. Consistent with this finding,

170

mutations affecting

rh1

(

ninaE

rh3

rh4

did not alter the impact of light on the

171

aversion to BA (Figure 3C). In contrast, flies lacking

rh7

showed no difference in

172

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aversion between dark and UV conditions, although the overall responses for one of two

173

alleles tested (

rh7

) was slightly lower than exhibited by control flies (Figure 3C).

174

However, we did not detect

rh7

-promoter-driven GFP staining in the antenna (Figure

175

S2), suggesting that the effect of this light sensor in modifying the ORN response to BA

176

is independent of the antenna.

177

178

Cryptochrome required for UV and blue light suppression of olfactory avoidance

179

Cryptochrome (Cry), which detects UV/blue light in

Drosophila

central pacemaker

180

neurons in the brain,

46,47

is also expressed in the antenna.

Therefore, Cry is an

181

excellent candidate sensor for functioning in the light-dependent modification of the

182

response to BA in the antenna. We discovered that a null mutation in

cry

(

cry

)

183

eliminated the suppression of the olfactory avoidance to BA by UV or blue light (Figure

184

3D). We observed a similar phenotype when we placed the

cry

mutation

in trans

with

185

a deficiency that uncovered the

cry

locus indicating that the phenotype is due to

cry

186

rather than a background mutation (Figure 3D). Thus, we conclude that Cry is required

187

for the UV- and blue-induced suppression of BA repulsion.

188

To determine whether

cry

impacts the electrical response to BA in the antenna,

189

we performed EAGs. As mentioned above, light suppressed the BA response in wild-

190

type antennas significantly (Figures 3A and B). In contrast,

cry

flies still exhibited a

191

robust BA response under blue light (Figures 3E and F), although there was a relatively

192

small reduction in the peak EAG amplitude, which was not significant (81.5 ±8.4% of the

193

peak in the dark). Thus, these data indicate that

cry

provides a large fraction of the

194

inhibition in odor response by light in the antenna.

195

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196

cry is expressed and required in non-neuronal cells in olfactory sensilla

197

The two main olfactory organs are the bilaterally symmetrical 3

antennal

198

segments and the maxillary palps.

These organs are decorated with bristles, referred

199

to as olfactory sensilla, which harbor one to four ORNs, and three types of accessory

200

cells. These include the trichogen (shaft), tormogen (socket), and thecogen (sheath).

201

To visualize the cells that express

cry

, we took advantage of a

cry

reporter (

cry-GAL4

)

202

to drive expression of

UAS-mCD8::GFP

. The reporter stained cells in both the 3

203

antennal segment and the maxillary palp (Figures 4 and S3).

204

Cry is expressed in central pacemaker neurons,

32,34,50

suggesting that in olfactory

205

organs it may also be expressed in neurons. To address whether

cry

is expressed in

206

ORNs, we performed double-labeling experiments using the

cry

GAL4

in combination

207

with a marker specific for ORNs,

orco-RFP,

which expresses

RFP

under control of the

208

orco

promoter.

Surprisingly, we did not detect co-staining with the

orco-RFP

either in

209

the antenna (Figures 4A

–D) or in the maxillary palp (Figures S3A–D). Moreover, there

210

were no

cry

-positive axonal processes extending into any glomeruli in the antennal

211

lobes in the brain (Figures S3E

–G). Together, these data indicate that the

cry

reporter is

212

not expressed in ORNs. To address whether Cry is expressed in support cells, we took

213

advantage of the GFP::Cry fusion protein,

which stained the same set of cells as the

214

cry-GAL4

(Figure S3H

–J). We found that nearly all of the GFP::Cry positive cells in the

215

antenna and maxillary palps were also stained with the

lush-GAL4

(Figures 4E

–G

and

216

S3K

–M), which labels a large population of support cells throughout the antenna. These

217

experimental observations align well with our analysis of transcript data from the

218

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FlyCellAtlas that is consistent with

cry

expression in support cells in olfactory sensilla

219

(Figures S4A

–F).

220

To identify the types of support cells that express

cry

, we used

GAL4

s to drive

221

expression in tormogen cells (

ase5

), thecogen cells (

nompA

), and glia

(

repo

222

Consistent with the extensive overlap of the

cry

and

lush

reporters, we found that the

223

GFP::Cry showed considerable overlap with all three cell types (Figures S4G

–O). To

224

determine whether

cry

is sufficient in olfactory support cells, we performed rescue

225

experiments using the

UAS-cry

and the

lush-GAL4

, and found that this recapitulated the

226

reduced aversion to BA in the presence of UV light (Figure 5A).

227

228

Role of reactive oxygen species in modulating odor response

229

The surprising finding that Cry expression

in support cells is sufficient for UV-

230

induced suppression of BA repulsion raises the question as to the underlying

231

mechanism. One possibility is that Cry in support cells leads to release of a diffusible

232

product that impacts ORN function. Activation of Cry by short-wavelength light changes

233

the oxidative state of the flavin FADH that is bound to Cry, resulting in the production of

234

reactive oxygen species (ROS).

The ROS could in principle affect the activity of the

235

ORNs.

236

To test whether activation of Cry by UV light increases production of ROS in the

237

antennae, we used the ROS sensor, 2′,7′-dichlorofluorescein diacetate

(

DCFDA), as

238

described previously.

We found that when we surgically removed antennae from

239

control flies and incubated them with DCFDA, they showed a significant increase in

240

fluorescence response after 1 second of UV exposure (Figures 5B, and S5). In contrast,

241

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the response was reduced in

cry

mutant antennae (Figures 5B and S5). The residual

242

fluorescence response was likely an effect of UV on other endogenous photosensitizers

243

in the tissue.

54,55

To determine whether induction of oxidative stress in

lush

- and

cry

244

expressing olfactory support cells is sufficient to reduce aversion to BA, we used the

245

lush-GAL4

to express dual oxidase (

UAS-duox

), which encodes a protein that

246

generates hydrogen peroxide. We found that this was sufficient to attenuate the

247

repulsion to BA in the dark (Figure 5C).

248

249

TRPA1-C is required for avoidance to benzaldehyde

250

The observation that

cry

is required for UV-induced ROS production in the

251

antenna raises the question as to the molecular target for the ROS. ROS including

252

, which are produced by UV light, activate

Drosophila

TRPA1

56,57

and we have

253

demonstrated previously that a

trpA1

reporter is expressed in ORNs in the 3

antennal

254

segment.

The

trpA1

gene is expressed as four primary isoforms (A-D).

36,58-60

Both

255

trpA1-A

and

trpA1-B

use one promoter, and

trpA1-C

and

trpA1-D

share an alternative

256

promoter. To address which isoform-pair is expressed in ORNs, we used

GAL4

lines

257

that we previously created that are specific to the

isoforms (

trpA1-AB

GAL4

) and the

258

isoforms (

trpA1-CD

GAL4

The axons of ORNs extend into different glomeruli in the

259

olfactory lobes. Thus, using

UAS-mCD8::GFP

, which labels the cell surfaces of cell

260

bodies and neuronal processes, we could determine which driver is expressed in ORNs

261

by examining labeling of the 58 glomeruli in each olfactory lobe.

62-65

Using the

trpA1-

262

GAL4

, we did not detect significant staining of olfactory glomeruli (Figure 6A). In

263

contrast, the

trpA1-CD-GAL4

labeled ≥13 glomeruli in each lobe (Figures 6B and S6).

264

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To determine whether the

trpA1-C

or the

trpA1-D

isoform is predominantly expressed in

265

ORNs we employed the

trpA1-C-T2A-GAL4

and

trpA1-D-T2A-GAL4

We detected

266

significant staining in the antennal lobe driven by the

trpA1-C-T2A-GAL4

but

not by the

267

trpA1-D-T2A-GAL4

(Figures 6C and D).

268

Due to the observation that the

trpA1-C

reporter is expressed in ORNs, we set

269

out to determine whether the

trpA1-C

isoform is sufficient for BA avoidance using two

270

lines of flies. First, we analyzed

trpA1-C

knockin flies (

trpA1-C-KI

which express

271

trpA1-C

in the absence of any other isoform, and found that the

trpA1-C-KI

flies

272

exhibited BA avoidance similar to control flies (Figure 7A). In addition, we performed

273

rescue experiments by expressing either the

UAS-trpA1-C

or the

UAS-trpA1-D

274

transgene in a

trpA1

null mutant background under control of the

trpA1-CD-GAL4

275

Expression of

trpA1-C

rescued the

trpA1

mutant phenotype, while there was only a

276

small effect of expressing the

trpA1-D

isoform, which was not significant (Figure 7B).

277

Furthermore, when we inactivated

trpA1-C

ORNs by expressing an inwardly rectifying

278

channel (

UAS-kir2.1

) under control of a

trpA1-C-T2A-GAL4

, the repulsion to 1% BA

279

was reduced significantly (Figure 7C).

280

To determine whether TRPA1-C is directly activated by BA, we expressed

281

TRPA1-C in an insect cell line (S2 cells) and performed whole-cell recordings. We

282

applied BA dissolved in the extracellular bath solution, and terminated each recording

283

with an activator of TRPA1 channels, allyl isothiocyanate (AITC).

66-68

We found that BA

284

activated TRPA1-C with an average current of 389.1 ±132.6 pA (Figures 7D and F). The

285

TRPA1-D isoform was also activated by BA, but the peak current was much smaller

286

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(128.1 ±39.1 pA; Figures 7E and F). Therefore, we conclude that the TRPA1-C channel

287

is critical for conferring repulsion to BA.

288

Next, we asked whether exposing cells expressing TRPA1-C to H

would

289

prevent TRPA1-

C from being activated by BA. We found that 100 μM H

robustly

290

activated TRPA1-C-dependent currents (Figures 7G-I). When we applied BA to the

291

same cells that had been exposed to H

for 2 minutes, we found that BA did not

292

result in any further activation of TRPA1-C (Figures 7H-I).

293

294

Discussion

295

We provide the first demonstration that light sensation in an olfactory organ

296

impacts odor detection. Specifically, we show that the flies’ avoidance of certain

297

botanically-derived chemicals, such as BA, IVA, and lemongrass is reduced in the

298

presence of light. Flies sleep to a greater extent at night than during the day, especially

299

compared to dawn and dusk. We suggest that the reduced avoidance in a light

300

environment results because the flies become more active, and need to begin to forage

301

after the onset of light.

69-72

Therefore, the barrier to feeding may be much higher in the

302

dark, and should decrease in the light. Food options are complex mixtures of many

303

chemicals, some of which are attractive on their own, while others are aversive.

304

Therefore, in the light, when the barrier to feeding declines, we suggest that light

305

decreases the flies’ aversion to some repulsive volatile chemicals in a potential food,

306

thereby allowing them to land on the substrate and use contact chemosensation and

307

other senses to further evaluate the food.

308

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Surprisingly, the mechanism for the light-induced reduction in olfactory avoidance

309

depends on light sensation in the antenna by Cry, which was originally identified due to

310

its role in light detection in circadian pacemaker neurons in the fly’s brain.

32-35

Also,

311

unexpected is the observation that Cry functions in support cells in olfactory sensilla,

312

rather than in neurons. The finding that Cry is a light sensor in the antenna provides an

313

explanation for the salience of UV and blue light, rather than longer wavelengths, for

314

suppressing olfactory aversion, since Cry is activated by these shorter wavelengths.

46,47

315

Moreover, this work indicates that support cells in olfactory sensilla function as a new

316

class of light sensitive cell.

317

Four rhodopsins (Rh1, Rh3, Rh4 and Rh7) sense short wavelength light.

42-45

318

However, mutations disrupting the

rh1

(

ninaE

rh3

and

rh4

genes do not diminish the

319

suppression of olfactory repulsion by light. The Rh1, Rh3 and Rh4 rhodopsins couple to

320

a PLC

encoded by the

norpA

locus, and consistent with the findings with these

321

mutants, a null mutation in

norpA

also does not reduce the light-induced suppression of

322

the BA repulsion. However, disruption of

rh7

does diminish the effect of light.

323

Nevertheless, we do not detect

rh7

reporter expression in the antenna, indicating that it

324

does not contribute to the effect of light on the olfactory response in the antenna.

325

A key question concerns the mechanism through which expression of Cry in

326

antennal support cells leads to suppression of olfactory avoidance in ORNs. It has been

327

shown previously that UV/blue light activation of Cry leads to ROS production,

and

328

using a ROS sensor we found that short-wavelength light generated ROS in the

329

antenna, which was reduced in the

cry

mutant background. Conversely, expression of

330

DuOx in support cells, which promotes production of hydrogen peroxide, reduces BA

331

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repulsion in the absence of UV/blue light. Of note,

Drosophila

TRPA1 is activated by

332

ROS,

56,57

and we found that TRPA1-C is expressed in ORNs. Moreover, we showed

333

that TRPA1-C contributes to olfactory repulsion of BA, and is directly activated by BA.

334

We propose the model that ROS produced by light activation of Cry in support cells

335

leads to desensitization of TRPA1-C in ORNs, which in turn attenuates the aversion to

336

BA. These findings also highlight a role that support cells play in actively modulating the

337

response to an odor. Although we were not able to maintain our whole-cell recordings

338

sufficiently long to determine if H

results in complete return to the baseline

339

(desensitization), we found that within a couple of minutes of H

exposure, the current

340

began to decline, and was not enhanced upon application of BA.

341

342

Limitation of the study

343

We discovered that light can modify olfactory reception and behavior, and this

344

occurs through direct light activation of cells in olfactory sensilla. We suggest that light is

345

detected by support cells in olfactory sensilla, since Cry is expressed and required in

346

these cells for modification of the olfactory response by light. Thus, in addition to

cry

347

dependent light sensing neurons in the central brain,

Drosophila

appears to be

348

equipped with

cry

-dependent light-sensitive non-neuronal cells in olfactory sensilla.

349

Nevertheless, we did not provide electrophysiological evidence that support cells

350

respond to light. Moreover, since we used field recordings (EAGs) in this analysis, our

351

data do not discriminate between whether light causes fewer ORNs to respond to BA, or

352

simply lowers the spike frequency within a specific population. Single sensillum

353

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recordings would not only be useful in addressing this question, but help clarify the

354

specific population of TRPC1-C-expressing ORNs that respond to BA.

355

While we show that Cry expression in the antenna is important for the UV and

356

blue light-induced suppression of the BA response, Rh7 also impacts the BA sensitivity.

357

Even though according to the FlyCellAtlas there may be

rh7

transcripts in antennal glia,

358

we did not detect

rh7

expression in the antenna, raising the question as to the tissue

359

where light activation of

rh7

suppresses BA responsiveness.

rh7

is expressed in the

360

brain, and at low levels compound eyes.

43,73-75

Therefore, its contribution to the light-

361

induced suppression of olfactory repulsion may reflect either a function in higher order

362

neurons in the brain, or light reception in the eyes.

363

According to our model, a simple prediction is that light would suppress the

364

repulsion to all aversive compounds that activate TRPA1-C. However, light did not

365

reduce the repulsion to AITC, even though it is a well-established activator of TRPA1 in

366

Drosophila

36,40

and of homologs in other animals.

66-68

This raises the possibility that

367

TRPA1-C is not the only receptor for AITC. Consistent with this possibility, repulsion of

368

volatile AITC depends on an odorant receptor, OR42a.

Moreover, since volatile AITC

369

is highly toxic to

Drosophila

a reduction in repulsion to this chemical would reduce

370

survival, and so the observation that repulsion to AITC is not reduced by light is

371

consistent with a likely requirement to strongly avoid AITC at all times. Citronellal is a

372

relatively weak activator of

Drosophila

TRPA1, yet light does not attenuate the repulsion

373

to this chemical either. This may be because citronellal is also detected through

374

olfactory receptors (ORs).

Benzaldehyde can also be sensed in part through ORs.

375

We suggest that may account for our findings that the olfactory repulsion to BA is

376

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reduced but not eliminated in the

trpA1

mutant, and that UV/blue light attenuates rather

377

than completely suppresses the olfactory repulsion to BA.

378

379

RESOURCE AVAILABILITY

380

Lead contact

381

Inquiries should be directed to the lead contact, Craig Montell

382

(cmontell@ucsb.edu).

383

384

Materials availability

385

No reagents were generated as part of this study.

386

387

Data and Code availability

388

•

All raw data and videos are available on Dryad:

389

https://doi.org/10.5061/dryad.ffbg79d5n.

390

•

The codes (CoM_tracker.py and PI_tracker.py) are available on Dryad:

391

https://doi.org/10.5061/dryad.ffbg79d5n.

392

•

The 3D printer files (Funnel.stl, Imaging-Chamber.stl, and Odorant-cap.stl) are

393

available on Dryad:

https://doi.org/10.5061/dryad.ffbg79d5n.

394

395

Acknowledgments

396

This work was supported by grants to C.M. from the National Institute on

397

Deafness and Other Communication Disorders (DC007864 and DC016278) and the

398

Journal Pre-proof

National Institute of Allergy and Infectious Diseases (AI165575). We thank Matthieu

399

Louis, Angela Bontempo and David Tadres for technical suggestions.

400

401

Author contributions

402

D.T. and C.M. designed experiments. D.T. designed the DART2 assay and wrote

403

the code for the analysis. D.T., S.H., T.T., M.P., and J.M.R. performed behavioral

404

experiments. D.T. also performed the EAGs, immunohistochemistry, DCFDA imaging,

405

and the patch clamp experiments. D.T. and C.M. wrote the first draft and edited the

406

manuscript.

407

408

Declaration of interests

409

The authors declare no competing interests.

410

411

Declaration of generative ai and ai-assisted technologies

412

Neither generative AI nor AI-assisted technologies were used in the preparation

413

of this manuscript.

414

415

STAR

★

Methods

416

KEY RESOURCES TABLE

417

Attached as KeyResourcesTable.docx

418

419

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

420

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Flies were reared at 25 °C on normal cornmeal food under 12 hr light/12 hr dark

421

cycles. The control flies carried

and the 2

and 3

chromosomes were from

1118

422

(BDSC #5905). All mutants were outcrossed to

1118

for five generations. The following

423

lines were used (BDSC stock numbers):

trpA1

(26504),

trpA1-AB

GAL4

(67131),

trpA1-

424

GAL4

(67133)

, cry-GAL4

(24514),

cry

(27922)

, cry

orco-RFP

(63045),

orco-GAL4

425

(23292),

ase5-GAL4

(93029),

nompA-GAL4

(605353),

repo-GAL4

(7415),

UAS-hDuOx

426

(78412),

UAS-Cry

(86263),

UAS-mCD8::GFP

(5137),

10xUAS-IVS-mCD8::RFP

427

13xLexAop2-mCD8::GFP

(32229),

norpA

P24

(9048),

ninaE

I17

(5701),

rh3

rh4

rh7

428

rh7

(rh7-LexA)

UAS-dsRed

Y. Xiang provided

trpA1-KO, trpA1-C-KI, trpA1-C-T2A-

429

GAL4

and

trpA1-D-T2A-GAL4

W. D. Tracey provided

UAS-trpA1-C, UAS-trpA1-D

430

D. Smith provided the

lush-GAL4

431

432

METHOD DETAILS

433

Chemicals

434

The following were from Sigma-Aldrich: benzaldehyde (B1334), propionic acid

435

(81910), isovaleric acid (129542), citronellal (27470), AITC (W203408), and poly-L-

436

ornithine (P4957). The sources of the following chemicals are as indicated: lemongrass

437

oil (Nature’s Alchemy Pure Essential Oils), R-limonene (Fluka, 62122), DMSO (JT

438

Baker, 67-68-5), and H2-DCFDA (Thermo Fisher Scientific, D399).

439

440

DART2 assays

441

We constructed each assay tube by assembling one 150 mm-long Pyrex tube

442

(10 mm outer diameter, 1 mm wall

Testing the impact of using

ness) capped at either end

443

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with an odorant carrying cap. The odorant carrying caps were designed using Microsoft

444

3D Builder and printed using a Formlabs II 3D printer and black resin (FLGPBK01). The

445

‘Odorant-cap.stl’ 3D printer file is available at Dryad:

446

https://doi.org/10.5061/dryad.ffbg79d5n. The caps were washed in isopropyl alcohol

447

for 1 hr., cured at 60 °C under violet light illumination in a Formlabs curing oven

448

(FormCure), washed with an odorless detergent (Alconox, Cat. no. 1104-1), followed by

449

a wash in ethanol, and then baked at 65 °C for 12-16 hours to remove residual odors.

450

To ensure that there was no contact-chemosensation in the assay, the cap had meshed

451

chambers to create an 8 mm-wide separation between the odorant and the flies in the

452

assay tube.

453

We reared all flies at 25 °C. To prepare the flies for the assays, we collected flies 1-3

454

days post-eclosion from culture vials using CO

, placed flies in individual vials

455

containing fresh food and allowed them to recover from the CO

exposure for 48-72

456

hours. In preparation for the assay, the flies were transferred to each tube by attaching

457

a 3D-printed custom funnel on the Pyrex tube and tapping the flies into the tube. The

458

‘Funnel.stl’ 3D printer file is available at Dryad:

459

https://doi.org/10.5061/dryad.ffbg79d5n. We used Clear resin (Formlabs FLGPCL04)

460

to print the funnel as we found that builds made out of clear resin offered better impact-

461

resistance. The flies were then distributed homogeneously in each tube by gently tilting

462

and tapping the tube. The flies were allowed to acclimatize in the tubes for 20 minutes.

463

5 μL of the solvent control and the test odor were added to filter paper rounds

464

(Whatman cat no. 1001-6508), and then placed in the external chambers of the odor

465

caps. The external chambers were then capped with lids broken off from 500 μL

466

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Eppendorf tubes. The tubes were immediately placed in a test chamber, similar to a

467

previously described four field chamber,

which we repurposed for these assays. The

468

test chamber was maintained at 23 °C, and the temperature was monitored using a

469

Thorlabs TSP-01 thermal probe. Flies were illuminated by an array of near infrared

470

LEDs. Fly locomotion was captured using a CCD camera equipped with a 850 nm cutoff

471

filter (Edmund Optics), at the rate of 1 frame/sec for 60 min.

472

The response to each odor was unique and therefore the time windows to pick

473

the average CoM were unique for every odor (Figure

S7)

ー

propionic acid: 5 - 25 mins,

474

R-limonene: 0 - 60 mins, AITC: 20 - 60 mins, citronellal: 20 - 60 mins, isovaleric acid: 5 -

475

25 mins, lemongrass: 20 - 60 min. All odorants were dissolved in DMSO, with the

476

exception of propionic acid, which was dissolved in distilled H

477

The odor caps and tubes were cleaned for reuse after every assay. The caps

478

and tubes were first soaked in distilled H

O saturated with Alconox odorless soap for 1

479

hour. The soap was then completely washed off, the caps and tubes were soaked in

480

50% ethanol for 1 hr, and washed in distilled H

O. The caps and tubes were then baked

481

for 20-24 hours at 65 °C.

482

Light intensities were measured with a Thorlabs PM100D power meter. LEDs

483

were mounted on heatsinks and placed above the assay tubes such that there was

484

near-uniform illumination of the entire tube length: UV (365 nm, 7021.365, Waveform

485

lighting), blue (450 nm; Samsung LM561C), green (530 nm, 7041.525, Waveform

486

lighting) and red (630 nm, 7041.630, Waveform lighting). T

he intensities measured at

487

either end of each tube were the same, while the intensity at the center of the tube was on

488

average ~10% lower than at either end of the tube.

The temperature inside the test

489

Journal Pre-proof

chamber was monitored using a Thorlabs TSP-01 probe and maintained by attaching a

490

24 V exhaust fan at the top of the enclosure. The test chamber was vented at the

491

bottom so that room air was able to flow in while stray light from the exterior was not

492

permitted in.

493

494

Immunohistochemistry

495

For reporter labeling of tissue, we used

UAS-GFP

UAS-RFP

and

LexAop-GFP

496

expressed under control of the indicated

GAL4

and

LexA

drivers at 25 °C. Flies were

497

fixed in 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS) for 30 minutes

498

at room temperature (RT, ~22 °C). The fixed animals were washed 6x with PBS

499

containing 0.3% Triton X-100 (PBS-T) at RT. Antennae and brains were dissected in

500

PBS-T. All tissue samples were washed 3x in PBS-T and blocked for 1 hr in PBS-T with

501

5% goat serum. The samples were incubated with primary antibodies diluted in blocking

502

solution for 48 hours at 4 °C: mouse nc82 (1:100, Developmental Study Hybridoma

503

Bank), chicken anti-GFP (1:1000, A-11122, Invitrogen), and rabbit anti-dsRed (1:1000,

504

632496, Clontech) or mouse anti-nc82 (DSHB). The samples were washed 3x for 20

505

min each in PBS-T and then incubated with secondary antibodies for 24 hrs at 4 °C:

506

Alexa Fluor 488 goat anti-chicken (1:200, A-11039, Invitrogen), and Alexa Fluor 568

507

goat anti-rabbit (1:200, A-11011, Invitrogen) or Alexa Fluor 568 goat anti-mouse (1:200,

508

A-11004, Invitrogen). The samples were washed 3x in PBS-T. Brain samples were

509

mounted with Vectashield mounting media (VectorLabs). Antenna and maxillary palp

510

samples were mounted in Rapiclear (Sunjin Lab). All samples were imaged on an LSM

511

Journal Pre-proof

700 or LSM 900 confocal microscope (Zeiss). Images were prepared using Zen2 (Zeiss)

512

or Fiji (ImageJ).

513

514

Electroantennography (EAG recordings)

515

Flies were trapped in 200 μL tips and mounted under an inverted microscope

516

(Nikon Eclipse FN1). A steady stream of humidified air was applied over the flies. The

517

ground and recording electrode pipets were pulled using a Sutter P97 puller using

518

1B150F-3 borosilicate glass (Warner Instruments) to obtain a tip diameter that showed

519

a resistance of 0.5 - 1 MOhms. Both the recording and reference pipettes were filled

520

with electrolyte solution (108 mM NaCl, 4 mM NaHCO

, 1 mM NaH

, 5 mM KCl, 2

521

mM CaCl

, 8.2 mM MgCl

, 5 mM HEPES, 10 mM sucrose, 5 mM trehalose). The pipet

522

tips were coated with conducting cream (Sigma Creme, AD Instruments). The reference

523

electrode was placed at the base of the antenna on the frons between the eyes, and the

524

recording electrode was placed at the tip of the antenna. 5 μL of each odorant was

525

applied to a filter paper round placed inside a 1 mL tip. Odor air puffs were applied for

526

500 ms through the 1 mL pipet tip positioned 1.5 cm from the fly, using a Syntech air

527

pump. The responses were recorded using EAGPro (Syntech Oeckenfels GmbH) at the

528

rate of 5kHz. We chose to perform EAG experiments with blue rather than UV light

529

since the EAGs were not within an enclosure. Consequently, the use of blue light

530

prevented exposing the experimenter to UV light. Moreover, our microscope lamp bulb

531

emission was low in the UV range. Blue light was applied through the objective of the

532

microscope using a 450 nm filter (Chroma Technology Corp.). To avoid adaptation to

533

the light stimulus we performed one trial per fly. For each trial, after recording the

534

Journal Pre-proof

response to benzaldehyde in the dark we allowed the fly to remain in the dark for 5

535

minutes to avoid adaptation to the odor, then applied blue light for 5 minutes before

536

applying benzaldehyde. For a few samples, the blue light stimulation was then switched

537

off and the preparation was allowed to recover in the dark for 5 minutes before applying

538

benzaldehyde in the dark. The application of benzaldehyde in the 2

dark-period

539

yielded EAG responses similar in amplitude to the response in the dark-period before

540

application of blue light, indicating that the antenna recovered its sensitivity to

541

benzaldehyde in the dark.

542

543

DCFDA imaging

544

Antennae were dissected and placed in artificial hemolymph solution (AHLS)

545

inside a 3D printer imaging chamber printed using black resin (Formlabs, FLGPBK01)

546

(ImagingChamber.stl 3D printer file;

https://doi.org/10.5061/dryad.ffbg79d5n). The

547

imaging chamber had a thin perforation at the bottom that allowed a single antenna to

548

be placed securely. Each antenna was then incubated for 45 min at RT with 2′,7′-

549

dichlorofluorescein diacetate

(

DCFDA) at a final concentration of 2 μM in AHLS,

550

washed 3x with ALHS, and imaged on a Zeiss LSM 900 confocal microscope. Baseline

551

fluorescence at 530 nm was recorded for 30 sec before application of the UV stimulus

552

(360 nm) by switching to the inbuilt DAPI filter in the LSM 900 for 1 sec. Following UV

553

stimulation, DCFDA fluorescence was recorded every 3 sec for 2 minutes, and the

554

steady-state peak fluorescence was used to compute the change in fluorescence from

555

the baseline reading.

556

557

Journal Pre-proof

Patch-clamp electrophysiology

558

S2 cells were cultured at RT in Schneider’s

Drosophila

medium (21720, Gibco)

559

and transfected using either Lipofectamine 2000 (Invitrogen) or X-tremeGENE HP DNA

560

Transfection Reagent (Roche) with the control plasmid (pAc5.1) encoding

GFP

only, or

561

the plasmid encoding

GFP

and either

trpA1-C

trpA1

. The transfected cells were

562

plated on poly-L-ornithine-coated 12 mm coverslips and assayed by whole-cell clamp

563

48 hours post-transfection. Drugs were dissolved in extracellular saline (140 mM NaCl,

564

5 mM KCl, 1 mM MgCl

, 2 mM CaCl

, 10 mM HEPES, 10 mM glucose), which was

565

buffered to pH 7.4 with NaOH and with an osmolarity adjusted to 300 mOsm with

566

mannitol. Cells were patched using borosilicate pipets (BF150-86-10, Sutter Instrument)

567

with a pipet resistance of 5-7 MOhms. The pipet solution contained 140 mM CsCl, 1 mM

568

MgCl

, 0.05 mM EGTA, 10 mM HEPES, which was buffered to pH 7.2 with CsOH and

569

with an osmolarity adjusted to 290 mOsm with mannitol. Both the extracellular and pipet

570

solutions were sterile filtered with 0.22

m sterile filters. Drugs were added to cells using

571

a gravity-driven perfusion system. Isolated cells were voltage-clamped in the whole-cell

572

mode using an Axon 200B amplifier and 1440A Digitizer (Molecular Devices). Currents

573

were recorded at 10 kHz. Cells were held at -60 mV with voltage ramps applied every 5

574

sec, which linearly increased from -80 to +80 mV within 200 ms to extract current vs.

575

voltage characteristics. To test for membrane resealing, a membrane test pulse of -10

576

mV was applied for 20 msec within each voltage-clamp cycle 100 msec before

577

application of the +80 to -80 mV voltage ramp. Cells that resealed were eliminated from

578

analysis.

579

580

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QUANTIFICATION AND STATISTICAL ANALYSIS

581

Fly movements were analyzed per-frame using a custom Python script. The

582

transverse components of the average position of the flies or the center of mass (CoM)

583

were calculated using CoM_tracker (

available as “CoM_tracker” at Dryad:

584

https://doi.org/10.5061/dryad.ffbg79d5n). The CoM per frame were calculated based

585

on the formula: CoM = Σm

/(M*L). We defined the terms as follows: m, the pixel

586

count that crossed a preset brightness threshold to distinguish from the background in

587

each video; x, the lengthwise coordinate of each count; M, the total number of counts; L,

588

the length of the tube in pixels.

589

The avoidance index was calculated using PI_tracker (code is available at Dryad

590

as “PI_tracker”: https://doi.org/10.5061/dryad.ffbg79d5n). We bisected each tube.

591

Flies in the half of the tube that was nearest the solvent were considered to be repelled

592

by the odor. Flies in the half of the tube nearest the side with the test odor dissolved in

593

the solvent were considered to be attracted to the test odor. The avoidance index per

594

frame was as follows:

595

A.I. = (Σflies in odorant side - Σflies in solvent side)/Σall flies in tube. Images were

596

captured at the rate of 1 frame/sec and the center of mass (CoM) of the flies was

597

analyzed at 1 frame/10 sec. The average CoM was obtained for the range of CoM

598

values per frame when the behavior response reached a maximum followed by an

599

approximately steady state. The number of flies used in each tube and the number of

600

experimental replicates are stated in the figure legend corresponding to each figure.

601

We used Mann-Whitney U test to compare means between two groups, and ANOVA

602

followed by Dunn’s multiple comparisons test when comparing means across more than

603

Journal Pre-proof

two groups. EAG responses were quantified as: amplitude of the peak response - mean

604

of baseline amplitude for 5 seconds preceding application of the odor puff, and

605

compared using paired Wilcoxon test. Recordings that showed no response (< 0.05 mV)

606

to odor application were excluded from analysis. DCFDA response was quantified as

607

steady state response after UV stimulation - steady-state baseline response. Voltage

608

clamp recordings from S2 cells were quantified as amplitude of the peak response -

609

mean of baseline amplitude for 5 seconds preceding application of the stimulus. Quality

610

of the whole-cell seal was monitored using a -10 mV step pulse applied every 5 s and

611

cells that showed re-sealing during the recording were excluded from analysis. Mean

612

response

s were compared using ANOVA followed by Dunn’s multiple comparison’s test.

613

Precise information about the analysis and statistical tests used in each panel can be

614

found in the corresponding figure legends. Unless otherwise indicated, in all figures,

615

error bars indicate mean ± SEMs. * p < 0.05. ** p < 0.01. *** p < 0.001. ns = not

616

significant.

617

618

References

619

Spence, C. (2015). Eating with our ears: assessing the importance of the sounds

620

of consumption on our perception and enjoyment of multisensory flavour

621

experiences. Flavour

4:3

622

Hochenberger, R., and Ohla, K. (2019). A bittersweet symphony: Evidence for

623

taste-sound correspondences without effects on taste quality-specific perception.

624

J. Neurosci. Res.

, 267-275. 10.1002/jnr.24308.

625

Journal Pre-proof

Tu, Y.J., Yang, Z., and Ma, C.Q. (2016). The taste of plate: how the spiciness of

626

food is affected by the color of the plate used to serve it. J. Sens. Stud.

, 50-

627

60. 10.1111/joss.12190.

628

Spence, C., Velasco, C., and Knoeferle, K. (2014). A large sample study on the

629

influence of the multisensory environment on the wine drinking experience.

630

Flavour

3:8

631

Oberfeld, D., Hecht, H., Allendorf, U., and Wickelmaier, F. (2009). Ambient

632

lighting modifies the flavor of wine. J. Sens. Stud.

, 797-832. 10.1111/j.1745-

633

459X.2009.00239.x.

634

Srivastava, S., Donaldson, L.F., Rai, D., Melichar, J.K., and Potokar, J. (2013).

635

Single bright light exposure decreases sweet taste threshold in healthy

636

volunteers. J. Psychopharm.

, 921-929. Doi 10.1177/0269881113499206.

637

van der Heijden, K., Festjens, A., and Goukens, C. (2021). On the bright side:

638

The influence of brightness on overall taste intensity perception. Food Qual.

639

Prefer.

. ARTN 104099 10.1016/j.foodqual.2020.104099.

640

Szczesniak, A.S. (2002). Texture is a sensory property. Food Qual. Prefer.

641

215-225. Pii S0950-3293(01)00039-8 Doi 10.1016/S0950-3293(01)00039-8.

642

Zhang, Y.V., Aikin, T.J., Li, Z., and Montell, C. (2016). The basis of food texture

643

sensation in

Drosophila

. Neuron

, 863-877. 10.1016/j.neuron.2016.07.013.

644

10.

Li, Q., DeBeaubien, N.A., Sokabe, T., and Montell, C. (2020). Temperature and

645

sweet taste integration in

Drosophila

. Curr. Biol.

, 2051-2067

646

10.1016/j.cub.2020.04.084.

647

Journal Pre-proof

11.

Li, Q., and Montell, C. (2021). Mechanism for food texture preference based on

648

grittiness. Curr. Biol.

, 1850-1861. 10.1016/j.cub.2021.02.007.

649

12.

Sánchez-Alcañiz, J.A., Zappia, G., Marion-Poll, F., and Benton, R. (2017). A

650

mechanosensory receptor required for food texture detection in

Drosophila

. Nat

651

Commun

, 14192. 10.1038/ncomms14192.

652

13.

Jeong, Y.T., Oh, S.M., Shim, J., Seo, J.T., Kwon, J.Y., and Moon, S.J. (2016).

653

Mechanosensory neurons control sweet sensing in

Drosophila

. Nat Commun

654

12872. 10.1038/ncomms12872.

655

14.

Talavera, K., Yasumatsu, K., Voets, T., Droogmans, G., Shigemura, N.,

656

Ninomiya, Y., Margolskee, R.F., and Nilius, B. (2005). Heat activation of TRPM5

657

underlies thermal sensitivity of sweet taste. Nature

438

, 1022-1025.

658

15.

Speed, L.J., de Valk, J., Croijmans, I., Huisman, J.L.A., and Majid, A. (2023).

659

Odor-color associations are not mediated by concurrent verbalization. Cogn. Sci.

660

, e13266. 10.1111/cogs.13266.

661

16.

Tamura, K., and Okamoto, T. (2023). Odor descriptive ratings can predict some

662

odor-color associations in different color features of hue or lightness. PeerJ

663

e15251. 10.7717/peerj.15251.

664

17.

Montell, C. (2021).

Drosophila

sensory receptors

—a set of molecular Swiss Army

665

Knives. Genetics

217

, 1-34. 10.1093/genetics/iyaa011.

666

18.

Enjin, A., Zaharieva, E.E., Frank, D.D., Mansourian, S., Suh, G.S., Gallio, M.,

667

and Stensmyr, M.C. (2016). Humidity sensing in

Drosophila

. Curr. Biol.

, 1352-

668

1358. 10.1016/j.cub.2016.03.049.

669

Journal Pre-proof

19.

Knecht, Z.A., Silbering, A.F., Ni, L., Klein, M., Budelli, G., Bell, R., Abuin, L.,

670

Ferrer, A.J., Samuel, A.D., Benton, R., and Garrity, P.A. (2016). Distinct

671

combinations of variant ionotropic glutamate receptors mediate thermosensation

672

and hygrosensation in

Drosophila

. eLife

, e17879. 10.7554/eLife.17879.

673

20.

Knecht, Z.A., Silbering, A.F., Cruz, J., Yang, L., Croset, V., Benton, R., and

674

Garrity, P.A. (2017). Ionotropic Receptor-dependent moist and dry cells control

675

hygrosensation in

Drosophila

. eLife

, e26654. 10.7554/eLife.26654.

676

21.

Eberl, D.F., Hardy, R.W., and Kernan, M.J. (2000). Genetically similar

677

transduction mechanisms for touch and hearing in

Drosophila

. J. Neurosci.

678

5981-5988.

679

22.

Göpfert, M.C., and Robert, D. (2001). Biomechanics. Turning the key on

680

Drosophila

audition. Nature

411

, 908. 10.1038/35082144.

681

23.

Yorozu, S., Wong, A., Fischer, B.J., Dankert, H., Kernan, M.J., Kamikouchi, A.,

682

Ito, K., and Anderson, D.J. (2009). Distinct sensory representations of wind and

683

near-field sound in the

Drosophila

brain. Nature

458

, 201-205.

684

10.1038/nature07843.

685

24.

Kamikouchi, A., Inagaki, H.K., Effertz, T., Hendrich, O., Fiala, A., Göpfert, M.C.,

686

and Ito, K. (2009). The neural basis of

Drosophila

gravity-sensing and hearing.

687

Nature

458

, 165-171. 10.1038/nature07810.

688

25.

Zars, T. (2001). Two thermosensors in

Drosophila

have different behavioral

689

functions. J. Comp. Physiol. A

187

, 235-242.

690

Journal Pre-proof

26.

Martin, F., Riveron, J., and Alcorta, E. (2011). Environmental temperature

691

modulates olfactory reception in

Drosophila melanogaster

. J. Insect Physiol.

692

1631-1642. 10.1016/j.jinsphys.2011.08.016.

693

27.

Riveron, J., Boto, T., and Alcorta, E. (2009). The effect of environmental

694

temperature on olfactory perception in

Drosophila melanogaster

. J. Insect

695

Physiol.

, 943-951. 10.1016/j.jinsphys.2009.06.009.

696

28.

Lou, L., Tu, Z.J., Lahondere, C., and Vinauger, C. (2024). Rhythms in insect

697

olfactory systems: underlying mechanisms and outstanding questions. J. Exp.

698

Biol.

227

, jeb244182. 10.1242/jeb.244182.

699

29.

Senthilan, P.R., Piepenbrock, D., Ovezmyradov, G., Nadrowski, B., Bechstedt,

700

S., Pauls, S., Winkler, M., Mobius, W., Howard, J., and Göpfert, M.C. (2012).

701

Drosophila

auditory organ genes and genetic hearing defects. Cell

150

, 1042-

702

1054. 10.1016/j.cell.2012.06.043.

703

30.

Krishnan, B., Levine, J.D., Lynch, M.K., Dowse, H.B., Funes, P., Hall, J.C.,

704

Hardin, P.E., and Dryer, S.E. (2001). A new role for cryptochrome in a

Drosophila

705

circadian oscillator. Nature

411

, 313-317. 10.1038/35077094.

706

31.

Ikeda, K., Kataoka, M., and Tanaka, N.K. (2022). Nonsynaptic transmission

707

mediates light context-dependent odor responses in

Drosophila melanogaster

. J.

708

Neurosci.

, 8621-8628. 10.1523/JNEUROSCI.1106-22.2022.

709

32.

Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager-Smith, K., Kay, S.A.,

710

Rosbash, M., and Hall, J.C. (1998). The

cry

mutation identifies cryptochrome as

711

a circadian photoreceptor in

Drosophila

. Cell

, 681-692.

712

Journal Pre-proof

33.

Emery, P., So, W.V., Kaneko, M., Hall, J.C., and Rosbash, M. (1998). CRY, a

713

Drosophila clock and light-regulated cryptochrome, is a major contributor to

714

circadian rhythm resetting and photosensitivity. Cell

, 669-679. S0092-

715

8674(00)81637-2 [pii].

716

34.

Emery, P., Stanewsky, R., Helfrich-Forster, C., Emery-Le, M., Hall, J.C., and

717

Rosbash, M. (2000).

Drosophila

CRY is a deep brain circadian photoreceptor.

718

Neuron

, 493-504.

719

35.

Ishikawa, T., Matsumoto, A., Kato, T., Jr., Togashi, S., Ryo, H., Ikenaga, M.,

720

Todo, T., Ueda, R., and Tanimura, T. (1999). DCRY is a

Drosophila

721

photoreceptor protein implicated in light entrainment of circadian rhythm. Genes

722

Cells

, 57-65. 10.1046/j.1365-2443.1999.00237.x.

723

36.

Kwon, Y., Kim, S.H., Ronderos, D.S., Lee, Y., Akitake, B., Woodward, O.M.,

724

Guggino, W.B., Smith, D.P., and Montell, C. (2010).

Drosophila

TRPA1 channel

725

is required to avoid the naturally occurring insect repellent citronellal. Curr. Biol.

726

, 1672-1678. S0960-9822(10)01006-7 [pii] 10.1016/j.cub.2010.08.016.

727

37.

Helfand, S.L., and Carlson, J.R. (1989). Isolation and characterization of an

728

olfactory mutant in

Drosophila

with a chemically specific defect. Proc. Natl. Acad.

729

Sci. USA

, 2908-2912. 10.1073/pnas.86.8.2908.

730

38.

Keene, A.C., Stratmann, M., Keller, A., Perrat, P.N., Vosshall, L.B., and Waddell,

731

S. (2004). Diverse odor-conditioned memories require uniquely timed dorsal

732

paired medial neuron output. Neuron

, 521-533.

733

10.1016/j.neuron.2004.10.006.

734

Journal Pre-proof

39.

Ramdya, P., Lichocki, P., Cruchet, S., Frisch, L., Tse, W., Floreano, D., and

735

Benton, R. (2015). Mechanosensory interactions drive collective behaviour in

736

Drosophila

. Nature

519

, 233-236. 10.1038/nature14024.

737

40.

Kang, K., Pulver, S.R., Panzano, V.C., Chang, E.C., Griffith, L.C., Theobald,

738

D.L., and Garrity, P.A. (2010). Analysis of

Drosophila

TRPA1 reveals an ancient

739

origin for human chemical nociception. Nature

464

, 597-600. nature08848 [pii]

740

10.1038/nature08848.

741

41.

Oyedele, A.O., Gbolade, A.A., Sosan, M.B., Adewoyin, F.B., Soyelu, O.L., and

742

Orafidiya, O.O. (2002). Formulation of an effective mosquito-repellent topical

743

product from lemongrass oil. Phytomedicine

, 259-262. 10.1078/0944-7113-

744

00120.

745

42.

Montell, C. (2012).

Drosophila

visual transduction. Trends Neurosci.

, 356-363.

746

10.1016/j.tins.2012.03.004.

747

43.

Ni, J.D., Baik, L.S., Holmes, T.C., and Montell, C. (2017). A rhodopsin in the

748

brain functions in circadian photoentrainment in

Drosophila

. Nature

545

, 340-

749

344. 10.1038/nature22325.

750

44.

Sakai, K., Tsutsui, K., Yamashita, T., Iwabe, N., Takahashi, K., Wada, A., and

751

Shichida, Y. (2017).

Drosophila melanogaster

rhodopsin Rh7 is a UV-to-visible

752

light sensor with an extraordinarily broad absorption spectrum. Sci. Rep.

, 7349.

753

10.1038/s41598-017-07461-9.

754

45.

Sharkey, C.R., Blanco, J., Leibowitz, M.M., Pinto-Benito, D., and Wardill, T.J.

755

(2020). The spectral sensitivity of

Drosophila

photoreceptors. Sci. Rep.

756

18242. 10.1038/s41598-020-74742-1.

757

Journal Pre-proof

46.

Fogle, K.J., Parson, K.G., Dahm, N.A., and Holmes, T.C. (2011).

758

CRYPTOCHROME is a blue-light sensor that regulates neuronal firing rate.

759

Science

331

, 1409-1413. 10.1126/science.1199702.

760

47.

Baik, L.S., Fogle, K.J., Roberts, L., Galschiodt, A.M., Chevez, J.A., Recinos, Y.,

761

Nguy, V., and Holmes, T.C. (2017). CRYPTOCHROME mediates behavioral

762

executive choice in response to UV light. Proc. Natl. Acad. Sci. USA

114

, 776-

763

781. 10.1073/pnas.1607989114.

764

48.

Vosshall, L.B., and Stocker, R.F. (2007). Molecular architecture of smell and

765

taste in

Drosophila

. Annu. Rev. Neurosci.

, 505-533.

766

49.

Zhao, J., Kilman, V.L., Keegan, K.P., Peng, Y., Emery, P., Rosbash, M., and

767

Allada, R. (2003).

Drosophila

clock can generate ectopic circadian clocks. Cell

768

113

, 755-766. 10.1016/s0092-8674(03)00400-8.

769

50.

Emery, P., Stanewsky, R., Hall, J.C., and Rosbash, M. (2000). A unique

770

circadian-rhythm photoreceptor. Nature

404

, 456-457. 10.1038/35006558.

771

51.

Kwon, J.Y., Dahanukar, A., Weiss, L.A., and Carlson, J.R. (2011). Molecular and

772

cellular organization of the taste system in the

Drosophila

larva. J. Neurosci.

773

15300-15309. 31/43/15300 [pii] 10.1523/JNEUROSCI.3363-11.2011.

774

52.

Agrawal, P., Houl, J.H., Gunawardhana, K.L., Liu, T., Zhou, J., Zoran, M.J., and

775

Hardin, P.E. (2017).

Drosophila

CRY entrains clocks in body tissues to light and

776

maintains passive membrane properties in a non-clock body tissue independent

777

of light. Curr. Biol.

, 2431-2441. 10.1016/j.cub.2017.06.064.

778

53.

Arthaut, L.D., Jourdan, N., Mteyrek, A., Procopio, M., El-Esawi, M., d'Harlingue,

779

A., Bouchet, P.E., Witczak, J., Ritz, T., Klarsfeld, A., et al. (2017). Blue-light

780

Journal Pre-proof

induced accumulation of reactive oxygen species is a consequence of the

781

Drosophila

cryptochrome photocycle. PLoS One

, e0171836.

782

10.1371/journal.pone.0171836.

783

54.

Fang, X., Ide, N., Higashi, S., Kamei, Y., Toyooka, T., Ibuki, Y., Kawai, K., Kasai,

784

H., Okamoto, K., Arimoto-Kobayashi, S., and Negishi, T. (2014). Somatic cell

785

mutations caused by 365 nm LED-UVA due to DNA double-strand breaks

786

through oxidative damage. Photochem. Photobiol. Sci.

, 1338-1346.

787

10.1039/c4pp00148f.

788

55.

Negishi, T., Xing, F., Koike, R., Iwasaki, M., Wakasugi, M., and Matsunaga, T.

789

(2023). UVA causes specific mutagenic DNA damage through ROS production,

790

rather than CPD formation, in

Drosophila

larvae. Mutat. Res. Genet. Toxicol.

791

Environ. Mutagen.

887

, 503616. 10.1016/j.mrgentox.2023.503616.

792

56.

Guntur, A.R., Gu, P., Takle, K., Chen, J., Xiang, Y., and Yang, C.H. (2015).

793

Drosophila

TRPA1 isoforms detect UV light via photochemical production of

794

. Proc. Natl. Acad. Sci. USA

112

, E5753-5761. 10.1073/pnas.1514862112.

795

57.

Du, E.J., Ahn, T.J., Wen, X., Seo, D.W., Na, D.L., Kwon, J.Y., Choi, M., Kim,

796

H.W., Cho, H., and Kang, K. (2016). Nucleophile sensitivity of

Drosophila

TRPA1

797

underlies light-induced feeding deterrence. eLife

, e18425.

798

10.7554/eLife.18425.

799

58.

Zhong, L., Bellemer, A., Yan, H., Honjo, K., Robertson, J., Hwang, R.Y., Pitt,

800

G.S., and Tracey, W.D. (2012). Thermosensory and non-thermosensory isoforms

801

Drosophila melanogaster

TRPA1 reveal heat sensor domains of a thermoTRP

802

channel. Cell Rep.

, 43-55. 10.1016/j.celrep.2011.11.002.

803

Journal Pre-proof

59.

Kang, K., Panzano, V.C., Chang, E.C., Ni, L., Dainis, A.M., Jenkins, A.M.,

804

Regna, K., Muskavitch, M.A., and Garrity, P.A. (2012). Modulation of TRPA1

805

thermal sensitivity enables sensory discrimination in

Drosophila

. Nature

481

, 76-

806

80. 10.1038/nature10715.

807

60.

Gu, P., Gong, J., Shang, Y., Wang, F., Ruppell, K.T., Ma, Z., Sheehan, A.E.,

808

Freeman, M.R., and Xiang, Y. (2019). Polymodal nociception in

Drosophila

809

requires alternative splicing of

TrpA1

. Curr. Biol.

, 3961-3973.

810

10.1016/j.cub.2019.09.070.

811

61.

Luo, J., Shen, W.L., and Montell, C. (2017). TRPA1 mediates sensation of the

812

rate of temperature change in

Drosophila

larvae. Nat. Neurosci.

, 34-41.

813

10.1038/nn.4416.

814

62.

Gao, Q., Yuan, B., and Chess, A. (2000). Convergent projections of

Drosophila

815

olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci.

816

780-785. 10.1038/77680.

817

63.

Grabe, V., Strutz, A., Baschwitz, A., Hansson, B.S., and Sachse, S. (2015).

818

Digital

in vivo

3D atlas of the antennal lobe of

Drosophila melanogaster

. J. Comp.

819

Neurol.

523

, 530-544. 10.1002/cne.23697.

820

64.

Laissue, P.P., Reiter, C., Hiesinger, P.R., Halter, S., Fischbach, K.F., and

821

Stocker, R.F. (1999). Three-dimensional reconstruction of the antennal lobe in

822

Drosophila melanogaster

. J. Comp. Neurol.

405

, 543-552.

823

65.

Bates, A.S., Schlegel, P., Roberts, R.J.V., Drummond, N., Tamimi, I.F.M.,

824

Turnbull, R., Zhao, X., Marin, E.C., Popovici, P.D., Dhawan, S., et al. (2020).

825

Journal Pre-proof

Complete connectomic reconstruction of olfactory projection neurons in the fly

826

brain. Curr. Biol.

, 3183-3199. 10.1016/j.cub.2020.06.042.

827

66.

Story, G.M., Peier, A.M., Reeve, A.J., Eid, S.R., Mosbacher, J., Hricik, T.R.,

828

Earley, T.J., Hergarden, A.C., Andersson, D.A., Hwang, S.W., et al. (2003).

829

ANKTM1, a TRP-like channel expressed in nociceptive neurons, Is activated by

830

cold temperatures. Cell

112

, 819-829.

831

67.

Bandell, M., Story, G.M., Hwang, S.W., Viswanath, V., Eid, S.R., Petrus, M.J.,

832

Earley, T.J., and Patapoutian, A. (2004). Noxious cold ion channel TRPA1 is

833

activated by pungent compounds and bradykinin. Neuron

, 849-857.

834

68.

Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M.,

835

Högestätt, E.D., Meng, I.D., and Julius, D. (2004). Mustard oils and cannabinoids

836

excite sensory nerve fibres through the TRP channel ANKTM1. Nature

427

, 260-

837

265.

838

69.

Hall, J.C. (2003). Genetics and molecular biology of rhythms in Drosophila and

839

other insects. Adv. Genet.

, 1-280. 10.1016/s0065-2660(03)48000-0.

840

70.

Konopka, R.J., and Benzer, S. (1971). Clock mutants of

Drosophila

841

melanogaster

. Proc. Natl. Acad. Sci. USA.

, 2112-2116.

842

71.

Rosbash, M. (2021). Circadian rhythms and the transcriptional feedback loop

843

(Nobel Lecture). Angew. Chem. Int. Ed. Engl.

, 8650-8666.

844

10.1002/anie.202015199.

845

72.

Young, M.W. (2018). Time travels: a 40-year journey from Drosophila's clock

846

mutants to human circadian disorders (Nobel Lecture). Angew. Chem. Int. Ed.

847

Engl.

, 11532-11539. 10.1002/anie.201803337.

848

Journal Pre-proof

73.

Grebler, R., Kistenpfennig, C., Rieger, D., Bentrop, J., Schneuwly, S., Senthilan,

849

P.R., and Helfrich-Forster, C. (2017). Drosophila Rhodopsin 7 can partially

850

replace the structural role of Rhodopsin 1, but not its physiological function. J.

851

Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 10.1007/s00359-

852

017-1182-8.

853

74.

Kistenpfennig, C., Grebler, R., Ogueta, M., Hermann-Luibl, C., Schlichting, M.,

854

Stanewsky, R., Senthilan, P.R., and Helfrich-Forster, C. (2017). A new rhodopsin

855

influences light-dependent daily activity patterns of fruit flies. J. Biol. Rhythms,

856

748730417721826. 10.1177/0748730417721826.

857

75.

Meyerhof, G.T., Easwaran, S., Morales, A.E., Montell, C., and Montell, D.J.

858

(2024). Altered circadian rhythm, sleep, and rhodopsin 7-dependent shade

859

preference during diapause in

Drosophila melanogaster

. Proc. Natl. Acad. Sci.

860

U.S.A.

121

, e2400964121. 10.1073/pnas.2400964121.

861

76.

Matsunaga, T., Reisenman, C.E., Goldman-Huertas, B., Rajshekar, S., Suzuki,

862

H.C., Tadres, D., Wong, J., Louis, M., Ramirez, S.R., and Whiteman, N.K.

863

(2024). Odorant receptors tuned to isothiocyanates in

Drosophila melanogaster

864

and their evolutionary expansion in herbivorous relatives. bioRxiv.

865

10.1101/2024.10.08.617316.

866

77.

Hallem, E.A., Ho, M.G., and Carlson, J.R. (2004). The molecular basis of odor

867

coding in the

Drosophila

antenna. Cell

117

, 965-979. 10.1016/j.cell.2004.05.012

868

S0092867404004982 [pii].

869

78.

Vasiliauskas, D., Mazzoni, E.O., Sprecher, S.G., Brodetskiy, K., Johnston Jr,

870

R.J., Lidder, P., Vogt, N., Celik, A., and Desplan, C. (2011). Feedback from

871

Journal Pre-proof

rhodopsin controls rhodopsin exclusion in

Drosophila

photoreceptors. Nature

872

479

, 108-112. nature10451 [pii] 10.1038/nature10451.

873

79.

Dolezelova, E., Dolezel, D., and Hall, J.C. (2007). Rhythm defects caused by

874

newly engineered null mutations in Drosophila's

cryptochrome

gene. Genetics

875

177

, 329-345. 10.1534/genetics.107.076513.

876

80.

Ye, B., Zhang, Y., Song, W., Younger, S.H., Jan, L.Y., and Jan, Y.N. (2007).

877

Growing dendrites and axons differ in their reliance on the secretory pathway.

878

Cell

130

, 717-729. 10.1016/j.cell.2007.06.032.

879

81.

Ha, T.S., Sengupta, S., Powell, J., and Smith, D.P. (2023). An angiotensin

880

converting enzyme homolog is required for volatile pheromone detection, odorant

881

binding protein secretion and normal courtship behavior in

Drosophila

882

melanogaster

. Genetics

224

, iyad109. 10.1093/genetics/iyad109.

883

82.

Lin, C.C., Prokop-Prigge, K.A., Preti, G., and Potter, C.J. (2015). Food odors

884

trigger

Drosophila

males to deposit a pheromone that guides aggregation and

885

female oviposition decisions. eLife

, e08688. 10.7554/eLife.08688.

886

83.

Hamada, F.N., Rosenzweig, M., Kang, K., Pulver, S.R., Ghezzi, A., Jegla, T.J.,

887

and Garrity, P.A. (2008). An internal thermal sensor controlling temperature

888

preference in

Drosophila

. Nature

454

, 217-220.

889

890

Figure legends

891

Figure 1. DART2 assay for assaying olfactory behavior

892

Journal Pre-proof

(A) A two-way choice DART2 assay set up using glass tubes with meshed chambers

893

carrying odorants or solvents. Flies were imaged using reflected near-infrared (N-IR)

894

light.

895

(B) Calibration of the number of flies used per tube. Summary of variance and effect

896

size plotted against number of flies per tube, which were exposed to 1% BA. Each

897

population test was repeated 5

– 6 times.

898

(C-I) Testing the impact of using different numbers of flies in the DART2 assay on the

899

variability of the CoM. (C) 1 fly per tube. (D) 2 flies per tube. (E) 4 flies per tube. (F) 8

900

flies per tube. (G) 16 flies per tube. (H) 32 flies per tube. (I) 64 flies per tube.

901