PIIS2589004224010411-html.html

TITLE

Photoreceptors for immediate effects of light on circadian behaviour

AUTHORS

Daniel Bidell

, Natalie-Danielle Feige

, Tilman Triphan

, Claudia Müller

, Dennis Pauls

Charlotte Helfrich-Förster

and Mareike Selcho

1,4,*

AFFILIATIONS

Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany

Department of Genetics, Institute of Biology, Leipzig University, Leipzig, Germany

Neurobiology and Genetics, Biocentre, University of Würzburg, Würzburg, Germany

Lead contact

Corresponding author: MS (mareike.selcho@uni-leipzig.de)

SUMMARY

Animals need to sharpen behavioural output in the adaptation to a variable environment.

Hereby, light is one of the most pivotal environmental signals and thus behavioural plasticity

in response to light can be observed in diurnal animals, including humans. Furthermore,

light is the main entraining signal of the clock, yet immediate effects of light enhance or

overwrite circadian output and thereby mask circadian behaviour.

Drosophila

, such masking effects are most evident as a lights-on response in two

behavioural rhythms

– the emergence of the adult insect from the pupa, called eclosion, and

the diurnal rhythm of locomotor activity. Here, we show that the immediate effect of light on

eclosion depends solely on R8 photoreceptors of the eyes. In contrast, the increase in

activity by light at night is triggered by different cells and organs, that seem to compensate

for the loss of each other, potentially to ensure behavioural plasticity.

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INTRODUCTION

An appropriate daily timing of behaviour is of critical importance for animal fitness

1,2

. Light

shapes the daily timing in two ways: (1) the cyclic change in light intensity acts as an

entraining signal, synchronizing the endogenous (circadian) timing system to appropriately

adjust an organism to the 24h environmental period; (2) light directly modulates behaviour

i.e. increases alertness, locomotor activity, body temperature and heart rate in diurnal

animals including humans

–7

while light supresses activity and promotes sleep in nocturnal

animals

8,9

. These immediate light effects often obscure circadian behaviours, and are

referred to as

“masking”

. The immediate light effects are essential for appropriate

responses of the animal to changes in the environment and for sharpening the behavioural

output. Interestingly, light responses can even provoke quasi-wildtype activity rhythms in

clock-less and thus arrhythmic fruit flies and mice under natural-like conditions

–13

. These

results were obtained in studies either carried out in the wild under natural light and

temperature cycles

11-10

, or in the laboratory under constant temperature but with natural-

like light cycles (simulating dawn and dusk)

. The latter study clearly underlines the

importance of light for normal behaviour.

Drosophila melanogaster

, the immediate light effects are most evident as a lights-on

response in two well described behavioural rhythms of the fly

– the emergence rhythm of

the adult insect from the pupa, called eclosion, and the adult activity rhythm

–17

. Eclosion

is gated by the circadian clock to the early day, most probably to prevent desiccation and

enhance survival rate. A light stimulus induces a rapid increase in eclosion rate (lights-on

effect) that is eliminated in flies without eyes and potentially in mutants lacking ocelli

14,15

Even though the lights-on effect is gone, eclosion remains synchronized to the light-dark

cycle in eyeless flies by the circadian blue-light photopigment Cryptochrome (CRY;

14,18,19

reviewed by

). Similarly, the locomotor activity rhythm of adult eyeless flies remains

synchronized to the light-dark cycle due to entrainment by CRY (reviewed by

), but the

immediate increase of activity after lights-on at ZT0

, also known as “startle response”,

disappears after elimination of the eyes

16,22

. Subsequent studies showed that several

rhodopsins contribute to this immediate light effect in adult flies

23,24

Flies receive light information via two external organs, the compound eyes and ocelli, as

well as the internal extraretinal Hofbauer-Buchner (H-B) eyelets. In addition, a small subset

of central brain neurons express the blue-light sensitive photopigment CRY

18,19

. The fruit

fly’s compound eye consists of around 800 ommatidia, each of them equipped with six outer

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(R1-6) and two inner (R7, R8) photoreceptor cells. While the outer photoreceptor cells

express the photoreceptor protein Rhodopsin 1 (Rh1), the inner ones express specific

combinations of Rh3, Rh4, Rh5 and Rh6 (reviewed by

). Photoreceptor proteins are

specifically expressed in two main ommatidia types: pale ommatidia express Rh3 in R7 and

Rh5 in R8 photoreceptors and yellow ommatidia Rh4 in R7 and Rh6 in R8 cells. A third type,

the DRA ommatidia, positioned in the dorsal rim area (DRA) expresses Rh3 in R7 and R8

cells. The photoreceptors R1-6 were shown to be involved in dim light vision and the

perception of motion

, while R7/R8 are involved in colour vision

–28

and DRA

photoreceptors are involved in polarization vision

. Besides the large compound eyes, fruit

flies contain three simple dorsal eyes called ocelli. Ocelli consist of only one photoreceptor

type, containing Rh2

30,31

. The ocelli do not form an image or perceive objects in the

environment, instead they are sensitive to changes in light intensities and seem to respond

to polarized light. As Rh2 is highly sensitive to UV light, the ocelli provide information to

distinguish between sky and the ground (reviewed by

). This enables the fly to maintain its

orientation in space. The H-B eyelets evolve from the larval Bolwig

’s organ and express Rh6

20,33

–35

. These extraretinal light detecting cells are involved in entrainment at high light

intensities

. In addition to the light detecting organs,

Drosophila

contains CRY expressing

neurons in the brain and compound eyes, sensitive to blue light

–40

. Beside the compound

eyes, CRY is the main photoreceptor involved in light entrainment of the circadian clock

16,41

In this study, we aim to identify the photoreceptors necessary for the immediate effect of

light on eclosion. In addition, we investigate if the same light detecting cells are involved in

the increase of locomotor activity to unexpected light at night. The locomotor response to

light is time-of-day dependent with increase in activity at night and absence of increase at

daytimes

42,43

. We provide evidence that the light effect on eclosion is mediated by Rh5-

positive R8 photoreceptor neurons of the compound eyes with potential contribution of Rh6-

positive R8 photoreceptor cells. In contrast, the ocelli, the H-B eyelets, as well as light

sensitive CRY-positive cells are not required to elicit this lights-on response. These results

are in line with previous studies showing that the masking effect of light depends on the

intact eyes

. Interestingly, the increase in locomotor activity in response to light at night

remains in flies without eyes, ocelli or CRY-positive cells. We hypothesize redundant

signalling pathways and propose that light perceiving cells and organs compensate the loss

of each other, enabling flies to react to unexpected changes in their environment. Thus,

100

masking effects of light on circadian behaviours seem to depend on different underlying

101

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neuronal networks potentially dependent on the time of day, type of behaviour, or

102

developmental stage of the animal.

103

104

RESULTS

105

Light elicits an immediate increase in eclosion at lights-on in fly populations monitored under

106

14:10 light:dark cycles (Fig.1A). This immediate effect is also visible in flies kept under

107

constant darkness when a light pulse is given at times around circadian time 0 (CT0, Fig.1B-

108

D). In detail, in line with previous studies

, we could show that a twenty-minute white light

109

pulse (I = 4,1 W/m

) one hour before or after CT0 elicits an immediate eclosion response in

110

control flies (Fig.1B-D and Fig.S1A). The light response thus enables the flies to eclose

111

promptly in response to their environment, reinforcing the behaviour controlled by the clock.

112

Next, we tested whether the pigmentation of the eyes plays a role in the detection of light

113

necessary to elicit immediate eclosion. For this, we monitored eclosion in red-eyed CantonS

114

(CS) and white-eyed (

1118

) flies. Both groups showed a clear eclosion peak in response to

115

light, even though the distribution within the twenty minutes is different (Fig.1C,D and

116

Fig.S1). Nevertheless, the loss of eye pigmentation does not influence the ability to react to

117

light.

118

Further, we tested different mutants to discover the light perceiving cells and organs

119

triggering immediate eclosion. The lights-on response is gone in flies lacking the compound

120

eyes (

cli

eya

, Fig.2B and Fig.S1C;

14,15

) and in

norpA

(

no receptor potential

) mutants with

121

disturbed phospholipase C function (

norpA

p41

, Fig.2C and Fig.S1D). In contrast, flies with

122

disabled light perception in photoreceptors of the ocelli (

rh2

, Fig.2D and Fig.S1E),

cry

123

positive cells (

cry

, Fig.2E and Fig.S1E) or the Rh6-positive H-B eyelets (

rh6

, Fig.S2),

124

respectively, showed an immediate increase in eclosion in response to light indicating the

125

exclusive requirement of the compound eyes in the immediate effect of light on eclosion.

126

127

Our data suggest that the compound eyes are required for the immediate light effect on

128

eclosion. Light information is received by photoreceptor cells of the compound eyes and

129

converted into neuronal activity. The outer photoreceptor cells R1-R6 contain Rh1, the inner

130

R7 cells express Rh3 (R7pale) or Rh4 (R7yellow) and proximal R8 cells express either Rh5

131

(R8pale) or Rh6 (R8yellow), while DRA ommatidia express Rh3 in R7 and R8. Thus, the

132

different photoreceptor cells respond to light of a specific spectrum as the five different

133

rhodopsin’s of the compound eyes have different absorption spectra

–46

. Therefore,

134

illumination by monochromatic light of different wavelength will allow to address specific sets

135

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of Rh cells only and thereby give insights into the sufficiency of the different photoreceptor

136

cells in this context. Flies showed an immediate behavioural response to twenty-minute blue

137

(455

– 475 nm, I = 3,6 W/m

), green (510

– 545 nm, I = 2 W/m

) and red (625

– 642 nm, I =

138

2,3 W/m

) light pulses (Fig.3A-C and Fig.S1G-I). In contrast to mammals, where immediate

139

light-effects depend exclusively on blue light-sensitive melanopsin-expressing retinal

140

ganglion cells

–51

, flies are able to additionally respond to longer wavelengths. As red light

141

of 633nm could only be absorbed by Rh1 or Rh6, we monitored the immediate light

142

responses in flies without Rh1 (

ninaE

), flies that lack Rh6 (

rh6

) and double mutants

143

without both of these rhodopsins (

ninaE

; rh6

). As expected, the lights-on response to red

144

light is missing in the

rh1,rh6

double mutant flies (Fig.3F and Fig.S1L). Lacking just one of

145

the red light detecting rhodopsins leads to a strongly reduced lights-on response that was

146

not significantly different from the controls in the

rh6

mutants (Fig.3D,E and Fig.S1J,K). This

147

indicates at least a contribution of Rh6 to the immediate effect of red light. However, flies

148

lacking Rh1 and Rh6 show an immediate behavioural response to a twenty-minute white

149

light pulse (I = 4,1 W/m

, Fig.3G and Fig.S1M), indicating that both rhodopsins are

150

dispensable for the immediate effect of white light on eclosion. White light includes short-

151

wavelength light, which is mainly detected by the inner photoreceptor cells R7 and R8

44-46

152

To further disentangle the role of the R7, R8 cells, we screened rhodopsin and photoreceptor

153

mutants for their behavioural response to white light (Fig.3G-J). The quadruple mutant

154

(

rh5

;rh3

,rh4

,rh6

)

lacks all rhodopsins of the inner photoreceptors, so that light perception

155

is only possible via R1-R6. The lack of the lights-on response (Fig.3H and Fig.S1N) indicates

156

that (1) R7 and/ or R8 transmit light information for the lights-on response and (2) functional

157

outer photoreceptors are not sufficient to trigger immediate eclosion. To address if R7 is

158

involved in the immediate effect of light, we monitored eclosion in

sevenless

mutants (

sev

LY3

159

Fig.3I and Fig.S1O). Flies without R7 photoreceptor cells show an immediate response,

160

suggesting that R8 cells are responsible for transmitting light information. Since the absence

161

of Rh6 has no effect on the immediate response to white light (Fig.3G and Fig.S2), we tuned

162

our attention to Rh5, which is also expressed in R8 cells. As expected,

rh5

mutants (

rh5

)

163

show no lights-on response (Fig.3J and Fig.S1P). Thus, pale R8 ommatidia expressing

164

functional Rh5 turned out to be essential for the immediate response to white light. In line

165

with this hypothesis, we optogenetically activated

rh5

-expressing R8 neurons (Fig.3K,L and

166

Fig.S2C).

Photostimulation

rh5

-positive

neurons

via

Channelrhodopsin-2

XXL

167

(

rh5

>chop2

XXL

, Fig.3K;

52,53

) using a short blue light pulse of 2 min triggered immediate

168

eclosion within the first 10 min, while this short light pulse was not sufficient to cause a

169

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significant increase in eclosion in control flies (Fig.3L and Fig.S1Q,R). This demonstrates

170

that activation of

rh5

-positive R8 cells can trigger immediate eclosion. Our data demonstrate

171

that Rh5-expressing R8 cells are necessary and sufficient for the immediate effect of white

172

light on eclosion. We cannot completely rule out a contribution from the other

173

photoreceptors, Rh6 in particular seems to mediate the immediate effects of red light on

174

eclosion.

175

176

In a next step we aimed to investigate whether the same photoreceptors that mediate

177

immediate light effects on eclosion are generally responsible for mediating immediate light

178

effects. For this, we focussed on immediate light effects on locomotor activity. Unlike

179

eclosion, which occurs only once in a fly’s lifetime, activity can be recorded in individual flies,

180

and consequently the immediate effects of light on activity can be studied multiple times per

181

the 24h day

42-44

. This allowed us to test the effects of completely unexpected light by

182

administering the light pulse during the night to flies that are otherwise normally entrained

183

to light-dark cycles. Previous studies have shown that flies respond strongly to unexpected

184

light during the night

42,43,54

. In contrast, responses to light during the subjective day are

185

strongly modulated by the circadian clock and thus can be either suppressed or promoted

186

42-44

. Importantly, the effects of light administered at CT 0 are difficult to distinguish from the

187

activity-promoting effects of the circadian clock in the morning. Therefore, we chose a

188

different experimental paradigm from that used for the experiments on eclosion and

189

administered a twenty-minute white light pulse (I = 0.923 W/m

) at ZT22, i.e. two hours

190

before the expected onset of light at ZT0. This paradigm even allowed us to distinguish the

191

flies’ responses to unexpected light from the expected lights-on response observed at the

192

transition from night to day at ZT0 (startle response, Fig.4A). It has been shown that this

193

startle response depends on the compound eyes

. As expected, the light pulse at ZT22

194

elicits an immediate increase in locomotor activity in control flies (

1118

; Fig.4A). Surprisingly,

195

the immediate light response in locomotor activity is also present in eyeless flies, that in

196

addition lack functional CRY (

cli

eya

;cry

; Fig.4B). Thus, neither photoreceptor cells of the

197

compound eyes nor CRY are required for the increase in activity in response to light at night

198

(Fig.4B and Fig.S3 for

and

norpA

mutants). Nevertheless, these photoreceptors might

199

contribute to the response to unexpected light, as the increase of locomotor activity in the

200

mutants was lower than in

1118

flies (compare Fig. 4A to Fig. 4B). In addition, we analysed

201

locomotor activity in two different

cry

mutants to clarify the role of photosensitive neurons

202

outside the visual system (

cry

and

cry

). Flies without functional CRY responded to light at

203

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ZT22 with an immediate increased locomotor activity that was lower than that of the controls

204

(Fig.4C and Fig.S3A). Further, immediate light effects can be seen in flies with disabled light

205

perception in photoreceptors of the ocelli (

rh2

, Fig.4D) and flies without Rh5 and Rh6 and

206

therefore without functional eyelets (

rh5

, rh6

; Fig.4E). Thus, all the different cells and

207

organs that perceive light appear to mediate its immediate effects and compensate for the

208

failure of individual photoreceptors, so that flies can respond immediately to a light stimulus

209

at night. In contrast, flies without a functional histidine decarboxylase (

hdc

JK910

), the enzyme

210

necessary for histamine synthesis, show no increase in locomotor activity in response to

211

light (Fig.4F). Histamine is the main transmitter of the photoreceptor cells of the eyes, ocelli

212

and eyelets

, leaving the hypothesis that

cry

-positive cells alone may not be sufficient to

213

elicit the lights-on response. A closer examination of the activity pattern suggests that

214

hdc

JK910

mutants display a reduced activity during the light pulse and increase it after the

215

light-pulse has ended. CRY thus appears to be involved in the response to unexpected light,

216

but may act in the opposite direction to the rhodopsins of the photoreceptors. Contrasting

217

effects of CRY and the visual system on fly activity have also been observed previously

218

and merit further investigation by future studies.

219

220

In summary, our data provide evidence that photoreceptors of the compound eyes are

221

involved in the masking effect of expected light, while unexpected light at night is mediated

222

by histamine-positive cells and therefore a combination of the eyes, H-B eyelets and

223

potentially the ocelli and CRY.

224

225

DISCUSSION

226

The immediate effect of light on locomotor activity is evident every morning in flies under LD

227

rhythms at ZT0

16,17

. Interestingly, former studies showed, that the immediate increase in

228

locomotion at ZT0 depends on the compound eyes and is completely absent in eyeless

229

flies

. Here, we observed the same: eyeless flies (

cli

eya

;cry

mutants, which lack functional

230

CRY in addition to the eyes) as well as flies without histamine (

hdc

JK910

mutants) lack the

231

startle response at ZT0 (first column, Fig.4B,F). Nevertheless, a light pulse during the night

232

two hours before lights-on at ZT22, provokes an immediate increase in activity in

cli

eya

;cry

233

mutants (Fig.4B). Only the disruption of neuronal communication of photoreceptor cells of

234

the eyes, ocelli and eyelets by the loss of histamine (

hdc

JK910

mutants) prevents a startle

235

response to light at night (ZT22, Fig. 4F). The immediate increase in activity in response to

236

unexpected light at ZT22 is thus mediated by functionally redundant photoreceptor cells,

237

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whereas the startle response at ZT0 depends purely on the receptors in the eyes. The

238

immediate effect of light on locomotion in

Drosophila

is modulated in a time-of-day-

239

dependent manner: light at night increases activity, while activity is suppressed during the

240

day

42,43

. The switch in behaviour seems to depend on the daily morphological changes of

241

central clock neurons

and on a light-mediated circuit switching in the

Drosophila

neuronal

242

clock network

. The differences in the photoreceptor requirements observed between ZT22

243

and ZT0 in this study might reflect the time of day and resulting morphological changes in

244

the downstream neuronal network.

245

The functional redundancy of photoreceptors was also described in nocturnal mice. Here,

246

bright light at night leads to an inhibition of locomotor activity, whereas dim light increases

247

activity

–60

. Mice with degenerated retina (

rd/rd

mice), devoid of rods and cones, lack the

248

increase of activity by dim light, but still show activity inhibition by bright light. In addition,

249

mice lacking the photopigment melanopsin (

Opn4

-/-

) in the retinal ganglion cells also show

250

inhibition of activity by bright light, while double mutants (

Opn4

-/-

; rd/rd

) lose the ability to

251

respond to light

49,50,61

. Thus, also in mice, several photopigments contribute to the

252

immediate light responses and these can compensate to a certain degree the loss of each

253

other to ensure immediate responses to unexpected external stimuli.

254

Interestingly, recent data suggests that CRY suppresses activity during the night in flies,

255

while the absence of CRY enhances activity during moonlit nights

. Flies were as active

256

during moonlit nights as they were during the day, suggesting that CRY is important for

257

distinguishing nocturnal low light of moonlight intensity from day light. Interestingly, this is

258

consistent with data from marine bristle worms

. If CRY is present, it seems to suppress

259

activity during the night in the diurnal

D. melanogaster

and to suppress swarming activity

260

during the day in the nocturnal marine bristle worms. This finding for

Drosophila

261

corroborated by the present study:

cry

mutants show strong immediate light effects upon a

262

light pulse, but flies that lack the function of all photoreceptors except of CRY (

hdc

JK910

263

mutants) have decreased responses to unexpected light. In summary, we conclude that the

264

immediate light effects of adult flies in response to nocturnal light are mediated by all

265

rhodopsins (those in the compound eyes, the H-B eyelets and putatively also the ocelli),

266

while they are inhibited by CRY. This is again different for the startle response at ZT0. Here

267

we could not see any inhibiting effect of CRY.

268

The immediate effect of white light on eclosion depends on pale R8 cells in the compound

269

eyes. White light elicits eclosion in flies without functional ocelli, H-B eyelets and

cry

-positive

270

photoreceptive cells, while flies with impaired phospholipase C activity and without eyes or

271

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Rh5 lack the lights-on response. Masking by red light, however, depends on Rh6-positive

272

yellow R8 cells. Rh6 is also expressed in the H-B eyelet of adult flies

63,35

. During

273

metamorphosis four larval Rh5 photoreceptors switch rhodopsin expression and become

274

the adult Rh6-positive eyelet

. It was demonstrated that these extraretinal photoreceptors

275

are involved in entrainment, but they only fulfil this function a few days after eclosion

276

because the H-B eyelets need several days to become mature

. Therefore, it is unlikely

277

that the H-B eyelets contribute to the light effects during eclosion.

278

279

Limitations of the study

280

Even though the context dependent behavioural plasticity depends on the circadian clock,

281

the increase in locomotor activity in response to light is still visible in flies without functional

282

internal clocks

42,43,64

. We cannot completely exclude that contextualization of the light

283

stimulus is disturbed in

hdc

JK910

mutants leading to a decrease in activity in response to light

284

at ZT22. In any case, the experiments using the

hdc

JK910

mutants show that the immediate

285

effects of light on locomotion are mediated via neurotransmission through histamine, which

286

is the main transmitter of the compound eyes, ocelli and H-B eyelets

. The H-B eyelets

287

use additionally acetylcholine (ACh) as a neurotransmitter

and a recent study shows that

288

this is also true for the inner photoreceptors R8

. Most interestingly, ACh appears to

289

transmit photic input to the circadian clock, while histamine transmits visual signals for image

290

detection and motion. Thus, consistent with our results histamine seems to be the

291

transmitter for the direct light responses.

292

For the immediate effect of white light on eclosion, only Rh5 in R8 cells of the compound

293

eyes is needed. Possibly, not all photoreceptors are yet mature at the time of eclosion. This

294

is certainly true for the H-B eyelets that are fully functional only several days after eclosion

295

. For the other photoreceptors such a delayed maturation is not known. R8 photoreceptors

296

develop first, but retina formation is completed in pupal state P13/P14 including

297

photoreceptor differentiation and Rhodopsin expression

66,67,68

. Nevertheless, it is possible

298

that not all the connections to the central brain, particularly those that connect to activity-

299

promoting centres, are fully established.

300

301

ACKNOWLEDGMENTS

302

We thank Claude Desplan, Craig Montell, Christopher Schnaitmann and Ralf Stanewsky

303

for providing flies and Simon Sprecher for sharing antibodies. This work was supported by

304

grants from the German Research Foundation (DFG) to MS (PA3241/2-1) and DP

305

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(PA1979/2-1, PA1979/5-1). Stocks obtained from the Bloomington Drosophila Stock

306

Center (NIH P40OD018537) were used in this study.

307

308

AUTHOR CONTRIBUTIONS

309

Conceptualization, M.S.; Methodology, D.P., C.H.-F., and M.S.; Software, T.T.;

310

Investigation, D.B., N.D.F., and C.M.; Writing, C.H.-F., D.P., and M.S.; Visualization, D.B.,

311

and M.S.; Funding Acquisition, C.H.-F., and M.S.

312

313

DECLERATION OF INTERESTS

314

The authors declare no competing interests

315

316

FIGURE LEGENDS

317

Fig.1: The immediate light effect on eclosion behaviour.

(A)

Eclosion pattern of

318

Drosophila

flies in ten minutes intervals at the times around lights-on. Light elicits an

319

immediate increase in eclosion.

(B,C)

The lights-on response is visible in flies perceiving a

320

twenty-minute light pulse (B) one hour before (-1) or (C) one hour after (1) expected lights-

321

on at CT (circadian time) 0.

(D-

D’)

Wildtype CantonS (CS) flies show an immediate response

322

to light (

D), while flies in darkness lack the eclosion peak (D’).

(D’’)

The third plot combines

323

(D) and (D’) to visualize the immediate light response in comparison to the appropriate

324

controls monitored in darkness. Grey bars: % eclosion in dark phase, yellow bars: %

325

eclosion in light phase; dashed bars: % eclosion in controls. n= 384 (A), 516 (B), 524 (C),

326

649 (D), 636 (D’). See also Figure S1.

327

328

Fig.2: The immediate light effect on eclosion behaviour requires the compound eyes

329

and phospholipase C activity.

(A-E)

Eclosion pattern in ten minutes intervals at the times

330

around circadian time (CT) 0. Each plot visualizes the results for the experimental (grey,

331

yellow bars) and control groups (dashed bars) as shown in Fig.1D-

D’’.

(B,C)

Flies without

332

eyes (B,

cli

eya

) and impaired phospholipase C activity (C,

norpA

p41

) lack the lights-on

333

response.

(D,E)

Flies lacking the rhodopsin (Rh) of the ocelli photoreceptor cells (D,

rh2

)

334

or the photoprotein Cryptochrome (E,

cry

) respond to light. Grey bars: % eclosion in dark

335

phase, yellow bars: % eclosion in light phase; dashed bars: % eclosion in darkness controls.

336

exp

, n

ctrl

= 524, 544 (A); 502, 508 (B); 520, 539 (C); 604, 584 (D); 510, 556 (E). See also

337

Figure S1.

338

339

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Fig.3: The immediate light effect on eclosion behaviour depends on R8 cells.

(A-J)

340

Eclosion pattern in ten minutes intervals at the times around circadian time (CT) 0. Each

341

plot visualizes the results for the experimental (grey, yellow bars) and control groups

342

(dashed bars) as shown in Fig.1D-

D’’.

(A-C)

Twenty-minute blue (A, 455

– 475 nm, I = 3,6

343

W/m

), green (B, 510

– 545 nm, I = 2 W/m

) and red (C, 625

– 642 nm, I = 2,3 W/m

) light

344

pulses elicit immediate eclosion.

(D-F)

The eclosion response to red light is visible in

rh1

(D,

345

ninaE

) but gone in

rh6

mutants (E,

rh6

) and

rh1,rh6

double mutants (F,

ninaE

; rh6

(G)

346

In contrast,

rh1,rh6

double mutants (

ninaE

; rh6

) respond with an increase of eclosion to

347

white light (I = 4,1 W/m

(H)

The quadruple mutant (

rh5

;

rh3

rh4

rh6

), lacking all

348

rhodopsins of the inner photoreceptors, shows no reaction to light.

(I)

The lights-on response

349

in flies without R7 cells (

sev

LY3

(J)

Flies lacking Rh5 (

rh5

) do not respond to light with

350

increased eclosion.

(K)

Optogenetic activation of

rh5

-positive neurons with a 2 min blue light

351

pulse (blue line; 455-475nm, I = 3,41



W/mm

) one hour after expected lights-on elicits

352

eclosion.

(L)

Control flies without Gal4 expression (

1118

; chop2

XXL

) do not respond to the 2

353

min light pulse. n

exp

ctrl

= 534, 1543 (A); 521, 1543 (B); 547, 1543 (C); 511, 512 (D); 579,

354

731 (E); 604, 567 (F); 561, 567 (G); 595, 518 (H), 525, 531 (I); 585, 506 (J); n= 516 (K), 515

355

(L). See also Figure S1.

356

357

Fig. 4: The immediate effect of light on locomotor activity is visible in flies without

358

functional eyes, photosensation in cry-positive cells or ocelli.

(A-F)

Activity pattern in

359

ten minutes intervals at the time around Zeitgeber time (ZT) 0. First and second column

360

show bar plots of mean



SEM activity at the day the light pulse was applied (second column)

361

and the activity of the same flies on the previous day (first column). The third column

362

visualizes the comparison between the mean activity at ZT22 in 10 minutes intervals (0’-10’

363

and 10’-20’) during the light pulse (L) and the previous control day in darkness (D). Data are

364

presented as mean (bar plot) and individual values (dots).

(A)

Control flies (

1118

) and

(B)

365

eyeless (

cli

eya

;

cry

) flies,

(C)

flies lacking Cryptochrome (

cry

(D)

flies without functional

366

ocelli photoreceptors (

rh2

(E)

flies

without Rh5 and Rh6 (

rh5

;

rh6

)

increase activity in

367

response to light at ZT22.

(F)

The light response is absent in flies that lack the histidine

368

decarboxylase (

hdc

JK910

) and therefore histamine, the transmitter of photoreceptor cells. n =

369

27- 32; asterisks denote level of significance: *p



0.05, **p



0.01, ***p



0.001, ****p



0.0001.

370

See also Figure S3 and Table S1.

371

372

Journal Pre-proof

STAR Methods

373

RESOURCE AVAILABILITY

374

Lead contact

375

Further information and requests for resources and reagents should be directed to and will

376

be fulfilled by the lead contact, Mareike Selcho (mareike.selcho@uni-leipzig.de).

377

378

Materials availability

379

This study did not generate new unique reagents.

380

381

Data and code availability

382

The datasets generated and analysed during the current study are available from the lead

383

contact on reasonable request.

384

All code and further information is publicly accessible and can be found at

385

https://zenodo.org/records/10985365.

386

Any additional information required to reanalyse the data reported in this paper is available

387

from the lead contact upon request.

388

389

Experimental Model and Study Participant Details

390

Fly stocks

391

Flies (

Drosophila melanogaster

) were raised on standard cornmeal and molasses medium

392

at 25°C and 65% relative humidity at a 14:10 light-dark (L:D) cycle unless otherwise stated.

393

For optogenetic experiments, vials have been covered with a light filter foil (Nr. 026; LEE

394

Filters Worldwide, UK). The following

flies were used in this study:

cli

eya

(

cry

(

cry

395

(

, kind gift of R. Stanewsky),

hdc

JK910

(

nina

E17

(

73,74

norpA

p41

(

75,76

, kind gift of R.

396

Stanewsky),

rh2

(

, kind gift of C. Montell),

rh5

(

, kind gift of R. Stanewsky),

rh6

(

, kind

397

gift of C. Montell),

nina

E17

;

rh6

(

sev

LY3

(

) and the following combinations of the mutants

398

were used:

cli

eya

; cry

and

rh5

; rh6

and

rh5

; rh3

, rh4

, rh6

. The Gal4- and UAS- lines

399

used:

rh5

(

, kind gift of C. Montell; BDSC #66671),

UAS-chop2

XXL

(

10xUAS-IVS-

400

myr::GFP

(

; BDSC #32197).

401

Seven to nine days old pupae, of both sexes were used for eclosion experiments and two

402

to four days old flies of both sexes were used for locomotor activity measurements.

403

404

METHOD DETAILS

405

Eclosion behaviour

406

Journal Pre-proof

To investigate the immediate light effect on eclosion behaviour, an eclosion monitor based

407

on the WEclMon-System

83,84

was used (Fig.S4A). Experiments were performed under

408

constant temperature (24.5°C



0.2°C) and humidity (around 65% RH). Temperature

409

fluctuations upon light exposure were below 0.2°C. Seven to nine days old pupae, of both

410

sexes, were placed onto a transparent acrylic plate. This plate was placed on an area light

411

with an RGB or White-LED illumination (LED-color:



(blue)= 455

– 475 nm,



(green)= 510

412

– 545 nm,



(red)= 625

– 642 nm; white LED (





430

– 730 nm, Fig.S4B); Hansen GmbH,

413

Germany, Luminous Panel (RGBW)) and an additional IR LED strip was installed at the

414

bottom of the lighting unit (SOLAROX® LED,



(infrared) = 850nm, IR1-60-850, Winger

415

Electronics GmbH & Co. KG, Germany) and covered with an aluminium box. Flies were kept

416

in darkness and received a twenty-minute light pulse one hour after expected lights-on (CT1)

417

on the second day, or remained in darkness (darkness control). The eclosion monitor is

418

equipped with a camera (DMK 27AUC02 with a TPL 0420 6MP objective; DMK 37BUX287

419

with a TPL 0620 6MP objective, The Imaging Source Europe GmbH) that took images every

420

two minutes (IC Capture 64bit, V2.5.1547.4007, The Imaging Source Europe GmbH,

421

Germany). We developed a Python-script for Fiji (V2.9.0,

) that was able to independently

422

scan a selected image sequence for eclosion events (detailed description below).

423

424

Eclosion data analysis

425

The detection of hatching events is based on the difference in brightness between a pupa

426

and an empty pupal case. The latter is almost transparent while a late pupa is considerably

427

darker (Fig.S4C, empty pupal case marked with an asterisk). In the first step of the analysis

428

all pupae are detected. The pupae are darker than the background and can be easily

429

extracted in a single image. After setting a threshold (Fig.S4D), objects are detected, and

430

their outlines stored. Objects that are outside of the parameters defined for a pupa (area too

431

big or too small) are excluded. Note the empty pupal case that is excluded from further

432

analysis. As the pupae are stationary, we can follow them through time and calculate the

433

median grey value of the area contained in their respective outlines (Fig.S4E) for each

434

frame. Alternatively, the average or mode (i.e. most frequent) grey values can be used. A

435

large jump in brightness indicates an eclosion event (Fig.S4F-H). The median grey values

436

are more than doubled from around 40 to almost 100. For each eclosion event a few frames

437

before and after the event are extracted to help with manual confirmation of real hatching

438

events or to exclude inaccurate detections (Fig.S4G). The time for each eclosion event is

439

Journal Pre-proof

stored in a csv file for further analysis. Additional checks are implemented to reduce the

440

number of incorrect detections and handle changes in lighting. See the commented source

441

code for details. The complete workflow is implemented as a Python script for Fiji. Specific

442

parameters that depend on camera resolution, optics and lighting can be set manually (e.g.

443

area of the pupae in pixel, difference in brightness for full and empty pupae, etc.). All code

444

and further information can be found at https://zenodo.org/records/10985365.

445

For the evaluation of eclosion events, a time window of five hours was chosen, from two

446

hours before expected lights-on to three hours after expected lights-on. To calculate the

447

eclosion percentage (% eclosion), the number of flies eclosed in a 10 min time interval was

448

normalized to the number of flies eclosed during the 5 h time window. The bar plots in

449

Fig.1D’’, Fig.2 and Fig.3 visualize the eclosion of flies perceiving a 20 min light pulse and of

450

appropriate control flies kept in darkness (Fig.1D-

D’’). n refers to the number of individuals

451

tested.

452

To analyse changes in eclosion in response to light, eclosion percentage of experimental

453

(L) and control (D) flies in the first 10 min (0’-10’) and second 10 min (10’-20’) interval after

454

lights-on at CT1 was compared. Data was analysed with Excel (Microsoft) and Prism 8.2

455

(GraphPad). The Shapiro-Wilk test was used to analyse normal distribution and data were

456

compared by an unpaired two-tailed t-test. Not normally distributed data were compared by

457

a nonparametric Mann-Whitney rank sum test. Prism was used to plot the results (Fig.S1

458

and S2). Barplots show the mean and individual values in the first and second ten minutes

459

interval at CT1 in light and darkness. Significance levels refer to the raw p values obtained

460

in the statistical tests. N refers to the eclosion experiments analysed.

461

462

Optogenetics

463

For optogenetic activation, Channelrhodopsin-2

XXL

(UAS-

chop2

XXL

) has been used to

464

depolarize

rh5

-positive neurons by blue light. Pupae have been collected under red light

465

during their subjective day to monitor eclosion. One hour after expected lights-on pupae

466

received a 2 min blue light stimulus (455

– 475 nm,



= 3,41



W/mm

) and eclosion was

467

monitored as described above. n refers to the number of individuals tested. Barplots show

468

the mean and individual values in the ten minutes interval before and under/after the 2 min

469

light pulse at CT1. Significance levels refer to the raw p values obtained in the statistical

470

tests. N refers to the eclosion experiments analysed.

471

472

Locomotor Activity

473

Journal Pre-proof

Flies were entrained to a 12:12 LD rhythm to be able to compare results to the data by

42,43

474

To investigate the lights-on effect on locomotor activity, we placed two to four days old flies

475

of both sexes in small glass tubes with 2% agarose and 4% sugar on one side into the

476

Drosophila

Activity Monitoring System (DAM, V2, TriKinetics Inc., USA) and recorded

477

locomotor activity for the next four night-day cycles under constant temperature and humidity

478

(24.9



0.1°C,



65% RH). In the first three night-day cycles the light regime did not differ

479

from the entrained rhythm (12:12 LD). On the fourth night, a 20 min light pulse (





435

–

480

780 nm, I = 0.923 W/m



100 lux) was given two hours before normal lights-on at ZT22.

481

The data received from the DAM system was analysed by taking the number of counts of a

482

10 min interval of each tube and calculating the average activity. This was done for both the

483

6 h time window (-3 h to +3 h from ZT0) in which the 20 min light pulse was given and the

484

same time window on the day before, which was used as control. To analyse changes in

485

locomotor activity in response to light, the activity at ZT22 under light (L) was compared to

486

the activity at ZT22 in darkness (D) the day before (third column Fig.4, S3). Data was

487

analysed with Excel (Microsoft) and Prism 8.2 (GraphPad). The Shapiro-Wilk test was used

488

to analyse normal distribution and data was compared by a paired two-tailed t-test and for

489

not normally distributed data by a Wilcoxon signed rank test. Prism was used to plot data.

490

Activity levels in the first and second ten minutes interval at ZT22 are presented as bar plots

491

showing the mean and individual values (Fig.4, S3). Significance levels refer to the raw p

492

values obtained in the statistical tests.

493

494

Immunohistochemistry

495

rh5

> 10xmyrGFP

pharate flies have been used to confirm GAL4 expression in Rh5-positive

496

R8 cells (Fig. S2C). Whole heads have been fixed for 2 hours in 4% PFA. After washing in

497

PBS (Phosphate Buffered Saline) specimens have been embedded in 7% agarose and cut

498

into 70-100



m sections using a vibratome (Leica VT1000S;

). Sections have been washed

499

three times for 10 min in 0.3% PBT (PBS with 0.3% TritonX-100), blocked for 1,5 h in 5%

500

normal goat serum in PBT. Afterwards the first antibody solution was incubated overnight at

501

4°C. Specimens were washed six times and the second antibody solution was added and

502

incubated overnight at 4°C. After another washing step, specimens were mounted in

503

Vectashield (Vector Laboratories) and stored at 4°C until scanning. Probes have been

504

imaged using a LSM 800 (Zeiss). Afterwards the images have been edited for brightness

505

and contrast using FIJI and Adobe Photoshop (V23.5.3).

506

Journal Pre-proof

The following antibodies were used: rabbit-

α-GFP (1:1000; ThermoFisher Scientific,

507

A11122), 3C11

α-Synapsin (1:50,

), goat-

α-rabbit AlexaFluor 488 (1:250, ThermoFisher

508

Scientific , A11034) and goat-

α-mouse STAR RED (1:250, Abberior, STRED-1001).

509

510

QUANTIFICATION AND STATISTICAL ANALYSIS

511

All statistical analysis and p-values are included in TableS1. Eclosion behaviour: the

512

Shapiro-Wilk test was used to analyse normal distribution and data were compared by an

513

unpaired two-tailed t-test. Not normally distributed data were compared by a

514

nonparametric Mann-Whitney rank sum test. Prism was used to calculate p-values and

515

plot the results (Fig.S1 and S2). Locomotor activity: the Shapiro-Wilk test was used to

516

analyse normal distribution and data was compared by a paired two-tailed t-test and for

517

not normally distributed data by a Wilcoxon signed rank test. Prism was used to calculate

518

p-values and plot the results (Fig.4 and S3).

519

520

521

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Journal Pre-proof

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

rabbit-

α-GFP

ThermoFisher Scientific

CAT#A11122,
RRID:AB_221569

3C11

Klagges et al.

RRID:AB_2313617

goat-

α-rabbit AlexaFluor 488

ThermoFisher Scientific

CAT#A11034,
RRID:AB_2576217

goat-

α-mouse STAR RED

Abberior

CAT#STRED-1001,
RRID:AB_3068620

Chemicals, Peptides, and Recombinant Proteins

normal goat serum

Sigma-Aldrich

CAT#G9023

Vectashield

Vector Laboratories

CAT#H-1000

Deposited Data

Eclosion Detector, Python script
for Fiji

Tilman Triphan,

https://zenodo.org/records/10985365

DOI:
10.5281/zenodo.10985365

Experimental Models: Organisms/Strains

Drosophila melanogaster: w

1118

Kittel lab

N/A

Drosophila melanogaster: cli

eya

Bonini et al.

N/A

Drosophila melanogaster: cry

Dolezelova et al.

N/A

Drosophila melanogaster: cry

Stanewsky lab; Stanewsky et al.

N/A

Drosophila melanogaster:
hdc

JK910

Burg et al.

N/A

Drosophila melanogaster:
nina

E17

Helfrich-

Förster lab; O’Tousa et al.

N/A

Drosophila melanogaster:
norpA

p41

Stanewsky lab

N/A

Drosophila melanogaster: rh2

Montell lab; Sokabe et al.

N/A

Drosophila melanogaster: rh5

Stanewsky lab; Yamaguchi et al.

N/A

Drosophila melanogaster: rh6

Montell lab; Cook et al.

N/A

Drosophila melanogaster: rh5

;

rh6

Yamaguchi et al.

N/A

Drosophila melanogaster:
nina

E17

;

rh6

Hanai et al.

N/A

Drosophila melanogaster: sev

LY3

Stark et al.

N/A

Drosophila melanogaster: rh5

;

rh3

, rh4

, rh6

Helfrich-Förster lab

N/A

Drosophila melanogaster: rh5

Montell lab; Sokabe et al.,

BDSC #66671

Drosophila melanogaster: UAS-
chop2

XXL

Dawydow et al.

N/A

Drosophila melanogaster:
10xUAS-IVS-myr::GFP

Pfeiffer et al.

BDSC #32197

Software and Algorithms

IC Capture 64bit

The Imaging Source Europe GmbH RRID: SCR_016047

Journal Pre-proof