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SIFamide-dependent Synaptic Plasticity in Male-specific GABAergic Neurons

Underlies Experience-Modulated Mating Behaviors

Tianmu Zhang, Hongyu Miao, Yutong Song, Zekun Wu, Woo Jae Kim

PII:

S2589-0042(25)01777-8

DOI:

https://doi.org/10.1016/j.isci.2025.113516

Reference:

ISCI 113516

To appear in:

iScience

Received Date: 10 April 2025
Revised Date: 3 July 2025
Accepted Date: 3 September 2025

Please cite this article as: Zhang, T., Miao, H., Song, Y., Wu, Z., Kim, W.J., SIFamide-dependent

Synaptic Plasticity in Male-specific GABAergic Neurons Underlies Experience-Modulated Mating

Behaviors,

iScience

(2025), doi:

https://doi.org/10.1016/j.isci.2025.113516

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TITLE

SIFamide-dependent Synaptic Plasticity in Male-specific GABAergic Neurons

Underlies Experience-Modulated Mating Behaviors

SHORT TITLE

SIFa signaling modulate fru-positive neurons of male Drosophila

AUTHORS

Tianmu Zhang

(

张天目

)

, Hongyu Miao

(

苗鸿钰

)

, Yutong Song

(

宋雨桐

)

, Zekun

(

吴泽坤

)

, and Woo Jae Kim

(

金佑宰

김우재

)

1, 2, 3, *

AFFILIATIONS

HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute

of Technology, Harbin 150001, China

Medical and Health Research Institute , Zhengzhou Research Institute of HIT

Zhengzhou

，

Henan 450000

，

China

Lead contact

Correspondence to: wkim@hit.edu.cn

TZ: 22B928013@stu.hit.edu.cn

HM: 20210125L@stu.hit.edu.cn

YT: 22S028048@stu.hit.edu.cn

Journal Pre-proof

ZW:

1192800118@stu.hit.edu.cn

ABSTRACT

Drosophila melanogaster

relies on complex brain circuits and neuromodulation for

experience-dependent sexual behavior, yet the role of neuropeptide signaling in

behavioral refinement remains unclear. We investigate SIFamide (SIFa) and its receptor

(SIFaR) in regulating male

Drosophila

sexual behavior. SIFaR is essential for these

behaviors, particularly interval timing. Knockdown in fru-positive neurons impairs

courtship index (CI) and mating duration (MD) but not copulation latency (CL).

Critically, SIFaR in GABAergic mAL neurons (fru

, dsx

) specifically controls MD and

CI, not CL. Additionally, SIFa signaling in mAL neurons of experienced males

increases presynaptic terminals, indicating context-dependent synaptic plasticity. These

results reveal a complex SIFa/SIFaR interaction with specific neurons in modulating

male behavior. The distinct roles of SIFaR in fru-positive versus other neurons,

alongside its plasticity function, provide new insights into neural mechanisms of

context-dependent behavioral adaptation.

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INTRODUCTION

Neuropeptides (NPs) participate in a variety of functions, including hormone control,

metabolic processes, and behavior

. Neuropeptides have a significant role in sexual

dimorphism, the phenomenon in which the sexes of the same animal exhibit unique

physical and/or behavioral characteristics. In certain species, the expression of specific

neuropeptides varies between sexes, which may result in distinct behavioral or physical

features. For example, corticotropin-releasing factor (CRF) is a neuropeptide that is

involved in the regulation of stress and fear responses, and it is present at different

concentrations in males and females. In males, CRF is involved in the regulation of

aggression, while in females it is involved in the regulation of maternal behavior

2,3

Neuropeptide receptors (NPRs) with sexually dimorphic activity can be found in many

different types of organisms, including mammals. The GnRH receptor is an example of

a neuropeptide receptor that is expressed differently in males and females of the same

species. This receptor is more common in male than in female, and it is thought to play

a role in how reproductive physiology and behavior are controlled

4,5

. The corazonin

receptor (CrzR) in

Drosophila

serves as a notable example of a sexually dimorphic

NPR. This receptor exhibits higher expression levels in males compared to females and

is believed to play a crucial role in regulating male-specific behaviors, including

courtship and aggression

6,7

SIFamide (SIFa) is a neuropeptide expressed in a small group of neurons in the

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posterior-interior (PI) cluster of the central brain of the fruit fly

Drosophila

melanogaster

. SIFa regulates multiple behaviors, including sleep, feeding, and

reproduction. The SIFa receptor (SIFaR) is known to regulate behaviors such as

circadian rhythms, food intake, and metabolic processes. It is also known to play a role

in the regulation of neuronal communication and excitability, with studies indicating

that it has a role in modulating the activity of some neurotransmitters, such as dopamine

and serotonin, as well as in influencing the release of hormones. Additionally, SIFaR is

thought to be involved in the regulation of complex behaviors such as learning, memory,

and decision making

9–15

Emerging evidence suggests that SIFaR-expressing neurons, rather than SIFa-

producing neurons, may contribute to the development of sex-specific circuitry within

fru

-defined neuronal populations

. SIFaR knockdown in

fru

-positive cells led to male-

male courtship

. Although the potential association between SIFaR signaling and

fru

positive sexually dimorphic circuits is identified, the intricacies of sexually dimorphic

circuits linked to specific behavior have not yet been described. Our previous research

has established that the activity of SIFa and the synaptic plasticity resulting from the

extensive branching of SIFa neurons are vital for regulating energy balance behaviors

in male

Drosophila

, mediated by long-range neuropeptide signaling through SIFaR

17,18

In this work, we reveal that SIFa inputs to SIFaR receptors present in

fru

-expressing

mAL neurons are essential for the modulation of energy balance behaviors driven by

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SIFa. We hypothesize that SIFa inputs play a key role in orchestrating the activity of

fru

-positive pC1 and mAL neurons via SIFaR expression, thereby enabling the

processing of information necessary for adjusting internal states related to various

energy balance behaviors. This study builds on our earlier findings, highlighting the

importance of SIFa signaling in modulating energy balance behaviors through its

interactions with SIFaR in specific neuronal populations, particularly those expressing

fruitless

RESULTS

Diverse Input-Output Combinations of SIFa Neurons Regulate Male Sexual

Behavior Repertoire.

SIFa has been recognized as a multifunctional peptide, but it was first identified for its

involvement in male-male courtship behavior

. Research indicates that the removal of

SIFa-expressing neurons or the reduction of SIFa peptide levels can lead to an increase

in male-to-male courtship behaviors

. To gain insights into the mechanisms by which

SIFa neurons modulate sexual behaviors, we initially attempted to alter neuronal

activity in SIFa-positive neurons using various activity modifiers under the

GAL4

SIFa.PT

100

driver. Notably, expressing TNT (tetanus toxin light chain), potassium channels

101

KCNJ2/OrkΔC

, or the bacterial sodium channel NaChBac within SIFa neurons did

102

not significantly affect courtship indices when compared to control flies (Fig. 1A).

103

Conversely, a reduction in SIFa peptide levels via RNA interference (RNAi) led to a

104

substantial decrease in male courtship indices toward females compared to control

105

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groups (Fig. 1B).

106

107

The use of TNT has proven effective in inhibiting neurotransmitter release across

108

several studies; KCNJ2/OrkΔC prevents membrane depolarization, while NaChBac

109

induces sodium conductance that results in cell depolarization

. Thus, our research

110

indicates that the neuropeptide SIFa's release is more essential for normal male

111

courtship behavior than the neuronal activity of SIFa-expressing neurons themselves.

112

113

Next, we examined another aspect of male sexual behavior: copulation (or mating)

114

latency (CL). CL, defined as the time it takes for a male to initiate copulation with a

115

female, is a critical behavioral trait that can significantly impact reproductive success

116

in various species, including

Drosophila melanogaster

. From an evolutionary

117

perspective, CL can be influenced by several factors, including competition among

118

males and the quality of potential mates. For instance, males exposed to rival males

119

often exhibit shorter mating latencies, suggesting an adaptive response to increased

120

competition

21–23

. This phenomenon indicates that males may prioritize rapid mating to

121

maximize reproductive opportunities when faced with competitors. CL reflects an

122

integrated behavioral output influenced by male courtship intensity, female receptivity,

123

and copulatory performance. To specifically dissect male contributions to CL, all assays

124

utilized SPR-deficient virgin females (

SPR

-/-

), which exhibit constitutively high

125

receptivity and minimal rejection behavior

. This approach standardizes female

126

responsiveness, thereby isolating genetic and neuronal mechanisms underlying male

127

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courtship initiation, persistence, and copulation efficiency. Under these controlled

128

conditions, CL serves as a quantifiable proxy for male eagerness and performance.

129

130

Inhibiting SIFa neuronal activity by expressing TNT and KCNJ2/OrkΔC led to a

131

notable delay in CL, while hyperactivation of SIFa neurons through NaChBac did not

132

influence CL (Fig. 1C). Furthermore, reducing SIFa levels via RNAi also failed to affect

133

CL (Fig. 1D). Unlike the data for CI, these results imply that the active states of SIFa

134

neurons are more essential for sustaining rapid CL than the release of the SIFa

135

neuropeptide. Overall, our findings reveal that CI and CL, two interconnected facets of

136

male sexual behavior, are governed by different neural mechanisms associated with

137

SIFa neurons.

138

139

Numerous studies have indicated that SIFa neurons predominantly employ glutamate

140

(Glu) and dopamine (DA) as their main neurotransmitters to regulate sleep and interval

141

timing behaviors

11,18,25

. To ascertain which neurotransmitter system contributes to CI

142

and CL, we conducted RNAi to knock down essential genes involved in the production

143

of each neurotransmitter. We targeted

VGlut-RNAi

(vesicular glutamate transporter)

144

to impair the glutamatergic (Glu) system,

ple-RNAi

(pale)

for the dopaminergic (DA)

145

system,

Gad1-RNAi

(glutamic acid decarboxylase 1)

for the GABAergic (GABA)

146

system,

ChaT-RNAi

(choline acetyltransferase)

for the cholinergic (Cha) system,

147

Tdc2-RNAi

(tyrosine decarboxylase 2)

for the octopaminergic/tyraminergic (OA/TA)

148

system and

Trhn-RNAi

(Tryptophan hydroxylase neuronal)

for the serotoninergic (5-

149

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HT) system. Our findings from RNAi-mediated knockdown experiments indicate that

150

the DA system is essential for facilitating normal CI behavior in SIFa-positive neurons

151

(Fig. 1E). Conversely, our results suggest that CL is largely dependent of Glu and

152

OA/TA systems produced by SIFa neurons (Fig. 1F). These observations imply that

153

while CI is dependent on SIFa and DA transmission, CL may function independently

154

through Glu and OA/TA synaptic transmission mechanisms instead

155

156

The SIFa neurons are known to integrate signals from various peptidergic pathways

157

such as Crz, dilp2, Dsk, sNPF, MIP, and hugin

1,14,31

. To determine which neuropeptide

158

inputs are crucial for CI and CL, we conducted a systematic analysis of each

159

neuropeptide receptor's role within SIFa-positive cells. A reduction in InR levels within

160

SIFa neurons led to a notable increase in CI compared to control flies. In contrast, the

161

knockdown of PK2-R2 (hugin receptor) and sNPF-R levels resulted in a significant

162

decrease in CI relative to control groups (Fig. 1G). Furthermore, reducing CrzR and

163

sNPF-R levels in SIFa neurons caused delays in CL (Fig. 1H). These results suggest

164

that sNPF signaling is vital for modulating both CI and CL, while insulin and hugin

165

signaling specifically regulate CI, with Crz signaling exclusively influencing CL (Fig.

166

1G-H). Overall, our findings indicate that CI and CL rely on distinct input-output

167

pathways from SIFa neurons for their appropriate generation

26–28

168

169

Male-Specific SIFaR-Expressing Neurons Differentially Mediate SIFa Signaling

170

to Generate Internal States for Varied Sexual Behaviors.

171

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It is well established that SIFa modulates sexual behavior through its action on

fruitless

172

neurons. In

Drosophila melanogaster

, the

fruitless

gene encodes male-specific

173

transcription factors that delineate distinct neuronal circuits involved in courtship

174

behavior. Mutations in the

fruitless

gene can lead to bisexual courting behaviors in

175

males, where they exhibit courtship towards both females and other males

. Utilizing

176

RNAi to specifically diminish SIFaR expression within these neurons, the authors

177

showed that this alteration resulted in heightened male-male courtship behaviors

. This

178

evidence underscores the significant role of SIFa signaling through

fruitless

neurons in

179

shaping sexual behaviors in

Drosophila

180

181

Previous research has shown that SIFa inputs specifically target various SIFaR neuronal

182

circuits, which are integral to the modulation of interval timing behaviors

17,25,33

. We

183

suggest that the chain reaction of peptidergic signaling from SIFa to SIFaR circuits

184

establishes context-sensitive internal states, facilitating the regulation of a multifaceted

185

behavioral repertoire (Fig. S1A). The reduction of SIFaR levels via RNAi specifically

186

in neurons results in a marked decrease in CI and a delay in CL (Fig. 2A-B), indicating

187

that neuronal expression of SIFaR is critical for regulating both behaviors, regardless

188

of their dependency on SIFa.

189

190

Knockdown of SIFaR in neurons marked by

fru-GAL4

dsx-GAL4

(which target

191

overlapping populations) significantly reduced male CI (Fig. 2C) but did not affect CL

192

(Fig. 2D). Given the known overlap between

fru

- and

dsx

-expressing neurons

32,34,35

193

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these results indicate that CI requires SIFaR expression in the shared or distinct

194

neuronal populations defined by these markers, while CL operates independently of

195

SIFaR in these cell groups. In previous studies, we demonstrated that the

SIFaR

24F06

196

GAL4

driver labels distinct neuronal populations in the SOG (subesophageal ganglion),

197

OL (optic lobe), and AG (abdominal ganglion) regions, which overlap with those

198

labeled by the

SIFaR

-GAL4

line (generated using the chemoconnectome (CCT)

199

knock-in method)

. Additionally, we found that SIFaR

24F06

neurons are negative for

200

both

fru

and

dsx

expression

. Blocking synaptic transmission through TNT expression

201

in either

fru

-negative SIFaR

24F06

neurons, all SIFaR neurons, or specifically within

fru

202

positive SIFaR neurons severely reduces CI and leads to an absence of successful

203

mating attempts by males (Fig. 2E-F). Thus, although CI and CL are differentially

204

modulated by the expression of SIFaR in

fru

-positive neurons, both behaviors may

205

necessitate the proper functioning of

fru

-positive SIFaR neurons.

206

207

Notably, inhibiting synaptic transmission in

fru

-positive SIFaR neurons does not affect

208

the climbing ability of male flies (Fig. S2A), suggesting that the decreased courtship

209

activity or failure to mate is not attributable to locomotor impairments. It has also been

210

reported that inhibiting

fruitless

neurons impacts courtship behavior without

211

influencing locomotion

. Considering that genetic controls exhibit normal sexual

212

behavior (Fig. S2B-C), these results imply that SIFaR-positive neurons are essential for

213

generating both CI and CL (Fig. 2C-F). Furthermore, while SIFaR function is necessary

214

for CI in both

fru

-positive and -negative neurons, it is only required in

fru

-negative

215

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neurons for CL generation (Fig. 2E-G).

216

217

Male’s mating duration (MD) in

Drosophila melanogaster

is a critical aspect of

218

reproductive fitness, influenced by evolutionary pressures that shape mating strategies

219

and behaviors. The MD in male fruit flies reflects adaptive responses to varying levels

220

of competition and environmental contexts. Males exposed to high competition tend to

221

exhibit longer mating durations, which can enhance their reproductive success by

222

increasing the likelihood of successful fertilization as we reported previously

22,38

. This

223

plasticity in mating duration allows males to adjust their reproductive investment based

224

on social dynamics, optimizing their fitness in different mating scenarios. Our previous

225

research

39–42

and that of others

43,44

has established various frameworks for investigating

226

MD through advanced genetic techniques, allowing us to analyze the neural circuits

227

that govern interval timing. Notably, males exhibit longer mating durations (LMD

228

behavior) when faced with rival males, indicating an adaptive strategy to enhance

229

reproductive success

45–50

. Conversely, under sustained female-rich conditions (e.g., 1:2

230

male-to-female ratio), males exhibit Shorter Mating Duration (SMD) behavior—

231

reducing copulation time to conserve reproductive effort for additional mating

232

opportunities

51,52

(See

Quantification and Interpretation of Mating Duration

233

Behaviors

STAR Methods

section).

234

235

Our recent findings indicate that the long-range neuropeptide relay from SIFa to SIFaR

236

plays a critical role in mediating LMD and SMD behaviors

. To further explore

237

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whether SIFaR expression in

fru

-positive neurons contributes to LMD/SMD behaviors,

238

we utilized RNAi to decrease SIFaR levels. The results showed that reducing SIFaR

239

levels specifically within

fru

-positive neurons completely disrupted LMD/SMD

240

behaviors when compared to genetic controls (Fig. 3A-B). Recent single-cell RNA

241

sequencing data from a fly cell atlas have identified several neuropeptide receptors

242

(NPRs) that are highly enriched in

fru

- and

dsx

-positive neurons, suggesting their

243

involvement in sexually dimorphic behaviors

53,54

. Among these NPRs, AstC-R1 is

244

particularly enriched in

fru

-positive neurons and has been associated with regulating

245

insulin-producing cells (IPCs) that influence female egg-laying rhythms

. Both AstC-

246

R1 and AstC-R2 serve as receptors for the neuropeptide AstC; notably, AstC-R2 has

247

been identified as a modulator of evening locomotor activity

and is involved in food

248

intake and metabolic homeostasis during nutrient stress in the gut

249

250

Despite both AstC-R1 and AstC-R2 being highly expressed in

fru

-positive neurons

251

compared to SIFaR levels (Supporting Information 1), knocking down AstC-R1 did not

252

alter LMD/SMD behaviors (Fig. 3C), while knocking down AstC-R2 specifically

253

affected SMD behavior (Fig. 3D). These data indicate that the expression of AstC-R1

254

fru

-positive neurons is not necessary for LMD/SMD behaviors. Additionally,

255

neuronal knockdown of AstC significantly delayed CL compared to controls or flies

256

with reduced SIFa levels (Fig. 3E), indicating that AstC release from these neurons

257

contributes to CL phenotype. Interestingly, knockdown of both AstC-R1 and AstC-R2

258

did not impact CL phenotype relative to control or SIFaR knockdown flies, which

259

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exhibited significant delays in CL (Fig. 3F), suggesting that signaling from AstC to its

260

receptors for CL may involve non-neuronal pathways.

261

262

Previous studies have indicated that the functions of AstC-R1/R2 may extend into the

263

gut for behavioral modulation

57,58

. Furthermore, we found that many neurons

264

expressing SIFaR also co-express FRU within the male brain (Fig. 3G), reinforcing the

265

notion that NPR expression levels do not necessarily correlate with specific fru-

266

mediated functions (Supporting information 1). Overall, considering genetic controls

267

for mating duration, these findings imply that the function of SIFaR within

fru

-positive

268

neurons regarding sex-specific behaviors is distinctively unique (Fig. S3A-D).

269

270

Fru-Specific SIFaR Expression in P1 and mAL Neuronal Populations Specifically

271

Modulates Mating Duration, Not Copulation Latency.

272

To identify the minimal region necessary for labeling GAL4 drivers that assess the

273

functionality of SIFaR-mediated mating duration behaviors, we examined four custom

274

GAL4 drivers targeting the SIFaR enhancer region (Fig. 4A)

Our previous work with

275

these modified drivers demonstrated that co-expression of

SIFaR-RNAi

with either

276

SIFaR

57F10

-GAL4

SIFaR

24F06

-GAL4

led to a complete loss of LMD and SMD

277

behaviors, highlighting the essential role of SIFaR expression in these GAL4 driver-

278

expressing cells for mating duration behaviors

279

280

Among these drivers, only inhibiting SIFaR

57F10

-positive and

fru

-positive neurons

281

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through TNT expression abolished both LMD and SMD behaviors compared to genetic

282

controls (Fig. 4B-E and Fig. S4A-E), suggesting that these specific neurons are crucial

283

for modulating LMD/SMD behaviors.

Notably, CL was unaffected by either the

284

inhibition of SIFaR

57F10

- and

fru

-positive neurons (Fig. 4F) or by knocking down SIFaR

285

in these neuronal populations (Fig. S4F), indicating their lack of involvement in CL

286

regulation.

287

288

Visualization of SIFaR

57F10

-positive and

fru

-positive neurons revealed the presence of

289

fruP1 and mAL neurons in the male central brain (CB) (top panels in Fig. 4G and Fig.

290

S4G). Previous studies have established that fruP1-expressing neurons are vital for

291

complex male courtship displays

32,60

, while also directing female reproductive

292

behaviors

. In females, the neuronal populations labeled by SIFaR

57F10

closely

293

resemble those expressing fruP1 and mAL neurons (bottom panels in Fig. 4G and Fig.

294

S4G). The membrane arborization coverage, fluorescence intensity, number of cells,

295

and cell size of fru-positive neurons labeled by SIFaR

57F10

are similar between males

296

and females (Fig. 4H-L), suggesting shared neural circuits of

fru

-positive SIFaR

297

neurons encoding sexually dimorphic behaviors in

Drosophila

298

299

SIFaR Expression in GABAergic fru-Positive mAL Neurons Regulates MD and

300

CI but Not CL.

301

Since the SIFaR promoter region drives SIFaR expression in the fruP1 and mAL regions,

302

we systematically confirmed the fru-positive expression of SIFaR neurons utilizing a

303

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gene-specific T2A-GAL4 construct,

SIFaR

T2A

-GAL4

36,62,63

. As anticipated, the

304

SIFaR

T2A

-GAL4

driver labeled

fru

-positive P1 and mAL neurons in the male brain (Fig.

305

5A). Notably, we observed that while the

dsx

-positive

SIFaR

T2A

-GAL4

driver marked

306

P1 neurons, it did not label mAL neurons in the male brain (Fig. 5B). This data indicates

307

that SIFaR is expressed in

fru

-positive and

dsx

-positive P1 neurons but is restricted to

308

fru

-positive and

dsx

-negative mAL neurons.

309

310

We further validated that the expression patterns of the fruP1 and mAL-specific drivers

311

exclusively colocalize with the

fru

-positive SIFaR

T2A

driver, while showing no overlap

312

with the

fru

-negative SIFaR

24F06

driver (Fig. S5A, B and F). These findings align with

313

our previous orthogonal verification using anti-FruM antibodies and nuclear markers

314

as well as

fru

FLP

intersectional methods, confirming SIFaR

24F06

neurons lack FruM

315

protein expression

. Given that mAL neurons are GABAergic and therefore exhibit

316

characteristically low intracellular calcium concentrations under basal conditions, their

317

Ca²⁺ levels fall below the minimum detection threshold of the CaLexA imaging system

318

(Fig. S5G). Consequently, these drivers enable us to explore the specific functions of

319

SIFaR within both

fru

-positive and

fru

-negative interneuronal circuits. By utilizing

320

these specific drivers, we can elucidate how SIFaR influences neural circuitry related

321

to mating investment in a sexually dimorphic context.

322

323

Knockdown of SIFaR using fruP1- and mAL-specific

GAL4

drivers resulted in a

324

complete disruption of both LMD/SMD behaviors compared to genetic controls (Fig.

325

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5C-F). To determine whether ventral nerve cord (VNC) neurons modulate sexual

326

behavior, we combined

tsh-GAL80

with

SIFaR

knockdown in fruP1 neurons. Notably,

327

this manipulation did not alter key behavioral parameters—including mating duration,

328

courtship index, or copulation latency—suggesting that VNC neurons are not critically

329

involved in regulating these aspects of sexual behavior (Fig. S5C-E). Notably, when

330

SIFaR levels were specifically reduced in mAL neurons, MD in sexually experienced

331

males was significantly longer compared to that of naïve males (Fig. 5D). The mAL

332

neurons are GABAergic interneurons involved in courtship regulation. Recent studies

333

have demonstrated that mAL neurons are activated by both male and female

334

pheromones, underscoring their involvement in social interactions and behavioral

335

responses to environmental stimuli. Based on their anatomical structure and

336

neurotransmitter profile, mAL neurons have been proposed as sexually dimorphic

337

GABAergic interneurons that convey inhibitory courtship signals to higher brain

338

centers in males

339

340

Activation of the mAL cluster inhibits male courtship behavior towards females

65–67

341

At the circuit level, this regulation is mediated through GABA release from the mAL

342

cluster into the main courtship command center of fruP1 neurons within the fly brain

343

This circuit receives inputs from interactions with females, which subsequently activate

344

male-specific interneurons that trigger stereotypical courtship behavior in

345

Drosophila

. Our previous findings suggest that taste and pheromonal inputs via male-

346

specific Gr5a/ppk25/ppk29 neurons into the brain are critical for generating SMD

347

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behavior

. Thus, we hypothesize that the reversed SMD behavioral phenotype

348

resulting from SIFaR knockdown in mAL neurons arises from inhibited GABAergic

349

function within these neurons, leading to disinhibition (Fig. 5I).

350

351

Interestingly, the knockdown of SIFaR in either fruP1 or mAL neurons resulted in a

352

reduction of the CI (Fig. 5J), without producing the increased courtship activity as

353

shown in SMD behavior (Fig. 5D). This finding suggests that MD may rely on

354

disinhibitory circuits for male decision-making, whereas CI does not depend on the

355

same disinhibitory pathways for context-dependent regulation (Fig. 5J). Additionally,

356

when monitoring fruP1 calcium dynamics using CalexA system, we observed that Ca

357

levels were significantly lower in single reared and sexually experienced males

358

compared to naive controls (Fig. 5G, 5H), further supporting the role of activity-

359

dependent plasticity in these circuits. Knockdown of SIFaR in both fruP1 and mAL

360

neurons did not affect CL (Fig. 5K), indicating that CL is likely modulated by

fru

361

negative SIFaR-expressing neurons. Collectively, these results imply that various

362

mating behaviors in males can be regulated by the same SIFa-to-SIFaR circuitry

363

through different regional expressions of SIFaR and distinct circuit combinations (Fig.

364

5I).

365

366

To identify the specific neuronal populations responsible for modulating CL without

367

affecting CI or MD, we conducted a mini screen of previously reported

368

neuropeptidergic circuits. We discovered that knocking down SIFaR in neuropeptide

369

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tachykinin (Tk)-positive neurons specifically delayed CL without impacting

370

LMD/SMD behaviors (Fig. S5H-I). Tk neurons have been shown to promote aggression

371

in male

Drosophila

. When Tk neuropeptide was knocked down in SIFaR-positive

372

neurons, LMD/SMD behaviors remained intact, but CL was significantly delayed (Fig.

373

S5J-K), suggesting that the SIFa-to-SIFaR/Tk pathway specifically modulates CL (Fig.

374

5I). These findings highlight the nuanced roles of different neuropeptidergic circuits in

375

regulating distinct aspects of mating behavior. By elucidating these pathways, we can

376

gain deeper insights into how neuropeptidergic signaling influences mating investment

377

and behavioral outcomes in

Drosophila

378

379

The Synaptic Alterations Observed in mAL Neurons are Associated with Distinct

380

Social Contexts.

381

To understand how mAL neurons differentially regulate LMD and SMD behaviors, we

382

conducted a genetic intersection analysis to visualize the presynaptic and postsynaptic

383

terminals of

fru

-positive and

SIFaR

-positive neurons

. Notably, the presynaptic

384

terminals of

SIFaR

/fru

mAL neurons exhibited a significant increase in sexually

385

experienced males (Fig. 6A-B). In contrast, the postsynaptic terminals of mAL neurons

386

were enhanced in both socially isolated and sexually experienced males (Fig. 6C-D).

387

We also found that both social isolation and sexual encounters precipitated an

388

augmentation in the synaptic connectivity between these neuronal populations (Fig. 6E-

389

F). The intensified DsCam-GFP, nSyb-GFP, and tGRASP signals in experienced males

390

(Fig. 6A-F) may arise in part from elevated SIFaR expression induced by social and

391

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sexual experience, consistent with our prior transcriptomic data

18,72

. Thus, while

392

indicative of enhanced connectivity, these results cannot exclude contributions from

393

expression-level changes of the receptor.

394

395

We identified that only one cell in each hemisphere of the mAL neurons expresses

396

SIFaR (Fig. S6A). To investigate the synaptic architecture linking SIFa and mAL

397

neurons within the brain under diverse social conditions, we observed that the signal

398

was strictly restricted in the SIFaR

/mAL

cells as Fig. S6A demonstrated. To validate

399

the specificity of our newly generated

lexA

SIFa.PT

driver, we compared its expression

400

pattern with that of

GAL4

SIFa.PT

using a membrane marker in the fly brain and VNC.

401

Although

lexA

SIFa.PT

exhibited weaker expression in some regions, the four

SIFa

402

expressing cells showed complete overlap between the two drivers (Fig. S6B). This

403

precise colocalization confirms the successful generation of

lexA

SIFa.PT

and supports the

404

reliability of our experimental results.

405

406

To investigate whether mAL neurons respond to SIFa neuron activation, we performed

407

live calcium (Ca

) imaging in mAL neurons using two-photon microscopy. A

408

genetically encoded calcium indicator, GCaMP7s, was expressed in mAL neurons

409

under the control of the

lexA

SIFa.PT

driver. To selectively activate SIFa neurons, we

410

employed an optogenetic approach using the red-light-sensitive channelrhodopsin

411

CsChrimson, expressed via

lexA

SIFa.PT

(Fig. 6G). Upon optogenetic stimulation of SIFa

412

neurons, we observed no significant change in Ca

activity in mAL neurons, which

413

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remained stable and comparable to control flies lacking all-trans retinal (ATR) (Fig.

414

6H-I). These results indicate that mAL neurons do not respond to SIFa neuron

415

activation, likely due to their GABAergic nature, which may inhibit downstream

416

signaling. These findings suggest that SIFa inputs to this unique

fru

/SIFaR

mAL

417

neuron create context-specific internal states by modulating synaptic plasticity within

418

these neurons.

419

420

DISCUSSSION

421

The results of our study reveal that SIFa neurons play a critical role in orchestrating

422

male sexual behaviors, specifically CI, CL, and MD. We found that reducing SIFa

423

peptide levels through RNA interference significantly decreased male CI towards

424

females, indicating the importance of SIFa release for normal courtship behavior. In

425

contrast, altering neuronal activity in SIFa-positive neurons did not affect CI,

426

suggesting that the neuropeptide's release is more crucial than the activity of SIFa-

427

expressing neurons themselves. Regarding CL, we observed that inhibiting SIFa

428

neuronal activity led to delays in copulation initiation, while hyperactivation of these

429

neurons had no effect. This indicates that the active states of SIFa neurons are essential

430

for maintaining rapid CL, unlike CI, which is dependent on synaptic transmission

431

involving dopamine. Our analysis of neuropeptide receptors indicated that sNPF

432

signaling is vital for both CI and CL modulation, while insulin and hugin signaling

433

specifically regulate CI, and Crz signaling influences CL.

434

435

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We also established that SIFaR expression in

fru

-positive neurons is critical for

436

regulating MD and CI but not CL. Knockdown of SIFaR in fruP1 and mAL neurons

437

disrupted both LMD and SMD behaviors, highlighting their essential role in mating

438

investment. Importantly, sexually experienced males exhibited longer MD when SIFaR

439

levels were reduced specifically in mAL neurons compared to naïve males. Overall,

440

these findings underscore the distinct yet interconnected neural mechanisms underlying

441

male sexual behavior, emphasizing the role of SIFa and its receptors in shaping

442

reproductive strategies in

Drosophila melanogaster

. This research highlights the

443

intricate relationship between sensory input and behavioral output in

Drosophila

mating

444

strategies. The results of our study reveal that both social isolation and instances of

445

sexual activity result in enhanced synaptic connectivity within the SIFa to mAL

446

neuronal pathway (Fig. 6E-F). This observation implies that synaptic plasticity is a

447

pivotal mechanism in adapting the SIFa-mAL circuitry based on varying social

448

environments. The modulation of synaptic plasticity in response to contextual cues

449

emphasizes the adaptability of neural circuits involved in mating behaviors. Our

450

findings contribute to a deeper understanding of how neuropeptidergic signaling can

451

fine-tune behavioral responses based on social and sexual experiences. We further

452

propose that SIFa-to-SIFaR signaling induces distinct internal brain states in CI and CL,

453

potentially mediated by varying neuropeptidergic input thresholds to SIFa neurons

73–75

454

differential reliance on neurotransmitter systems, and downstream neuronal regulation

455

of SIFaR-expressing neurons. However, additional experiments are required to validate

456

this hypothesis and elucidate the precise mechanisms involved (Fig. 1I).

457

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458

Our results reveal a functional segregation within SIFa-positive neurons that CI

459

depends exclusively on DA signaling, while CL requires Glu/OA/TA transmission. To

460

explain the SIFa's release independence and the apparent discrepancy in

ple

knockdown

461

effects, we propose a mechanistic model. We hypothesize that SIFa, as a neuropeptide,

462

is released via dense-core vesicles (DCVs) through calcium-dependent but SNARE-

463

independent mechanisms

76,77

, rendering it resistant to TNT or depolarization-blocking

464

agents like KCNJ2. This non-canonical release mechanism enables SIFa to regulate

465

neuronal activity independent of conventional vesicular fusion machinery, while still

466

maintaining the capacity to modulate CI through specific neural circuits.

467

468

In this dual-transmitter architecture, SIFa acts as a master regulator biasing co-released

469

neurotransmitters toward distinct behavioral outputs. Regarding CI, SIFa potentiates

470

DA signaling via autocrine feedback mechanisms, likely through SIFa receptor

471

(SIFaR)-mediated enhancement of tyrosine hydroxylase activity or dopamine vesicle

472

loading. This is consistent with our previous demonstration of SIFaR expression in SIFa

473

neurons themselves

. This explains why

ple

knockdown (disrupting DA synthesis)

474

impairs CI (Fig. 1E) while CL remains unaffected (Fig. 1F). For CL, SIFa

475

independently potentiates Glu/OA/TA transmission through ionotropic receptor

476

modulation. Regarding the query about TNT/KCNJ2 effects on DA release (Fig. 1A),

477

we posit that SIFa’s TNT-resistant release compensates by either: (i) directly activating

478

postsynaptic DA receptors via volume transmission, or (ii) sustaining baseline DA tone

479

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through enhanced

ple

-mediated synthesis, even when exocytosis is impaired. Thus,

480

while

ple

knockdown causes irreversible DA depletion, TNT/KCNJ2 may only

481

transiently block exocytosis, leaving SIFa-mediated DA signaling partially functional.

482

483

This model predicts that SIFa knockout would abolish both CI and CL, whereas

484

TNT/KCNJ2 expression in SIFa neurons would impair CL (via disrupted Glu/OA/TA

485

release) but spare CI due to SIFa’s compensatory actions. Future studies should validate

486

these predictions through intersectional manipulations of SIFa and specific

487

neurotransmitter systems. Collectively, our findings suggest SIFa neurons employ

488

hierarchical release strategies, with neuropeptide-mediated gating ensuring robust

489

behavioral modulation independent of conventional release pathways.

490

491

The P1 (pC1) neurons in

Drosophila melanogaster

are a pivotal cluster of male-specific

492

neurons that integrate sensory information and modulate behaviors related to mating

493

and aggression. Recent studies have highlighted the role of neuropeptidergic inputs in

494

regulating the activity of these neurons, thereby influencing male mating investment

495

and behavioral outcomes. P1 neurons express a diverse array of neuropeptides that play

496

critical roles in modulating their activity. For instance, recent transcriptional profiling

497

revealed that these neurons are enriched in genes encoding neuropeptides such as

498

Neuropeptide F (NPF), Drosulfakinin (Dsk), and others that are implicated in

499

behavioral regulation

53,78

. The activation of P1 neurons can lead to both aggressive and

500

mating behaviors, suggesting that the balance of neuropeptidergic signaling is crucial

501

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for determining behavioral outcomes in different social contexts

. Moreover, P1

502

neurons have been shown to be influenced by external factors such as food availability

503

and social interactions. High-quality food and starvation conditions modulate P1 neuron

504

activity through insulin and tyramine signaling pathways, indicating a sophisticated

505

interplay between nutritional state and mating drive

. This modulation underscores the

506

context-dependent nature of neuropeptide signaling in shaping persistent internal states

507

for mating investment.

508

509

SIFa neurons exhibit extensive branching throughout the central nervous system and

510

integrate diverse inputs from multiple neuropeptide signals, playing a crucial role in

511

regulating internal states related to hunger, satiety, and social status

1,14,18

. Our study

512

provides significant insights into how SIFa inputs to fruP1 central neurons can

513

orchestrate various sexual behaviors in a context-dependent manner. Specifically, we

514

found that the modulation of MD and CI is influenced by SIFa signaling, with distinct

515

neural mechanisms governing these behaviors. The results indicate that while SIFaR

516

expression in

fru

-positive neurons is essential for CI, it does not impact CL.

517

Additionally, the differential responses of mAL neurons to social contexts further

518

underscore the complexity of SIFa's role in shaping male sexual behaviors. Overall, our

519

findings shed light on the mechanistic details of prior research indicating that SIFa

520

influences sexual behavior through its action on

fruitless

neurons via SIFaR

15,16

521

522

One intriguing finding is that CL is not influenced by SIFa release (Fig. 1D) or NT

523

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release from SIFa neurons (Fig. 1F), yet it is affected by the neuronal knockdown of

524

SIFaR (Fig. 2B). In fact, CL relies on specific neuropeptide inputs to SIFa neurons,

525

particularly Crz and sNPF (Fig. 1H). This raises the question of how CL can be

526

modulated by SIFa inputs and neuronal SIFaR expression while remaining unaffected

527

by SIFa expression or NT release from SIFa neurons. We propose that non-synaptic

528

transmission from SIFa to

fru

-negative SIFaR neurons may account for this

529

phenomenon. Neurons can indeed influence behavior independently of traditional

530

neurotransmitter systems

81–83

531

532

Disinhibition, characterized by the alleviation of inhibitory constraints, allows for the

533

activation of neural circuits typically suppressed. This mechanism is vital for shaping

534

behavioral patterns and facilitating the sequential progression of actions. By promoting

535

selective neuronal activation while concurrently inhibiting competing neural pathways,

536

disinhibition enables the brain to dynamically assess and maintain necessary behavioral

537

states while facilitating transitions to subsequent behavioral phases

. It is also

538

recognized that

Drosophila

neural circuits exhibit disinhibition phenotypes related to

539

light preference and ethanol sensitization

85,86

540

541

Additionally, research has shown that certain behaviors can remain intact even when

542

specific neurotransmitter systems are genetically disrupted. For example, studies on

543

dopamine neurons have indicated that while glutamatergic inputs are important for

544

certain effort-related tasks, other aspects of behavior may not be significantly affected

545

Journal Pre-proof

by the absence of these inputs

. This suggests that neurons can utilize various signaling

546

modalities to influence behavior, including non-neurotransmitter signaling pathways.

547

Moreover, the presence of ‘non-neurotransmitter signaling’ pathways, such as

548

neuroinflammatory responses and other chemical mediators such as nitric oxide, further

549

illustrates how neuronal and non-neuronal interactions contribute to behavioral

550

outcomes

. Thus, it is clear that while neurotransmitters play a vital role in neuronal

551

communication and behavior, there are multiple mechanisms through which neurons

552

can affect behavior independently of traditional neurotransmitter systems

88–91

. The

553

stable phenotype of CL may suggest the involvement of non-synaptic transmission

554

mechanisms as regulatory controls.

555

556

It is well established that mAL neurons receive pheromonal inputs from both female

557

and male sources through vAB3 neuronal circuits and inhibit fruP1 neurons via GABA

558

release

. Integrating these observations with our data, we propose that the differential

559

pheromonal inputs from males and females may be orchestrated by specific SIFaR

560

expression in targeted P1 and mAL neurons to elicit distinct behavioral responses (Fig.

561

S6C)

562

563

While our RNAi experiments implicate the genes we tested in regulating LMD/SMD

564

and CI/CL behaviors, we note that RNAi-mediated knockdown may not achieve

565

complete loss-of-function. The lines used in our study were pre-validated in both prior

566

studies

26,27,93–105

and our lab controls (

UAS-RNAi

/+,

GAL4

/+), showing consistent

567

Journal Pre-proof

phenotypic effects. We interpret reduced effects as "attenuation" rather than abolition

568

unless supported by null mutants or rescue experiments. Future studies using CRISPR

569

or tissue-specific rescue could further validate these findings, but our results remain

570

robust within RNAi limitations.

571

572

In conclusion, our study elucidates the critical role of SIFa neurons in orchestrating

573

male sexual behaviors through distinct neuropeptidergic signaling pathways. By

574

demonstrating how SIFa influences various sexual behaviors via different mechanisms,

575

we provide valuable insights into the complex interplay between neuropeptides and

576

sexual behavior, underscoring the importance of context-dependent regulation in

577

Drosophila melanogaster

578

579

Journal Pre-proof

SUPPLEMENTAL INFORMATION

580

RESOURCE AVAILABILITY

581

LEAD CONTACT

582

Requests for further information and resources should be directed to and will be

583

fulfilled by the lead contact, Woo Jae Kim (email: wkim@hit.edu.cn).

584

585

MATERIALS AVAILABILITY

586

Strains are available upon request.

587

588

DATA AND CODE AVAILABILITY

589

⚫

All data reported in this paper will be shared by the lead contact upon

590

request.

591

⚫

This paper does not report original code. The URL of the codes used in this

592

paper are listed in the key resources table.

593

⚫

Any additional information required to reanalyze the data in this paper is

594

available from the lead contact upon request.

595

596

LIMITATIONS OF THE STUDY

597

While our study provides insights into the role of SIFa/SIFaR signaling in male mating

598

behaviors, several limitations should be acknowledged. First, SIFa might influence

599

copulation latency through non-synaptic volume transmission remains hypothetical

600

without direct evidence of neuropeptide diffusion dynamics in vivo. Cutting-edge tools

601

like genetically encoded neuropeptide sensors (e.g., GRAB reporters) combined with

602

Journal Pre-proof

in vivo imaging could experimentally test this proposed mechanism. Second, while we

603

standardized behavioral assays using SPR

-/-

females, environmental variability might

604

still influence results. Future studies could employ connectomics, or real-time

605

neuropeptide imaging to address these gaps.

606

607

ACKNOWLEDGMENTS

608

We thank Dr. Jan A. Veenstra (University of Bordeaux) for sharing

SIFa

-GAL4

driver,

609

Dr. Young-Joon Kim (GIST) for sharing

UAS-stop-nSyb-GFP

and

UAS-stop-

610

Dscam17.1-EGFP

fly strains. We gratefully acknowledge Dr. Yufeng Pan (Southeast

611

University) for generously providing the fruP1 and mAL driver lines, as well as for

612

sharing valuable insights into sexually dimorphic circuit analysis. We gratefully

613

acknowledge Dr. Wei Zhang (Tsinghua University) for his valuable guidance and expert

614

advice on our GCaMP live imaging experiments. We have greatly benefited from the

615

resources provided by the FlyBase website in our genetic research endeavors. We are

616

grateful for the ongoing efforts of the FlyBase staff in maintaining this comprehensive

617

Drosophila

database

106–110

. This research was supported by Startup funds from HIT

618

Center for Life Science to WJK. The funder had no role in study design, data collection

619

and analysis, decision to publish, or preparation of the manuscript.

620

621

DECLARATION OF INTERESTS

622

The authors declare no competing interests.

623

624

Journal Pre-proof

AUTHOR CONTRIBUTIONS

625

Conceptualization:

Woo Jae Kim.

626

Data curation:

Tianmu Zhang, Hongyu Miao, Yutong Song, Zekun Wu, Woo Jae Kim.

627

Formal analysis:

Tianmu Zhang, Hongyu Miao, Yutong Song, Zekun Wu, and Woo

628

Jae Kim.

629

Funding acquisition:

Woo Jae Kim.

630

Investigation:

Woo Jae Kim.

631

Methodology:

Woo Jae Kim.

632

Project administration:

Woo Jae Kim.

633

Resources:

Woo Jae Kim.

634

Supervision:

Woo Jae Kim.

635

Validation:

Tianmu Zhang, Hongyu Miao, Yutong Song, Woo Jae Kim.

636

Visualization:

Zekun Wu, Woo Jae Kim.

637

Writing – original draft:

Woo Jae Kim.

638

Writing – review & editing:

Tianmu Zhang, Woo Jae Kim.

639

640

STATEMENT OF ANIMAL RESEARCH COMPLIANCE

641

All animal experiments reported in this manuscript were conducted in compliance with

642

the ARRIVE guidelines and adhered to the U.K. Animals (Scientific Procedures) Act,

643

1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or

644

the National Research Council's Guide for the Care and Use of Laboratory Animals.

645

646

Journal Pre-proof

DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES

647

IN THE WRITING PROCESS

648

During the creation of this work, the author(s) utilized deepseek AI

649

(https://chat.deepseek.com/) to rephrase English sentences, verify English grammar,

650

and detect plagiarism, as none of the authors of this paper are native English speakers.

651

After using this tool/service, the author(s) reviewed and edited the content as needed

652

and take(s) full responsibility for the content of the publication.

653

654

655

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FIGURE TITLES AND LEGENDS

1119

Fig.1 SIFa neuron activity and SIFa levels influence male sexual behavior

1120

differently

1121

Journal Pre-proof

(A) Courtship index (CI) of group-housed males expressing

GAL4

SIFa.PT

driver together

1122

with

UAS-GFP.nls, UAS-TNT, UAS-KCNJ2, UAS-OrkΔC

and

UAS-NaChBac

. CI was

1123

measured by [courtship time/ (mating time-courtship time) *100%]. For detailed

1124

methods, see the

STAR Methods

for a detailed description of the CI assays used in this

1125

study. Statistical significance determined by one-way ANOVA followed by Tukey’s

1126

multiple comparisons test. p = 0.2167 (one-way ANOVA, F = 1.45, R² = 0.01; Tukey's

1127

post hoc). The ns represents non-significant differences. Sample sizes (n) are indicated

1128

in the figure panels. Data are represented as mean +/- SEM.

1129

(B) CI of group-housed males expressing

GAL4

SIFa.PT

driver together with

SIFa-RNAi

1130

Flies expressing

GAL4

empty

with

SIFa-RNAi

and

empty-RNAi

with

SIFa-RNAi

as control.

1131

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1132

comparisons test. p < 0.0001 (one-way ANOVA, F = 37.03, R² = 0.28; Tukey's post

1133

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1134

the figure panels. Data are represented as mean +/- SEM.

1135

GAL4

SIFa.PT

driver

1136

together with

UAS-GFP.nls, UAS-TNT, UAS-KCNJ2, UAS-OrkΔC

and

UAS-NaChBac

1137

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1138

comparisons test. p < 0.0001 (one-way ANOVA, F = 29.49, R² = 0.13; Tukey's post

1139

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1140

the figure panels. For detailed methods, see the

STAR Methods

for a detailed

1141

description of the CL assays used in this study. Data are represented as mean +/- SEM.

1142

(D) CL of group-housed males expressing

GAL4

SIFa.PT

driver together with

SIFa-RNAi

1143

Journal Pre-proof

Flies expressing

GAL4

empty

with

SIFa-RNAi

and

empty-RNAi

with

SIFa-RNAi

as control.

1144

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1145

comparisons test. p = 0.9779 (one-way ANOVA, F = 0.02, R² = 0.00023; Tukey's post

1146

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1147

the figure panels. Data are represented as mean +/- SEM.

1148

(E) CI of group-housed males expressing

GAL4

SIFa.PT

driver together with

empty-RNAi,

1149

ChaT-RNAi, Gad1-RNAi, VGlut-RNAi

ple-RNAi, Tdc2-RNAi and Trhn-RNAi

1150

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1151

comparisons test. p < 0.0001 (one-way ANOVA, F = 8.69, R² = 0.11; Tukey's post hoc).

1152

The ns represents non-significant differences. Sample sizes (n) are indicated in the

1153

figure panels. Data are represented as mean +/- SEM.

1154

(F) CL of group-housed males expressing

GAL4

SIFa.PT

driver together with

empty-RNAi,

1155

ChaT-RNAi, Gad1-RNAi, VGlut-RNAi

ple-RNAi, Tdc2-RNAi and Trhn-RNAi

1156

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1157

comparisons test. p = 0.4548 (one-way ANOVA, F = 0.96, R² = 0.0085; Tukey's post

1158

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1159

the figure panels. Data are represented as mean +/- SEM.

1160

(G) CI of group-housed males expressing

GAL4

SIFa.PT

driver together with

empty-RNAi,

1161

CrzR-RNAi, CCHa1-R-RNAi, InR-RNAi, Lgr3-RNAi, Lgr4-RNAi, Pk2-R1-RNAi, PK2-

1162

R2-RNAi, CCKLR17D1-RNAi, CCKLR17D3-RNAi

and

sNPF-R-RNAi

. Statistical

1163

significance determined by one-way ANOVA followed by Tukey’s multiple

1164

comparisons test. p < 0.0001 (one-way ANOVA, F = 5.66, R² = 0.086; Tukey's post

1165

Journal Pre-proof

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1166

the figure panels. Data are represented as mean +/- SEM.

1167

(H) CL of group-housed males expressing

GAL4

SIFa.PT

driver together with

empty-RNAi,

1168

CrzR-RNAi, CCHa1-R-RNAi, InR-RNAi, Lgr3-RNAi, Lgr4-RNAi, Pk2-R1-RNAi, PK2-

1169

R2-RNAi, CCKLR17D1-RNAi, CCKLR17D3-RNAi

and

sNPF-R-RNAi

. Statistical

1170

significance determined by one-way ANOVA followed by Tukey’s multiple

1171

comparisons test. p < 0.0001 (one-way ANOVA, F = 25.55, R² = 0.13; Tukey's post

1172

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1173

the figure panels. Data are represented as mean +/- SEM.

1174

(I) Schematic representation of SIFa neurons and SIFaR neurons regulating internal

1175

states

1176

1177

Fig.2 SIFaR expression in male-specific neurons is crucial for male sexual

1178

behavior

1179

(A) CI of group-housed males expressing

empty-GAL4, repo-GAL4

and

nSyb-GAL4

1180

together with

SIFaR-RNAi

. Statistical significance determined by one-way ANOVA

1181

followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F =

1182

43.66, R² = 0.31; Tukey's post hoc). The ns represents non-significant differences.

1183

Sample sizes (n) are indicated in the figure panels. Data are represented as mean +/-

1184

SEM.

1185

(B) CL of group-housed males expressing

empty-GAL4, repo-GAL4

and

nSyb-GAL4

1186

together with

SIFaR-RNAi

. Statistical significance determined by one-way ANOVA

1187

Journal Pre-proof

followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F = 9.39,

1188

R² = 0.064; Tukey's post hoc). The ns represents non-significant differences. Sample

1189

sizes (n) are indicated in the figure panels. Data are represented as mean +/- SEM.

1190

fru-GAL4

and

dsx-GAL4

driver together with

1191

empty-RNAi

and

SIFaR-RNAi

. Statistical significance determined by one-way ANOVA

1192

followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F

1193

=14.82, R² = 0.13; Tukey's post hoc). The ns represents non-significant differences.

1194

Sample sizes (n) are indicated in the figure panels. Data are represented as mean +/-

1195

SEM.

1196

(D) CL of group-housed males expressing

fru-GAL4

and

dsx-GAL4

driver together with

1197

empty-RNAi

and

SIFaR-RNAi

. Statistical significance determined by one-way ANOVA

1198

followed by Tukey’s multiple comparisons test. p = 0.046 (one-way ANOVA, F = 2.70,

1199

R² = 0.029; Tukey's post hoc). The ns represents non-significant differences. Sample

1200

sizes (n) are indicated in the figure panels. Data are represented as mean +/- SEM.

1201

(E) CI of group-housed males expressing

empty-GAL4

with

UAS-TNT/tub-GAL80

1202

SIFaR

24F06

with

UAS-TNT/tub-GAL80

, SIFaR

-GAL4

with

UAS-GFP. nls

SIFaR-

1203

GAL4

with

UAS-TNT/tub-GAL80

empty-GAL4

with

UAS-stop-TNT, fru

FLP

and

1204

SIFaR

-GAL4

with

UAS-stop-TNT, fru

FLP

. Statistical significance determined by one-

1205

way ANOVA followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way

1206

ANOVA, F = 584.9, R² = 0.86; Tukey's post hoc). The ns represents non-significant

1207

differences. Sample sizes (n) are indicated in the figure panels. Data are represented as

1208

mean +/- SEM.

1209

Journal Pre-proof

(F) CL of group-housed males expressing

empty-GAL4

with

UAS-TNT/tub-GAL80

1210

SIFaR

24F06

with

UAS-TNT/tub-GAL80

, SIFaR

-GAL4

with

UAS-GFP. nls

SIFaR-

1211

GAL4

with

UAS-TNT/tub-GAL80

empty-GAL4

with

UAS-stop-TNT, fru

FLP

and

1212

SIFaR

-GAL4

with

UAS-stop-TNT, fru

FLP

. Statistical significance determined by one-

1213

way ANOVA followed by Tukey’s multiple comparisons test. p = 0.3251 (one-way

1214

ANOVA, F = 1.16, R² = 0.018; Tukey's post hoc). The ns represents non-significant

1215

differences. Sample sizes (n) are indicated in the figure panels. Data are represented as

1216

mean +/- SEM.

1217

(G) Schematic representation of SIFa neurons regulating courtship index and courtship

1218

latency

1219

1220

Fig.3 SIFaR and AstC signaling in fru-positive neurons regulate mating duration

1221

(A) Mating Duration (MD) assays of flies expressing the

fru-GAL4

driver together with

1222

SIFaR-RNAi

. light grey violin plot denotes males that were group-reared (or sexually

1223

naïve), whereas blue violin plot signifies males that were singly reared, and pink violin

1224

plot signify males that were sexual experienced. The dot plots represent the MD of each

1225

male fly. The mean value and standard error are labeled within the dot plot (black lines).

1226

Asterisks represent significant differences. For detailed methods, see the

STAR

1227

Methods

for a detailed description of the mating duration assays used in this study.

1228

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1229

comparisons test. p = 0.5881 (one-way ANOVA, F = 0.53, R² = 0.0073; Tukey's post

1230

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1231

Journal Pre-proof

the figure panels. In the framework of our investigation, the routine application of

1232

internal controls is employed for the vast majority of experimental procedures, as

1233

delineated in the "

Mating Duration Assay

" and "

Statistical Tests

" subsections of the

1234

STAR Methods

section. The identical analytical approach employed for the MD assays

1235

is maintained for the subsequent data presented. Data are represented as mean +/- SEM.

1236

(B) MD assays of flies expressing the

fru-GAL4

driver together with

empty-RNAi

1237

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1238

comparisons test. p < 0.0001 (one-way ANOVA, F = 16.42, R² = 0.20; Tukey's post

1239

hoc). Sample sizes (n) are indicated in the figure panels. Data are represented as mean

1240

+/- SEM.

1241

fru-GAL4

driver together with

AstC-R1-RNAi.

1242

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1243

comparisons test. p < 0.0001 (one-way ANOVA, F = 10.72, R² = 0.13; Tukey's post

1244

hoc). Sample sizes (n) are indicated in the figure panels. Data are represented as mean

1245

+/- SEM.

1246

(D) MD assays of flies expressing the

fru-GAL4

driver together with

AstC-R2-RNAi.

1247

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1248

comparisons test. p = 0.0286 (one-way ANOVA, F = 3.65, R² = 0.05; Tukey's post hoc).

1249

The ns represents non-significant differences. Sample sizes (n) are indicated in the

1250

figure panels. Data are represented as mean +/- SEM.

1251

(E) CL of group-housed males expressing

elav

c155

together with

empty-RNAi, AstC-

1252

RNAi

and

SIFa-RNAi.

Statistical significance determined by one-way ANOVA

1253

Journal Pre-proof

followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F =

1254

38.92, R² = 0.30; Tukey's post hoc). The ns represents non-significant differences.

1255

Sample sizes (n) are indicated in the figure panels. Data are represented as mean +/-

1256

SEM.

1257

(F) CL of group-housed males expressing

elav

c155

together with

empty-RNAi, AstC-R1-

1258

RNAi, AstC-R2-RNAi

and

SIFaR-RNAi.

Statistical significance determined by one-way

1259

ANOVA followed by Tukey’s multiple comparisons test. p < 0.0001(one-way ANOVA,

1260

F = 20.99, R² = 0.28; Tukey's post hoc). The ns represents non-significant differences.

1261

Sample sizes (n) are indicated in the figure panels. Data are represented as mean +/-

1262

SEM.

1263

(G) Male flies expressing

GAL4

SIFa.2A

and

fru-lexA

together with

UAS-Stinger

and

1264

LexAop-tdTomato.nls

were immunostained with anti-GFP (green), anti-RFP (red), nc82

1265

(blue) antibodies. Scale bars represent 100 µm. For detailed methods, see the

STAR

1266

Methods

for a detailed description of the immunostaining procedure used in this study.

1267

1268

Fig.4 Specific SIFaR-expressing neurons regulate mating duration

1269

(A) Diagram of the captured enhancer region within each

SIFaR-GAL4

construct.

1270

(B) MD assays of flies expressing the

SIFaR

R23G06

-GAL4

driver together with

UAS-

1271

stop-TNT; fru

FLP

Statistical significance determined by one-way ANOVA followed by

1272

Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F = 23.86, R² = 0.22;

1273

Tukey's post hoc). Sample sizes (n) are indicated in the figure panels. Data are

1274

represented as mean +/- SEM.

1275

Journal Pre-proof

SIFaR

R24A12

-GAL4

driver together with

UAS-

1276

stop-TNT; fru

FLP

Statistical significance determined by one-way ANOVA followed by

1277

Tukey’s multiple comparisons test. p = 0.5266 (one-way ANOVA, F = 0.6439, R² =

1278

0.0079; Tukey's post hoc). Sample sizes (n) are indicated in the figure panels. Data are

1279

represented as mean +/- SEM.

1280

(D) MD assays of flies expressing the

SIFaR

R24F06

-GAL4

driver together with

UAS-

1281

stop-TNT; fru

FLP

Statistical significance determined by one-way ANOVA followed by

1282

Tukey’s multiple comparisons test. p < 0.0001 (one-way ANOVA, F = 18.41, R² = 0.24;

1283

Tukey's post hoc). Sample sizes (n) are indicated in the figure panels. Data are

1284

represented as mean +/- SEM.

1285

(E) MD assays of flies expressing the

SIFaR

R57F10

-GAL4

driver together with

UAS-stop-

1286

TNT; fru

FLP

. Statistical significance determined by one-way ANOVA followed by

1287

Tukey’s multiple comparisons test. p = 0.3654 (one-way ANOVA, F = 1.01, R² = 0.015;

1288

Tukey's post hoc). The ns represents non-significant differences. Sample sizes (n) are

1289

indicated in the figure panels. Data are represented as mean +/- SEM.

1290

(F) CL of group-housed males expressing

empty-GAL4

together with

UAS-stop-TNT;

1291

fru

FLP

and males expressing

SIFaR

R23G06

-GAL4, SIFaR

R24A12

-GAL4, SIFaR

R24F06

-GAL4

1292

and

SIFaR

R57F10

-GAL4

driver together with

UAS-stop-TNT; fru

FLP

. Statistical

1293

significance determined by one-way ANOVA followed by Tukey’s multiple

1294

comparisons test. p = 0.0778 (one-way ANOVA, F = 2.11, R² = 0.012; Tukey's post

1295

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1296

the figure panels. Data are represented as mean +/- SEM.

1297

Journal Pre-proof

(G) Male(upper) and female(below) flies expressing

SIFaR

R57F10

and

fru

FLP

together

1298

with

UAS-RedStinger

and

UAS-stop-mCD8GFP

were immunostained with anti-GFP

1299

(green), anti-RFP (red), nc82 (blue) antibodies. The left two panels are presented as a

1300

gray scale to clearly show the GFP and RFP signal. Scale bars represent 50 µm.

1301

(H-L) Quantification of GFP fluorescence (H and I) and RFP fluorescence (E and J-L)

1302

in the male and female fly CB expressing

SIFaR

R57F10

and

fru

FLP

together with

UAS-

1303

RedStinger

and

UAS-stop-mCD8GFP

. (I) Quantification of GFP fluorescence in male

1304

and female CB. Bars represent the mean GFP fluorescence level with error bars

1305

representing SEM. Asterisks represent significant differences, as revealed by the

1306

Student’s t test and ns represents non-significant difference (

*p<0.05, **p<0.01,

1307

***p< 0.001

). The same symbols for statistical significance are used in all other Figures.

1308

See the

STAR Methods

for a detailed description of the colocalization analysis used in

1309

this study. (J) Quantification of relative value for RFP intensity between male and

1310

female flies. See the

STAR Methods

for a detailed description of the fluorescence

1311

intensity analysis used in this study. (K-L) Quantification of particle number (K) and

1312

cell size (L) between male and female flies. See the

STAR Methods

for a detailed

1313

description of the particle analysis used in this study. Sample sizes (n) are indicated in

1314

the figure panels. Data are represented as mean +/- SEM.

1315

1316

Fig.5 Fru-specific SIFaR expression in P1 and mAL neurons modulates mating

1317

duration

1318

(A) Male flies expressing

SIFaR

T2A

and

fru

FLP

together with

UAS-stop-mCD8GFP

were

1319

Journal Pre-proof

immunostained with anti-GFP (yellow), nc82 (blue) antibodies.

1320

(B) Male flies expressing

SIFaR

T2A

and

dsx

FLP

together with

UAS-stop-mCD8GFP

were

1321

immunostained with anti-GFP (yellow), nc82 (blue) antibodies.

1322

fruP1-GAL4

driver together with

SIFaR-RNAi

1323

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1324

comparisons test. p = 0.9192 (one-way ANOVA, F = 0.084, R² = 0.0019; Tukey's post

1325

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1326

the figure panels. Data are represented as mean +/- SEM.

1327

(D) MD assays of flies expressing the

GAL4

R43D01

driver together with

SIFaR-RNAi

1328

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1329

comparisons test. p < 0.0001 (one-way ANOVA, F = 44.07, R² = 0.34; Tukey's post

1330

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1331

the figure panels. Data are represented as mean +/- SEM.

1332

(E) MD assays of flies expressing the

fruP1-GAL4

driver together with

empty-RNAi

1333

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1334

comparisons test. p < 0.0001 (one-way ANOVA, F = 49.56, R² = 0.38; Tukey's post

1335

hoc). Sample sizes (n) are indicated in the figure panels. Data are represented as mean

1336

+/- SEM.

1337

(F) MD assays of flies expressing the

GAL4

R43D01

driver together with

empty -RNAi

1338

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1339

comparisons test. p < 0.0001 (one-way ANOVA, F = 47.72, R² = 0.41; Tukey's post

1340

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1341

Journal Pre-proof

the figure panels. Data are represented as mean +/- SEM.

1342

(G) Different levels of neural activity of the brain as revealed by the CaLexA system in

1343

naïve, single and experienced flies. Male flies expressing

fruP1

GAL4

along with

1344

LexAop-CD2-GFP, UAS-mLexA-VP16-NFAT and LexAop-CD8-GFP-A2-CD8-GFP

1345

were dissected after 5 days of growth (mated male flies had 1-day of sexual experience

1346

with virgin females). The dissected brains were then immunostained with anti-GFP

1347

(green) and anti-nc82 (blue). GFP is pseudo-colored as “red hot”. Scale bars represent

1348

50 μm.

1349

(H) Quantification of relative intensity value for GFP fluorescence. Statistical

1350

significance determined by one-way ANOVA followed by Tukey’s multiple

1351

comparisons test. p < 0.0001 (one-way ANOVA, F = 97.94, R² = 0.93; Tukey's post

1352

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1353

the figure panels. Data are represented as mean +/- SEM.

1354

(I) Diagram of interconnection between SIFaR and fru/dsx neurons along with

1355

interconnected networks formed by GAL4

24F06

1356

(J) CI of group-housed males expressing

fruP1-GAL4

together with

empty-RNAi

1357

SIFaR-RNAi

and males expressing

GAL4

R43D01

with

empty-RNAi

SIFaR-RNAi

1358

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1359

comparisons test. p < 0.0001 (one-way ANOVA, F = 70.43, R² = 0.30; Tukey's post

1360

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1361

the figure panels. Data are represented as mean +/- SEM.

1362

(K) CL of group-housed males expressing

fruP1-GAL4

together with

empty-RNAi

1363

Journal Pre-proof

SIFaR-RNAi

and males expressing

GAL4

R43D01

with

empty-RNAi

SIFaR-RNAi

1364

Statistical significance determined by one-way ANOVA followed by Tukey’s multiple

1365

comparisons test. p = 0.2605 (one-way ANOVA, F = 1.34, R² = 0.007; Tukey's post

1366

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1367

the figure panels. Data are represented as mean +/- SEM.

1368

1369

Fig.6 Synaptic alterations in mAL neurons correlate with social contexts

1370

(A) The SIFaR

/fru

presynaptic terminals visualized by males expressing

SIFaR

1371

GAL4, fru

FLP

together with

UAS-stop-Dscam-GFP

in naïve, single and exp. male flies.

1372

Scale bars represent 100 µm.

1373

(B) Quantification of relative value for presynaptic terminals intensity. Statistical

1374

significance determined by one-way ANOVA followed by Tukey’s multiple

1375

comparisons test. p < 0.0001 (one-way ANOVA, F = 70.55, R² = 0.92; Tukey's post

1376

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1377

the figure panels. Data are represented as mean +/- SEM.

1378

/fru

postsynaptic terminals visualized by males expressing

SIFaR

1379

GAL4, fru

FLP

together with

UAS-stop-nSyb-GFP

in naïve, single and exp. male flies.

1380

Scale bars represent 100 µm.

1381

(D) Quantification of relative value for postsynaptic terminals intensity. Statistical

1382

significance determined by one-way ANOVA followed by Tukey’s multiple

1383

comparisons test. p < 0.0001 (one-way ANOVA, F = 59.83, R² = 0.91; Tukey's post

1384

hoc). The ns represents non-significant differences. Sample sizes (n) are indicated in

1385

Journal Pre-proof

the figure panels. Data are represented as mean +/- SEM.

1386

(E) The synaptic interactions visualized utilizing the tGRASP system in naïve, single

1387

and exp. male flies.

1388

(F) Quantification of synaptic puncta formed between

SIFa

lexA.PT

and

GAL4

R43D01

1389

brain between naïve, single and exp. male flies. Statistical significance determined by

1390

one-way ANOVA followed by Tukey’s multiple comparisons test. p < 0.0001 (one-way

1391

ANOVA, F = 202.1, R² = 0.96; Tukey's post hoc). The ns represents non-significant

1392

differences. Sample sizes (n) are indicated in the figure panels. Data are represented as

1393

mean +/- SEM.

1394

(G-I) mAL neurons don't respond to SIFas activation. Fluorescence changes (Δ

) of

1395

GCaMP7s in mALs after optogenetic stimulation of SIFas (two-tailed unpaired

-test).

1396

In all plots and statistical tests. Data are presented as mean ± s.e.m. ns = not significant

1397

(

p>0.05

*p<0.05, **p<0.01, ***p< 0.001, ****p< 0.0001

. DBMs represent the

1398

'difference between means' for the evaluation of estimation statistics (See

STAR

1399

Methods

). Sample sizes (n) are indicated in the figure panels. Data are represented as

1400

mean +/- SEM. LED light was fired 2 s after 30 s of dark. Red arrows indicate the time

1401

point of light stimulation.

= 4 to 9 in each group. Red lines and bars indicate

1402

experimental group fed with ATR; black lines and bars indicate control group fed

1403

without ATR. See the

STAR Methods

for a detailed description of the two-photon

1404

calcium imaging used in this study.

1405

1406

Table 1. Genotypes of Flies Used for Experiments in This Study.

1407

Journal Pre-proof

Figure panel

Genotype

Fig. 1A

GAL4

SIFa. PT

/ UAS-GFP. nls

GAL4

SIFa. PT

/ UAS-TNT

GAL4

SIFa. PT

/ UAS-KCNJ2

GAL4

SIFa. PT

/ UAS-OrkΔC

GAL4

SIFa. PT

/ UAS-NaChBac

Fig. 1B

GAL4

empty

/ SIFa-RNAi

GAL4

SIFa. PT

/ empty-RNAi

GAL4

SIFa. PT

/ SIFa-RNAi

Fig. 1C

Same as Fig. 1A

Fig. 1D

Same as Fig. 1B

Fig. 1E

GAL4

SIFa. PT

/ empty-RNAi

GAL4

SIFa. PT

/ Chat-RNAi

GAL4

SIFa. PT

/ Gad1-RNAi

GAL4

SIFa. PT

/ VGlut-RNAi

GAL4

SIFa. PT

/ ple-RNAi

GAL4

SIFa. PT

/ Tdc2-RNAi

GAL4

SIFa. PT

/ Trhn-RNAi

Fig. 1F

Same as Fig. 1E

Fig. 1G

GAL4

SIFa. PT

/ empty-RNAi

GAL4

SIFa. PT

/ CrzR-RNAi

GAL4

SIFa. PT

/ CCHa1-R-RNAi

GAL4

SIFa. PT

/ InR-RNAi

GAL4

SIFa. PT

/ Lgr3-RNAi

GAL4

SIFa. PT

/ Lgr4-RNAi

GAL4

SIFa. PT

/ PK2-R1-RNAi

GAL4

SIFa. PT

/ PK2-R2-RNAi

Journal Pre-proof

GAL4

SIFa. PT

/ CCKLR17D1-RNAi

GAL4

SIFa. PT

/ CCKLR17D3-RNAi

GAL4

SIFa. PT

/ sNPR-R-RNAi

Fig. 1H

Same as Fig. 1G

Fig. 2A

GAL4

empty

/ SIFaR-RNAi

repo-GAL4 / SIFaR-RNAi

nSyb-GAL4 / SIFaR-RNAi

Fig. 2B

Same as Fig. 2A

Fig. 2C

fru-GAL4/ empty-RNAi

fru-GAL4 / SIFaR-RNAi

dsx-GAL4 / empty-RNAi

dsx-GAL4 / SIFaR-RNAi

Fig. 2D

Same as Fig. 2C

Fig. 2E

GAL4

empty

/ UAS-TNT, tub-GAL80

SIFaR

24F06

/ UAS-TNT, tub-GAL80

SIFaR

/ UAS-GFP

SIFaR

/ UAS-TNT, tub-GAL80

GAL4

empty

/ UAS-stop-TNT, fru

FLP

SIFaR

/ UAS-stop-TNT, fru

FLP

Fig. 2F

Same as Fig. 2E

Fig. 3A

fru-GAL4 / SIFaR-RNAi

Fig. 3B

fru-GAL4 / empty-RNAi

Fig. 3C

fru-GAL4 / AstC-R1-RNAi

Fig. 3C

fru-GAL4 / AstC-R2-RNAi

Fig. 3E

elav

c155

/ SIFaR-RNAi

elav

c155

/ AstC-RNAi

elav

c155

/ SIFa-RNAi

Journal Pre-proof

Fig. 3F

elav

c155

/ empty-RNAi

elav

c155

/ AstC-R1-RNAi

elav

c155

/ AstC-R2-RNAi

elav

c155

/ SIFaR-RNAi

Fig. 3G

SIFaR

-GAL4, fru-lexA/ UAS-Stinger

lexAop-tdTomato.nls

Fig. 4B

SIFaR

R23G06

/ UAS-stop-TNT, fru

FLP

Fig. 4C

SIFaR

R24A12

/ UAS-stop-TNT, fru

FLP

Fig. 4D

SIFaR

R24F06

/ UAS-stop-TNT, fru

FLP

Fig. 4E

SIFaR

R57F10

/ UAS-stop-TNT, fru

FLP

Fig. 4F

GAL4

empty

/ UAS-stop-TNT, fru

FLP

SIFaR

R23G06

/ UAS-stop-TNT, fru

FLP

SIFaR

R24A12

/ UAS-stop-TNT, fru

FLP

SIFaR

R24F06

/ UAS-stop-TNT, fru

FLP

SIFaR

R57F10

/ UAS-stop-TNT, fru

FLP

Fig. 4G-L

SIFaR

R57F10

, fru

FLP

/ UAS-stop-mCD8GFP, UAS-RedStinger

Fig. 5A

SIFaR

, fru

FLP

/ UAS-stop-mCD8GFP

Fig. 5B

SIFaR

, dsx

FLP

/ UAS-stop-mCD8GFP

Fig. 5C

fruP1-GAL4/ SIFaR-RNAi

Fig. 5D

GAL4

R43D01

/ SIFaR-RNAi

Fig. 5E

fruP1-GAL4/ empty-RNAi

Fig. 5F

GAL4

R43D01

/ empty-RNAi

Fig. 5G-H

GAL4

fruP1

/ lexAop-CD2-GFP, UAS-mLexA-VP16-NFAT,

lexAop-CD8-GFP-A2-CD8-GFP

Fig. 5J-K

fruP1-GAL4/ empty-RNAi

fruP1-GAL4/ SIFaR-RNAi

GAL4

R43D01

/ empty-RNAi

GAL4

R43D01

/ SIFaR-RNAi

Journal Pre-proof

Fig. 6A-B

SIFaR

, fru

FLP

/ UAS-stop-Dscam-GFP

Fig. 6C-D

SIFaR

, fru

FLP

/ UAS-stop-nSyb-GFP

Fig. 6E-F

SIFa

lexA.PT

, GAL4

R43D01

UAS-post-t-GRASP, lexAop2-pre-t-

GRASP

Fig. 6G-I

SIFa

lexA.PT

, GAL4

R43D01

/lexAop-CsChrimson, UAS-GCaMP7s

Fig. S2A-C

GAL4

empty

/ UAS-stop-TNT, fru

FLP

SIFaR

/ UAS-stop-TNT, fru

FLP

Fig. S3A-D

GAL4

empty

/ AstC-R1-RNAi

GAL4

empty

/ AstC-R2-RNAi

dsx-GAL4 / empty-RNAi

GAL4

empty

/ SIFaR-RNAi

Fig. S4A-F

GAL4

empty

/ UAS-stop-TNT, fru

FLP

SIFaR

R23G06

/ UAS-stop-TNT, fru

FLP

SIFaR

R24A12

/ UAS-stop-TNT, fru

FLP

SIFaR

R24F06

/ UAS-stop-TNT, fru

FLP

SIFaR

R57F10

/ UAS-stop-TNT, fru

FLP

Fig. S4G

SIFaR

R57F10

, fru

FLP

/ UAS-stop-mCD8GFP, UAS-RedStinger

Fig. S5A

fruP1-GAL4, SIFaR

R24F06

-lexA/

UAS-mCD8RFP, lexAop-

mCD8GFP

Fig. S5B

fruP1-GAL4,

SIFaR

T2A

-lexA/

UAS-mCD8RFP,

lexAop-

mCD8GFP

Fig. S5C

fruP1-GAL4, Tsh-GAL80/SIFaR-RNAi

Fig. S5D-E

fruP1-GAL4, Tsh-GAL80/empty-RNAi

fruP1-GAL4, Tsh-GAL80/SIFaR-RNAi

Fig. S5F

GAL4

R43D01

, SIFaR

R24F06

-lexA/

UAS-mCD8RFP,

lexAop-

mCD8GFP

Fig. S5G

GAL4

R43D01

/lexAop-CD2-GFP,

UAS-mLexA-VP16-NFAT,

Journal Pre-proof

lexAop-CD8-GFP-A2-CD8-GFP

Fig. S5H-I

Tk-GAL4 / SIFaR-RNAi

Fig. S5J-K

SIFaR-GAL4 / Tk-RNAi

Fig. S6A

SIFaR

-lexA, GAL4

R43D01

/ UAS-Stinger

lexAop-tdTomato.nls

Fig. S6B

GAL4

SIFa.PT

, lexA

SIFa.PT

/UAS-mCD8RFP, lexAop-mCD8GFP

1408

STAR

★

METHODS

1409

KEY RESOURCES TABLE

1410

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Chicken polyclonal anti-GFP

Invitrogen

RRID: AB_2534023

Donkey Alexa-488 anti-chicken

Jackson

ImmunoResearch

RRID: AB_2340375

Goat Alexa-555 anti-rabbit

Invitrogen

RRID: AB_2535849

Goat Alexa-647 anti-mouse

Jackson

ImmunoResearch

RRID: AB_2338902

Mouse polyclonal anti-nc82

Developmental Studies

Hybridoma Bank

RRID: AB_2314866

Rabbit polyclonal anti-RFP

Rockland

Immunochemicals

RRID: AB_2209751

Bacterial and virus strains

pBPLexA::p65Uw

Gerald Rubin

Addgene 26231

E2 Enhancer-lexA

Qidong Fungene

Biotechnology

N/A

DH5α Chemically Competent Cell

Weidibio

DL1001

Chemicals, peptides, and recombinant proteins

Triton X-100

Alphabio

A2576

4% Polyformaldehyde

Alphabio

A1283

Journal Pre-proof

Mounting solution

Solarbio

S2100-5

PBS

Solarbio

P1022-500

Critical commercial assays

FastDigest EcoRI

Thermo Scientific

FD0274

XbaI

NEB

R0145S

DNAzol BD

Invitrogen

10974020

PrimeSTAR HS DNA Polymerase

Takara

R010A

Light Cloning kit

Biodragon (China)

BDIT0014

Experimental models: Organisms/strains

D. melanogaster: Deficiency females:

Df(1)

Exel6234

Bloomington Drosophila

Stock Center

RRID: BDSC_7708

D. melanogaster: genetic control:

UAS-GFP.nls

Bloomington Drosophila

Stock Center

RRID: BDSC_4775

D. melanogaster: pan neuronal driver:

elav

c155

; UAS-Dicer

Bloomington Drosophila

Stock Center

RRID:

BDSC_25750

D. melanogaster: CalexA system:

lexAop-CD8GFP; UAS-mLexA-VP16-

NFAT, lexAop-rCD2-GFP

Bloomington Drosophila

Stock Center

RRID:

BDSC_66542

D. melanogaster: tGRASP system:

UAS-post-t-GRASP, lexAop2-pre-t-

GRASP

Bloomington Drosophila

Stock Center

RRID:

BDSC_79039

D. melanogaster:SIFa-RNAi:

SIFa-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_60484

D. melanogaster: SIFa.PT-GAL4 driver:

SIFa-GAL4

Jan A Veenstra

N/A

D. melanogaster: stop-GFP marker:

UAS-FRT-stop-FRT-mCD8GFP

Bloomington Drosophila

Stock Center

RRID:

BDSC_30125

D. melanogaster: SIFa.PT-lexA driver:

SIFa-lexA

This paper

STaR Methods

Journal Pre-proof

D. melanogaster: pan glial driver:

repo-GAL4

Bloomington Drosophila

Stock Center

RRID: BDSC_7415

D. melanogaster: control GAL4:

empty-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_68384

D. melanogaster: control RNAi:

empty-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_36304

D. melanogaster: SIFaR-23G06-GAL4

driver:

GAL4

23G06

Bloomington Drosophila

Stock Center

RRID:

BDSC_49041

D. melanogaster: SIFaR-24A12-GAL4

driver:

GAL4

24A12

Bloomington Drosophila

Stock Center

RRID:

BDSC_49061

D. melanogaster: SIFaR-24F06-GAL4

driver:

GAL4

24F06

Bloomington Drosophila

Stock Center

RRID:

BDSC_49087

D. melanogaster: SIFaR-57F10-GAL4

driver:

GAL4

57F10

Bloomington Drosophila

Stock Center

RRID:

BDSC_46391

D. melanogaster: SIFaR-T2A-GAL4

driver:

SIFaR-GAL4

T2A

Qidong Fungene

Biotechnology

RRID: FBF00102

D. melanogaster: SIFaR-24F06-lexA

driver:

lexA

24F06

Bloomington Drosophila

Stock Center

RRID:

BDSC_52695

D. melanogaster: SIFaR-T2A-lexA

driver:

SIFaR-lexA

T2A

Qidong Fungene

Biotechnology

RRID: FBF00086

D. melanogaster: Open rectifier K

channel 1:

UAS-Ork1.Delta-C

Bloomington Drosophila

Stock Center

RRID: BDSC_6586

Journal Pre-proof

D. melanogaster: ple-RNAi:

ple-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25796

D. melanogaster: NaChBac:

UAS-NaChBac

Bloomington Drosophila

Stock Center

RRID: BDSC_9467

D. melanogaster: ChaT-RNAi

ChaT-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25856

D. melanogaster: double sex driver:

dsx-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_30027

D. melanogaster: Gad1-RNAi:

Gad1-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_51794

D. melanogaster: nucleus fluorescent

marker:

UAS-RedStinger

Bloomington Drosophila

Stock Center

RRID: BDSC_8546

D. melanogaster: Nucleus fluorescent

marker:

UAS-Stinger, lexAop-tdTomato.nls

Bloomington Drosophila

Stock Center

RRID:

BDSC_66680

D. melanogaster: CrzR-RNAi:

CrzR-RNAi

JF02042

Bloomington Drosophila

Stock Center

RRID:

BDSC_26017

D. melanogaster: VGlut-RNAi:

VGlut-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_27538

D. melanogaster: Lgr4-RNAi:

Lgr4-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_28655

D. melanogaster: potassium channel

activator:

UAS-KCNJ2

Bloomington Drosophila

Stock Center

RRID: BDSC_6596

D. melanogaster: membrane marker:

UAS-mCD8RFP, lexAop-mCD8GFP

Bloomington Drosophila

Stock Center

RRID:

BDSC_32229

D. melanogaster: chemical synaptic

transmission blocker:

UAS-TNT

Bloomington Drosophila

Stock Center

RRID:

BDSC_28838

Journal Pre-proof

D. melanogaster: Lgr3-RNAi:

Lgr3-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_28789

D. melanogaster: CCKLR-17D3-RNAi:

CCKLR-17D3-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_28333

D. melanogaster: CCKLR-17D1-RNAi:

CCKLR-17D1-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_27494

D. melanogaster: sNPF-RNAi:

sNPF-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_27507

D. melanogaster: fruitless driver:

fru-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_30027

D. melanogaster: fruitless driver:

fru-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_30027

D. melanogaster: fruitless driver:

fru-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_30027

D. melanogaster: temperature sensitive

tub-GAL80:

tub-GAL80

Bloomington Drosophila

Stock Center

RRID: BDSC_7017

D. melanogaster: fruP1 driver:

fruP1-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_66696

D. melanogaster: fru-FLP:

fru-FLP

Bloomington Drosophila

Stock Center

RRID:

BDSC_66870

D. melanogaster: UAS-stop-TNT:

UAS-stop-TNT

Bloomington Drosophila

Stock Center

RRID:

BDSC_28842

D. melanogaster: UAS-stop-TNT

inactive form:

UAS-stop-TNT

Bloomington Drosophila

Stock Center

RRID:

BDSC_28844

D. melanogaster: AstC-R2-RNAi:

AstC-R2-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25940

D. melanogaster: mAL driver:

R43D01-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_64345

Journal Pre-proof

D. melanogaster: Tk driver:

Tk-GAL4

Bloomington Drosophila

Stock Center

RRID:

BDSC_93412

D. melanogaster: Trhn-RNAi:

Trhn-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25842

D. melanogaster: Tdc2-RNAi:

Tdc2-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25871

D. melanogaster: Tk-RNAi:

Tk-RNAi

Bloomington Drosophila

Stock Center

RRID:

BDSC_25800

D. melanogaster: CCHa1-R-RNAi:

CCHa1-R-RNAi

Vienna

Drosophila

Resource Center

RRID:

VDRC_103055

D. melanogaster: InR-RNAi:

InR-RNAi

Vienna

Drosophila

Resource Center

RRID: VDRC_991

D. melanogaster: PK2-R1-RNAi:

PK2-R1-RNAi

Vienna

Drosophila

Resource Center

RRID:

VDRC_103822

D. melanogaster: PK2-R2-RNAi:

PK2-R2-RNAi

Vienna

Drosophila

Resource Center

RRID:

VDRC_44871

D. melanogaster: AstC-R1-RNAi:

AstC-R1-RNAi

Vienna

Drosophila

Resource Center

RRID:

VDRC_13560

D. melanogaster: nSyb GFP marker:

UAS-stop-nSyb-GFP

Young-Joon Kim

N/A

D. melanogaster: Dscam GFP marker:

UAS-stop-Dscam17.1-EGFP

Young-Joon Kim

N/A

Oligonucleotides

Primer: SIFa Forward:

GCCAATTGGCTGAATCTCCTGACCCT

Jan A Veenstra

N/A

Primer: SIFa Reverse:

GCAGATCTCTTGCAGTTTTCGGTGAG

Jan A Veenstra

N/A

Journal Pre-proof

Software and algorithms

Fiji/ImageJ

Schindelin et al., 2012

https://fiji.sc

Prism 9.0

GraphPad

https://www.graphp

ad.com

Adobe illustrator 2020

Adobe.com

N/A

SCOPE

Fly Cell Atlas

https://scope.aertsl
ab.org/#/FlyCellAtla
s/*/welcome

Seurat

Yuhan et al., 2023

https://github.com/s

atijalab/seurat

R v4.2.2

R Core Team

https://www.R-

project.org/

QuillBot AI

QuillBot Core Team

https://quillbot.com/

Microsoft Office Excel

Microsoft

https://www.micros

oft.com/en-

au/microsoft-

365/excel

1411

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

1412

Drosophila

1413

Drosophila melanogaster were cultured under standard laboratory conditions at

1414

℃

. Samples were prepared as described in the Methods Details. All fly strains

1415

are listed in the key resources table.

1416

1417

METHOD DETAILS

1418

Fly Stocks and Husbandry

1419

Drosophila melanogaster

were raised on cornmeal-yeast medium at similar densities to

1420

Journal Pre-proof

yield adults with similar body sizes. For 20 liters of fly food, the composition includes

1421

188 grams of agar, 262 grams of inactive yeast, 930 grams of maize flour, 120 grams

1422

of soy flour, 1400 milliliters of molasses, 140 milliliters of Tegosept solution, 50

1423

milliliters of propionic acid, and 10 milliliters of phosphoric acid. Flies were kept in 12

1424

h light: 12 h dark cycles (LD) at 25℃ (ZT 0 is the beginning of the light phase at 8:00

1425

AM, ZT 12 is the beginning of the dark phase at 8:00 PM) except for some experimental

1426

manipulation. For temperature-controlled experiments, including those utilizing the

1427

temperature-sensitive tub-GAL80

driver, the flies were initially crossed and

1428

maintained at a constant temperature of 22℃ within an incubator. The temperature shift

1429

was initiated post-eclosion. Once the flies had emerged, they were transferred to an

1430

incubator set at an elevated temperature of 29℃ for a defined period, after which the

1431

experimental protocols were carried out. Wild-type flies were

Canton-S

(

). The

1432

genotypes of the flies used in each figure were listed in Table 1.

1433

1434

Validation of RNAi Lines

1435

All RNAi lines used in this study were selected based on prior validation of their

1436

efficacy and specificity in published work. This includes all lines targeting

SIFa-

1437

RNAi

, SIFaR-RNAi

, Chat-RNAi

, VGlut-RNAi

, CrzR-RNAi

, ple-RNAi

, Gad1-

1438

RNAi

, Tdc2-RNAi

Trhn-RNAi

, Lgr4-RNAi

111

, Lgr3-RNAi

111

, CCKLR-17D3-

1439

RNAi

, CCKLR-17D1-RNAi

, sNPFR-RNAi

, Tk-RNAi

112

, AstC-R2-RNAi

1440

CCHa1-R-RNAi

100

, InR-RNAi

101

, PK2-R1-RNAi

102

, PK2-R2-RNAi

103

and

AstC-R1-

1441

RNAi

104

. These independent studies confirmed target gene knockdown using methods

1442

Journal Pre-proof

including qRT-PCR, Western blotting, and/or phenotypic rescue with multiple RNAi

1443

strains. The control genotype (

empty-RNAi

105

) was similarly validated to lack non-

1444

specific effects.

1445

1446

Courtship Assays for Copulation Latency (CL) and Courtship Index (CI)

1447

Courtship assay was performed as previously described

113

under normal light

1448

conditions in circular courtship arenas 11 mm in diameter, from noon to 4 pm. We

1449

utilized the time at which copulation (mating) was initiated to quantify copulation

1450

latency (CL). The statistical methodology applied for the analysis of CL is identical to

1451

that employed for the MD assay. Upon the onset of courtship behavior, the courtship

1452

index (CI) was computed as the proportion of time a male dedicated to courtship-

1453

related behaviors within a 10-minute observation window or until the initiation of

1454

mating. The initiation of mating is defined as the moment at which male flies

1455

successfully achieve mounting on females. All copulation latency (CL) and mating

1456

behavior assays were performed using

SPR

-/-

null mutant females,

Df(1)

exel6234

1457

standardize female receptivity

. This approach eliminates variation from female

1458

rejection behaviors, constitutively elevates baseline receptivity (>95% acceptance

1459

rate, and enables isolation of male-specific contributions to CL.

1460

1461

Mating Duration Assays

1462

The mating duration assay in this study has been reported

45,46,52

. To enhance the

1463

efficiency of the mating duration assay, we utilized the

Df(1)

Exel6234

(DF here after)

1464

Journal Pre-proof

genetic modified fly line in this study, which harbors a deletion of a specific genomic

1465

region that includes the sex peptide receptor (SPR)

114,115

. Previous studies have

1466

demonstrated that virgin females of this line exhibit increased receptivity to males

115

1467

We conducted a comparative analysis between the virgin females of this line and the

1468

CS virgin females and found that both groups induced SMD. Consequently, we have

1469

elected to employ virgin females from this modified line in all subsequent studies. For

1470

group reared (naïve) males, 40 males from the same strain were placed into a vial with

1471

food for 5 days. For single reared males, males of the same strain were collected

1472

individually and placed into vials with food for 5 days. For experienced males, 40

1473

males from the same strain were placed into a vial with food for 4 days then 80 DF

1474

virgin females were introduced into vials for last 1 day before assay. 40 DF virgin

1475

females were collected from bottles and placed into a vial for 5 days. These females

1476

provide both sexually experienced partners and mating partners for mating duration

1477

assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin

1478

females were mildly anaesthetized by CO

. After placing a single female into the

1479

mating chamber, we inserted a transparent film then placed a single male to the other

1480

side of the film in each chamber. After allowing for 1 h of recovery in the mating

1481

chamber in 25℃ incubators, we removed the transparent film and recorded the mating

1482

activities. Only those males that succeeded to mate within 1 h were included for

1483

analyses. Initiation and completion of copulation were recorded with an accuracy of

1484

10 sec, and total mating duration was calculated for each couple

40,93,94,97,98

. Genetic

1485

controls for LMD and SMD behaviors were incorporated exclusively when assessing

1486

Journal Pre-proof

novel fly strains that had not previously been examined. In essence, internal controls

1487

were predominantly employed in the experiments, as LMD and SMD behaviors

1488

exhibit enhanced statistical significance when internally controlled. Within the LMD

1489

assay, both group and single conditions function reciprocally as internal controls. A

1490

significant distinction between the naïve and single conditions implies that the

1491

experimental manipulation does not affect LMD. Conversely, the lack of a significant

1492

discrepancy suggests that the manipulation does influence LMD. In the context of

1493

SMD experiments, the naïve condition (equivalent to the group condition in the LMD

1494

assay) and sexually experienced males act as mutual internal controls for one another.

1495

A statistically significant divergence between naïve and experienced males indicates

1496

that the experimental procedure does not alter SMD. Conversely, the absence of a

1497

statistically significant difference suggests that the manipulation does impact SMD.

1498

Hence, we incorporated supplementary genetic control experiments solely if they

1499

deemed indispensable for testing. All assays were performed from noon to 4 PM. We

1500

conducted blinded studies for every test.

1501

1502

Quantification and Interpretation of Mating Duration Behaviors

1503

Long Mating Duration (LMD): Males exposed to rivals exhibit significantly

1504

prolonged MD compared to socially naive control males (no rival exposure). Intact

1505

LMD is defined as a statistically significant increase (p < 0.05) in MD in rival-

1506

exposed males versus controls

45–48,50

1507

Journal Pre-proof

Short Mating Duration (SMD): Sexually experienced males exhibit significantly

1508

reduced MD compared to sexually naive control males. Intact SMD is defined as a

1509

statistically significant decrease (p < 0.05) in MD in experienced males versus

1510

controls

51,52,116–118

1511

Intact Behavior Criteria: LMD or SMD is considered

intact

when rival-exposed males

1512

show a statistically significant increase (p < 0.05) in MD versus controls (LMD),

1513

experienced males show a statistically significant decrease (p < 0.05) in MD versus

1514

controls (SMD).

1515

Abolished Behavior Criteria: LMD or SMD is considered

abolished

when

1516

experimental manipulations eliminate the adaptive duration difference, resulting in no

1517

statistically significant difference (p ≥ 0.05) in MD between rival-exposed males

1518

versus controls (LMD),

experienced males versus controls (SMD).

1519

Note: "Abolished" indicates loss of context-dependent mating duration modulation;

1520

mating initiation and completion persist in all groups.

1521

1522

Generation of Transgenic Flies

1523

To generate the

SIFa

-lexA

driver, the putative promotor sequence of the gene was

1524

amplified by PCR using wild-type genomic DNA as a template with the following

1525

primers GCCAATTGGCTGAATCTCCTGACCCTCA and

1526

GCAGATCTCTTGCAGTTTTCGGTGAGC as mentioned before

119

. The amplified

1527

DNA fragment (1482 base pairs located immediately upstream of the

SIFa

coding

1528

sequence) was inserted into the E2 Enhancer-lexA vector. This vector, supplied by

1529

Journal Pre-proof

Qidong Fungene Biotechnology Co., Ltd. (http://www.fungene.tech/), is a derivative

1530

of the pBPLexA::p65Uw vector (available at https://www.addgene.org/26231). The

1531

insertion was achieved by digesting the fragment and the vector with EcoRI and XbaI

1532

restriction enzymes to create compatible sticky ends. The genetic construct was

1533

inserted into the

attp2

site on chromosome III to generate transgenic flies using

1534

established techniques, a service conducted by Qidong Fungene Biotechnology Co.,

1535

Ltd.

1536

1537

Immunostaining

1538

The dissection and immunostaining protocols for the experiments are described

1539

elsewhere

. After 5 days of eclosion, the

Drosophila

brain was taken from adult flies

1540

and fixed in 4% formaldehyde at room temperature for 30 minutes. The sample was

1541

washed three times (5 minutes each) in 1% PBT and then blocked in 5% normal goat

1542

serum for 30 minutes. The sample next be incubated overnight at 4

℃

with primary

1543

antibodies in 1% PBT, followed by the addition of fluorophore-conjugated secondary

1544

antibodies for one hour at room temperature. The brain was mounted on plates with an

1545

antifade mounting solution (Solarbio) for imaging purposes.

1546

1547

Confocal imaging was conducted utilizing a ZEISS LSM880 microscope. GFP

1548

detection was accomplished through immunostaining with chicken anti-GFP primary

1549

antibody (1:500; A10262, Invitrogen) and Alexa Fluor 488-conjugated donkey anti-

1550

chicken IgG secondary antibody (1:200, Jackson ImmunoResearch), facilitating the

1551

Journal Pre-proof

visualization of: (i) native GFP expression, (ii) CalexA GFP signals, and (iii) t-GRASP

1552

reconstitution GFP signals based on previous studies

120–122

. For anatomical reference

1553

and supplementary labeling, the following antibodies were used: (i) mouse anti-

1554

Bruchpilot (nc82; 1:50, DSHB) detected by Alexa Fluor 647-conjugated goat anti-

1555

mouse IgG (1:200, Jackson ImmunoResearch) to accentuate neuropil architecture, and

1556

(ii) rabbit anti-RFP (1:500, Rockland Immunochemicals) with Alexa Fluor 555-

1557

conjugated goat anti-rabbit IgG (1:200, Invitrogen) for RFP detection.

1558

1559

Quantitative Analysis of Fluorescence Intensity

1560

To ascertain calcium levels and synaptic intensity from microscopic images, we

1561

dissected and imaged five-day-old flies of various social conditions and genotypes

1562

under uniform conditions. For group reared (naïve) flies, the flies were reared in group

1563

condition and dissect right after 5 days of rearing without any further action. For single

1564

reared flies, the flies were reared in single condition and dissect at the same time as

1565

group reared flies right after 5 days of rearing without any further action. For sexual

1566

experienced flies, the flies were reared in group condition after 4 days of rearing and

1567

will be given virgins to give them sexual experience for one day, those flies will also be

1568

dissected at the same time as group and single reared flies after one day. The GFP signal

1569

in the brains and VNCs was amplified through immunostaining with chicken anti-GFP

1570

primary antibody. Image analysis was conducted using ImageJ software. For CaLexA

1571

signal quantification, we adhered to protocols detailed by Kayser et al.

123

, which

1572

involve measuring the ROI's GFP-labeled area by summing pixel values across the

1573

Journal Pre-proof

image stack. This method assumes that changes in the GFP-labeled area are indicative

1574

of alterations in the CaLexA signal, reflecting synaptic activity. ROI intensities were

1575

background-corrected by measuring and subtracting the fluorescent intensity from a

1576

non-specific adjacent area, as per Kayser et al.

123

. For the analysis of GRASP or

1577

tGRASP signals, a sub-stack encompassing all synaptic puncta was thresholded by a

1578

genotype-blinded investigator to achieve the optimal signal-to-noise ratio. The

1579

fluorescence area or ROI for each region was quantified using ImageJ, employing a

1580

similar approach to that used for CaLexA quantification

124

1581

1582

Colocalization Analysis

1583

Before the colocalization analysis, an investigator, blinded to the fly's genotype,

1584

thresholded the sum of all pixel intensities within a sub-stack to optimize the signal-to-

1585

noise ratio, following established methods

124

. To perform colocalization analysis of

1586

multi-color fluorescence microscopy images in this study, we employed ImageJ

1587

software

125

. In brief, we merged image channels to form a composite with accurate color

1588

representation and applied a threshold to isolate yellow pixels, signifying colocalization.

1589

The measured “area” values represented the colocalization zones between fluorophores.

1590

To determine the colocalization percentage relative to the total area of interest (e.g.,

1591

GFP), we adjusted thresholds to capture the full fluorophore areas and remeasured to

1592

obtain total areas. The quantification of the overlap was performed using confocal

1593

images with projection by standard deviation function provided by ImageJ to ensure

1594

precise measurements and avoid pixel saturation artifacts. The colocalization efficiency

1595

Journal Pre-proof

was calculated by dividing the colocalized area by the total fluorophore area. All

1596

samples were imaged uniformly.

1597

1598

Particle Analysis

1599

Before the particle analysis, an investigator, blinded to the fly's genotype, thresholded

1600

the sum of all pixel intensities within a sub-stack to optimize the signal-to-noise ratio,

1601

following established methods

124

. To quantitatively measure particle intensity of cell

1602

number and synaptic puncta in microscopic images, we applied ImageJ software.

1603

Initially, the image is converted to grayscale to reduce complexity and enhance contrast.

1604

Subsequently, thresholding techniques are employed to binarize the image,

1605

distinguishing particles from the background. This binarization can be achieved through

1606

automated thresholding algorithms or manual adjustment to optimize the segmentation.

1607

The results of these measurements are then available for review by conducting

1608

"Analyze Particles" function of ImageJ. All specimens were imaged under identical

1609

conditions.

1610

1611

Climbing Assay

1612

For climbing assay, we modified the conventional RING assay

126,127

. In brief, 40-50 20-

1613

day-aged flies were placed in an empty vial and were tapped to the bottom of the tube.

1614

After tapping of flies, we recorded 10 seconds of video clip. This experiment was done

1615

five times with 5-minute intervals. With recorded video files, we captured the position

1616

of flies 10 seconds after tapping the vial. This captured image file was then loaded in

1617

Journal Pre-proof

ImageJ to perform particle analysis. For quantifying the location of flies inside a vial,

1618

we used the “analyze particles” function of ImageJ

128

. The position of pixels was

1619

normalized by height of vial then only the particles above the midline (4 cm) of vial

1620

were counted.

1621

1622

Feeding of Retinal

1623

All trans-retinal powder (Sigma) was dissolved in EtOH as a 100 mM stock solution.

1624

200



l of this stock solution was diluted in 50 mL of melted normal food to prepare 400

1625



M of all trans-retinal (ATR) food. For CsChrimson experiments, male flies of proper

1626

genotype were selected and separated into two groups (with and without ATR) after 5

1627

days of eclosion for at least 3 days prior to any optogenetic experiments.

1628

1629

Two-photon Calcium Imaging

1630

To measure the neuron activity in AG region, the flies were carefully sedated with ice

1631

and then placed in an inverted posture on a plastic plate coated with UV glue, which

1632

securely held flies in the middle. Whole fly was exposed to AHL for red light activation.

1633

The plate was filled with

Drosophila

Adult Hemolymph-Like Saline (AHLS) buffer

1634

[108 mM NaCl, 5 mM KCl, 4 mM NaHCO

, 1 mM NaH

, 15 mM ribose, 5 mM

1635

Hepes (pH 7.5), 300 mosM, CaCl

(2 mM), and MgCl

(8.2 mM)] to ensure the flies’

1636

neurons remained in an active state throughout the experiment

26,129

. Subsequently, the

1637

flies were dissected to specifically expose the VNC for detailed examination. Calcium

1638

imaging was performed using Zeiss LSM880 microscope with a 20x water immersion

1639

Journal Pre-proof

objective. Images were acquired at 2 frames per second at a resolution of 512 × 512

1640

pixels. GCaMP7 slow (GCaMP7s) signals were recorded with an 880 nm laser and

1641

optogenetic stimulation was achieved with a 590-595 nm 20 W red light pulses at 50-

1642

60Hz (0.16mW/mm2), LED stimulation last 2 s. ROIs were manually selected from the

1643

cell body in AG area with ImageJ.

1644

1645

= (

−

× 100% and

Peak ΔF

= (

Peak F

–

× 100% were used to

1646

determine the fluorescence change, where Ft is the fluorescence at time point n and

1647

is the fluorescence from the average intensity of 10 frames fluorescence before

1648

optogenetic stimulation. To quantify neuronal activity in the brain region, the brains of

1649

male flies with the appropriate genotype were dissected and secured in a plastic dish.

1650

All subsequent steps were conducted as previously described

26,129

1651

1652

scRNA-seq Data Analysis

1653

We utilized the single-cell RNA-seq data from

https://flycellatlas.org

130

, and the UMIs

1654

(Unique Molecular Identifiers) data are generated from the loom file of 10x VSN All

1655

(Stringent), which comprises a total of 506,660 cells. The data were subsequently

1656

analyzed by Seurat (v4.3.0)

131

. And we used the 'NormalizeData' function to

1657

automatically normalize the data. The Dot plot is based on the function 'Dotplot', the

1658

cells were first divided according to the 'annotation' which refers to the cell type, then

1659

the table of dot plot of one gene was calculated by the function 'Dotplot', and finally,

1660

the organization category was placed on the x-axis.

1661

Journal Pre-proof

1662

Statistical Tests

1663

Statistical analysis of mating duration assay was described previously

45,46,52

. More than

1664

50 males (naïve, experienced and single) were used for mating duration assay. Our

1665

experience suggests that the relative mating duration differences between naïve and

1666

experienced condition and singly reared are always consistent; however, both absolute

1667

values and the magnitude of the difference in each strain can vary. So, we always

1668

include internal controls for each treatment as suggested by previous studies

132

1669

Therefore, statistical comparisons were made between groups that were naïvely reared,

1670

sexually experienced and singly reared within each experiment. As mating duration of

1671

males showed normal distribution (Kolmogorov-Smirnov tests, p > 0.05), we used two-

1672

sided Student’s

-tests. As mating duration of males showed normal distribution

1673

(Kolmogorov-Smirnov tests, p > 0.05), we used two-sided Student’s t tests. The mean

1674

± standard error (s.e.m) (

**** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p <

1675

0.05

). All analysis was done in GraphPad (Prism). Individual tests and significance are

1676

detailed in figure legends.

1677

1678

Besides traditional

-test for statistical analysis, we added estimation statistics for all

1679

MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple

1680

framework that—while avoiding the pitfalls of significance testing—uses familiar

1681

statistical concepts: means, mean differences, and error bars. More importantly, it

1682

focuses on the effect size of one’s experiment/intervention, as opposed to significance

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testing

133

. In comparison to typical NHST plots, estimation graphics have the following

1684

five significant advantages such as (1) avoid false dichotomy, (2) display all observed

1685

values (3) visualize estimate precision (4) show mean difference distribution. And most

1686

importantly (5) by focusing attention on an effect size, the difference diagram

1687

encourages quantitative reasoning about the system under study

134

. Thus, we conducted

1688

a reanalysis of all our two group data sets using both standard

tests and estimate

1689

statistics. In 2019, the Society for Neuroscience journal eNeuro instituted a policy

1690

recommending the use of estimation graphics as the preferred method for data

1691

presentation

135

1692

1693

For comparisons involving three or more experimental groups (e.g., CI and CL

1694

measurements), we conducted one-way analysis of variance (ANOVA). When ANOVA

1695

indicated significant main effects (p < 0.05), we performed Tukey's honestly significant

1696

difference (HSD) post hoc tests to control for multiple comparisons while maintaining

1697

the family-wise error rate at



= 0.05. The effect size for the overall ANOVA model

1698

was quantified using the coefficient of determination (R²), representing the proportion

1699

of variance in the dependent variable explained by group differences. We interpreted R²

1700

values using Cohen's conventional thresholds: 0.01 ≤ R² < 0.09 (small effect), 0.09 ≤

1701

R² < 0.25 (medium effect), and R² ≥ 0.25 (large effect).

1702

1703

QUANTIFICATION AND STATISTICAL ANALYSIS

1704

All statistical analyses were performed using GraphPad Prism 10 unless otherwise

1705

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indicated. The statistical details for each experiment, including the test used, sample

1706

size (n), definition of n, and measures of central tendency and variability, are provided

1707

in the figure legends. In general, n represents the number of animals (flies) tested for

1708

behavioral assays (mating duration assays and courtship assays) and the number of cells

1709

(neurons) analyzed for neuronal morphology quantification. Data are expressed as

1710

mean ± SEM unless otherwise stated. DBM (Difference Between Means) plots show

1711

the mean difference with 95% confidence intervals estimated by bootstrap resampling.

1712

For pairwise comparisons, two-tailed Student’s t-tests were used; for multiple group

1713

comparisons, one-way ANOVA followed by Tukey’s post hoc test was applied. All tests

1714

assumed normal distribution of the data (Kolmogorov–Smirnov test). Exact statistical

1715

test used in each assay are indicated in each figure legend.

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