A novel male accessory gland peptide reduces female post-mating receptivity in the brown planthopper

16 Mating in insects commonly induces a profound change in the physiology and 17 behavior of the female that serves to secure numerous and viable offspring and to 18 ensure paternity for the male by reducing receptivity of the female to further mating 19 attempts. Here, we set out to characterize the post-mating response (PMR) in a pest 20 insect, the brown planthopper (BPH) Nilaparvata lugens and to identify a functional 21 analog of sex peptide ( SP) and/or other seminal fluid factors that contribute to the 22 PMR in Drosophila. We find that BPHs display a distinct PMR that lasts for about 4 23 days and includes a change in female behavior with decreased receptivity to males 24 and increased oviposition. Extract from male accessory glands (MAG) injected into 25 virgin females triggers a similar PMR, lasting about 24h. Since SP does not exist in 26 BPHs, we screened for candidate mediator s by performing a transcriptional and 27 proteomics analysis of MAG extract. We identified a novel 51 amino acid peptide 28 present only in the MAG and not in female BPHs. This peptide, that we designate 29 maccessin (macc), affects the female PMR. Females mated by males with macc 30 knockdown display receptivity to wild type males in a second mating, which does not 31 occur in controls. However, oviposition is not affected. Injection of recombinant macc 32 reduces female receptivity, with no effect on oviposition. Thus, macc is so far the only 33 candidate seminal fluid peptide that promotes a PMR in BPHs. Our analysis suggests 34 that the gene encoding the macc precursor is restricted to species closely related to 35 BPHs. 36 37

post-mating response; bioactive peptide; male accessory gland; seminal 38 fluid proteins; brown planthopper; egg-laying 39 40 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Author summary 41 In insects, mating often induces a long -lasting change in the female behavior and 42 physiology, called a post-mating response (PMR). This ensures numerous and viable 43 offspring, but also serves to secure paternity for the male by inhibiting the female 44 receptivity to further mating attempts. Here, we demonstrate that a pest insect, the 45 brown planthopper (BPH) Nilaparvata lugens, also displays a PMR with decreased 46 receptivity to further mating and increased egg laying. We furthermore find that 47 seminal fluid extracted from the male accessory gland of BPHs injected into females 48 generates a PMR. Next, we identified a novel peptide u nique to the male accessory 49 gland (designated maccessin) and demonstrate that this peptide is responsible for the 50 reduced receptivity in the PMR, but does not affect egg laying. The gene encoding 51 maccessin appears unique to close relatives of N. lugens . Th is is similar to a 52 Drosophila male accessory gland factor, sex peptide, which is known to induce a PMR, 53 and occurs only in a limited number of Drosophila species. 54 55 56 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

57 Mating in insects commonly leads to a profound change in the physiology and 58 behavior of the female that serves to secure a viable offspring and also to ensure 59 paternity for the male by reducing receptivity of the female to further mating attempts 60 [1-3]. This phenotypic switch has been especially well documented in Drosophila 61 where the post -mating response (PMR) includes not only an increase in egg 62 production, but also a reduced receptivity to courting males and changes in feeding, 63 metabolism, and sleep pattern that lasts about a week [1, 3-10]. The trigger of this 64 behavior switch is transferred from the male with the semen during copulation, and in 65 Drosophila a major factor is a secreted 36 amino acid peptide, designated sex peptide 66 (SP) [4, 6, 7, 9]. This male -specific peptide, produced in the male accessory gland 67 (MAG) acts primarily on a set of sensory neurons in the female reproductive tract 68 known to express the sex -determination gene fruitless and connect to higher order 69 brain circuitry consisting of doublesex expressing neurons [11-14]. Thus, transfer of 70 SP and activation of sex-specific neuronal circuits under lie part of the P MR in 71 Drosophila females. 72 Interestingly, SP and the related peptide DUP99B have only been identified in the 73 genomes of a small set of Drosophila species and not in other insects [15]. The 74 receptor for SP (SPR) [16] was found to be promiscuous and is additionally activated 75 by myoinhibitory peptide (MIP), also known as allatostatin -B [17, 18]. These authors 76 suggested that MIPs are the ancestral ligands of the SPR, but it is noteworthy that 77 MIPs do not activate the PMR in Drosophila [17, 18]. MIPs can be found in most insect 78 species together with its receptor (MIPR). We he nceforth use MIPR for this receptor 79 and the Drosophila SPR. Although it is possible that MIPs could act as mediators of 80 the PMR in insects that lack SP, there is so far no evidence for this [19]. 81 However, in some insects, it seems that the MIPR is involved in a portion of the 82 PMR as a target of other hitherto unidentified ligands [19]. Examples are the oriental 83 fruitflies Bactrocera oleae and Bactrocera dorsalis [20-22] and the cotton bollworm 84 Helicoverpa armigera [23, 24] where post -mating oviposition is affected by MIPR 85 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint knockdown. Diminishment of his receptor also affects oviposition in Tobacco cutworm, 86 Spodoptera litura, but has no effect on the PMR [25]. The identity of the authentic 87 MIPR ligand(s) remains to be identified in these species. 88 In other species investigated, the MIPR seems not to be involved in the PMR. 89 The mosquito Aedes aegypti is one such case [19]. Interestingly, however, a 90 male-specific peptide was found in the A. aegypti MAG and shown to be transferred to 91 females at mating [26]. This decapeptide, Aedes head peptide (HP-1), does not act on 92 the MIPR [19, 26, 27]. Instead the HP-1 receptor was identified as a short neuropeptide 93 F receptor (NPYLR1) [28], and interestingly HP-1 induces a life-long refractoriness to 94 insemination by other males [27]. Hence, the mosquito HP -1/NPYLR1 underlies a 95 post-mating change in mate receptivity in females, but has no impact on fecundity, 96 host-seeking or blood -feeding [27], suggesting that this peptide signaling is not fully 97 equivalent to the SP-MIPR axis in Drosophila. Finally, there is evidence for a 98 non-peptidergic signal inducing PMR in the malaria mosquito Anopheles gambiae [29, 99 30]. In this species a male-specific form of 20-hydroxyecdysone is sexually transferred 100 to females to induce mating refractoriness [29]. 101 We are interested in the molecular mechanisms and signaling pathway 102 responsible for a possible PMR in a pest insect, the brown planthopper (BPH) , 103 Nilaparvata lugens . The mating behavior of BPHs has been investigated in some 104 detail [31-34], but it is not yet clear whether females display a post -mating switch in 105 physiology and behavior. Our study identifies a distinct PMR in BPHs with a change in 106 female receptivity to males and an increase in oviposition, lasting for about 4 days . 107 We found that extract from MAGs injected into virgin females induced a similar PMR, 108 although lasting only for about 24h. To screen for a seminal fluid factor responsible for 109 this PMR we performed a transcriptional and proteomics analysis of MAG extract. We 110 identified a novel peptide precursor that turns out to be specific to the MAG in males 111 and not foun d in female BPHs. The ma ture 51 amino acid peptide of this precursor 112 was designated maccessin (macc). When exposing females to a second mating after 113 first being mated with males where the maccessin (macc) gene was knocked down 114 we did not observe any change in receptivity in contrast to controls. However, 115 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint oviposition is not affected. Injection of recombinant macc peptide reduces female 116 receptivity, but also here there is no effect on oviposition. Thus, we propose that this 117 novel MAG peptide is transferred via seminal fluid to females during copulation and 118 induces a post-mating change in female behavior. Importantly, the gene encoding the 119 macc precursor can only be found in insect species closely related to BPHs. It can be 120 noted that we and a previous study identified another peptide precursor transcript in 121 the MAG [35]. This encodes an isoform (splice variant) of an ion transport peptide 122 (ITPL-1). However, the same ITPL-1 peptide can also be produced by another splice 123 variant in females. We found that knockdown of this peptide in males and injection of 124 recombinant ITPL-1 in females affected the female PMR similar to macc. 125 126 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

127 Brown planthopper females display a distinct post-mating response 128 Previous studies have described the mating behavior of the brown planthopper (BPH) 129 in some detail [31-34]. During courtship in BPH s the males perform most of the 130 behavioral steps while females only perform a few. The sequence of behaviors 131 includes male abdominal vibration , virgin female abdominal vibration, then male s 132 performing following female, wing extension and abdomen vibration, followed by 133 tapping, attempted copulation, copulation and terminated copulation (Figure S1A-H 134 and Video S1). However, there are no reports about a post-mating switch in female 135 behavior and physiology in the BPH . Hence, we first asked whether BPH females 136 display a post mating response (PMR) similar to that observed in Drosophila [1, 6, 7, 137 36, 37], malaria mosquito [29, 30], and other insect species [38-40]. Indeed, we found 138 that once a virgin female BPH has been mated, she is unwilling to accept another 139 courting male (Figure 1A) and lays more eggs than virgin females (Figure 1B). The 140 decreased receptivity of mated females is maintained for at least four days following 141 copulation (Figure 1A). Furthermore, we noted that the PMR in female BPHs includes 142 specific behaviors such as female abdominal vibration and extrusion of the ovipositor 143 towards the courting males, which is also observed in the PMR of Drosophila [41, 42] 144 (Figure S1I and J and Video S2). Our data, thus, show that female BPHs exhibit a 145 distinct PMR. 146 147 Seminal fluid proteins induce a post-mating response in N. lugens 148 Transfer of s eminal fluid proteins (SFP s) into virgin females of different insect 149 species, known to display a PMR , results in the repression of female sexual 150 receptivity and stimulates their oviposition to levels similar to those of mated females 151 [2, 5, 7, 16, 37, 43-45]. Hence, we asked whether seminal fluid proteins, transferred into 152 female reproductive organs during copulation could induce a PMR also in BPH s. 153 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Seminal fluid proteins are primarily produced in the male accessary gland (MAG) and 154 ejaculatory duct (Figure 1C) and transferred into female reproductive organs such as 155 copulatory bursa and spermatheca (Figure 1D) during copulation. 156 Indeed, we found that injection of SFP s, extracted from MAGs of BPHs , into 157 abdomens of virgin females , significantly diminished receptivity to courting males 158 (Figure 1E). These females frequently displayed rejection behavior, e.g. abdominal 159 vibration and ovipositor extrusion, typical of mated females ( Video S3). This effect 160 lasts at least 24 hours after injection of SFPs, and thus is shorter than the PMR seen 161 after mating (Figure 1B and 1E). This suggests that there could be other factors that 162 play important role s in long-term PMR in the BPH. Another possibility is that SFPs 163 need to be associated with (bound to) sperm to ensure gradual release of SFPs over 164 a longer duration as was shown in Drosophila [46]. We furthermore observed that 165 injection of SFPs into virgin females stimulate s egg-laying and leads to an increased 166 percentage (40%) of SFP-injected females ovipositing compared with solvent-injected 167 controls (2%) (Figure 1F and G). Taken together, our results demonstrate that BPHs 168 display a distinct PMR and that SFPs play an important role in this response. 169 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 1 170 171 Fig. 1. Brown planthoppers display a distinct post mating response and seminal 172 fluid proteins induces a post-mating response. 173 A. Female receptivity changes after a first mating . Graph shows the proportion of 174 courted females that respond to males as virgins and mated insects over five days. 175 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Rates of courtship differs significantly between mated and virgin females for each of 176 the initial four days . The numbers below the bars denote total number of animals. 177 ****P < 0.0001, **P 0.05; Mann–Whitney test. B. 178 Numbers of eggs laid per female. Note that wild type BPH females are known to mate 179 less with age. The small circles and the numbers below the bars denote total number 180 of animals. Data are shown as mean ± s.e.m. Student’s t -test. *P < 0.05, **P < 0.01, 181 ***P 0.05, for comparisons against virgin in A-B. 182 C. The reproductive system of the male brown planthopper includes the testes, vas 183 deferens, accessory glands and ejaculatory ducts. D. The reproductive system of the 184 female brown planthopper includes the ovary, oviduct, spermatheca and copulatory 185 bursa. E. Receptivity to mating in virgin females after injection of male accessory 186 gland proteins extracted from the male BPH, measured as percentage of females that 187 copulated within 30 min . The number of refractory females is high 3 -6 h after SFP 188 injection, but then declines. The numbers in brackets denote total number of animals. 189 *P < 0.05, **P < 0.01, ***P < 0.001, for comparisons against control; Mann–Whitney 190 test. F. Numbers of eggs laid per female in 24 h. The small circles and the numbers 191 below the bars denote total number of animals. ***P < 0.001, Student’ t-test. G. 192 Percentage of virgins laying eggs during 24 h after SFP injection. The numbers below 193 the bars denote total number of animals. ***P < 0.001, Mann –Whitney test (At least 194 four biological replicates with at least five insects per replicate for each experiment). 195 196 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 1-figure supplement 1 197 198 Figure 1 - figure supplement 1. Mating and post -mating behavior of brown 199 planthoppers. A-H: The mating behavior sequence includes eight steps ( A-H, 200 following, wing extension, abdominal vibration, abdominal rubbing, attempted 201 copulation, copulation, terminated copulation and leaving). I and J: The post-mating 202 response includes two behaviors, female abdominal vibration and ovipositor extrusion. 203 The bigger insect is the female and smaller is the male. 204 Video S1 205 Video S1. Courtship behavior of brown planthopper. 206 Video S2 207 Video S2. Post-mating behavior of brown planthopper. 208 Video S3 209 Video S3. Post-mating behavior of virgin brown planthopper after injection with 210 seminal fluid proteins (SFPs). 211 212 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Transcriptome and proteome analysis of male accessory gland (MAG) 213 To search for factors that may be transferred with the seminal fluid to induce a 214 PMR, we next performed transcriptional (RNA -seq quanti fication) and proteomics 215 analyses of MAGs from N. lugens (Figure 2-figure supplement 1A). 216 Illumina sequencing libraries were constructed by using mRNA from the MAG of 217 brown planthoppers. We obtained 54,867,464.7 clean reads on average from 218 samples of MAG ( Figure 2-Table S1). After removing low -quality regions, adapters, 219 and possible contamination, we obtained more than 6 giga base clean bases with a 220 Q20 percentage over 98%, Q30 percentage over 94%, and a GC percentage between 221 38.94 and 41.58% ( Table S1). After alignment by Bowtie, 61.01 –65.66% and 61.97–222 66.21% unique reads were mapped into the reference genome of N. lugens. All of the 223 RNA sequence data in this article have been deposited in the China National Center 224 for Bioinformation database and are accessible in CRA019725. To identify the 225 putative function of assembled transcripts, sequence similarity search was conducted 226 against the NCBI non -redundant (NR) and Swiss -Prot protein databases using 227 BLASTx search with a cut-off E value of 10−5. 228 Proteomic analysis was performed using Label-free. Production data was 229 searched against brown planthopper MAG transcriptome database s using the 230 Proteome Discoverer 2.2 (PD2.2, Thermo) and identical search parameters. 231 Searching against the de novo assembled N. lugens MAG transcriptome, proteomics 232 analysis identified 366,540 total spectra, 101,639 spectra after cleaning and quality 233 checks, 27.73 percent in the total spectra, with the identified 28998 peptides and 3951 234 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint proteins. The mass spectrometry proteomics data have been deposited to the Omix of 235 the China National Center for Bioinformation database (https://ngdc.cncb.ac.cn/omix/) 236 via the PRIDE partner repository with the dataset identifier OMIX007634. 237 Gene ontology (GO), an international standardized gene functional classification 238 system, was used to classify the function of the predicted brown planthopper genes. 239 Based on sequence homology, a total of 16,367 transcripts (36.78%) and 1911 240 proteins could be categorized into three main categories: biological process, cellular 241 component, and molecular func tion, with 112 function groups in the transcripts and 242 112 in the protein s (Figure 2). Genes and proteins involved in oxidation -reduction 243 process was the largest category in biological processes, including 1121 (15.3%) 244 transcripts, 113 (12.9%) proteins. The re were 459 (6.3%) transcripts and 78 (8.9%) 245 proteins involved in proteolysis, 72 proteins involved in translation and 69 involved in 246 metabolic process, and the number of proteins involved in signal transduction and 247 transport reached 630 and 644 transcript s, respectively (Figure 2A). In the cellular 248 component category, proteins involved i n integral component of membrane (1750, 249 27.7% transcripts and 79, 14.2% proteins respectively) and membrane (1509, 23.9% 250 transcripts and 59, 10.6% proteins) were all prominently represented (Figure 2C). The 251 genes and proteins associated with ‘binding’ were 70.2% and 65.7% respectively in 252 the molecular function category ( Figure 2B). This pattern of distribution is typically 253 seen in the transcriptome of samples undergoing development processes [47]. In our 254 database, 586 transcripts were annotated as related to metabolic processes, which 255 suggests that this analysis provides abundant information on novel genes involved in 256 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint metabolic pathways, including secondary metabolism. 257 The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was utilized 258 to categorize gene function and pathways. There were 10,582 transcripts mapped into 259 229 KEGG pathways. The maps with the highest t ranscripts representation were 260 signal transduction (1565 transcripts, 14.8%), followed by endocrine system (942 261 transcripts, 8.9%), carbohydrate metabolism (795 transcripts, 7.5%), and amino acid 262 metabolism (584 transcripts, 5.5%) (Figure 2-figure supplement 2A). The presence of 263 abundant metabolic pathways has also been found in the proteomics of accessory 264 gland of brown pla nthopper [48]. There were 1047 proteins that mapped into 122 265 KEGG pathways, with the highest protein representation in global and overview maps 266 (344, 16.5%), followed by carbohydrate metabo lism (228, 10.9%), transport and 267 catabolism (202, 9.7%), folding, sorting and degradation (202, 9.7%), translation (195, 268 9.3%), overview (160, 7.7%), amino acid metabolism (122, 5.8%) (Figure 2-figure 269 supplement 2B). 270 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 2 271 272 Figure 2. GO classification of brown planthopper MAG transcripts and proteins. 273 Genes and proteins are classified according to gene ontology annotations, and the 274 proportions of each category are shown in terms of percentages of (A) biological 275 processes, (B) molecular functions, and (C) cell components. Outer ring, transcript, 276 inner ring, protein. 277 278 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 2-figure supplement 1 279 280 Figure 2-figure supplement 1. (A) Workflow for identification and quantitation of 281 seminal fluid proteins in the brown planthopper, N. lugens . Dissected accessory 282 glands were used for extraction and subjected to transcriptome and proteome 283 analysis using liquid chromatography (LC) and mass spectrometry (MS/MS). (B) Venn 284 diagram of the numbers of predicted seminal fluid proteins comparing transcriptome 285 prediction and MS identification. 286 287 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 2-figure supplement 2 288 289 Figure 2-figure supplement 2. Pathway assignment based on KEGG analysis. 290 (A) Classification based on transcript. (B) Classification based on protein. 291 292 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Identification of seminal fluid proteins of N. lugens 293 Genes encoding seminal fluid proteins were predicted using both the N. lugens 294 transcriptome assembly and proteomic analysis. We identified a total of 373 putative 295 SFPs from the MAG transcriptome data. Of these, 209 sequences were confirmed by 296 proteomic analysis (Figure 2-figure supplement 1B and Table S2). Among of these, 297 131 putative SFPs have signal peptides and are likely to be secreted by the MAG 298 (Table S 2). One of these gene transcript s in the MAG encodes an ion transport 299 peptide-like peptide precursor (ITPL-1) (Table S2). It had previously been reported 300 that one splice isoform of this gene is specifically expressed in the MAG of BPHs [35, 301 48]. However, the same mature peptide (ITPL -1) could be produced from another 302 splice variant of the same gene in females [35, 48], suggesting that this ITPL-1 303 signaling can also be endogenous to females. 304 Interestingly, we discovered a hitherto unknown peptide precursor in the MAG by 305 transcriptome and mass spectrometric analysis ( Table S2). This is encod ed by the 306 gene BAO00947, which has been previously annotated as a peptide precurs or [47]. 307 The peptide encoded on this precursor i s 91 amino acids and the mature peptide 308 between the KR-cleavage sites is 51 amino acids long, and has six cysteines that can 309 form three disulfide bridges, spaced in a fashion resembling ion transport peptides [49, 310 50]. There is no C -terminal amidation signal suggesting that the peptide is 311 non-amidated. We designate this peptide maccessin (male accessory gland peptide; 312 macc). In Figure 3A, we show the amino acid sequence of the predicted neuropeptide 313 precursor encoded by BAO00947, including the signal peptide and cleavage sites . A 314 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint macc peptide fragment (ATLGEYTY) could also be detected in MAG tissue extract in 315 our proteomics analysis (Table S 2). We performed a semi-quantitative RT -PCR 316 analysis of macc and found that it is only expressed in the MAG, and cannot be found 317 in males with the MAG removed, or in females (Figure 3B). Thus, macc is a male and 318 tissue-specific peptide. 319 The macc gene could not be detected in related insect species, such as small 320 brown rice planthopper, Laodelphax striatellus , and whitebacked planthopper, 321 Sogatella furcifera or other more distantly related insects including fruit flies and 322 mosquitos (Figure 3-figure supplement 1A). However, we found a gene homologous 323 to macc in the closely related species Nilaparvata muiri. Thus, the macc peptide is 324 well conserved between Nilaparvata muiri and Nilaparvata lugens (Figure 3 -figure 325 supplement 1B). 326 327 The novel peptide maccessin reduces female receptivity but does not 328 induce oviposition in N. lugens 329 Next, we asked whether the novel peptide macc plays a role in the PMR of BPHs. 330 We diminished the expression of the macc gene in males by injection of dsRNA with a 331 knockdown efficiency of more than 90% (Figure 3C), and found that a significant 332 number of females that had been mated with these males would mate again with wild 333 type male s (Figure 3D). Such re -mating is never observed in the control 334 (dsgfp-injected) group. However, the re-mating rate is small with only 7 percent of the 335 females courted in the secondary mating being receptive (Figure 3D). The number of 336 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint eggs laid per female displayed no difference between females mated with dsgfp- and 337 dsmacc- injected males (Figure 3E). 338 If macc is transferred from a male to a female during copulation it could enforce 339 his paternity by reducing receptivity of the female to further mating attempts. A similar 340 response should be seen after macc peptide has been injected into a virgin female. To 341 test this, we generated recombinant macc for injections. Next, we injected individual 342 wild-type virgin females with either buffer or mature macc peptide and allowed them to 343 recover for 6 hr in groups. This extended recovery time was required because virgins 344 tested shortly after injection did not mate regardless of the substance injected. After 345 recovery, injected females were exposed to wild-type males for 30 min, an exposure 346 time that was sufficient for nearly all control females to show receptivity. We found that 347 injection of macc significantly reduces virgin female receptivity (Figure 3F). Besides 348 this, we observed an obvious post mating response in virgin females 6 hours after 349 injection with macc, such as ovipositor extrusion, which is never seen in PBS injected 350 females. However, injection o f macc did not induce oviposition i n virgin female s 351 (Figure 3G and H ). In summary, our data show that females mated with males with 352 diminished macc will mate again and that injection of macc in female s reduces 353 receptivity, but does not induce oviposition of virgin BPHs. 354 355 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 3 356 357 Figure 3. Maccessin reduces female receptivity but not ovipostion in brown 358 planthopper. 359 A. Amino acid sequence of the maccessin precursor in brown planthopper. Yellow 360 marker indicates signal peptide sequence. The six cysteines (blue marker) can form 361 three disulfide bonds. The mature peptide is shown in gray background. The 362 red-marked KR sites indicate cleavage sites for mature peptide. Note that there is no 363 C-terminal amidation signal. The amino acids marked in red font indicate the peptide 364 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint detected by mass spectrometry. B. The tissue distribution of maccessin was analyzed 365 by semi-quantitative RT-PCR. Whole animals (female, male), male accessory glands 366 (AG) and whole males with accessory glands removed (Male –AG), were assayed. C. 367 The gene-silencing efficacy of Maccessin in male insects following dsRNA injection 368 assayed by qPCR. The small circles denote the number of replicates. Data are shown 369 as mean ± s.e.m. Student’s t -test. ***P < 0.001. D. Receptivity of virgin and mated 370 females, scored as the percentage of females that copulated within 30 min. The small 371 circles denote the number of replicates ; the numbers below the bars denote total 372 number of animals. Data are shown as mean ± s.e.m. Mann–Whitney test. *P 0.05. E. Number of eggs laid per female in 48 h. The 374 dsRNA was injected in males at the end of the fifth instar, and the first mating took 375 place after two days of eclosion. The numbers below the bars denote total number of 376 animals. At least four biological replicates with at least five insects per replicate for 377 each experiment . Data are shown as mean ± s.e.m. Student’s t -test. ns 378 (non-significant), P > 0.05. F. Mating receptivity shown as percentage of virgins that 379 copulated within 30 min and tested six hours after injection. Each virgin was injected 380 with 30 nl of 1×PBS or 100 μmol/L maccessin. The small circles denote the number of 381 replicates; the numbers below the bars denote total number of animals . Data are 382 shown as mean ± s.e.m. Mann–Whitney test. *P < 0.05. G. Number of eggs laid after 383 injection of maccessin in virgin females. Data are shown as mean ± s.e.m. Student’s 384 t-test. ***P < 0.001, *P 0.05, for comparisons 385 against dsgfp injected in C -G. The numbers below the bars denote total number of 386 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint animals. At least four biological replicates with at least five insects per replicate for 387 each experiment . Each virgin was injected with 30 nl of 1×PBS or 100 μmol/L 388 maccessin. H. Percentage of virgins laying eggs during 48 h after maccessin injection. 389 The numbers below the bars denote total number of animals. At least four biological 390 replicates with at least five insects per replicate for each experiment. Each virgin was 391 injected with 30 nl of 1×PBS or 100 μmol/L maccessin. Chi-square test with the Yates’ 392 correction ns (not significant), P > 0.05, for comparisons against PBS injection. 393 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 3-figure supplement 1. 394 395 396 Figure 3 - figure supplement 1. (A) A gene encoding maccessin precursors was 397 detected in Nilaparvata muiri and Nilaparvata lugens , but not present in other 398 planthoppers, or other insects such as fruit flies, mosquito and silkworm . (B) The 399 alignment of maccessin protein sequence between Nilaparvata muiri (upper) and 400 Nilaparvata lugens (lower). 401 402 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint An ITP-like peptide (ITPL-1) also reduces female receptivity but does not induce 403 oviposition in N. lugens 404 Previous work identified another ITP precursor gene (Accession number: 405 XP_039277955) in the BPH that gives rise to an amidated ITP peptide (ITPa) and four 406 distinct ITPL transcripts (ITPL-1-4) containing different 5’ UTRs [48]. The open reading 407 frame (ORF) and the 3’ UTR regions of the four ITPL transcripts are equivalent and 408 encode identical non-amidated ITPL peptides (Figure 4-figure supplement 1) [35, 48]. 409 The four ITPL transcripts display differential spatio -temporal expression patterns, 410 where ITPL -1 is exclusively expressed in males, and specifically only in the male 411 reproductive system [35]. However, the three other splice forms itpl-2-4 are expressed 412 in other tissues in both males and females [35]. We confirmed these findings by 413 RT-PCR and qPCR and found that itpl-1 is exclusively expressed in the MAG and not 414 in females (Figure 4-figure supplement 2A-C). 415 As noted above, the mature ITPL that can be generated from itpl-1 transcript is 416 identical to the ones derived from itpl -2-4, suggesting that this peptide can be 417 produced also in females. Nevertheless, we next asked whether male-derived ITPL-1 418 plays a role in the PMR of BPHs. Thus, we injected dsRNA that target itp/itpl (it is not 419 possible to only silence itp) or only itpl (dsRNA to target exon 3) to determine whether 420 peptide-deficient males can induce a PMR in female BPHs. Our data show that 421 dsRNA significantly reduced the transcript levels of itp and the four splice forms itpl1-4 422 in BPH (Figure 4-figure supplement 2D-G). Like for macc, we observed that a number 423 of females who first had mated with dsitp/itpl and dsitpl males did remate with wildtype 424 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint males, which does not occur after first mating with the dsgfp control males (Figure 4A). 425 However, this remating rate is lower than 20 perce nt of the females courted in the 426 secondary mating ( Figure 4A ). The number of eggs laid per female displayed no 427 difference between females mated with dsgfp, dsitp/itpl and dsitpl injected males 428 (Figure 4B). To specifically silence the itpl-1 isoform, we synthesized small interfering 429 RNAs (siRNAs), which target the exon 1a (Figure 4-figure supplement 1). We again 430 observed that a number of females who first mated with itpl-1 siRNA males did remate 431 with wild type males, which was not seen in controls ( Figure 4C ). However, the 432 number of eggs laid was not changed after copulating with itpl-1 siRNA males (Figure 433 4D). In summary, females who mated with itpl-1 knockdown males displayed low rate 434 of receptivity in the second mating (similar to the novel macc). 435 Next, we used recombinant amidated ITP (ITPa) and non-amidated ITPL-1 to test 436 the effect of injection in females on the PMR. Individual wild-type virgin females were 437 injected with either buffer, mature ITPa or ITPL -1 peptide. We found that injection of 438 ITPL-1 reduced the receptivity of virgin females (Figure 4E). However, ITPa only has a 439 weak effect on receptivity of virg in females (Figure 4E). We hypothesize that since 440 ITPa is not produced in the MAG, the effect of injected peptide on female receptivity 441 might reflect the action of endogenous female ITPa in post -mating physiology . 442 Furthermore, since also peptides identical to ITPL -1 ( ITPL-2-4) could be produced 443 endogenously i n females , we cannot exclude that also injected ITPL -1 mimics 444 endogenous peptide, at least partly . We did not find that oviposition increased after 445 ITPa and ITPL injection into virgin females (Figure 4F and G). 446 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 4 447 448 Figure 4 . ITPL-1 also reduces female receptivity but not ovipostion in brown 449 planthopper. 450 A. Receptivity of virgin and mated females after itp/itpl and itpl knockdown by dsRNA 451 injections, scored as the percentage of females that copulated within 30 min. The 452 small circles denote the number of replicates; the numbers below the bars denote 453 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint total number of animals. Data are shown as mean ± s.e.m. Mann –Whitney test, for 454 comparisons against dsgfp injection control. *P 455 0.05. B. Number of eggs laid per female in 48 h. dsRNA was injected in males at the 456 end of the fifth instar, and the first mating took place two days after eclosion. The 457 small circles and the numbers below the bars denote total number of animals . Data 458 are shown as mean ± s.e.m. Student’s t -test. ns (not significant), P > 0.05, for 459 comparisons against dsgfp injected. C. The mating receptivity rates of female to NC 460 (negative control) and Nlitpl1-siRNA injected male courtship. The Nlitpl1-siRNA is 461 designed to target a sequence specific to Nlitpl1, differentiating it from the other four 462 spliceosome components. The small circles denote the number of replicates; the 463 numbers below the bars denote total number of animals. Data are shown as mean ± 464 s.e.m. Mann–Whitney test. *P 0.05. D. Number of 465 eggs laid per female in 48 h. The experimental protocol and symbols are the same as 466 Fig. 4B. The small circles and the numbers below the bars denote total number of 467 animals. Data are shown as mean ± s.e.m. Student’s t-test. ns (not significant), P > 468 0.05, for comparisons against NC injected. E. Mating receptivity shown as percentage 469 of virgins that copulated within 30 min as tested six hours after injection. Each virgin 470 was injected with 30 nl of 1×PBS , 400 μmol/L ITP or 400 μmol/L ITPL. The numbers 471 below the bars denote total number of animals. Data are shown as mean ± s.e.m. 472 Groups that share at least one letter are statistically indistinguishable; Kruskal–Wallis 473 test followed by Dunn’s multiple comparisons test with P < 0.05. F. Number of eggs 474 laid after injection of ITP a or ITPL-1 peptide in virgin females. The small circles and 475 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint the numbers below the bars denote total number of animals. Data are shown as mean 476 ± s.e.m. Student’s t-test. Ns (not significant), P > 0.05, for comparisons against PBS 477 injected. G. Percentage of virgins laying eggs during 48 h after ITP or ITPL injection. 478 The numbers below the bars denote total number of animals. Chi-square test with the 479 Yates’ correction ns (not significant), P > 0.05, for comparisons against PBS injection. 480 481 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 4-figure supplement 1 482 483 Figure 4 - figure supplement 1. Sequence analysis of ITP/ITPL. 484 A. The identified ion transport peptide/ ITP -like (ITPa/ITPL) transcripts. The coloured 485 blocks represent exons within the N. lugens ITP/ITPL transcripts. Exons 1a, 1b, 1c 486 and 1d are alternative 5’ untranslated regions used by ITP and ITPLs. * denote the 487 start codon and stop codons of the transcripts. B. Amino acid sequence of ITP and 488 ITPL of brown planthopper . Orange indicates sequence of signal peptide; green 489 indicates mature peptide sequence; red indicates difference sequence of ITP and 490 ITPL. Note that four slice forms of itpl are known (itpl-1-4), which all could give rise to 491 the same mature ITPL peptide. C. Multiple comparison s of ITP and ITPL mature 492 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint peptides in brown planthopper and other species. The red frames indicate conserved 493 cysteines. Deduced ITP and ITPL sequences are shown for Manduca sexta (Manse, 494 AY950500, AY950501), Bombyx mori (Bommo, AY950502, AY950503), Schistocerca 495 gregaria (Schgr), Apis mellifera (Apime), Aedes aegypti (AY950504, AY950505, 496 AY950506), Anopheles gambiae (Anoga), Drosophila melanogaster (Drome), and 497 Tribolium castaneum (Trica, EFA07585). 498 499 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 4-figure supplement 2. 500 501 Figure 4 -figure supplement 2. Distribution of itpl-1 transcript in different 502 genders and tissues of brown planthopper. A. Relative expression of itpl-1 gene in 503 N. lugens at different genders. Data are shown as mean ± s.e.m. Student’s t-test. ****, 504 P < 0.0001, for comparisons against dsgfp injected. B. Relative expression of itpl-1 505 gene in N. lugens at different tissues in male reproductive system. Data are shown as 506 mean ± s.e.m. Groups that share at least one letter are statistically indistinguishable; 507 Kruskal–Wallis test followed by Dunn’s multiple comparisons test with P < 0.05. C. 508 The tissue distribution of itpl-1 was analyzed by semi -quantitative RT -PCR. RNA 509 samples from adult females, adult males, male accessary gland (AG) alone and adult 510 male without accessary gland (male -AG). D-G. Relative expression of different 511 spliceosomes of itpl-1-4 gene in males injected with dsRNA. Data are shown as mean 512 ± s.e.m. Groups that share at least one letter are statistically indistinguishable; 513 Kruskal–Wallis test followed by Dunn’s multiple comparisons test with P < 0.05. 514 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Myoinhibitory peptides (MIPs) and Drosophila sex peptide (SP) do not trigger a 515 post mating response in N. lugens 516 We found a seminal fluid -derived peptide, macc, that can trigger a PMR in the 517 female BPH, but since it is an “orphan” peptide unrelated to previously known ones 518 the receptor is unknown. T hus, we asked whether the MIPR previously implicated in 519 Drosophila and other insects may act as a receptor of macc. However, first, we asked 520 whether the known MIPR ligands SP or MIPs play any role in the PMR of BPHs. We 521 thus tested whether Drosophila SP can induce a PMR in BPH. As a control, we 522 showed that injection of SP significantly inhibits receptivity of virgin female Drosophila 523 (Figure 5A). However, injection of Drosophila SP does not diminish receptivity of virgin 524 BPHs (Figure 5B). 525 MIPs have been reported as the ancestral ligands of the promiscuous Drosophila 526 SP receptor (also known as MIPR) [17, 18, 51, 52], but do not induce a PMR in 527 Drosophila [17, 18]. Since MIP signaling is present ubiquitously in insects ( Kim et al., 528 2010; Poels et al., 2010 ;) and the MIPR has been implicated in the PMR in a few insect 529 species [see [19]], we asked whether MIP signaling is involved in the PMR in BPHs. 530 First, we cloned the mip gene of the BPH and found that it encodes four mature MIP 531 peptides, MIP1 - MIP4 ( Figure 5-figure supplement 1). Of these, MIP2 is 532 predominantly expressed in BPH with eight paracopies in the precursor ( Figure 533 5-figure supplement 1). Next, we examined the expression pattern of mip in BPHs. 534 Investigating different developmental stages by real -time PCR, we found that mip 535 transcript levels are boosted in third instar larvae and adult males. Transcripts are 536 more abundant in adult males than in females. Of the different tissues, mip was 537 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint detected in highest levels in the head of both male and female BPHs ( Figure 5-figure 538 supplement 2A and B). 539 We synthesized four mature MIP peptides for testing a possible role in the PMR in 540 BPHs. For this test, we injected a mix of MIPs (MIP1 – MIP4) at different doses into 541 the abdominal hemocoel of virgin females and allowed them to recover for 6 hr in 542 groups. This extended recovery time was required because virgins tested shortly after 543 injection did not mate regardless of the substance injected. After recovery, injected 544 females were expos ed to wild -type males for 30 min, an exposure time that was 545 sufficient for nearly all control females to show receptivity. Similar to results in 546 Drosophila [17, 18], we found that injecting a mix of MIPs into the abdominal hemocoel 547 of virgin females does not decrease their receptivity (Figure 5C) or induce oviposition 548 (Figure 5D and E). 549 550 The MIP receptor is not involved in the post mating response of BPHs 551 The MIP receptor (MIPR) has been reported to be involved in the PMR of 552 Drosophila [16], tobacco cutworm [25] and cotton bollworm [24]. However, a recent 553 study indicated that MIPR is not required for refractoriness to remating or induction of 554 egg laying in Aedes aegypti [53]. The MIPR has been identif ied in most of insect 555 species, including BPHs [17, 18, 47] (Figure 5-figure supplement 3). The MIPR of 556 BPHs is orthologous to the Drosophila MIPR (Figure 5-figure supplement 3). Hence, 557 we asked whether the MIPR mediates the post -mating switch in BPH behavior. As a 558 first step to address this question, we cloned the mipr gene of BPH ( Figure 5-figure 559 supplement 4). The MIPR of the BPH displays a typical seven transmembrane 560 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint domain and it clusters with MIPR of other insect species ( Figure 5-figure supplement 561 4A and B). We furthermore investigated the expression pattern of mipr in tissues of 562 the BPH. The mipr is foun d throughout the nymphal and adult stages, and is 563 expressed more predominantly in the head than other tissues ( Figure 5-figure 564 supplement 5A and B). 565 Next, we asked whether the MIPR plays a role in t he PMR of BPHs using RNAi 566 technique. The efficacy of the R NAi was tested by qPCR of whole animals and we 567 found that the expression of mipr was significantly reduced (Figure 5G). We used a 568 protocol in which individual virgin females ( dsgfp- or controls that were 569 dsmipr-injected) were first tested for receptivity towards a naive male ( Figure 5F). 570 Those females that mated were then allowed to lay eggs for 48 h before being 571 retested for receptivity with a second naive male ( Figure 5F). In the initial mating 572 assays, virgin mipr RNAi females were as receptive as the control females ( Figure 573 5H). When testing mated females for a second mating we did not detect any 574 difference between controls and BPHs with mipr knockdown, suggesting that t he 575 MIPR has no effect on the refractoriness to remating (Figure 5H). 576 Next, we tested whether increased post-mating oviposition requires MIPR 577 signaling by applying mipr RNAi. We found that both dsgfp-injected and 578 dsmipr-injected females laid very few eggs if they were not mated ( Figure 5I). We 579 hypothesized that if the MIPR is required for post mating oviposition, dsmipr-injected 580 females would lay few or no eggs even after mating. However, the number of eggs 581 laid by mated dsmipr-injected females was not significantly different from that laid by 582 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint those that were dsgfp-injected (Figure 5I). We thus conclude that neither MIPs nor the 583 MIPR are required for increased post mating oviposition in N. lugens, and the MIPR is 584 not likely to be the receptor of the novel peptide macc. 585 586 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5 587 588 Fig. 5. MIP and MIPR are not involved in the post -mating response of brown 589 planthoppers. A. Mating receptivity shown as percentage of virgins that copulated 590 within 30 min as tested six hours after sex peptide (SP) injection in Drosophila 591 melanogaster. The small circles denote the number of replicates; the numbers below 592 the bars d enote total number of animals. Data are shown as mean ± s.e.m. Mann –593 Whitney test. ***P < 0.001. B. Mating receptivity shown as percentage of virgins that 594 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint copulated within 30 min as tested six hours after SP injection in Nilaparvata lugens. 595 Each virgin was injected with 30 nl of 600 μmol/L SP. Data are shown as mean ± 596 s.e.m. Mann–Whitney test. ns: no significant. C. MIP mixture injection does not affect 597 female receptivity. Mating receptivity shown as percentage of virgins that copulated 598 within 30 min as tested six hours after MIPs injection. Each virgin was injected with 30 599 nl of 1×PBS or 600 μmol/L MIPs. MIPs are a blend of MIP1, MIP2, MIP3 and MIP4. 600 The small circles denote the number of replicates; the numbers below the bars denote 601 total number of animals. Mann–Whitney test. ns: no significant. D. Number of eggs 602 laid after injection of MIPs in virgin females. The small circles denote the total number 603 of animals. Ns: no significant P > 0.05; Student’s t test. E. Percentage of virgins laying 604 eggs during 48 h after MIP injection. The numbers below the bars denote total number 605 of animals. Chi-square test with the Yates’ correction ns (not significant), P > 0.05. F. 606 Protocol for behavioral experiments in G and H. G. Downregulation of mipr gene using 607 mipr-RNAi leads to a reduction in mRNA expression level. ****P < 0.0001; Student’s t 608 test. The small circles denote the replicates. H. Receptivity of virgin and mated 609 females, scored as the percentage of females that copulated within 30 min. Data are 610 shown as mean ± s.e.m. Mann–Whitney test. Ns (not significant), P > 0.05. The small 611 circles denote the number of replicates; the numbers below the bars denote total 612 number of animals. I. Number of eggs laid per female in 48 h. The small circles denote 613 the total number of animals . Data are shown as mean ± s.e.m. Student’s t-test. Ns 614 (not significant), P > 0.05. 615 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5-figure supplement 1 616 617 Figure 5 - figure supplement 1. Nucleotide and amino acid sequence of the BPH 618 mip precursor gene (mip). The four distinct mature peptides —MIP1 (green), MIP2 619 (yellow), MIP3 (purple), and MIP4 (cyan) are distinguished by unique colors. 620 Cleavage sites (KR) are denoted by rectangular boxes, while the glycine residues (G) 621 essential for amidation are highlighted by double underlining. 622 623 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5 - figure supplement 2 624 625 Figure 5 - figure supplement 2. Relative quantification of mip transcript levels in 626 different developmental stages (A) and tissues (B) of N. lugens . A. Relative 627 expression of Nlmip gene in N. lugens at different developmental stages. B. Relative 628 expression of Nlmip gene in N. lugens in different tissues. M: male; F: female. Data 629 are shown as mean ± s.e.m. 630 631 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5 - figure supplement 3 632 633 Figure 5 - figure supplement 3. The presence of SPR/MIPR and MIP is widespread 634 across the genomes of most insects that have been sequenced, while the presence of 635 SP is restricted to certain species within the Drosophila genus. The brown 636 planthopper (marked in red) lacks SP . In Hymenopteran insects (marked in green), the 637 honey bee Apis mellifera and the parasitic wasp Nasonia vitripennis, neither SP , MIP, 638 nor SPR/MIPR are found. The "+" symbol represents presence, while the " -" symbol 639 indicates absence. The phylogenetic tree of different insect species has been 640 downloaded and modified from http://flybase.org/blast/. 641 642 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5-figure supplement 4 643 644 Figure 5- figure supplement 4. Sequence analysis of MIPRs. A Multiple 645 comparison of MIPR in brown planthopper (NLMIPR) and four other insect species 646 (Bombyx mori, Drosophila melanogaster , Amyelois transitella and Tribolium 647 castaneum). The black lines (TM1 -TM7) depict the transmembrane domains. B. 648 Phylogenetic analysis of MIPRs in different insect species. 649 650 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Figure 5-figure supplement 5 651 652 Figure 5-figure supplement 5. MIP receptor expression in BPH : r elative 653 quantification of mipr transcript levels in different developmental stages (A) 654 and tissues (B) of N. lugens. A. Relative expression of Nlmipr gene in N. lugens at 655 different developmental stages. B. Relative expression of the Nlmipr gene in different 656 tissues of adult N. lugens. M: male; F: female. Data are shown as mean ± s.e.m. 657 658 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

659 A mating-induced switch in female behavior and phys iology to ensure a numerous 660 and viable offspring, as well as to secure paternity, is common in insects, but only in 661 Drosophila melanogaster the underlying mechanisms have been clarified in detail and 662 the MAG-derived peptide SP identified as the main secreted signal [3, 4, 6, 7, 9, 11-13]. 663 Another closely related M AG-derived peptide, DUP99B, is also contributing to the 664 PMR in D. melanogaster [54, 55]. Since SP and DUP99B can be found only in a few 665 Drosophila species, we set out to identify factors in the MAG of the brown planthopper 666 (BPH) that might play a role similar to SP in inducing a PMR. First we established that 667 BPH females display a distinct change in behavior and physiology after mating. This is 668 manifested in a reduction in receptivity to mating males and an increase in ovulation 669 lasting four days. Next, we showed that extract from MAG injected into female BPHs 670 induced a significant decrease in receptivity, lasting about 24 h, and a sign ificant 671 increase oviposition. Then we asked what the active factor in MAG extract that 672 induces the PMR might be. We therefore went on to perform a transcriptional analysis 673 of MAG extract. As expected SP and MIPs were not detected, bu t one splice form of 674 ion transport peptide, ITPL, and a novel 51 amino acid peptide were identified. A 675 fragment of t he latter was also detected by mass spectr ometry. While the ITPL was 676 identified also in other tissues of b oth males and females, (see also [35, 48]) we 677 focused on the novel peptide, maccessin, which is male specific and only found in the 678 MAG. 679 Virgin female BPHs m ated to males with macc knockdown do not display a 680 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint repression of the propensity to re -mate, whereas injections of recombinant macc 681 peptide into virgin females render them less rece ptive to courting males. However , 682 oviposition was not affected by these manipulations. Thus, we propose that macc 683 mediates a male signal transferred in seminal fluid to reduce female receptivity to 684 further courting males. In our experiments this signal onl y mediates the receptivity 685 part of the total PMR seen after regular mating and the duration of the effect is shorter 686 (only 24 h instead of 4 d). We speculate that the partial and shorter PMR effect seen 687 in our macc experiments could be for the followin g reasons: (1) in Drosophila SP to 688 exert its full effect over about a week needs to be bound to sperm when transferred to 689 the female and thereafter gradually released [46], an d thus macc injection without 690 sperm may be less efficient and the peptide exposed to protease degradation, (2) it is 691 likely that macc is not the only MAG-secreted factor required for a full PMR effect and 692 therefore macc RNAi in males is not sufficient. In fact, we did identify another peptide, 693 ITPL-1 in the MAG of BPHs and found that it also induces a partial effect on the 694 female PMR. 695 Since the receptor of SP in Drosophila [16] can be activated also by MIPs [8, 17, 696 18], and the MIPR was implicated in the PMR in some insects [25] [24], we tested 697 whether SP , MIPs or their receptor (MIPR) affects the PMR in BPHs. We found no 698 effect of manipulating MIP signaling or injections of Drosophila SP and planthopp er 699 MIPs. The outcome of the MIPR knockdown experiment also suggest s that this 700 receptor is not required for macc signaling. Thus, since macc is a novel peptide with 701 no sequence relation to previously identified peptides, its receptor is still unknown, 702 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint and we were unable to manipulate this part of the signal pathway in female tissues for 703 further tests. 704 It is intrigu ing that SP and Dup99B are found only in the genomes of a few 705 Drosophila species related to D. melanogaster [15, 17, 56]. Fu rthermore, other 706 peptides regulating a PMR response have not yet been unequivocally identified in any 707 other insect, except the mosquito Aedes aegypti where post -mating receptivity to 708 males was found affected by a MAG -derived peptide, HP-1 [27], as detailed below. 709 Since SP acts on the MIPR several studies investigated the involvement of MIP 710 signaling in a PMR in various insects. However, MIP activated MIPR signaling seems 711 not to regulate PMR in the insects studied (i ncluding the present study), although the 712 MIPR and some unidentified ligand affects oviposition and ovary development in 713 some insects [20-22, 25, 53]. In mosquitos a post -mating decline in female receptivity 714 to further mating attempts is mediated by the MAG -derived head peptide, HP -1 (a 715 form of short neu ropeptide F, sNPF) and the sNPF receptor NPYLR1 [27]. However, 716 the HP-1 signaling does not affect fecundity, host -seeking or blood -feeding. This is 717 similar to the BPH where macc is MAG-derived signal that reduces female receptivity, 718 but not fecundity. Thus, mosquito HP -1 and BPH macc may be partial functional 719 analogs of SP . 720 What SP, HP-1 and macc have in common is that they are MAG-derived peptides 721 that appear to be restricted p hylogenetically. We found a macc precursor transcript 722 only in the genomes of Nilaparvata lugens and Nilaparvata muiri, but not in the related 723 small brown rice planthopper, Laodelphax striatellus, or the white-backed planthopper, 724 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Sogatella furcifera, or other more distantly related insects . Apparently MAG -derived 725 secretory peptides undergo rapid evolution in certain species and in Drosophila SP 726 repurposes an already existing receptor (MIPR) for distantly related MIP 727 neuropeptides [15, 17, 18, 56]. Similarly, Aedes HP-1, has adopted an sNPF receptor 728 [27]. Thus, SP displays some sequence similarities to MIPs [17, 18] and HP -1 is 729 sNPF-like, while the macc sequence is unique making it a more complex task to select 730 known GPCRs (or orphan receptors) for screening. The role of ITPL -1 needs to be 731 further investigated. Since it also seems to be produced in female BPHs from the 732 other splice variants itpl-2-4 it may act both via transfer from males at copulation and 733 as an endogenously secreted peptides in females. Recept ors for ITPa and ITPL 734 peptides have been identified in the moth Bombyx mori and the fly Drosophila [57, 58]. 735 Thus, receptor knockdown could be attempted in females for tests of PMR in future. 736 In summary, we identified a novel peptide, macc, in the MAG of BPHs that 737 induces a PMR in mated females rendering them less receptive to further mating 738 attempts. It remains to identify a receptor for this novel peptide and to characterize 739 target cir cuits in the central nervous system that modulate the female behavior. 740 Additionally, the role of ITPL-1 in the PMR should be further investigated, including the 741 possible role of endogenous female ITPL -1 in regulating reproductive behavior and 742 physiology, and a search for additional factors that ensures the fecundity in mated 743 females and leads to a more complete PMR resembling that seen after mating. 744 745 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

746 Experimental insects and husbandry 747 The brown planthopper N. lugens was reared on ‘Taichung Native 1’ (TN1) rice 748 (Oryza sativa L.) seedlings in the laboratory and maintained at 27 ± 1 ∘C, with 70 ± 10% 749 relative humidity, under a 16 h: 8 h light dark photoperiod [59]. The brown planthopper 750 sensitive strain w as originally supplied in 1995 by Zhejiang Chemical Technology 751 Group Co., LTD. 752 Extraction of male accessory glands proteins 753 300 pairs of male accessory glands were dissected in phosphate buffer at pH 7.2 754 and were transferred into 100 μl of 80% methyl alcohol on the ice. These samples 755 were treated by ultrasonic homogenization. After centrifugation supernatants were 756 collected and the precipitate washed twice with 80% methanol. The supernatant was 757 mixed and freeze dried in a Speed Vac Vacuum concentrator. 758 Mass spectrometry 759 Total protein extraction: 760 We dissected the male accessory glands of brown planthoppers in PBS buffer, 761 and collected 300 glands for each replicate (for a total of three biological replicates ). 762 The male accessory glands were quickly frozen in liquid nitrogen, ground into powder 763 at low temperature, and quickly transfer red it to a centrifuge tube pre -cooled with 764 liquid nitrogen. We added an appropriate amount of protein lysis buffer (100 mM 765 ammonium bicarbonate, 8M urea, 0.2% SDS, pH=8), mixed well and sonicated in an 766 ice-water bath for 5 minutes to e nsure complete lysis. Centrifugation was performed 767 at 4°C and 12000 g for 15 minutes, and the supernatant collected. A final 768 concentration of 10 mM DTT was added to the supernatant and reacted at 56°C for 1 769 hour. After that, an adequate amount of IAM was added and react ed at room 770 temperature in the dark for 1 hour. Four times the volume of pre-cooled acetone was 771 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint added at -20°C to precipitate for at least 2 hours at -20°C, then centrifugated at 4°C 772 and 12000 g for 15 minut es to collect the precipitate. The precipitate was 773 resuspended and washed with 1 mL of pre -cooled acetone at -20°C, centrifuged at 774 4°C and 12000 g for 15 minutes, and the precipitate collected. The precipitate was 775 air-dried, and then the protein precipitate dissolved by adding an appropriate amount 776 of protein solubilization buffer (6M urea, 100 mM TEAB, pH=8.5). 777 Protein quality inspection: 778 We u sed the Bradford protein assay kit to prepare a BSA standard protein 779 solution according to the instructions, with a concentration gradient ranging from 0 to 780 0.5 µg/µL. We took different concentrations of the BSA standard protein solution and 781 different dilution s of the test sample solution and add ed them to a 96 -well plate, 782 making up the volume to 20 µL, with each gradient repeated 3 times. We q uickly 783 added 180 µL of G250 staining solution, let it stand at room temperature for 5 minutes, 784 and measure d the absorba nce at 595 nm. We plotted a standard curve using the 785 absorbance of the standard protein solution and calculate d the protein concentration 786 of the test samples. We took 20 µg of protein test samples for 12% SDS -PAGE gel 787 electrophoresis, with the conditions for the stacking gel electrophoresis being 80 V for 788 20 minutes, and the conditions for the separating gel electrophoresis being 120 V for 789 90 minutes. After the electrophoresis is completed, we stained with Coomassie 790 Brilliant Blue R-250, and destained until the bands are clear. 791 Proteolysis: 792 We took 120 µg of protein samples, added protein solubilization buffer to make 793 up the volume to 100 µL, added 1.5 µg of trypsin and 500 µL of 100 mM TEAB buffer, 794 mixed well, and digested at 37°C for 4 hours. We then added 1.5 µg of trypsin and 795 CaCl2 for overnight digestion. We adjusted the pH to less than 3 with formic acid, 796 mixed well, and centrifuged at room temperature at 12000 g for 5 minutes, took the 797 supernatant, and slowly passed it through a C18 desalting column. After that, we used 798 a washing solution (0.1% formic acid, 3% acetonitrile) to wash continuously three 799 times, then added an appropriate amount of elution solution (0.1% formic acid, 70% 800 acetonitrile), collected the filtrate, and freeze-dried it. 801 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Liquid quality detection: 802 We prepared mobile phase A solution (100% water, 0.1% formic acid) and B solution 803 (80% acetonitrile, 0.1% formic acid). We dissolved the freeze-dried powder with 10 µL 804 of A solution, centrifuged at 4°C at 14000 g for 20 minutes, and took the supernatant 805 for sample injection, with 1 µg of sample for liquid chromatography -mass 806 spectrometry (LC-MS) analysis. We used the EASY -nLCTM 1200 nanoflow UHPLC 807 system, equipped with a homemade pre -column (2 cm × 75 µm, 3 µm) and a 808 homemade analytical column (15 cm × 150 µm, 1.9 µm). The liquid chromatography 809 elution conditions were as depicted in Table S4. We employed the Q ExactiveTM HF-X 810 mass spectrometer with a Nanospray Flex™ (ESI) ion source, set the ion spray 811 voltage to 2.3 kV, and the ion transfer tube temperature to 320°C. The mass 812 spectrometer operated in data-dependent acquisition mode, with a full scan range of 813 m/z 350-1500, a primary mass spectrometry resolution set to 60000 (at 200 m/z), a 814 maximum C -trap capacity of 3×10 6, and a maximum injection time of 20 ms. We 815 selected the top 40 parent ions with the highest intensity in the full scan for 816 fragmentation using high -energy collision dissociation (HCD) for second ary mass 817 spectrometry analysis. We set the secondary mass spectrometry resolution to 15000 818 (at 200 m/z), a maximum C -trap capacity of 1×10 5, and a maximum injection time of 819 45 ms. The peptide fragmentation collision energy was set to 27%, the threshold 820 intensity was set to 2.2×10 4, and the dynamic exclusion window was set to 20 821 seconds. We generated raw mass spectrometry data (.raw). 822 Data analysis: 823 We used the brown planthopper protein database to search all the result spectra 824 with the search software Prote ome Discoverer 2.2 (PD2.2, Thermo). We set the 825 search parameters as follows: the mass tolerance for precursor ions was 10 ppm, and 826 the mass tolerance for fragment ions was 0.02 Da. The fixed modification was 827 carbamidomethylation of cysteine, the variable m odification was oxidation of 828 methionine, and the N-terminus was acetylated. We allowed up to 2 missed cleavage 829 sites. 830 We enhanced the quality of the analytical results by further filtering the search 831 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

using the PD2.2 software: Peptide Spectrum Match es (PSMs) with a 832 confidence level above 99% were considered reliable PSMs, and proteins that 833 contained at least one unique peptide were considered reliable proteins. We retained 834 only the reliable PSMs and proteins, and performed a False Discovery Rate (FDR ) 835 validation to remove peptides and proteins with an FDR greater than 1%. 836 We used the InterProScan software for GO and IPR functional annotation, which 837 included databases such as Pfam, PRINTS, ProDom, SMART, ProSite, and 838 PANTHER. We performed functional pr otein family and pathway analysis on the 839 identified proteins using COG and KEGG. We conducted volcano plot analysis, 840 clustering heatmap analysis, and pathway enrichment analysis for GO, IPR, and 841 KEGG on Differentially Expressed Proteins (DPE). Additionally, we predicted potential 842 protein-protein interactions using the STRING DB software (http://STRING.embl.de/). 843 Gene cloning and sequence analysis 844 We used the NCBI database and BLAST programs for sequence alignment and 845 analysis. Then we used EditSeq to predict Open Reading Frames (orfs). The primers 846 were designed by tool s in NCBI. According to the manufacturer's instructions, total 847 RNA was extracted by TRIzol reagents (Inv itrogen, Carlsbad, CA , USA). We used 848 HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) reverse 849 transcription kit to synthesize cDNA templates for cloning, and stored the synthesized 850 cDNA templates at -20℃. 851 We predicted protein transmembrane fragments and topological structures 852 through TMHMM v2.0 ( http://www.cbs.dtu.dk/services/TMHMM-2.0/) (Krough et al., 853 2001). Multiple alignments on the complete amino acid sequence s were performed 854 using ClustalX (http://www.clustal.org/clustal2/). The phylogenetic tree was 855 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint constructed using MEGA 10.0 software and the Maximum Likelihood Method, with 856 1000 repeated starts. 857 858 Gene expression profile analysis 859 For the stage -specific expression study of mip and mipr, total RNA were 860 extracted from pools of thirty individuals from the following developmental stages: 1 st 861 to 5th instar nymphs, adult male and female insects. For the tissue-specific expression 862 study of mip and mipr, total RNA was isolated from various tissues including he ad, 863 thorax and abdomen of three-day-old male adults, and head, thorax and abdomen of 864 three-day-old virgin female adults. For conducting a ti ssue-specific expression 865 analysis of maccessin, total RNA was extracted from pooled samples of multiple 866 individuals across the following phases: virgin females and males three days 867 post-emergence, male accessory glands, and male bodies with removed accesso ry 868 glands. All samples were extracted by using TRIzol reagent (Invitrogen). 869 870 Quantitative RT-PCR 871 The first-strand cDNA was synthesized with HiScript® II Q RT SuperMix for qPCR 872 (+gDNA wiper) kit (Vazyme, Nanjing, China) using an oligo(dT)18 primer and 500 n g 873 total RNA template in a 10 μl reaction, following the instructions. Real-time qPCRs of 874 the various samples used the UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, 875 China). The PCR was performed in 20 μl mixture including 4 μl of 10 -fold diluted 876 cDNA, 1μl of each primer (10 μM), 10 μl 2 × UltraSYBR Mixture, and 6 μl RNase-free 877 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint water. The PCR conditions used were as follows: initial incubation at 95˚C for 10 min, 878 followed by 40 cycles of 95˚C for 10 s and 60˚C for 45 s. N. lugens 18S rRNA was 879 used as an internal control. Relative quantification was performed via the comparative 880 2−△△CT method [60]. 881 882 RNA interference 883 For lab-synthesized dsRNA, gfp, mipr, itp, itp/itpl and Maccessin fragments were 884 amplified by PCR using specific primers conjugated with the T7 RNA polymerase 885 promoter (primers listed in Supplementary Table S3). The dsRNA was synthesized by 886 a kit (MEGAscri pt T7 transcription kit, Ambion) according to the manufacturer’s 887 instructions. The integrity and quantity of the double-stranded RNA (dsRNA) products 888 were confirmed using 1% agarose gel electrophoresis and a Nanodrop 1000 889 spectrophotometer. Subsequently, the samples were stored at -70°C until further use. 890 In order to achieve the effect of silencing target genes, 5 μg/μl dsRNA was 891 injected into brown planthopper, male 40nl, female 50nl, and control group the same 892 amount of dsgfp. Total RNA was individually c ollected from each insect on the day 893 after reception assay , followed by extraction. The efficiency of gene silencing was 894 subsequently assessed through qPCR. 895 Peptide synthesis 896 Peptides were synthesized by Genscript (Nanjing, China) Co., Ltd. Myoinhibitory 897 Peptides and Sex Peptide mass was confirmed by MS and the amount of peptide was 898 quantified by amino acid analysis. Ion transport peptides and maccessin peptide were 899 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint immune recombinant proteins expressed in CHO cell. These proteins were purified by 900 AmMagTM Ni Magnetic Beads. The amino acid sequence of the peptides used in this 901 study are: N. lugens Myoinhibitory Peptide 1: (MIP1): AWRDLQSSWamide; 902 Myoinhibitory Peptide 2: (MIP2): GWQDMPSSGWamide; Myoinhibitory Peptide 3 903 (MIP3): GWQDLQGGWamide; Myoinhibitory Peptide 4 (MIP2): AWSSLRGTWamide; 904 D. melanogaster Sex Peptide: (SP): 905 WEWPWNRK{Hyp}TKF{Hyp}I{Hyp}S{Hyp}N{Hyp}RDKWCRLNLGPAWGGRC. 906 Mature proteins ITPa (comprising amino acids 23-113), ITPL (comprising amino acids 907 23-117), and maccessin (comprising amino acids 20-91), each fused with a 6xHis tag 908 at their C-termini (designated as protein-His tag), were expressed in Chinese Hamster 909 Ovary (CHO) cells. These proteins were subsequently purified using AmMagTM Ni 910 Magnetic Beads. Additionally, the mature ITPa protein with an amide modification at 911 the C -terminus (referred to as ITPa -amide) was also expressed in CHO cells. All 912 these proteins were codon-optimized for expression in mammalian cells. 913 914 Behaviour assays 915 1. Post mating response 916 For first mating assay, a couple of virgin females and males (3 days after eclosion) 917 were kept for 30 min in a 24 mm (diameter) × 95 (height) mm transparent circular tube 918 with rice s eedlings. The mated females were subjected to re -mating assay every 24 919 hours for 1 -5 days after first mating, while the virgin females of the same age were 920 used as controls. 921 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint For egg laying, virgin or mated fema les (3 days after eclosion) were kept in a 24 922 mm × 95 mm transparent circular tube with rice seedling. Each female was numbered 923 and moved into a new tube every 24 hours. The number of eggs in the rice seedlings 924 was counted. 925 2. Tests if SFPs induce a post-mating response 926 For experiments to test SFPs-injected females, virgin females that had eclosed 3 927 days earlier were selected for injection. Reception assays were performed with males 928 of the same age placed in a single pair in the tube 3h, 6h, 12h, 24h and 36h after 929 injection. Each female was injected with the equivalent of half of accessory gland. 930 On the day after eclosion, each virgin was injected with 30 nl SFPs or solvent and 931 placed in the transparent circular tube with rice seedling to lay eggs for 24 h. 932 3. Peptide injection to test virgin receptivity 933 For exp eriments using peptide -injected females (3 days after eclosion) , the 934 mating experiment was conducted 6 hours after injection, when females had fully 935 recovered from the wound. 936 On the day after eclosion, each virgin female was administered an 937 intra-abdominal injection of 30 nl of mature peptide or PBS. Subsequently, they were 938 housed in a transparent cylindrical tube with a rice seedling, where they were allowed 939 to oviposit for a period of 48 hours. 940 4. Effect of silencing female mipr gene on post-mating response 941 For the effect of sile ncing the female brown planthopper mipr gene on the 942 post-mating response, the experimental protocol is shown in Figure 2D. After 943 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint recovering for 1 day, the virgin females were mated with wild type males. The mated 944 females were re-mated with virgin males 2 days after the first reception assay. 945 Between the first reception and the re-mating assay, mated females were placed in 946 tubes with rice seedlings to lay eggs. 947 5. Effect of silencing the male maccessin gene on post-mating response 948 As for experiments using dsRNA-injected males, co-caging with female virgins of 949 the same age was 3-4 days after injection, when the maccessin gene was silenced to 950 the greatest extent. Mated females were collected and remated with wild -type virgin 951 males 2 days later. Between the reception and remating assays, the mated females 952 were introduced into tubes with rice seedlings to facilitate oviposition. 953 Following the daily activity of the brown planthopper, the mating experiment s 954 were scheduled between 3 p.m. to 7 p.m. The male and female insects were kept in 955 the tubes for half an hour to see if mating took place. Each expe riment was repeated 956 no less than 3 times, with at least 10 insects per repetition. 957 958 RNA-seq analysis 959 Total RNA from 150 virgin male accessory glands was isolated three days 960 post-emergence using TRIzol reagent (Invitrogen), following the manufacturer's 961 protocol. Library construction and sequencing was performed by Novogene with 962 Illumina HiSeq2000 platform (Novogene Bioinformatics Technology Co.Ltd, Beijing, 963 China). 964 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint After filtering out low-quality sequences, the raw data were subjected to analysis. 965 Sequence alignment was conducted aga inst the Nilaparvata lugens genome, 966 accessible via the NCBI database 967 (https://www.ncbi.nlm.nih.gov/genome/?term=Nilaparvata+lugens), using Hisat2 968 v2.0.5. The gene expression levels derived from RNA sequencing data were 969 normalized using the FPKM method, which accounts for both sequencing depth and 970 gene length in the calculation of read counts, making it a widely adopted approach for 971 estimating gene expression levels. Differential gene expression analysis was 972 executed with the DESeq2 R package (version 1.16.1). Subsequently, Gene Ontology 973 (GO) enrichment and KEGG pathway analyses were performed using the 974 clusterProfiler R package. 975 976 Proteome analysis 977 The tissues were isolated from 150 virgin male accessory glands three days 978 post-emergence. T he label -free quantitative method involves mass spectrometry 979 analysis of enzymatically digested protein s. Utilizing raw mass spectrometry data, a 980 search was conducted against the RNA -seq database to identify proteins based on 981 the search outcomes. Subsequently, an association analysis with the transcriptome 982 data was conducted to elucida te the relationships between protein expression and 983 gene sequences. 984 985 Statistics 986 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint We employed GraphPad Prism 9 software for data visualization and statistical 987 analysis. Data presented in this study were first verified for normal distribution by 988 D’Agostino– Pearson normality test. If normally distributed, Student’s t test was used 989 for pairwise comparisons, and one-way ANOVA was used for comparisons among 990 multiple groups, followed by Tukey’s multiple comparisons. If not normally distributed, 991 Mann–Whitney test was used for pairwise comparisons, and Kruskal–Wallis test was 992 used for comparisons among multiple groups, followed by Dunn’s multiple 993 comparisons. All data are presented as mean ± s.e.m. All data are collected from at 994 least four independent experiments. Every independent experiment used at least five 995 insects. 996 ACKNOWLEDGMENTS. 997 This research was supported by the National Key Research and Development 998 Program of China (2022YFD1700200) , the National Natural Science Foundation of 999 China (No. 32472542), the Guidance Foundation of the Sanya Institute of Nanjing 1000 Agricultural University (NAUSYMS15) and the Fundamental Research Funds for the 1001 Central Universities (No. KJJQ2024016). We thank Dr. Mariana Wolfner for 1002 commenting on an earlier version of this paper. 1003 Supporting information 1004 Author Contributions 1005 Conceptualization: Shun-Fan Wu, Dick R. Nässel. 1006 Data curation: Yi-Jie Zhang, Ning Zhang, Ruo-Tong Bu, Shun-Fan Wu. 1007 Formal analysis: Yi-Jie Zhang, Shun-Fan Wu. 1008 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint Funding acquisition: Shun-Fan Wu, Cong-Fen Gao. 1009 Investigation: Yi-Jie Zhang, Ning Zhang, Ruo-Tong Bu. 1010 Supervision: Shun-Fan Wu, Cong-Fen Gao, Dick R. Nässel. 1011 Validation: Yi-Jie Zhang, Ning Zhang, Ruo-Tong Bu. 1012 Writing – original draft: Yi-Jie Zhang, Shun-Fan Wu, Dick R. Nässel. 1013 Writing – review & editing: Shun-Fan Wu, Dick R. Nässel. 1014 1015 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 27, 2024. ; https://doi.org/10.1101/2024.10.26.620448doi: bioRxiv preprint

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