Galactoside in the male ejaculate evaluated as a nuptial gift by the female nutrient sensing neurons

doi:10.21203/rs.3.rs-2137467/v1

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Galactoside in the male ejaculate evaluated as a nuptial gift by the female nutrient sensing neurons

https://doi.org/10.21203/rs.3.rs-2137467/v1

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Nuptial gift-giving or courtship feeding is prevalent across. In an extreme case, females eat male body parts; in another case, females derive nutrients from males’ seminal fluids1. The nursery web spider, Pisaura mirabilis, females store more sperm from nuptial gift-giving males than those offering no gift with the identical copulation duration2. However, it is not known how females evaluate nuptial gifts and adjust sperm storage. Here, we show that Drosophila melanogaster females hold the ejaculate of starved males for shorter period and store less number of their sperm. A knock-down of the sugar transporter Tret1-1 in neurons that express the neuropeptide diuretic hormone 44 (Dh44-PI) critical for the ejaculate holding3 and detection of the nutritional value of sugar4 impairs the female’s ability to adjust the ejaculate holding period according to the male energy status. A galactoside selectively present in the male ejaculate activates Dh44-PI neurons in the brain to secrete Dh44, and starvation reduces its production by approximately 50%. RNAi-knockdown of glycosyltransferases, including UGT305A1, allows males to produce significantly lower amounts of the galactoside without starvation. Females mated with UGT305A1 knockdown males hold the ejaculate shorter, store less sperm and lay fewer eggs. The seminal galactoside enters the female hemocoel during mating, stimulates the activity of Dh44-PI neurons in the brain, augments the secretion of Dh44, and increase the ejaculate holding period, thereby enabling the uptake of injected nutrients and sperm. These evidence suggest that the galactoside signals reflecting the quality of male partners increase sperm storage and may be a source of nutrients used for egg production. Together, this study provides a molecular and cellular framework for understanding how females evaluate courtship feeding and optimize sexual selection after mating.

Biological sciences/Neuroscience/Sexual behaviour

Biological sciences/Evolution/Sexual selection

In nuptial gift-giving species, starvation often leads polyandrous females to accept more partners, thus acquiring additional nutrients to ensure reproductive fitness^5,6. In this situation, females should be able to evaluate nuptial gifts as an energy source. To understand the neural and molecular basis of this female behavior, we set out to test whether females in the genetically amenable insect Drosophila melanogaster are able to modify their receptiveness to nuptial gifts according to their energy needs. D. melanogaster females acquire nutrients from the male ejaculate⁷, but can mate only few times because sex peptide in the ejaculate suppresses further mating⁸. Inferred from this observation, the female energy status would influence the ‘ejaculate holding period (EHP),’ the period from the end of copulation to the expulsion of ejaculate, including unstored sperm, from the uterus, which serves as a time window for females to absorb nutrients from the ejaculate. Because a longer EHP is considered to absorb greater amounts of nutrients, we reasoned that starved females would increase EHP. To test this, we subjected wild-type Canton-S (CS) or w¹¹¹⁸ females to various periods of starvation prior to mating with well-nourished CS males. Up to 24 hours of starvation had no impact on female mating receptivity and copulation duration (Extended Data Fig. 1a, b), but longer durations of starvation increased EHP (Fig. 1a, Extended Data Fig. 1c). We found no significant difference in EHP between flies fed a standard diet (SD) or a carbohydrate-only diet (CD), suggesting that the CD is sufficient to meet the nutritional requirement for EHP (Extended Data Fig. 1d). We also found that either 12 hours of SD or 12 hours of CD, but not 12 hours of a protein-only diet (PD), can rescue the increase of EHP that was induced by 12 hours of starvation (Extended Data Fig. 1e). Starvation resulted in a decrease in the sugar levels in the body and internal milieu (Extended Data Fig. 1f, g), but also caused a plethora of other physiological changes. To determine the relationship between glycaemia and EHP, we directly injected 1 μg of trehalose, a major insect blood sugar, into the hemocoel of females 4 hours before mating. This sugar injection shortened EHP by approximately 20 minutes (Fig. 1b).

Nuptial gifts in general are energetically costly to produce⁹. Thus, males in inhospitable environments would produce and transfer less amount of nuptial gifts during copulation. When we allowed starved females to copulate with males that had been starved for 24 hours, the resulting EHPs were 23 minutes shorter than those in females mated with well-nourished males (Fig. 1c). Well-nourished females, however, did not alter EHP in response to starvation of their mates. Furthermore, we found that starved females store 19.4% less sperm from males starved for 24 hours than from well-nourished males (Fig. 1d). In contrast, well-nourished females did not reduce sperm storage in response to starvation of their mates. Energy-state dependent acceptance of nuptial gifts by females was also reported in other Drosophila species. For D. subobscura, where males regurgitate food to feed females during courtship, starved females prefer to mate with gift-giving males over none-gift-giving males, whereas well-nourished females do not discriminate between these males¹⁰.

We previously reported that Dh44-PI, a group of six Dh44-expressing cells in the brain pars intercerebralis, regulates both EHP and nutrient sensing^3,4. Thus, we examined its role in adjusting EHP in response to ejaculate quality. Dh44-PI neurons are essential for the post-ingestive selection of nutritive sugars⁴. These neurons generate Ca²⁺ oscillations in response to nutritive D-glucose, but not to nonnutritive L-glucose. The sugar transporter blocker phlorizin can suppress nutritive sugar-induced Ca²⁺ oscillations⁴. Thus, we reasoned that sugar transporter(s) in Dh44-PI critical for post-ingestive nutrient selection may also be important for EHP. When we knocked down 23 Drosophila sugar uniporters and sodium co-transporters in Dh44-PI neurons using transgenic RNAi, we found that female flies depleted for trehalose transporter 1-1 (Tret 1-1) were impaired in selecting nutritive D-glucose over nonnutritive L-glucose in a two-choice assay (Extended Data Fig. 1h). Tret1-1 transports both glucose and trehalose¹¹, suggesting that its knockdown would result in an underestimation of glycaemia, mimicking the effect of starvation. Indeed, flies in which Tret1-1 was knocked down showed an increase in EHP (Fig. 1e, Extended Data Fig. 1i). Furthermore, Tret1-1 knockdown flies appeared to be largely insensitive to the energy levels of their mates. Starved males did not lower the EHP of Tret1-1 knockdown flies (Fig. 1f). Thus, Dh44-PI neurons seem to be essential for adjusting EHP in response to ejaculate quality, particularly when females are low on energy.

Flies lacking Dh44 in Dh44-PI neurons exhibited an extremely short EHP (<5 minutes)³. Mating episodes lead to the secretion of Dh44 that is required for establishing normal EHP (~90 minutes). To understand how Dh44-PI and its secretory responsiveness to nutritive sugars enable starved females to modulate EHP, we sought to determine Dh44 levels in starved females. Starvation significantly increased Dh44 protein levels in Dh44-PI cells (Fig. 2a). Because the hemolymph sugars activate Dh44-PI⁴, hypoglycaemia induced by a prolonged period of starvation predictably reduces Dh44-PI neural activity, leading to accumulation of Dh44 peptide inside Dh44-PI cells. This hypothesis suggests that Dh44-PI neurons continuously respond to the hemolymph sugar to regulate their secretion of Dh44 in vivo. Because Ca²⁺ activity in Dh44-PI neurons is directly associated with Dh44 secretion⁴, we monitored Ca²⁺-dependent changes in GCaMP6m fluorescence through a small window on the head capsule of a live fly¹². Using this preparation, we were able to observe robust and spontaneous [Ca²⁺]_i oscillations in Dh44-PI neurons (Fig. 2b). Notably, well-nourished females produced oscillations with larger amplitude than starved females (Fig. 2b, c). Thus, well-nourished females secrete more Dh44 peptide leaving less Dh44 in Dh44-PI neurons. This difference in the basal rate of Dh44 secretion is consistent with the observation that less Dh44 is found in well-nourished females.

Next, we examined Dh44 secretion in starved and fed females 4 hours after mating. We observed a marked reduction in the levels of Dh44 peptide in mated females, compared with those in virgin females (Fig. 2d, e). This mating-induced Dh44 reduction was pronounced in both starved and well-nourished females. Because [Ca²⁺]_i is positively correlated with secretory activity in many neuropeptide neurons¹³, we next examined whether mating increases [Ca²⁺]_i in Dh44-PI. To compare [Ca²⁺]_i in Dh44-PI before and after mating, we employed an end-point Ca²⁺ monitoring tool called TRIC (i.e., Transcriptional Reporter of Intracellular Ca²⁺) that induces EGFP expression in proportion to [Ca²⁺]_i¹⁴. First, we conducted control experiments by comparing TRIC activity in fed versus starved condition. Consistent with our in vivo Ca²⁺ imaging results, well-nourished virgin females showed stronger TRIC activity than starved virgin females (Extended Data Fig. 2a). We further examined TRIC activity in Dh44-PI 5 hours after mating, and found that TRIC activity in mated females was 14 ± 2.7% higher than that in age-matched virgin females (Extended Data Fig. 2b). This increase in TRIC was significant in starved condition, but not in fed condition (Extended Data Fig. 2c), likely due to a ceiling effect of saturated TRIC activity in fed condition. Hypoglycaemia during a period of starvation reduces the baseline Dh44-PI release and therefore, increases the Dh44 pool. By contrast, hyperglycaemia increases the baseline Dh44-PI release and decreases the Dh44 pool. Thus, females starved before mating had a larger Dh44 pool and showed a longer EHP than females well-nourished.

We further investigated the relationship between the Dh44 pool and EHP by chronically inhibiting or activating Dh44-PI prior to mating. To inhibit the activity of Dh44-PI, we expressed the light-gated anion channel Guillardia theta anion channelrhodopsin 1 (GtACR1)¹⁵ in Dh44-PI neurons. Compared with the controls that did not receive all-trans-retinal (ATR), 18 hours of optogenetic inhibition increased the Dh44 peptide levels by 67 ± 21.3% and EHP by 24 minutes (Fig. 2f). In contrast, thermal activation of Dh44-PI via the warmth-gated cation channel Drosophila transient receptor potential A1 (dTrpA1)¹⁶ reduced Dh44 peptide levels by 42 ± 9.6% and EHP by 56 minutes, compared to the controls (Fig. 2g). These results suggest that an increase in Dh44-PI activity and a decrease in the Dh44 pool before mating shortens EHP, whereas a decrease in Dh44-PI activity and an increase in the Dh44 pool prolongs EHP.

In some organisms, the male ejaculate has been shown to be rich in nutritive sugars: fructose and glucose in humans and fructose, glucose and trehalose in bees^17,18. As such, seminal sugars may serve as a nuptial gift that contributes, at least in part, to an increase in EHP. We next employed the mass spectrometry to identify a sugar or a sugar-derivative in the male ejaculate that enters the female circulation and eventually activates Dh44-PI neurons and Dh44 secretion after mating. We found no evidence in the male ejaculate eluent of mass units that correspond to glucose, trehalose, or fructose. Instead, we observed a robust signal at a molecular weight of 377, [M-H]^-= 376.10142 (Fig. 3a). In subsequent examinations of male and female reproductive tissue extracts, we found that this peak was present exclusively in males, especially in male accessory glands (MAG) (Fig. 3b, Extended Fig. 3a-c). Hereafter, we refer to this substance as the Drosophila Seminal Galactoside of molecular weight 377 (SG377). SG377 was the major peak in the hydrophilic eluent of MAG extracts (Fig. 3b). Trace amounts of SG377 were also present in male ejaculatory ducts and testes (Extended Data Fig. 3c). We found that starved males produced only half amount of SG377 compared to well-nourished males (Fig. 3c), indicating that energy state strongly affects the level of SG377 in the male ejaculate. Finally, we detected SG377 in hemolymph collected from females 4 hours after mating, but not from virgin females (Fig. 3d, Extended Data Fig. 3d). In D. melanogaster, copulation induces physical damage to the female reproductive system, allowing components of the ejaculate to diffuse passively into the hemocoel¹⁹. This may be a plausible route by which SG377 enters the female hemolymph and influences the neuronal activity in the brain.

We deduced the molecular formula of SG377—C₁₁H₂₄O₁₁NP—via electrospray ionization high-resolution mass spectrometry ([M-H]^- C₁₁H₂₃O₁₁NP^-, calculated m/z 376.10142, found 376.10139, Δ 0.08 ppm mass error). We proceeded to a structural identification of SG377 via ¹H, ¹³C, and ³¹P NMR and 2D NMR spectral analysis (Supplementary information). The combined ¹H-¹H COSY, ¹³C-¹H HMQC, and HMBC spectra revealed the structure of SG377 as 1-O-[4-O-(2-aminoethylphosphate)-β-_D-galactopyranosyl]-x-glycerol (Fig. 3e), which was previously identified in D. melanogaster male accessory glands, but had not yet been associated with a biological function²⁰. The ³¹P-NMR analysis of the MAG extracts found that a male fly produces approximately 1.2 nmol of SG377. In a single mating, the male transfers 39 ± 3.6% of the SG377 pool to the female, and the SG377 pool is restored after ~24 hours (Extended Data Fig. 3e).

Because SG377 enters female hemolymph after mating, we asked whether it could generate Ca²⁺ oscillations in Dh44-PI neurons expressing GCaMP6m. In our brain preparations that contain tetrodotoxin (TTX) to eliminate synaptic inputs of Dh44-PI neurons, ~30% of Dh44-PI responded to an application of MAG extract corresponding to 0.48 mM SG377 (Fig. 3f, Extended Data Fig. 4a). Dh44-PI also responded to 1 mM glucose with a similar frequency. Notable qualitative differences, however, were found in the Ca²⁺ transients induced by glucose versus MAG extract. Glucose generated oscillatory responses with higher frequency and shorter duration than MAG extract (Fig. 3f). The oscillation durations induced by 1 mM and 2 mM glucose were 13 ± 3 and 19 ± 6 seconds, respectively, whereas the oscillation duration induced by MAG extract (i.e., 0.48 mM SG377) was 53 ± 25 seconds. Because MAG extract contains other substances besides SG377, we synthesized SG377 and found that the application of 1 mM synthetic SG377 induced a Ca²⁺ response in ~40% of Dh44-PI (Extended Data Fig. 4b). Like MAG extract, synthetic SG377 also produced longer-lasting Ca²⁺ dynamics than glucose (Fig. 3f), with the oscillation duration of 89 ± 23 (for 0.1 mM) and 128 ± 45 (for 2 mM) seconds. Isolated brains treated with as low as 2 mM synthetic SG377 exhibited a significant decrease in Dh44 protein levels in Dh44-PI neurons, indicating that SG377 induces Dh44 secretion (Fig. 3g). Because MAG extracts from individual males contain ~1.2 nmol of SG377 and a male transfers ~ 40% of the MAG content during a single mating (Extended Data Fig. 3e), SG377 levels would reach ~6 mM in the female hemolymph. Notably, 2 mM of SG377 robustly stimulated Ca²⁺ oscillations in Dh44-PI neurons with a latency of 8.3 ± 1.4 minutes. During a copulation episode lasting ~15 minutes, the ejaculate transfer is completed within 7-8 minutes after copulation²¹. Thus, Dh44-PI neurons can initiate Dh44 secretion roughly by the end of copulation. Together, we concluded that the sugar-derivative SG377 transferred from male partners stimulates Dh44-PI neuronal activity and promotes Dh44 secretion upon copulation.

Sugar-induced Ca²⁺ oscillations in Dh44-PI were suppressed by the sugar transporter blocker phlorizin⁴. We found that pre-treatment with phlorizin also abolished Ca²⁺ transients induced by MAG extract (Extended Data Fig. 4c). Since Ca²⁺ oscillations induced by either glucose or SG377 require a sugar transporter, we therefore asked whether Tret1-1 knockdown eliminates the SG377-induced Ca²⁺ oscillations (Fig. 3h). Consistent with the result obtained using phlorizin, Tret1-1 knockdown significantly reduced Ca²⁺-transients induced by SG377. Tret1-1 knockdown also attenuated the SG377-induced Dh44 secretion (Fig. 3i). That the effect of SG377 on Dh44-PI requires the sugar transporter Tret1-1 suggests that Dh44-PI neurons import SG377 through this transporter. Glucose metabolites such as glucose-6-phosphate are involved in generating glucose-induced Ca²⁺ oscillations in Dh44-PI, which were blocked by the hexokinase blocker alloxan⁴. Alloxan pretreatment, however, had a limited effect on SG377-induced Ca²⁺ transients (Extended Data Fig. 4c), suggesting that the intracellular pathway activated by SG377 is distinct from the pathway triggered by glucose or other hemolymph sugars.

SG377 carries b-galactose attached to glycerol, a moiety commonly found in glycoglycerolipids. MAG is rich in lipids and lipases²². Thus, we speculated that SG377 production from MAG would require a glycosyltransferase capable of transferring galactose or other hexoses to a lipid acceptor with a glycerol moiety (e.g., diacylglycerol). We searched the Carbohydrate-Active enZymes (CAZy) database^23,24 and found the GT28 gene family has 1,2-diacylglycerol 3-b-galactosyl-transferase activity²⁵ (Supplementary Table S1). The D. melanogaster genome contains 18 genes with GT28 domain, 6 of which are expressed in MAG (Supplementary Table S2 and S3; Extended Data Fig. 5a). MAG-specific RNAi knockdown showed that one or more RNAi against UGT305A1, UGT302C1, UGT86Dj, and UGT36Dc significantly reduced SG377 content (Fig 4a; Extended Data Fig. 5b). This indicates functional redundancy between these enzymes important for SG377 production. In D. melanogaster, MAG matures over 4-5 days after emergence²⁶. The SG377 pool is small at the emergence and then increases rapidly (Extended Data Fig. 5c). We investigated the mRNA levels of five MAG-expressing GT28 genes during the first two days after emergence. As with the SG377 pool, we found a significant increase in UGT305A1 expression during this period (Extended Data Fig. 5a; Supplementary Table S3).

We employed UGT305A1 knockdown males, which produce ~40% less SG377 than control males, to investigate the role of SG377 in EHP regulation and sperm storage (Fig. 4b, c). Similar to those mated with starved males, starved females mated with UGT305A1 knockdown fed males had shorter EHP and stored less sperm than those mated with control males. This is consistent with the results of starved males, which produce ~50% less SG377 compared to well-nourished males (Fig. 1c, d). Consistent with the previous result, well-nourished females showed no changes in EHP and sperm storage when mated with UGT305A1 knockdown males.

Finally, we investigated the role of SG377 in female fertility. Females mated with UGT305A1 knockdown males were significantly less fertile than females mated with control males, with 40-52% reductions in cumulative egg-laying over 7 days (Fig. 4d). In this experiment, the effect of SG377 on sperm storage was minimal because females were well-nourished prior to mating. Also, a decrease in SG377 in the male ejaculate did not significantly affect sperm vigor and viability of eggs and offspring produced by recipient females (Extended Data Fig. 5d, e). Egg development requires a significant amount of phosphorus. D. melanogaster females incorporate phosphorus from the male ejaculate into DNA and RNA in their ovaries and eggs⁷. Thus, SG377, which has a phosphoethanolamine moiety and is highly abundant in the ejaculate, could also be a good source of male-derived phosphorus, with Dh44-PI in females directing the uptake of more SG377. As can be inferred from these evidence, SG377 may improve female fertility, at least in part, by supplying nutrients necessary for ovarian or oocyte development (Fig. 4e).

In our model, glycaemia continuously drives the basal levels of Dh44 secretion, whereas SG377 transferred from male partners triggers massive Dh44 secretion, the amount of which determines the duration of EHP (Fig. 2; Extended Data Fig. 6a). As such, the glycaemia-dependent baseline secretion of Dh44 determines the size of the Dh44 pool in Dh44-PI neurons that is available for the SG377-induced Dh44 secretion. The Dh44 levels in Dh44-PI neurons, particularly in starved females who had been mated with well-nourished males, were large enough to sustain the massive Dh44 secretion induced by SG377. In this situation, the size of the Dh44 pool is a major determinant of EHP duration (Extended Data Fig. 6b, c). By contrast, females mated with starved males who produce ~50% of the normal SG377 level receive considerably less SG377. In this situation, the amount of SG377 in the ejaculate would determine EHP, particularly in starved females with a larger Dh44 pool in their Dh44-PI neurons (Extended Data Fig. 6d, e). These would explain why females starved before mating can change EHP more readily than well-nourished females.

Nuptial gifts, in general, convey the information about male attractiveness or quality, and females often prefer gift-giving males^10,27 There is also a clear link between nuptial gifts and sexual selection that occurs after mating². Like D. melanogaster, the female red flour beetle Tribolium castaneum store more sperm when mating with well-fed males than when mating with starved males²⁸. Because SG377 prolongs EHP and promotes the uptake of sperm by activating Dh44-PI in the female brain, it presents a novel inter-sexual signaling mechanism that signals male quality and promotes sperm storage (Fig. 4e).

Data availability statement: All data generated during this study will be included in this published article (and its supplementary information files).

Acknowledgements: We thank Drs. H. i. Jung (Yonsei University) and S. J. Moon (Yonsei University) for the statistical analysis advice, Drs. M-R. Song (GIST) and Y. Jun (GIST) for the insightful discussion, and K. Kim (GIST) for the graphical illustration. We also thank B. Park and S-H. Kim for excellent technical assistance. This work was supported by the GIST Research Institute (GRI) in 2022, and by Basic Science Research Programs (NRF-2018R1A2A1A05079359, NRF-2019R1A4A1029724, and NRF- 2022M3E5E8081194, and NRF-2022R1A2C3008091) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP) of the Republic of Korea. Stocks were obtained from the Korea Drosophila Resource Center (NRF-2022M3H9A1085169), the Bloomington Drosophila Stock Center, and the Vienna Drosophila Resource Center.

Author Contributions: K-M.L., S-J.K., S.H.P., T.Y., I.S., M.Y., C.Z. and M.D. performed the experiments. S.H.P., Z-Y.P., S.L., J-I.K., G.S.B.S. and Y-J.K. analyzed data. Y-J.K. supervised the project and wrote the manuscript with inputs from G.S.B.S.

Fly stocks.

Flies were cultured on a standard diet (SD) containing dextrose, cornmeal, and yeast at room temperature in a 12 hours:12 hours light:dark cycle. All behavioural assays were performed at 25°C in Zeitgeber time (ZT) 5:00-11:00. Virgin males and females were collected at eclosion. Males were aged individually for 4-6 days; females were aged for 3-4 days in groups of 15-20. Starved flies were reared on 1% agar (Nacalai Tesque, 01056-15). For a carbohydrate-only diet (CD), 4% sucrose was added to 1% agar media. For a protein-only diet (PD), 6% HuntAA was added to 1% agar media²⁹. The following transgenic lines have been described previously: Canton-S (CS)³, P_Dh44-Gal4⁴, UAS-GtACR1¹⁵, UAS-dTrpA1¹⁶, and protamine-GFP³⁰. Cha-Gal80;Dh44-Gal4 is from the Korea Drosophila Resource Center (stock number, 10333). The following stocks are from the Bloomington Drosophila Stock Center: UAS-GCaMP6.0m (42747), w[*] P{y[+t7.7] w[+mC]=10XUAS -IVS-mCD8::RFP}attP18 P{y[+t7.7] w[+mC]=13XLexAop2-mCD8::GFP}su(Hw)attP8; P{y[+t7. 7] w[+mC]=nSyb-MKII::nlsLexADBDo}attP24/CyO; M{UAS-p.65AD::CaM}ZH-86Fb (61679, for TRIC), cn bw (264), Prd-F-Gal4 (1947), UAS-Ugt35E2-IR1 (67307), UAS-Alg13-IR1 (67799), UAS-Ugt304A1-IR1 (62258), and UAS-Ugt37D1-IR1 (64567). The following RNAi stocks are from the Vienna Drosophila Resource Center; w¹¹¹⁸ (stock number, 60000), UAS-Tret1-1-IR1 (103045), UAS-Tret1-1-IR2 (8126), UAS-Tret1-1-IR3 (52361), UAS-Tret1-1-IR4 (52360), UAS-Ugt305A1-IR1 (108715), UAS-Ugt305A1-IR2 (45926), UAS-Ugt302C1-IR1 (8573), UAS-Ugt302C1-IR2 (105923), UAS-Ugt304A1-IR2 (8577), and UAS-Ugt37D1-IR2 (47655).

Behavior assays.

For determining the ejaculate-holding period (EHP), defined as the period between the end of copulation and sperm ejection, virgin females were paired individually with naïve males in 10 mm diameter chambers. Immediately after copulation concluded (usually within 30 minutes), males were removed and females were videotaped with a digital camcorder (SONY, DCR-SR47). EHP was normalized to yield DEHP by subtracting by the mean of the reference EHP. We assayed EHP in the absence of food because Dh44-PI neurons regulate feeding⁴, and therefore mating-induced Dh44-PI activation (see main text) likely stimulates feeding activity. If females access food, the potential interaction between Dh44-PI neurons and the feeding activity may produce a longer EHP. For sperm storage analysis, virgin females mated with naïve protamine-GFP males the sperm heads of which express GFP were frozen by transferring to -80°C. Then the seminal receptacles of females 6 hours post-mating were dissected in PBS and the number of sperm was manually counted using a fluorescence microscope (Olympus IX71). For mating assays, the virgin females were paired individually with naïve CS males and videotaped for 1 hour. For the two-choice preference assay, we used the method described previously³¹. Briefly, approximately 40 male flies 4-8 days old were starved for 18-22 hours and then given a choice between 200 mM D-glucose (Sigma, 49139) and 200 mM L-glucose (Carbosynth, MG05247), each colour-coded with tasteless food dyes (McCormick), for 2 hours. Food preference was scored as percent preference index (% PI), shown below: %PI = ((# flies that ate food 1 + 0.5 * # flies ate both) – (# flies that ate food 2 + 0.5 * # flies that ate both)) / (total # flies that ate). For egg-laying assays, a previously described protocol was followed³². Virgin w¹¹¹⁸ females were aged in groups for 3 days after eclosion, and virgin males were aged individually for 5 days after eclosion. For mating, virgin females were paired individually with naïve males in 10 mm diameter chambers. Each female was allowed to lay eggs in SD vials for 7 days, and the number of eggs laid was counted every 24 hours. For egg-to-pupa viability analysis, the mated females lay eggs in SD vials for 2 days, and the number of pupae that emerged from the eggs was counted on the 5-6 days after laying eggs. For sperm competition analysis, we modified a method described previously³³. Virgin cn bw females were aged in groups for 3 days after eclosion, and virgin males were aged individually for 5 days after eclosion. For defense assays, cn bw females were individually mated with test males in SD vials for 2 hours. After 3 days, the females were individually re-mated with cn bw males for 12 hours in the same vial and transferred to a new SD vial. For offense assay, cn bw females we mated with cn bw males and 3 days later with test males. Paternity was determined by allowing double mating females to lay eggs for 3 days and examining eye colour of the offspring.

Optogenetic silencing and thermal activation experiments

For optogenetic silencing with GtACR1, freshly eclosed females were fed SD containing 1 mM all-trans-retinal (ATR) (Sigma, R2500) in constant darkness for 2-3 days. The flies then were exposed to light from a blue LED (460 nm, 24mW/mm²) for 18 hours prior to behavioral analysis. For thermal activation with dTrpA1, freshly eclosed females were stored in SD vials for 1 day at 20°C and then transferred to 20°C or 30°C for 2 days prior to behavioral analysis.

Sugar quantification.

To measure whole body sugar, we modified a method described previously³⁴, in which 15-20 female flies were collected and homogenized in 200 µl 0.25 M Na₂CO₃ buffer and incubated at 95 °C for 2 hours. 120 µl 1 M acetic acid and 500 µl 0.25 M Na-acetate (pH 5.2) were added and the solution was centrifuged (12,000 rpm, 24 °C, 10 minutes). 200 µl of supernatant were incubated overnight at 37 °C with 2 µl porcine kidney trehalase (Sigma, T8778) to convert trehalose into glucose. Hemolymph was collected, using a modified method described previously³⁵. 30-40 female flies were punched on the thorax with a tungsten needle and were collected in a small 0.5ml tube with a hole underneath. The small tube was then placed in 1.7 ml tubes and centrifuged (5000 rpm, 4 °C, 5 minutes). 1 µl hemolymph was diluted in 50 µl of H₂O and incubated overnight at 37°C with 0.5 µl porcine kidney trehalase. A 10 µl sample of the solution was added to 90 µl of buffer from a glucose assay kit (Sigma, GAHK20), and its glucose concentration was quantified at 340 nm with a spectrophotometer (Molecular Devices, FlexStation 3).

Immunohistochemistry.

Brains were dissected under PBS (pH 7.4) and fixed for 30 minutes at room temperature in 4% paraformaldehyde in PBS. After washing and blocking, the brains were incubated in primary antibody for 24 hours at 4 °C, and in secondary antibody for 24 hours at 4 °C. The antibodies used were: rabbit anti-Dh44 (1:2000)³⁶ and Alexa 488-conjugated goat anti-rabbit (1:1000; Invitrogen, A11008). To quantify SG377-induced Dh44 secretion, the dissected brains were incubated with 100 µl of sugar-free adult hemolymph-like Ringer’s saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 8.2 mM MgCl₂, 4 mM NaHCO₃, 1 mM NaH₂PO₄, 5 mM HEPES)³⁷ with or without synthetic SG377 for 30 minutes before the fixation and anti-Dh44 staining. For Ca²⁺ monitoring with TRIC, dissected brains from females carrying Dh44-Gal4, Cha-Gal80, nSyb-MKII::nlsLexADBD, UAS-p65AD::CaM, and LexAop-mCD8::GFP were mounted after fixation without antibody staining. Tissues were mounted with Vectashield (Vector Laboratories, H-1000). Images were acquired with an LSM 700/Axiovert 200M with 40x lens (Zeiss) and processed in Image J³⁸. For each sample, 15 confocal images of 5 µm thickness were captured to produce a Z-stacked image with averaged intensity.

Calcium imaging.

For in vivo live calcium imaging, a cold anesthetized virgin female carrying P_Dh44-Gal4 and UAS-GCaMP6.0m was fixed on a metal plate by light-curing instant adhesive (ThreeBond, TB1773E). A piece of fly head cuticle was cut and removed with the blade of a disposable needle (Kovax, 25G 5/8") under sugar-free adult hemolymph-like Ringer’s saline³⁷. The brain was observed with an Axio Examiner A1 (Zeiss) equipped with a 40x water immersion lens (Zeiss, 421767-9970), Luca^EM R 604 camera (ANDOR technology, DL-00419), and LED light source (CoolLED, 244-1400). During recording, the brain was bathed in sugar-free adult hemolymph-like Ringer’s saline. To normalize the fluorescent value of each fly, Dh44-PI neurons expressing both the Ca²⁺ indicator GCaMP6.0m and the nucleus-targeted red fluorescent protein RedStinger was recorded under RFP excitation light (565 nm) for 60 frames (one frame per second) and then recorded for an additional 100 frames under GFP excitation light (470 nm). The normalized relative fluorescence was measured as (F_gi- F_bi)/F_r , where F_gi is the GCaMP fluorescence of a ROI (i.e., Region of interest) on cell body for the i^th frame, F_bi is the GCaMP fluorescence value of a ROI on the background, and F_r is the mean RFP fluorescence value of the first 60 frames. To calculate the area under the curve, the relative fluorescence values from all 5-6 visible cells in a brain preparation were averaged, integrated for 100 frames, and then normalized with the mean of the data from the fed group (n = 5). Image analyses and processing were done with Metamorph (Molecular Devices) and Exel (Microsoft).

For ex vivo live calcium imaging analysis, 4-5 days old virgin females carrying P_Dh44-Gal4 and UAS-GCaMP6.0m were dissected under sugar-free adult hemolymph-like Ringer’s saline. The brain was mounted on a cover slide with a 40 µl drop of saline bath containing 0.5 µM TTX and observed with a confocal microscope LSM 700/Axiovert 200M with 20x lens (Zeiss). For treatment with phlorizin or alloxan, the brain was mounted in a 40 µl saline bath containing 0.5 µM TTX and 1.25 µM phlorizin or 10 µM alloxan. The brain was recorded under GFP excitation light (470 nm) during 150 frames (1 frame per 2 seconds). Subsequently, 10 µl of D-glucose or SG377 in saline was added and the recording was resumed for an additional 750 frames. Image analyses and processing were performed using Image J and OriginPro9.1. F₀ is calculated by averaging fluorescence values from 10 consecutive frames that show baseline levels of GCaMP fluorescence. Ca²⁺ transients were defined as a peak reaching >50% of their maximum ∆F/F₀ within a 10-frame-window. % Responders is the proportions of cells that have shown more than one Ca²⁺ transient. Max. peak amplitude is the maximum value of ∆F/F₀. The oscillation number is the number of Ca²⁺ transients observed in each cell. The oscillation duration is calculated from the Ca²⁺ oscillation or peak with the maximum peak intensity in each cell and is defined as the time interval between 50% of the rising phase and 50% of the decaying phase to the peak ∆F/F₀. The area under the curve is calculated by integrating ∆F/F₀ values over baseline after D-glucose or SG377 application. Activity onset latency is defined as the time point when the first Ca²⁺ transient reaches 50% of its peak ∆F/F₀ in each cell. Cells that showed a strong baseline increase (> 50% ∆F) without a Ca²⁺ transient during the total recording period were excluded from the dataset (n = 226/514).

SG377 extraction and enrichment.

To collect the male ejaculate for biochemical analysis, we used Dh44-RNAi females, which expel a white spermatophore-like sac made of semen and sperm shortly after mating³. The spermatophore-like sac or reproductive organs were homogenized in 10 µl of 0.1% formic acid. After spin-down (12,000 rpm, 4 °C, 10 minutes), the supernatant was filtered by Zip-Tip C₁₈ (Sigma, ZTC18S096) to remove protein and other hydrophobic substances. For the comparison of SG377 contents in male reproductive tissues, dissected tissues of 10 males were collected and homogenized in 10 µl of 0.1% formic acid. After spin-down, 10 µl supernatant was mixed in 10 µl of 5.55 mM glucose as an internal standard. For the relative quantification of SG377 in hemolymph, 1 µl hemolymph was mixed in 5 µl of 0.1% formic acid and 1 µl of 2.68 mM ¹³C glucose as an internal standard. To avoid contaminations from the male ejaculate that might remain in the females, we collected hemolymph from females 4 hours after mating when sperm ejection was completed.

Mass Spectrometry and NMR spectroscopy.

Mass Spectrometric analyses were performed using a high mass accuracy, high resolution mass spectrometer (Q Exactive Plus mass spectrometer; Thermo Fisher Scientific, Bremen, Germany) and a linear trap mass spectrometer (LTQ; Thermo Fisher, Bremen, Germany) with a microspray ESI source operated in a full mass spectral acquisition mode for (+) and (-) MS scan in the mass range of m/z 100-500. NMR Spectroscopy– ¹H, ¹³C, ³¹P, and 2D-NMR experiments were performed using either a Varian Unity INOVA 500 or a Varian VNMRS 600 MHz NMR spectrometer. MAG extract collected from 200 males was analyzed with the NMR Spectroscopy. The ³¹P-NMR signal intensities of MAG extract collected from 200 males were compared with those of 1mM Na₃PO₄ standard to estimate the SG377 content in MAG extract from a single male.

Synthetic SG377.

1-O-[4-O-(2-aminoethylphosphate)-β-_D-galactopyranosyl]-x-glycerol in a R and S isomer mix was custom synthesized by WuXi AppTec Co. (China).

Carbohydrate-Active enZymes (CAZy) annotation

To annotate proteins with the CAZy domain, the peptide sequences of Drosophila melanogaster (BDGP6.32 assembly version, Ensembl database)³⁹ were mapped with profile hidden Markov models (HMM) of CAZy families, which were deposited at dbCAN, a CAZy domain HMM database²³ through HMMER v3.3, a homologs searching tool (hmmer.org) (e-values < 10-5) (See annotations in Supplementary Table S1).

QuantSeq 3’mRNA-seq.
Total RNA was extracted from 50-75 MAG for each sample using TRIZOL (Invitrogen, Korea) according to the manufacturer’s instructions. The construction of library was performed using QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer’s instructions. In brief, each 500 ng total RNA were prepared and an oligo-dT primer containing an Illumina-compatible sequence at its 5’ end was hybridized to the RNA and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5’ end. The double-stranded library was purified by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library is purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc., USA). QauntSeq 3’ mRNA-Seq Library generation and NGS run (>10M read/sample) were performed by ebiogen Inc (Korea). Raw sequencing reads were mapped on the transcript sequence of Drosophila melanogaster (released version 6.42, FlyBase database)⁴⁰ using kallisto gene expression quantification program⁴¹ to generate count value and transcript per million base pair (tpm) value for each transcript (see expression quantification table in Supplementary Table S2 and S3).

Statistics and reproducibility.

Experimental flies and genetic controls were tested under the same condition, and data are collected from at least two independent experiments. Statistical analyses were performed using GraphPad Prism Software version 8.00 (GraphPad Software) or Stata 17 (StataCorp. 2021). For one-way ANOVA, Bonferroni post-hoc analysis was used if at least 50% of the comparison groups passed the Shapiro-Wilk normality test, and Kruskal-Wallis test with Dunn post-hoc analysis was used otherwise. Two-way ANOVA was used if at least 50% of the comparison groups passed the Shapiro-Wilk normality test, and Robust ANOVA was used otherwise. All data subjected to the unpaired t-test passed the Shapiro-Wilk normality test. Data shown in Extended Data Fig. 1d failed the Shapiro-Wilk normality test, and was subjected to Mann Whitney test. The statistical methods used are shown in the figure legend and in Supplementary Table S4.

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Galactoside in the male ejaculate evaluated as a nuptial gift by the female nutrient sensing neurons

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