A female’s reproductive state influences her perception of odors and tastes along with her changed behavioral state and physiological needs. The mechanism that modulates chemosensory processing, however, remains largely elusive. Using Drosophila, we have identified a behavioral, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality perception, to the physiological needs of a gravid female. Pungent smelling polyamines, such as putrescine and spermidine, are essential for cell proliferation, reproduction, and embryonic development in all animals. A polyamine-rich diet increases reproductive success in many species, including flies. Using a combination of behavioral analysis and in vivo physiology, we show that polyamine attraction is modulated in gravid females through a G-protein coupled receptor, the sex peptide receptor (SPR), and its neuropeptide ligands, MIPs (myoinhibitory peptides), which act directly in the polyamine-detecting olfactory and taste neurons. This modulation is triggered by an increase of SPR expression in chemosensory neurons, which is sufficient to convert virgin to mated female olfactory choice behavior. Together, our data show that neuropeptide-mediated modulation of peripheral chemosensory neurons increases a gravid female’s preference for important nutrients, thereby ensuring optimal conditions for her growing progeny.

Food choices often correlate with nutritional needs or physiological states of an animal. For instance, during pregnancy, women frequently report that their food preferences change—sometimes dramatically. In part, this change in preference is brought about by a change in the perception of smells and tastes. Research has shown that female insects also change their food and egg-laying site preferences depending on their reproductive state. However, the mechanisms that trigger these changes are not understood in either mammals or insects. We have unraveled a mechanism that changes a mated female’s perception of odors and tastes and thereby adapts her choices to her reproductive state. Using the model fly Drosophila melanogaster, we show that mating increases females’ interest in sources of specific beneficial nutrients: polyamines such as spermine and putrescine. Polyamine levels in the body are maintained by diet, microorganisms in the gut, and own synthesis. Increased levels are required during pregnancy and reproduction. Indeed, mated females were more attracted to the taste and smell of polyamines than virgins were. We found that this behavioral modulation is regulated through a secreted peptide and its receptor, whose expression rises markedly in sensory organs upon mating. This signal appears to change the intensity of how polyamine taste or smell information reaches the brain and ultimately elicits a choice. Given that odor and taste processing in mammals and insects are similar, our findings in flies can lead to a better understanding of how dynamic physiological states affect our perception of the environment and lead us to adapt our choices of food and other relevant decisions.

Funding: This study was generously supported by a Boehringer Ingelheim Exploration Grant to ICGK ( http://www.boehringer-ingelheim-stiftung.de/de/was-wir-foerdern/exploration-grants.html ), the Max Planck Society to ICGK ( www.mpg.de ), an ERC starting grant, Grant number: 637472 to ICGK, ( https://erc.europa.eu/funding-and-grants/funding-schemes/starting-grants ), and the EMBO Young Investigator award to ICGK ( www.embo.org ) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we show that the olfactory and gustatory perception of polyamines is modulated by the female’s reproductive state and guides her choice behavior accordingly. This sensory and behavioral modulation depends on SPR and its conserved ligands, the MIPs that act directly on the chemosensory neurons themselves. Together, our results suggest that mating-state-dependent neuropeptidergic modulation of chemosensory neurons matches the female fly’s decision-making to her physiological needs.

This beneficial role of polyamines has a well-characterized biological basis: polyamines are essential for basic cellular processes such as cell growth and proliferation, and are of specific importance during reproduction [ 35 ]. They enhance the quality of sperm and egg and are critical during embryogenesis and postnatal development [ 32 , 36 ]. While the organism can generate polyamines, a significant part is taken in with the diet [ 37 , 38 ]. Moreover, endogenous synthesis of polyamines declines with ageing and can be compensated for through a polyamine-rich diet [ 32 ]. Therefore, these compounds represent a sensory cue as well as an essential component of the diet of a gravid female fly.

We have examined the causative mechanisms that integrate reproductive state into preference behavior and chemosensory processing. We have focused on the perception of another class of byproducts of fermenting fruits, polyamines. Polyamines such as putrescine, spermine, and spermidine are important nutrients that are associated with reproductive success across animal species [ 32 ]. A diet high in polyamines indeed increases the number of offspring of a fly couple, and female flies prefer to lay their eggs on polyamine-rich food [ 33 ]. Importantly, we have previously characterized the chemosensory mechanisms flies use to find and evaluate polyamine-rich food sources and oviposition sites. In brief, volatile polyamines are detected by OSNs on the fly’s antenna, co-expressing two ionotropic receptors (IRs), IR41a and IR76b [ 33 , 34 ]. Interestingly, the taste of polyamines is also detected by IR76b in labellar gustatory receptor neurons (GRNs) [ 33 ].

To identify optimal food and oviposition sites, female flies rely strongly on their sense of smell and taste [ 26 – 29 ]. D. melanogaster females prefer to oviposit in decaying fruit and use byproducts of fermentation such as ethanol and acetic acid to choose oviposition sites [ 29 , 30 ]. Their receptivity to these byproducts is enhanced by their internal state [ 29 , 31 ]. It was shown, for instance, that the presence of an egg about to be laid results in increased attraction to acetic acid [ 31 ]. Yet the mechanisms linking reproductive state to the modulation of chemosensory processing remain unknown.

Additional SPR ligands have been identified that are not required for the canonical post-mating switch, opening the possibility that this receptor is involved in the neuromodulation of other processes [ 19 – 22 ]. These alternative ligands, the myoinhibitory peptides (MIPs)/allatostatin-Bs, unlike SP, have been found outside of drosophilids, in many other insect species such as the silkmoth (Bombyx mori), several mosquito species, and the red flour beetle (Tribolium castaneum) [ 19 ]. They are expressed in the brain of flies and mosquitoes, including in the centers of olfactory and gustatory sensory neuron projections, the antennal lobe (AL), and the subesophageal zone (SEZ), respectively [ 19 , 23 , 24 ]. Although these high-affinity SPR ligands have recently been implicated in the control of sleep in Drosophila males and females [ 25 ], nothing thus far suggests a function in reproductive behaviors [ 19 ].

The neuronal underpinnings of mating and its consequences on female behaviors have arguably been best characterized in the fruit fly Drosophila melanogaster [ 7 , 8 ]. Shortly after copulation, female flies engage in a series of post-mating behaviors contrasting with those of virgins: their sexual receptivity decreases, and they feed to accumulate essential resources needed for the production of eggs [ 9 – 12 ]; finally, they lay their eggs. This suite of behaviors results from a post-mating trigger located in the female’s reproductive tract [ 12 ]. Sensory neurons extending their dendrites directly into the oviduct are activated by a component of the male’s ejaculate, the sex peptide (SP) [ 13 , 14 ]. Sex peptide receptor (SPR) expressed by these sensory neurons triggers the post-mating switch [ 15 ]. Mated females mutant for SPR produce and lay fewer eggs while maintaining a high sexual receptivity [ 13 – 15 ]. In addition to SP, male ejaculate contains more than 200 proteins, which are transferred along with SP into the female. These have been implicated in conformational changes of the uterus, induction of ovulation, and sperm storage [ 7 , 16 – 18 ].

The behavior of females in most animal species changes significantly as a consequence of mating. Those changes are interpreted from an evolutionary standpoint as the female’s preparation to maximize the fitness of her offspring. In general, they entail a qualitative and quantitative change in her diet, as well as the search for an optimal site where her progeny will develop. In humans, the eating behavior and perception of tastes and odors of a pregnant woman are modulated in concert with altered physiology and the specific needs of the embryo [ 1 – 3 ]. While several neuromodulatory molecules such as noradrenaline are found in the vertebrate olfactory and gustatory systems, little is known about how reproductive state and pregnancy shape a female’s odor and taste preferences [ 4 , 5 ]. Very recent work in the mouse showed that olfactory sensory neurons (OSNs) are modulated during the estrus cycle [ 6 ]. Progesterone receptor expressed in OSNs decreases the sensitivity of pheromone-detecting OSNs and thereby reduces the non-sexually receptive female’s interest in male pheromones. The mechanisms of how mating, pregnancy, and lactation shape the response of the female olfactory and gustatory systems remain poorly understood.

Results

Mating State Modulates the Perception of Polyamines Males and female flies are strongly attracted to polyamines [33]. The perception of sensory stimuli, however, can be modulated and depends on behavioral context [39]. Given that polyamine-rich foods increase the number of progeny [33], we wondered whether mating state influences the perception of these important molecules. To test this, we compared olfactory and oviposition behaviors of mated to virgin female flies. In an olfactory choice assay, the T-maze, mated females showed a strong attraction to volatile polyamines, which requires their sense of smell, as we have shown in the companion paper and as previously suggested by Silbering et al. [33,34]. Virgin flies displayed a significantly altered preference for the polyamines putrescine and cadaverine compared to mated flies (Fig 1A). While mated females preferred relatively high concentrations of polyamines typically present in fermenting fruit (1 mM or 10 ppm, [36,37]), virgin females showed strong attraction to only the lowest levels and increasing avoidance of higher levels of these odors (Fig 1B). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Mating state modulates the perception of polyamines. (A) Virgin flies are less attracted to a high polyamine concentration of 1 mM (10 ppm) as compared to mated flies. Olfactory preference index of Canton S mated (♀) and Canton S virgin (☿) females in the T-maze assay. Violet and green bars represent putrescine and cadaverine, respectively. (n = 8, 60 mated (♀) or virgin (☿) flies/trial). (B) Mated females, unlike virgins, preferred relatively high concentrations of polyamines, naturally present in fermenting fruit (1 mM or 10ppm). By contrast, virgin females were most attracted to very low concentrations of polyamine. Line graph shows dose-dependent olfactory preference index of mated (♀) and virgin (☿) flies to polyamines. (n = 8 ± SEM, 60 mated (♀) or virgin (☿) flies/trial). (C) Virgin flies show no preference between polyamines (putrescine or cadaverine, 1 mM) and 1% low melting agarose, and deposited their low number of eggs on either site of the assay. (n = 8, 60 mated (♀) or virgin (☿) flies/trial). (D) Polyamine preference appears to correlate with the female’s egg-laying activity. Graphs show olfactory preference index of females 2 d post-mating (♀, 2 d), virgin females (☿), and females 14 d post-mating (♀, 14 d) to 10 ppm of polyamine. (n = 8, 60 mated (♀, 2 d), virgin (☿) and mated (♀, 14 d) female flies/trial). (E) Mated but sterile ovoD1 mutant females (ovoD1/+, Canton S) show similar attraction to polyamine odor compared to wildtype controls (+/+, Canton S). (F) Mated sex peptide receptor mutant (SPR-/-) female flies display a significantly reduced attraction to polyamine odor (n = 8, 60 flies/trial). (G) Oviposition preference index of mated sex peptide receptor mutant (SPR-/-) females. Mated SPR mutant females show indifference to polyamines. (n = 8, 60 mated (♀) flies/ trial). (H) Olfactory preference for 10 ppm polyamine of SPR-/- male flies is comparable to control males. Box plots show median and upper/lower quartiles (n = 8, 60 flies/trial). All p-values were calculated via two-way ANOVA with the Bonferroni multiple comparison post-hoc test, with the exception of (E), where p-values were calculated with an unpaired t-test (ns > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). https://doi.org/10.1371/journal.pbio.1002455.g001 We next analyzed whether virgin flies would make different egg-laying choices compared to mated flies. Mated females taste polyamines with taste sensilla on their labellum and use this information during egg-laying decisions [33]. Although egg-laying substrates containing just polyamines are avoided as egg-laying substrates because of their bitter taste, polyamine-rich sugary substrates such as decaying fruit are strongly preferred over fresh fruit [33]. To assay the egg-laying preferences, we used a simple oviposition assay consisting of a plate with a plain agarose substrate (1%) that was on one-half of the egg-laying plate supplemented with the polyamines, putrescine or cadaverine (1 mM, Fig 1C, see Materials and Methods). Consistent with our dissection of polyamine perception [33], mated flies displayed a strong preference and laid the majority of their eggs on plain agarose (Fig 1C). By contrast, virgin females, albeit laying very few (and unfertilized) eggs, distributed their eggs equally between polyamine and control sides (Figs 1C and S1A). Therefore, we concluded that, while mated females actively develop a choice behavior, virgin females are indifferent to polyamines as an egg-laying substrate. Taken together, odor as well as taste perception of polyamines strongly depends on the female fly’s mating state. We have shown that a polyamine-rich diet increases the number of offspring of a fly couple [33]. These data could potentially indicate that needs arising through egg production and laying, and not exclusively or primarily through mating, drive a female to seek polyamines. We therefore first tested whether polyamine choice behavior correlated with the female’s egg-laying activity and time after mating. This appeared to be the case, because mated females that had ceased to lay eggs at 14 d after mating returned to their pre-mating preference behavior and made choices that resembled the choices of virgin flies (Fig 1D). This return to virgin behavior could be due to the time elapsed after mating or to a reduction in egg-laying. To dissect the relative contribution of egg-laying activity and mating, we analyzed the preference behavior of mated ovoD1 mutant females [40]. These females are sterile due to an atrophy of the ovaries. Mated ovoD1 mutant females showed the same preference to polyamines in the T-maze compared to control mated females (Fig 1E). From these data, it appears that mating itself provides a key signal that changes the female’s perception and stimulates her to seek polyamines. While previous research has shown that mating state and egg-laying activity influence the choice behavior of female flies when selecting food or oviposition substrates [9,29,31], how mating state modulates neural sensitivity and processing of sensory information remains not understood. Having defined the gustatory and olfactory receptors and sensory neurons for the detection of polyamines [33], we sought to identify the mechanism that modulates this detection and processing in a mating state-dependent manner. SPR and SP are required for the classical post-mating switch (see Introduction) and changes in feeding behavior [9,10,41]. To test the role of SPR in mating-state-dependent polyamine choice behavior, we initially examined the olfactory preference and oviposition behavior of SPR mutant females (Df(1)Exel6234) [15]. Mated SPR mutant females showed a significantly reduced preference behavior in the T-maze (odor) as well as in oviposition assays (taste) compared to that of mated control females (Fig 1F and 1G). Importantly, SPR mutant males maintained the same level of attraction as wildtype control males, possibly representing the constant need of polyamines such as spermine and spermidine for sperm production (Fig 1H). These results indicated that the SPR pathway is part of the mechanism that controls mating-induced changes in the perception of the smell and taste of polyamines.

G-Protein Coupled Receptor (GPCR) Signaling in Chemosensory Neurons Modulates Female Perception Increasing evidence in different model organisms indicates that chemosensory neurons themselves are potent targets for neuromodulation [6,42–44]. Although SPR is required in specific internal sensory neurons in the female reproductive tract for the canonical post-mating switch, its rather broad expression in the nervous system, including chemosensory organs and their projection zones in the brain [15,45], prompted us to ask whether SPR signaling was acting directly in peripheral chemosensory neurons. Previous work successfully employed RNA interference (RNAi) directed against SPR to identify the set of sensory neurons in the female reproductive tract sufficient to trigger two important post-mating behaviors: increased egg-laying and rejection of males [13,14]. We induced RNAi against SPR (UAS-SPRi) specifically in olfactory and gustatory neurons that sense polyamines, using the driver IR76b-Gal4 [33]. Importantly, this driver was not expressed in the internal sensory neurons that require SPR to induce the mating switch (S2A and S2C Fig). Mated females of the genotype IR76b-Gal4;UAS-SPRi showed a significantly reduced attraction to polyamine odor in the T-maze assay as compared to controls (Fig 2A). Remarkably, this reduction was similar to the reduction seen in SPR mutants (see Fig 1). Importantly, SPR RNAi did not reduce the attraction of virgin females further, showing that the regulation by SPR is indeed mating-state-dependent (Fig 2B). Similarly, expression of SPR RNAi in IR76b neurons fully abolished the taste-dependent egg-laying preference behavior of mated females (Figs 2C and S1C). We then refined the experiment with another, significantly more specific Gal4 driver, IR41a-Gal4, targeting only the small number of olfactory neurons sensing polyamine odor (IR41a-Gal4;UAS-SPRi). We observed a similar reduction in attraction to polyamine odor in the T-maze compared to knockdown with IR76b-Gal4 in mated females (Fig 2D). By contrast, egg-laying preference was comparable to control mated females (Figs 2E and S1D). This result was consistent with the absence of IR41a-Gal4 expression in IR76b gustatory neurons [33]. These data were consistent with the hypothesis that SPR in chemosensory neurons is necessary to modulate the attraction of females to the smell and taste of polyamines after mating. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. GPCR signaling in chemosensory neurons modulates female perception. (A) Knockdown of SPR in IR76b polyamine chemosensory neurons using RNAi (IR76b-Gal4;UAS-SPRi) significantly reduces olfactory preference to 10 ppm putrescine or cadaverine in mated females as compared to mated controls. (n = 8, 60 mated (♀) flies/trial). (B) The effect of SPRi in IR76b neurons is mating state-dependent, as knockdown of SPR (IR76b-Gal4;UAS-SPRi) does not further decrease the olfactory attraction of virgin females to polyamines compared to control virgins. (n = 8, 60 mated (♀) or virgin (☿) flies/trial). (C) Oviposition avoidance of a 1 mM polyamine/agarose substrate compared to a plain agarose substrate is strongly reduced upon knockdown of SPR in IR76b neurons (IR76b-Gal4;UAS-SPRi) (n = 8, 60 mated (♀) flies/trial). (D) Knockdown of SPR in IR41a neurons (IR41a-Gal4;UAS-SPRi) leads to a similar decrease in attraction to the odor of polyamine (10 ppm) in the T-maze as compared to knockdown of SPR with IR76b-Gal4, suggesting that SPR is required in olfactory neurons to enhance the attraction of mated females to the polyamine odors (n = 8, 60 mated (♀) flies/trial). (E) Knockdown of SPR in IR41a neurons (IR41a-Gal4;UAS-SPRi) did not affect oviposition behavior, and female behavior remained like their genetic controls. This result is consistent with the lack of expression of IR41a in taste neurons. (F) Re-expression of SPR using either IR41a-Gal4 or IR76b-Gal4 neurons fully rescued the SPR mutant phenotype of mated females in olfaction behavior to 10 ppm polyamines. (n = 8, 60 flies/trial). (G) Re-expression of SPR in IR76b taste neurons using IR76b-Gal4 fully rescued the SPR mutant phenotype of mated females in oviposition behavior. Conversely, re-expression of SPR in IR41a olfactory neurons or GR66a bitter taste neurons did not rescue oviposition preference behavior. (n = 8, 60 flies/trial). Box plots show median and upper/lower quartiles. All p-values were calculated via two-way ANOVA with the Bonferroni multiple comparison post-hoc test (ns > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). https://doi.org/10.1371/journal.pbio.1002455.g002 Given the central role of SPR in the classical post-mating switch, we asked whether SPR in chemosensory neurons was not only necessary but also sufficient to modulate their sensitivity. To this end, we re-expressed SPR in SPR mutant females in all IR76b neurons (IR76b-Gal4, polyamine taste and olfaction), in bitter taste neurons (GR66a-Gal4), or just in the olfactory subset of IR76b-expressing neurons that express IR41a (IR41a-Gal4) and assayed olfactory behavior (T-maze) and taste-dependent oviposition behavior. We found that re-expression of SPR in IR76b neurons fully rescued the SPR mutant phenotype of mated females in olfaction as well as in oviposition behavior (Fig 2F and 2G). Expression of SPR in GR66a bitter neurons, by contrast, had no effect on the SPR mutant phenotype in either of the two choice behaviors (Fig 2F and 2G). Re-expression of SPR selectively in IR41a OSNs did not rescue oviposition behavior of SPR mutant females, consistent with the fact that the egg-laying choice is mediated by taste neurons (Fig 2G). It did, however, rescue the olfactory attraction of SPR mutant females to polyamine odor in the T-maze (Fig 2F). This suggests that SPR plays a cell-autonomous role in a specific set of peripheral chemosensory neurons independent of its function in the cells in the female reproductive system. Altogether, based on these data, we propose that SPR regulates choice behavior in a mating-state-dependent manner directly in chemosensory neurons, providing a mechanistic link between mating state and the neurons that process odors and taste.

Mating and SPR Signaling Enhance Sensitivity of Gustatory Neurons SPR signaling in chemosensory neurons appears to be required for the change in choice behavior after mating. This genetic mechanism could influence neuronal physiology at several levels of olfactory and taste processing starting at the peripheral level. We have previously shown that IR76b taste neurons on the labellum are of particular importance for egg-laying choices on polyamine substrates [33]. Loss of IR76b completely abolishes the egg-laying preference of a mated female [33]. To test whether mating modulates the sensitivity of gustatory neurons, we examined the activity of IR76b chemosensory neurons by recording their Ca2+ responses to polyamines at the level of their axon terminals in the SEZ of the central brain (Fig 3A). Because mating induces short-term (<24 h) and long-term (~1 wk) effects [46,47], we performed these experiments at two different time points: at 1–6 h or at 1 wk post-mating (Fig 3B–3F). We measured Ca2+ increases by recording GCaMP6f signals in IR76b axon terminals in the SEZ (IR76b-Gal4;UAS-GCaMP6f), which we divided based on the innervation pattern of IR76b neuron subsets into two broader innervation zones, region of interest (ROI) 1 and ROI 2 (Fig 3A). At 1–6 h post-mating, labellar IR76b neurons projecting to ROI 1, the primary response area for polyamines [33], responded significantly more strongly to a putrescine taste solution in mated females than in virgin females (Fig 3C and 3D). Interestingly, this was not the case for ROI 2, which responded significantly only to higher concentrations of putrescine (10–100 mM). IR76b neurons projecting to this region of the SEZ of virgin and mated females showed a similar response (Fig 3E). Interestingly, at the later time point (1 wk post-mating), the difference observed for axons projecting to ROI 1 was no longer significant. Hence, we conclude that mating transiently increases the sensitivity of polyamine-detecting IR76b labellar taste neurons after mating. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Mating increases sensitivity of taste neurons through SPR. (A) Scheme of the SEZ in vivo calcium imaging setup (top). Illustration of the SEZ area showing the innervation pattern of IR76b taste neuron axons (bottom). ROI 1 and ROI 2 delineate the regions of interest (ROI) used for quantification of the relative change in GCaMP-fluorescence (%ΔF/F). (B) Representative images of SEZ imaging of IR76b-Gal4; UAS-GCaMP6f mated and virgin female flies stimulated with distilled water (0 mM), 1 mM putrescine (1 mM), 10 mM putrescine (10 mM), and 100 mM putrescine (100 mM), respectively. (C) IR76b taste neuron terminals of mated females show a significantly increased response to putrescine after mating. While the response is highly significant at 1–6 h post-mating, it remains only a trend at 1 wk post-mating (n = 7). (D) Females at 1–6 h post-mating show higher IR76b taste neuron responses. GCaMP6f-fluorescence peak responses were quantified (in %ΔF/F) in the ROI 1 area. Flies were stimulated with increasing concentrations of putrescine (n = 7). (E) IR76b taste neurons of the same females as in (D) show no difference in the ROI 2 area. (F) Average response trace of the ROI 1 area (n = 7). The gray bar illustrates the stimulation period. The dark colored line in the middle presents the average value, and the light shade presents the SEM. (G) Representative images of IR76b GRN axons in the SEZ of test (IR76b-Gal4,UAS-SPRi;UAS-GCaMP5) and control (IR76b-Gal4;UAS-GCaMP5) females at 1–6 h post-mating. Flies were stimulated with distilled water (0 mM) and 10 mM putrescine (10 mM). (H) Quantification of peak responses (in %ΔF/F) of IR76b axon terminals of IR76b-Gal4,UAS-SPRi;UAS-GCaMP5 and control (IR76b-Gal4;UAS-GCaMP5) females at 1–6 h post-mating (n = 8). Box plots show median and upper/lower quartiles, and whiskers show minimum/maximum values. (I) Average response trace of ROI 1 and ROI 2 area of IR76b axons in the SEZ of IR76b>SPRi and control females (n = 8). All p-values were calculated using an unpaired t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). https://doi.org/10.1371/journal.pbio.1002455.g003 Is this shift of sensitivity in the GRNs mediated by SPR signaling directly in chemosensory neurons as the behavioral data suggests? To answer this, we recorded GCaMP signals from polyamine-sensitive taste neurons of mated females, in which we triggered RNAi against SPR. Knock-down of SPR in IR76b GRNs (IR76b-Gal4,UAS-GCaMP5;UAS-SPRi) of mated females led to a significant decrease in the presynaptic calcium increase of these neurons in response to polyamine taste compared to the response of mated controls (Fig 3G–3I). Notably, SPR knockdown had no effect on the response of IR76b neurons projecting to the ROI 2 region of the SEZ. These neurons responded like control neurons (Fig 3H and 3I), suggesting that SPR modulation only occurred in neurons that were affected by the mating state. These results provide a mechanistic explanation for behavioral change occurring in the oviposition choice behavior of females upon mating, and they are consistent with our model that SPR in GRNs directly modulates sensory neuron sensitivity and thereby regulates choice behavior.

Mating and SPR Signaling Decreases Responsiveness of Olfactory Neurons to Polyamines Olfactory preference behavior appears to undergo a similar shift as gustatory preference behavior after mating. We therefore carried out a set of experiments in the olfactory system similar to those described above. Axons of OSNs project centrally to the AL, the functional equivalent of the vertebrate olfactory bulb. This first-order olfactory information is further processed by local interneurons and then transferred by projection neurons (PNs) to higher brain centers (Fig 4A) [48]. Recent studies have shown that hunger enhances olfactory sensitivity to food odor by increasing presynaptic responses of OSNs via the OSN-resident short neuropeptide F (sNPF) and its receptor, sNPFR [42,43]. Metabolic state thereby regulates the efficacy of the synapse between OSN and PN similarly to what we have observed for mating state and GRNs. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. SPR decreases sensitivity of olfactory neurons to polyamines after mating. (A) Schematic diagram of a fly brain and its antennal appendages with olfactory sensory neurons (OSNs). OSNs project into the antennal lobe (AL), where they innervate a specific glomerulus (green). Projection neurons (PN) send the information mainly to two higher brain centers, the mushroom body (MB) and the lateral horn (LH) (top). Illustrative confocal image stack showing the IR41a and IR76b OSN innervation in the AL (bottom). VC5 is the glomerulus innervated by the polyamine-responding IR41a/IR76b sensory neurons. (B) Illustration of the in vivo calcium imaging setup. (C–E) In vivo calcium imaging of IR41-Gal4;UAS-GCaMP6f flies stimulated with water and 10 ppm putrescine, respectively. Mated females’ OSN axon terminals show a significant reduction in their sensitivity to putrescine at 1–6 h post-mating. (C) Quantification of peak ΔF responses (in %ΔF/F) in virgin and mated females. Boxes show median and upper/lower quartiles, and whiskers show minimum/maximum values. *p < 0.05, unpaired t test (n = 8). (D) Representative pseudo-color images showing the response to water and 10 ppm putrescine in virgin and mated flies at 1–6 h and 1 wk post-mating. (E) Average response trace (in %ΔF/F) of the VC5 glomerulus peak response at 1–6 h and 1 wk post-mating compared to traces from virgin females. The dark colored line in the middle presents the average value and the light shade presents the SEM. (F–H) In vivo calcium imaging of test (IR41a-Gal4,UAS-SPRi;UAS-GCaMP5) and control (IR41a-Gal4;UAS-GCaMP5) mated female flies. OSN axon terminals of IR41a>SPRi females show significantly enhanced responses to putrescine compared to control females. (F) Representative pseudo-color images showing the response to water and 10 ppm putrescine in IR41a>SPRi and control females, respectively. (G) Average response trace of the VC5 glomerulus in IR41a>SPRi and control females at 1–6 h post-mating for 10 ppm putrescine. (E,G) The gray column represents the 0.5 s stimulation period. Dark colored line is the average response and the light shade is the SEM. (H) Quantification of peak ΔF responses (in %ΔF/F) in IR41a>SPRi and control females for 0 ppm, 6 ppm, 8 ppm, and 10 ppm putrescine, respectively (n = 7 ± SEM). All p-values were calculated using an unpaired t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). https://doi.org/10.1371/journal.pbio.1002455.g004 To test whether OSNs are modulated in a similar manner as GRNs, we imaged calcium increases of IR41a axon terminals at the level of the AL (IR41a-Gal4;UAS-GCaMP6f) (Fig 4B). Surprisingly, we observed that mating significantly decreased the response of these neurons to behaviorally relevant concentrations of polyamines (Fig 4C–4E). As in the gustatory system, this decrease was strongly significant at 1–6 h and remained only a trend at 1 wk post-mating (Fig 4C). In contrast to GRNs, however, mating transiently suppresses the sensitivity of OSNs. How does this result explain the behavioral shift toward higher polyamine levels after mating? Virgins show highest attraction to very low levels of polyamines and reduced attraction or enhanced aversion at levels preferred by mated females (Fig 1B). By contrast, mated females show the highest attraction to relatively high amounts of polyamine, which roughly corresponds to decaying fruit (10 ppm/1 mM; Fig 1B). It was previously shown that different odor concentrations can have differential behavioral effects and can even recruit different PNs downstream of the same OSNs [49,50]. Such a mechanism could also explain the change of behavior to polyamines, whereby a reduction of olfactory sensitivity may change higher olfactory processing and consequently shift the mated female’s preference to increased levels of beneficial polyamines for egg-laying. Again, we asked whether this change in sensitivity was mediated by SPR signaling in OSNs themselves, as the behavioral data would suggest. As in the gustatory system, this appeared to be the case for the olfactory system, as knockdown of SPR in IR41a OSNs resulted in a significant change in presynaptic calcium responses of these neurons (Fig 4F–4H). As predicted from the comparison of mated and virgin OSN responses to putrescine, we observed a greater increase of GCaMP fluorescence in OSN axon terminals of mated females with SPR knockdown (IR41a-Gal4,UAS-GCaMP5;UAS-SPRi) compared to mated genetic controls (Fig 4H). Together, we interpret these data to mean that SPR in chemosensory neurons regulates the sensitivity of OSNs and GRNs to polyamines directly at the level of these chemosensory neurons. This change in sensitivity follows two different neural mechanisms, i.e., increased calcium responses of GRN and decreased responses of OSN axon terminals. This, in turn, appears to alter the mated female’s perception and adjusts her choice behavior to polyamines.

Myoinhibitory Peptides Regulate Polyamine Sensitivity in the Context of Mating We showed that polyamine perception changes upon mating and that this change is mediated by SPR signaling in chemosensory neurons. How SPR signaling is triggered in chemosensory neurons, however, remains unclear. The best-characterized SPR ligand is SP itself. A role for SP in feeding behavior was demonstrated previously. For instance, SP provided by the male stimulates feeding in mated females, and SP mutant male-mated females do not show this increase [10]. Furthermore, the mated female’s feeding preference for yeast and salt depends on SP provided by the male during mating [9,41]. Here, SP activates the canonical SPR pathway through ppk-positive SPR neurons in the female’s oviduct, which leads to a change in feeding preference. Whether and how mating and/or SP alter the sensitivity of taste neurons to yeast or salt or their higher-order chemosensory processing is not known. Furthermore, in the present context, if SP were to act directly on the chemosensory neurons, some SP would have to be transferred from its point of delivery, the female reproductive tract, to SPR in chemosensory neurons on the head. To test the requirement of SP in the sensitivity to polyamines, we crossed males that were mutant for the SP gene (SP0), and thus lacking SP from their semen, to wild-type virgin females [51]. We compared the behavior of these females to that of females mated to wild-type males. Interestingly, the attraction of females mated to SP0 males to polyamine odor in the T-maze was not significantly different from females mated to wild type males (Fig 5A). This suggested that SP was not the key to mating-state-dependent olfactory sensitivity modulation. Furthermore, it also indicated that changes in feeding behavior as reported by Carvalho et al. [10] are not necessary for the observed olfactory modulation. We also analyzed the contribution of SP to oviposition preference. SP0 mated females appeared to show the same lack of preference as virgin flies and laid their very few eggs on either side of the assay (Fig 5B). Nevertheless, the olfactory preference data as well as the site of action of SP indicated that another additional ligand was involved in mating-state-dependent chemosensory changes in females. Moreover, this result was in agreement with our data showing that re-expression of SPR in gustatory or olfactory neurons was sufficient to modulate their responses to polyamines. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Myoinhibitory peptides regulate polyamine sensitivity in the context of mating. (A) Loss of sex peptide (SP) in the sperm of the male does not significantly affect chemosensory attraction of mated females to 10 ppm of polyamines. Wild-type (wt) Canton S females mated to wild-type or sex peptide mutant (SP0) males do not show a significantly altered level of attraction to the odor of putrescine or cadaverine. (n = 8, 60 flies/trial). (B) SP0 male-mated Canton S females lay their low numbers of eggs on either site of the oviposition assay and show no preference behavior. (n = 8, 60 flies/trial). (C) Myoinhibitory peptide (MIP) expression in the AL and SEZ regions in the female brain. In the AL, the glomerulus innervated by IR41a OSNs is displayed (IR41a-Gal4;UAS-mCD8GFP). Note that MIP staining is detected in close proximity to IR41a axon terminals. In the SEZ, anti-MIP staining (green) localizes close to IR76b neuron axons and axon terminals (magenta) consistent with MIPs being secreted by IR76b neurons (IR76b-QF;QUAS-mtd-tomato) (see arrowheads). (D,E) MIPs modulate olfactory attraction to polyamines selectively in mated females but not males. RNAi-mediated knockdown of MIPs with four different RNAi transgenic lines in IR76b neurons (IR76b-Gal4;UAS-MIPi) selectively reduces the olfactory preference of mated females but not of males to 10 ppm of putrescine (D) or 10 ppm of cadaverine (E) (n = 8, 60 flies/trial). (F) The effect of MIP knockdown (IR76b-Gal4;UAS-MIPi) depends on the mating state of the female, as the low attraction of virgin females to 10 ppm polyamine odor was not further reduced in virgin females with MIP knockdown compared to virgin controls without RNAi against MIPs. Box plots show median and upper/lower quartiles (n = 8, 60 flies/trial). (G) Knockdown of MIPs in IR76b neurons abolishes oviposition preference to 1 mM putrescine and cadaverine using three different MIPi transgenic lines (IR76b-Gal4;UAS-MIPi). Females laid their eggs on either side of the assay. All box plots show median and upper/lower quartiles (n = 8, 60 flies/trial). All p-values were calculated via two-way ANOVA with the Bonferroni multiple comparison post-hoc test (ns > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). https://doi.org/10.1371/journal.pbio.1002455.g005 We therefore asked whether MIPs could be the functional ligands of SPR at the level of the chemosensory neuron central projections and could mediate the modulation of polyamine behavior. The expression of MIPs in the vicinity of IR41a axon terminals in the AL and in the vicinity of IR76b axons and axon terminals in the SEZ (Fig 5C) is consistent with their possible requirement in the chemosensory neurons themselves. We employed four different, independent RNAi-triggering transgenic lines to knockdown the expression of MIPs in IR76b-positive sensory neurons and tested fly behavior in the T-maze (olfaction) and oviposition (taste) assays. RNAi-mediated suppression of MIP expression in chemosensory neurons (IR76b-Gal4;UAS-MIPi) reduced the expression of MIP in chemosensory processing centers, but not in the rest of the brain as compared to controls or knockdown with a pan-neural driver (S5A Fig). Importantly, this manipulation (IR76b-Gal4;UAS-MIPi) also significantly lowered the attraction of mated females to polyamines in the T-maze as compared to genetic controls (Fig 5D and 5E). Notably, although MIP expression appears highly similar between males and females (S4 Fig) [52], this reduced olfactory attraction was only observed in females, but not in male flies (Fig 5D and 5E). These data mirror the lack of olfactory phenotype in the SPR mutant male (see Fig 1G) and further supported our model of a gender-specific role for SPR signaling. Furthermore, similar to what was observed upon SPR knockdown (see Fig 2C), virgin female attraction to polyamines was not further decreased when MIPs were down-regulated by RNAi, showing that the effect of MIP was mating-state-dependent (Fig 5F). Finally, a similar analysis in the context of oviposition behavior showed that knockdown of MIPs in IR76b neurons (IR76b-Gal4;UAS-MIPi) had the same effect on female oviposition behavior as knockdown of SPR (Fig 5G). Female flies laid their eggs in equal numbers on polyamine-rich and control substrates (S5B Fig). These data describe a role for MIPs in female reproductive behavior and indicate that they regulate polyamine-mediated chemosensory behavior presumably as ligands for SPR. Furthermore, similar to sNPF and its receptor [43], MIPs and SPR appear to be required directly in gustatory and olfactory neurons. In contrast to sNPF and sNPFR, SPR and MIPs are only required in the female.