Sexual Failure Decreases Sweet Taste Perception in Male Drosophila via Dopaminergic Signaling

Reviewer #1 (Public review):

Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the subesophageal zone that interacts with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet-taste sensitivity and sugar-feeding behavior in male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.

Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the subesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar-feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. found that DopR1 and Dop2R, but not DopEcR, are involved in the sexual failure-induced decrease in sugar sensitivity in these neurons. I wish the authors had further explored or discussed this discrepancy, as it is unclear how dopamine release selectively engages different receptors to modulate neuronal sensitivity in a context-dependent manner.

The data presented by Wang et al. are solid and effectively support their conclusions. However, certain aspects of their experimental design, data analysis, and interpretation warrant further review, as outlined below.

(1) The authors did not explicitly indicate the feeding status of the flies, but it appears they were not starved. However, the naive and satisfied flies in this study displayed high feeding and PER baselines, similar to those observed in starved flies in other studies. This raises the concern that sexually failed flies may have consumed additional food during the 4.5-hour conditioning period, potentially lowering their baseline hunger levels and subsequently reducing PER responses. This alternative explanation is worth considering, as an earlier study demonstrated that sexually deprived males consumed more alcohol, and both alcohol and food are known rewards for flies. To address this concern, the authors could remove food during the conditioning phase to rule out its influence on the results.

(2) Figure 1B reveals that approximately half of the males in the Failed group did not consume sucrose, yet Figure 1-S1A suggests that the total volume consumed remained unchanged. Were the flies that did not consume sucrose omitted from the dataset presented in Figure 1-S1A? If so, does this imply that only half of the male flies experience sexual failure, or that sexual failure affects only half of males while the others remain unaffected? The authors should clarify this point.

(3) The evidence linking TH-GAL4 labeled dopaminergic neurons to reduced sugar sensitivity in Gr5a+ neurons in sexually failed males could be further strengthened. Ideally, the authors would have activated TH-GAL4 neurons and observed whether this restored GCaMP responses in Gr5a+ neurons in sexually failed males. Instead, the authors performed a less direct experiment, shown in Figures 3-S1C and D. The manuscript does not describe the condition of the flies used in this experiment, but it appears that they were not sexually conditioned. I have two concerns with this experiment. First, no statistical analysis was provided to support the enhancement of sucrose responses following activation of TH-GAL4 neurons. Second, without performing this experiment in sexually failed males, the authors lack direct evidence to confirm that the dampened response of Gr5a+ neurons to sucrose results from decreased activity in TH-GAL4 neurons.

(4) The statistical methods used in this study are poorly described, making it unclear which method was used for each experiment. I suggest that the authors include a clear description of the statistical methods used for each experiment in the figure legends. Furthermore, as I have pointed out, there is a lack of statistical comparisons in Figures 3-S1C and D, a similar problem exists for Figures 6E and F.

(5) The experiments in Figure 5 lack specificity. The target neurons in this study are Gr5a+ neurons, which are directly involved in sugar sensing. However, the authors used the less specific Dop1R1- and Dop2R-GAL4 lines for their manipulations. Using Gr5a-GAL4 to specifically target Gr5a+ neurons would provide greater precision and ensure that the observed effects are directly attributable to the modulation of Gr5a+ neurons, rather than being influenced by potential off-target effects from other neuronal populations expressing these dopamine receptors.

(6) I found the results presented in Fig. 6F puzzling. The knockdown of Dop2R in Gr5a+ neurons would be expected to decrease sucrose responses in naive and satisfied flies, given the role of Dop2R in enhancing sweet sensitivity. However, the figure shows an apparent increase in responses across all three groups, which contradicts this expectation. The authors may want to provide an explanation for this unexpected result.

(7) In several instances in the manuscript, the authors described the effects of silencing dopamine signaling pathways or knocking down dopamine receptors in Gr5a neurons with phrases such as 'no longer exhibited reduced sweet sensitivity' (e.g., L269 and L288), 'prevent the reduction of sweet sensitivity' (e.g., L292), or 'this suppression was reversed' (e.g. L299). I found these descriptions misleading, as they suggest that sweet sensitivity in naive and satisfied groups remains normal while the reduction in failed flies is specifically prevented or reversed. However, this is not the case. The data indicate that these manipulations result in an overall decrease in sweet sensitivity across all groups, such that a further reduction in failed flies is not observed. I recommend revising these descriptions to accurately reflect the observed phenotypes and avoid any confusion regarding the effects of these manipulations.

Reviewer #2 (Public review):

Summary:

The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.

Strengths:

The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.

Weaknesses:

The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.

Reviewer #3 (Public review):

Summary

In this work, the authors asked how mating experience impacts reward perception and processing. For this, they employ fruit flies as a model, with a combination of behavioral, immunostaining, and live calcium imaging approaches.

Their study allowed them to demonstrate that courtship failure decreases the fraction of flies motivated to eat sweet compounds, revealing a link between reproductive stress and reward-related behaviors. This effect is mediated by a small group of dopaminergic neurons projecting to the SEZ. After courtship failure, these dopaminergic neurons exhibit reduced activity, leading to decreased Gr5a+ neuron activity via Dop1R1 and Dop2R signaling, and leading to reduced sweet sensitivity. The authors therefore showed how mating failure influences broader behavioral outputs through suppression of the dopamine-mediated reward system and underscores the interactions between reproductive and reward pathways.

Concern

My main concern regarding this study lies in the way the authors chose to present their results. If I understood correctly, they provided evidence that mating failure induces a decrease in the fraction of flies exhibiting PER. However, they also showed that food consumption was not affected (Fig. 1, supplement), suggesting that individuals who did eat consumed more. This raises questions about the analysis and interpretation of the results. Should we consider the group as a whole, with a reduced sensitivity to sweetness, or should we focus on individuals, with each one eating more? I am also concerned about how this could influence the results obtained using live imaging approaches, as the flies being imaged might or might not have been motivated to eat during the feeding assays. I would like the authors to clarify their choice of analysis and discuss this critical point, as the interpretation of the results could potentially be the opposite of what is presented in the manuscript.

Author response:

Reviewer #1 (Public review):

Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the suboesophageal zone that interacts with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet-taste sensitivity and sugar-feeding behavior in male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.

Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the suboesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar-feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. found that DopR1 and Dop2R, but not DopEcR, are involved in the sexual failure-induced decrease in sugar sensitivity in these neurons. I wish the authors had further explored or discussed this discrepancy, as it is unclear how dopamine release selectively engages different receptors to modulate neuronal sensitivity in a context-dependent manner.

Our immunostaining experiments showed that three dopamine receptors, DopR1, Dop2R, and DopEcR were expressed in Gr5a⁺ neurons in the proboscis, which was consistent with previous findings by using RT-PCR (Inagaki et al 2012). As the reviewer pointed out, we found that DopR1 and Dop2R were required for courtship failure-induced suppression of sugar sensitivity, whereas Marella et al 2012 and Inagaki et al 2012 found that Dop2R and DopEcR were required for starvation-induced enhancement of sugar sensitivity. These results may suggest different internal states (courtship failure vs. starvation) modulate peripheral sensory system via different signaling pathways (e.g. different subsets of dopaminergic neurons; different dopamine release mechanisms; and different dopamine receptors). We will further discuss these possibilities in the revised manuscript.

The data presented by Wang et al. are solid and effectively support their conclusions. However, certain aspects of their experimental design, data analysis, and interpretation warrant further review, as outlined below.

(1) The authors did not explicitly indicate the feeding status of the flies, but it appears they were not starved. However, the naive and satisfied flies in this study displayed high feeding and PER baselines, similar to those observed in starved flies in other studies. This raises the concern that sexually failed flies may have consumed additional food during the 4.5-hour conditioning period, potentially lowering their baseline hunger levels and subsequently reducing PER responses. This alternative explanation is worth considering, as an earlier study demonstrated that sexually deprived males consumed more alcohol, and both alcohol and food are known rewards for flies. To address this concern, the authors could remove food during the conditioning phase to rule out its influence on the results.

We think this is a valid concern. We will conduct courtship conditioning in the absence of food and test if courtship failure can still suppress sugar sensitivity in the revised manuscript.

(2) Figure 1B reveals that approximately half of the males in the Failed group did not consume sucrose yet Figure 1-S1A suggests that the total volume consumed remained unchanged. Were the flies that did not consume sucrose omitted from the dataset presented in Figure 1-S1A? If so, does this imply that only half of the male flies experience sexual failure, or that sexual failure affects only half of males while the others remain unaffected? The authors should clarify this point.

Here is a brief clarification of our experimental design and we will further clarify the details in the revised manuscript:

After the behavioral conditioning, male flies were divided for two assays. On the one hand, we quantified PER responses of individual flies. As shown in Figure 1C, Failed males exhibited decreased sweet sensitivity (as demonstrated by the right shift of the response curve).

On the other hand, we sought to quantify food consumption of individual flies by using the MAFE assay (Qi et al 2005). When presented with 400 mM sucrose, approximately 100% of the flies in the Naïve and Satisfied groups, and 50% of the flies in the Failed group, extended their proboscis and started feeding (Figure 1B). For these flies, we could quantify the consumed volumes and found there was no change (Figure 1, S1A). We should also note the consistency of these two experiments, e.g. in Figure 1C, only 50-60% of Failed males responded to 400 mM stimulation.

These two experiments in combination suggest that sexual failure suppressed sweet sensitivity of the Failed males. Meanwhile, as long as they still initiated feeding, the volume of food consumption remained unchanged. These results led us to focus on the modulatory effect of sexual failure on the sensory system, the main topic of this present study.

In addition, to further clarify the potential misunderstanding, we plan to examine food consumption by using 800 mM sucrose in the revised manuscript. As shown in Figure 1C, 800 mM sucrose was adequate to induce feeding in ~100% of the flies.

(3) The evidence linking TH-GAL4 labeled dopaminergic neurons to reduced sugar sensitivity in Gr5a+ neurons in sexually failed males could be further strengthened. Ideally, the authors would have activated TH-GAL4 neurons and observed whether this restored GCaMP responses in Gr5a+ neurons in sexually failed males. Instead, the authors performed a less direct experiment, shown in Figures 3-S1C and D. The manuscript does not describe the condition of the flies used in this experiment, but it appears that they were not sexually conditioned. I have two concerns with this experiment. First, no statistical analysis was provided to support the enhancement of sucrose responses following activation of TH-GAL4 neurons. Second, without performing this experiment in sexually failed males, the authors lack direct evidence to confirm that the dampened response of Gr5a+ neurons to sucrose results from decreased activity in TH-GAL4 neurons.

We think this is also a valid suggestion. We will directly examine whether activating TH⁺ neurons in sexually conditioned males would enhance sugar responses of Gr5a⁺ neurons in sexually failed males. We will also add in statistical analysis.

Nevertheless, we would still argue our current experiments using Naive males (Figure 3, S1C-D) are adequate to show a functional link between TH⁺ neurons and Gr5a⁺ neurons. Combining with the results that these neurons form active synapses (Figure 3, S1B) and that the activity of TH⁺ neurons was dampened in sexually failed males (Figure 3G-I), our current data support the notion that sexual failure suppresses sweet sensitivity via TH-Gr5a circuity.

(4) The statistical methods used in this study are poorly described, making it unclear which method was used for each experiment. I suggest that the authors include a clear description of the statistical methods used for each experiment in the figure legends. Furthermore, as I have pointed out, there is a lack of statistical comparisons in Figures 3-S1C and D, a similar problem exists for Figures 6E and F.

We will add detailed information of statistical analysis in each figure legend.

(5) The experiments in Figure 5 lack specificity. The target neurons in this study are Gr5a+ neurons, which are directly involved in sugar sensing. However, the authors used the less specific Dop1R1- and Dop2R-GAL4 lines for their manipulations. Using Gr5a-GAL4 to specifically target Gr5a+ neurons would provide greater precision and ensure that the observed effects are directly attributable to the modulation of Gr5a+ neurons, rather than being influenced by potential off-target effects from other neuronal populations expressing these dopamine receptors.

We agree with the reviewer that manipulating Dop1R1 and Dop2R genes (Figure 4) and the neurons expressing them (Figure 5) might have broader impacts. In fact, we have also tested the role of Dop1R1 and Dop2R in Gr5a⁺ neurons by RNAi experiments (Figure 6). As shown by both behavioral and calcium imaging experiments, knocking down Dop1R1 and Dop2R in Gr5a⁺ neurons both eliminated the effect of sexual failure to dampen sweet sensitivity, further confirming the role of these two receptors in Gr5a⁺ neurons.

(6) I found the results presented in Fig. 6F puzzling. The knockdown of Dop2R in Gr5a+ neurons would be expected to decrease sucrose responses in naive and satisfied flies, given the role of Dop2R in enhancing sweet sensitivity. However, the figure shows an apparent increase in responses across all three groups, which contradicts this expectation. The authors may want to provide an explanation for this unexpected result.

We agree that there might be some potential discrepancies. However, our current data are not adequate for the clarification given the experiments shown in Figure 6E-F and the apparent control (Figure 3C) were not conducted under identical settings at the same (that’s why we did not directly compare these results). One way to address the issues is to conduct these calcium imaging experiments again with a head-to-head comparison with the control group (Gr5a-GCaMP, +/- Dop1R1 and Dop2R RNAi). We will conduct the experiments and present the data in the revised manuscript.

(7) In several instances in the manuscript, the authors described the effects of silencing dopamine signaling pathways or knocking down dopamine receptors in Gr5a neurons with phrases such as 'no longer exhibited reduced sweet sensitivity' (e.g., L269 and L288), 'prevent the reduction of sweet sensitivity' (e.g., L292), or 'this suppression was reversed' (e.g. L299). I found these descriptions misleading, as they suggest that sweet sensitivity in naive and satisfied groups remains normal while the reduction in failed flies is specifically prevented or reversed. However, this is not the case. The data indicate that these manipulations result in an overall decrease in sweet sensitivity across all groups, such that a further reduction in failed flies is not observed. I recommend revising these descriptions to accurately reflect the observed phenotypes and avoid any confusion regarding the effects of these manipulations.

We will change our expressions in the revised manuscript. In brief, we think that these manipulations (suppressing Dop1R1⁺ and Dop2R⁺ neurons) have two consequences: suppressing the overall sweet sensitivity and eliminating the effect of sexual failure.

Reviewer #2 (Public review):

Summary:

The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.

Strengths:

The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.

Weaknesses:

The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.

We agree with the reviewer that in the current study, we did not examine how mating experience suppressed the activity of dopaminergic neurons in the SEZ. The current study mainly focused on the behavioral characterization (sexual failure suppresses sweet sensitivity) and the downstream mechanism (TH-Gr5a pathway). We think that examining the upstream modulatory mechanism may be more suitable for a separate future study.

We believe that a sustained reduction in sweet sensitivity (not limited to sucrose but extend to other sweet compounds, Figure 1, S1B-C) upon sexual failure suggests a generalized and sustained consequence on reward-related behaviors. Sexual failure may thus resemble a state of “primitive emotion” in fruit flies. We will further discuss this possibility in the revised manuscript.

Reviewer #3 (Public review):

Summary

In this work, the authors asked how mating experience impacts reward perception and processing. For this, they employ fruit flies as a model, with a combination of behavioral, immunostaining, and live calcium imaging approaches.

Their study allowed them to demonstrate that courtship failure decreases the fraction of flies motivated to eat sweet compounds, revealing a link between reproductive stress and reward-related behaviors. This effect is mediated by a small group of dopaminergic neurons projecting to the SEZ. After courtship failure, these dopaminergic neurons exhibit reduced activity, leading to decreased Gr5a+ neuron activity via Dop1R1 and Dop2R signaling, and leading to reduced sweet sensitivity. The authors therefore showed how mating failure influences broader behavioral outputs through suppression of the dopamine-mediated reward system and underscores the interactions between reproductive and reward pathways.

Concern

My main concern regarding this study lies in the way the authors chose to present their results. If I understood correctly, they provided evidence that mating failure induces a decrease in the fraction of flies exhibiting PER. However, they also showed that food consumption was not affected (Fig. 1, supplement), suggesting that individuals who did eat consumed more. This raises questions about the analysis and interpretation of the results. Should we consider the group as a whole, with a reduced sensitivity to sweetness, or should we focus on individuals, with each one eating more? I am also concerned about how this could influence the results obtained using live imaging approaches, as the flies being imaged might or might not have been motivated to eat during the feeding assays. I would like the authors to clarify their choice of analysis and discuss this critical point, as the interpretation of the results could potentially be the opposite of what is presented in the manuscript.

Here is a brief clarification of our experimental design and we will further clarify the details in the revised manuscript:

After the behavioral conditioning, male flies were divided for two assays. On the one hand, we quantified PER responses of individual flies. As shown in Figure 1C, Failed males exhibited decreased sweet sensitivity (as demonstrated by the right shift of the response curve).

On the other hand, we sought to quantify food consumption of individual flies by using the MAFE assay (Qi et al 2005). When presented with 400 mM sucrose, approximately 100% of the flies in the Naïve and Satisfied groups, and 50% of the flies in the Failed group, extended their proboscis and started feeding (Figure 1B). For these flies, we could quantify the consumed volumes and found there was no change (Figure 1, S1A). We should also note the consistency of these two experiments, e.g. in Figure 1C, only 50-60% of Failed males responded to 400 mM stimulation.

These two experiments in combination suggest that sexual failure suppressed sweet sensitivity of the Failed males. Meanwhile, as long as they still initiated feeding, the volume of food consumption remained unchanged. These results led us to focus on the modulatory effect of sexual failure on the sensory system, the main topic of this present study.

In addition, to further clarify the potential misunderstanding, we plan to examine food consumption by using 800 mM sucrose instead. As shown in Figure 1C, 800 mM sucrose was adequate to induce feeding in ~100% of the flies.