Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone

Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small-molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-specific response to ascr#8. Concurrent biochemical studies confirmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a specific NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-specific and core locomotory circuitries.

This is a very interesting paper by Reilly et al. where they find that several peptides encoded by the flp-3 gene determine the valence of the pheromone component ascr#8 in C. elegans males. One major strength of the work is the breadth of approaches they employ. They use a combination of new behavioural assays, genetics, gene expression analysis, genome editing, biochemistry and rescue-byfeeding to show that the peptides flp-3.2 and 3.9 act through their receptors npr-10 and frpr-16 to switch the behaviour of males towards ascr#8 from repulsion to attraction. In addition, they authors are able to dissect which peptides and perhaps even aminoacids are important for a two-step process of suppression of avoidance and promotion of attraction. How sensory perception and valence of stimuli are encoded in the nervous system at the molecular and cellular level is a major question in neurobiology and one that this journal's broad readership will be interested in.
Major coments: -The authors refer to supplemental figure 2B to state that only a small percentage of animals (30-45% ) exhibit attractive visits to the cue but the graph shows % of attractive visits per worm not % of worms visiting the spot. Where is the data showing % animals?
And also, what is the % of animals showing avoidance? This data seems important because the authors state that the CEMs are the only sensors of ascr#8 (for both attraction and avoidance) and that the 35-40% responses matches well the rate of CEM calcium transients upon ascaroside exposure.
-The authors conclude that the CEM neurons are the only source of ascr#8 chemosensation based on the fact that ceh-30 mutants (which lack CEMs) are neither attracted to nor repelled by ascr#8. This conclusion is an overstatement. The experiment shows that the CEMs are indeed required for sensation, however, it doesn't exclude the possibility that other neurons may also be required (despite not being sufficient to drive behavioural responses in the absence of the CEMs). Furthermore, because npr-10, one of the receptors for flp-3 which is important for the behavioural responses to ascr#8, is expressed in ADL, a neuron shown to sense other ascarosides in a sexually dimorphic manner, the authors should test the requirement of ADL in ascr#8 sensation.
-Through the rescue-by-feeding experiments, the authors very nicely show that some peptides specifically drive attraction and others suppress avoidance. Based on the sequence of such peptides, they propose that a Threonin in the 9th position from the C terminus of the peptide is critical for suppression. This conclusion could be further strengthened with a direct manipulation: by testing whether switching the E in that position by a T in flp-3.7 and feeding it, rescues the avoidance phenotype of flp-3 mutants.
-Lines 301-302, fig. 5E -the npr-10 and frpr-16 double mutant has a somewhat surprising phenotype where the avoidance phenotype of single mutants is supressed. The authors only state that the phenotype is not additive and therefore the receptors are not redundant. However, here or in the discussion, they should provide some potential explanations for the wildtype-like phenotype of the double mutant.
Minor comments: -When they describe the expression pattern of flp-3 for the first time (p11 and 12) it is not clear whether Il1 and SPD are the only sites of expression and whether this is male-specific expression or whether hermaphrodites also have expression in IL1.
-The figures need to be better labelled stating whether the data refers to males or hermaphrodites; to ascr-8 or ascr-3; the expression patterns are of the ligand or the receptors; etc. Otherwise it gets a bit confusing.
-Line 212 -PQR is a sensory neuron, not an interneuron Reviewer #2 (Remarks to the Author): Males and hermaphrodites have differential responses to several pheromones in the nematode C. elegans. Males have an attractive response to the pheromone ascaroside #8 (ascr#8), while hermaphrodites show avoidance. Neuropeptides, such as FLP-3, 6, 12, and 19, are expressed in several male, sex-specific neurons. The authors premise that some pheromone responses are mediated through neuropeptide signaling. Response to ascr#8 was examined in this paper.
Animals were tested for their dwell time on and attraction to ascr#8. Wild-type males dwell on the ascr#8 spot for significantly more time than hermaphrodites, which avoid ascr#8. flp-6, 12, and 19 male mutants showed similar dwell times as wild-type males. By contrast, flp-3 male mutants showed significantly lower dwell times on and avoided ascr#8, suggesting that FLP-3 peptides act to suppress ascr#8 avoidance. The response of flp-3 mutants to ascr#3, a different chemoattractive pheromone, was unaffected, nicely showing that responses to ascr#8 and #3 are mediated by different pathways.
CEH-30 transcription factor is necessary for specification of the male-specific CEM neurons. Knockout (KO) of the CEMs in males did not result in an avoidance response to ascr#8 in a wild-type or flp-3 mutant background, suggesting that FLP-3 peptides are released from CEMs to suppress the avoidance response.
Several FLP-3 receptors have been previously identified: NPR-4, NPR-5, and NPR-10; the authors also classify the Drosophila homologue FRPR-16 as a FLP-3 receptor. Loss of npr-4 and npr-5 did not induce ascr#8 avoidance; by contrast, KO of npr-10 showed avoidance to ascr#8, although the avoidance was not as robust as in the flp-3 KOs, suggesting other FLP-3 receptors are aiding in the suppression response. Using CRISPR/Cas, the authors generated a frpr-16 KO, which also showed avoidance to ascr#8, although again, not as robustly as the flp-3 KO. A double npr-10; frpr-16 double knockout, surprisingly, showed no avoidance response; this result is particularly confounding as the double KOs had a decreased dwell time. This discrepancy should be addressed further in the Discussion.
To confirm that FLP-3 peptides could activate NPR-10 and FRPR-16, the npr-10 and frpr-16 constructs were transfected into CHO cells and different FLP-3 peptides applied. With the exception of FLP-3-6 and FLP-3-10, the remaining FLP-3 peptides could activate the receptors at nM concentrations; FRPR-16 could be activated at lower FLP-3 concentrations than NPR-10. However, these results are somewhat different than what was found in the peptide rescue experiments, but the discrepancy was not addressed in the Discussion.
Lastly, the authors used an innovative method to determine whether the chemoattractive response to ascr#8 could be rescued by feeding bacteria that contained different FLP-3 constructs. However, it is somewhat confusing from these data (and the referenced paper) how the peptides are generated. Bacteria are transformed with the flp-3 pGEX constructs, which were not described in Materials and Methods and described cursorily in the legend. The bacteria presumably generate the propeptide, but the construct does not have a signal peptide and it is unclear how the protein gets properly modified (cleaved and amidated) and released. Are the authors assuming that intestinal digestion of bacteria release the propeptides for modification by C. elegans? For a new technique, there should be more details and indication that the peptides are made (e.g., do bacterial lysates bind an anti-FLP antibody on a dot blot?). The FLP-3 constructs have a His tag, but it is unclear how the His tag was used. While the data looked promising and potentially a novel method of applying neuropeptides, more controls and explanations need to be included. The conclusions that only specific FLP-3 neuropeptides could rescue assumes that all neuropeptides have equal turnover and degradation rates. The N-terminal of each FLP-3 peptide differs slightly and could affect its protection from proteases, turnover rate, and/or degradation rates, thereby leading to lack of rescue.
The legends are all somewhat skimpy; additional details could be added to all. Legends that are particularly lacking are indicated below. Overall, the experiments are very cleverly designed and provide an excellent basis for understanding sex-specific behaviors. The data are well-presented but some need further clarifications.
Minor comments: 1. The differences between the S (spatial) and V (vehicle=water) controls were not explicitly delineated. Both S and V were used as controls and should presumably have the same values, yet in many instances were significantly different from each other. The vehicle control added liquid to the spot, whereas no liquid was added to the spatial controls, so it is unclear why there were differences between the two. For instance, why did osm-3 male mutants and him-5 hermaphrodite mutants dwell significantly longer on the vehicle than the spatial control (Figs. 1B and 1D)?

Response to Reviewers' comments: Reviewer #1 (Remarks to the Author):
This is a very interesting paper by Reilly et al. where they find that several peptides encoded by the flp-3 gene determine the valence of the pheromone component ascr#8 in C. elegans males. One major strength of the work is the breadth of approaches they employ. They use a combination of new behavioural assays, genetics, gene expression analysis, genome editing, biochemistry and rescue-by-feeding to show that the peptides flp-3.2 and 3.9 act through their receptors npr-10 and frpr-16 to switch the behaviour of males towards ascr#8 from repulsion to attraction. In addition, they authors are able to dissect which peptides and perhaps even amino acids are important for a two-step process of suppression of avoidance and promotion of attraction. How sensory perception and valence of stimuli are encoded in the nervous system at the molecular and cellular level is a major question in neurobiology and one that this journal's broad readership will be interested in.µ

Major comments:
Reviewer Comment-1: The authors refer to supplemental figure 2B to state that only a small percentage of animals (30-45% )

exhibit attractive visits to the cue but the graph shows % of attractive visits per worm not % of worms visiting the spot. Where is the data showing % animals? And also, what is the % of animals showing avoidance? This data seems important because the authors state that the CEMs are the only sensors of ascr#8 (for both attraction and avoidance)
and that the 35-40% responses matches well the rate of CEM calcium transients upon ascaroside exposure.
Author Response-1:We appreciate the reviewer's critical analysis of our data and interpretation. In Supp. Figure 2B, we show that males exhibit 30-45% attractive visits to ascr#8. As the reviewer points out, this is calculated as the percentage of attractive visits per worm, not the percentage of animals visiting the spot.
We have reanalyzed our data sets to investigate the percent of worms that attracted to the spot (below, Supp. Fig 2C,F). Similar to the percent attractive visits per worm, all wildtype strains how a higher percentage of attraction to ascr#8 over the vehicle control.
The Avoidance Index (AI) data displayed in the main text and figures is a direct correlate of the % of animals exhibited avoidance to ascr#8. The AI is calculated as the number of animals avoiding ascr#8 divided by the total number of animals tested. As such, the AI is merely the percent of animals avoiding divided by 100.
Reviewer Comment-2: The authors conclude that the CEM neurons are the only source of ascr#8 chemosensation based on the fact that ceh-30 mutants (which lack CEMs) are neither attracted to nor repelled by ascr#8. This conclusion is an overstatement. The experiment shows that the CEMs are indeed required for sensation, however, it doesn't exclude the possibility that other neurons may also be required (despite not being sufficient to drive behavioural responses in the absence of the CEMs). Furthermore, because npr-10, one of the receptors for flp-3 which is important for the behavioural responses to ascr#8, is expressed in ADL, a neuron shown to sense other ascarosides in a sexually dimorphic manner, the authors should test the requirement of ADL in ascr#8 sensation.
Author Response-2: We thank the reviewer for pointing out the interpretation of results to our ceh-30 lof experiments. We agree that other neurons may be indirectly involved and have changed the wording of the results (lines 231-232) to refrain from excluding the possibility of the involvement of other neurons, as they may be unable to elicit behavioral responses on their own.
Original text: "confirming that the CEM neurons are the sole source of ascr#8 chemosensation in male C. elegans." Changed text: "confirming that the CEM neurons are the primary route of ascr#8 chemosensation which results in the male C. elegans behavioral response to ascr#8." However, we argue that testing the involvement of the ADL sensory neuron will not provide an additional meaningful outcome within the scope of this paper. We have shown that (1) loss of the CEM neurons (ceh-30) abolishes the ability of males to respond attractively to ascr#8, and that (2) a ceh-30;flp-3 double mutant still does not avoid the pheromone.
While ADL does expresses NPR-10, a receptor we pose serves to propagate the flp-3 signal, ablation of the ADL neurons (and therefore NPR-10) would not add to our understanding of regulating the avoidance of ascr#8, as ceh-30;flp-3 animals already do not avoid. We feel that additional inquiry into the involvement of ADL in acsr#8 sensation, while beneficial in understanding sexually dimorphic pheromone sensation, will not add to this manuscript's insights regarding flp-3 gene's regulation of innate behaviors.
Reviewer Comment-3: Through the rescue-by-feeding experiments, the authors very nicely show that some peptides specifically drive attraction and others suppress avoidance. Based on the sequence of such peptides, they propose that a Threonine in the 9th position from the C terminus of the peptide is critical for suppression. This conclusion could be further strengthened with a direct manipulation: by testing whether switching the E in that position by a T in flp-3.7 and feeding it, rescues the avoidance phenotype of flp-3 mutants.
Author Response-3: We thank the reviewer for suggesting this vital experiment, to verify the role of the Threonine in mediating avoidance to the small-molecule ascr#8. We generated the variants using site-directed mutagenesis and fed these variants to the flp-3 lof worms. We discovered that while FLP-3-7 does not rescue either attraction or avoidance behavior, FLP-3-7T (the same peptide, with a Threonine replacing a Glutamate) is indeed able to suppress the avoidance behavior. These data are depicted in panels Figure6 E, F, G, H.
Simultaneously, these experiments serve to lend credence to our second hypothesis that the terminal NPEND sequence of FLP-3-9 is what drives attraction: FLP-3-7 contains amino acids prior to the threonine and is unable to drive attraction.
These data have been added as Figure 6 E-H, and Supp. Figure 13. Similarly, the text has been updated in all relevant sections.  Figure 5E -the npr-10 and frpr-16 double mutant has a somewhat surprising phenotype where the avoidance phenotype of single mutants is suppressed. The authors only state that the phenotype is not additive and therefore the receptors are not redundant. However, here or in the discussion, they should provide some potential explanations for the wildtype-like phenotype of the double mutant.
Author Response-4: We have adjusted the text within the results section to correct this statement.
Original text: "However, a double mutant containing both npr-10 and frpr-16 null alleles did not result in an additive effect in the avoidance phenotype, suggesting that these receptors are non-redundant in their functions ( Figure 5E, Supp. Figure 11C)." Changed Text: " Interestingly, a double mutant containing both npr-10 and frpr-16 null alleles not only did not result in an additive effect in the avoidance phenotype, but in fact suppressed the avoidance phenotype, suggesting that these receptors are non-redundant in their functions ( Figure  5E, Supp. Figure 11C)." In the discussion section, we have also elaborated further on this, as while one mutant results in a skewed behavioral response, loss of both abolishes the behavioral response: New text: "We argue that is the loss of both of these modules that underlies the lack of ascr#8 behavioral response in npr-10;frpr-16 double mutant animals (Figure 3, 5)."

Minor comments:
Reviewer Comment-5: When they describe the expression pattern of flp-3 for the first time (p11 and 12) it is not clear whether Il1 and SPD are the only sites of expression and whether this is male-specific expression or whether hermaphrodites also have expression in IL1.
Author Response-5: We apologize for the lack of clarity in our description of the flp-3 expression pattern. Within the male C. elegans, IL1 and SPD are the only sites of expression -which is now clarified in the text. Characterizing the expression pattern of the translational fusion of flp-3 in hermaphrodites indicates that flp-3 expressed in the amphid IL1 neurons. Comparing the expression patterns in the two sexes suggests that flp-3 expression in the male-specific neuron SPD making flp-3 tail expression sex-specific.
The revised version of the manuscript now includes an image depicting hermaphrodite flp-3 localization within IL1 neurons, alongside the supplementary figure depicting the same for the male (Supp. Figure 6). We have also updated the text describing the expression pattern of flp-3 to reflect this clearer finding (Lines 216-226).
Supp. Figure 6 E, F: (E, F) IL1 expression of pflp-3::flp-3::mCherry. mCherry is faintly observed in the IL1 soma (arrows). The fluorescent protein is also observed in the dendritic cilia of the IL1 neurons (bars), as well as in punctate vesicles along the dendrites (arrowheads).

Reviewer Comment-6: The figures need to be better labelled stating whether the data refers to males or hermaphrodites; to ascr-8 or ascr-3; the expression patterns are of the ligand or the receptors; etc. Otherwise, it gets a bit confusing.
Author Response-6: We thank the reviewer for pointing out this lack of clarity in the expression image panels. We have now added male and hermaphrodite symbols to all figure panels depicting data (schematic panels do no include symbols). We have also edited the text of Supp. Figure 5 denoting that the data is relating to ascr#3, not ascr#8, for that figure only.

Reviewer Comment-7: Line 212 -PQR is a sensory neuron, not an interneuron
Author Response-7: We appreciate the reviewer noting this oversight and have since updated the text to correct this mistake.
Reviewer Comment-1: Males and hermaphrodites have differential responses to several pheromones in the nematode C. elegans. Males have an attractive response to the pheromone ascaroside #8 (ascr#8), while hermaphrodites show avoidance. Neuropeptides,6,12,and 19, are expressed in several male, sex-specific neurons. The authors premise that some pheromone responses are mediated through neuropeptide signaling. Response to ascr#8 was examined in this paper.
Animals were tested for their dwell time on and attraction to ascr#8. Wild-type males dwell on the ascr#8 spot for significantly more time than hermaphrodites, which avoid ascr#8. flp-6, 12, and 19 male mutants showed similar dwell times as wild-type males. By contrast, flp-3 male mutants showed significantly lower dwell times on and avoided ascr#8, suggesting that FLP-3 peptides act to suppress ascr#8 avoidance. The response of flp-3 mutants to ascr#3, a different chemoattractive pheromone, was unaffected, nicely showing that responses to ascr#8 and #3 are mediated by different pathways.

CEH-30 transcription factor is necessary for specification of the male-specific CEM neurons. Knockout (KO) of the CEMs in males did not result in an avoidance response to ascr#8 in a wildtype or flp-3 mutant background, suggesting that FLP-3 peptides are released from CEMs to suppress the avoidance response.
Author Response-1: We apologize for the lack of clarity in the text describing CEM neurons. We do not believe that the CEM neurons are responsible for releasing FLP-3 peptides. Based on our expression pattern analyses, the flp-3 translational fusion does not express within CEM neurons, but instead the IL1 neurons of the amphid region. (Figure number ….) We instead argue that the CEM neurons are a major component of the ascr#8 sensory network, and that this sensory network is upstream of a behavioral network that is modulated by FLP-3 signaling.
Original text: "confirming that the CEM neurons are the sole source of ascr#8 chemosensation in male C. elegans." Changed text: "confirming that the CEM neurons are the primary route of ascr#8 chemosensation which results in the male C. elegans behavioral response." Reviewer Comment-2: Several FLP-3 receptors have been previously identified: NPR-4, NPR-5, and NPR-10; the authors also classify the Drosophila homologue FRPR-16 as a FLP-3 receptor. Loss of npr-4 and npr-5 did not induce ascr#8 avoidance; by contrast, KO of npr-10 showed avoidance to ascr#8, although the avoidance was not as robust as in the flp-3 KOs, suggesting other FLP-3 receptors are aiding in the suppression response. Using CRISPR/Cas, the authors generated a frpr-16 KO, which also showed avoidance to ascr#8, although again, not as robustly as the flp-3 KO. A double npr-10; frpr-16 double knockout, surprisingly, showed no avoidance response; this result is particularly confounding as the double KOs had a decreased dwell time. This discrepancy should be addressed further in the Discussion.
Author Response-2: We agree with the reviewer's suggestion. In our revised version, we have elaborated our explanation as to how NPR module loss affects the behavioral response on further in the Discussion. (Lines 412-217).
New text: "We argue that it is the loss of both of these modules that underlies the lack of ascr#8 behavioral response in npr-10;frpr-16 double mutant animals (Figure 3, 5); the loss of one module results in a skewed behavioral response, while the loss of both NPR-10 and FRPR-16 modules abolishes the ability of the animal to response to ascr#8. Future studies incorporating cell-specific rescue of both NPR-10 and FRPR-16 will further elucidate this circuitry." Reviewer Comment-3: To confirm that FLP-3 peptides could activate NPR-10 and FRPR-16, the npr-10 and frpr-16 constructs were transfected into CHO cells and different FLP-3 peptides applied. With the exception of FLP-3-6 and FLP-3-10, the remaining FLP-3 peptides could activate the receptors at nM concentrations; FRPR-16 could be activated at lower FLP-3 concentrations than NPR-10. However, these results are somewhat different than what was found in the peptide rescue experiments, but the discrepancy was not addressed in the Discussion.
Author Response-3: We appreciate the Reviewer's comment on our biochemical data and its relevant conclusions. We would like to clarify our interpretation of the in-vitro binding efficiency of the receptor to the ligand and we believe that this binding efficiency does not require perfect correlation to in vivo potencies.
In the heterologous system employed in our manuscript as well as other manuscripts that have used this technology (Nelson et al., PLos One, 2015;Iannacone et al., Elife, 2017, Van Sinay et al., PNAS, 2017Peymen et al, PLoS Genetics, 2019), a worm receptor is expressed in mammalian cells. These cells inherently may differ in their innate G protein coupling systems, co-factors, and even the functionality of the receptor. To obtain EC50 values for the most receptors, the human G alpha 16 protein is used.
However, while we agree that in vivo, these receptors may couple with other G proteins at varying levels of efficiency, the EC50 values obtained in our in-vitro binding studies should be considered as indicative of the physiological relevance of the interaction: nM affinities tend to be functionally relevant.
The behavioral experiments in this study are not a verification of binding affinity, but rather a confirmation of ligand-receptor activity in vivo. Though FRPR-16 may exhibit higher affinities than NPR-10 in vitro, this may change in vivo. Furthermore, the full ascr#8 behavioral circuitry is much more complex than the valence-control mechanism elucidated in this study.
Reviewer Comment-4: Lastly, the authors used an innovative method to determine whether the chemoattractive response to ascr#8 could be rescued by feeding bacteria that contained different FLP-3 constructs. However, it is somewhat confusing from these data (and the referenced paper) how the peptides are generated. Bacteria are transformed with the flp-3 pGEX constructs, which were not described in Materials and Methods and described cursorily in the legend. The bacteria presumably generate the propeptide, but the construct does not have a signal peptide and it is unclear how the protein gets properly modified (cleaved and amidated) and released. Are the authors assuming that intestinal digestion of bacteria release the propeptides for modification by C. elegans? For a new technique, there should be more details and indication that the peptides are made (e.g., do bacterial lysates bind an anti-FLP antibody on a dot blot?). The FLP-3 constructs have a His tag, but it is unclear how the His tag was used. While the data looked promising and potentially a novel method of applying neuropeptides, more controls and explanations need to be included. The conclusions that only specific FLP-3 neuropeptides could rescue assumes that all neuropeptides have equal turnover and degradation rates. The N-terminal of each FLP-3 peptide differs slightly and could affect its protection from proteases, turnover rate, and/or degradation rates, thereby leading to lack of rescue.
Author Response-4:We appreciate the reviewer's comment on the lack of clarity in the manuscript of our novel rescue paradigm. In the revised version, we added more details to provide a clear explanation of the method. To clarify the reviewer's comment, we would like to highlight the some of the important aspects of the technique: 1. We did not use pGEX, but instead generated peptide-encoding plasmids using Gateway Cloning technology: first into a p1-p2 vector, and finally pDEST527: a bacterial expression vector with a T7 promoter which is induced by IPTG. 2. While we agree that it is likely that the bacteria produces the propeptide, we believe that it is the mRNA that is taken up by the nematode for functional translation. 3. The His tag is a a component of the expression vector used previously (Xu et al., Peptides, 2017, doi: 10.1016/j.peptides.2017.01.003). We did not alter the vector used in our technique. While we agree that it would be interesting to understand the mechanisms of the feeding protocol in this manuscript, we have now submitted for review a manuscript that provides more detail on the mechanisms underlying the technique DiLoreto, Reilly and Srinivasan (Scientific Reports in review, biorxiv, doi: 10.1101/2021. Out studies suggest that the mechanism of uptake is via mRNA uptake over direct peptide uptake.
Reviewer Comment-5: The legends are all somewhat skimpy; additional details could be added to all. Legends that are particularly lacking are indicated below. Overall, the experiments are very cleverly designed and provide an excellent basis for understanding sex-specific behaviors. The data are well-presented but some need further clarifications.
Author Response-5: The revised version of the manuscript contains detailed description of each of the figure legends and we thank the reviewer for pointing out the lack of description in our figure captions.

Minor comments:
Reviewer Comment-6: The differences between the S (spatial) and V (vehicle=water) controls were not explicitly delineated. Both S and V were used as controls and should presumably have the same values, yet in many instances were significantly different from each other. The vehicle control added liquid to the spot, whereas no liquid was added to the spatial controls, so it is unclear why there were differences between the two. For instance, why did osm-3 male mutants and him-5 hermaphrodite mutants dwell significantly longer on the vehicle than the spatial control (Figs. 1B and 1D)?
Author Response-6: We thank the reviewer for pointing out our lack of elaboration on the differences between spatial and vehicle controls. We have since added to the text our explanations (Lines 123-126).
Original text: "In this assay, individual animals are placed directly into the spot of the ascaroside cue while simultaneously removing any potential of male-male contact." Revised text: "In this assay, individual animals are placed directly into the spot of the ascaroside cue while simultaneously removing any potential of male-male contact. A spatial control is included throughout the assay plate to allow us to investigate any innate differences in the number of visits to the well center, or the time spent therein, of which we have only found one strain to date with differences in male dwell time ( Figure 1B)." In summary, some mutant stains exhibit 'edge effects', we wanted to be able to spot differences in how often strains visited the center of the well with no controls (spatial), and what affect water (vehicle) had on that. osm-3 mutant males (Figure 1B) is the only strain that statistically exhibited an increase in vehicle dwell time over the spatial control. Figure 1D displays hermaphrodite data. Hermaphrodites repeatedly exhibit differences in spatial vs. vehicle dwell time (which is abolished in ascr#8 conditions). But as this study is focused on investigating male attraction to ascr#8, we have not determined the mechanisms driving the increase in hermaphrodite vehicle dwell time.
Reviewer Comment-7: Figure 2 legend: A bit too concise. Should be fleshed out more to understand what was done.
Author Response-7: The revised manuscript expands the description of this figure legend to add more clarity and aid the reader in more fully understanding what was done in these experiments.
Reviewer   Author Response-8: We thank the reviewer for bringing this inconsistency to our attention. We have since corrected the values for C16D6.2 and Y58G8A.4 to reflect the correct micromolar affinities in the revised manuscript.
Reviewer  The authors assume that all FLP-3 peptides have the same function. However, different FLP-3 peptides could have different functions, some of which are not related to the pheromone response.
Author Response-9: We agree with the reviewer that the other peptides can have functions not related to pheromone responses. Our data clearly shows that FLP-3-2 suppresses the avoidance response, while FLP-3-9 drives attraction ( Figure 6). Since the other peptides did not affect ascr#8 responses, we interpreted that these peptides do not play a role in pheromone responses at all. We have added textual clarification throughout the revised text to imply the same.
Reviewer Comment-10: Supp. Figure 5 legend: The legend is confusing. Is this figure just showing data for ascr#3? If so, remove mention of ascr#8 from the legend.
Author Response-10: We have changed the title of the figure legend to read, "Loss of flp-3 does not affect male behavioral response to acsr#3." We have additionally added text over each panel clarifying that this figure is depicting data for ascr#3.
Reviewer Comment-11: Figure 6 legend: As with Materials & Methods, there needs to be more explanation.
Author Response-11: We have added description to include more detail to the figure legend in the revised version of the manuscript.