The Neuropeptide Sulfakinin, a peripheral regulator of insect behavioral switch between mating and foraging

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study characterizes the role of sulfakinin and the sulfakinin receptor 1 in changes in olfactory responses associated with foraging versus mating behavior in the oriental fruit fly (Bactrocera dorsalis), a significant agricultural pest. This pathway regulates food consumption and mating receptivity in other species; here the authors use genetic disruption of sulfakinin and sulfakinin receptor 1 to provide strong evidence that changes in sulfakinin signaling modulate antennal responses to food versus pheromonal cues and alter the expression of ORs that detect relevant stimuli.

Strengths:

The authors utilize multiple complementary approaches including CRISPR/Cas9 mutagenesis, behavioral characterization, electroantennograms, RNA sequencing and heterologous expression to convincingly demonstrate the involvement of the sulfakinin pathway in the switch between foraging and mating behaviors. The use of both sulfakinin peptide and receptor mutants is a strength of the study and implicates specific signaling actors.

Weaknesses:

The authors demonstrate that SKR is expressed in olfactory neurons, however there are additional potential sites of action that may contribute to these results.

Author response:

The following is the authors’ response to the original reviews.

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study investigated the role of the neuropeptide, sulfakinin, and of its receptor, the sulfakinin receptor 1 (SkR1), in mediating this switch in the oriental fruit fly, Bactrocera dorsalis. The authors use genetic disruption of sulfakinin and of SkR1 to provide strong evidence that changes in sulfakinin signaling alter odorant receptor expression profiles and antennal responses and that these changes mediate the behavioral switch. The combination of molecular and physiological data is a strength of the study. Additional work would be needed to determine whether the physiological and molecular changes observed account for the behavioral changes observed.

Strengths:

(1) The authors show that sulfakinin signaling in the olfactory organ mediates the switch between foraging and mating, thereby providing evidence that peripheral sensory inputs contribute to this important change in behavior.

(2) The authors' development of an assay to investigate the behavioral switch and their use of different approaches to demonstrate the role of sulfakinin and SkR1 in this process provides strong support for their hypothesis.

(3) The manuscript is overall well-organized and documented.

Weaknesses:

(1) The authors claim that sulfakinin acts directly on SkR1-positive neurons to modulate the foraging and mating behaviors in B. dorsalis. The authors also indicated in the schematic that satiation suppresses SkR1 expression. Additional experiments and more a detailed discussion of the results would help support these claims.

(2) The findings reported could be strengthened with additional experimental details regarding time of day versus duration of starvation effects and additional genetic controls, amongst others.

Recommendations for the authors:

Major issues

(1) As written the introduction is somewhat fragmented and does not lay out a clear rationale for the current study in the species used by the authors. Others, including Guo et al. (2021) and Wang et al. (2022), have previously shown that sulfakinin signaling pathways are important for feeding and receptivity regulation in D. melanogaster. Thus, the novelty of this study should be more clearly articulated.

The introduction in the revision is significantly changed to improve the description for the rationale of study (lines 60-66 in the revision).

(2) In addition, the Introduction should provide more specific background information on the pheromonal activity of oriental fruit fly body extract, the odor-preferences, and the sex pheromone of this species compared to that of model insects such as Drosophila melanogaster.

The revision contains a paragraph of introduction for chemical ecology of oriental fruit fly that is related to this study (lines 67-75).

(3) It isn't clear what the first image in Figure 1C represents - is this a schematic of the area or does it represent data?

The Fig 1C and the associated figure caption are revised. The figure is more visible by changing the track colors. The figure caption is revised as “Representative foraging trajectories in the 100 mm diameter arenas within a 15-min observation period of flies starved for different durations.”

(4) The authors should include examples of the EAG recordings following the stimulation with food volatiles or pheromones, not only the results of their analyses. This could be included in the main figures or even in supporting information.

As suggested, we added the examples of the EAG recordings following the stimulation with food odors and body extracts in the Figure 1 and Figure 3.

(5) The demonstration that removal of the antennae severely impairs mating is dispensable because the antennae are required for other functions in addition to olfaction.

We agree that the roles of the antennae are likely more than the olfactory function. As suggested, we removed the data.

(6) It is currently difficult to understand how the authors measured successful rates of foraging. Please provide more details.

In the revision, we added a sentence describing the method for measuring in detail. See line 269-273.

(7) The expression of sulfakinin does not change significantly in the antennae following starvation (Figure 2A). Do the authors know whether they change in the central nervous system under these conditions? Have the authors (or has anyone else) checked the expression pattern of sulfakinin in the antennae? This information would help determine whether the sulfakinin signal that acts on SkR1 is released from neurons in the central nervous system (Figure S4C) or whether it is also released from the neurons in the olfactory organs. Based on the immunochemistry results shown in Figure S4C, it would also be interesting to determine whether the intensity of anti-sulfakinin immunoreactivity changes before versus after starvation. This could help establish whether sulfakinin is released during starvation.

We added the expression data showing the the mRNA level of Sk in the head that is higher after refeeding in Fig. S3. The change in the expression of Sk is also added in the text (lines 107-110). We were unable to identify the Sk neurons in the antennae suggesting possibility of the direct action of humoral Sk on the antennae.

(8) In Figure 2A, the authors show that the expression levels of some neuropeptides system components change during starvation. However, it would be helpful if the authors could include more detailed information on how the results are shown in the figure legends (e.g., the expression level of each candidate in fed flies was set as 1, etc).

We revised the figure caption to explain the Figure 2 with the expression values in the figure legend.

(9) In Figure 2D, null mutant males of sulfakinin and SkR1 consume more food at all times compared to the wild type. However, the corresponding mutant females consume more food only at night. Is this because the wild-type female flies eat more food during the day? In a related issue, Figure 2D shows differences in food consumption measured at different times of day, however, this is not directly addressed in the text, which instead mentions that "the amount of excess food consumed by the mutants was dependent on the duration of the starvation period in both sexes".

Thank you for the important suggestions. We speculate that the difference of feeding amounts of females occurring only at night is due to the high basal feeding rate of females during the daytime, masking the increase in feeding in the knockout of Sk signaling. As suggested, we have added a relevant description of the difference in food consumption. In addition, we changed the Y-axis scale in the figure for a justified comparison between males and females. See line 123-128.

(10) It isn't clear how the time of day relates to the duration of starvation. This suggests that mutant females only consume more at 21:00 (presumably at night) whereas males consume more throughout the day. Does this suggest an interaction with the circadian system? What is the duration of starvation in Figure 3A? In a related issue, in Figure 4 it would be useful to know what time of day the EAG analysis was done because the data shown in Figure 2D suggests that the time of day significantly impacts behavioral responses. And does the red versus blue color scheme of the OR subunits represent up/downregulated levels in wild-type animals? Please define this for the reader.

In addition to the response to the point 9, responding to the issue of feeding amount in females. As the reviewer noted, there was indeed a diurnal difference in food amount consumed by B. dorsalis. However, whether this is related to circadian rhythms is something we haven't studied for further in-depth. Measuring food intake at these 3 times of day, we all ensured that the duration of starvation was the same 12 h. The duration of starvation in Figure 3A is 12h. We have mentioned this in the manuscript. See line 267-268.

The EAG for sex pheromones and body surface extracts were measured form 21:00-23:00, and food odor was measured from 9:00-11:00. The times of the experiments are described in the revision. See line 309-311.

Accordingly, we made a revision of the figure caption for explaining the colored fonts. Red color represents a set of ORs related with foraging and blue color is for a set of ORs related with mating. Therefore, the ORs with red color were upregulated in starved wild-type animals and the ORs with blue color were downregulated in starved wild-type flies. We have defined this in the revised manuscript. See line 672-673.

(11) The authors convincingly show that SKR1 is present in the antennae and is co-expressed with orco. It would be useful to discuss whether this receptor is also expressed in other tissues where there may be additional sites of action of this pathway.

Indeed, SkR1 is also expressed in the Drosophila brain. We added the discussion on the expression and additional sites of action of SKR1 within the central nervous system. See line 200-205.

(12) It isn't clear what the dotted arrows in the model shown in Figure 5 represent.

Dashed arrows represent the additional possible pathways that have not been tested in this study, but not excluded in the model. Please see the discussion for details of additional possible factors modulating odorant sensitivity relevant to satiety. See line 210-229.

(13) In Figure 5, the authors indicate that satiation suppresses SkR1 expression. It would be helpful if the authors tested the expression level of SkR1 in re-fed flies (by feeding the flies after 12h starvation) to see whether levels of expression are rapidly restored to the levels seen in satiated animals. Such a result could further support the claims made by the authors.

Thank for your suggestions. Indeed, refeeding after 12h starvation significantly decreased SkR1. We added the result in supporting information (Fig. S3). See line 713. Results see line 107-110.

(14) The authors show that locomotor activity is unaffected in the mutants but body size comparison would be more useful here since this could also contribute to baseline differences in meal size.

In the revision, we provided a comparison between WT and Sk-/- in the supplementary data. Results showed that mutant flies have the same body size as the WT flies. (Fig. S7) See line 742. Results see line 120-121.

(15) Have the authors tested the behavioral phenotypes of heterozygotes mutant of both Sk and SkR1 flies? This may reveal whether a reduced expression of Sk-SkR1 will also cause significant changes in the foraging and mating behaviors seen during starvation.

We tested the behavioral phenotypes of heterozygous mutant of Sk knockout flies. The results showed that foraging and mating behaviors of Sk heterozygous mutants were unaffected during starvation, suggesting the mutants are completely recessive. We have added the results in supporting information (Fig. S8). See line 746. Results see line 132-135.

(16) It would be useful to provide information about which SK peptide is detected by the antibody used in Figure S4C. In Figures S4C and S5D, it would be useful to include a counterstain to show that the general morphology is unaffected in the mutants.

As suggested, we added a detailed description for rabbit anti-BdSk antibody. See line 362-363. We have improved the background image to be available to show the general structure. So counter staining would not be essential.

(17) The figure legends for supporting figures need to be improved as they are currently difficult to understand. For example, in S2: what is the meaning of "different removal of antennae"? In S3: it isn't clear how the authors evaluated the responses in EAG experiments; in S4A: there are several DNA sequences that do not appear in the main text of the manuscript; in S4C: the meaning of the boxes and the dots is unclear, as is the figure to the left; in S5D, the authors explain only the suppression of SKR1, yet the figure indicates some images for SKR IHC. These are only a few examples; we ask that the authors revise and improve the legends for supporting figures.

For S2, we removed the data as suggested. For S3, we added a sentence describing the method for measuring in detail. See line 707-709. For S4, the figure in the revision is significantly changed and added a detailed description in the legend (lines 717-724 in the revision). For S5, we have improved our description. See line 731-734. In addition, we have checked all the figure legends of our manuscript and changes were displayed in track version.

Minor issues

(1) It isn't clear what the meaning of "the complexity of sulfakinin pathways" is. Please explain.

We have rewritten the sentence in the revised manuscript by adding the description as “…complexity of Sk pathways, special and temporal dynamics and multiple ligands and receptors, is…”. See line 61-65.

(2) Please double-check the calls to the various figures in the text.

We have double-checked the calls to all the figures in the text to make sure they were correct.

(3) L125: What is the meaning of "olfactory reprogramming"? Please explain.

We rephrased it to “alteration of olfactory sensitivities”. See line 145.

(4) L135: After mentioning qRT-PCR the authors should include a call to a figure that shows these results.

Thank you for your suggestion, the qRT-PCR results are shown in Figure 4B, and we have added it as suggested. See line 154.

(5) L270: Details are provided for the extraction of the pheromone. However, more details are needed on how the EAG and other functional assays were done.

We have described the assay procedures in detail in the materials and method part. See line 298-311.

(6) Figure 2B. Please remove the period(".") at the C-terminal end of WT sk.

We are sorry for our mistake. We have corrected it.