Microbiota impact Drosophila ageing via Acetobacter, Tachykinin, and TkR99D

  1. Diana Marcu
  2. David R Sannino
  3. Anthony J Dornan
  4. Rita Ibrahim
  5. Atharv Kapoor
  6. Miriam Wood
  7. Adam J Dobson

  • Curated by eLife

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    eLife Assessment

    This important study demonstrates that in Drosophila melanogaster, tachykinin (Tk) expression is regulated by the microbiota. The authors present convincing evidence that axenic flies raised with no microbiota are longer-lived than conventionally reared animals, and that Tk expression and Tk receptors in the nervous system are required for this effect. They further test individual bacterial strains for their role in these effects and connect the effect to loss of lipid stores and suggest that FOXO may be involved in the phenotype, results that are of interest to the fields of environmental perception, host microbiome interactions, and geroscience.

    [Editors' note: this paper was reviewed by Review Commons.]

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Abstract

Gut microbiota exert an evolutionarily conserved influence on ageing, from invertebrates to humans. How do microbes that are physically confined to the gut lumen affect the systemic physiological process of ageing? In female Drosophila, we show that microbiota increase expression of the peptide hormone Tachykinin (Tk), which corresponds to reduced lifespan. Tk is required for microbiota to shorten lifespan, with knockdown rendering flies constitutively long-lived even in the presence of an intact microbiota. This lifespan extension does not come with canonical costs to fecundity or feeding, but impacts on triacylglyceride (TAG) storage suggest adaptive functions in metabolic homeostasis. In flies with defined (gnotobiotic) microbiotas, we show that we can model Tk-dependent effects of microbiota on lifespan and TAG by monoassociation with Acetobacter pomorum. These effects require Tk in the midgut, and the cognate TK receptor TkR99D in neurons, implicating a microbiota-gut-neuron relay. This relay also appears to compromise gut barrier function in aged flies, indicating roles in healthspan as well as lifespan. However, the effect of TkR99D is independent of its reported role in insulin signalling and adipokinetic hormone signalling which, respectively, are canonical regulators of lifespan and TAG metabolism, suggesting a non-canonical role for TkR99D elsewhere in the nervous system. Altogether our results implicate a microbiota-gut-neuron axis in ageing, via a specific bacterium modulating activity of a specific and evolutionarily-conserved hormone.

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  1. Version published to 10.7554/elife.109547.1 on eLife
  2. Version published to 10.7554/elife.109547 on eLife
  3. eLife

    eLife Assessment

    This important study demonstrates that in Drosophila melanogaster, tachykinin (Tk) expression is regulated by the microbiota. The authors present convincing evidence that axenic flies raised with no microbiota are longer-lived than conventionally reared animals, and that Tk expression and Tk receptors in the nervous system are required for this effect. They further test individual bacterial strains for their role in these effects and connect the effect to loss of lipid stores and suggest that FOXO may be involved in the phenotype, results that are of interest to the fields of environmental perception, host microbiome interactions, and geroscience.

    [Editors' note: this paper was reviewed by Review Commons.]

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    In this study the authors use a Drosophila model to demonstrate that Tachykinin (Tk) expression is regulated by the microbiota. In Drosophila conventionally reared (CR) flies are typically shorter lived than those raised without a microbiota (axenic). Here, knockdown of Tk expression is found to prevent lifespan shortening by the microbiota and the reduction of lipid stores typically seen in CR flies when compared to axenic counterparts. It does so without reducing food intake or fecundity which are often seen as necessary trade-offs for lifespan extension. Further, the strength of the interaction between Tk and the microbiota is found to be bacteria specific and is stronger in Acetobacter pomorum (Ap) mono-associated flies compared to Levilactobacillus brevis (Lb) mono-association. The impact on lipid storage was also only apparent in Ap-flies.

    Building on these findings the authors show that gut specific knockdown is largely sufficient to explain these phenotypes. Knockdown of the Tk receptor, TkR99D, in neurons recapitulates the lifespan phenotype of intestinal Tk knockdown supporting a model whereby Tk from the gut signals to TkR99D expressing neurons to regulate lifespan. In addition, the authors show that FOXO may have a role in lifespan regulation by the Tk-microbiota interaction. However, they rule out a role for insulin producing cells or Akh-producing cells suggesting the microbiota-Tk interaction regulates lifespan through other, yet unidentified, mechanisms.

    Major comments:

    Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. In particular, the impact of TkRNAi on lifespan and lipid levels, the central finding in this study, has been demonstrated multiple times in different experiments and using different genetic tools. As a result, I don't feel that additional experimental work is necessary to support the current conclusions.

    However, I find it hard to assess the robustness of the lifespan data from the other manipulations used (TkR99DRNAi, TkRNAi in dFoxo mutants etc.) because information on the population size and whether these experiments have been replicated is lacking. Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done? For all other data it is clear how many replicates have been done, and the methods give enough detail for all experiments to be reproduced.

    Significance:

    Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. We have known that the microbiota influence lifespan for some time but the mechanisms by which they do so have remained elusive. This study identifies one such mechanism and as a result opens several avenues for further research. The Tk-microbiota interaction is shown to be important for both lifespan and lipid homeostasis, although it's clear these are independent phenotypes. The fact that the outcome of the Tk-microbiota interaction depends on the bacterial species is of particular interest because it supports the idea that manipulation of the microbiota, or specific aspects of the host-microbiota interaction, may have therapeutic potential.

    These findings will be of interest to a broad readership spanning host-microbiota interactions and their influence on host health. They move forward the study of microbial regulation of host longevity and have relevance to our understanding of microbial regulation of host lipid homeostasis. They will also be of significant interest to those studying the mechanisms of action and physiological roles of Tachykinins.

    Field of expertise: Drosophila, gut, ageing, microbiota, innate immunity

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    The main finding of this work is that microbiota impacts lifespan though regulating the expression of a gut hormone (Tk) which in turn acts on its receptor expressed on neurons. This conclusion is robust and based on a number of experimental observations, carefully using techniques in fly genetics and physiology: 1) microbiota regulates Tk expression, 2) lifespan reduction by microbiota is absent when Tk is knocked down in gut (specifically in the EEs), 3) Tk knockdown extends lifespan and this is recapitulated by knockdown of a Tk receptor in neurons. These key conclusions are very convincing. Additional data are presented detailing the relationship between Tk and insulin/IGF signalling and Akh in this context. These are two other important endocrine signalling pathways in flies. The presentation and analysis of the data are excellent.

    There are only a few experiments or edits that I would suggest as important to confirm or refine the conclusions of this manuscript. These are:

    (1) When comparing the effects of microbiota (or single bacterial species) in different genetic backgrounds or experimental conditions, I think it would be good to show that the bacterial levels are not impacted by the other intervention(s). For example, the lifespan results observed in Figure 2A are consistent with Tk acting downstream of the microbes but also with Tk RNAi having an impact on the microbiota itself. I think this simple, additional control could be done for a few key experiments. Similarly, the authors could compare the two bacterial species to see if the differences in their effects come from different ability to colonise the flies.

    (2) The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this? Better clarification is required.

    (3) With respect to insulin signalling, all the experiments bar one indicate that insulin is mediating the effects of Tk. The one experiment that does not is using dilpGS to knock down TkR99D. Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this, but as a minimum I would be a bit more cautious with the interpretation of these data.

    (4) Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned? This would further clarify that there are no off-target effects that can account for the phenotypes.

    There are a few other experiments that I could suggest as I think they could enrich the current manuscript, but I do not believe they are essential for publication:

    (5) The manuscript could be extended with a little more biochemical/cell biology analysis. For example, is it possible to look at Tk protein levels, Tk levels in circulation, or even TkR receptor activation or activation of its downstream signalling pathways? Comparing Ax and CR or Ap and CR one would expect to find differences consistent with the model proposed. This would add depth to the genetic analysis already conducted. Similarly, for insulin signalling - would it be possible to use some readout of the pathway activity and compare between Ax and CR or Ap and CR?

    (6) The authors use a pan-acetyl-K antibody but are specifically interested in acetylated histones. Would it be possible to use antibodies for acetylated histones? This would have the added benefit that one can confirm the changes are not in the levels of histones themselves.

    (7) I think the presentation of the results could be tightened a bit, with fewer sections and one figure per section.

    Significance:

    The main contribution of this manuscript is the identification of a mechanism that links the microbiota to lifespan. This is very exciting and topical for several reasons:

    (1) The microbiota is very important for overall health but it is still unclear how. Studying the interaction between microbiota and health is an emerging, growing field, and one that has attracted a lot of interest, but one that is often lacking in mechanistic insight. Identifying mechanisms provides opportunities for therapies. The main impact of this study comes from using the fruit fly to identify a mechanism.

    (2) It is very interesting that the authors focus on an endocrine mechanism, especially with the clear clinical relevance of gut hormones to human health recently demonstrated with new, effective therapies (e.g. Wegovy).

    (3) Tk is emerging as an important fly hormone and this study adds a new and interesting dimension by placing TK between microbiota and lifespan.

    I think the manuscript will be of great interest to researchers in ageing, human and animal physiology and in gut endocrinology and gut function.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    Marcu et al. demonstrate a gut-neuron axis that is required for the lifespan-shortening effects mediated by gut bacteria. They show that the presence of commensal bacteria-particularly Acetobacter pomorum-promotes Tk expression in the gut, which then binds to neuronal tachykinin receptors to shorten lifespan. Tk has also recently been reported to extend lifespan via adipokinetic hormone (Akh) signaling (Ahrentløv et al., Nat Metab 7, 2025), but the mechanism here appears distinct. The lifespan shortening by Ap via Tk seems to be partially dependent on foxo and independent of both insulin signaling and Akh-mediated lipid mobilization.

    Although the detailed mechanistic link to lifespan is not fully resolved, the experiment and its results clearly show the involvement of the molecules tested. This work adds a valuable dimension to our growing understanding of how gut bacteria influence host longevity. However, there are some points that should be addressed.

    (1) Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.

    (2) In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary.

    (3) If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.

    (4) Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association and/or (2) compare the results using different genes as internal standard.

    (5) While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (i.e.. Tk transcription or EE activity upregulation) by the microbiome on males.

    (6) We also had some concerns regarding the wording of the title.
    Fig6B and C suggests that TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D.
    The difference between "aging" and "lifespan" should also be addressed. While the study shows a role for Tk in lifespan, assessment of aging phenotypes (e.g. Climbing assay, ISC proliferation) beyond the smurf assay is required to make conclusions about aging.

    (7) The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference, if available.

    Significance:

    General assessment: The main strength of this study is the careful and extensive lifespan analyses, which convincingly demonstrate the role of gut microbiota in regulating longevity. The authors clarify an important aspect of how microbial factors contribute to lifespan control. The main limitation is that the study primarily confirms the involvement of previously reported signaling pathways, without identifying novel molecular players or previously unrecognized mechanisms of lifespan regulation.

    Advance: The lifespan-shortening effect of Acetobacter pomorum (Ap) has been reported previously, as has the lifespan-shortening effect of Tachykinin (Tk). However, this study is the first to link these two factors mechanistically, which represents a significant and original contribution to the field. The advance is primarily mechanistic, providing new insight into how microbial cues converge on host signaling pathways to influence ageing.

    Audience: This work will be of particular interest to a specialized audience of basic researchers in ageing biology. It will also attract interest from microbiome researchers who are investigating host-microbe interactions and their physiological consequences. The findings will be useful as a conceptual framework for future mechanistic studies in this area.

    Field of expertise: Drosophila ageing, lifespan, microbiome, metabolism

  7. eLife

    Author response:

    (1) General Statements

    The goal of our study was to mechanistically connect microbiota to host longevity. We have done so using a combination of genetic and physiological experiments, which outline a role for a neuroendocrine relay mediated by the intestinal neuropeptide Tachykinin, and its receptor TkR99D in neurons. We also show a requirement for these genes in metabolic and healthspan effects of microbiota.

    The referees' comments suggest they find the data novel and technically sound. We have added data in response to numerous points, which we feel enhance the manuscript further, and we have clarified text as requested. Reviewer #3 identified an error in Figure 4, which we have rectified. We felt that some specific experiments suggested in review would not add significant further depth, as we articulate below.

    Altogether our reviewers appear to agree that our manuscript makes a significant contribution to both the microbiome and ageing fields, using a large number of experiments to mechanistically outline the role(s) of various pathways and tissues. We thank the reviewers for their positive contributions to the publication process.

    (2) Description of the planned revisions

    Reviewer #2:

    Not…essential for publication…is it possible to look at Tk protein levels?

    We have acquired a small amount of anti-TK antibody and we will attempt to immunostain guts associated with A. pomorum and L. brevis. We are also attempting the equivalent experiment in mouse colon reared with/without a defined microbiota. These experiments are ongoing, but we note that the referee feels that the manuscript is a publishable unit whether these stainings succeed or not.

    (3) Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #1:

    Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done?

    We have incorporated the requested information into legends for lifespan experiments.

    Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader.

    We read these statements differently. The only experiment in which a genetic intervention prevented lifespan extension by axenic conditions is neuronal TkR86C knockdown (Figure 6B-C). Otherwise, microbiota shortened lifespan relative to axenic conditions, and genetic knockdowns extend blocked this effect (e.g. see lines 131-133). We have ensured that the framing is consistent throughout, with text edited at lines 198-199, 298-299, 311-312, 345-347, 407-408, 424-425, 450, 497-503.

    TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing

    We have added text at lines 219-222 to address this point. We agree that this effect is hard to interpret biologically, since expressing RNAi in axenics has no additional effect on Tk expression (Figure S7). Consequently we can only interpret this unexpected effect as a possible off-target effect of RU feeding on TAG, specific to axenic flies. However, this possibility does not void our conclusion, because an off-target dimunition of TAG cannot explain why CR flies accumulate TAG following TkRNAi induction. We hope that our added text clarifies.

    I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader.

    We have added the requested statement at lines 423-424, explaining that we expected the IPC ablation to render flies constitutively long-lived and non-responsive to A pomorum.

    Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses?

    We have added our logic at lines 449-454. In brief, we conclude involvement for insulin signalling because FoxO mutant lifespan does not respond to TkRNAi, and diminishes the lifespan-shortening effect of A. pomorum. However, we cannot state that the effects are direct because we do not have data that mechanistically connects Tk/TkR99D signalling directly in insulin-producing cells. The current evidence is most consistent with insulin signalling priming responses to microbiota/Tk/TkR99D, as per the newly-added text.

    Typographical errors

    We have remedied the highlighted errors, at lines 128-140.

    Reviewer #2:

    it would be good to show that the bacterial levels are not impacted [by TkRNAi]

    We have quantified CFUs in CR flies upon ubiquitous TkRNAi (Figure S5), finding that the RNAi does not affect bacterial load. New text at lines 138-139 articulates this point.

    The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this?

    As per response to Reviewer #1, we have added text at lines 219-222 to address this point.

    Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned?

    We have added another experiment showing longevity upon knockdown in conventional flies, using an independent TkRNAi line (Figure S3).

    Reviewer #3:

    In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary.

    We thank the reviewer sincerely for their keen eye, which has highlighted an error in the previous version of the figure. In revisiting this figure we have noticed, to our dismay, that the figures for GFP quantification were actually re-plots of the figures for (ac)K quantification. This error led to the discrepancy between statistics and graphics, which thankfully the reviewer noticed. We have revised the figure to remedy our error, and the statistics now match the boxplots and results text.

    Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association

    We selected Adh on the basis of our RNAseq analysis, which showed it was not different between AX and CV guts, whereas many commonly-used “housekeeping” genes were. We have now added a plot to demonstrate (Figure S2).

    The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference

    We have added the requested reference (Hung et al, 2020) at line 86.

    (4) Description of analyses that authors prefer not to carry out

    Reviewer #1:

    I'd encourage the authors to provide lifespan plots that enable comparison between all conditions

    We have avoided this approach because the number of survival curves that would need to be presented on the same axis (e.g. 16 for Figure 5) is not legible. However we have ensured that axes on faceted plots are equivalent and with grid lines for comparison. Moreover, our approach using statistical coefficients (EMMs) enables direct quantitative comparison of the differences among conditions.

    Reviewer #2:

    Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this

    This comment relates to Figure 7G. We do see an effect of the knockdown in this experiment, so we believe that the knockdown is effective. However the direction of response is not consistent with our hypothesis so the experiment is not informative about the role of these cells. We therefore feel there is little to be gained by testing efficacy of knockdown, which would also be technically challenging because the cells are a small population in a larger tissue which expresses the same transcripts elsewhere (i.e. necessitating FISH).

    Would it be possible to use antibodies for acetylated histones?

    The comment relates to Figure 4C-E. The proposed studies would be a significant amount of work because, to our knowledge, the specific histone marks which drive activation in TK+ cells remain unknown. On the other hand, we do not see how this information would enrich the present story, rather such experiments would appear to be the beginning of something new. We therefore agree with Reviewer #1 (in cross-commenting) that this additional work is not justified.

    Reviewer #3:

    Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.

    We agree with reviewers 1-2 (in cross-commenting) that this proposal is non-trivial and not justified by the additional insight that would be gained. As described above, we are attempting to immunostain Tk, which if successful will provide a third line of evidence for regulation of Tk+ cells. However we note that we already have the strongest possible evidence for a role of these cells via genetic analysis (Figure 5).

    While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.

    As the reviewer recognises, maintaining axenic experiments for months on end is not trivial. Given the tendency for males either to simply mirror female responses to lifespan-extending interventions, or to not respond at all, we made the decision in our work to only study females. We have instead emphasised in the manuscript that results are from female flies.

    TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D.

    We disagree with this interpretation: the results do not show that TkR86C-RNAi recapitulates the effect of enteric Tk-RNAi. A potentially interesting interaction is apparent, but the data do not support a causal role for TkR86C. A causal role is supported only for TkR99D, knockdown of which recapitulates the longevity of axenic flies and TkRNAi flies_._ Therefore we feel that our current title is therefore justified by the data, and a more generic version would misrepresent our findings.

    The difference between "aging" and "lifespan" should also be addressed.

    The smurf phenotype is a well-established metric of healthspan. Moreover, lifespan is the leading aggregate measure of ageing. We therefore feel that the use of “ageing” in the title is appropriate.

    If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.

    Foxo nuclear localisation has already been shown in axenic flies (Shin et al, 2011). We have added text and citation at lines 401-402.

  8. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    We thank the referees for taking time to review our manuscript. These reviews are positive, highlighting the novelty of our findings. The majority of comments are cosmetic, and we have added data in response to some technical points. We feel that some of the additional experiments proposed would not add significant methodological depth, and cross-commenting suggests that our referees agree. At present we are attempting antibody staining to quantify Tk peptide retention in the midgut, as per suggestion by reviewer #2.

    We enclose our point-by-point response to each referee's points, below.

    __Reviewer #1 __

    • Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done?

    • We have incorporated the requested information into legends for lifespan experiments.

    • Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader.

    • We read these statements differently. The only experiment in which a genetic intervention prevented lifespan extension by axenic conditions is neuronal *TkR86C *knockdown (Figure 6B-C). Otherwise, microbiota shortened lifespan relative to axenic conditions, and genetic knockdowns extend blocked this effect (e.g. see lines 131-133). We have ensured that the framing is consistent throughout, with text edited at lines 198-199, 298-299, 311-312, 345-347, 408-409, 424-425, 450, 497-503.

    • TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing

    • We have added text at lines 219-222 to address this point. We agree that this effect is hard to interpret biologically, since expressing RNAi in axenics has no additional effect on *Tk *expression (Figure S7). Consequently we can only interpret this unexpected effect as a possible off-target effect of RU feeding on TAG, specific to axenic flies. However, this possibility does not void our conclusion, because an off-target dimunition of TAG cannot explain why CR flies accumulate TAG following TkRNAi We hope that our added text clarifies.

    • *I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader. *

    • We have added the requested statement at lines 423-424, explaining that we expected the IPC ablation to render flies constitutively long-lived and non-responsive to A pomorum.

    • *Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses? *

    • We have added our logic at lines 449-454. In brief, we conclude involvement for insulin signalling because FoxO mutant lifespan does not respond to *TkRNAi, and diminishes the lifespan-shortening effect of * pomorum. However, we cannot state that the effects are direct because we do not have data that mechanistically connects Tk/TkR99D signalling directly in insulin-producing cells. The current evidence is most consistent with insulin signalling priming responses to microbiota/Tk/TkR99D, as per the newly-added text.

    • Typographical errors

    • We have remedied the highlighted errors, at lines 128-140.

    • I'd encourage the authors to provide lifespan plots that enable comparison between all conditions

    • We have plotted our figures in faceted boxes, because the number of survival curves that would need to be presented on the same axis (e.g. 16 for Figure 5) would not be intellegible. However we have ensured that axes on faceted plots are equivalent and with grid lines for comparison. Moreover, our approach using statistical coefficients (EMMs) enables direct quantitative comparison of the differences among conditions.

    Reviewer #2

    • Not…essential for publication…is it possible to look at Tk protein levels?

    • We have acquired a small amount of anti-TK antibody and we will attempt to immunostain guts associated with * pomorum *and L. brevis. We are also attempting the equivalent experiment in mouse colon reared with/without a defined microbiota. These experiments are ongoing, but we note that the referee feels that the manuscript is a publishable unit whether these stainings succeed or not.

    • it would be good to show that the bacterial levels are not impacted [by TkRNAi]

    • We have quantified CFUs in CR flies upon ubiquitous TkRNAi (Figure S5), finding that the RNAi does not affect bacterial load. New text at lines 138-139 articulates this point.

    • The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this?

    • As per response to Reviewer #1, we have added text at lines 219-222 to address this point.

    • Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned?

    • We have added another experiment showing longevity upon knockdown in conventional flies, using an independent *TkRNAi *line (Figure S3).

    • Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this

    • This comment relates to Figure 7G. We do see an effect of the knockdown in this experiment, so we believe that the knockdown is effective. However the direction of response is not consistent with our hypothesis so the experiment is not informative about the role of these cells. We therefore feel there is little to be gained by testing efficacy of knockdown, which would also be technically challenging because the cells are a small population in a larger tissue which expresses the same transcripts elsewhere (i.e. necessitating FISH).

    • Would it be possible to use antibodies for acetylated histones?

    • The comment relates to Figure 4C-E. The proposed studies would be a significant amount of work because, to our knowledge, the specific histone marks which drive activation in TK+ cells remain unknown. On the other hand, we do not see how this information would enrich the present story, rather such experiments would appear to be the beginning of something new. We therefore agree with Reviewer #1 (in cross-commenting) that this additional work is not justified.

    Reviewer #3

    • *In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary. *

    • We thank the reviewer sincerely for their keen eye, which has highlighted an error in the previous version of the figure. In revisiting this figure we have noticed, to our dismay, that the figures for GFP quantification were actually re-plots of the figures for (ac)K quantification. This error led to the discrepancy between statistics and graphics, which thankfully the reviewer noticed. We have revised the figure to remedy our error, and the statistics now match the boxplots and results text.

    • Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association

    • We selected *Adh *on the basis of our RNAseq analysis, which showed it was not different between AX and CV guts, whereas many commonly-used “housekeeping” genes were. We have now added a plot to demonstrate (Figure S2).

    • The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference

    • We have added the requested reference (Hung et al, 2020) at line 86.

    • Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.

    • We agree with reviewers 1-2 (in cross-commenting) that this proposal is non-trivial and not justified by the additional insight that would be gained. As described above, we are attempting to immunostain Tk, which if successful will provide a third line of evidence for regulation of Tk+ cells. However we note that we already have the strongest possible evidence for a role of these cells via genetic analysis (Figure 5).

    • While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.

    • As the reviewer recognises, maintaining axenic experiments for months on end is not trivial. Given the tendency for males either to simply mirror female responses to lifespan-extending interventions, or to not respond at all, we made the decision in our work to only study females. We have instead emphasised in the manuscript that results are from female flies.

    • TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D.

    • We disagree with this interpretation: the results do not show that TkR86C-RNAi recapitulates the effect of enteric Tk-RNAi. A potentially interesting interaction is apparent, but the data do not support a causal role for TkR86C. A causal role is supported only for *TkR99D, *knockdown of which recapitulates the longevity of axenic flies and *TkRNAi flies. *Therefore we feel that our current title is therefore justified by the data, and a more generic version would misrepresent our findings.

    • The difference between "aging" and "lifespan" should also be addressed.

    • The smurf phenotype is a well-established metric of healthspan. Moreover, lifespan is the leading aggregate measure of ageing. We therefore feel that the use of “ageing” in the title is appropriate.

    • If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.

    • Foxo nuclear localisation has already been shown in axenic flies (Shin et al, 2011). We have added text and citation at lines 402-403.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    Marcu et al. demonstrate a gut-neuron axis that is required for the lifespan-shortening effects mediated by gut bacteria. They show that the presence of commensal bacteria-particularly Acetobacter pomorum-promotes Tk expression in the gut, which then binds to neuronal tachykinin receptors to shorten lifespan. Tk has also recently been reported to extend lifespan via adipokinetic hormone (Akh) signaling (Ahrentløv et al., Nat Metab 7, 2025), but the mechanism here appears distinct. The lifespan shortening by Ap via Tk seems to be partially dependent on foxo and independent of both insulin signaling and Akh-mediated lipid mobilization. Although the detailed mechanistic link to lifespan is not fully resolved, the experiment and its results clearly shows the involvement of the molecules tested. This work adds a valuable dimension to our growing understanding of how gut bacteria influence host longevity. However, there are some points that should be addressed.

    1. Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.
    2. In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary.
    3. If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.
    4. Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association and/or (2) compare the results using different genes as internal standard.
    5. While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.
    6. We also had some concerns regarding the wording of the title. Fig6B and C suggests that TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D. The difference between "aging" and "lifespan" should also be addressed. While the study shows a role for Tk in lifespan, assessment of aging phenotypes (eg. Climbing assay, ISC proliferation) beyond the smurf assay is required to make conclusions about aging.
    7. The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference, if available.

    Referees cross-commenting

    I agree with the other reviewers that the study has been done very well and hence additional experiments are not mandatory to be published such as calcium imaging. However, I still believe that testing Tk's elevation by the Ap in males should greatly increase the generality of the finding, no matter what the outcome would be. Too many studies use only females.

    Significance

    General assessment

    The main strength of this study is the careful and extensive lifespan analyses, which convincingly demonstrate the role of gut microbiota in regulating longevity. The authors clarify an important aspect of how microbial factors contribute to lifespan control. The main limitation is that the study primarily confirms the involvement of previously reported signaling pathways, without identifying novel molecular players or previously unrecognized mechanisms of lifespan regulation.

    Advance

    The lifespan-shortening effect of Acetobacter pomorum (Ap) has been reported previously, as has the lifespan-shortening effect of Tachykinin (Tk). However, this study is the first to link these two factors mechanistically, which represents a significant and original contribution to the field. The advance is primarily mechanistic, providing new insight into how microbial cues converge on host signaling pathways to influence ageing.

    Audience

    This work will be of particular interest to a specialized audience of basic researchers in ageing biology. It will also attract interest from microbiome researchers who are investigating host-microbe interactions and their physiological consequences. The findings will be useful as a conceptual framework for future mechanistic studies in this area.

    Field of expertise

    Drosophila ageing, lifespan, microbiome, metabolism

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    Referee #2

    Evidence, reproducibility and clarity

    The main finding of this work is that microbiota impacts lifespan though regulating the expression of a gut hormone (Tk) which in turn acts on its receptor expressed on neurons. This conclusion is robust and based on a number of experimental observation, carefully using techniques in fly genetics and physiology: 1) microbiota regulates Tk expression, 2) lifespan reduction by microbiota is absent when Tk is knocked down in gut (specifically in the EEs), 3) Tk knockdown extends lifespan and this is recapitulated by knockdown of a Tk receptor in neurons. These key conclusions are very convincing. Additional data are presented detailing the relationship between Tk and insulin/IGF signalling and Akh in this context. These are two other important endocrine signalling pathways in flies. The presentation and analysis of the data are excellent.

    There are only a few experiments or edits that I would suggest as important to confirm or refine the conclusions of this manuscript. These are:

    1. When comparing the effects of microbiota (or single bacterial species) in different genetic backgrounds or experimental conditions, I think it would be good to show that the bacterial levels are not impacted by the other intervention(s). For example, the lifespan results observed in Figure 2A are consistent with Tk acting downstream of the microbes but also with Tk RNAi having an impact on the microbiota itself. I think this simple, additional control could be done for a few key experiments. Similarly, the authors could compare the two bacterial species to see if the differences in their effects come from different ability to colonise the flies.
    2. The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this? Better clarification is required.
    3. With respect to insulin signalling, all the experiments bar one indicate that insulin is mediating the effects of Tk. The one experiment that does not is using dilpGS to knock down TkR99D. Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this, but as a minimum I would be a bit more cautious with the interpretation of these data.
    4. Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned? This would further clarify that there are no off-target effects that can account for the phenotypes.

    There are a few other experiments that I could suggest as I think they could enrich the current manuscript, but I do not believe they are essential for publication:

    1. The manuscript could be extended with a little more biochemical/cell biology analysis. For example, is it possible to look at Tk protein levels, Tk levels in circulation, or even TkR receptor activation or activation of its downstream signalling pathways? Comparing Ax and CR or Ap and CR one would expect to find differences consistent with the model proposed. This would add depth to the genetic analysis already conducted. Similarly, for insulin signalling - would it be possible to use some readout of the pathway activity and compare between Ax and CR or Ap and CR?
    2. The authors use a pan-acetyl-K antibody but are specifically interested in acetylated histones. Would it be possible to use antibodies for acetylated histones? This would have the added benefit that one can confirm the changes are not in the levels of histones themselves.
    3. I think the presentation of the results could be tightened a bit, with fewer sections and one figure per section.

    Referees cross-commenting

    Reviewer 1

    I generally agree with this reviewer but for

    "I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here."

    To the best of my knowledge, Foxo has only been shown to be required for lifespan extension/modulation by a reduction in insulin-like signalling. I.e. it does respond to other pathways but this is the only one where Foxo activity is known to modulate lifespan.

    Reviewer 3

    I agree with reviewer 1 that point raised under (1) does not appear strictly required for the conclusions of the manuscript.

    Both reviewers 1 and 3:

    I have a different take on the results of experiments where IPCs are manipulated. To me, Figure 7D and E show that ablating the IPCs removes the difference between Ax and Ap i.e. the IPCs are involved and insulin-like signalling is likely involved. The fact that RNAi against the TKR99D receptor does not have the same effect, does not matter (the sensing could happen in different neurons). Similarly, dilp expression is only a minor readout of what is happening with insulin-like signalling - dilps are controlled at the level of secretion.

    However, I would be happy for the authors to present different arguments and make a reasonable conclusion, which may differ from mine. But I think the arguments I present above should be taken into account.

    Significance

    The main contribution of this manuscript is the identification of a mechanism that links the microbiota to lifespan. This is very exciting and topical for several reasons:

    1. The microbiota is very important for overall health but it is still unclear how. Studying the interaction between microbiota and health is an emerging, growing field, and one that has attracted a lot of interest, but one that is often lacking in mechanistic insight. Identifying mechanisms provides opportunities for therapies. The main impact of this study comes from using the fruit fly to identify a mechanism.

    2. It is very interesting that the authors focus on an endocrine mechanism, especially with the clear clinical relevance of gut hormones to human health recently demonstrated with new, effective therapies (e.g. Wegovy).

    3. Tk is emerging as an important fly hormone and this study adds a new and interesting dimension by placing TK between microbiota and lifespan.

    I think the manuscript will be of great interest to researchers in ageing, human and animal physiology and in gut endocrinology and gut function.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this study the authors use a Drosophila model to demonstrate that Tachykinin (Tk) expression is regulated by the microbiota. In Drosophila conventionally reared (CR) flies are typically shorter lived than those raised without a microbiota (axenic). Here, knockdown of Tk expression is found to prevent lifespan shortening by the microbiota and the reduction of lipid stores typically seen in CR flies when compared to axenic counterparts. It does so without reducing food intake or fecundity which are often seen as necessary trade-offs for lifespan extension. Further, the strength of the interaction between Tk and the microbiota is found to be bacteria specific and is stronger in Acetobacter pomorum (Ap) monoassociated flies compared to Levilactobacillus brevis (Lb) monoassociation. The impact on lipid storage was also only apparent in Ap-flies. Building on these findings the authors show that gut specific knockdown is largely sufficient to explain these phenotypes. Knockdown of the Tk receptor, TkR99D, in neurons recapitulates the lifespan phenotype of intestinal Tk knockdown supporting a model whereby Tk from the gut signals to TkR99D expressing neurons to regulate lifespan. In addition, the authors show that FOXO may have a role in lifespan regulation by the Tk-microbiota interaction. However, they rule out a role for insulin producing cells or Akh-producing cells suggesting the microbiota-Tk interaction regulates lifespan through other, yet unidentified, mechanisms.

    Major comments:

    Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. In particular, the impact of TkRNAi on lifespan and lipid levels, the central finding in this study, has been demonstrated multiple times in different experiments and using different genetic tools. As a result, I don't feel that additional experimental work is necessary to support the current conclusions. However, I find it hard to assess the robustness of the lifespan data from the other manipulations used (TkR99DRNAi, TkRNAi in dFoxo mutants etc.) because information on the population size and whether these experiments have been replicated is lacking. Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done? For all other data it is clear how many replicates have been done, and the methods give enough detail for all experiments to be reproduced.

    Minor comments:

    While I feel the conclusions of this study are well supported by the data I found this to be a complex read and in places hard to follow. I feel some work is necessary in the writing to help the reader follow the authors logic. Below I describe some of the issues that confused me and provide some suggestions that I hope the authors will find helpful.

    Survival curves The authors state that the lifespan difference between CR and axenic flies disappears with TkRNAi because TkRNAi CR flies are longer lived, rather than because TkRNAi axenic flies are shorter lived. Is this consistent in every TkRNAi experiment? It's hard for the reader to assess this because the relevant lifespan curves are presented on separate plots. I'd encourage the authors to provide lifespan plots that enable comparison between all conditions. For example, in figures 2 and 6 the reader wants to directly compare between RU- and RU+ but can't easily do so. Additional plots could be made available in the supplementary figures showing the comparisons that are not easy to make on the main figures.

    Consistent framing of the data Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader. For example, line 325 says TkR86CRNAi prevents lifespan extension in axenic flies. Given the framing in the previous sections, it might be clearer to say that TkR86CRNAi shortens the lifespan of axenic flies to that of CR flies in contrast to TkRNAi and TkR99DRNAi which don't.

    The impact of TkRNAi on lipid levels in axenic flies TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing for the reader because it is inconsistent with the microbiota driving both higher Tk expression and higher lipid storage. If the microbiota increases Tk expression and this results in reduced lipid storage, why does reduced Tk expression also result in reduced lipid storage in axenic flies? This could further highlight the unique impact that the interaction between TkRNAi and the microbiota has on lipid storage, given it reverses both the impact of the microbiota alone and TkRNAi alone. I feel this aspect of the data should be given more attention in the text both for clarity and because it may be telling us something important about the function of Tk. The current framing around pleiotropic effects is valid, and the impact of Tk on lipid storage is clearly independent of its impact on lifespan and so is not central to this study. However, I feel a short additional paragraph to acknowledge this nuance of the data is needed. It can be made clear in the text that further exploration is beyond the scope of the current study.

    Role of insulin signalling and insulin producing cells I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here.

    I would expect ablation of IPCs to have the opposite effect to foxo mutation and to increase FOXO activity throughout the organism due to a reduction in Dilp levels and so reduced insulin signalling. So, I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader. They find that TkRNAi still prevents lifespan shortening by Ap when IPCs are ablated and that TkR99DRNAi in IPCs also doesn't block lifespan shortening by Ap despite reducing the expression of dilp3 and dilp5. To me these data rule out a role for insulin signalling despite the requirement for FOXO and yet the authors conclude that insulin signalling is involved in the response to Ap and TkRNAi, although not obligately (lines 420 - 422 and 511 - 512). Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses? The potential involvement of other signalling inputs to FOXO activity, e.g. immune signalling and JNK, should be acknowledged and warrants some discussion.

    Typographical errors:

    Incomplete sentence line 121 to 122 - starting "Cox proportional hazards.... and posthoc tests (Fig 2b).

    Line 123 "EMMs" - define abbreviation on first use

    References to Fig 2b (first given on line 122), should be capitalised to Fig 2B for consistency.

    Lines 231 and 317 - the phrase "steady state (microbiota independent) expression" in reference to flyATLAS 2 data could be misleading. The term "microbiota independent" could suggest that expression levels have been shown not to be regulated by the microbiota and this is not the case. The authors should change this to simply state they are referring to steady state expression in conventionally reared flies.

    Referees cross-commenting

    Below are brief comments on the revision suggestions that reviewers 2 and 3 have requested.

    Reviewer 2

    1. I agree that confirmation that TkRNAi doesn't impact microbial levels could be helpful and would be straightforward for the authors to do. However, I don't feel it's essential to support the central claims of the paper.
    2. I agree.
    3. I don't feel that any of these experiments supports a role for insulin signalling, so I don't feel that this additional control is needed.
    4. It would be a good addition to have lifespan data from a separate knockdown line for corroboration. However, this has already been done in several different genetic backgrounds through crosses with different driver lines in multiple tissues, so I feel it's unnecessary given the time and resources that lifespan experiments take. There's also the caveat that different RNAi lines can knockdown to different extents so that would have to be assessed as well and if there's a difference it may mean that ultimately not much can be concluded from this additional experiment.
    5. A good suggestion, but not straightforward and depends on the availability of the necessary tools, or possibly the generation of new tools. One for a follow up study.
    6. I feel this is not important enough to the central findings of the study to warrant the extra work.
    7. I agree.

    Reviewer 3

    1. Imaging calcium signalling is not straightforward unless a lab already has the tools available and optimised. If Tk+ EEs show changes in calcium signalling I'm not convinced that this tells us anything specific to the Tk-microbiota interaction. The point is the role of Tk itself, not the broader activity of the cells that express it.
    2. I agree this needs clarification.
    3. I agree that this would add depth, if feasible, but feel it's not essential to support the current conclusions.
    4. This is a minor point and given the RT-qPCR data and the RNAseq data corroborate each other I'm convinced that Tk levels are elevated.
    5. I feel exploring this in males is opening an additional line of enquiry beyond the scope of the current study. Either the phenotypes are the same - in which case what is added? - or they are different but there's no scope to assess why. A good suggestion for a follow up study.
    6. No comment.
    7. Agreed.

    One final comment. It's true that FOXO has only been shown to regulate lifespan in the context of insulin signalling. However, as far as I'm aware it hasn't been shown not to regulate lifespan downstream of it's other activators, this simply hasn't been explored due to the historical focus on insulin signalling in this field. In the context of host-microbiota interactions considering other pathways the activate FOXO, such as immune and JNK signals, would make sense.

    Reviewed by Dr Rebecca Clark, Department of Biosciences, Durham University

    Significance

    Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. We have known that the microbiota influence lifespan for some time but the mechanisms by which they do so have remained elusive. This study identifies one such mechanism and as a result opens several avenues for further research. The Tk-microbiota interaction is shown to be important for both lifespan and lipid homeostasis, although it's clear these are independent phenotypes. The fact that the outcome of the Tk-microbiota interaction depends on the bacterial species is of particular interest because it supports the idea that manipulation of the microbiota, or specific aspects of the host-microbiota interaction, may have therapeutic potential.
    These findings will be of interest to a broad readership spanning host-microbiota interactions and their influence on host health. They move forward the study of microbial regulation of host longevity and have relevance to our understanding of microbial regulation of host lipid homeostasis. They will also be of significant interest to those studying the mechanisms of action and physiological roles of Tachykinins.

    Field of expertise: Drosophila, gut, ageing, microbiota, innate immunity

  12. Version published to 10.1101/2025.07.31.667994 on bioRxiv