Microbiota impact Drosophila ageing via Acetobacter, Tachykinin, and TkR99D

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

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.

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

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 Tk^RNAi 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 Tk^RNAi, 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 Tk^RNAi 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.