Auxin Exposure Disrupts Feeding Behavior and Fatty Acid Metabolism in Adult Drosophila

Reviewer #1 (Public Review):

Recently the auxin-inducible gene expression system (AGES) has been frequently used for inducing target protein degradation acutely in Drosophila and other organisms. This study investigated the effects of auxin exposure on Drosophila adults, focusing on their feeding behavior, fatty acid metabolism, and oogenesis. The authors have provided strong evidence that high levels of auxin exposure perturb feeding behavior, survival rates, lipid metabolism, and gene expression patterns, providing a cautionary note for the field in using this technology.

This study documented the auxin feeding-induced effects in adult Drosophila, with a design with temporally controlled gene expression using a modified Gal4/Gal80 system. Due to the widespread usage of the auxin-mediated method, it is important to address whether the application of auxin itself causes any physiological changes.

Overall, the experiments were suitably designed with appropriate sample size and data analysis methods. The authors reported evidence of several auxin-induced effects, including strong evidence that high levels of auxin exposure perturb feeding behavior, survival rates, lipid metabolism, and gene expression patterns. For example, they found that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes in genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes.

However, this work can be improved based on the following recommendations:

1. Although authors showed that auxin causes gene expression changes including the possible alteration of Gal4 expression levels, no cell-type-specific data is provided. It would be informative to the Drosophila field if the authors could examine major Gal4 drivers in their expression levels, such as the ones used in studying metabolism and oogenesis.

2. Although the authors briefly mentioned aging research, feeding behavior, and lipid metabolism, RNA-seq data are provided only for short-term treatment (2 days). The ovary phenotype was examined with long-term treatment (15 days). It would be informative if the authors could also show other long-term treatment data.

3. The auxin used in this work is a more water-soluble version and at a high concentration (10 mM). In the C. elegans system, researchers are using a much lower concentration of auxin typically at 1 mM. Therefore, the discussion of their results in terms of potential impacts on other experimental systems should be done carefully. It would be helpful to know what impacts might be observed at a lower concentration of auxin. The recommendation would be that the authors add the 1 mM auxin data point to key elements of their analysis.

4. Another related question is whether these detected changes are reversible or not after exposure to auxin at different concentrations. This would be informative for researchers to better design their temporally controlled experiments.

5. It would also be helpful to know whether spermatogenesis is affected or not.

6. A few other points include changing the nomenclature and validating some of the key genes shown in Figure 3 using quantitative RT-PCR experiments with the tissues where the affected genes are known to be expressed and functional.

Reviewer #2 (Public Review):

Summary:

In recent years, Auxin treatment has been frequently used for inducing targeted protein degradation in Drosophila and various other organisms. This approach provides a way to acutely alter the levels of specific proteins. In this manuscript, the authors examine the impact of Auxin treatment and provide strong evidence that Auxin treatment elicits alterations in feeding activity, survival rates, lipid metabolism, and gene expression patterns. Researchers should carefully consider these effects to design experiments and interpret their data.

Strengths:

Regarding the widespread usage of Auxin mediated gene manipulation method, it is important to address whether the application of Auxin itself causes any physiological changes. The authors provide evidence of several Auxin effects. Experiments are suitably designed with appropriate sample size and data analysis methods.

Weaknesses:

The provided information is limited and not very helpful for many applications. For example, although authors briefly mentioned aging research, feeding behavior, and lipid data, RNA seq data are provided only for short-term (48 hours) treatment. Especially, since ovary phenotype was examined with long-term treatment (15 days), authors should also show other data for long-term treatment as well.

Although the authors show that Auxin causes a change in gene expression patterns and suggests the possible alteration of Gal4 expression levels, no cell-type-specific data is provided. It would be informative if the authors could examine the expression level of major Gal4 drivers.
Authors should discuss how severe these changes are by comparing them with other treatments or conditions, such as starvation or mutant data (ideally, comparing with reported data or their own data if any?).

Reviewer #3 (Public Review):

In this study, Fleck and colleagues investigate the effects of auxin exposure on Drosophila melanogaster adults, focusing their analysis on feeding behavior, fatty acid metabolism, and oogenesis. The motivation for the study is that auxin-inducible transcription systems are now being used by Drosophila researchers to drive transcription using the Gal4-UAS system as a complement to Gal80ts versions of the system. I found the study to be carefully done. This study will be of interest to researchers using the Drosophila system, especially those focusing on fatty acid metabolism or physiology. The authors might address the following minor points.

1. Auxin, actually 1-naphthaleneaceid acid here, which is a more water-soluble version of auxin (indole-3-acetic acid) is used at what I consider to be a high concentration-10 mM. The problem I have is that the authors are discussing their results in terms of potential impacts on other experimental systems. At least for C. elegans, I think this is not a reasonable extension of the current dataset. In the C. elegans system, researchers are using 1 mM auxin. The authors note that their RNA-seq results suggest a xenobiotic response. Could this apparent xenobiotic response be due to a metabolic byproduct following auxin administration at high concentrations? Figure S1 A shows that there is quite a robust transcriptional response at 1 mM auxin. It would be helpful to know what impacts might be observed at this lower concentration in which the transcriptional induction could be used in the context of biologically meaningful experiments. The recommendation would be that the authors add the 1 mM auxin data point to key elements of their analysis.

2. This reviewer was confused by the genetic nomenclature the authors use. The authors have chosen to use the designation 3.1Lsp2-Gal4 (3.1Lsp2-Gal4AID). I think this is potentially confusing because a reader might think that it is the Gal4 transcription factor that is the direct target of auxin- and TIR1-mediated protein degradation, as I initially did. Rather, it is the Gal80 repressor protein that is the direct target. The authors might consider a nomenclature that is more reflective of how this system works. It would also be helpful if the full genotypes of strains were included in each figure legend.

3. The RNA-seq dataset does not appear to be validated by RT-PCR experiments. The authors should consider validating some of the key genes shown in Figure 3 using quantitative RT-PCR experiments, potentially adding a 1 mM auxin data point.

Reviewer #4 (Public Review):

This work by Fleck et al. and colleagues documented the auxin feeding-induced effects in adult flies, since auxin could be used in temporally controlled gene expression using a modified Gal4/Gal80 system. Overall, the experiments were well-designed and carefully executed. The results were quantified with appropriate statistical analyses. The paper was also well-written and the results were presented logically. The findings demonstrate that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes in genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes.