• I might be missing something, but I don’t question that (some of) the developmental plasticity you observe is mediated by microbes to some extent: indeed, in my view the mircobial composition affects the rearing environment, which in turn affects developmental plasticity.

• I still think the MANOVA analysis doesn’t test what you want to test. In the ANCOVA I suggested you can test how microbe treatment/environment and food treatment affects the correlation between body size and development time by entering a three-way (or four-way) treatmentsizedevtime interaction.

• You can test for overall effects of symbiont treatment crossed with medium/grape food treatment all right. But within any treatment combination you cannot separate the (presumably random) effect of the food vs. the microbe environment, if this is what you are arguing. Again, I might be missing something.

• What remains is an acceptable descriptive paper on microbiome effects on developmental plasticity that is worth being published and will interest especially the community of scientists interested in the microbiome. I can give my OK for that.

Wolf Blanckenhorn

It is well known that the rearing environment has strong effects on life history and fitness traits of organisms. Microbes are part of every environment and as such likely contribute to such environmental effects. Gut bacteria are a special type of microbe that most animals harbor, and as such they are part of most animals’ environment. How much do selected gut bacteria affect the organismal phenotype, in terms of life history and larval foraging traits, of the fruit fly Drosophila melanogaster, a common laboratory model species in biology? This was the main question underlying the laboratory study by Guilhot et al., which was previously assessed by two reviewers and myself for PCI.
To investigate the above question, the authors isolated 4 types/species of bacteria from the gut of their lab strain of Drosophila, and subsequently let Drosophila eggs and larvae develop in both the usual artificial laboratory medium as well as grapes (a natural habitat for Drosophila larvae) inoculated with theses bacteria, singly and in combination, plus a bacteria-free control. By investigating various relevant developmental traits, the authors found that adding particularly Enterobacteria had some visible improving effects on several traits (with three other types of bacteria showing only minor or even no effects), both upward (indicting improvement) and downward (being detrimental). In general, the grape medium reduced performance relative to the standard lab medium. Strongest interactive effects occurred for development time and body size, with lesser such effects on some related feeding behavioral traits (Figs. 2,3).
The study overall was conducted correctly. In response to the previous reviews the Introduction was improved in the revised version to make clearer the general objectives of the study. The study remains largely descriptive in that no particular a priori hypotheses or predictions in relation to the particular bacteria isolated were tested, not least because the bacteria were somewhat arbitrarily chosen and there were apparently no particular prior studies from which to derive concrete predictions. Overall, the results of this study should be of interest to the community of evolutionary ecologists, at least those working on nutritional and microbiome effects on animal life histories. As the strictly evolutionary content is limited (to non-existent), this paper would be best suited for a (microbial or behavioral or physiological) ecology outlet.
The inclusion of a natural medium (grapes), in addition to the artificial lab medium, must be commended, because this should permit inferences and conclusions for at least one natural environment, as inferences drawn from laboratory studies for the natural situation are typically limited. The choice of bacteria isolated and used remains arbitrary, though there are many. Nevertheless a good start. It could be asked why not natural but artificial lab populations of Drosophila were used for this experiment, if the aim is to draw inferences for the wild situation. But again: a good start.
While these specific data are novel, they are not very surprising. If we grow animals in different environments we can expect some detectable effects of these environments, including the bacterial (microbiome) environment, on the hosts life history. The standard and predicted (Stearns & Koella 1986 Evolution) life history response of Drosophila melanogaster (but not all insects: Blanckenhorn 1999: Evol Ecol) facing stressful nutritional environments is to extend development but come out smaller in the end. This is what happened here (Figs. 3,4): size was smaller and development time longer in the grape medium, and some of the bacteria also weakly induced this (or the opposite) response. The biological interpretation is that individuals have more trouble ingesting and/or digesting the nutrients (thus prolonging their foraging period and development) and still cannot convert the nutrients effectively into body size increments (hence emerging smaller). This is what the authors here refer to as developmental plasticity, which is, still, ultimately nutritionally mediated. That is, the conclusion that this is not mediated by resource acquisition is misguided.
I point out that the main and strongest interactive effects between medium and bacterial type are really only apparent for the enterobacteria, and they probably also mediate the overall effect of the mixture. For simplicity, I therefore suggest to initially present all data, but, in a second step, to only analyse control, enterobacteria & mixture, which would make plots and analyses much simpler and clearer. I say this because the new analyses presented in response to our previous statistical comments are actually no improvement whatsoever, but worse than before.
In general, more encompassing and increased questions in this context to be researched in the future could be: 1) are these effects predictable (not (yet) at this point, or so it seems), and 2) how strong are these environmental bacterial effects relative to other, more standard effects (e.g. relative to genetic variation, population variation, etc., or relative to other types of environmental effects like, say, temperature)? Although Genotype x Environment effects are invoked, they were not tested here due to lacking genetically different Drosophila families or populations tested. I consider this a major weakness of the study because it does not allow comparisons with other environments or situations. So, while interesting in principle, the study appears a bit basic in the end.
As mentioned before, and outlined above, the negative correlation between development time and body size is expected in stressful nutritional environments, which the enterobacteria apparently create. The multivariate analysis presented (which in the end analyses the effects of fruit salad instead of apples and oranges separately) is not what should be done; authors need to test for a correlation (between size and development time) within treatments properly with ANCOVA, entering ALL main factors (medium, bacteria, sex, plus interactions; cf. Table 1), such that the remaining correlation is indeed tested WITHIN treatment combinations. The negative regression ACROSS treatments is not really relevant because the tested environments are arbitrary. Suffice it to show that body size declines relative to control, while development time prolongs much more in relation (Fig. 4). That this somewhat differs between the two nutritional treatments is interesting, of course. I don’t see much “accelerated” but mostly decelerated development, in standard medium females in particular. The flies grow better in standard medium than on grapes (Fig. 4), which is perhaps a bit surprising. The reversal of sexual size dimorphism across bacterial treatments in the grape environment visible in Fig. 3 is interesting too, though I don’t understand why this happens. If I understand correctly your sample size should be 15 (replicates) x 2 nutritional media x 6 bacteria treatments = 180. When analysing sexes separately this number doubles. The latter should be the magnitude of the error term in your full analyses. I don’t see a need to analyse treatment means (you can plot them all right!); just present the overall ANOVAs of Table 1 (plus a corresponding ANCOVA; see above). (And reduce the data set to 3 bacteria treatments to do the same analysis, as suggested above.) I found it difficult to edit the tracked-changes version, which did not save my comments after all. In summary, some further revisions are in order and then the paper could be recommended by PCI EvolBiol.

Wolf Blanckenhorn, University of Zurich

It is well known that the rearing environment has strong effects on life history and fitness traits of organisms. Microbes are part of every environment and as such likely contribute to such environmental effects. Gut bacteria are a special type of microbe that most animals harbor, and as such they are part of the animal's environment. How much do selected gut bacteria affect the phenotype, in terms of life history and larval foraging traits, of the fruit fly Drosophila melanogaster, a common laboratory model species in biology? This was the main aim of the laboratory study by Guilhot et al. that was herewith assessed by two reviewers.
To investigate the above question, the authors isolated 4 types/species of bacteria from the gut of their lab strain of Drosophila, and subsequently let Drosophila eggs and larvae develop in both the usual artificial laboratory medium and grapes (a natural habitat for Drosophila larvae) inoculated with theses bacteria, singly and in combination, plus a bacteria-free control. By investigating various relevant developmental traits, the authors found that adding the bacteria DID have some visible (significant) effects on various traits, both upward (indicting improvement) and downward (being detrimental).
The study overall appears well conducted and presented. The text is largely clear, though the reviewers point out to some unclarities, which can and should be fixed. There are no major errors. As is, the study is largely descriptive, i.e. does not test particular hypotheses or predictions in relation to the particular bacteria isolated, not least because the bacteria were chosen ad (post) hoc and there were apparently no particular prior studies from which to derive concrete predictions. The inclusion of a natural medium (grapes), in addition to the artificial lab medium, must be commended, because this should permit inferences and conclusions for at least one natural environment, as inferences drawn from laboratory studies for the natural situation are typically limited. Nevertheless, it remains unclear why these, and not other bacteria were chosen, further limiting general inferences. It also remains unclear why not natural but artificial lab populations of Drosophila were used, if the aim is to draw inferences for the wild situation. But this is a good first start.
While the data are novel, the fact that some detectable effects of the bacterial environment on life history traits is present is not really surprising, as the reviewers point out. More interesting questions would be: 1) are these effects predictable (not (yet) at this point, or so it seems), and 2) how strong are these environmental bacterial effects relative to other, more standard effects (e.g. relative to genetic variation, population variation (cf. reviewer 2), or relative to other types of environmental effects like, say, temperature)? Although Genotype x Environment effects are invoked, they were not tested here due to lacking genetically different Drosophila families or populations tested. I consider this a major weakness of the study because it does not allow comparisons to other environments or situations. So, while interesting in principle, the study appears a bit arbitrary in the end.
The reviewers point out a number of weaknesses and unclarities, to which I add my own. Many will be fixable, and should be fixed, in a revision. But to make this study more meaningful and interesting for the community it would be best to add some additional treatment levels as suggested above (different families, populations, perhaps another environmental factor, etc.). The questions addressed are not laid out clearly in the Introduction. What WERE the main questions/expectations/predictions? Why these and not other bacteria? The role of the "evolutionary" part regarding one bacterium class is not related to the rest of the study, which is not evolutionary at all but mainly physiological or developmental. Hence the title is a misnomer. The negative correlation between development time and body size needs to be tested properly with ANCOVA, entering ALL main factors (medium, bacteria, sex, plus interactions; cf. Table 1), such that the remaining correlation is indeed tested WITHIN treatment combinations. Fig. 3 clearly indicates that the negative regression line largely results from the two medium treatments (red vs. blue dots; lab vs. grape). I don't think this is what the authors are saying or testing.
Overall, this descriptive study is worth publishing after major revision as indicated by the reviewers and myself above. Better still, adding further treatment levels would make this work more interesting and meaningful.

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