Within-population variability in a moth sex pheromone blend, part 2: selection towards fixation

To understand how variation in sexual communication systems evolves, the genetic architecture underlying sexual signals and responses needs to be identified. Especially in animals where mating signals are important for mate recognition, and signals and responses are governed by independently assorting genes, it is difficult to envision how signals and preferences can (co)evolve. Moths are a prime example of such animals. In the noctuid moth Heliothis virescens, we found within-population variation in the female pheromone. In previous selection experiments followed by quantitative trait locus (QTL) analysis and expression analysis of candidate desaturase genes, we developed a model involving a trans-acting repressor of the delta-11-desaturase. In our current study with new selection lines, we fixed the most extreme phenotype and found a single underlying mutation: a premature stop codon in the first coding exon of delta-11-desaturase, which we could trace back to its origin in the laboratory. Interestingly, we found no pleiotropic effects of this knock-out mutation on the male physiological or behavioural response, or on growth or fertility. This finding is in contrast to Drosophila melanogaster, where a single desaturase gene affects both female pheromone production and male behavioural response, but similar to other Lepidoptera where these traits are under independent genetic control. To our knowledge, this is the first time that a single point mutation has been identified that underlies the phenotypic variation in the pheromone signal of a moth.


Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept as is

Comments to the Author(s)
The authors have in my opinion responded well to the critique of the reviewers, save some little misunderstanding. I attach a file with some responses and minor points. Nice if the authors can accommodate these (Appendix A).

31-Jan-2019
Dear Dr Groot On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-182050 entitled "Within-population variability in a moth sex pheromone blend, part 2: Selection towards fixation" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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Kind regards, Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Professor Kevin Padian (Subject Editor) openscience@royalsociety.org Associate Editor Comments to Author: The reviewers are largely satisfied with your changes, but recommend some additional modifications to hone an otherwise promising piece of work -good luck and we look forward to receiving the revision.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) I begin by reiterating my previous assessment that this is a very well-constructed manuscript that describes an excellent series of experiments. The core result is the discovery of a stop codon mutation in a desaturase gene that has a profound effect on female pheromone blend but no detectable effect on male response or other fitness components. This will certainly be of interest in the field of pheromone communication and the wider field of signal-response evolution. My principal concern with the version that I saw previously was a tendency to over-state the significance of the results. This arose largely through implying extension of the observations on this laboratory mutation to variation in natural populations. With some reluctance, the authors have adjusted the MS appropriately, in most places, as well as responding to my other minor comments and those of the second reviewer. I have a few remaining issues. They are all small wording changes, in a sense, but I think they are significant in terms of getting the message right. Final sentence of abstract -"… a single point mutation that underlies the phenotypic variation…". This is still inclined to give the wrong impression because 'the phenotypic variation' can easily be read as the variation present in nature. " … a single point mutation that alters the pheromone signal of a moth" would be preferable. 69 -I suggest replacing 'within-population variation' with 'difference between lines'. Then on line 73, make clear that this is 99% of the variance in the QTL cross, not in the source population and on line 75 replace 'contributes to the within-population female pheromone variation' with 'can contribute to female pheromone variation'. 258-260 -is 'X-square' here the same as 'Chi-square'? If so, the numbers look to be incorrect. If not, please explain the test. I don't agree that p=0.066 is 'marginal significant' (now line 318), even if the authors 'prefer' this wording (see response). The authors actually describe it as 'just missed significance' on line 225. If the cut-off is p=0.05, this is simply not significant. 331-2 -"These results confirm that in Heliothis virescens, independent and unlinked genes govern the variation in the female pheromone signal and the male pheromone response." I disagree. The results reveal a single mutation in a single gene that influences pheromone blend but not male response. This may tend to support a general picture of independent genetic determination but it does not 'confirm' it. 339-348 -this is a useful addition. I think that the genetic independence of pheromone blend and male response in Ostrinia should also be mentioned at this point. It would also be good to mention here the evidence in Heliconius (Lepidoptera) for pleiotropic effects on preference of major genes influencing visual signals. [cited as ref. 24] In response to Rev. 2, the authors say: "Loss of a functional delta-11 desaturase activity in the female pheromone gland of H. gelotopoeon is the only possible explanation of this phenomenon. Therefore it is appropriate to retain the reference to saltational evolution at this point of the Discussion." The implication is that loss of function must be saltational (now line 370). I don't agree. The end result of loss of function can be reached in a series of small steps. Decision letter (RSOS-182050.R1)

18-Feb-2019
Dear Dr Groot, I am pleased to inform you that your manuscript entitled "Within-population variability in a moth sex pheromone blend, part 2: Selection towards fixation" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. , which i leave to the authors to identify and correct. Answer: In the pheromone literature, there is general agreement on calling a gland chemical a pheromone component when an attractive response of conspecific males has been shown, and a pheromone compound when this is not so clear. This is thus the nomenclature that we are using throughout this manuscript.
I suggest to change pheromone compound to pheromone GLAND compound/component. The difference between component/compound is not so generally understood. I would strongly advise to revise so no-one gets confused.
-line 27 : the dynamic mechanisms. I am not sure if one should call the mechanisms dynamic or rather diverse, the first implying that the mechanisms (eg selected genes that induce change) are themselves dynamic .. well you get the idea Answer: We don't understand the reviewer's point here, but we have removed the word "dynamic".
to clarify: dynamic mechanisms imply that the mechanisms themselves are dynamic instead of (what is meant) that mechanisms cause pheromone communication to be dynamic (as implied by evolution).
-line 298: does amount of precursor being generated (in this case 16Ald) depend on the activity of the enzyme that converts it to the pheromone (in this case z11-16Ald)? This is the case in many microbes. I mention this as inthe low lines increasing the amount of Z11-16Ald (eg higher enzymatic conversion) could simply lead to more 16Ald be produced (maybe with pleiotropic effects on Z9-14Ald, see also fig 1a and comments below) and thus not change the ratio. In that light it could be good if the authors not only present relative amounts, but also absolute quantities (if the the above would be true one would expect more pheromone compounds and precursors in the low lines) Answer: We do not understand the reviewer's point, as he writes that an increasing amount of Z11-16:Ald can lead to more 16:Ald, which is opposite to our observations. Moreover, we have not assayed enzyme activity directly. It is the ratio between 16:Ald and Z11-16:Ald that is changed, as is written throughout the manuscript (i.e the ratio being < 1 or > 1), meaning that more 16:Ald is directly related to less Z11-16:Ald. To explain: In biological systems, depleting a substrate by higher enzymatic activity can lead to an increased production of the substrate. In this case, when selecting for lower 16:Ald, the slection lines may have favored increased enzymatic activity in the gland. This may have caused more 16:ald being produced (nd converted into pheromone components). In effect there is no change in ratio. However, there may be a change in absolute amount of pheromone components being produced. However, the authors did now mentioned that the absolute quantity in the low lines was lower than the high lines, which shows that the above is not the case. Please insert a line in the results on the absolute quantity