Peer review of: Early-exposure to new sex pheromone blends alters mate preference in female butterflies (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This paper describes interesting learning experiments with the butterfly Bicyclus anynana to show that 
pre-exposure to pheromone affects female choice, even in the second generation. Overall, I think this 
is a really interesting paper. However, I hope that Caroline Nieberding has also been consulted as 
reviewer for this paper, as she is the expert on the sex pheromone communication system in this 
species. In addition, I have the following questions and comments that I hope the authors can address 
before publication: 
 
1. First of all, I think it is important to point out that B. anynana is different from other moth species 
in that it is relatively long-lived, which makes learning a likely strategy. This is in contrast to most 
other moth species that generally live only a few weeks. 
 
2. line 89: the wet season form: explain this form for outsider audience; with or without eyespots? 
 
3. line 104: immature females: please explain what you mean with this. How long after hatching are 
females of B. anynana immature and when are they mature? The exposure experiments are done with 
females directly after hatching, at day 0, so does this mean that these females are still immature? At 
what age do females start to mate? 
 
4. line 112 “MSP2 was previously suggested to be the most relevant pheromone to female choice”; 
this is a very strange sentence, as one component of the pheromone blend is not the pheromone of 
the species. In general, it is not one component, but the ratio between components that is important 
 
5. lines 278-279: “This treatment prevents the emission of MSP2 produced by hindwing glands only”. 
So where are MSP1 and MSP3 emitted from in addition to the hindwing glands? 
 
6. line 284: 5 ug of MSP2 was applied; this is a huge amount, as the authors also write. What was the 
purity of this compound? Please specify as much as possible, as it is possible that small ‘contaminants’ 
may have an effect as well. 
 
7. I can’t figure out how long after the perfuming of the male the mate choice experiments took place. 
Were the same perfumed males used in the female exposure assays and the mate choice assays? In 
that case, males were perfumed and exposed to females on day 0 and then used in the mate choice 
trials on day 2? If so, did the authors check the amount of pheromone (MSP1, 2 and 3) on day 2, i.e. 
right before and/or after the mate choice trials? 
 
8. The females were pre-exposed to males. Were the males that were used for these pre-exposure 
experiments used also in the mate choice tests or not? If so, then could this pre-exposure have 
influenced the males in the mate choice trials? 
 
9. line 313: “Males were checked for presence of powder every 2 hours to prevent multiple mating.” 
How long does one mating last? Why were matings not observed e.g. every 30 min? 
 
10. I wonder whether the term ‘choosing’ is completely correct throughout the manuscript. The 
authors used mating as the proxy, which may have been due to female choice and/or to interactions 
between the (competing) males. 
 
Minor comments: 

-line 86: sex pheromone is singular; the sex pheromone of a species consists of a number of sex 
pheromone components (when behavioral response has been established) or compounds (when 
behavioral significance is not clear or not proven) 
-line 94: the authors write “we investigated how pheromone blends vary across closely related 
species’, but this ms deals only with B. anynana, so this should be changed. 
-line 322: choose should chose, as it is the past tense. 
 
Hopefully these comments are helpful. 
Kind regards, Astrid Groot 
 
 
 
Reviewer #2: 
Remarks to the Author: 
COMMENTS TO THE AUTHOR 
 
This is a well written, nicely organized and presented study. Their experimental manipulations of 
natural pheromone blends are elegant. I think this component of the manuscript is strong. The 
possibility of generational transfer of preference for learned pheromone blends is intriguing, but I am 
not convinced that the evidence here is convincing enough for publication. 
 
I am concerned that it looks as if the females in the second generation did not behave significantly 
differently from each other or the naïve females in the first generation. I think that given that the 
transgenerational learned preference is a fairly extraordinary claim, it needs incontrovertible evidence, 
and I am not sure you have that here. I would like to see the preferences of these females compared 
to the preferences of naïve females, or even better a control group where the mothers had not been 
exposed to any male pheromones other than the Wt they mated with. 
 
 
MINOR COMMENTS 
 
Lines 26 – 27: the statement that females are able to “pass that learned preference down to the next 
generation” needs more clarification and explanation if it is to be included in the abstract. 
 
Lines 111-118: My impression from this section is that NB1 males had overall reduced pheromone 
amounts (including no MSP2 and 70% and 60% of MSP1 and MSP3). NB2 males on the other hand 
just had more MSP2, which has already been shown to be more attractive to females (lines 112-113). 
 
Please add an explanation of what the Wt1 and Wt2 control blends are in the text 
 
Line 129: “these variable” do you mean male age? Clarify 
 
Lines 111-154: Please list your sample sizes for each experiment throughout the results (especially 
since you mention effect size in line 151). 
 
Lines 301-302: sentence is awkward – please reword 
 
 
 
Reviewer #3: 
Remarks to the Author: 

It seems to me that the phenotype of the F1 sons (brothers to the daughters with inherited 
preferences) is critical to this story. If they do not possess off-ratio blends to mirror the inherited 
preferences of their sisters, it seems that the alternate outcomes of introgression or extinction are 
more likely. 
 
It is also not clear to me why learning would alter female preference functions. What is the selective 
advantage? 
 
I do not like the bioassay. The dose (line 284 = 5 micrograms) seems very high. I realize this was 
necessary to try to have altered male phenotypes throughout the bioassay period, which is another 
problem. I question the significance of data generated by an 8-hour endpoint bioassay in a very small 
arena where females cannot escape male attention and none of the participants can leave. In nature 
it’s quite improbable that males and females would remain in such close proximity for 8 hours. Why 
didn’t the authors observe the interactions? The temporal elements associated with this bioassay (how 
often, when in the 8 hour window, etc.) seem more relevant than the outcome after such a long 
period. I also think it is problematic that the modified male phenotype is dynamic during the bioassay 
interval, returning to wild type phenotype by the end of the 8 hour interval (if I read the MS 
correctly), particularly since it was an endpoint bioassay and the mating could have happened near 
the end of the 8 hour period, raising questions about what the preference of these females really is. 
 
Specific 
Line 50 – I don’t like the term “originating.” Perhaps facilitating would be better? 
Line 86 – there is one pheromone and three pheromone components. 
Line 95 – I do not see this citation (29) as a good supporting reference for this statement. 
Line 99 – I think it would have been more meaningful if the variation reflected intraspecific variation 
rather than variation among congener a novels. 
 
Overall the authors have proposed a novel series of mechanisms that would in their view facilitate the 
rapid evolution of new pheromone communication systems (at least in butterflies that use a strongly 
pheromone-biased system). In is incumbent of them to provide definitive proof of the feasibility of 
their model. For the reasons above, publication ought to await further confirmatory experiments that 
support their proposed step-wise mechanisms and correct the bioassay’s deficiencies. Furthermore, 
the authors should recognize that although their tests achieved statistical significance, a change in the 
behavior of a relative small number of butterflies would have eliminated any significance by chi-
squared tests.