Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps
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eLife assessment
This important study reveals the genetic regulation of changes in cuticular hydrocarbon profiles in a Hymenopteran insect and links these changes with courtship behaviour and sexual attractiveness. It provides convincing empirical evidence, spanning genetic, chemical, and behavioural data. It adds valuable new perspectives on the mechanisms that underlie chemical recognition and communication systems in nature.
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Abstract
Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes that most likely evolved by tandem gene duplication and that simultaneously impact sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knockdown in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male mating response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in complex cuticular hydrocarbon (CHC) profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.
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Author Response
Reviewer #1 (Public Review):
The authors convincingly show in this study the effects of the fas5 gene on changes in the CHC profile and the importance of these changes toward sexual attractiveness.
The main strength of this study lies in its holistic approach (from genes to behaviour) showing a full and convincing picture of the stated conclusions. The authors succeeded in putting a very interdisciplinary set of experiments together to support the main claims of this manuscript.
We appreciate the kind comments from the reviewer.
The main weakness stems from the lack of transparency behind the statistical analyses conducted in the study. Detailed statistical results are never mentioned in the text, nor is it always clear what was compared to what. I also believe that some tests that were conducted are not adequate …
Author Response
Reviewer #1 (Public Review):
The authors convincingly show in this study the effects of the fas5 gene on changes in the CHC profile and the importance of these changes toward sexual attractiveness.
The main strength of this study lies in its holistic approach (from genes to behaviour) showing a full and convincing picture of the stated conclusions. The authors succeeded in putting a very interdisciplinary set of experiments together to support the main claims of this manuscript.
We appreciate the kind comments from the reviewer.
The main weakness stems from the lack of transparency behind the statistical analyses conducted in the study. Detailed statistical results are never mentioned in the text, nor is it always clear what was compared to what. I also believe that some tests that were conducted are not adequate for the given data. I am therefore unable to properly assess the significance of the results from the presented information. Nevertheless, the graphical representations are convincing enough for me to believe that a revision of the statistics would not significantly affect the main conclusions of this manuscript.
We apologize for neglecting a detailed description of statistical tests that were performed. We wrote additional paragraphs in the method part specifically explaining the statistical analyses (line 435-445; 489-502; 559-561; 586-591).
The second major problem I had with the study was how it brushes over the somewhat contradicting results they found in males (Fig S2). These are only mentioned twice in the main text and in both cases as being "similarly affected", even though their own stats seem to indicate otherwise for many of the analysed compound groups. This also should affect the main conclusion concerning the effects of fas5 genes in the discussion, a more careful wording when interpreting the results is therefore necessary.
Thank you for pointing this out. Though our focus clearly lay on the female CHC profiles as a function in sexual signaling has only been described thus far for them, we now elaborated the result and discussion for the fas5 RNAi male part (line 167-178; 258-268).
Reviewer #2 (Public Review):
Insects have long been known to use cuticular hydrocarbons for communication. While the general pathways for hydrocarbon synthesis have been worked out, their specificity and in particular the specificity of the different enzymes involved is surprisingly little understood. Here, the authors convincingly demonstrate that a single fatty acid synthase gene is responsible for a shift in the positions of methyl groups across the entire alkane spectrum of a wasp, and that the wasps males recognize females specifically based on these methyl group positions. The strength of the study is the combination of gene expression manipulations with behavioural observations evaluating the effect of the associated changes in the cuticular hydrocarbon profiles. The authors make sure that the behavioural effect is indeed due to the chemical changes by not only testing life animals, but also dead animals and corpses with manipulated cuticular hydrocarbons.
I find the evidence that the hydrocarbon changes do not affect survival and desiccation resistance less convincing (due to the limited set of conditions and relatively small sample size), but the data presented are certainly congruent with the idea that the methyl alkane changes do not have large effects on desiccation.
We appreciate the kind comments from the reviewer.
Reviewer #3 (Public Review):
In this manuscript, the authors are aiming to demonstrate that a fatty-acyl synthase gene (fas5) is involved in the composition of the blend of surface hydrocarbons of a parasitoid wasp and that it affects the sexual attractiveness of females for males. Overall, the manuscript reads very well, it is very streamlined, and the authors' claims are mostly supported by their experiments and observations.
We appreciate the kind comments from the reviewer.
However, I find that some experiments, information and/or discussion are absent to assess how the effects they observe are, at least in part, not due to other factors than fas5 and the methyl-branched (MB) alkanes. I'm also wondering if what the authors observe is only a change in the sexual attractiveness of females and not related to species recognition as well.
We appreciate the interesting point that the reviewer raises in sexual attractiveness and species recognition and now expand upon this potential aspect in the discussion (lines 327-330). However, in this manuscript, we very much focused on the effect of fas5 knockdown on the conveyance of female sexual attractiveness in a single species (Nasonia vitripennis). Therefore, we argue that species recognition constitutes a different communication modality here, and we currently cannot infer whether and how species recognition is exactly encoded in Nasonia CHC profiles despite some circumstantial evidence for species-specificity (Buellesbach et al. 2013; Mair et al. 2017). Thus, we would like to refrain from any further speculation on species recognition before this can be unambiguously demonstrated, and remain within the mechanism of sexual attractiveness within a single species which we clearly show is mediated by the female MB-alkane fraction governed by the fatty acid synthase genes. We however still consider potential alternative explanations (e.g., n-alkenes acting as a deterrent of homosexual mating attempts).
The authors explore the function of cuticular hydrocarbons (CHCs) and a fatty-acyl synthase in Nasonia vitripennis, a parasitic wasp. Using RNAi, they successfully knockdown the expression of the fas5 gene in wasps. The authors do not justify their choice of fatty-acyl synthase candidate gene. It would have been interesting to know if that is one of many genes they studied or if there was some evidence that drove them to focus their interest in fas5.
In a previous study, 5 fas candidate genes orthologous to Drosophila melanogaster fas genes were identified and mapped in the genome of Nasonia vitripennis (Buellesbach et al. 2022). We actually investigated the effects of all of these fas genes on CHC variation, but only fas5 led to such a striking, traceable pattern shift. We are currently preparing another manuscript discussing the effects of the other fas genes, but decided to focus exclusively on fas5 here, due to its significance for revealing how sexual attractiveness can be encoded and conveyed in complex chemical profiles, maintained and governed by a surprisingly simple genetic basis.
The authors observe large changes in the cuticular hydrocarbons (CHC) profile of male and females. These changes are mostly a reduction of some MB alkanes and an increase in others as well as an increase of n-alkene in fas5 knockdown females. For males fas5 knockdowns, the overall quantity of CHC is increased and consequently, multiple types of compounds are increased compared to wild-type, with only one compound appearing to decrease compared to wild-type. Insects are known to rely on ratios of compounds in blends to recognize odors. Authors address this by showing a plot of the relative ratios, but it seems to me that they do show statistical tests of those changes in the proportions of the different types of compounds. In the results section, the authors give percentages while referring to figures showing the absolute amount of CHCs. They should also test if the ratios are significantly different or not between experimental conditions. Similar data should be displayed for the males as well.
We appreciate your suggestions. We kindly refer you to our response to reviewer 1, where we addressed the statistical tests. Specifically, we generated separate subplots to display the proportions of different compound classes and performed statistical tests to compare these proportions between different treatments for both males and females. Additionally, we have revised the results section to replace relative abundances with absolute quantity, as depicted in Figure 2C-G.
Furthermore, the authors didn't use an internal standard to measure the quantity of CHCs in the extracts, which, to me, is the gold standard in the field. If I understood correctly, the authors check the abundance measured for known quantities of n-alkanes. I'm sure this method is fine, but I would have liked to be reassured that the quantities measured through this method are good by either testing some samples with an internal standard, or referring to work that demonstrates that this method is always accurate to assess the quantities of CHC in extracts of known volumes.
We actually did include 7,5 ng/μl dodecane (C12) as an “internal” standard in the hexane resuspensions of all of our processed samples (line 456, Materials and Methods). This was primarily done to allow for visually inspecting and comparing the congruence of all chromatograms in the subsequent data analysis and immediately detect any variation from sample preparation, injection process and instrument fluctuation. In our study, we have a very elaborate and standardized CHC extraction method that the volume of solvent and duration for extraction are strictly controlled to minimize the variation from sample preparation steps. Furthermore, we calibrated each individual CHC compound quantity with a dilution series of external standards (C21-C40) of known concentration. By constructing a calibration curve based on this dilution series, we achieved the most accurate compound quantification, also taking into account and counteracting the generally diminishing quantities of compounds with higher chain lengths.
The authors provide a sensible control for their RNAi experiments: targeting an unrelated gene, absent in N. vitripennis (the GFP). This allows us to see if the injection of RNAi might affect CHC profiles, which it appears to do in some cases in males, but not in females. The authors also show to the reader that their RNAi experiments do reduce the expression of the target gene. However, one of the caveats of their experiments, is that the authors don't provide evidence or information to allow the (non-expert) reader to assess whether the fas5 RNAi experiments did affect the expression of other fatty-acyl synthase genes. I'm not an expert in RNAi, so maybe this suggestion is not relevant, but it should, at least, be addressed somewhere in the manuscript that such off-target effects are very unlikely or impossible, in that case, or more generally.
We acknowledge the reviewer’s concern about potential off-target effect of the fas5 knockdown. We actually did check initially for off-target effects on the other four previously published fas genes in N. vitripennis (Lammers et al. 2019; Buellesbach et al. 2022) and did not find any effects on their respective expressions. We now include these results as supplementary data (Figure 2-figure supplement 1). However, as mentioned in the cover letter to the editor, we discovered a previously uncharacterized fas gene in the most recent N. vitripennis genome assembly (NC_045761.1), fas6, most likely constituting a tandem gene duplication of fas5. These two genes turned out to have such high sequence similarity (> 90 %, Figure 2-figure supplement 2) that both were simultaneously downregulated by our fas5 dsRNAi construct, which we confirmed with qPCR and now incorporated into our manuscript (Fig. 2H). Therefore, we now explicitly mention that the knockdown affects both genes, and either one or both could have the observed phenotypic effects. Recognizing this RNAi off-target effect, we have now also incorporated a discussion of this issue in the appropriate section of the manuscript (line 364-377), as well as the potential off-target effects of our GFP dsRNAi controls (line 262-274).
The authors observe that the modified CHCs profiles of RNAi females reduce courtship and copulation attempts, but not antennation, by males toward live and (dead) dummy females. They show that the MB alkanes of the CHC profile are sufficient to elicit sexual behaviors from males towards dummy females and that the same fraction from extracts of fas5 knockdown females does so significantly less. From the previous data, it seems that dummy females with fas5 female's MB alkanes profile elicit more antennation than CHC-cleared dummy females, but the authors do not display data for this type of target on the figure for MB alkane behavioral experiments.
Actually similar proportions of males performed antennation behavior towards female dummies with MB alkane fraction of fas5 RNAi females and CHC-cleared female dummies (55% and 50%, respectively, see Author response image 1 for the corresponding parts of the sub-figures 3 E and 4 D). We did not deem it necessary to show the same data on CHC-cleared female dummies in Figure 3 as well.
Author response image 1.
Unfortunately, the authors don't present experiments testing the effect of the non-MB alkanes fractions of the CHC extracts on male behavior toward females. As such, they are not able to (and didn't) conclude that the MB-alkane is necessary to trigger the sexual behaviors of males. I believe testing this would have significantly enhanced the significance of this work. I would also have found it interesting for the authors to comment on whether they observe aggressive behavior of males towards females (live or dead) and/or whether such behavior is expected or not in inter-individual interactions in parasitoids wasps.
In our experiment, we focus on the function of the MB-alkane fraction in female CHC profiles, and we comprehensibly demonstrate in figure 4 that the MB-alkane fraction from WT females alone is sufficient to trigger mating behavior coherent with that on alive and untreated female dummies. Therefore, we do not completely understand the reviewer’s concern about us not being ” able to (and didn't) conclude that the MB-alkane is necessary to trigger the sexual behaviors of males”. We appreciate the suggestion from the reviewer of testing the non-MB alkanes (n-alkanes and n-alkenes). However, due to the experimental procedure of separating the CHC compound class fractions through elution with molecular sieves, it was not possible for us to retrieve either the whole n-alkane or n-alkene fraction remaining bound to the sieves after separation). The role of n-alkenes in N. vitripennis is however considered in the discussion, as a deterrent for homosexual interactions between males (Wang et al. 2022a). Moreover, we did not observe aggressive behavior of males towards live or dead females.
CHCs are used by insects to signal and/or recognize various traits of targets of interest, including species or groups of origin, fertility, etc. The authors claim that their experiments show the sexual attractiveness of females can be encoded in the specific ratio of MB alkanes. While I understand how they come to this conclusion, I am somewhat concerned. The authors very quickly discuss their results in light of the literature about the role of CHCs (and notably MB alkanes) in various recognition behaviors in Hymenoptera, including conspecific recognition. Previous work (cited by the authors) has shown that males recognize males from females using an alkene (Z9C31). As such, it remains possible that the "sexual attractiveness" of N. vitripennis females for males relies on them not being males and being from the right species as well. The authors do not address the question of whether the CHCs (and the MB alkanes in particular) of females signal their sex or their species. While I acknowledge that responding to this question is beyond the scope of this work, I also strongly believe that it should be discussed in the manuscript. Otherwise, non-specialist readers would not be able to understand what I believe is one of the points that could temper the conclusions from this work.
We acknowledge the reviewer’s insight about the MB alkanes in signaling sex or species in N. vitripennis, and now include this aspect in our revised discussion (line 324-330). Moreover, we clearly demonstrate that n-alkenes have been reduced to minute trace components after our compound class separation, and the males still do not display courtship and copulation behaviors similar to WT females, thus strongly indicating that the n-alkenes do not play a role when relying solely on the changed MB-alkane patterns, further strengthening our main argument.
References
Benjamini, Y. and D. Yekutieli. 2001. The control of the false discovery rate in multiple testing under dependency. Ann. Stat. 29:1165-1188.
Buellesbach, J., J. Gadau, L. W. Beukeboom, F. Echinger, R. Raychoudhury, J. H. Werren, and T. Schmitt. 2013. Cuticular hydrocarbon divergence in the jewel wasp Nasonia: Evolutionary shifts in chemical communication channels? J. Evol. Biol. 26:2467-2478.
Buellesbach, J., C. Greim, and T. Schmitt. 2014. Asymmetric interspecific mating behavior reflects incomplete prezygotic isolation in the jewel wasp genus Nasonia. Ethology 120:834-843.
Buellesbach, J., H. Holze, L. Schrader, J. Liebig, T. Schmitt, J. Gadau, and O. Niehuis. 2022. Genetic and genomic architecture of species-specific cuticular hydrocarbon variation in parasitoid wasps. Proc. R. Soc. B 289:20220336.
Engl, T., N. Eberl, C. Gorse, T. Krüger, T. H. P. Schmidt, R. Plarre, C. Adler, and M. Kaltenpoth. 2018. Ancient symbiosis confers desiccation resistance to stored grain pest beetles. Mol. Ecol. 27:2095-2108.
Ferveur, J. F., J. Cortot, K. Rihani, M. Cobb, and C. Everaerts. 2018. Desiccation resistance: effect of cuticular hydrocarbons and water content in Drosophila melanogaster adults. Peerj 6.
Lammers, M., K. Kraaijeveld, J. Mariën, and J. Ellers. 2019. Gene expression changes associated with the evolutionary loss of a metabolic trait: lack of lipogenesis in parasitoids. BMC Genom. 20:309.
Mair, M. M., V. Kmezic, S. Huber, B. A. Pannebakker, and J. Ruther. 2017. The chemical basis of mate recognition in two parasitoid wasp species of the genus Nasonia. Entomol. Exp. Appl. 164:1-15.
Wang, Y., W. Sun, S. Fleischmann, J. G. Millar, J. Ruther, and E. C. Verhulst. 2022a. Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males. Proc. R. Soc. B 289:20212002.
Wang, Z., J. P. Receveur, J. Pu, H. Cong, C. Richards, M. Liang, and H. Chung. 2022b. Desiccation resistance differences in Drosophila species can be largely explained by variations in cuticular hydrocarbons. eLife 11:e80859.
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eLife assessment
This important study reveals the genetic regulation of changes in cuticular hydrocarbon profiles in a Hymenopteran insect and links these changes with courtship behaviour and sexual attractiveness. It provides convincing empirical evidence, spanning genetic, chemical, and behavioural data. It adds valuable new perspectives on the mechanisms that underlie chemical recognition and communication systems in nature.
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Reviewer #1 (Public Review):
The authors convincingly show in this study the effects of the fas5 gene on changes in the CHC profile and the importance of these changes toward sexual attractiveness.
The main strength of this study lies in its holistic approach (from genes to behaviour) showing a full and convincing picture of the stated conclusions. The authors succeeded in putting a very interdisciplinary set of experiments together to support the main claims of this manuscript.
The main weakness stems from the lack of transparency behind the statistical analyses conducted in the study. Detailed statistical results are never mentioned in the text, nor is it always clear what was compared to what. I also believe that some tests that were conducted are not adequate for the given data. I am therefore unable to properly assess the …
Reviewer #1 (Public Review):
The authors convincingly show in this study the effects of the fas5 gene on changes in the CHC profile and the importance of these changes toward sexual attractiveness.
The main strength of this study lies in its holistic approach (from genes to behaviour) showing a full and convincing picture of the stated conclusions. The authors succeeded in putting a very interdisciplinary set of experiments together to support the main claims of this manuscript.
The main weakness stems from the lack of transparency behind the statistical analyses conducted in the study. Detailed statistical results are never mentioned in the text, nor is it always clear what was compared to what. I also believe that some tests that were conducted are not adequate for the given data. I am therefore unable to properly assess the significance of the results from the presented information. Nevertheless, the graphical representations are convincing enough for me to believe that a revision of the statistics would not significantly affect the main conclusions of this manuscript.
The second major problem I had with the study was how it brushes over the somewhat contradicting results they found in males (Fig S2). These are only mentioned twice in the main text and in both cases as being "similarly affected", even though their own stats seem to indicate otherwise for many of the analysed compound groups. This also should affect the main conclusion concerning the effects of fas5 genes in the discussion, a more careful wording when interpreting the results is therefore necessary.
-
Reviewer #2 (Public Review):
Insects have long been known to use cuticular hydrocarbons for communication. While the general pathways for hydrocarbon synthesis have been worked out, their specificity and in particular the specificity of the different enzymes involved is surprisingly little understood. Here, the authors convincingly demonstrate that a single fatty acid synthase gene is responsible for a shift in the positions of methyl groups across the entire alkane spectrum of a wasp, and that the wasps males recognize females specifically based on these methyl group positions. The strength of the study is the combination of gene expression manipulations with behavioural observations evaluating the effect of the associated changes in the cuticular hydrocarbon profiles. The authors make sure that the behavioural effect is indeed due to …
Reviewer #2 (Public Review):
Insects have long been known to use cuticular hydrocarbons for communication. While the general pathways for hydrocarbon synthesis have been worked out, their specificity and in particular the specificity of the different enzymes involved is surprisingly little understood. Here, the authors convincingly demonstrate that a single fatty acid synthase gene is responsible for a shift in the positions of methyl groups across the entire alkane spectrum of a wasp, and that the wasps males recognize females specifically based on these methyl group positions. The strength of the study is the combination of gene expression manipulations with behavioural observations evaluating the effect of the associated changes in the cuticular hydrocarbon profiles. The authors make sure that the behavioural effect is indeed due to the chemical changes by not only testing life animals, but also dead animals and corpses with manipulated cuticular hydrocarbons.
I find the evidence that the hydrocarbon changes do not affect survival and desiccation resistance less convincing (due to the limited set of conditions and relatively small sample size), but the data presented are certainly congruent with the idea that the methyl alkane changes do not have large effects on desiccation.
-
Reviewer #3 (Public Review):
In this manuscript, the authors are aiming to demonstrate that a fatty-acyl synthase gene (fas5) is involved in the composition of the blend of surface hydrocarbons of a parasitoid wasp and that it affects the sexual attractiveness of females for males. Overall, the manuscript reads very well, it is very streamlined, and the authors' claims are mostly supported by their experiments and observations. However, I find that some experiments, information and/or discussion are absent to assess how the effects they observe are, at least in part, not due to other factors than fas5 and the methyl-branched (MB) alkanes. I'm also wondering if what the authors observe is only a change in the sexual attractiveness of females and not related to species recognition as well.
The authors explore the function of cuticular …
Reviewer #3 (Public Review):
In this manuscript, the authors are aiming to demonstrate that a fatty-acyl synthase gene (fas5) is involved in the composition of the blend of surface hydrocarbons of a parasitoid wasp and that it affects the sexual attractiveness of females for males. Overall, the manuscript reads very well, it is very streamlined, and the authors' claims are mostly supported by their experiments and observations. However, I find that some experiments, information and/or discussion are absent to assess how the effects they observe are, at least in part, not due to other factors than fas5 and the methyl-branched (MB) alkanes. I'm also wondering if what the authors observe is only a change in the sexual attractiveness of females and not related to species recognition as well.
The authors explore the function of cuticular hydrocarbons (CHCs) and a fatty-acyl synthase in Nasonia vitripennis, a parasitic wasp. Using RNAi, they successfully knockdown the expression of the fas5 gene in wasps. The authors do not justify their choice of fatty-acyl synthase candidate gene. It would have been interesting to know if that is one of many genes they studied or if there was some evidence that drove them to focus their interest in fas5. The authors observe large changes in the cuticular hydrocarbons (CHC) profile of male and females. These changes are mostly a reduction of some MB alkanes and an increase in others as well as an increase of n-alkene in fas5 knockdown females. For males fas5 knockdowns, the overall quantity of CHC is increased and consequently, multiple types of compounds are increased compared to wild-type, with only one compound appearing to decrease compared to wild-type. Insects are known to rely on ratios of compounds in blends to recognize odors. Authors address this by showing a plot of the relative ratios, but it seems to me that they do show statistical tests of those changes in the proportions of the different types of compounds. In the results section, the authors give percentages while referring to figures showing the absolute amount of CHCs. They should also test if the ratios are significantly different or not between experimental conditions. Similar data should be displayed for the males as well. Furthermore, the authors didn't use an internal standard to measure the quantity of CHCs in the extracts, which, to me, is the gold standard in the field. If I understood correctly, the authors check the abundance measured for known quantities of n-alkanes. I'm sure this method is fine, but I would have liked to be reassured that the quantities measured through this method are good by either testing some samples with an internal standard, or referring to work that demonstrates that this method is always accurate to assess the quantities of CHC in extracts of known volumes.
The authors provide a sensible control for their RNAi experiments: targeting an unrelated gene, absent in N. vitripennis (the GFP). This allows us to see if the injection of RNAi might affect CHC profiles, which it appears to do in some cases in males, but not in females. The authors also show to the reader that their RNAi experiments do reduce the expression of the target gene. However, one of the caveats of their experiments, is that the authors don't provide evidence or information to allow the (non-expert) reader to assess whether the fas5 RNAi experiments did affect the expression of other fatty-acyl synthase genes. I'm not an expert in RNAi, so maybe this suggestion is not relevant, but it should, at least, be addressed somewhere in the manuscript that such off-target effects are very unlikely or impossible, in that case, or more generally.
The authors observe that the modified CHCs profiles of RNAi females reduce courtship and copulation attempts, but not antennation, by males toward live and (dead) dummy females. They show that the MB alkanes of the CHC profile are sufficient to elicit sexual behaviors from males towards dummy females and that the same fraction from extracts of fas5 knockdown females does so significantly less. From the previous data, it seems that dummy females with fas5 female's MB alkanes profile elicit more antennation than CHC-cleared dummy females, but the authors do not display data for this type of target on the figure for MB alkane behavioral experiments. Unfortunately, the authors don't present experiments testing the effect of the non-MB alkanes fractions of the CHC extracts on male behavior toward females. As such, they are not able to (and didn't) conclude that the MB-alkane is necessary to trigger the sexual behaviors of males. I believe testing this would have significantly enhanced the significance of this work. I would also have found it interesting for the authors to comment on whether they observe aggressive behavior of males towards females (live or dead) and/or whether such behavior is expected or not in inter-individual interactions in parasitoids wasps.
CHCs are used by insects to signal and/or recognize various traits of targets of interest, including species or groups of origin, fertility, etc. The authors claim that their experiments show the sexual attractiveness of females can be encoded in the specific ratio of MB alkanes. While I understand how they come to this conclusion, I am somewhat concerned. The authors very quickly discuss their results in light of the literature about the role of CHCs (and notably MB alkanes) in various recognition behaviors in Hymenoptera, including conspecific recognition. Previous work (cited by the authors) has shown that males recognize males from females using an alkene (Z9C31). As such, it remains possible that the "sexual attractiveness" of N. vitripennis females for males relies on them not being males and being from the right species as well. The authors do not address the question of whether the CHCs (and the MB alkanes in particular) of females signal their sex or their species. While I acknowledge that responding to this question is beyond the scope of this work, I also strongly believe that it should be discussed in the manuscript. Otherwise, non-specialist readers would not be able to understand what I believe is one of the points that could temper the conclusions from this work.
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