RETRACTED: Amino acid synthesis loss in parasitoid wasps and other hymenopterans

Acceptance summary:

How the co-evolution of parasites and hosts impacts the content of their genomes is a major question of great fundamental and applied interest. In this work, the authors combine metabolomic, comparative genomic, and experimental approaches to understand how parasitoid wasps have gained or lost core metabolic machinery involved in amino acid synthesis to adapt and co-evolve their interactions with their host, and more generally understand how these interactions have shaped the evolution of these biochemical processes in hymenopteran insects.

Decision letter after peer review:

Thank you for submitting your article "Amino acid synthesis loss in parasitoid wasps and other hymenopterans" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Patricia Wittkopp as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

The relationship between parasite and host is an interaction that has universal applicability across the life sciences by providing a model for addressing fundamental ecological, evolutionary, and developmental biology questions as well as solutions for applied problems like biological control agents and pest management. With this manuscript, the authors combine metabolomic, comparative genomic, and experimental approaches to understand how parasitoid wasps have gained or lost core metabolic machinery involved in amino acid synthesis to adapt and co-evolve their interactions with their host, and more generally understand how these interactions have shaped the evolution of these biochemical processes in hymenopteran insects.

Essential revisions:

1) Please use color-blind friendly color schemes and/or something other than color to differentiate between items in figures.

2) Figure 1D: The synteny analysis needs to be clarified. In particular, it is not clear what Figure 1D is showing. Do the heavy bars represent a chromosome? Or all of the sequence for the given species arranged linearly?

3) Subsection “Influences of parasitism on host amino acid synthesis, gene expression, and free amino acid content in host hemolymph”, last paragraph: The logic in the discussion of free amino acid levels in the host is not clear. It sounds like the rationale is that the parasitoid decreases amino acid synthesis in the host to ensure adequate levels of amino acids. Or is the idea that the protease activity is driving amino acid availability and the effect of infection on host gene expression is less/un- important?

4) Figure 3B: Why are only some enzymes shown? Are the others unchanged or were they not tested? Please clarify this to allow for better interpretation of the results.

5) Subsection “Amino acid transport in the parasitoid wasp C. chilonis”: We do not understand the comparison being made. Several of the genes appear to be more highly expressed in pupae than larvae. The figure legend acknowledges this but the text seems to just focus on larvae, although it is unclear what is meant by "other stage".

6) Subsection “Amino acid transport in the parasitoid wasp C. chilonis”: How would host AA transporter expression increase parasitoid AA uptake? This needs some explanation.

7) Twice in the paper, the authors over-interpret a statistical result that is in fact statistically insignificant (subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, second paragraph and subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, last paragraph; and to a lesser extent in the first paragraph of the subsection “Influences of parasitism on host amino acid synthesis, gene expression, and free amino acid content in host hemolymph”).

8) Based on the flow of logic of the paper, we suggest putting the host-parasite experiment after the comparative genomics data/findings. This might make it easier to interpret the host-parasite experiments.

9) Regarding the interpretations (in the Discussion) of the host-parasite experiment and the resulting increase/decrease of amino acids (Discussion, first paragraph), the authors describe how the concentrations of the 8 key amino acids were varied, when in fact only two really change (Tyrosine goes up, Lysine goes down). The authors should revise these statements accordingly and zoom in on these two amino acids and their resulting opposite directionality (going up or down in concentration), and interpret this based on their data and the literature.

10) Discussion, last paragraph: In the paper Summary/Conclusion, the authors omit a recap of the amino acid transporter analysis and its relevance to the findings of the paper. Again, it might be worth the authors doing a larger taxonomic sampling (currently 3 species) of these amino acid transporters, as this might be very illuminating for the parasite-host relationship investigated here.

11) There were several issues with the statistical analyses:

a) What correction for multiple testing was applied to the statistics? (e.g. Figure 2C)

b) What stats were used for determining differential gene expression? Fold change alone is not sufficient to conclude up or down regulation without a test that takes variation in

to account.

c) Subsection “Disruptions in amino acid biosynthetic pathways in Hymenoptera”, eighth paragraph: What are the stats for these proportions?

d) Here are two occurrences (subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, second paragraph and subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, last paragraph) where the authors over-interpret statistically insignificant results – please revise accordingly.

12) While the amino acid biosynthesis pathway disruption/capacity analyses are very interesting and relevant for both the parasites analyzed as well as insects in general, the analyses on amino acid transporters (and their expansion) would offer far greater insight in terms of a specific understanding of the parasite studied. The manuscript currently is focused on the parasite-host question, and secondarily on metabolic pathway evolution across hymenopterans. The authors should either re-frame the manuscript more explicitly on hymenopterans, or they go should go deeper with their analyses of amino acid transporters of the parasite. If the authors do expand their study to incorporate more regarding amino acid transport (and they find something of further interest), the title might benefit from reflecting that aspect as well.

Essential revisions:

1) Please use color-blind friendly color schemes and/or something other than color to differentiate between items in figures.

Thanks for this suggestion. We carefully checked the colors of all the figures in our manuscript (including the Supplementary files) and changed them to the colorblind friendly color schemes.

2) Figure 1D: The synteny analysis needs to be clarified. In particular, it is not clear what Figure 1D is showing. Do the heavy bars represent a chromosome? Or all of the sequence for the given species arranged linearly?

We revised the statement of synteny analysis in the figure legend. Because there are no chromosome-level genome assemblies for these three wasps, the heavy bars in Figure 1D represent all the scaffolds linked together in an artificial order.

3) Subsection “Influences of parasitism on host amino acid synthesis, gene expression, and free amino acid content in host hemolymph”, last paragraph: The logic in the discussion of free amino acid levels in the host is not clear. It sounds like the rationale is that the parasitoid decreases amino acid synthesis in the host to ensure adequate levels of amino acids. Or is the idea that the protease activity is driving amino acid availability and the effect of infection on host gene expression is less/un- important?

Thanks for this suggestion. We prefer the second option, and revised the manuscript, “Our results suggest that C. chilonis venom and/or larvae feeding may finely regulate the host’s protease activity to re-allocate the amino acids into the hemolymph, to ensure adequate nutrition for the parasitoid. However, we have so far not been able to separate the effects of venom from actions of feeding larvae in this endoparasitoid wasp”. As we found that only 11 genes in the amino acid biosynthetic pathway of the host significantly changed their expression after parasitism, we concluded that the influence of parasitism on the host’s amino acid biosynthetic pathways might be less important.

4) Figure 3B: Why are only some enzymes shown? Are the others unchanged or were they not tested? Please clarify this to allow for better interpretation of the results.

In Figure 3B (Figure 5B in revised manuscript), we only showed the genes with significantly expression changes between parasitism and non-parasitism. We now indicate this in the figure legend.

5) Subsection “Amino acid transport in the parasitoid wasp C. chilonis”: We do not understand the comparison being made. Several of the genes appear to be more highly expressed in pupae than larvae. The figure legend acknowledges this but the text seems to just focus on larvae, although it is unclear what is meant by "other stage".

Apologies, we compared the gene expressions among difference development stages. Other stage meant pupal and adult stages. We have now revised the statement: “Two APC genes were highly expressed in the larval stage relative to the pupal and adult stages. Five APC genes were highly expressed in the larval and pupal stages relative to the adult stage”.

6) Subsection “Amino acid transport in the parasitoid wasp C. chilonis”: How would host AA transporter expression increase parasitoid AA uptake? This needs some explanation.

We have added the following to the Discussion: “Up-regulation of host genes involved in amino acid transport could contribute to release free amino acids into the hemolymph, and therefore their role in amino acid availability for parasitoid larvae warrants future investigation.”

7) Twice in the paper, the authors over-interpret a statistical result that is in fact statistically insignificant (subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, second paragraph and subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, last paragraph; and to a lesser extent in the first paragraph of the subsection “Influences of parasitism on host amino acid synthesis, gene expression, and free amino acid content in host hemolymph”).

Thanks for this suggestion. We have removed these over-interpreted statements.

8) Based on the flow of logic of the paper, we suggest putting the host-parasite experiment after the comparative genomics data/findings. This might make it easier to interpret the host-parasite experiments.

Agree. As you suggested, we reordered the whole paper and put the comparative genomics data/findings in front of the host-parasite experiment. We also moved a part of the details of basic genome information on C. chilonis (genome sequencing, assembly, and annotation) from the Results section to the Materials and methods section. This allows us to get more directly into the main points of the paper.

The logic of our current manuscript is that: we first conducted genome sequencing of C. chilonis and analyzed its genome evolution with other hymenopterans. We next examined the C. chilonis’s genomic repertoire for amino acid synthesis pathway genes and place these results in an evolutionary context by a comparative genomics analysis of 38 hymenopteran species (three sawflies, 17 aculeates and 18 parasitoids), and compared to a set of 13 other holometabolous and hemimetabolous arthropods. We then returned to C. chilonis to conduct a set of experiments to investigate the amino acid requirements of their larvae, in light of the pathways predicted to be disrupted by the genomic analysis.

In addition, we also reordered the Abstract, Introduction and Discussion to make sure they are consistent with our revised logic. We checked all the transition sentences based on this change in order.

9) Regarding the interpretations (in the Discussion) of the host-parasite experiment and the resulting increase/decrease of amino acids (Discussion, first paragraph), the authors describe how the concentrations of the 8 key amino acids were varied, when in fact only two really change (Tyrosine goes up, Lysine goes down). The authors should revise these statements accordingly and zoom in on these two amino acids and their resulting opposite directionality (going up or down in concentration), and interpret this based on their data and the literature.

Agree. We have revised these statements and added a discussion about these two changes in concentration; it now reads: “For the eight amino acids that C. chilonis cannot synthesize, the concentrations of lysine and tyrosine were found to change significantly in host hemolymph after parasitism (tyrosine went up, lysine went down). […] These interpretations are speculative, and would require further detailed analysis to determine the contributions of modifications induced by wasp venom, teratocytes, and feeding larvae on amino acid levels.”

10) Discussion, last paragraph: In the paper Summary/Conclusion, the authors omit a recap of the amino acid transporter analysis and its relevance to the findings of the paper. Again, it might be worth the authors doing a larger taxonomic sampling (currently 3 species) of these amino acid transporters, as this might be very illuminating for the parasite-host relationship investigated here.

Agree. We have added the findings about amino acid transporter analysis to the summary section: “Expansion of amino acid transporters and their increased expression in the larval stage indicate that they might play important roles in nutrition interaction between parasitoid and host; however, this has not been as extensively investigated”.

As suggested, we did a larger taxonomic sampling of amino acid transporters in hymenopteran genomes. However, the results didn’t support the view that transporter genes are expanded basally in the parasitoid wasps. Instead, the result indicates that gene expansion of ABC transporter genes in C. chilonis was an independent expansion event. We have added this result to our revision which now reads: “We next searched for these transporter genes in a large collection of hymenopteran genomes, but the results didn’t support the view that these transporter genes are expanded basally in the parasitoid wasps (Figure 6—figure supplement 2). […] The expansion events also independently occurred in the sawfly Cephus cinctus, some ants, Megachile bees and Bombus bees.”

The evolution of transporter genes and their functions on parasite-host interaction might be complex, and we don’t have enough genomic and transcriptomic data to explain it in this study. So, we opted to leave that for the future.

11) There were several issues with the statistical analyses:

a) What correction for multiple testing was applied to the statistics? (e.g. Figure 2C)

Thanks. For the survival rate statistical analyses in Figure 2C (now Figure 4C), we now used a Benjamini-Hochberg correction method to correction for multiple test comparison. We found that the results are not changed.

b) What stats were used for determining differential gene expression? Fold change alone is not sufficient to conclude up or down regulation without a test that takes variation in

to account.

We used edgeR version 3.11 to identify differential gene expression. For the accuracy of the improved differentially expressed genes, we defined the fold change of gene expression ≥ 2 and FDR adjusted (Benjamini-Hochberg correction) P value < 0.05 as the criteria for significant differences in gene expression levels. We have revised our method to make it clearer to the readers that 2-fold expression was not our sole criterion, but also an FDR adjusted P < 0.05 was required.

c) Subsection “Disruptions in amino acid biosynthetic pathways in Hymenoptera”, eighth paragraph: What are the stats for these proportions?

To test if gene loss is more likely to occur in the disrupted pathway, we calculated a random expected proportion based on the gene number of each pathway in the ancestral state of Hymenoptera. We used Chi-square to perform the statistical test, using custom expected frequencies for the gene loss event. We now make this more clear in the Materials and methods section.

d) Here are two occurrences (subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, second paragraph and subsection “Amino acid biosynthetic pathway disruptions and gene losses in Hymenoptera”, last paragraph) where the authors over-interpret statistically insignificant results – please revise accordingly.

We have removed these over-interpreted statements.

12) While the amino acid biosynthesis pathway disruption/capacity analyses are very interesting and relevant for both the parasites analyzed as well as insects in general, the analyses on amino acid transporters (and their expansion) would offer far greater insight in terms of a specific understanding of the parasite studied. The manuscript currently is focused on the parasite-host question, and secondarily on metabolic pathway evolution across hymenopterans. The authors should either re-frame the manuscript more explicitly on hymenopterans, or they go should go deeper with their analyses of amino acid transporters of the parasite. If the authors do expand their study to incorporate more regarding amino acid transport (and they find something of further interest), the title might benefit from reflecting that aspect as well.

We agree that the analyses on amino acid transporters (and their expansion) would offer useful insights. In our revision, we did a larger taxonomic sampling of amino acid transporters in hymenopterans. The results did not support that amino acid transporters are expanded basally in the parasitoid wasps comparing to other hymenopterans. We have added this finding in our revision. Because we found the expansion of ABC transporter genes in C. chilonis, but not in the closely related species, we have thus added this sentence, “This result suggests that the expansion of ABC transporters in C. chilonis is an independent event , as it was not found in close relatives.”. We hope to eventually go deeper on the analyses of amino acid transporters of the parasitoids. Unfortunately, the limited transcriptome data for parasitoid wasps (especially for early embryos and larvae) could be a problem for deeper analyses at this time. So, we opted to leave that for the future. Here, we re-structured the manuscript more explicitly on the metabolic pathway evolution of hymenopterans as we described above in the response to comment 8.