Endoparasitoid lifestyle promotes endogenization and domestication of dsDNA viruses
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eLife assessment
This important manuscript employs a rigorous and multi-pronged comparative genomics approach to unravel how lifestyle modulates the acquisition and domestication of viral genetic elements in the genomes of hymenopteran insects. Using an extensive dataset of over 120 hymenopteran genomes, the authors provide convincing evidence that endoparasitism (where parasite development occurs within hosts) facilitates the uptake and domestication of double-stranded DNA viral elements.
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Abstract
The accidental endogenization of viral elements within eukaryotic genomes can occasionally provide significant evolutionary benefits, giving rise to their long-term retention, that is, to viral domestication. For instance, in some endoparasitoid wasps (whose immature stages develop inside their hosts), the membrane-fusion property of double-stranded DNA viruses have been repeatedly domesticated following ancestral endogenizations. The endogenized genes provide female wasps with a delivery tool to inject virulence factors that are essential to the developmental success of their offspring. Because all known cases of viral domestication involve endoparasitic wasps, we hypothesized that this lifestyle, relying on a close interaction between individuals, may have promoted the endogenization and domestication of viruses. By analyzing the composition of 124 Hymenoptera genomes, spread over the diversity of this clade and including free-living, ecto, and endoparasitoid species, we tested this hypothesis. Our analysis first revealed that double-stranded DNA viruses, in comparison with other viral genomic structures (ssDNA, dsRNA, ssRNA), are more often endogenized and domesticated (that is, retained by selection) than expected from their estimated abundance in insect viral communities. Second, our analysis indicates that the rate at which dsDNA viruses are endogenized is higher in endoparasitoids than in ectoparasitoids or free-living hymenopterans, which also translates into more frequent events of domestication. Hence, these results are consistent with the hypothesis that the endoparasitoid lifestyle has facilitated the endogenization of dsDNA viruses, in turn, increasing the opportunities of domestications that now play a central role in the biology of many endoparasitoid lineages.
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Author Response
Reviewer #1 (Public Review):
This manuscript represents a substantial and well-executed body of work that contributes new data on 32 hymenopteran genomes, systematically identifies viral endogenization and domestication events, and tests whether this phenomenon is more common in hymenopteran species with specific lifestyles, eg. endoparasitism. The authors developed a pipeline to identify endogenization that improves upon previously described pipelines and is more comprehensive for the identification of endogenization events from a variety of virus types. Significant findings include the identification of previously undocumented cases of viral endogenization in several hymenopteran species and also moderate statistical support for a higher rate of dsDNA virus endogenization and domestication in endoparasitoids.
- The …
Author Response
Reviewer #1 (Public Review):
This manuscript represents a substantial and well-executed body of work that contributes new data on 32 hymenopteran genomes, systematically identifies viral endogenization and domestication events, and tests whether this phenomenon is more common in hymenopteran species with specific lifestyles, eg. endoparasitism. The authors developed a pipeline to identify endogenization that improves upon previously described pipelines and is more comprehensive for the identification of endogenization events from a variety of virus types. Significant findings include the identification of previously undocumented cases of viral endogenization in several hymenopteran species and also moderate statistical support for a higher rate of dsDNA virus endogenization and domestication in endoparasitoids.
- The authors have tested whether the lifestyle of hymenopteran species (endoparasitism, ectoparasitism, or free-living) is related to the incidence of virus endogenization and domestication. Addressing this kind of question has only become possible with the availability of genome sequences from many taxa so that any results can be statistically supported by appropriate sample sizes. It appears that the authors have not included new genomic data from hymenopteran genomes that have been published since 2019, which are of similar or better quality than the data used in this manuscript. A number of taxa with endogenous viruses (and also without) have become available since then. The best solution would be for the authors to use their pipeline to incorporate the new data, which may have an impact on their findings and could even strengthen their conclusions about virus domestication being more common in endoparasitoids. If this is not possible, the authors should at least justify their decision not to include the most recent data and discuss how it could affect their results.
The first step of our pipeline is to extract all candidate loci from each genome. Then all these loci are clustered and further analyzed to infer endogenization and domestication (sequence alignments, phylogeny, dN/dS, genomic context, mapping…). Thus, adding new genomes requires re-run the whole analysis from the very beginning which represents a huge amount of work and computational resources together with their associated carbon costs. Additionally, this work was part of Benjamin Guinet’s Phd project which was defended on 21 Marsh 2023. In conclusion, we will not be able run again the whole pipeline in all the genomes published since 2019.
- Please summarize in the main manuscript (results or discussion) what the limitations of the pipeline to detect EVEs and dEVEs are - what are important factors to consider, including the availability of closely related "free-living" viruses, and of closely related wasp species for dN/dS analyses.
We added a paragraph in the discussion section to discuss the limitations of our pipeline as follow:
“Because the identification of EVEs necessitates the availability of related viruses in the database, we should see these numbers as an underestimation of the real number of EVEs. In addition, our pipeline necessitates the availability of either related species sharing the same EVEs (or at least the presence of paralogs within a single species) or the availability of RNAseq data to infer domestication. Because these last conditions were only met for 701 out 1261 EVEs, the results we obtained here regarding domestication should be seen as an underestimation of the prevalence of the phenomenon.”
- In this manuscript, a description of the methods that precede the results would make it much easier to appreciate the results shown. It appears that this is allowed in cases where it makes sense, according to the author's instructions.
The first paragraph of the result section was intended to give this overview on the methods used. However, we tried to give more details on the methods in this paragraph. We hope that this will increase readability of the paper.
- The sensitivity and specificity of methods analysis are commendable, as is the availability of substantial supplementary data and scripts on GitHub. However, more effort could be made to align numbers reported in the text and in figures so that readers can verify support for the conclusions described.
To align numbers reported in the text and the figures, we added a new excel sheet within the supplementary file 6 named “Figure_data” in which we report the data used to build the figures 2A, 3A and 3B.
Reviewer #2 (Public Review):
Guinet et al address the question of whether the divergent lifestyles in hymenopteran insects determine the rates of acquisition and domestication of viral genetic elements. As endoparasitoids are intimately associated with their hosts and often develop as broods herein, they predicted that the acquisition rate is higher compared to free-living and ectoparasitoid hymenopterans. Following viral domestication in the new recipient wasp genome, these viral elements have been shown to contribute to endoparasitism by promoting the delivery of secreted compounds in insect hosts (where immature wasps develop). Because of this functional importance, the authors predicted that the rate of domestication is also higher in endoparasitoid wasps. I was impressed with the solid and rigorous approach that was followed to test these two hypotheses. The authors carefully ruled out confounding factors, including contamination of genome assemblies. Previously characterized hymenopteran genomes were included as positive controls to assess the developed pipelines. There was also great merit in using a Bayesian model to study endogenization within the phylogenetic framework. To summarize, this multi-pronged strategy to mine animal genomes for viral genetic elements has the potential of becoming a new benchmark for future studies.
Although the authors do partially achieve their aim of coupling endogenization with an endoparasitoid lifestyle, I am afraid some of the assumptions and generalizations hinder a more solid conclusion. I feel that categorizing hymenopterans either as free-living, endoparasitoids, or ectoparasitoids is an oversimplification. Many of the authors' arguments to associate endogenization with endoparasitoids also apply to free-living eusocial hymenopterans. Both endoparasitoid and eusocial insects can be relatively more exposed to viruses because of intimate conspecific interactions within confined spaces. As endoparasitoids intimately interact with their host, so do eusocial insects with their social guests (melittophiles, myrmecophiles, and termitophiles). Perhaps, you could even argue that some gregarious insects also fit the bill. I would be interested to see whether the conclusions hold when "free-living" is further subdivided and "eusocial" is a separate category.
To answer this question, we reran the study by separating the free-living category into "eusocial" and "free-living" subcategories. All of the new eusocial assignations and their accompanying bibliographies have been added to the Supplemental file 1 under the columns "lifestyle2" and "ref-lifestyle2". All of the new GLM results have been added to the Supplemental file 6. We also made a new violin plot figure names “Figure 4-figure supplement 3” which contains the GLM coefficients distribution of the model run on A only and A to D scaffolds.
We also added a few lines in the M&M to explain this analysis “The same analysis was carried out by splitting the free-living category into two sub-categories, namely eusocial and free-living. A new glm model was then built (GLM(Number EVEs ~ free-living + eusocial + endoparasitoid + ectoparasitoid * Branch_length, family = zero inflated neg binomial). (Lines 754-756).
Overall, the new models that included free-living eusocial hymenopterans revealed the exact same patterns as found in the main analysis. We added a new section entitled “Conclusions hold when eusociality is taken into account” to report the results.
In conclusion, when "free-living" is further subdivided, the mains findings still hold.
Second, I wonder why the authors did not include Wolbachia infection as an explanatory variable to explain the endogenization rate. Wolbachia bacteria infect the insect germline and are often associated with phages. These phages could thus be a major source of viral genetic elements. Having said that, I do not see any Symbioviridae, the phylogenetic clade in which these phages reside (https://doi.org/10.1371/journal.pgen.1010227), in Figure 2B - so perhaps this is a minor point.
In this study we chose to concentrate our attention on eukaryotic viruses, since we reasoned that they have better opportunities to integrate into the insect genomes du to their intimate relationship. This is the reason why we eliminated from our database all phage proteins (as specified in line 511).
Finally, in addition to the dsDNA virus - endoparasitoids relationship, the authors also detect a link between ssRNA viruses and free-living hymenopterans. (Maybe eusociality is biasing these results?)
Thanks to reviewer’s comment, we realize that the sentence referring to this point was misleading. In our initial analysis, ectoparasitoid species showed in fact less domestication events involving ssRNA viruses compared to all other lifestyles (Figure 4-figure supplement 1-L). We clarified this sentence in the main text as follow: “except for a lower rate of domestication of ssRNA viruses in ectoparasitoids compared to other lifestyles (Figure 4-figure supplement 1-L)” (lines 195-196).
The same effect was observed when including eusociality (see Figure 4-figure supplement 3-L).
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eLife assessment
This important manuscript employs a rigorous and multi-pronged comparative genomics approach to unravel how lifestyle modulates the acquisition and domestication of viral genetic elements in the genomes of hymenopteran insects. Using an extensive dataset of over 120 hymenopteran genomes, the authors provide convincing evidence that endoparasitism (where parasite development occurs within hosts) facilitates the uptake and domestication of double-stranded DNA viral elements.
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Reviewer #1 (Public Review):
This manuscript represents a substantial and well-executed body of work that contributes new data on 32 hymenopteran genomes, systematically identifies viral endogenization and domestication events, and tests whether this phenomenon is more common in hymenopteran species with specific lifestyles, eg. endoparasitism. The authors developed a pipeline to identify endogenization that improves upon previously described pipelines and is more comprehensive for the identification of endogenization events from a variety of virus types. Significant findings include the identification of previously undocumented cases of viral endogenization in several hymenopteran species and also moderate statistical support for a higher rate of dsDNA virus endogenization and domestication in endoparasitoids.
1. The authors have …
Reviewer #1 (Public Review):
This manuscript represents a substantial and well-executed body of work that contributes new data on 32 hymenopteran genomes, systematically identifies viral endogenization and domestication events, and tests whether this phenomenon is more common in hymenopteran species with specific lifestyles, eg. endoparasitism. The authors developed a pipeline to identify endogenization that improves upon previously described pipelines and is more comprehensive for the identification of endogenization events from a variety of virus types. Significant findings include the identification of previously undocumented cases of viral endogenization in several hymenopteran species and also moderate statistical support for a higher rate of dsDNA virus endogenization and domestication in endoparasitoids.
1. The authors have tested whether the lifestyle of hymenopteran species (endoparasitism, ectoparasitism, or free-living) is related to the incidence of virus endogenization and domestication. Addressing this kind of question has only become possible with the availability of genome sequences from many taxa so that any results can be statistically supported by appropriate sample sizes. It appears that the authors have not included new genomic data from hymenopteran genomes that have been published since 2019, which are of similar or better quality than the data used in this manuscript. A number of taxa with endogenous viruses (and also without) have become available since then. The best solution would be for the authors to use their pipeline to incorporate the new data, which may have an impact on their findings and could even strengthen their conclusions about virus domestication being more common in endoparasitoids. If this is not possible, the authors should at least justify their decision not to include the most recent data and discuss how it could affect their results.
2. Please summarize in the main manuscript (results or discussion) what the limitations of the pipeline to detect EVEs and dEVEs are - what are important factors to consider, including the availability of closely related "free-living" viruses, and of closely related wasp species for dN/dS analyses.
3. In this manuscript, a description of the methods that precede the results would make it much easier to appreciate the results shown. It appears that this is allowed in cases where it makes sense, according to the author's instructions.
4. The sensitivity and specificity of methods analysis are commendable, as is the availability of substantial supplementary data and scripts on GitHub. However, more effort could be made to align numbers reported in the text and in figures so that readers can verify support for the conclusions described.
-
Reviewer #2 (Public Review):
Guinet et al address the question of whether the divergent lifestyles in hymenopteran insects determine the rates of acquisition and domestication of viral genetic elements. As endoparasitoids are intimately associated with their hosts and often develop as broods herein, they predicted that the acquisition rate is higher compared to free-living and ectoparasitoid hymenopterans. Following viral domestication in the new recipient wasp genome, these viral elements have been shown to contribute to endoparasitism by promoting the delivery of secreted compounds in insect hosts (where immature wasps develop). Because of this functional importance, the authors predicted that the rate of domestication is also higher in endoparasitoid wasps. I was impressed with the solid and rigorous approach that was followed to …
Reviewer #2 (Public Review):
Guinet et al address the question of whether the divergent lifestyles in hymenopteran insects determine the rates of acquisition and domestication of viral genetic elements. As endoparasitoids are intimately associated with their hosts and often develop as broods herein, they predicted that the acquisition rate is higher compared to free-living and ectoparasitoid hymenopterans. Following viral domestication in the new recipient wasp genome, these viral elements have been shown to contribute to endoparasitism by promoting the delivery of secreted compounds in insect hosts (where immature wasps develop). Because of this functional importance, the authors predicted that the rate of domestication is also higher in endoparasitoid wasps. I was impressed with the solid and rigorous approach that was followed to test these two hypotheses. The authors carefully ruled out confounding factors, including contamination of genome assemblies. Previously characterized hymenopteran genomes were included as positive controls to assess the developed pipelines. There was also great merit in using a Bayesian model to study endogenization within the phylogenetic framework. To summarize, this multi-pronged strategy to mine animal genomes for viral genetic elements has the potential of becoming a new benchmark for future studies.
Although the authors do partially achieve their aim of coupling endogenization with an endoparasitoid lifestyle, I am afraid some of the assumptions and generalizations hinder a more solid conclusion. I feel that categorizing hymenopterans either as free-living, endoparasitoids, or ectoparasitoids is an oversimplification. Many of the authors' arguments to associate endogenization with endoparasitoids also apply to free-living eusocial hymenopterans. Both endoparasitoid and eusocial insects can be relatively more exposed to viruses because of intimate conspecific interactions within confined spaces. As endoparasitoids intimately interact with their host, so do eusocial insects with their social guests (melittophiles, myrmecophiles, and termitophiles). Perhaps, you could even argue that some gregarious insects also fit the bill. I would be interested to see whether the conclusions hold when "free-living" is further subdivided and "eusocial" is a separate category. Second, I wonder why the authors did not include Wolbachia infection as an explanatory variable to explain the endogenization rate. Wolbachia bacteria infect the insect germline and are often associated with phages. These phages could thus be a major source of viral genetic elements. Having said that, I do not see any Symbioviridae, the phylogenetic clade in which these phages reside (https://doi.org/10.1371/journal.pgen.1010227), in Figure 2B - so perhaps this is a minor point.
Finally, in addition to the dsDNA virus - endoparasitoids relationship, the authors also detect a link between ssRNA viruses and free-living hymenopterans. (Maybe eusociality is biasing these results?) In any case, I realize the manuscript is already heavy in content but it would be interesting to also dissect these observations in a bit more detail.
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Reviewer #3 (Public Review):
In this manuscript, Guinet and colleagues explore the impact of endoparasitoid lifestyle in Hymenopterans on endogenization and domestication of viruses. Using a well-structured bioinformatic pipeline, they show that an endoparasitoid lifestyle promotes viral endogenization and domestication, particularly for dsDNA viruses. In their discussion, they provide multiple discussion points to hypothesize why this could be the case. It is, to my knowledge, one of the first to link life history traits of insects to particular bias in the genomic endogenization of viruses, which has implications for virology and host-parasite interaction at large.
The manuscript is well-written and structured. The amount of data generated and analyzed is impressive, and the authors have carefully set up their analysis. I have no …
Reviewer #3 (Public Review):
In this manuscript, Guinet and colleagues explore the impact of endoparasitoid lifestyle in Hymenopterans on endogenization and domestication of viruses. Using a well-structured bioinformatic pipeline, they show that an endoparasitoid lifestyle promotes viral endogenization and domestication, particularly for dsDNA viruses. In their discussion, they provide multiple discussion points to hypothesize why this could be the case. It is, to my knowledge, one of the first to link life history traits of insects to particular bias in the genomic endogenization of viruses, which has implications for virology and host-parasite interaction at large.
The manuscript is well-written and structured. The amount of data generated and analyzed is impressive, and the authors have carefully set up their analysis. I have no reasons to doubt any of the analyses the authors have conducted on the output of the screening pipeline set up to discover and characterize endogenous viral elements. I would, however, have appreciated a more thorough investigation on the impact of the scoring system for EVE detection (Scaffold endogenization score), which strongly shapes the dataset used for the analysis, and thus might introduce biases. While I completely understand the need for a scoring system and agree that the parameters used seem reasonable, these are new for the field, and their impact has not been properly explored here. The authors have chosen to focus on a conservative threshold of EVEs scored above D (see Table S2): I wonder what the picture would be if they included all potential EVEs, even poorly scored. How dependent are the results of this unvalidated scoring system? I know several proven EVEs in mosquitoes (confirmed in vivo) that would have been poorly scored and excluded here. By being sure to exclude false positives, the authors may have biased their dataset in ways that influence the results.
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