sign in sign up
<<
Home , Aculeata, Amino acid synthesis, Cephoidea, Cephus, Cephus cinctus, Ceratosolen

This article has been retracted. Read the retraction notice

RESEARCH ARTICLE

Amino acid synthesis loss in and other hymenopterans Xinhai Ye1,2†, Shijiao Xiong1†, Ziwen Teng1, Yi Yang1, Jiale Wang1, Kaili Yu1, Huizi Wu1, Yang Mei1, Zhichao Yan1, Sammy Cheng2, Chuanlin Yin1, Fang Wang1, Hongwei Yao1, Qi Fang1, Qisheng Song3, John H Werren2‡*, Gongyin Ye1‡*, Fei Li1‡*

1State Key Laboratory of Rice Biology & Ministry of Agricultural and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and , Institute of Sciences, Zhejiang University, Hangzhou, China; 2Department of Biology, University of Rochester, Rochester, United States; 3Division of Plant Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, United States

Abstract Insects utilize diverse food resources which can affect the evolution of their genomic repertoire, including leading to losses in different nutrient pathways. Here, we investigate gene loss in synthesis pathways, with special attention to hymenopterans and parasitoid wasps. Using comparative genomics, we find that synthesis capability for , , , and was lost in holometabolous insects prior to hymenopteran divergence, while , , and were lost in the common ancestor of . *For correspondence: Subsequently, multiple loss events of synthesis occurred independently in the Parasitoida [email protected] (JHW); and . Experiments in the parasitoid Cotesia chilonis confirm that it has lost the ability to [email protected] (GY); synthesize eight amino acids. Our findings provide insights into amino acid synthesis evolution, and [email protected] (FL) specifically can be used to inform the design of parasitoid artificial diets for . †These authors contributed equally to this work ‡These authors also contributed equally to this work Introduction Competing interests: The The Hymenoptera contain diverse insects (e.g. , wasps, , and ) which utilize a wide authors declare that no variety of food resources (Quicke, 1997; Peters et al., 2017). Among the Hymenoptera, competing interests exist. account for about 75% of and 10 ~ 20% of all insect species (Pennacchio and Strand, 2006). Funding: See page 20 They are also important biological control agents in integrated pest management (IPM) (Bale et al., Received: 08 June 2020 2008). Female parasitoid wasps attack hosts and lay their eggs upon (ectoparasitoid) or Accepted: 17 October 2020 within (endoparasitoid) them, where the offspring feed and develop, eventually causing death. Published: 19 October 2020 Therefore, parasitoids feed on a food resource rich in , , and other nutrients. In addi- tion, parasitoids can manipulate the nutritional value of the hosts through effectors injected into the Reviewing editor: Antonis Rokas, Vanderbilt University, host, such as proteins, , and molecular factors produced by parasitoid cells United States (teratocytes) that are either injected into the host or produced by feeding larvae (Pennacchio and Strand, 2006; Pennacchio et al., 2014). As well as inhibiting host immunity and alterning host Copyright Ye et al. This article development, these mechanisms alter host in ways that mobilize nutrients from host tis- is distributed under the terms of sues to meet the demands of developing larvae (Mrinalini et al., 2015; Pennacchio et al., 1995; the Creative Commons Attribution License, which Rivers and Denlinger, 1994). permits unrestricted use and We originally began this project to investigate how the -rich diet of parasitoids and their redistribution provided that the ability to manipulate amino acid availability in hosts, has affected their genomic repertoire in amino original author and source are acid synthesis pathways. Our hypothesis was that parasitoids would show extensive loss of in credited. amino acid synthesis pathways due to the availability of amino acids in their diet.

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 1 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Gene loss in nutritional biosynthetic pathways has been described in several insect groups. For instance, some hemipteran insects have lost genes in amino acid biosynthetic pathways, apparently because they can obtain the nutrition from their endosymbionts (Douglas, 2006; Feldhaar, 2011). In some cases, this has been confirmed by annotation of the complete pathway of the endosymbiont and incomplete pathway of the insect, for instance in (Richards et al., 2010), planthoppers (Xue et al., 2014), leafhoppers (McCutcheon and Moran, 2007), and mealybugs (Husnik and McCutcheon, 2016; Gil et al., 2018). Many of these studies involve herbivorous insects that feed on plant sap. However, hymenopteran insects have diverse food resources. Thus, it is reasonable to assume that changes involved in some biosynthetic pathways occurred during the evolution of hyme- nopteran insects. In particular, carnivorous parasitoid wasps feed on a protein and -rich food resource of hosts, which could have resulted in genomic changes. This prompted us to investigate whether some gene losses occur in the nutritional biosynthetic pathways and how parasitoid wasps exploit the nutrition of hosts. The amino acid, , and lipid requirements of several parasitoid wasps have been eval- uated using the traditional nutrient removal method (Thompson, 1986), that is removing particular components from an artificial diet. Parasitoid wasps can manipulate their hosts to produce a nutri- tionally favorable environment for parasitoid development, and there is considerable evidence that they do so through and teratocytes injected by the mother into the host and via modifica- tions induced by feeding larvae (Nakamatsu and Tanaka, 2003; Nakamatsu and Tanaka, 2004; Pennacchio et al., 2014). For example, parasitoid venoms induce a higher concentration of lipids, which is confirmed by in vitro injection of venom into the host (Nakamatsu and Tanaka, 2003; Nakamatsu and Tanaka, 2004). Detailed transcriptomic and metabolomic analyses of venom injected hosts of Nasonia vitripennis reveal dramatic alterations in host (Martinson et al., 2014), sugar, , and , as well as elevation of free amino acid levels (Mrinalini et al., 2015). To meet the demands of the developing larvae, nutritional com- ponents such as proteins, acylglycerols and free amino acids change in the of the para- sitized pea Acyrthosiphon pisum during development of Aphidius ervi larvae (Pennacchio et al., 1995; Rahbe´ et al., 2002). Previous studies showed that at early stages, endo- parasitoid wasp larvae absorb nutrients from host hemolymph through thin exoskeletons and epider- mis, whereas they absorb nutrients mainly through gut epithelium at later stages (Giordana et al., 2003; Caccia et al., 2005; Grimaldi et al., 2006; Pennacchio et al., 2014). Although parasitoids develop on a nutritionally rich food resource and manipulate the nutritional qualities of the host, there have been very few studies on how this relationship impacts their genomic evolution. Although some studies have investigated lipid utilization and in parasitoids (Visser and Ellers, 2008; Visser et al., 2010; Visser et al., 2012; Lammers et al., 2019), there has been very little research on changes in amino acid biosynthetic pathways. To investigate the idea that parasitoids have lost synthesis genes due to their amino-acid-rich food resource, we first conducted genome sequencing of Cotesia chilonis and exam- ined its genomic repertoire for amino acid synthesis pathway genes. To place these results in an evo- lutionary context, we next examined the genomes of 38 hymenopteran species (3 sawflies, 17 aculeates and 18 parasitoids) for which well assembled and annotated genomes are available, and compared these to a set of 13 other holometabolous and hemimetabolous . 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. To investi- gate the effects of parasitoid venom and feeding larvae on host amino acids, changes in these host nutrition components in host hemolymph were analyzed after by this wasp, using UPLC- MS/MS (ultra-performance liquid chromatography tandem mass spectrometry). Finally, the in vitro deletion method was used to determine which essential amino acids developing wasps require from its host. Here, we consider three kinds of losses relevant to amino acid metabolism: gene loss in amino acid pathways, pathway disruption due to gene loss, and loss of synthesis ability for different amino acids. It is noteworthy that pathway disruption for a particular amino acid does not always mean loss of the ability to synthesize that amino acid, because there are alternative pathways for synthesis of some amino acids. Our results indicate a disruption of 16 amino acid pathways at the base of the branch leading to holometabolous insects, which disrupted the synthetic capability for four amino acids (tryptophan, phenylalanine, tyrosine and histidine). Additional disruption of seven pathways

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 2 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Figure 1. Hymenoptera genome evolution and comparative genomic analysis. (A) A female C. chilonis attacking its host C. suppressalis and the cocoons of C. chilonis.(B) Hymenoptera phylogeny and orthology assignment based on genome data. The was based on 2291 single-copy proteins. Red flour T. castaneum was used as the outgroup. Divergence time for each node is represented by gray bars at the node, the range of the bar indicated the 95% confidence interval of the divergence time. Bars are subdivided to represent different types of orthology clusters, as indicated. Universal single-copy genes are single-copy across all species analyzed by us, and absence or duplication in a single genome was allowed; Universal multiple-copy genes represent other universal genes; Specific duplication represents specific duplication genes; Species-specific genes represent species-specific genes with only one copy in the genome; Dispensable clusters represent the remaining genes. (C) Amino acid identity of pairwise species. (D) Gene collinearity analysis between three braconid wasps (using scaffolds that contain more than five genes) with MCScanX. The heavy bars represent all the scaffolds linked together in an artificial order. In C. chilonis and M. demolitor pair, 6335 genes constituted 497 synteny blocks; In C. chilonis and M. cingulum pair, 946 genes constituted 127 synteny blocks. The online version of this article includes the following source data for figure 1: Source data 1. This file includes the phylogeny tree file in Figure 1B. Source data 2. This table includes the orthology assignment based on genome data. Source data 3. This table includes the amino acid identity of pairwise species. Source data 4. This table includes the gene collinearity pairs among C. chilonis, M. cingulum and M. demolitor.

occurred basally in the Hymenoptera, which caused the loss of synthesis capability for three addi- tional amino acids (valine, leucine, and isoleucine). The result indicates that hymenopterans have lost the ability to synthesize seven amino acids. Subsequently during the evolution of Hymenoptera, independent pathway disruptions related to the biosynthesis of two amino acids (lysine and ) were found. The lysine pathway disruptions caused several independent losses of synthesis capability for lysine both in members of the Aculeate and Parasitoida infraorders. The disruptions in the cyste- ine pathway were only found in the Parasitoida infraorder, and they did not disrupt the synthesis capability of cysteine because alternative pathways for cysteine synthesis remained. C. chilonis shows the seven expected amino acid synthesis losses in Hymenoptera, based on the phylogenetic analysis, and an additional loss of lysine synthesis found in C. chilonis and close relatives relative to other hymenopterans. Our nutritional experiments show that C. chilonis, as expected, has lost the ability to synthesize these eight amino acids.

Results Genome evolution of C. chilonis The evolution of hymenopteran insects has attracted increasing research interest. Many phylogenetic analyses have been conducted using transcriptome data (Bank et al., 2017; Peters et al., 2017; Peters et al., 2018). Since genomic sequences contain more information than transcriptomes, we used the available hymenopteran genomes to infer the phylogenetic relationships between C. chilo- nis and 13 other hymenopteran species (8 parasitoids and 5 non-parasitoids). The protein sequences of 2291 single-copy genes were used for phylogenetic inference and the red flour beetle Tribolium

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 3 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics castaneum was used as the outgroup. Phylogenetic analysis by the maximum likelihood phylogenetic method showed that C. chilonis clusters with four braconid wasps, as expected, to form a group in the family , a member of the superfamily (Figure 1B). As previously established (Heraty, 2009; Misof et al., 2014; Peters et al., 2017), the superfamily Ichneumonoi- dea is a sister group to the superfamily Chalcidoidea, and the parasitoid wasps in Braconidae and Chalcidoidea are clustered together, and are members of the infraorder Parasitoida (Peters et al., 2017), which is a sister group to the infraorder Aculeata (containing vespid wasps, ants, and bees). The Parasitoida contains a number of superfamilies in addition to the Ichneumonoids and Chalci- doids, such as Cynipoids, Platygastroids, and Protrotrupoids. Most species are parasitoids, with some reversions to plant feeding, such as in some cynipoids and in pollinating fig wasps (Heraty, 2009; Peters et al., 2017). C. chilonis has a close relationship with another braconid wasp, M. demolitor, with 75% amino acid identity in orthologous proteins (Figure 1C). We chose scaffolds that contain more than five genes for synteny analysis among three braconid wasps, and found that chromosomal rearrange- ment frequently occurred in various wasp species, especially after the divergence of C. chilonis and M. cingulum (Figure 1D). C. chilonis was estimated to have diverged from M. demolitor approxi- mately 61.05 (31.74–108.70) million years ago.

Figure 2. Pathway disruptions and independent gene losses in the amino acid biosynthetic pathways and loss of amino acid biosynthetic capability in Hymenoptera. (A) Phylogenetic tree of 38 hymenopteran insects and pathway disruptions during the Hymenoptera evolution. In total, we have documented 14 independent disruptions of the amino acid synthetic pathways in hymenopterans, 10 in Ichneumonoid/Chalcidoid and four in the Aculeata clade. These independent pathway disruptions were showed as triangles on the branches. (B) Independent gene losses in amino acid biosynthetic pathways (white, present; black, lost). In Hymenoptera, 164 independent gene losses were found, 77 in the Chalcidoid/Ichneumonoid clade and 63 in the Aculeate clade. (C) Amino acid biosynthetic capability of each species was evaluated in terms of combined metabolic pathways for each amino acid (white, present; black, lost). The most recent common ancestor (MRCA) states of Hymenoptera (HYM) and Holometabola (HOL) were reconstructed using 13 additional outgroups to Hymenoptera. The online version of this article includes the following source data for figure 2: Source data 1. This file includes the phylogeny tree file in Figure 2A. Source data 2. Genes in amino acid biosynthetic pathways, ’+’ means present, ’x’ means lost. Source data 3. Amino acid biosynthetic capability of each species, ’+’ means present, ’x’ means lost.

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 4 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Disruptions in amino acid biosynthetic pathways in hymenoptera We next conducted a comparative analysis in hymenopterans for gene loss and pathway disruptions in amino acid biosynthesis (Figure 2, Figure 3), and loss of synthesis capability for different amino acids (Figure 2C). KEGG modules (or sub-pathways) for amino acid biosynthesis were used for our analyses, which were also used in the pathway study of parasitic worms (Coghlan et al., 2018). We define a pathway disruption as the loss of one or more genes required for amino acid synthesis in the pathway. The loss of synthesis capability to an amino acid occurs when all synthetic pathways for this amino acid are disrupted in such a way that there is no complete path to synthesis of the amino acid based on the currently known pathways. It should be noted that the pathway disruption and loss of synthesis capability are different, because there are alternative pathways for synthesis of some amino acids. Two major superfamilies of parasitoid wasps, Ichneumonoidea and Chalcidoidea (in the infraorder Parasitoida, Peters et al., 2017) were examined, along with members of the infraorder Aculeata. Species were selected that have well-assembled genomes. For Parasitoida, there are 2 in the Ichneu- monidae, 8 in the Braconidae, and 7 in the Chalcidoidea. For the Aculeata, there is 1 paper wasp (), 8 ants (Formicidae), and 8 bees (Anthophila). Both of these infraorders have additional superfamilies, but we focused our analysis on this set because they have high-quality genome assem- blies, which are necessary for reliable identification of gene loss. The more basal hymenopteran saw- A. rosae (), lecontei (Tenthredinoidea), cinctus (), and parasitic wood wasp O. abietinus (Orussoidea) were also used for comparisons. In total, 10 ich- neumonoid wasps, 7 chalcidoid wasps, 1 paper wasp, 8 ants, 8 bees, 1 parasitic wood wasp, and 3 sawflies were included in this analysis. In addition, to learn more about the ancestral state of Hyme- noptera, we used 13 species in orders outside of Hymenoptera (2 from Coleoptera, 2 from Lepidop- tera, 3 from Diptera, 2 from , 1 from Thysanoptera, 1 from Collembola, and 1 each from arthropod taxa Cladocera and Trombidiformes) to evaluate the ancestral amino acid synthesis reper- toire (Supplementary file 1 – Table 1).

Figure 3. Amino acid biosynthetic pathways that have been disrupted in the most recent common ancestor (MRCA) of Holometabola, MRCA of Hymenoptera, MRCA of Braconidae and MRCA of Cotesia. We defined a pathway disruption as the loss of one or more genes required for amino acid synthesis in the pathway (KEGG modules). The KEGG modules in black box mean the novel disruptions compared to the outgroups. The amino acid biosynthetic pathways are redrawn from KEGG pathway, map01230. The gene losses caused the pathway disruptions during the Hymenoptera evolution are shown in colorful arrows, corresponding to the pathway disruption events shown in Figure 2A.

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 5 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics After filtering potential contaminated bacterial sequences using a pipeline modified from Wheeler et al., 2013 (see Materials and methods), the BlastKOALA web server and iPathCons tool were used to reconstruct the amino acid biosynthetic pathway in each genome. For each species, we identified losses of genes and pathway disruptions in the pathways and synthesis capability to amino acids (Figure 2, Supplementary file 1 – Table 1). The ancestral amino acid biosynthetic path- ways of Hymenoptera, holometabolous insects were constructed based on the gene losses in the pathways and species phylogenetic positions. Pathway disruption is not the same as a loss of capability to synthesize an amino acid, because alternative pathways are available for some amino acids (Figure 2). We first examined gene losses, pathway disruptions, and amino acid synthesis loss in the species set (Figure 2A, Figure 2B, Supplementary file 1 – Table 1). Seven pathway disruptions and 16 gene losses have occurred in the common ancestor of Hymenoptera (Figure 2B, Figure 3) after their divergence from other holo- metabolous insects. These disrupted pathways are related to synthesis of cysteine (M00021 and M00609), valine/leucine/isoleucine (M00019, M00432 and M00570) and (M00844 and M00029). Within the Hymenoptera, we have documented 14 independent disruptions of the amino acid synthetic pathways, 10 in the Ichneumonoid/Chalcidoid clade and 4 in the Aculeata clade, based on the genes involved and phylogenetic position of the species (Figure 2A). Both aculeates and parasi- toids show pathway disruptions in amino acid biosynthesis. There is no significantly difference between the observed proportion of pathway disruptions in the Ichneumonoid/Chalcidoid clade (71%) and the expected proportion (55%) (p=0.695, Fisher’s exact test, N = 14). We also compared the frequency of gene losses within pathways, and identified a total of 164 independent gene losses, 63 in the Aculeate clade and 77 in the Chalcidoid/Ichneumonoid clade (Figure 2B, Supplementary file 1 – Table 1). The number of independent gene losses is higher than the number of independent pathway disruptions in Hymenoptera evolution. This is mainly due to two reasons: (1) there are many gene losses in the pathways which had already been disrupted in the common ancestor of Hymenoptera or earlier; (2) there can be more than one gene loss in the same pathway during the Hymenoptera evolution. Total gene losses show a similar frequency between the Ichnuemonoid/Chalcidoid and Aculeate based on branch lengths (55% observed vs 55% expected compared to 45% vs 45% expected (p=1, Chi-square test, Supplementary file 2 – Table 1)), indicating that neither clade is enriched for gene losses relative to the other. Three pathways (M00030, M00433 from lysine synthetic pathways and the pathway from 3-phos- phoserine to cysteine) were disrupted during hymenopteran evolution in some lineages, with 14 independent events (Figure 2A, Figure 3). Loss of M00030 is the direct cause of loss of capability to synthesize lysine, which independently happened nine times in Hymenoptera, five in the Ichneumo- noid/Chalcidoid clade and four in the Aculeata clade. All these are due to loss of the same gene in the M00030 pathway, aminotransferase I [EC:2.6.1.57 2.6.1.39 2.6.1.27 2.6.1.5] (ARO8, K00838) (Figure 2B, Figure 3). In the Ichneumonoidea, the ARO8 gene was lost in nine wasps, but retained in the braconid wasp Fopius arisanus. Their phylogeny positions suggest there are four independent loss events (Figure 2A). This gene can be found in the earlier branch of Chalci- doidea, Trichogramma pretiosum, but cannot be detected in any other chalcidoid wasps in this anal- ysis. This result suggests a gene loss event in the common ancestor of six chalcidoid wasps (Figure 2A). In the eight ants in this analysis, this gene was lost in four ants, including Harpegnathos saltator, Solenopsis invicta, Atta cephalotes and Acromyrmex echinatior. This result suggests that this gene was independently lost in the H. saltator and the common ancestor of S. invicta, A. cepha- lotes and A. echinatior (Figure 2A). In bees, two independent losses of this gene were found in the Dufourea novaeangliae and the common ancestor of Eufriesea mexicana, Apis mellifera, Melipona quadrifasciata and two Bombus bees (Figure 2A). The pattern suggests that this gene is prone to independent loss during evolution. Another lysine biosynthetic pathway module (M00433) was inde- pendently lost in the ichneumonid wasp Venturia canescens and braconid wasp Aphidius ervi due to the loss of homocitrate synthase [EC:2.3.3.14] (LYS21, K01655). This gene can be found in many other hymenopteran insects in this analysis (Figure 2A). Disruption of M00433 only happened in par- asitoid wasps, and occurred after lysine synthesis capability was lost due to disruption of the M00030 pathway. We also identified another gene, cysteine synthase [EC:2.5.1.47 2.5.1.65 4.2.1.22] (cysO, K10150) that converts 3-phosphoserine to cysteine, which was independently lost in the V. canescens, the common ancestor of two aphid parasitoids (A. ervi and Lysiphlebus fabarum) and the

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 6 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics common ancestor of four pteromalid wasps (Figure 2). However, loss of cysO doesn’t disrupt the cysteine biosynthesis because cysteine can be converted from L- by cystathionine gamma-lyase [EC:4.4.1.1] (CTH, K01758) in M00338 (Figure 3). During the evolution of Hymenoptera, we found many gene losses in pathways which had already been disrupted in the common ancestor of Holometabola and the common ancestor of Hymenop- tera. Examples include the synthetic pathway for histidine (M00026) and aromatic amino acids (M00024 and M00025). Examination of pathway completeness for seven species from three different orders of Holometabola (Coleoptera, , and Diptera) revealed that 16 pathways were dis- rupted in the common ancestor of Holometabola (causing loss of biosynthesis in four amino acids). Seven pathways were newly disrupted in the Hymenoptera after their divergence from other holo- metabolous insects (Figure 3), leading to a synthesis loss for three amino acids. There is a total of 125 (76%) independent gene losses in Hymenoptera for pathways that had already been disrupted in the common ancestor of Holometabola. This is significantly higher than the random expected pro- portion of gene losses (52%) estimated based on the gene number of each pathway in the ancestral state of Hymenoptera (p<0.01, df = 2, Chi-square test, Supplementary file 2 – Table 2). This pattern is expected, given that once the pathway is disrupted, selection to maintain other genes in the path- way would be diminished, unless the gene had functions in other pathways as well. Indeed, the per- sistence of some genes in these pathways implies that they have other functions that have resulted in their retention in evolution. Only 15 (9%) of independent gene losses occurred in pathways that had been disrupted in the common ancestor of Hymenoptera, which is lower than the expected pro- portion of 30%. Twenty-four (15%) independent gene losses happened in the pathways that were functional in the common ancestor of Hymenoptera, which is similar to the expected proportion of 18%. This result suggested that gene losses are more likely to occur in pathways which have already been disrupted in the common ancestor of Holometabola. We also compared the gene losses between the Ichnuemonoid/Chalcidoid and Aculeate clades, in the pathways that are complete in the common ancestor of Hymenoptera. We documented 17 independent gene loss events in the Ichnuemonoid/Chalcidoid clade and 6 in the Aculeate clade. There is no significant difference between the observed proportion of pathway disruptions in the Ich- neumonoid/Chalcidoids (74%) and the expected proportion (55%) based on branch lengths (see Materials and methods) (p=0.207, Chi-square test, Supplementary file 2 – Table 3). As more well- assembled genomes come available for these taxa, the question can be revisited.

Changes in amino acid synthesis capability in the hymenoptera A disruption of an individual pathway does not necessarily lead to loss of amino acid synthesis capa- bility, due to redundancy in pathways for some amino acids. Based on the pathway completeness (i.e. coverage of reference pathway in KEGG, map01230), we next evaluated how the capability to synthesize amino acids has changed during evolution. Hymenoptera is the basal order for the holo- metabolous insects (Savard et al., 2006; Misof et al., 2014). The capabilities to synthesize histidine, tryptophan, tyrosine, and phenylalanine appear to have been lost early in holometabolous insects, prior to divergence of the basal Hymenoptera (Figure 2C). The capabilities to synthesize valine, leu- cine, and isoleucine were subsequently lost in the common ancestor of Hymenoptera after their divergence from other holometabolous insects (Figure 2C). This was due to the losses of two key genes in the valine/leucine/isoleucine pathway, ketol-acid reductoisomerase [EC:1.1.1.86] (ilvC, K00053) and I/III small subunit [EC:2.2.1.6] (ilvH, K01653) (Figure 2B). As a result, our data show that synthesis capability of four amino acids (tryptophan, phenylalanine, tyro- sine, and histidine) was lost earlier in holometabolous insects, and synthesis capability of three amino acids (valine, isoleucine and leucine) was lost in the common ancestor of Hymenoptera. Otherwise, the capability to synthesize amino acids in Hymenoptera is largely conserved, despite the disruptions of three pathways with 14 independent events. This is due to the redundancy in synthesis for certain amino acids. We only detected losses of the capability to synthesize lysine within different lineages of hymenopterans, all of which are due to independent gene loss in the same gene, ARO8 (K00838) in the M00030 pathway. After the loss of capability to synthesize lysine caused by disruption of M00030, a gene in the M00433, LYS21 (K01655), was lost independently in ichneumonid wasp V. canescens and braconid wasp A. ervi (Figure 2A). In this analysis, 25 hymenopteran species lost the capability to synthesize lysine, 15 in the Ichneumonoid/Chalcidoid clade and 10 in the Aculeata clade (Figure 2C). Their phylogeny positions suggest these are due to nine independent loss events, five

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 7 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics in the Ichneumonoid/Chalcidoid clade and four in the Aculeata clade. We did not find synthesis loss in Orussus or the sawflies. Disruptions of the pathway from 3-phosphoserine to cysteine in some par- asitoid lineages caused by single gene loss does not disrupt the cysteine biosynthesis because it can be converted from L-cystathionine by CTH (K01758) in M00338 (Figures 2 and 3). These are in addi- tion to the disruption of 7 amino acid pathways (which resulted in synthesis capability loss of valine, leucine and isoleucine) in the common ancestor of the Hymenoptera, and 16 pathway disruptions in the common ancestor of the Holometabola (which resulted in synthesis capability loss of histidine, tryptophan, tyrosine, and phenylalanine). Therefore, different hymenopteran species in our data set show a total of 23–26 pathway disruptions, depending on whether loss of the lysine (M00030, M00433) or/and cysteine (from 3-phosphoserine to cysteine) pathways occurred within its lineage. The number of synthesis capability losses ranged from seven to eight, depending on whether lysine synthesis capability, due to disruption in the lysine pathway M00030, occurred in a particular lineage. In C. chilonis, our analysis showed 24 pathway disruptions, 23 of them occurred basally in Hyme- noptera or earlier, and the remaining one (M00030) happened in the common ancestor of Cotesia, Microplitis and Macrocentrus. These pathway losses disrupted the biosynthesis of eight amino acids (lysine, tryptophan, phenylalanine, tyrosine, isoleucine, leucine, valine, and histidine) (Figure 4A, Supplementary file 2 – Table 4, Table 5). We next examined this result by amino acid depletion feeding assays and confirmed for all eight in this study (see below).

Loss of the ability to synthesize eight amino acids in C. chilonis Although many efforts have been devoted to elucidate the immune manipulation of hosts by parasit- oid wasps, relatively less is known about the nutrition interactions between the host and parasitoid (Pennacchio et al., 2014). A high-quality genome of the host C. suppressalis has previously been reported (Yin et al., 2014; Ma et al., 2020), so C. chilonis-C. suppressalis is an excellent model sys- tem to investigate the genetic basis of nutrition utilization of the host by parasitoid wasps, and its implications to gene gains and losses in the parasitoid. Our analysis above has shown that the C. chi- lonis has experienced pathway and gene losses in its evolutionary history that have disrupted the biosynthesis of eight amino acids (lysine, tryptophan, phenylalanine, tyrosine, isoleucine, leucine, valine, and histidine) (Figure 4A, Supplementary file 2 – Table 4, Table 5). All these are essential amino acids for parasitoids except tyrosine, which can be synthesized from phenylalanine (Thomp- son, 1981). The genes related to the biosynthesis of three other essential amino acids (arginine, and ) were not lost. For methionine and threonine, although the biosynthetic pathways from aspartate were broken by some gene loss, these amino acids can be synthesized from another non-essential amino acid, (Figure 4—figure supplement 1). The expression of genes in the pathway from serine to methionine and threonine also were examined using RNA-seq data, and it was found that all genes could be expressed in at least one stage during wasp develop- ment. Interestingly, the 5-methyltetrahydrofolate- methyltransferase (metH; EC: 2.1.1.13) gene in the last step to synthesize methionine was only expressed in later larvae and adults, which means C. chilonis cannot synthesize methionine by itself in early larval and pupal stages (Fig- ure 4—figure supplement 1). For arginine, the biosynthetic pathway from glutamate to arginine (KEGG module: M00845) was broken, but the pathway from to arginine (KEGG module: M00844) was complete with gene expression at different life stages (Figure 4—figure supplement 1). These results indicate that, from a genomic point of view, C. chilonis can synthesize arginine, threonine, and methionine (but for methionine, only at later larval and adult stages).

In vitro verification of requirements for different amino acids To study how the loss of the capability to synthesize certain amino acids influences larval develop- ment, we in vitro reared 5-day-old C. chilonis larvae in the chemically defined medium (Figure 4B; see Materials and methods). The Grace’s Insect Medium (see Materials and methods for detail com- ponents and concentrations), which contains 20 amino acids was used as a positive control, and the baseline medium which deleted eight amino acids (lysine, tryptophan, phenylalanine, tyrosine, leu- cine, isoleucine, valine, and histidine) that C. chilonis cannot synthesize based on the genomic analy- sis, was used as a negative control. In addition, eight different media with exclusion of each individual amino acid were also tested. The results indicated that the absence of any of the eight

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 8 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Figure 4. C. chilonis loses the ability to synthesize eight amino acids. (A) Gene loss in amino acid biosynthetic pathways in C. chilonis. BlastKOALA web server and iPathCons tool were used for re-constructing the amino acid biosynthetic pathway in C. chilonis with annotated C. chilonis protein coding genes. For those genes missing in the first step, we searched them in whole genome assembly and PacBio long reads by TBLASTN (see Materials and methods). In total, 46 genes were found to be missing in the amino acid biosynthesis pathway for C. chilonis, thus disrupting the biosynthesis of eight amino acids (Lys, Trp, Phe, Tyr, Ile, Leu, Val, His). Full line indicates the reaction exists in C. chilonis, while dotted line indicates the interaction cannot be found. Amino acid names in red can be synthesized by C. chilonis, names in gray with strikethrough cannot be synthesized due to the lost genes on each dotted line. (B) In vitro rearing of C. chilonis. Five days after parasitism, larvae were put on the membrane of a transwell chamber; then the transwell was placed in the well containing 250 ml of Cotesia rearing medium so that the wasp larvae could reach the nutrients (see Materials and methods). (C) Survival rates of C. chilonis larvae developed on 11 different rearing media, respectively. Positive control: Grace’s Insect Medium, containing 20 amino acids (see Materials and methods, n = 30); Negative control uses positive control medium minus the eight amino acids that C. chilonis cannot synthesize, Lys, Trp, Phe, Tyr, Ile, Leu, Val, His, n = 30; Single amino acid deficiencies use control media minus only one amino acid, indicated by ’-’ superscript, for example Gly deficiency (Gly-) indicates excluding only. Gehan-Breslow-Wilcoxon test was used for survival rate statistical analyses. The Benjamini-Hochberg method was used for multiple testing correction. The statistical results of pairwise group comparisons are indicated. The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. Genes in amino acid biosynthetic pathways of C. chilonis. Source data 2. This table includes the survival rates of C. chilonis larvae developed on 11 different rearing media. Figure supplement 1. Activation of amino acid biosynthetic pathways.

amino acids mentioned above led to developmental arrest of parasitoid larvae, demonstrating that C. chilonis larvae cannot survive in the medium without each of these eight amino acids. However, they could survive in the medium without glycine, presumably because they can synthesize it (based on the pathway analysis, Figure 4C).

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 9 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Influences of parasitism on host amino acid synthesis, gene expression, and free amino acid content in host hemolymph Our comparative genomics analyses and feeding assays have showed that C. chilonis has lost the ability to synthesize eight amino acids that are essential for parasitoid survival. The next step was to test whether these eight amino acids were available in host hemolymph, and if concentrations were altered in parasitized host hemolymph. To this end, we conducted a metabolomic analysis of the fourth-instar larval hemolymph of C. suppressalis. In the non-parasitized C. suppressalis larvae, all eight amino acids were represented in the hemolymph. Histidine was the most abundant at a con- centration of 4.455 mg/ml, followed by at a concentration of 3.262 mg/ml (Figure 5A). In the parasitized larvae, the amounts of four amino acids (arginine, serine, tyrosine, and ) were significantly increased and four amino acids (5-hydroxylysine, , methionine, and lysine) were significantly decreased in the first 3 days after parasitism (p<0.05, Student’s t-test). Among the eight amino acids that C. chilonis cannot synthesize by itself, lysine was found to be significantly decreased in the host hemolymph in the first 3 days after parasitism, likely resulting from the absorp- tion of lysine by the wasp larvae. However, tyrosine was significantly increased in the first 3 days after parasitism, while levels of six other amino acids (valine, tryptophan, histidine, phenylalanine, isoleucine, and leucine) were not significantly changed in the hemolymph. These findings suggest that parasitism by C. chilonis manipulates the C. suppressalis larvae to store or release amino acids into the hemolymph, possibly for the first instar larvae of C. chilonis to absorb the free amino acids, such as lysine, from the host hemolymph (Figure 5A). Next, we used the previously reported transcriptome of host fat body and hemolymph in unpara- sitized and post-parasitized hosts to examine differential gene expression (Wu et al., 2013). It was found that 16 protease genes were significantly upregulated (4–59-fold) in fat body and hemolymph of the host following parasitism (Supplementary file 2 – Table 6). Most were the genes encoding serine proteases, trypsins and carboxypeptidases, which play important roles in . These data suggest that parasitoid wasps can stimulate the expression of key proteases in the host to help hydrolyze proteins to free amino acids. In addition, parasitism does not have significant influence on host’s amino acid biosynthetic pathways, as the expression of most genes (82%) in the pathways were not changed significantly after parasitism (Figure 5B, Figure 5—figure supplement 1). Only six genes were found to be significantly downregulated in the fat body. Interestingly, we found that five genes in the amino acid biosynthetic pathway were significantly upregulated after parasitism. All of them are also belong to (M00002) and citrate cycle (M00010) (Figure 3B). This finding indicates that parasitism may activate the host’s . 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.

Amino acid transport in the parasitoid wasp C. chilonis We also explored the repertoire of amino acid transporters, which recognize and transport free amino acids across the plasma membranes of cells (Wolfersberger, 2000). The free amino acids in host hemolymph are absorbed by early instar larvae through both the gut epithelium and thin exoskeleton, and only through the gut epithelium in late-instar larvae (Giordana et al., 2003; Caccia et al., 2005; Grimaldi et al., 2006; Pennacchio et al., 2014). However, it is uncertain whether the amino acid absorption is correlated with enhanced transport ability. To investigate this possibility, we examined transporter gene families associated with amino acid transport, including ATP-binding cassette transporters (ABC transporters) and amino acid transporters (AATs). ABC transporters comprise an extensive and variable transporter superfamily and play a role in transfer- ring a variety of compounds across cellular membranes, including amino acids, sugars, lipids, and other xenobiotics (Dermauw and Van Leeuwen, 2014). AATs are more specifically for transferring amino acids (Wolfersberger, 2000). In total, 103 ABC transporter genes were identified in the C. chilonis genome. Compared with other hymenopteran insects, the ABC transporters gene family showed expansion in C. chilonis, as revealed by maximum likelihood phylogenetic analysis (Figure 6A, Supplementary file 2 – Table 7). In contrast, M. demolitor, the closest relative of C. chi- lonis in our study, only has 51 ABC transporters in the genome. This result indicates that the

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 10 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Figure 5. Parasitism by C. chilonis influences the host amino acid synthetic pathway and free amino acid levels in host hemolymph. (A) Free amino acids levels in host hemolymph were changed after parasitism. UPLC-MS/MS analysis was used. Host hemolymph was collected 3 days after parasitism. The detection for each treatment were repeated 10 times. Student’s t-test was used for statistical analysis of amino acid changes. (B) Gene expression of amino acid biosynthetic pathways was changed 2 days after parasitism. Black line indicates the reaction exists in host, while grey line indicates the interaction cannot be found. Up- and down-regulated genes were considered if there was a fold change 2 and p-adjusted <0.05 in host fat body and hemocyte transcriptome data. Only the genes with significantly expression changes are showed. The asterisk indicates that the concentration of amino acids was significantly changed three days after parasitism. *Significant difference at p<0.05, **at p<0.01. The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. This table includes the free amino acids levels in host hemolymph. Source data 2. This table includes the host’s differentially expressed genes of amino acid biosynthetic pathways after parasitism. Figure supplement 1. Influence of parasitism on gene expression of the amino acid biosynthetic pathways in fat body and hemocytes of the host C. suppressalis.

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 11 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Figure 6. Amino acid transport genes in parasitoid wasp C. chilonis.(A) ABC transporter genes of C. chilonis.A total of 103 ABC transporter genes were identified in the C. chilonis genome. The ABC transporter gene family was significantly expanded in C. chilonis as revealed by phylogenetic comparison with the honeybee A. mellifera and the A. rosae. Heatmap showed the expression patterns of these 103 ABC transporter genes at different developmental stages. (B) Amino acid/polyamine/organocation (APC) family of C. chilonis. A total of 10 APC genes were found in C. chilonis genome. Phylogenetic analysis showed APCs were not expanded in C. chilonis compared with honeybee and sawfly. Transcriptome analysis showed 7 APCs were highly expressed at both larval and pupal stages, suggesting the amino acid transport ability was active at these two development stages. Yellow represents higher expression values while dark blue represents lower expression. The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. This file includes the phylogeny tree file of ABC transporter genes in Figure 6A. Source data 2. This file includes the phylogeny tree file of APC genes in Figure 6B. Source data 3. This file includes the gene expression levels (FPKM) of ABC transporter genes in C. chilonis. Source data 4. This file includes the gene expression levels (FPKM) of APC genes in C. chilonis. Figure supplement 1. Phylogenetic relationships of the AAAP family and gene expression in C. chilonis at different development stages. Figure supplement 2. Number of predicted ABC transporter genes, APC family and AAAP family across hymenopteran lineages.

expansion of ABC transporters occurred after divergence of C. chilonis and M. demolitor (Supplementary file 2 – Table 7). We found 36 ABC transporters were more highly expressed in the larval stage relative to the pupal stage (FoldChange (FPKM) >2). The amino acid/polyamine/organo- cation (APC) family and the amino acid/auxin permease (AAAP) gene family are closely associated with nutrition transport in insects (Colombani et al., 2003; Price et al., 2014). Ten putative APCs and eight putative AAAPs were identified in the C. chilonis genome (Supplementary file 2 – Table 8). Although phylogenetic analysis showed that the APC gene family was not significantly expanded, transcriptome analysis results indicate that two APC genes were highly expressed in the larval stage

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 12 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics relative to the pupal and adult stages. Five APC genes were highly expressed in the larval and pupal stages relative to the adult stage (Figure 6B). However, only one AAAP gene was highly expressed in larvae (Figure 6—figure supplement 1). We next searched for these transporter genes in a large collection of hymenopteran genomes, but the results did not support the view that these transporter genes are expanded basally in the parasitoid wasps (Figure 6—figure supplement 2). This finding suggests that the expansion of ABC transporters in C. chilonis is an independent event, as it was not found in close relatives. The expansion events also independently occurred in the sawfly , some ants, Megachile bees and Bombus bees. Interestingly, some host amino acid trans- porters, such as vacuolar amino acid transporters, vesicular glutamate transporters, and proton-cou- pled amino acid transporter-like proteins, were significantly upregulated after parasitism (Supplementary file 2 – Table 9). These data show that the amino acid transport ability in C. chilonis may be significantly enhanced by gene family expansion and increased expression. Upregulation 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.

Discussion Our original motivation for this study was to investigate amino acid pathway gene loss in the parasit- oid Hymenoptera, with the expectation that they would show greater loss rates than other hymenop- terans, due to their amino-acid-rich diet. This expectation was not met. Rather we detected major pathway losses basally in the Hymenoptera (and Holometabolous insects), and additional amino acid losses scattered independently in both the aculeates and parasitoids, without strong evidence of an accelerated rate in Parasitoida. More extensive whole genome sequencing will help reveal what eco- logical and dietary features may be associated with these losses, as well as potential roles for associ- ated microbiomes as sources for amino acids. Our comparative pathway analysis shows that hymenopterans’ loss of biosynthetic capability for several amino acids was caused by degradation of one or several key genes in the relevant path- ways, and this trait loss occurs in both parasitoid wasps and aculeate species (Figure 2, Supplementary file 1 – Table 1). However, the majority of amino acid synthesis losses occurred basally in the holometabolous insects and early in hymenopteran evolution. Trait loss is widely reported across diverse taxonomic groups, and can occur when their ecologically associated species (e.g. prey, plants, or microbial symbionts) provide the necessary resources or functions (Ellers et al., 2012). Gene losses have been identified in amino acid biosynthesis pathways for other insects, such as ants, aphids, planthoppers, and mealybugs, indicating that gene loss in amino acid biosynthesis pathways is relatively common for insects. However, these gene losses may be due to very different factors in different . For example, almost all phloem-sapping insects cannot synthesize some essential amino acids, but rely on endosymbionts for nutrition compensation (Douglas, 2006; McCutcheon and Moran, 2007; Douglas, 2009; Richards et al., 2010; Feldhaar, 2011; Xue et al., 2014; Husnik and McCutcheon, 2016; Gil et al., 2018). In contrast to phloem-sapping insects, which feed on a nutrient-poor diet, parasitoid wasps receive a diet rich in proteins and lipids from their host. Previous studies showed the lack of in most parasitoid wasps (Visser and Ellers, 2008; Visser et al., 2010). However, no extensive losses of genes involved in lipogenesis were noted (Visser et al., 2012; Lammers et al., 2019). In this study, we found that all hymenopter- ans appear to lack the capability to synthesize seven amino acids (four were lost earlier in holome- tabolous insects and three were lost in the common ancestor of Hymenoptera), and some parasitoids and aculeates have additionally lost the capability to synthesize lysine. The trait loss of lysine biosynthesis in parasitoid wasps may implicate their amino-acid-rich diet combined with the capability of parasitoids to manipulate the nutritional quality of hosts through venoms and other means. The trait loss in aculeates also may reflect their specialized diets. For example, bees feed on amino acid rich pollen and ants have a wide range of food sources (Rabie et al., 1983; Nicolson and Human, 2013). Another possibility is that symbiotic can provide essential nutrients to host insects, as reported in Cephalotes ants by Hu et al., 2018 and as well known in other insects such as aphids (Wilson et al., 2010; Feng et al., 2019).

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 13 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Previous studies indicate that intracellular bacteria Buchnera (symbiotic bacteria of aphids) signifi- cantly contributes to parasitoid host nutritional suitability (Pennacchio et al., 1999; Rahbe ´ et al., 2002). In our study, the synthesis capability of lysine was found to be lost in the common ancestor of two aphid parasitoids A. ervi and L. fabarum. This phenomenon may be associated with their parasit- oid relationship with aphids, which possess intracellular bacteria that provide amino acids to the host (Wilson et al., 2010; Feng et al., 2019). Further study is needed to extend taxon sampling and determine how amino acid synthetic pathways have evolved in aphid parasitoids, as well as how they have evolved in other parasitoid-host-symbiont systems (e.g. parasitoids of mealybugs). Also, it would be interesting to study how they have evolved in plant-feeding members of the Parasitoida (e.g. fig wasps) (Xiao et al., 2013; Peters et al., 2017). We also point out that our conclusions are based on the canonical amino acid synthesis pathways that have been characterized (Kanehisa et al., 2017). We cannot rule out that unidentified pathways exist in insects that can ‘res- cue’ individual amino acid biosynthesis, or that some genes have evolved so quickly that they are no longer recognized as canonical amino acid synthesis genes. Parasites are ubiquitous in nature, and nutrition exploitation is one of the most important aspects of parasitism (Pennacchio et al., 2014). Understanding the genetic basis of nutrition exploitation by parasitoid insects can be of importance for understanding the parasitism strategy and also for cus- tomizing an artificial diet to rear parasitoids for use in biological control. Previous studies have reported that parasitism can alter the host’s metabolic system and release nutrients into hemolymph to increase nutritional suitability for parasitoid through venoms, teratocytes and parasitoid larval feeding (Digilio et al., 2000; Nakamatsu and Tanaka, 2003; Nakamatsu and Tanaka, 2004; Caccia et al., 2005; Falabella et al., 2005; Falabella et al., 2007; Falabella et al., 2009; Caccia et al., 2012; Mrinalini et al., 2015). Many previous studies have demonstrated in vitro that some essential amino acids, which are of considerable importance in the nutritional and metabolic adaptations of parasitoid wasps, are supplied by the host (Giordana et al., 2003; Caccia et al., 2005). Here, we present genome-level evidence that C. chilonis has lost the capability for de novo biosynthesis of eight amino acids. Among these eight amino acids, the capability to synthesize four amino acids (histidine, tryptophan, tyrosine, and phenylalanine) was lost early in holometabolous insects, prior to divergence of the basal Hymenoptera. The synthetic capability of additional three amino acids (valine, leucine and isoleucine) was subsequently lost in the common ancestor of Hyme- noptera. The capability to synthesize lysine was lost in the common ancestor of Cotesia, Microplitis and Macrocentrus (Figure 2). This was also confirmed in vitro by rearing C. chilonis larvae in the medium deleting one or more specific amino acids. In addition, we noted significant increases in the number of amino acid transporter genes in C. chilonis (Figure 6), although the biological significance of this finding remains unclear. Our results are consistent with previous studies on the requirements of essential amino acids for parasitoid wasps and also explained why parasitoid wasps cannot survive on the chemically defined media without one or more kinds of these critical nutrients (Thomp- son, 1976; Thompson, 1981; Thompson, 1986; Bale et al., 2008). The metabolomic analysis showed that parasitism by C. chilonis significantly changed the levels of various amino acids in host hemolymph (Figure 5A). For the eight amino acids that C. chilonis cannot synthesize, the concentra- tions of lysine and tyrosine were found to change significantly in host hemolymph after parasitism (tyrosine went up, lysine went down). This result suggests that early larvae parasitoids may largely absorb lysine in host hemolymph, since lysine is essential for parasitoid development. In addition, venom and/or other effectors produced by parasitoids (i.e. PDV, teratocytes), and/or by larvae feed- ing significantly increase the concentration of tyrosine in host hemolymph, but parasitoid larvae uti- lize only a small amount of tyrosine at early stages (3 days after parasitism). These interpretations are speculative, and would require further detailed analysis to determine the contributions of modifi- cations induced by wasp venom, teratocytes, and feeding larvae on amino acid levels. Based on results from previous studies of the same parasitoid-host system (Hang and ZQ, 1991), it is likely that parasitism first increases amino acid levels in host hemolymph, then newly hatched wasp larvae begin to absorb and consume these amino acids and continue to do so at specific times during their development. To build a bigger picture of how parasitoid influence the amino acid lev- els in host hemolymph, more intensive sampling is required. The roles of venoms, PDV, teratocytes, and larval feeding in nutrition exploitation of C. chilonis in amino acid production need to be explored in the future.

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 14 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Many parasitoid wasps are important natural enemies of agriculture and forestry pests and have been used as biological control agents for a long time (Quicke, 1997; Beckage and Gelman, 2004), such as Trichogrammatid wasps (Knutson, 1998; Lindsey et al., 2018). The use of artificial diets for mass rearing of parasitoids is an important aspect of increasing their utility and cost effectiveness for augmentative biological control. With the results in this study, we provide information on which amino acids need to be supplemented to artificial diets according to the pathway completeness of each parasitoid wasp. The approach suggests one utility of parasitoid genome sequences in advanc- ing cost-effective biological control methods. In summary, comparative genomic analysis of two superfamilies of parasitoid wasps, non-parasit- oid hymenopterans (sawflies, paper wasps, ants, and bees), seven additional holometabolous insects in three orders, and six more basal arthropods provides genome-wide evidence that the synthesis capability for tryptophan, phenylalanine, tyrosine, and histidine was lost in holometabolus insects prior to hymenopteran divergence, and the synthesis capability of valine, leucine, and isoleucine pre- dicted by pathway analysis was lost in the common ancestor of Hymenoptera. Loss of synthesis capa- bility of lysine subsequently occurs during the Hymenoptera evolution by independent pathway disruptions. The loss of synthesis capability of amino acids was demonstrated by amino acid deple- tion feeding assays in C. chilonis. Metabolomic analysis provides an explanation that the required nutritional resource of parasitoid wasps is increased in parasitized host insects through host manipu- lation by venoms, teratocyte and/or parasitoid larval feeding. 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 investi- gated. Our finding also provides key information for designing artificial diets for mass-production of parasitoids as cost-effective biological control agents.

Materials and methods Insect rearing The parasitoid wasp C. chilonis and its host C. suppressalis were initially collected from fields in the experimental farmland of Zhejiang University, Hangzhou, China in 2012, and reared under laboratory conditions as previously described (Teng et al., 2016; Teng et al., 2017). Both laboratory colonies were maintained in an environmental chamber with constant conditions of 28 ± 1˚C, about 70% RH and 16 L: 8 D photoperiod.

Genome sequencing and assembly Adopting a whole-genome shotgun sequence strategy and next-generation sequencing technolo- gies, we used Illumina HiSeq 2000 and Pacbio platforms to sequence the genome of C. chilonis, sup- ported by Novogene Bioinformatics Institute (Beijing, China). DNA was extracted from 300 haploid third-instar male wasp larvae. We prepared sequencing libraries with insert sizes of 250 bp, 2 Kb, 5 Kb, and 10 Kb for paired-end reads. Finally, we generated about 64.44 Gb of Illumina reads and 7.63 Gb of Pacbio reads (~380 X coverage, Supplementary file 2 – Table 10). Based on Overlap-Lay- out-Consensus, we de novo assembled the genome with Pacbio data. Then, we used PILON soft- ware (Walker et al., 2014) for error correction with Illumina data. Finally, the consensus sequences were assembled to the genome using SSPACE software (version 3) (Boetzer et al., 2011). The final assembly yielded 189 Mb of the reference genomic sequence with a scaffold N50 of 2.2 Mb.

Transcriptome sequencing In total, we sequenced six transcriptomes for assisting genome annotation and further analysis. Total RNA was isolated from early larvae (3 days after parasitism), later larvae (9 days after parasitism), male pupae, female pupae, male adults, and female adults of C. chilonis using the TRIzol protocol (Life Technologies, USA). RNA sequencing libraries were constructed using the Illumina mRNA-Seq Prep Kit (Illumina, USA). Briefly, oligo (dT) magnetic beads were used to purify poly(A)-containing mRNA molecules. The mRNA was further fragmented and randomly primed during first-strand syn- thesis by reverse . This procedure was followed by second-strand synthesis with DNA polymerase I to create double-stranded cDNA fragments. The double-stranded cDNA was subjected to end repair by Klenow and T4 DNA polymerases, and A-tailed by Klenow lacking exonuclease

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 15 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics activity. We then ligated the cDNA to Illumina Paired-end Sequencing adapters, performed size selection by gel electrophoresis, and then PCR amplification to complete library preparation. The libraries were sequenced using Illumina HiSeq 2000 (101 bp at each end). The RNA-seq reads were either de novo assembled using Trinity (Haas et al., 2013) or mapped to the C. chilonis genome using HISAT2 (Kim et al., 2015) with default parameters.

Genome assembly assessment We ran the core eukaryotic genes mapping approach (CEGMA) (version 2.4) to estimate the gene space (Parra et al., 2007), showing that 237 (95.6%) out of 248 CEGMA genes were represented in the genome assembly. Evaluation using Benchmarking Universal Single-Copy Orthologs (BUSCO) (version 3)confirmed the high quality of genome assembly (Sima ˜o et al., 2015), showing that 96.6% of 1658 Single-Copy BUSCOs (insecta_odb9) were complete in length (Supplementary file 2 – Table 11). Default parameters of CEGMA and BUSCO software were used.

Genome annotation The C. chilonis genome was annotated using the OMIGA genome annotation pipeline (OMIGA) (Liu et al., 2014), which is an optimized Maker-based insect genome annotation workflow. First, we identified repeat sequences. The repeat library was constructed using RepeatModeler software (ver- sion 1.0.7). Transposable elements (TEs) were predicted in the assemblies by homology searching against RepBase using RepeatMasker software (version 4.0.5) (Tempel, 2012) with default parame- ters. In total, we predicted 353,649 repeat sequences in a total of 68 Mb, which constitutes 36.18% of the C. chilonis genome (Supplementary file 2 – Tables 11–12). Second, we mapped RNA-seq raw data to the genome. Six transcriptomes from different development stages were applied as the evi- dence of gene expression. Trimmomatic software (version 0.36) (Bolger et al., 2014) was used for quality filtering of all the RNA-seq raw data, then Bowtie software (version 2.2.5) (Langmead et al., 2009) was used to map RNA-seq data to the genome. Next, we used HISAT2 (Kim et al., 2015) and StringTie (version 1.3.4) (Pertea et al., 2015) to obtain putative transcripts (Pertea et al., 2016). Default parameters were used for all the above programs. To ensure high accuracy of gene prediction, we re-trained the de novo gene prediction software before genome annotation. We selected transcripts from the StringTie genes for training. TransDe- coder (version 2.0.1) was used to identify candidate-coding regions in transcript sequences. To improve sensitivity, we also applied BLAST against UniProtKB/Swiss-Prot proteins (E < 10À5) and searched Pfam to identify protein domain information (E < 10À5). Only genes with complete ORFs were regarded as candidates. If a gene had multiple transcripts, only the longest was chosen. After TransDecoder software filtering, the gene candidates were used to re-train the Augustus (version 3.1) (Stanke et al., 2004) and SNAP (version 2006-07-28) (Korf, 2004) prediction software. For GeneMark-ET (Suite 4.21) (Lomsadze et al., 2014), more than 10 Mb of the genome sequence was used to re-train the software. The default parameters were used for training. Three kinds of evidence were applied to annotate the protein-coding genes in the C. chilonis genome: homology-based predictions, de novo predictions and transcriptome-based predictions. MAKER pipeline (version 2.31) (Campbell et al., 2014) was used to annotate protein-coding genes of the C. chilonis genome. In the MAKER pipeline, sequences of homologous proteins from the NCBI RefSeq were used. Three gene prediction programs including Augustus, SNAP and GeneMark-ET, which had all been re-trained, were used to predict coding genes. Additionally, the RNA-Seq data were mapped to the genome using HISAT2, and StringTie was used to assemble transcripts to the gene models. All gene sequences predicted from the above three approaches were combined by MAKER into a weighted and non-redundant consensus of gene structures. All the MAKER parameters were default settings. In total, OMIGA identified 14,142 protein-coding genes in the C. chilonis genome (Supplementary file 2 – Table 11).

Ortholog analysis and comparative genomics We used OrthoMCL (Li, 2003) to found orthologous groups from protein sequences of fourteen Hymenoptera insects (A. rosae, O. abietinus, M. cingulum, M. demolitor, C. chilonis, D. alloeum, F. arisanus, C. floridanum, T. pertiosum, C. solmsi, N. vitripennis, S. invicta, A. cephalotes and A. melli- fera), of which the genome sequence data quality met the requirements for ortholog analysis, and

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 16 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics one Coleoptera species (T. castaneum)(Supplementary file 2 – Table 13). We used the default parameters settings and identified 2291 single-copy protein-coding genes from 17,248 OrthoMCL clusters using a custom perl script. The distribution of pairwise amino acid identity was measured for each ortholog protein by the needle module in the EMBOSS packages (version 6.6.0) (Olson, 2002). In total, there were 6431 orthologs between C. chilonis and A. mellifera, 6596 orthologs between C. chilonis and D. alloeum, 6640 orthologs between C. chilonis and F. arisanus, 5902 orthologs between C. chilonis and M. cingulum, 7017 orthologs between C. chilonis and M. demolitor, 5908 orthologs between C. chilonis and N. vitripennis, 7114 orthologs between C. floridanum and T. pre- tiosum, 7104 orthologs between N. vitripennis and C. solmsi, 7554 orthologs between A. cephalotes and S. invicta, and 6810 orthologs between A. mellifera and S. invicta. These ortholog groups were used for pairwise amino acid identity analysis.

Synteny analysis We used the MCScanX software package (Wang et al., 2012) to perform synteny analysis between two pairs of braconid wasps including C. chilonis vs. M. cingulum and C. chilonis vs. M. demolitor. The scaffolds that contained more than five genes were considered for gene collinearity analysis. We found orthologous counterparts between the two pairs by BLASTP (E < 10À10). Specifically, syntenic blocks were defined when at least five orthologous counterparts were both clustered and located in continuous loci in a single scaffold for each species in each pair.

Phylogenetic analysis We reconstructed a phylogeny of 14 Hymenopteran and 1 Coleopteran species from genomic data, using 2291 single-copy protein-coding genes, and rooted on the red flour beetle T. castaneum. The single-copy protein-coding genes were obtained from the OrthoMCL results. The protein sequences of single-copy protein-coding genes were aligned using MAFFT (version 7) (Katoh et al., 2002) with the default parameters. Then we filtered the saturated sites and poorly aligned regions using trimAl (Capella-Gutie´rrez et al., 2009) and concatenated to one super-sequence for each spe- cies for the phylogenetic analysis. The phylogenetic tree was reconstructed using RAxML (version 8.2.10) (Stamatakis, 2015), and IQ-TREE ModelFinder software (Kalyaanamoorthy et al., 2017) was used to select the best substi- tution model. Specifically, we used the ‘LG+I+F+G4’ model, and values of statistical support were obtained from 1000 replicates of bootstrap analysis. MCMCtree within the PAML software package (version 4.9 hr) (Yang, 1997) was used to estimate the divergence time between different species. Three calibration time points based on fossil records and previous studies, Khutelchalcis gobiensis (125.0–129.4 Mya), Cretobraconus maculatus (145.0–152.1 Mya), stem Hymenoptera (237.0–228.0 Mya), were used for divergence time calibration (Supplementary file 2 – Table 14).

Gene family analysis First, we obtained each gene family’s reference protein sequences from the GenBank of NCBI, and manually confirmed that each reference was intact and absolutely correct. Then, BLASTP was used to obtain the homolog candidate sequences with E < 10À5. All the candidate sequences were fil- tered by HMMER (Meng and Ji, 2013)( E < 10À5) against the Pfam database (Finn et al., 2016) to ensure each sequence contained the iconic domain structures characteristic of the gene family, and the remaining sequences were considered to be the corresponding genes. All multiple-sequence alignments were performed using the MAFFT, and conservation blocks trimmed using trimAl soft- ware. All phylogenetic relationship trees were constructed using RAxML software with the appropri- ate model, as selected by the ModelFinder software and bootstrap 1000.

Amino acid reconstruction and comparative analysis For amino acid synthetic pathway evolution analysis in the Hymenoptera, we applied a pathway annotation pipeline to a large collection of hymenopteran genomes (38 genomes) and an expanded representation of outgroups (13 genomes) specifically to investigate patterns of the gene loss across major lineages of Hymenoptera (Supplementary file 1 – Table 1). First, to minimize the impact on this analysis of genome contamination with bacterial sequences in the assemblies, we used a modifi- cation of the pipeline to detect bacterial scaffolds and lateral gene transfers (originally developed by

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 17 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Wheeler et al., 2013 and subsequently refined as described in Furguson et al., 2020). Bacterial scaffold contamination containing amino acid synthesis genes was detected in Cotesia vestalis (Gen- Bank accession number: LQNH00000000), Diadromus collaris (GenBank accession number: LQNJ00000000), solmsi (GenBank accession number: ATAC00000000) and subse- quently removed from the analysis. Then, a pathway annotation tool BlastKOALA v2.2 (Kanehisa et al., 2016) was used to identify genes in amino acid biosynthetic pathway (Pathway name: 01230 Biosynthesis of amino acids). Another pathway annotation tool iPathCons (Zhang et al., 2014) was used to confirm the results. To avoid missing genes during annotation, we used TBLASTN to scan genes in genome assembly with E < 10À5 and coverage above 75%. For C. chilonis, we also checked the PacBio long reads by TBLASTN using the same cut-off values. To avoid the miss-annotation of some rapidly evolve genes, we also used TBLASTN (E < 10À5) to check the genome assembly with a protein from closely related species as the reference sequence. We reconstructed the hymenopteran phylogeny by joining three phylogenetic trees, two of them are from pervious researches (Branstetter et al., 2017; Peters et al., 2017) and one is from this study. Because of the absence of some of the taxa used in this study in the previously mentioned ones, we reconstructed a phylogenetic tree that contains all Parasitoida (Ichneumonid/Chalcidoid) species used here that have genome annotation information, sawfly Athalia rosae, wood wasp Orus- sus abietinus, paper wasp Polistes dominula, ants Harpegnathos saltator and , and Apis mellifera based on 2923 single-copy proteins obtained from OrthoMCL using the MAFFT- trimAl-ModelFinder-RAxML pipeline (described in Phylogenetic analysis section of Materials and methods). The divergence time was estimated using MCMCtree based on five calibra- tion time points with 95% confidence intervals from Peters et al., 2017, including the common ancestor of and bee (136–193 Mya), the common ancestor of ant, bee and vespid wasp (150–212 Mya), the common ancestor of chalcidoid wasps (105–159 Mya), the common ancestor of braconid wasps (116–177 Mya), and the common ancestor of (203–276 Mya). The pathway disruptions and gene losses were identified using a KEGG online tool, KEGG Map- per (https://www.kegg.jp/kegg/mapper.html), for all predicted pathway genes. We then docu- mented independent pathway disruption and gene loss events in the Ichneumonid/Chalcidoid and Aculeata clades based on the genes involved and phylogenetic positions. To test whether pathway disruptions in amino acid biosynthesis were increased in the Ichneumonid/Chalcidoid clade com- pared to the Aculeate clade, we regraded the percent of branches in Parasitoida and Aculeata rela- tive to the total of the two infraorders to generate a random expected proportion of disruption events if rates of disruption were the same in both infraorders, and then used Fisher’s exact test to perform the statistical test because of the small sample size (N = 14). The median value of the branch estimated by MCMCtree was used for branch length calculation. The online tool http://www. quantpsy.org/chisq/chisq.htm was used to calculate Chi-square values, using the custom expected frequencies for the gene loss calculations along branches. Amino acid biosynthetic capability was evaluated in terms of completeness. The loss of synthesis capability to a particu- lar amino acid occurs when all currently known synthetic pathways for the amino acid are disrupted. We reconstructed the ancestral amino acid biosynthetic pathways for Hymenoptera and Holome- tabola by comparing with the pathway completeness of species in the outgroups. If a complete pathway was found in at least one of the holemetabolous insects, it was assumed to be present in the common ancestor of the Holometabola, and similarly for the common ancestor of the Hymenop- tera. We then identified the pathways which have been disrupted in the common ancestor of Hyme- noptera and Holometabola, respectively. 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, then used Chi-square to perform the statistical test using cus- tom expected frequencies for the gene loss event.

Rearing C. chilonis larvae in vitro Based on the protocols for rearing other parasitoid wasps (Thompson, 1976; Thompson, 1981), the chemically defined rearing media were prepared in our laboratory following the composition of Grace’s Insect Medium (Thermo Fisher, catalog number: 11605; see detail components and concen- trations at Supplementary file 2 – Table 15). All chemicals were obtained from Sigma Chemical Company (Shanghai, China). To verify the requirements for eight different amino acids of C. chilonis, we in vitro reared wasp larvae in the different mediums. The Grace’s Insect Medium was used as a

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 18 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics positive control, and the baseline medium which deleted eight amino acids (lysine, tryptophan, phe- nylalanine, tyrosine, leucine, isoleucine, valine, and histidine) that C. chilonis cannot synthesize was used as a negative control. Nine other mediums were formulated by deleting only one amino acid (including eight amino acids which C. chilonis cannot synthesize and one amino acid (glycine) which C. chilonis can synthesize). The deletion was accompanied by proportional increases in the quantity of all the remaining amino acids to maintain a constant amino acid level through adding a single amino acid, namely, glutamate, as described previously (Thompson, 1976; Thompson, 1981). Each artificial rearing medium was then sterilized by passing through a 0.22 mm filter (Merck Millipore Ltd.; Tullagreen, Carrigtwohill, Co. Cork, IRL). Each solution was then stored at À20˚C until use. The protocol for rearing parasitoid wasps in vitro was based on the methods used for N. vitripen- nis (Brucker and Bordenstein, 2012; Shropshire et al., 2016). For each tested rearing medium, the larvae of C. chilonis were collected by dissecting the parasitized C. suppressalis larvae, which were cleaned with 70% ethanol for surface sterilization 5 days after parasitism. Ten wasp larvae were transferred onto a 3 mm pore transwell polyester membrane (Costar; Corning Incorporated, Corning, NY, USA) after washing with 1 Â phosphate buffer saline (PBS) three times. Then, the transwell insert was transferred to a well with 250 ml of rearing medium in a 24-well plate. All plates were stored in a sterile Tupperware box at 27 ± 1˚C for the duration of the experiment. To confirm whether the larvae were alive, body movement, gut movement and body color were considered as criteria. The rearing experiments were replicated three times. Photos were also taken every day and the larvae body lengths were measured using ImageJ software (version 1.47).

Metabolomics analysis for free amino acids in host hemolymph Larvae of parasitized and non-parasitized C. suppressalis were surface-sterilized with 75% ethanol. Their prolegs were then cut with a pair of scissors and 30 ml of hemolymph was collected using micropipette tips and transferred into a 1.5 ml Eppendorf tube containing 10 ml saturated a-phenyl- thiourea (PTU). After a brief centrifugation, 20 ml supernatant of hemolymph without hemocytes was collected and mixed with 80 ml of pre-cooled methanol. The mixture was vortexed for 1 min. After overnight incubation at 4˚C, the sample was centrifuged at 14,000 g for 15 min at 4˚C. The resulting supernatant (10 ml) was diluted 20-fold with 50% aqueous acetonitrile and subsequently mixed with an equal volume of internal standard solution (ISs) (100 ng/ml in 50% aqueous acetonitrile) prior to UPLC-MS/MS analysis with 1 ml of injection volume. The UPLC-MS/MS analysis was performed on a Waters Acquity UPLC system (Waters, Milford, MA) coupled to a Triple Quad 5500 tandem mass spectrometer (AB Sciex, Framingham, MA), and 3 ml of each sample or calibration curve sample was injected onto a Waters BEH Amide column (100 mm Â2.1 mm, 1.7 mm) at a flow rate of 0.4 ml/min. The mobile phase consisted of (A) water with 10 mM ammonium formate and 0.2% formic acid and (B) acetonitrile with 2 mM ammonium formate and 0.2% formic acid. The chromatographic separation was conducted with a gradient elution pro- gram as follows: 0 min, 90% B; 0.5 min, 90% B; 5.5 min, 75% B; 6.5 min, 50% B; 7.5 min, 50% B; 7.51 min, 90% B; 10 min, 90% B. The column temperature was maintained at 40˚C. The samples eluted from the column were ionized in an electrospray ionization source in positive mode (ESI+). Source temperature: 550˚C, curtain gas (CUR): 35 psi, ion source gas 1 (GS1): 50 psi, ion source gas 2 (GS2): 50 psi, collision gas (CAD): 8 psi, ion spray voltage (IS): 5500 V, entrance potential (EP): 10 V, collision exits potential (CXP1): 10 V. The scheduled multiple reaction moni- toring (sMRM) was used to acquire data in optimized MRM transition (precursor >product), declus- tering potential (DP), and collision energy (CE) as shown in Supplementary file 2 – Table 16. The test samples and standard curve samples were analyzed simultaneously. AB Sciex Analyst software (version 1.5.2) was used to control instruments and acquire data.

Transcriptome analysis and differential expression analysis We followed the standard protocol of differential gene and transcript expression analysis of RNA- seq experiments with HISAT2 and StringTie (Pertea et al., 2016). First, we used Trimmomatic to remove adapter and low-quality sequences in RNA-seq raw data. We then mapped the sequences to the genome using Bowtie. We used HISAT2 and StringTie to obtain putative transcripts. Raw counts for each predicted gene were derived from the read alignments and normalized to fragments per kilobase of exon model per million mapped fragments (FPKM) and differential expression

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 19 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics analyses were performed using RSEM (version 1.3.0) (Li and Dewey, 2011). Heatmaps were gener- ated using R and the package pheatmap v1.0.8. Differentially expressed genes were identified using edgeR (version 3.11) (Robinson et al., 2010). Benjamini-Hochberg correction was used to adjust p values for multiple testing (FDR adjusted). We defined the fold change of gene expression 2 and p-adjusted <0.05 as the criteria for significantly differential expression changes.

Acknowledgements This work was supported by the Major International (Regional) Joint Research Project of NSFC (Grant no. 31620103915 to GYY), the Key Program of National Natural Science Foundation of China (NSFC) (Grant no. 31830074 to GYY), the National Natural Science Foundation of China (NSFC) (Grant no. 31772238 to FL), and National Key Research and Development Program of China (Grant no. 2017YFD0200400 to HWY; Grant no. 2017YFD0200904 to FL). JHW thanks US National Science Foundation (IOS-1456233) and the Nathaniel and Helen Wisch Chair for support. XHY thanks ‘Aca- demic Star’ Program for Ph. D Student of Zhejiang University for support. XHY thanks the University of Rochester, where data analysis and writing of the manuscript were finalized. We thank Novogene Bioinformatics Institute (Beijing, China) for assistance in using Illumina HiSeq 2000 and Pacbio plat- form to sequence the genome of C. chilonis and transcriptomes, and Proflerder Biotech Co., Ltd (Shanghai, China) for their technical support on metabolomic analyses. We thank Mr. Xi Chen from Zhejiang University for his help on genome annotation. We thank Mr. Longfei Chen, Mr. Chaodu Ren, Miss Yupan Zhang and Miss Xingyu Gao from Zhejiang University for help with refining some figures.

Additional information

Funding Funder Grant reference number Author National Natural Science Major International Gongyin Ye Foundation of China (Regional) Joint Research Project of National Natural Science Foundation of China 31620103915 National Natural Science Key Program of National Gongyin Ye Foundation of China Natural Science Foundation of China 31830074 National Natural Science 31772238 Fei Li Foundation of China National Key Research and 2017YFD0200400 Hongwei Yao Development Program of Chi- na National Key Research and 2017YFD0200904 Fei Li Development Program of Chi- na National Science Foundation IOS-1456233 John H Werren

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author contributions Xinhai Ye, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualiza- tion, Methodology, Writing - original draft, Writing - review and editing; Shijiao Xiong, Validation, Investigation, Visualization, Methodology, Writing - original draft; Ziwen Teng, Resources, Formal analysis, Validation, Investigation, Methodology; Yi Yang, Software, Formal analysis, Validation, Investigation, Methodology; Jiale Wang, Chuanlin Yin, Formal analysis, Investigation; Kaili Yu, Resources, Formal analysis, Investigation; Huizi Wu, Zhichao Yan, Fang Wang, Investigation; Yang Mei, Software, Investigation; Sammy Cheng, Formal analysis; Hongwei Yao, Funding acquisition,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 20 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics Investigation; Qi Fang, Resources, Formal analysis, Investigation, Writing - review and editing; Qish- eng Song, Supervision, Writing - review and editing; John H Werren, Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing; Gongyin Ye, Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project adminis- tration, Writing - review and editing; Fei Li, Conceptualization, Resources, Data curation, Supervi- sion, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

Author ORCIDs Xinhai Ye https://orcid.org/0000-0002-0203-0663 Shijiao Xiong https://orcid.org/0000-0002-4142-5852 Yi Yang https://orcid.org/0000-0002-8352-2626 Qi Fang https://orcid.org/0000-0003-4150-3408 Gongyin Ye https://orcid.org/0000-0003-4937-8867 Fei Li https://orcid.org/0000-0002-8410-5250

Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.59795.sa1 Author response https://doi.org/10.7554/eLife.59795.sa2

Additional files Supplementary files . Supplementary file 1. Supplementary Table 1. Genes in amino acid biosynthetic pathways (KEGG map01230), ’+’ means present, ’x’ means lost. . Supplementary file 2. Supplementary Tables 1 – 16. . Transparent reporting form

Data availability All sequence data of the C. chilonis genome project have been deposited in GenBank under the accession code RJVT00000000. In addition, all the data in this paper have been deposited in the InsectBase (www.insect-genome.com/cotesia/).

The following dataset was generated:

Database and Author(s) Year Dataset title Dataset URL Identifier Ye X, Xiong S, 2020 Genome of C. chilonis https://www.ncbi.nlm. NCBI BioProject, Teng Z, Fang Q, nih.gov/bioproject/ PRJNA501840 Werren JH, Ye G, PRJNA501840 Li F

The following previously published datasets were used:

Database and Author(s) Year Dataset title Dataset URL Identifier Wu SF, Sun F, Qi Y, 2013 Parasitization by Cotesia chilonis https://trace.ncbi.nlm. NCBI SRA, SRR651040 Yao Y, Fang Q, influences gene expression in nih.gov/Traces/sra/?run= Huang J, Stanley G, fatbody and hemocytes of Chilo SRR651040 Ye G suppressalis Hall AB, Papatha- 2015 Radical remodeling of the Y https://www.ncbi.nlm. NCBI GenBank, nos P-A, Sharma A, chromosome in a recent species nih.gov/nuccore/ LCWJ00000000.1 Cheng C, Akbari radiation LCWJ00000000.1/ OS, Assour L, Bergman NH, Cag- netti A, Crisanti A, Dottorini T, Fioren- tini E, Galizi R,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 21 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Hnath J, Jiang X, Koren S, Nolan T, Radune D, Shara- khova M V, Steele A Kelkar YD 2018 Flesh genome submission https://www.ncbi.nlm. NCBI GenBank, nih.gov/nuccore/ QOCX00000000.1 QOCX00000000.1/ Hoskins RA, Carl- 2015 The Release 6 Reference Sequence https://www.ncbi.nlm. NCBI Assembly, son JW, Wan KH, of the nih.gov/assembly/GCF_ GCF_000001215.4 Park S, Mendez I, Genome 000001215.4 Galle SE, Booth BW, Pfeiffer BD, George RA, Svirs- kas R, Krzywinski R, Schein J, Accardo MC, Damia E, Messina G, Me´ n- dez-Lago M, de Pablos B, De- makova OV, An- dreyeva EN, Boldyreva LV, Mar- ra M, Carvalho BA, Dimitri P, Villasante A, Zhimulev IF, Rubin GM, Karpen GH, Celniker SE Lin Y 2019 Chilo suppressalis genome https://www.ncbi.nlm. NCBI GenBank, nih.gov/nuccore/ RSAL00000000.1 RSAL00000000.1/ Mita K, Xia Q 2008 The genome of a lepidopteran https://www.ncbi.nlm. NCBI GenBank, model insect, the silkworm Bombyx nih.gov/nuccore/ BABH00000000.1 mori BABH00000000.1/ Murali S, Richards 2017 Anoplophora glabripennis RefSeq https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, Genome sequencing nih.gov/nuccore/ AQHT00000000.2 Bellair M, Blanken- AQHT00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R SaddB M, Barri- 2011 The genomes of two key https://www.ncbi.nlm. NCBI GenBank, beau SM, Bloch G, species with primitive nih.gov/nuccore/ AEQM00000000.2 de Graaf DC, eusocial organization AEQM00000000.2/ Dearden P, Elsik CG, Gadau J, Grimmelikhuijzen CJ, Hasselmann M, Lozier JD, Robert- son HM, Smagghe G, Stolle E, Van Vaerenbergh M, Waterhouse RM, Bornberg-Bauer E, Klasberg S, Bennett AK, Camara F, Guigo R, Hoff K, Mariotti M, Munoz- Torres M, Murphy

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 22 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

T, Santesmasses D, Amdam GV, Beck- ers M, Beye M, Biewer M, Bitondi MM, Blaxter ML, Bourke AF, Brown MJ, Buechel SD, Cameron R, Cap- pelle K, Carolan JC, Christiaens O, Ci- borowski KL, Clarke DF, Colgan TJ, Collins DH, Cridge AG, Dalmay T, Dreier S, du Plessis L, Duncan E, Erler S, Evans J, Falcon T, Flores K, Freitas FC, Fuchikawa T, Gempe T, Hart- felder K, Hauser F, Helbing S, Humann FC, Irvine F, Jer- miin LS, Johnson CE, Johnson RM, Jones AK, Kado- waki T, Kidner JH, Koch V, Kohler A, Kraus FB, Lattorff HM, Leask M, Lockett GA, Mallon EB, Antonio DS, Marxer M, Meeus I, Moritz RF, Nair A, Napflin K, Nissen I, Niu J, Nunes FM, Oakeshott JG, Os- borne A, Otte M, Pinheiro DG, Rossie N, Rueppell O, Santos CG, Schmid-Hempel R, Schmitt BD, Schulte C, Simoes ZL, Soares MP, Swevers L, Winnebeck EC, Wolschin F, Yu N, Zdobnov EM, Aq- rawi PK, Blanken- burg KP, Coyle M, Francisco L, Her- nandez AG, Holder M, Hudson ME, Jackson L, Jaya- seelan J, Joshi V, Kovar C, Lee SL, Mata R, Mathew T, Newsham IF, Ngo R, Okwuonu G, Pham C, Pu LL, Saada N, Santiba- nez J, Simmons D, Thornton R, Venkat A, Walden KK, Wu YQ, Debyser G, Devreese B, Asher C, Blommaert J, Chipman AD, Chit- tka L, Fouks B, Liu J, O’Neill MP, Sumner S, Puiu D, Qu J, Salzberg SL, Scherer SE, Muzny DM, Richards S,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 23 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Robinson GE, Gibbs RA, Schmid- Hempel P, Worley KC Pan H, Kapheim K 2015 The genome of Melipona https://www.ncbi.nlm. NCBI GenBank, quadrifasciata nih.gov/nuccore/ LIRP00000000.1 LIRP00000000.1/ Wallberg A, Bunikis 2018 A hybrid de novo genome https://www.ncbi.nlm. NCBI GenBank, I, Pettersson OV, assembly of the honeybee, Apis nih.gov/nuccore/ QIUM00000000.2 Mosbech MB, mellifera, with chromosome-length QIUM00000000.2/ Childers AK, Evans scaffolds JD, Mikheyev AS, Robertson HM, Ro- binson GE, Web- ster MT Pan H, Kapheim K 2015 The genome of Eufriesea mexicana https://www.ncbi.nlm. NCBI GenBank, nih.gov/nuccore/ LLKC00000000.1 LLKC00000000.1/ Pan H, Kapheim K 2015 The genome of Habropoda https://www.ncbi.nlm. NCBI GenBank, laboriosa nih.gov/nuccore/ LHQN00000000.1 LHQN00000000.1/ Robinson GE, Ro- 2011 Megachile rotundata, whole https://www.ncbi.nlm. NCBI GenBank, bertson HM, Hud- genome shotgun sequencing nih.gov/nuccore/ AFJA00000000.1 son ME, Walden K, project AFJA00000000.1/ Fischman BJ, Pitts- Singer T, James R, Salzberg SL, Puiu D, Magoc T, Kelley D, Zimin AV Pan H, Kapheim K 2015 The genome of Dufourea https://www.ncbi.nlm. NCBI GenBank, novaeangliae nih.gov/nuccore/ LGHO00000000.1 LGHO00000000.1/ Nygaard S, Zhang 2011 The genome of the leaf-cutting ant https://www.ncbi.nlm. NCBI GenBank, G, Schiott M, Li C, Acromyrmex echinatior suggests nih.gov/nuccore/ AEVX00000000.1 Wurm Y, Hu H, key adaptations to advanced social AEVX00000000.1/ Zhou J, Ji L, Qiu F, life and fungus farming Rasmusse M, Pan H, Hauser F, Krogh A, Grimmelikhuij- zen CJ, Wang J, Boomsma JJ Suen G, Teiling C, 2011 The genome sequence of the leaf- https://www.ncbi.nlm. NCBI GenBank, Li L, Holt C, cutter ant Atta cephalotes reveals nih.gov/nuccore/ ADTU00000000.1 Abouheif E, Born- insights into its obligate symbiotic ADTU00000000.1/ berg-Bauer E, lifestyle Bouffard P, Caldera EJ, Cash E, Cava- naugh A, Denas O, Elhaik E, Fave MJ, Gadau J, Gibson JD, Graur D, Grubbs KJ, Hagen DE, Harkins TT, Helmkampf M, Hu H, Johnson BR, Kim J, Marsh SE, Moeller JA, Munoz- Torres MC, Murphy MC, Naughton MC, Nigam S, Overson R, Rajakumar R, Reese JT, Scott JJ, Smith CR, Tao S, Tsutsui ND, Vilja- kainen L, Wissler L, Yandell MD, Zim- mer F, Taylor J, Slater SC, Clifton SW, Warren WC,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 24 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Elsik CG, Smith CD, Weinstock GM, Gerardo NM, Cur- rie CR Wurm Y, Wang J, 2018 The genome of the fire ant https://www.ncbi.nlm. NCBI GenBank, Riba-Grognuz O, Solenopsis invicta nih.gov/nuccore/ AEAQ00000000.2 Corona M, Nygaard AEAQ00000000.2/ S, Hunt BG, Ingram KK, Falquet L, Ni- pitwattanaphon M, Gotzek D, Dijkstra MB, Oettler J, Comtesse F, Shih CJ, Wu WJ, Yang CC, Thomas J, Beaudoing E, Pra- dervand S, Flegel V, Cook ED, Fabbretti R, Stockinger H, Long L, Farmerie WG, Oakey J, Boomsma JJ, Pa- milo P, Yi SV, Heinze J, Goodis- man MA, Farinelli L, Harshman K, Hulo N, Cerutti L, Xe- narios I, Shoemaker D, Keller L Smith CR, Smith 2010 Draft genome of the red harvester https://www.ncbi.nlm. NCBI GenBank, CD, Robertson HM, ant Pogonomyrmex barbatus nih.gov/nuccore/ ADIH00000000.1 Helmkampf M, Zi- ADIH00000000.1/ min A, Yandell M, Holt C, Hu H, Abouheif E, Benton R, Cash E, Croset V, Currie CR, Elhaik E, Elsik CG, Fave MJ, Fernandes V, Gibson JD, Graur D, Gronenberg W, Grubbs KJ, Hagen DE, Viniegra AS, Johnson BR, John- son RM, Khila A, Kim JW, Mathis KA, Munoz-Torres MC, Murphy MC, Mus- tard JA, Nakamura R, Niehuis O, Ni- gam S, Overson RP, Placek JE, Rajaku- mar R, Reese JT, Suen G, Tao S, Torres CW, Tsutsui ND, Viljakainen L, Wolschin F, Gadau J Richards S, Gibbs 2014 The genome of the model beetle https://www.ncbi.nlm. NCBI GenBank, RA, Weinstock GM, and pest Tribolium castaneum nih.gov/nuccore/ AAJJ00000000.2 Brown SJ, Denell R, AAJJ00000000.2/ Beeman RW, Gibbs R, Bucher G, Frie- drich M, Grimmeli- khuijzen CJ, Klingler M, Loren- zen M, Richards S, Roth S, Schroder R, Tautz D, Zdobnov EM, Muzny D, Att- away T, Bell S, Buhay CJ, Chan-

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 25 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

drabose MN, Cha- vez D, Clerk-Blan- kenburg KP, Cree A, Dao M, Davis C, Chacko J, Dinh H, Dugan-Rocha S, Fowler G, Garner TT, Garnes J, Gnirke A, Hawes A, Hernandez J, Hines S, Holder M, Hume J, Jhangiani SN, Joshi V, Khan ZM, Jackson L, Kovar C, Kowis A, Lee S, Lewis LR, Margolis J, Morgan M, Na- zareth LV, Nguyen N, Okwuonu G, Parker D, Ruiz SJ, Santibanez J, Sa- vard J, Scherer SE, Schneider B, So- dergren E, Vattahil S, Villasana D, White CS, Wright R, Park Y, Lord J, Oppert B, Brown S, Wang L, Weinstock G, Liu Y, Worley K, Elsik CG, Reese JT, Elhaik E, Landan G, Graur D, Arensbur- ger P, Atkinson P, Beidler J, Demuth JP, Drury DW, Du YZ, Fujiwara H, Maselli V, Osanai M, Robertson HM, Tu Z, Wang JJ, Wang S, Song H, Zhang L, Werner D, Stanke M, Morgen- stern B, Solovyev V, Kosarev P, Brown G, Chen HC, Er- molaeva O, Hlavina W, Kapustin Y, Kiryutin B, Kitts P, Maglott D, Pruitt K, Sapojnikov V, Sou- vorov A, Mackey AJ, Waterhouse RM, Wyder S, Kri- ventseva EV, Kado- waki T, Bork P, Aranda M, Bao R, Beermann A, Berns N, Bolognesi R, Bonneton F, Bopp D, Butts T, Chau- mot A, Denell RE, Ferrier DE, Gordon CM, Jindra M, Lan Q, Lattorff HM, Laudet V, von Levetsow C, Liu Z, Lutz R, Lynch JA, da Fonseca RN, Posnien N, Reuter R, Schinko JB, Schmitt C, Schopp- meier M, Shippy TD, Simonnet F,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 26 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Marques-Souza H, Tomoyasu Y, Trau- ner J, Van der Zee M, Vervoort M, Wittkopp N, Wim- mer EA, Yang X, Jones AK, Sattelle DB, Ebert PR, Nel- son D, Scott JG, Muthukrishnan S, Kramer KJ, Arakane Y, Zhu Q, Hogen- kamp D, Dixit R, Jiang H, Zou Z, Marshall J, Elpidina E, Vinokurov K, Oppert C, Evans J, Lu Z, Zhao P, Su- mathipala N, Altin- cicek B, Vilcinskas A, Williams M, Hultmark D, Hetru C, Hauser F, Caz- zamali G, William- son M, Li B, Tanaka Y, Predel R, Neu- pert S, Schachtner J, Verleyen P, Ra- ible F, Walden KK, Angeli S, Foret S, Schuetz S, Maleszka R, Miller SC, Grossmann D Smith CD, Zimin A, 2011 Draft genome of the globally https://www.ncbi.nlm. NCBI GenBank, Holt C, Abouheif E, widespread and invasive Argentine nih.gov/nuccore/ ADOQ00000000.1 Benton R, Cash E, ant (Linepithema humile) ADOQ00000000.1/ Croset V, Currie CR, Elhaik E, Elsik CG, Fave MJ, Fer- nandes V, Gadau J, Gibson JD, Graur D, Grubbs KJ, Ha- gen DE, Helm- kampf M, Holley JA, Hu H, Viniegra AS, Johnson BR, Johnson RM, Khila A, Kim JW, Laird J, Mathis KA, Moeller JA, Munoz-Torres MC, Murphy MC, Nakamura R, Ni- gam S, Overson RP, Placek JE, Rajaku- mar R, Reese JT, Robertson HM, Smith CR, Suarez AV, Suen G, Suhr EL, Tao S, Torres CW, van Wilgenburg E, Viljakainen L, Wal- den KK, Wild AL, Yandell M, Yorke JA, Tsutsui ND McKenzie SK, Kro- 2018 The genomic architecture and https://www.ncbi.nlm. NCBI GenBank, nauer DJC molecular evolution of ant odorant nih.gov/nuccore/ QOIP00000000.1 receptors QOIP00000000.1/ Shields EJ, Sheng 2018 High-Quality Genome Assemblies https://www.ncbi.nlm. NCBI GenBank, L, Weiner AK, Reveal Long Non-coding RNAs nih.gov/nuccore/ QANH00000000.1 Garcia BA, Bonasio Expressed in Ant Brains QANH00000000.1/ R

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 27 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Patalano S, Vlasova 2014 Molecular signatures of plastic https://www.ncbi.nlm. NCBI GenBank, A, Wyatt C, Ewels phenotypes in two eusocial insect nih.gov/nuccore/ JPHQ00000000.1 P, Camara F, Fer- species with simple societies JPHQ00000000.1/ reira PG, Asher CL, Jurkowski TP, Se- gonds-Pichon A, Bachman M, Gon- zalez-Navarrete I, Minoche AE, Krue- ger F, Lowy E, Marcet-Houben M, Rodriguez-Ales JL, Nascimento FS, Balasubramanian S, Gabaldon T, Tarver JE, Andrews S, Himmelbauer H, Hughes WO, Guigo R, Reik W, Sumner S Standage DS, 2015 Genome, transcriptome, and https://www.ncbi.nlm. NCBI GenBank, Brendel VP, Toth methylome sequencing of a nih.gov/nuccore/ LMBU00000000.1 AL primitively eusocial wasp reveal a LMBU00000000.1/ greatly reduced DNA methylation system in a social insect Huang DW, Wang 2013 The genome reflects https://www.ncbi.nlm. NCBI GenBank, J, Cook J, Xiao JH, extreme specialisation to a benign nih.gov/nuccore/ ATAC00000000.1 Yue Z, Jia LY, Yang host ATAC00000000.1/ XH, Niu LH, Wang Z, Zhang P, Sun BF, He SM, Li Z, Xiong TL, Xin W, Gu HF, Wang B, Werren J, Wheeler D, Niu LM, Ma GC, Tang T, Bian SN, Wang NX, Yang CY, Wang N, Fu YG, Murphy R, Li WZ, Yi S.V, Yang XY, Zhou Q, Lu CX, Xu CY, He LJ, Yu LL, Chen M, Zheng Y, Wang SW, Zhao S, Li YH, Yu YY, Qian XJ, Cai Y, Bian LL, Zhang S, Wang JY, Yin Y, Xiao H, Wang GH, Yu H, Wu WS Werren JH, Ri- 2012 Functional and evolutionary https://www.ncbi.nlm. NCBI GenBank, chards S, Desjar- insights from the genomes of three nih.gov/nuccore/ AAZX00000000.1 dins CA, Niehuis O, parasitoid Nasonia species AAZX00000000.1/ Gadau J, Col- bourne JK, Werren JH, Beukeboom LW, Desplan C, Elsik CG, Grimme- likhuijzen CJ, Kitts P, Lynch JA, Mur- phy T, Oliveira DC, Smith CD, van de Zande L, Worley KC, Zdob- nov EM, Aerts M, Albert S, Anaya VH, Anzola JM, Barchuk AR, Behura SK, Bera AN, Beren- baum MR, Bertossa RC, Bitondi MM, Bordenstein SR, Bork P, Bornberg-

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 28 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Bauer E, Brunain M, Cazzamali G, Cha- boub L, Chacko J, Chavez D, Childers CP, Choi JH, Clark ME, Claudianos C, Clinton RA, Cree AG, Cristino AS, Dang PM, Darby AC, de Graaf DC, Devreese B, Dinh HH, Edwards R, Elango N, Elhaik E, Ermolaeva O, Evans JD, Foret S, Fowler GR, Gerlach D, Gibson JD, Gilbert DG, Graur D, Grunder S, Hagen DE, Han Y, Hauser F, Hultmark D, Hunter HC IV Hurst GD, Jhangian SN, Jiang H, Johnson RM, Jones AK, Junier T, Kadowaki T, Kamping A, Ka- pustin Y, Kechavarzi B, Kim J, Kiryutin B, Koevoets T, Kovar CL, Kriventseva EV, Kucharski R, Lee H, Lee SL, Lees K, Lewis LR, Loehlin DW, Logsdon JM Jr, Lopez JA, Lo- zado RJ, Maglott D, Maleszka R, Mayampurath A, Mazur DJ, McClure MA, Moore AD, Morgan MB, Muller J, Munoz-Torres MC, Muzny DM, Nazareth LV, Neu- pert S, Nguyen NB, Nunes FM, Oake- shott JG, Okwuonu GO, Pannebakker BA, Pejaver VR, Peng Z, Pratt SC, Predel R, Pu LL, Ranson H, Ray- choudhury R, Re- chtsteiner A, Reese JT, Reid JG, Riddle M, Robertson HM, Romero-Severson J, Rosenberg M, Sackton TB, Sattelle DB, Schluns H, Schmitt T, Schnei- der M, Schuler A, Schurko AM, Shu- ker DM, Simoes ZL, Sinha S, Smith Z, Solovyev V, Sou- vorov A, Springauf A, Stafflinger E, Stage DE, Stanke M, Tanaka Y, Tel- schow A, Trent C, Vattathil S, Verhulst EC, Viljakainen L,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 29 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Wanner KW, Waterhouse RM, Whitfield JB, Wilkes TE, Williamson M, Willis JH, Wolschin F, Wyder S, Yama- da T, Yi SV, Zecher CN, Zhang L, Gibbs RA Ye X, Ye G 2020 A chromosome level genome https://www.ncbi.nlm. NCBI GenBank, assembly of the parasitoid wasp nih.gov/nuccore/ VCDM00000000.2 Pteromalus puparum VCDM00000000.2/ Murali S, Richards 2017 floridanum Genome https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, sequencing nih.gov/nuccore/ JBOX00000000.2 Bellair M, Blanken- JBOX00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Murali S, Richards 2017 Genome sequencing of https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, Trichogramma pretiosum and its nih.gov/nuccore/ JARR00000000.2 Bellair M, Blanken- endosymbiont JARR00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Gao F, Gu QJ, Pan 2016 Cotesia vestalis teratocytes express https://www.ncbi.nlm. NCBI GenBank, J, Wang ZH., Yin a diversity of genes and exhibit nih.gov/nuccore/ LQNH00000000.1 CL, Li F, Song QS, novel immune functions in LQNH00000000.1/ Strand MR, Chen parasitism XX, Shi M Burke GR, Walden 2015 Widespread genome https://www.ncbi.nlm. NCBI GenBank, KK, Whitfield JB, reorganization of an obligate virus nih.gov/nuccore/ AZMT00000000.2 Robertson HM, mutualist AZMT00000000.2/ Strand MR Yin C, Hu J, Li, F 2017 Genome analysis enriches our https://www.ncbi.nlm. NCBI GenBank, understanding of wasp parasitic nih.gov/nuccore/ MVJL00000000.1 tactics MVJL00000000.1/ Robertson HM, 2019 Genome of the parasitoid wasp https://www.ncbi.nlm. NCBI GenBank, Walden KK, Tvedte Diachasma alloeum, an emerging nih.gov/nuccore/ LDKA00000000.2 ES., Hood GR, model for ecological speciation LDKA00000000.2/ Feder JL., Forbes and transitions to asexual AA, Logsdon JM, Mcelroy KE

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 30 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Geib S 2014 Fari WGS Assembly https://www.ncbi.nlm. NCBI GenBank, nih.gov/nuccore/ JRKH00000000.1 JRKH00000000.1/ BIPAA 2017 Aphidius ervi genome v3.0 https://bipaa.genouest. BIPAA, aphidius_ org/sp/aphidius_ervi/ ervi BIPAA 2017 Lysiphlebus fabarum genome v1.0 https://bipaa.genouest. BIPAA, lysiphlebus_ org/sp/lysiphlebus_fa- fabarum barum/ Ye X, Wang Z 2016 Genomic analysis reveals new https://www.ncbi.nlm. NCBI GenBank, insights into parasitoid-host nih.gov/nuccore/ LQNJ00000000.1 interaction LQNJ00000000.1/ BIPAA 2018 Venturia canescens genome v1.0 https://bipaa.genouest. BIPAA, venturia_ org/sp/venturia_canes- canescens cens/ Murali S, Richards 2017 Orussus abietinus isolate:OABI.00- https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, Male Genome sequencing and nih.gov/nuccore/ AZGP00000000.2 Bellair M, Blanken- assembly AZGP00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Linnen C 2019 Neodiprion pinetum draft genome https://www.ncbi.nlm. NCBI GenBank, nih.gov/nuccore/ SSWZ00000000.1 SSWZ00000000.1/ Robertson HM, Ro- 2012 The Genome of the Wheatstem https://www.ncbi.nlm. NCBI GenBank, binson DE, Wanner Sawfly, Cephus cinctus nih.gov/nuccore/ AMWH00000000.1 K, Walden KKO AMWH00000000.1/ Murali S, Richards 2017 Athalia rosae genome sequencing https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, and assembly nih.gov/nuccore/ AOFN00000000.2 Bellair M, Blanken- AOFN00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Murali S, Richards 2018 Whole genome assembly of https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, Oncopeltus fasciatus using multiple nih.gov/nuccore/ JHQO00000000.2 Bellair M, Blanken- sequencing technologies JHQO00000000.2/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 31 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Moran NA, Li Y, 2019 Chromosome-level assembly of the https://www.ncbi.nlm. NCBI GenBank, Park H. Smith TE pea aphid genome using Hi-C nih.gov/nuccore/ SDIB00000000.1 SDIB00000000.1/ Murali S, Richards 2019 Frankliniella occidentalis isolate: https://www.ncbi.nlm. NCBI GenBank, S, Bandaranaike D, FOCC.00 Genome sequencing nih.gov/nuccore/ JMDY00000000.3 Bellair M, Blanken- JMDY00000000.3/ burg K, Chao H, Dinh H, Doddapa- neni H, Dugan-Ro- cha S, Elkadiri S, Gnanaolivu R, Her- nandez B, Skinner E, Javaid M, Lee S, Li M, Ming W, Munidasa M, Muniz J, Nguyen L, Hughes D, Osuji N, Pu L.-L, Puazo M, Qu C, Quiroz J, Raj R, Weissenberger G, Xin Y, Zou X, Han Y, Worley K, Muzny D, Gibbs R Faddeeva A, Derks 2015 The genome of Folsomia candida https://www.ncbi.nlm. NCBI GenBank, MF, Anvar Y, Smit nih.gov/nuccore/ LNIX00000000.1 S, Van Straalen N, LNIX00000000.1/ Roelofs D Lee J-S, Lee B-Y, 2018 The genome of the water flea https://www.ncbi.nlm. NCBI GenBank, Choi B-S Daphnia magna nih.gov/nuccore/ QYSF00000000.1 QYSF00000000.1/ Grbic M, 2011 Genome assembly and annotation https://www.ncbi.nlm. NCBI GenBank, Van Leeuwen T, of the Tetranychus urticae genome nih.gov/nuccore/ CAEY00000000.1 Clark R, Rombauts CAEY00000000.1/ S, Rouze P, Grbic V, Osborne E, Dermauw W, Cao Thi Ngoc P, Ortego F, Hernan- dez-Crespo P, Diaz I, Martinez M, Na- vajas M, Sucena E, Magalhaes S, Nagy L, Pace R, Djura- novic S, Smagghe G, Iga M, Chris- tiaens O, Veenstra J.A, Ewer J, Mancilla Villalobos R, Hutter J.L, Hud- son S.D, Velez M, Yi S, Zeng J, Pires- DaSilva A, Roch F, Cazaux M, Navarro M, Zhurov V, Ace- vedo G, Mijanovic A, Fawcett J.A, Bonnet E, Martens C, Baele G, Wissler L, Sanchez-Rodri- guez A, Tirry L,

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 32 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Blais C, Demees- tere K, Henz S, Gregory R, Mathieu J, Verdon L, Far- inelli L, Schmutz J, Lindquist E, Feyer- eisen R, Van de Peer Y

References Bale JS, van Lenteren JC, Bigler F. 2008. Biological control and sustainable food production. Philosophical Transactions of the Royal Society B: Biological Sciences 363:761–776. DOI: https://doi.org/10.1098/rstb.2007. 2182 Bank S, Sann M, Mayer C, Meusemann K, Donath A, Podsiadlowski L, Kozlov A, Petersen M, Krogmann L, Meier R, Rosa P, Schmitt T, Wurdack M, Liu S, Zhou X, Misof B, Peters RS, Niehuis O. 2017. Transcriptome and target DNA enrichment sequence data provide new insights into the phylogeny of vespid wasps (Hymenoptera: aculeata: vespidae). Molecular Phylogenetics and Evolution 116:213–226. DOI: https://doi.org/10.1016/j. ympev.2017.08.020, PMID: 28887149 Beckage NE, Gelman DB. 2004. Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. Annual Review of Entomology 49:299–330. DOI: https://doi. org/10.1146/annurev.ento.49.061802.123324, PMID: 14651466 Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. DOI: https://doi.org/10.1093/bioinformatics/btq683 Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30:2114–2120. DOI: https://doi.org/10.1093/bioinformatics/btu170, PMID: 24695404 Branstetter MG, Danforth BN, Pitts JP, Faircloth BC, Ward PS, Buffington ML, Gates MW, Kula RR, Brady SG. 2017. Phylogenomic insights into the evolution of stinging wasps and the origins of ants and bees. Current Biology 27:1019–1025. DOI: https://doi.org/10.1016/j.cub.2017.03.027, PMID: 28376325 Brucker RM, Bordenstein SR. 2012. In vitro cultivation of the hymenoptera genetic model, Nasonia. PLOS ONE 7:e51269. DOI: https://doi.org/10.1371/journal.pone.0051269, PMID: 23227258 Caccia S, Leonardi MG, Casartelli M, Grimaldi A, de Eguileor M, Pennacchio F, Giordana B. 2005. Nutrient absorption by Aphidius ervi larvae. Journal of Insect Physiology 51:1183–1192. DOI: https://doi.org/10.1016/j. jinsphys.2005.06.010, PMID: 16085087 Caccia S, Grimaldi A, Casartelli M, Falabella P, de Eguileor M, Pennacchio F, Giordana B. 2012. Functional analysis of a fatty acid binding protein produced by Aphidius ervi teratocytes. Journal of Insect Physiology 58: 621–627. DOI: https://doi.org/10.1016/j.jinsphys.2011.12.019, PMID: 22226822 Campbell MS, Holt C, Moore B, Yandell M. 2014. Genome annotation and curation using MAKER and maker-p. Current Protocols in Bioinformatics 48:bi0411s48. DOI: https://doi.org/10.1002/0471250953.bi0411s48 Capella-Gutie´ rrez S, Silla-Martı´nez JM, Gabaldo´n T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973. DOI: https://doi.org/10.1093/bioinformatics/ btp348, PMID: 19505945 Coghlan A, Tyagi R, Cotton JA, Holroyd N, Rosa BA, Tsal IJ, Laetsch DR, Beech RN, Day TA, Hallsworth-Pepin K, International Helminth Genomes Consortium. 2018. Comparative genomics of the major parasitic worms. Nature Genetics 51:163–174. DOI: https://doi.org/10.1038/s41588-018-0262-1, PMID: 30397333 Colombani J, Raisin S, Pantalacci S, Radimerski T, Montagne J, Le´opold P. 2003. A nutrient sensor mechanism controls Drosophila growth. Cell 114:739–749. DOI: https://doi.org/10.1016/S0092-8674(03)00713-X, PMID: 14505573 Dermauw W, Van Leeuwen T. 2014. The ABC gene family in arthropods: comparative genomics and role in transport and resistance. Insect and Molecular Biology 45:89–110. DOI: https://doi. org/10.1016/j.ibmb.2013.11.001, PMID: 24291285 Digilio MC, Isidoro N, Tremblay E, Pennacchio F. 2000. Host castration by Aphidius ervi venom proteins. Journal of Insect Physiology 46:1041–1050. DOI: https://doi.org/10.1016/S0022-1910(99)00216-4, PMID: 10802117 Douglas AE. 2006. Phloem-sap feeding by : problems and solutions. Journal of Experimental Botany 57: 747–754. DOI: https://doi.org/10.1093/jxb/erj067, PMID: 16449374 Douglas AE. 2009. The microbial dimension in insect nutritional . Functional Ecology 23:38–47. DOI: https://doi.org/10.1111/j.1365-2435.2008.01442.x Ellers J, Kiers ET, Currie CR, McDonald BR, Visser B. 2012. Ecological interactions drive evolutionary loss of traits. Ecology Letters 15:1071–1082. DOI: https://doi.org/10.1111/j.1461-0248.2012.01830.x, PMID: 22747703 Falabella P, Perugino G, Caccialupi P, Riviello L, Varricchio P, Tranfaglia A, Rossi M, Malva C, Graziani F, Moracci M, Pennacchio F. 2005. A novel fatty acid binding protein produced by teratocytes of the aphid parasitoid Aphidius ervi. Insect Molecular Biology 14:195–205. DOI: https://doi.org/10.1111/j.1365-2583.2004.00548.x, PMID: 15796753 Falabella P, Riviello L, Caccialupi P, Rossodivita T, Teresa Valente M, Luisa De Stradis M, Tranfaglia A, Varricchio P, Gigliotti S, Graziani F, Malva C, Pennacchio F. 2007. A gamma-glutamyl transpeptidase of Aphidius ervi

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 33 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

venom induces apoptosis in the ovaries of host aphids. Insect Biochemistry and Molecular Biology 37:453–465. DOI: https://doi.org/10.1016/j.ibmb.2007.02.005, PMID: 17456440 Falabella P, Riviello L, De Stradis ML, Stigliano C, Varricchio P, Grimaldi A, de Eguileor M, Graziani F, Gigliotti S, Pennacchio F. 2009. Aphidius ervi teratocytes release an extracellular enolase. Insect Biochemistry and Molecular Biology 39:801–813. DOI: https://doi.org/10.1016/j.ibmb.2009.09.005, PMID: 19786101 Feldhaar H. 2011. Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology 36:533–543. DOI: https://doi.org/10.1111/j.1365-2311.2011.01318.x Feng H, Edwards N, Anderson CMH, Althaus M, Duncan RP, Hsu YC, Luetje CW, Price DRG, Wilson ACC, Thwaites DT. 2019. Trading amino acids at the aphid-Buchnera symbiotic interface. PNAS 116:16003–16011. DOI: https://doi.org/10.1073/pnas.1906223116, PMID: 31337682 Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador- Vegas A, Salazar GA, Tate J, Bateman A. 2016. The pfam protein families database: towards a more sustainable future. Nucleic Acids Research 44:D279–D285. DOI: https://doi.org/10.1093/nar/gkv1344 Furguson KB, Visser S, Dalı´kova´ M, Provaznı´kova´I, Urbaneja A, Pe´rez-Hedo M, Marec F, Werren JH, Zwaan BJ, Pannebakker BA, Verhulst EC. 2020. Jekyll or hyde? the genome (and more) of Nesidiocoris tenuis, a zoophytophagous predatory bug that is both a biological control agent and a pest. bioRxiv. DOI: https://doi. org/10.1101/2020.02.27.967943 Gil R, Vargas-Chavez C, Lo´pez-Madrigal S, Santos-Garcı´a D, Latorre A, Moya A. 2018. Tremblaya phenacola PPER: an evolutionary beta-gammaproteobacterium collage. The ISME Journal 12:124–135. DOI: https://doi. org/10.1038/ismej.2017.144, PMID: 28914880 Giordana B, Milani A, Grimaldi A, Farneti R, Casartelli M, Ambrosecchio MR, Digilio MC, Leonardi MG, de Eguileor M, Pennacchio F. 2003. Absorption of sugars and amino acids by the epidermis of Aphidius ervi larvae. Journal of Insect Physiology 49:1115–1124. DOI: https://doi.org/10.1016/j.jinsphys.2003.08.010, PMID: 14624883 Grimaldi A, Caccia S, Congiu T, Ferrarese R, Tettamanti G, Rivas-Pena M, Perletti G, Valvassori R, Giordana B, Falabella P, Pennacchio F, de Eguileor M. 2006. Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps. Journal of Insect Physiology 52:870–880. DOI: https://doi.org/10.1016/j.jinsphys.2006.05.011, PMID: 16843482 Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, et al. 2013. De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nature Protocols 8:1494–1512. DOI: https://doi.org/10.1038/nprot.2013.084, PMID: 23845962 Hang SB, ZQ L. 1991. Physiological and biochenical changes in the hemolymph of Chilo suppressalis larvae parasitized by apanteies chilonis. Acta Entomologica Sinica 34:427–432. Heraty J. 2009. Parasitoid diversity and insect pest management. In: Adler P. H, Foottit R. G (Eds). Insect : Science and Society. Wiley-Blackwell. p. 445–462. DOI: https://doi.org/10.1002/9781118945568. ch19 Hu Y, Sanders JG, Łukasik P, D’Amelio CL, Millar JS, Vann DR, Lan Y, Newton JA, Schotanus M, Kronauer DJC, Pierce NE, Moreau CS, Wertz JT, Engel P, Russell JA. 2018. Author correction: herbivorous turtle ants obtain essential nutrients from a conserved -recycling gut microbiome. Nature Communications 9:04935. DOI: https://doi.org/10.1038/s41467-018-04935-w Husnik F, McCutcheon JP. 2016. Repeated replacement of an intrabacterial symbiont in the tripartite nested mealybug symbiosis. PNAS 113:E5416–E5424. DOI: https://doi.org/10.1073/pnas.1603910113, PMID: 2757381 9 Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods 14:587–589. DOI: https://doi.org/10.1038/nmeth.4285, PMID: 28481363 Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: kegg tools for functional characterization of genome and metagenome sequences. Journal of Molecular Biology 428:726–731. DOI: https://doi.org/10.1016/j.jmb.2015.11.006, PMID: 26585406 Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. 2017. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Research 45:D353–D361. DOI: https://doi.org/10.1093/nar/ gkw1092, PMID: 27899662 Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast fourier transform. Nucleic Acids Research 30:3059–3066. DOI: https://doi.org/10.1093/nar/ gkf436, PMID: 12136088 Kim D, Langmead B, Salzberg SL. 2015. HISAT: a fast spliced aligner with low memory requirements. Nature Methods 12:357–360. DOI: https://doi.org/10.1038/nmeth.3317 Knutson A. 1998. The Trichogramma Manual: A Guide to the Use of Trichogramma for Biological Control with Special Reference to Augmentative Releases for Control of Bollworm and Budworm in Cotton: Texas Agricultural Extension Service, the Texas A&M University System. Korf I. 2004. Gene finding in novel genomes. BMC Bioinformatics 5:59. DOI: https://doi.org/10.1186/1471-2105- 5-59, PMID: 15144565 Lammers M, Kraaijeveld K, Marie¨ n J, Ellers J. 2019. Gene expression changes associated with the evolutionary loss of a metabolic trait: lack of lipogenesis in parasitoids. BMC Genomics 20:6. DOI: https://doi.org/10.1186/ s12864-019-5673-6, PMID: 31014246

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 34 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Langmead B, Trapnell C, Pop M, Salzberg SL. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10:R25. DOI: https://doi.org/10.1186/gb-2009-10-3-r25, PMID: 19261174 Li L. 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Research 13:2178– 2189. DOI: https://doi.org/10.1101/gr.1224503 Li B, Dewey CN. 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323. DOI: https://doi.org/10.1186/1471-2105-12-323, PMID: 21816040 Lindsey ARI, Kelkar YD, Wu X, Sun D, Martinson EO, Yan Z, Rugman-Jones PF, Hughes DST, Murali SC, Qu J, Dugan S, Lee SL, Chao H, Dinh H, Han Y, Doddapaneni HV, Worley KC, Muzny DM, Ye G, Gibbs RA, et al. 2018. Comparative genomics of the miniature wasp and pest control agent Trichogramma pretiosum. BMC Biology 16:9. DOI: https://doi.org/10.1186/s12915-018-0520-9, PMID: 29776407 Liu J, Xiao H, Huang S, Li F. 2014. OMIGA: optimized Maker-Based insect genome annotation. Molecular Genetics and Genomics 289:567–573. DOI: https://doi.org/10.1007/s00438-014-0831-7 Lomsadze A, Burns PD, Borodovsky M. 2014. Integration of mapped RNA-Seq reads into automatic training of eukaryotic gene finding algorithm. Nucleic Acids Research 42:e119. DOI: https://doi.org/10.1093/nar/gku557 Ma W, Zhao X, Yin C, Jiang F, Du X, Chen T, Zhang Q, Qiu L, Xu H, Joe Hull J, Li G, Sung WK, Li F, Lin Y. 2020. A chromosome-level genome assembly reveals the genetic basis of cold tolerance in a notorious rice insect pest Chilo suppressalis. Molecular Ecology Resources 20:268–282. DOI: https://doi.org/10.1111/1755-0998. 13078, PMID: 31482680 Martinson EO, Wheeler D, Wright J, Mrinalini , Siebert AL, Werren JH. 2014. Nasonia vitripennis venom causes targeted gene expression changes in its fly host. Molecular Ecology 23:5918–5930. DOI: https://doi.org/10. 1111/mec.12967, PMID: 25319487 McCutcheon JP, Moran NA. 2007. Parallel genomic evolution and metabolic interdependence in an ancient symbiosis. PNAS 104:19392–19397. DOI: https://doi.org/10.1073/pnas.0708855104, PMID: 18048332 Meng X, Ji Y. 2013. Modern computational techniques for the HMMER sequence analysis. ISRN Bioinformatics 2013:1–13. DOI: https://doi.org/10.1155/2013/252183, PMID: 25937944 Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, Rust J, Aberer AJ, Aspo¨ ck U, Aspo¨ ck H, Bartel D, Blanke A, et al. 2014. Phylogenomics resolves the timing and pattern of insect evolution. Science 346:763–767. DOI: https://doi.org/10.1126/science.1257570, PMID: 25378627 Mrinalini , Siebert AL, Wright J, Martinson E, Wheeler D, Werren JH. 2015. Parasitoid venom induces metabolic cascades in fly hosts. Metabolomics 11:350–366. DOI: https://doi.org/10.1007/s11306-014-0697-z, PMID: 27 867325 Nakamatsu Y, Tanaka T. 2003. Venom of ectoparasitoid, Euplectrus sp near plathypenae (Hymenoptera: ) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource. Journal of Insect Physiology 49:149–159. DOI: https://doi.org/10.1016/S0022-1910(02)00261-5, PMID: 12770008 Nakamatsu Y, Tanaka T. 2004. Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells. Journal of Insect Physiology 50:267–275. DOI: https://doi.org/10.1016/j.jinsphys.2003.12.005, PMID: 150 81819 Nicolson SW, Human H. 2013. Chemical composition of the ‘low quality’ pollen of sunflower (Helianthus annuus, Asteraceae). Apidologie 44:144–152. DOI: https://doi.org/10.1007/s13592-012-0166-5 Olson SA. 2002. EMBOSS opens up sequence analysis. European molecular biology open software suite. Briefings in Bioinformatics 3:87–91. DOI: https://doi.org/10.1093/bib/3.1.87, PMID: 12002227 Parra G, Bradnam K, Korf I. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23:1061–1067. DOI: https://doi.org/10.1093/bioinformatics/btm071, PMID: 17332020 Pennacchio F, Digilio MC, Tremblay E. 1995. Biochemical and metabolic alterations inAcyrthosiphon Pisum parasitized byAphidius ervi. Archives of Insect Biochemistry and Physiology 30:351–367. DOI: https://doi.org/ 10.1002/arch.940300405 Pennacchio F, Fanti P, Falabella P, Digilio MC, Bisaccia F, Tremblay E. 1999. Development and nutrition of the braconid wasp,Aphidius ervi in Aposymbiotic host aphids. Archives of Insect Biochemistry and Physiology 40: 53–63. DOI: https://doi.org/10.1002/(SICI)1520-6327(1999)40:1<53::AID-ARCH6>3.0.CO;2-J Pennacchio F, Caccia S, Digilio MC. 2014. Host regulation and nutritional exploitation by parasitic wasps. Current Opinion in Insect Science 6:74–79. DOI: https://doi.org/10.1016/j.cois.2014.09.018, PMID: 32846685 Pennacchio F, Strand MR. 2006. Evolution of developmental strategies in parasitic hymenoptera. Annual Review of Entomology 51:233–258. DOI: https://doi.org/10.1146/annurev.ento.51.110104.151029, PMID: 16332211 Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology 33:290–295. DOI: https://doi. org/10.1038/nbt.3122, PMID: 25690850 Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. 2016. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and ballgown. Nature Protocols 11:1650–1667. DOI: https://doi.org/10. 1038/nprot.2016.095, PMID: 27560171 Peters RS, Krogmann L, Mayer C, Donath A, Gunkel S, Meusemann K, Kozlov A, Podsiadlowski L, Petersen M, Lanfear R, Diez PA, Heraty J, Kjer KM, Klopfstein S, Meier R, Polidori C, Schmitt T, Liu S, Zhou X, Wappler T, et al. 2017. Evolutionary history of the hymenoptera. Current Biology 27:1013–1018. DOI: https://doi.org/10. 1016/j.cub.2017.01.027, PMID: 28343967

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 35 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Peters RS, Niehuis O, Gunkel S, Bla¨ ser M, Mayer C, Podsiadlowski L, Kozlov A, Donath A, van Noort S, Liu S, Zhou X, Misof B, Heraty J, Krogmann L. 2018. Transcriptome sequence-based phylogeny of chalcidoid wasps (Hymenoptera: chalcidoidea) reveals a history of rapid radiations, convergence, and evolutionary success. Molecular Phylogenetics and Evolution 120:286–296. DOI: https://doi.org/10.1016/j.ympev.2017.12.005, PMID: 29247847 Price DR, Feng H, Baker JD, Bavan S, Luetje CW, Wilson AC. 2014. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts. PNAS 111:320–325. DOI: https://doi.org/10.1073/pnas. 1306068111, PMID: 24367072 Quicke DLJ. 1997. Parasitic Wasps. London: Chapman & Hall. DOI: https://doi.org/10.1017/S0007485300026110 Rabie AL, Wells JD, Dent LK. 1983. The nitrogen content of pollen protein. Journal of Apicultural Research 22: 119–123. DOI: https://doi.org/10.1080/00218839.1983.11100572 Rahbe´ Y, Digilio MC, Febvay G, Guillaud J, Fanti P, Pennacchio F. 2002. Metabolic and symbiotic interactions in amino acid pools of the pea aphid, Acyrthosiphon pisum, parasitized by the braconid Aphidius ervi. Journal of Insect Physiology 48:507–516. DOI: https://doi.org/10.1016/S0022-1910(02)00053-7, PMID: 12770078 Richards S, Gibbs RA, Gerardo NM, Moran N, Nakabachi A, Stern D, Tagu D, Wilson ACC, Muzny D, Kovar C, International Aphid Genomics Consortium. 2010. Genome sequence of the pea aphid Acyrthosiphon pisum. PLOS Biology 8:e1000313. DOI: https://doi.org/10.1371/journal.pbio.1000313, PMID: 20186266 Rivers DB, Denlinger DL. 1994. Redirection of metabolism in the flesh fly, sarcophaga bullata, following envenomation by the ectoparasitoid Nasonia vitripennis and correlation of metabolic effects with the diapause status of the host. Journal of Insect Physiology 40:207–215. DOI: https://doi.org/10.1016/0022-1910(94)90044- 2 Robinson MD, McCarthy DJ, Smyth GK. 2010. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. DOI: https://doi.org/10.1093/bioinformatics/ btp616 Savard J, Tautz D, Richards S, Weinstock GM, Gibbs RA, Werren JH, Tettelin H, Lercher MJ. 2006. Phylogenomic analysis reveals bees and wasps (Hymenoptera) at the base of the radiation of holometabolous insects. Genome Research 16:1334–1338. DOI: https://doi.org/10.1101/gr.5204306, PMID: 17065606 Shropshire JD, van Opstal EJ, Bordenstein SR. 2016. An optimized approach to germ-free rearing in the jewel wasp Nasonia. PeerJ 4:e2316. DOI: https://doi.org/10.7717/peerj.2316, PMID: 27602283 FASima˜ o , Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31:3210–3212. DOI: https:// doi.org/10.1093/bioinformatics/btv351, PMID: 26059717 Stamatakis A. 2015. Using RAxML to infer phylogenies. Current Protocols in Bioinformatics 51:bi0614s51. DOI: https://doi.org/10.1002/0471250953.bi0614s51 Stanke M, Steinkamp R, Waack S, Morgenstern B. 2004. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Research 32:W309–W312. DOI: https://doi.org/10.1093/nar/gkh379, PMID: 15215400 Tempel S. 2012. Using and understanding RepeatMasker. Methods in Molecular Biology 859:29–51. DOI: https://doi.org/10.1007/978-1-61779-603-6_2, PMID: 22367864 Teng ZW, Xu G, Gan SY, Chen X, Fang Q, Ye GY. 2016. Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: braconidae) parasitism, venom, and Calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae. Journal of Insect Physiology 85:46–56. DOI: https://doi. org/10.1016/j.jinsphys.2015.11.014, PMID: 26685058 Teng Z-W, Xiong S-J, Xu G, Gan S-Y, Chen X, Stanley D, Yan Z-C, Ye G-Y, Fang Q. 2017. Protein discovery: combined transcriptomic and proteomic analyses of venom from the endoparasitoid Cotesia chilonis (Hymenoptera: braconidae). Toxins 9:135. DOI: https://doi.org/10.3390/toxins9040135 Thompson SN. 1976. The amino acid requirements for larval development of the hymenopterous parasitoid Exeristes roborator fabricius (hymenoptera: ). Comparative Biochemistry and Physiology Part A: Physiology 53:211–213. DOI: https://doi.org/10.1016/S0300-9629(76)80057-6 Thompson SN. 1981. Essential amino acid requirements of four species of parasitic hymenoptera. Comparative Biochemistry and Physiology Part A: Physiology 69:173–174. DOI: https://doi.org/10.1016/0300-9629(81) 90658-7 Thompson SN. 1986. Nutrition and in vitro culture of insect parasitoids. Annual Review of Entomology 31:197– 219. DOI: https://doi.org/10.1146/annurev.en.31.010186.001213 Visser B, Le Lann C, den Blanken FJ, Harvey JA, van Alphen JJ, Ellers J. 2010. Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle. PNAS 107:8677–8682. DOI: https://doi.org/10.1073/pnas. 1001744107, PMID: 20421492 Visser B, Roelofs D, Hahn DA, Teal PE, Marie¨ n J, Ellers J. 2012. Transcriptional changes associated with lack of lipid synthesis in parasitoids. Genome Biology and Evolution 4:864–874. DOI: https://doi.org/10.1093/gbe/ evs065, PMID: 22820524 Visser B, Ellers J. 2008. Lack of lipogenesis in parasitoids: a review of physiological mechanisms and evolutionary implications. Journal of Insect Physiology 54:1315–1322. DOI: https://doi.org/10.1016/j.jinsphys.2008.07.014, PMID: 18706420 Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLOS ONE 9:e112963. DOI: https://doi.org/10.1371/journal.pone.0112963, PMID: 25409509

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 36 of 37 This article has been retracted. Read the retraction notice Research article Genetics and Genomics

Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H, Kissinger JC, Paterson AH. 2012. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research 40:e49. DOI: https://doi.org/10.1093/nar/gkr1293, PMID: 22217600 Wheeler D, Redding AJ, Werren JH. 2013. Characterization of an ancient lepidopteran lateral gene transfer. PLOS ONE 8:e59262. DOI: https://doi.org/10.1371/journal.pone.0059262, PMID: 23533610 Wilson AC, Ashton PD, Calevro F, Charles H, Colella S, Febvay G, Jander G, Kushlan PF, Macdonald SJ, Schwartz JF, Thomas GH, Douglas AE. 2010. Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola. Insect Molecular Biology 19 Suppl 2: 249–258. DOI: https://doi.org/10.1111/j.1365-2583.2009.00942.x, PMID: 20482655 Wolfersberger MG. 2000. Amino acid transport in insects. Annual Review of Entomology 45:111–120. DOI: https://doi.org/10.1146/annurev.ento.45.1.111, PMID: 10761572 Wu SF, Sun FD, Qi YX, Yao Y, Fang Q, Huang J, Stanley D, Ye GY. 2013. Parasitization by Cotesia chilonis influences gene expression in fatbody and hemocytes of Chilo suppressalis. PLOS ONE 8:e74309. DOI: https:// doi.org/10.1371/journal.pone.0074309, PMID: 24086331 Xiao JH, Yue Z, Jia LY, Yang XH, Niu LH, Wang Z, Zhang P, Sun BF, He SM, Li Z, Xiong TL, Xin W, Gu HF, Wang B, Werren JH, Murphy RW, Wheeler D, Niu LM, Ma GC, Tang T, et al. 2013. Obligate within a host drives the extreme specialization of a fig wasp genome. Genome Biology 14:R141. DOI: https://doi.org/10. 1186/gb-2013-14-12-r141, PMID: 24359812 Xue J, Zhou X, Zhang C-X, Yu L-L, Fan H-W, Wang Z, Xu H-J, Xi Y, Zhu Z-R, Zhou W-W, Pan P-L, Li B-L, Colbourne JK, Noda H, Suetsugu Y, Kobayashi T, Zheng Y, Liu S, Zhang R, Liu Y, et al. 2014. Genomes of the rice pest Brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genome Biology 15:21. DOI: https://doi.org/10.1186/s13059-014-0521-0 Yang Z. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Bioinformatics 13: 555–556. DOI: https://doi.org/10.1093/bioinformatics/13.5.555, PMID: 9367129 Yin C, Liu Y, Liu J, Xiao H, Huang S, Lin Y, Han Z, Li F. 2014. ChiloDB: a genomic and transcriptome database for an important rice insect pest Chilo suppressalis. Database 2014:bau065. DOI: https://doi.org/10.1093/ database/bau065, PMID: 24997141 Zhang Z, Yin C, Liu Y, Jie W, Lei W, Li F. 2014. iPathCons and iPathDB: an improved insect pathway construction tool and the database. Database 2014:1–11. DOI: https://doi.org/10.1093/database/bau105

Ye, Xiong, et al. eLife 2020;9:e59795. DOI: https://doi.org/10.7554/eLife.59795 37 of 37