Two novel venom proteins underlie divergent parasitic strategies between a generalist and a specialist parasite

Overall similar genomic and functional repertoires of specialist and generalist wasps

We first sequenced and de novo assembled the reference genomes of Lh and Lb. The generalist species Lh was fully sequenced with PacBio long reads (Supplementary Table 1), which yielded a 487 Mb genome assembly with high continuity (N50 = 2.18 Mb; Supplementary Table 2). The specialist species Lb was sequenced and assembled with 170-fold Illumina read coverage and paired-end sequencing data from five long-insert libraries (up to 13 Kb; Supplementary Table 3). Although the assembled Lb genome had a lower N50 size (480 Kb) and smaller genome size (324 Mb), both the Lh and Lb genomes showed high completeness based on BUSCO and CEGMA assessments (Supplementary Table 2). The difference in genome sizes between Lh and Lb was likely determined by their repeat contents, as 51.47% of the Lh genome was annotated as repeat content in comparison with a 33.79% ratio in the Lb genome (Supplementary Table 4). The average GC contents of the Lh and Lb genomes were 27 and 26%, respectively, indicating that Leptopilina genomes are remarkably AT-rich. Interestingly, unlike the uniform distribution in Lb, the Lh genome shows a secondary peak enriched with scattered genomic windows of remarkably low GC content (16%; Supplementary Fig. 2).

We identified 11,881 and 11,054 protein-coding genes as the official gene sets for Lh and Lb, respectively (Supplementary Table 5). These gene sets were mainly generated by an integrated pipeline, and those retained had evidence from either a full-length transcriptome of pooled developmental stages or high-confidence homology with Insecta genes (see “Methods”). Ortholog analyses across 10 hymenopteran genomes suggested that the genomes of both these parasitoid species maintained a typical hymenopteran gene repertoire (Supplementary Fig. 3). Compared with those of other sequenced insects, the gene numbers of these two Leptopilina species are relatively small, largely due to their low proportions of patchy genes that were not vertically inherited along speciation (Supplementary Fig. 3). We manually annotated several representative gene families or pathways that are strongly associated with the biology of parasitoid wasps (Fig. 1c). The genomes of Leptopilina encode more olfactory receptor (OR) genes than those of other parasitoids except Nasonia vitripennis, while they encode the fewest gustatory receptor (GR) genes. Genes associated with metabolic and immune pathways in Leptopilina were shown to be more or less the same as those in other hymenopteran species. Because consistent patterns within a phylogenetic context were not available, we did not gain informative insights into the gene family variations underlying the divergence between Leptopilina and other Parasitoida species.

The divergence time between the two Leptopilina species was estimated to be approximately 40 Mya, and those between the sublineages of Parasitoida (e.g., Cynipoidea and Chalcidoidea) were estimated to be as distant as hundreds of millions of years (Fig. 1c), consistent with the tremendous diversification of parasitoids^17,18. Thus we focused on making detailed comparisons within Leptopilina. The gene repertoires of Lh and Lb shared 8050 (~75%) orthologous pairs (Fig. 1d) with an average sequence identity of 85.6% at the amino acid (aa) level. We calculated the ratio of synonymous-to-nonsynonymous substitutions (dN/dS) to characterize the genes and functional modules associated with the divergence between Lh and Lb. Only four pathways were found to have significantly elevated dN/dS values (Z-test P < 0.01), including the bacterial secretion system as the most extreme outlier and, unexpectedly, the circadian rhythm pathway (Supplementary Fig. 4). By contrast, we identified 25 pathways presenting dN/dS values of approximately zero, indicating strong purifying selection. Among these highly conserved pathways between Lh and Lb, we found several sensory systems, including taste transduction as the most conserved pathway and those associated with photoreception and olfaction (Supplementary Fig. 4 and Supplementary Data 1). Interestingly, we noted that the genome of the specialist Lb encoded more chemoreception genes, including ORs, GRs, and ionotropic receptors (IRs), than that of the generalist Lh (Fig. 1c). These genomic signatures did not provide evidence to support a role of environment-sensing modules, e.g., host seeking, in driving the change of host ranges in Leptopilina.

We further compared the gene repertoires of Lh and Lb at the level of expression. To profile a dynamic pattern, we sampled developmental stages across the entire lifecycle and subjected each sample to RNAseq (Supplementary Table 6). The VGs were independently sequenced, given their tiny volumes but remarkable roles in the biology of parasitoid wasps¹⁹. Hierarchical clustering analyses revealed that one-third of the 8050 orthologous genes were expressed across all developmental stages in both species, that most genes were co-expressed in both species at one or more corresponding stages, and that highly expressed genes were co-opted by all developmental stages of both species, except in the VGs (Fig. 1e). By clustering the stages based on their overall expression patterns, we found that the early developmental stages (e.g., the egg and larval periods) of Lh and Lb were intercrossed together, whereas the later stages, particularly the late pupal stage (P3), differed between the species (Fig. 1e and Supplementary Fig. 5). The VGs showed a different expression pattern with all developmental stages and a large amount of species-specific expression, indicative of a unique regulatory system and rapid turnover between species (Fig. 1e and Supplementary Fig. 5). Our results suggest that the lifecycles of Lh and Lb were synchronized at the beginning of development, followed by a shift ahead in the late pupal stage of Lh.

A particular role of VGs in the divergence between the two species

Of the approximately 3000 non-orthologous genes, Lh and Lb encoded 1058 and 972 species-specific genes, respectively, most of which lacked clear homology outside Leptopilina (Supplementary Data 2 and 3). We inferred their potential roles based on the transcriptome and paid close attention to those with evident expression. A total of 740 Lh-specific genes (70%) were expressed in at least one stage, including 88 at significantly high levels, whereas 529 Lb-specific genes (54%) were expressed, and only 12 were highly expressed. Generally, highly expressed genes are skewed toward a high proportion of universal genes²⁰. In comparison with the whole set, highly expressed genes were indeed underrepresented in the Lb-specific set across all developmental stages, though not in the VG. By contrast, we did not find evident underrepresentation in the Lh-specific set but were surprised to find significant overrepresentations in the adult stage and particularly in the VGs (Fig. 1f).

Based on the transcriptome analyses, 4274 and 4538 genes were found to be expressed in Lh VGs and Lb VGs, respectively, accounting for approximately one-third of the whole gene set. Of the 50 genes with significantly high expression in Lh VG (Z-test, P < 0.05), a majority (78%) were exclusively expressed in the late pupal stage (P3) and female adults (AF), when VGs initially formed and developed, respectively (Supplementary Fig. 6). These expression signals were very likely to be contributed by the inclusion of VGs, given that the proportion of genes with exclusively concurrent expression in P3 and AF was extremely low (0.83%) in the whole set (hypergeometric test, false discovery rate (FDR)-adjusted P < 0.001; Supplementary Fig. 7). By contrast, many (60%) highly expressed genes in Lb VGs were found to have broad expression across development (Supplementary Fig. 6); none of them were found to have specialized expression in P3 and AF (Supplementary Fig. 7). Furthermore, we found that a majority (62%) of highly expressed genes in the Lh VG were species specific, whereas many of the highly expressed genes in the Lb VG had well-annotated insect homology (Supplementary Figs. 8 and 9). All the described distinct patterns between Lh and Lb concordantly suggest remarkable expression specialization of novel venom genes in the unique adaptation of Lh.

Indeed, the need to adapt to overcoming host immune responses and changing host ranges drives parasitoid venom repertoires to evolve quickly and to acquire novel functions²¹. In Lh and Lb, venom is produced in the VG, mostly packaged in virus-like-particles (VLPs), and stored in the venom reservoir (VR)^22,23. These VLPs are then injected along with the wasp egg during oviposition. We further studied the relevant functions of Lh and Lb venoms, which participate in reproductive success. Similar to previous reports¹³, we confirmed the absence of lamellocytes in unchallenged D. melanogaster larvae (Fig. 2a). In contrast, large quantities of lamellocytes were produced 48 h after attack by Lb (Fig. 2a). As expected, oviposition by Lh led to no induction of lamellocytes in parasitized larvae, which appeared comparable to unchallenged Drosophila larvae (Fig. 2a). It is well known that lamellocytes are mostly produced by the lymph gland (the hematopoietic organ) to encapsulate large foreign bodies¹⁴. Thus we dissected the lymph glands from host larvae that were parasitized by the two Leptopilina wasp species. We found that Lh parasitization caused rapid lysis of the host lymph gland 24 h after wasp egg laying, while the lymph gland was intact in Lb-parasitized larvae 24 h post infection (Fig. 2b). To determine whether the Lh venom was responsible for the lysis of the host lymph gland, we isolated venom fluid from the VRs of the two wasps and injected each of them directly into a host. After 24 h, we found that the injection of Lh venom caused loss of the host lymph gland, while the venom of Lb showed no effect (Fig. 2c). In combination, these results indicate that the venom of Lh has evolved much greater virulence than that of Lb and subsequently leads to a nearly complete lack of circulating lamellocytes in the attacked hosts by inducing apoptosis in the lymph gland (Fig. 2e).

A novel venom protein allows Lh to overcome host immunity

Thus the venom of Lh is very likely to be responsible for its immune suppression strategy. Since the genes expressed in the VG are not necessarily translated and secreted into the reservoir, we further generated proteomes from both Lh and Lb venom samples and integrated them with the venom transcriptomes to characterize reliable venom proteins. As expected, only a small set of VG-expressed genes were found to have solid proteome evidence (Fig. 2d), and these were determined to be venom protein-coding genes (VPs; see “Methods” and Supplementary Data 4). Of all the identified VPs (135) in Lh, a pair of closely related paralogs (LhOGS04147 and LhOGS20123) attracted our attention due to their extremely high expression in the venom (the first and sixth highest of all VPs, respectively), their marked expression specialization, the signature of rapid evolution (the between-sample dN/dS is 0.8392, higher than 99.9% of Lh–Lb orthologs; Supplementary Fig. 10), and the absence of homologs outside Lh (Fig. 2d).

We did not characterize any known functions or conserved domains for these two genes (see “Methods”), while the three-dimensional (3D) modeling predicted a hydrolase-like structure (Supplementary Fig. 11). We explored their potential functions in vivo via RNA interference (RNAi) experiments. Despite a lower expression value, LhOGS20123 showed a more marked signature than LhOGS04147 in the proteome (Supplementary Data 4). The specialized expression of LhOGS20123 in VGs was also confirmed by immunohistochemistry and western blot analysis (Supplementary Fig. 12). Double-stranded RNAs (dsRNA) of LhOGS20123 were injected into fifth instar larvae of Lh, and quantitative real-time PCR (qRT-PCR) showed that gene expression decreased by 85% in emerged wasp adults (Supplementary Fig. 13a). In comparison to dsGFP-injected wasps, we found a significant suppression of apoptosis in the host lymph gland and a correspondingly large population of lamellocytes in host larvae when parasitized by LhOGS20123-knockdown wasps (Fig. 2e, f and Supplementary Fig. 14). Knocking down this gene in female Lh also largely resulted in the predomination of melanotic capsules in infected hosts and widespread encapsulation of wasp eggs, which was rarely observed in the hosts parasitized by dsGFP-injected Lh (Fig. 2f and Supplementary Fig. 14). Given its substantial role in the lysis of lymph glands, we named this gene Lar (lymph gland apoptosis-related protein). We then performed an assay to detect whether parasitic efficiency was impaired in dsLar-injected female wasps due to a lack of the ability to repress the host encapsulation response. We found that the oviposition ability of Lh was unaffected by dsLar treatment (Fig. 2g), whereas the parasitism rate was dramatically decreased, with only 27% of wasp offspring successfully emerging in Drosophila hosts compared to 77% when parasitized by either normal Lh or dsGFP-treated Lh females (Fig. 2h). The results of antibody staining showed that Lar was readily detected in host lymph glands 12 h after Lh parasitization but not in other tested tissues (Fig. 2i, j’ and Supplementary Fig. 15). Though it is still not clear how Lar is specifically delivered into the host lymph gland in such a short time, it is possible that VLPs that are present in the Lh female venom apparatus may help to facilitate its entrance. Interestingly, VLPs have been renamed mixed strategy extracellular vesicles (MSEVs) or venosomes because there is no solid evidence of a viral origin^22,24. It will be both interesting and urgently necessary to identify the key elements that are in charge of delivering VPs into specific host tissues rapidly and precisely.

Unlike Lar, its paralog, LhOGS04147 (referred to as Lar’), was not effective with respect to suppressing host encapsulation response. As with previous approaches, silencing Lar’ did not rescue the parasitization-induced apoptosis of the host lymph gland or lead to the development of obviously melanotic capsules in parasitized Drosophila larvae (Supplementary Fig. 13). Most importantly, the parasitism rate and wasp offspring emergence rate were not changed when Drosophila larvae were parasitized by Lar’-silenced Lh female wasps compared to control wasps with dsGFP treatment (Supplementary Fig. 13). We additionally knocked down the other seven genes out of the ten VP genes of highest expression (i.e., LhOGS06609, LhOGS10118, LhOGS01638, LhOGS01639, LhOGS01180, LhOGS02019, and LhOGS00546; see Supplementary Data 4) and two VP genes annotated with the highest peptide number (i.e., LhOGS20077 and LhOGS08557; see Supplementary Data 4). Although qRT-PCR showed that the expression levels of these nine genes were all significantly reduced upon the injection of corresponding dsRNAs (Supplementary Fig. 16a), the parasitism rate was not found impacted in the host larvae parasitized by eight dsRNA-treated lines of wasps, with the only exception in the dsLhOGS06609-treated line (Supplementary Fig. 16b). In comparison to the control, the parasitism rate was significantly but marginally reduced upon the parasitization of dsLhOGS06609-treated Lh (Supplementary Fig. 16b). However, we found that knockdown of LhOGS06609 did not relieve the apoptosis of the host lymph glands (Supplementary Fig. 16c).

Taken together, these results suggest that Lar plays an important and unique role in provoking cell death in the lymph gland and hence suppress the host encapsulation response, leading to successful parasitism, and that some other VPs have minor effect on the parasitic efficiency of Lh.

Recruitment of novel VPs arose from LGT followed by rapid evolution

We next traced the origin and genomic evolution of this functionally important Lh-specific gene. Microbial contamination may cause false positive results when identifying species-specific genes. However, Lar was unlikely to be identified as part of a chimeric scaffold, given that the genome of Lh was continuously assembled using long reads. In addition, two full-length transcripts revealed that Lar bears an 8.6 kb intron (Supplementary Fig. 17), whereas bacterial or phage genes are generally intronless^25,26. In fact, the complete structure of Lar was fully embedded in the first intron of another reverse-strand-located gene, RRP8 (Supplementary Fig. 17). RRP8 is widespread across insect species and occurs in a short microsynteny block within Leptopilina (Lh, Lb, and L. clavipes, Lc; Supplementary Fig. 18)²⁷. Despite its conserved exon–intron structure, the first intron of RRP8 showed diverse architectures among the three Leptopilina species (Supplementary Fig. 17): (1) the copy in Lc was the simplest; (2) the copy in Lb shared two homologous segments with that in Lc, but one was disrupted by both a segmental duplication and an unfilled gap (see the figure legend); (3) the copy in Lh was replaced with the complete structure of Lar and its flanking, highly repeated, highly AT-rich (91%) sequences. On the other hand, the paralog of Lar, Lar’, which has 73% sequence identity at the nucleotide level (Supplementary Fig. 10), was located on a different scaffold. The orthologs of the two genes adjacent to Lar’ were located on different scaffolds in Lb and Lc, but they were adjacent in a more distantly related wasp species (N. vitripennis, Chalcidoidea) (Supplementary Fig. 19); that is, the local synteny was of ancient origin (prior to the divergence between the two lineages of parasitoids, 160 Mya (Fig. 1c)) and then was broken during recent evolution within Leptopilina. Lar’ showed both the same exon–intron structure and the same expression pattern as Lar (Supplementary Fig. 10) and, more importantly, was also surrounded by dispersed repetitive sequence blocks. Repetitive sequences increase the potential for duplication, deletion, and transfer across genomes. Indeed, Lh greatly differed from Lb in having a larger genome size and a unique enrichment of AT-rich genomic regions (Supplementary Fig. 2), suggesting more rapid turnover in the genome of Lh (Supplementary Table 4). Hence, we infer that Lar and Lar’ originated recently from a common ancestor and were translocated into two separate repeat-rich genomic loci.

Where did the ancestral copies of these two novel genes come from? We searched for putative homologs in the NCBI nonredundant (NR) protein database and found only a few hypothetical proteins with low aa identities (20–30%). Beyond their scattered distribution in a few arthropods, all these hits belonged to microbial and protist species (Fig. 3a). This absence in all metazoans outside the arthropods indicates that these arthropod genes originated from one or more LGT events. Their patchy distribution across arthropods raised the possibility that homology might have been overlooked by protein-coding gene prediction, which might miss genes due to either degraded structures or a deficiency of strong homology. To characterize the traces of Lar as completely as possible, we directly searched for its potential homologs in the genomic sequences of Lh and Lb (see “Methods”). A total of 94 and 6 genomic loci were identified in the Lh and Lb genomes, respectively (Supplementary Data 5). Indeed, most of these loci were not predicted in our official gene sets due to a lack of sufficient evidence, although they shared a relatively conserved blocks with G1-motif signature (Supplementary Fig. 20)²². Despite the uncertainty that these sequences were true protein-coding genes, the presence of such widespread G1 motifs suggests a more ancient origin and broader distribution than we expected. We further performed the same iterative searches to determine whether these motifs were underrepresented across the deposited gene sets of other species. Out of the 236 Hexapoda genomes we examined (including three Leptopilina species; see “Methods”), two Collembola species and 19 insect species from four orders were found to harbor at least one gene encoding the G1 motif (Fig. 3a and Supplementary Fig. 21). 3D modeling further predicted a Lar-like structure between distantly related sequences (Supplementary Fig. 11).

We reconstructed a maximum-likelihood (ML) phylogeny using all the recovered sequences and representative hits from NR, with prokaryotic sequences as outgroups. The phylogeny resolved three main clades (Fig. 3b), of which one (clade III) contained most of the wasp sequences except 11 sequences from three Chalcidoidea species, one (clade II) contained all the sequences from a Trichoptera species, and one (clade I) contained all the remaining arthropod sequences. Of the 269 recovered genes, 58% were from parasitoid wasp species, with only a minor proportion, all from Chalcidoidea, being placed together with the other Arthropod ones in clade I (Fig. 3). Interestingly, the Chalcidoidea parasitoid, N. vitripennis, is the only species that simultaneously retains the copies of clades I and III. However, phylogenetic analysis separated these copies by non-Metazoan copies (Supplementary Fig. 21). The deep split between these clades and the phylogenetic conflict between gene and species tree topologies both strongly suggest that the scattered presence of these Arthropod homologs were likely to originate from multiple, independent LGTs and experience distinct fates. LGTs of clade I probably has an ancient origin of transferring to the common ancestor of Arthropods and underwent massive losses independently, whereas the clade III, which arose Lar, likely took place prior to the divergence between Cynipoidea and Chalcidoidea wasps (>200 Mya; Fig. 1c) and was largely retained in Cynipoidea lineage (Fig. 3b). Due to the substantial divergence, we cannot pinpoint a precise donor as the source of these foreign DNA.

According to the phylogeny within clade III, we found that the Lh genes with specialized expression in venom were remarkably separated from those with broad expression and that the sequences from all other parasitoid wasps were clustered with the latter (Fig. 3a and Supplementary Fig. 22). A possible explanation is that the LGT underwent massive duplication along with its regulatory elements as a whole; evolved variations might give rise to changes in expression pattern and hence result in a functional split. The sublineage with VG specialization was retained or even further expanded in Lh but completely lost in all other species, whereas the sublineage with broad expression had the potential for neofunctionalization and hence was modestly retained in most Cynipoidea wasps. Due to the lack of definitive support in the phylogeny, it was difficult to infer the chronological order of the duplication and functional split. From the expanded group of genes with specialized VG expression, we chose LhOGS20047, the most highly expressed member after Lar’ and Lar (Supplementary Data 5), to test whether it had a similar function to that of Lar. Despite the proteome evidence, RNAi of this gene, similar to that of Lar’, did not relieve the lysis of the host lymph gland or affect the rate of parasitism and wasp offspring emergence (Supplementary Fig. 13). Thus it seems that most members of the expanded gene family did not evolve with venom-related functions despite their specialized expression in the VGs, which might partially explain why they were completely lost in other species. Further specific functional studies would help understand how the unique sequence evolution in Lar contributes to the functionalization regarding immune suppression.

Acquisition of a set of mucin-binding domain-containing genes helps Lb evade host immunity

It is intriguing that Lh exclusively recruited an LGT to perform a novel venom function that confers a new strategy for parasitic success. We next explored whether the specialist, Lb, had evolved alternative strategies for parasitic success through the acquisition of novel genes. The massive expansion of Lar across the genome suggests a particular role for gene duplication associated with the acquisition of novel genes. A small set (126) of Lb-specific genes were multiple copy, and we noticed that 11 of these genes formed the largest paralogous group (Supplementary Data 3) and that 9 of them encoded the putative mucin-binding domain (mucin-bd, IPR004954). This domain was also the most extreme outlier in an expansion/contraction analysis across all annotated InterPro domains between Lh/Lb and other hymenopterans; it was present in nine Lb genes but absent in all ten of the other hymenopteran gene sets we examined (Fig. 4a and Supplementary Data 6). The mucin-bd was documented in 260 sequences from 85 species in the Pfam database (PF03272) and in 2444 sequences from 553 species in the InterPro database (as of Feb. 29, 2020) (Supplementary Data 7 and 8). Of these sequences, 97.9% were bacterial, 2% were from viruses or fungi, and only 3 sequences belonged to a metazoan species (Parasteatoda tepidariorum, Arachnida) (Supplementary Fig. 23). Domain alignment did not show any homologous segments (E < 1e−5) between the nine Lb genes and those from metazoan or fungi species. Correspondingly, BLAST searches against NR identified only bacterial hits, mainly as viral enhancing proteins or hypothetical proteins, with modest conservation, for these nine genes (as of Feb. 29, 2020; Fig. 3b and Supplementary Table 7). Unlike the scattered distribution of Lar across arthropods, the above patterns suggest a more straightforward LGT that resulted in the widespread presence of the mucin-bd domain across the Lb genome. Correspondingly, several genera of bacteria enriched with mucin-bd domains were able to be recovered at the genus level from the microbiota sequencing of Lb guts and female whole bodies (Supplementary Fig. 23 and Supplementary Data 9).

We hypothesized that this bacterial domain was recruited as a parasitic strategy for Lb to circumvent host immunity. This microbial domain represents a putative binding domain for the substrates of enhancin, which is involved in the disruption of the peritrophic membrane and the fusion of nucleocapsids with midgut cells²⁸. To test this hypothesis, we used the RNAi technique to knock down each of the nine mucin-bd domain genes individually. The qRT-PCR results showed that the expression levels of these nine genes were significantly reduced in Lb adults after injection of the corresponding dsRNA into fifth instar Lb larvae as previously described (Supplementary Fig. 24). We next used 3-day-old dsRNA-treated Lb female wasps to parasitize Drosophila host larvae, with dsGFP-injected wasps as a control. Similar to our previous findings, >95% of wasp eggs were gradually attached to the host internal tissues (mainly host gut) by 4 h after parasitization by dsGFP-injected Lb (Fig. 4b); this strategy provides passive, physical protection against complete encapsulation by host hemocytes, and thus the wasp larvae can escape from the attachment site during hatching. The attachment rate of Lb eggs showed a significant reduction in Drosophila hosts when parasitized by LbOGS06929 knockdown female wasps (Fig. 4b). Thus LbOGS06929 was named Warm (wasp egg adhesion-related protein with a mucin-bd domain). However, the attachment rates of Lb eggs laid by female wasps with silencing of the other eight mucin-bd domain-containing genes were not impaired and were comparable to those of the dsGFP-treated control (Fig. 4b). Meanwhile, a significantly decreased parasitism rate was found when the hosts were parasitized by dsWarm-injected Lb but not by other treated wasps (Fig. 4c). Examining the Lb venom proteome, we found that Warm was also a venom protein that could be injected into the host body along with the wasp eggs (Fig. 4d). In contrast to the findings that injected venoms often induce cytotoxic effects on hemocytes, our results suggest that Warm is responsible for the adhesion of parasitoid eggs to host tissues and helps Lb wasps adopt an immune evasion strategy to overcome the host encapsulation reaction for successful parasitism.

Why does only one of these mucin-bd-containing genes show functional importance? Similar to previously documented genes in Pfam, the nine mucin-bd-containing genes in Lb had diverse domain architectures and were highly variable (Fig. 4d), indicating rapid evolution. Two genes (LbOGS00358 and LbOGS05722) showed substantial sequence degradation (Fig. 4d). Of the seven remaining genes, five showed specialized expression in the early larval period (L1), and two others, including Warm, had strong expression in the venom, despite their higher values in L1 (Fig. 4d). However, only Warm was present in the proteome. Two of the five specialized genes, LbOGS02281 and LbOGS04370, retained the most complete architecture compared to their bacterial homologs, with orders of magnitude higher level of possibility than those of the other genes (Supplementary Table 7). Thus we propose a scenario in which the LGT was basally expressed specifically in early larvae and then underwent gene co-option by the venom, resulting in novel venom functions.

The diverse parasitic strategies between Lh and Lb may contribute to the shift in host range

Lh and Lb are closely sympatric species. Lb is an obligate parasitoid of D. melanogaster, whereas Lh is a generalist parasitoid with a wide host range within Drosophilidae¹². We have characterized Lar and Warm that enable Lh and Lb to overcome the immune response of D. melanogaster in dramatically different ways. We next tried to address whether these diverse parasitic strategies might contribute to their different host ranges. We tested the parasitic efficiencies of Lh and Lb in eight Drosophila species, including five species in the melanogaster subgroup (D. melanogaster, D. simulans, D. yakuba, D. santomea, and D. erecta) and three species outside the melanogaster subgroup (D. suzukii, D. pseudoobscura, and D. virilis). We found that both Lh and Lb female wasps oviposited in the larvae of all eight species, regardless of whether parasitoid development succeeded. As expected, Lh could successfully parasitize almost all the species we tested, with a parasitism rate ranging from 82 to 92% and an offspring emergence rate of >58% (Fig. 5a). The only exception was D. suzukii, in which Lh was unable to complete development (Fig. 5a), consistent with previously described results^12,29. As a specialist, Lb showed high parasitism rates (86 and 87%, respectively) and offspring emergence rates (69 and 62%, respectively) only in D. melanogaster and its close relative, D. simulans. Lb failed to develop in the other six Drosophila species, resulting in few Lb wasps, if any, emerging from these hosts (Fig. 5a).

Host immune responses can result in encapsulation of parasitoid eggs, which leads to the death of the parasitoid offspring¹⁴. Fly larvae generate the lamellocytes used for encapsulation after wasp infection. We demonstrated that Lh used an active strategy to suppress the host immune response by inducing apoptosis in the lymph glands of parasitized D. melanogaster. Accordingly, we found that the lymph glands disappeared in the Lh-parasitized larvae of all the other Drosophila species at 24 h after parasitization, except D. suzukii (Fig. 5b). We next used RNAi to test the role of Lar in the lysis of lymph glands in these other Drosophila species. Knocking down Lar resulted in a significant rescue of the loss of lymph glands, which subsequently resulted in strong encapsulation responses in parasitized species of the melanogaster subgroup (Fig. 5a, b). Therefore, silencing Lar in the VG resulted in a failure to parasitize these Drosophila hosts. Interestingly, no encapsulation responses were found in D. pseudoobscura and D. virilis, although their lymph glands were intact after parasitization by dsLar-treated Lh (Fig. 5a, b). Thus the high parasitic efficiency in D. pseudoobscura and D. virilis is probably because the lymph glands lacked the ability to differentiate lamellocytes. Indeed, we found that the lymph glands in D. pseudoobscura and D. virilis were small compared to those in the other species (Fig. 5b). Some studies have revealed that most Drosophila species outside the melanogaster subgroup usually do not differentiate lamellocytes upon wasp infection³⁰. Collectively, these results suggest an evolutionary scenario in which the emergence of encapsulation responses in a common ancestor of melanogaster subgroup flies might drive novel adaptive strategies in which the effective suppression of host cellular immune responses by Lh might contribute to its colonization of a wide range of hosts.

In contrast to Lh, the specialist species Lb has evolved a relatively passive strategy to parasitize D. melanogaster, i.e., through attaching the eggs to host tissues to escape encapsulation with the help of Warm. Our assay showed that both the parasitism rates and offspring emergence rates of Lb were as high as those of Lh when parasitizing D. melanogaster and D. simulans (Fig. 5a). When Lb parasitized other Drosophila species, we found that the encapsulation responses of the hosts were widely activated, except those of D. pseudoobscura and D. virilis, and that the eggs of Lb also attached to host tissues in all species (Fig. 5a). It is apparent that other factors hindered the further development of Lb wasp eggs in these species; these factors may be related to physiological unsuitability³¹. These observations suggest that the passive immune evasion strategy might enable parasitoids to adapt to a specialized host as successfully as the aggressive strategy but might not support extending the host range.

Source: Ecology - nature.com