Genomic architecture of endogenous ichnoviruses reveals distinct evolutionary pathways leading to virus domestication in parasitic wasps

Abstract

Background: Polydnaviruses (PDVs) are mutualistic endogenous viruses inoculated by some lineages of parasitoid wasps into their hosts, where they facilitate successful wasp development. PDVs include the ichnoviruses and bracoviruses that originate from independent viral acquisitions in ichneumonid and braconid wasps respectively. PDV genomes are fully incorporated into the wasp genomes and consist of (1) genes involved in viral particle production, which derive from the viral ancestor and are not encapsidated, and (2) proviral segments harboring virulence genes, which are packaged into the viral particle. To help elucidating the mechanisms that have facilitated viral domestication in ichneumonid wasps, we analyzed the structure of the viral insertions by sequencing the whole genome of two ichnovirus-carrying wasp species, Hyposoter didymator and Campoletis sonorensis.

Results: Assemblies with long scaffold sizes allowed us to unravel the organization of the endogenous ichnovirus and revealed considerable dispersion of the viral loci within the wasp genomes. Proviral segments contained species-specific sets of genes and occupied distinct genomic locations in the two ichneumonid wasps. In contrast, viral machinery genes were organized in clusters showing highly conserved gene content and order, with some loci located in collinear wasp genomic regions. This genomic architecture clearly differs from the organization of PDVs in braconid wasps, in which proviral segments are clustered and viral machinery elements are more dispersed.

Conclusions: The contrasting structures of the two types of ichnovirus genomic elements are consistent with their different functions: proviral segments are vehicles for virulence proteins expected to adapt according to different host defense systems, whereas the genes involved in virus particle production in the wasp are likely more stable and may reflect ancestral viral architecture. The distinct genomic architectures seen in ichnoviruses versus bracoviruses reveal different evolutionary trajectories that have led to virus domestication in the two wasp lineages.

Keywords: Campopleginae; Endogenous virus architecture; IVSPERs; Koinobiont; Parasitoid wasp; Polydnavirus.

Fig. 1

Genomic features of Campoletis sonorensis and Hyposoter didymator genomes. a BUSCO analysis of parasitoid wasp genomes (Insecta protein set with 1658 proteins). On the left, results using the genome assemblies; on the right, results using the predicted protein set. b Orthogroups analysis. Left panel: Barplots above each branch of the phylogenic tree indicate the number of orthogroups specific to each species or group of species; the color of the bar indicates the size range of the corresponding orthogroups. Phylogenetic tree was constructed by aligning the complete genomes with Cactus ([49]), converting the resulting HAL alignment to MAF and then to multi fastas with the requirement of full alignment (all taxa present); fasta files were then concatenated into a single matrix (620 kb) and used in a maximum likelihood analysis with RAxML [50] with 1000 fast bootstrap replicates. Asterisks indicate the species carrying polydnaviruses. Right panel: Number of genes for each species that were (i) specific to the species and present either as singletons or duplicates, (ii) present in ichneumonids, (iii) present in braconids, (iv) present in both ichneumonids and braconids, (v) present in all parasitoids, and (vi) present in all hymenoptera. c Heatmaps indicating, for each species pair, the mean number (#) of genes in synteny blocs (SB), the percentage (%) of genes in SBs, and the size of the genome (% nucleotides) in SBs. HDID, Hyposoter didymator (ichneumonid, with PDV); CSON, Campoletis sonorensis (ichneumonid, with PDV); VCAN, Venturia canescens (ichneumonid); MDEM, Microplitis demolitor (braconid, with PDV); FARI, Fopius arisanus (braconid); DALL, Diachasma alloeum (braconid)

Fig. 2

Distribution of ichnovirus sequences within Campoletis sonorensis and Hyposoter didymator genomes. A Schematic representation of ichnovirus sequences within wasp genome scaffolds. (a) C. sonorensis scaffolds containing viral loci. C. sonorensis ichnovirus (CsIV) segments are named CsA to CsX8. Segments CsP and CsL, located in short scaffolds, are not shown. IVSPER-1 to IVSPER-5 corresponds to clusters of replication genes; U36L and U37L to isolated replication genes. (b) H. didymator scaffolds containing viral loci. H. didymator ichnovirus (HdIV) segments are named Hd1 to Hd51. Segments Hd45.1, Hd46, and Hd51, located in short scaffolds, are not shown. IVSPER-1 to IVSPER-5 corresponds to clusters of replication genes; the isolated replication gene U37, located in a short scaffold, is not shown. Complete scaffold list available in Additional file 4: Table S5. B Segments duplicated in H. didymator genome. Segments Hd23 (Genbank# KJ586309.1), Hd44 (Genbank# KJ586285.1) and Hd45 (Genbank# KJ586284.1), described as part of the packaged HdIV genome in [36], have two copies (named Hd(n).1 and Hd(n).2) that are either in the same scaffold (Hd23.1 and Hd23.2, Hd44.1, and Hd44.2) but in different insertion sites or in two different scaffold (Hd45.1 and Hd45.2). Nucleotide percentage identity between the two related segment sequences is given on the right part of the figure. By contrast, Hd9 (Genbank# KJ586324.1), initially described as a separate segment, is located in a genomic locus composed of a tandemly duplicated sequence (“copy 1” and “copy 2” in the diagram). C FISH on H. didymator chromosomes using BAC genomic clones containing HdIV segments. Upper panel shows hybridization using the probes containing segments Hd11 (labeled with FITC) and Hd6 (labeled with rhodamine); lower panel the probes containing viral segments Hd30 (labeled with FITC) and Hd29 (labeled with rhodamine). Each of the probes hybridized with a different H. didymator chromosome: Hd11 hybridized with chromosome #12, Hd6 to a medium-sized chromosome (potentially #5), Hd30 with chromosome #2 and Hd29 with chromosome #11

Fig. 3

Segment DRJ variability in terms of excision sites in Hyposoter didymator. a Schematic representation of the homologous recombination between the two DRJs flanking the proviral sequence (left DRJL and right DRJR ends) to produce the circular molecule (segment) containing one recombined DRJ sequence. When excision sites are located at different positions in the DRJ, segments differing in their recombined DRJ sequence are generated. Excision occurs more frequently at some positions, resulting in different relative amounts of each isoform. On the right, rationale of the algorithm developed to identify the “break points.” Mapping of the segment sequence (DRJsegment) with the two parental DRJs, which differ in their sequences (nucleotide (nt) mismatches), allows identification of the regions where the switch from one parental DRJ to the other has occurred (in the diagram, between the first and second mismatch). b Prediction of putative recombination break points in H. didymator DRJs. Each graph corresponds to the left copy of the DRJ for a given segment (indicated below each graph). The X-axis is the position in the scaffold. The Y-axis indicates the number of reads (obtained from sequencing of the packaged circular DNA molecules) confirming that the circle has been recombined between these two positions, based on the observed mismatches at both end of the segment for each read. We observed between 1 and 80 reads per breakpoint region according to the analyzed segment

Fig. 4

Comparative analysis of Campoletis sonorensis and Hyposoter didymator IVSPERs. A Synteny between the IVSPERs identified in H. didymator (Hd) and C. sonorensis (Cs) genomes. B Synteny of H. didymator genomic regions containing IVSPERs compared with C. sonorensis and other parasitoid genomes. (a) Synteny for H. didymator genomic region containing IVSPER-1 and IVSPER-2 (genes from HD016092 to HD016153); no C. sonorensis scaffold corresponded to the H. didymator IVSPER insertion sites. (b) Synteny for H. didymator genomic region containing IVSPER-4 (genes from HD001703 to HD001771); H. didymator IVSPER-4 and C. sonorensis IVSPER-4 are inserted in the same genomic environment. c Synteny for H. didymator genomic region containing IVSPER-3 and IVSPER-5 (genes from HD002066 to HD002111); in the region where H. didymator IVSPER-3 is inserted, there is conservation in gene order compared to C. sonorensis but no viral insertion; conversely, H. didymator IVSPER-5 and C. sonorensis IVSPER-5 are inserted in the same genomic environment. H. didymator genes from HD010503 to HD010526. Hd: Hyposoter didymator; Cs: Campoletis sonorensis; Vc: Venturia canescens (ichneumonid that has lost the ichnovirus [22]); Md: Microplitis demolitor (braconid with a bracovirus); Fa: Fopius arisanus (braconid with virus-like particles). Numbers following the species name correspond to scaffold number for Hd, Cs, and Vc, NCBI project codes for Md and Fa. Triangles within genomic regions correspond to predicted genes; triangles of the same color correspond to orthologs; white triangles are singletons or orphan genes. For better visualization, the name of the gene is indicated only for some viral (in red for segments, in blue for IVSPERs) genes. See Additional file 13: Table S13, for H. didymator genes list

Fig. 5

Steps of virus domestication in ichneumonids. Following integration of an ancestral virus genome in a wasp chromosome, the viral sequences were maintained but underwent significant modifications over evolutionary time. The viral sequences including genes necessary to produce particles (IVSPERs) were conserved through evolution, although they have undergone fragmentation and gene duplications and have lost some genes, including the viral DNA polymerase. The encapsidated sequences (proviral segments) include virulence genes involved in promoting parasitism. Ichnovirus segments likely derive from ancestral viral sequences that have acquired virulence genes from the wasp. Both proviral segments and IVSPERs are amplified in the replicative tissue in a coordinated manner suggesting regulation by a common mechanism and that they may both derive from the ancestral virus. Following amplification, viral segments are excised via homologous recombination or single-strand annealing mechanism (depicted) involving the direct repeated junctions