Fig 1.
Genome of H. didymator and position of HdIV loci.
Position of the HdIV loci previously identified in [16] within the twelve large scaffolds obtained using Hi-C technology. The HdIV replication genes, organized in Ichnovirus Structural Protein Encoding Regions (IVSPERs) or that are isolated in other regions of the H. didymator genome, are highlighted in red while viral segments are indicated in black. The scaffold diagram was generated using Mapchart software [29]. At the bottom of the figure, diagrams illustrate the difference between the two types of viral loci: the IVSPER which are clusters of HdIV replication genes inherited from the IV proviral ancestor, and the viral segments flanked by direct repeats located on the left (DRL) and right (DRR) sides of segments, housing virulence genes expressed in the parasitized insect.
Fig 2.
DNA amplification of HdIV loci.
(A) qPCR analysis of selected IVSPER genes in calyx cells during wasp pupal development. Top panel. A schematic representation of H. didymator IVSPERs-1, -2, and -3 (GenBank GQ923581.1, GQ923582.1, and GQ923583.1); genes selected for qPCR assays are highlighted in white. U1-U24 are unknown protein-encoding genes, while IVSPs are members of a gene family encoding ichnovirus structural proteins. Bottom panel. Genomic (g) DNA amplification levels of IVSPER genes and wasp XRCC1 in calyx cells from pupal stage 1–4. The XRCC1 (X-Ray Repair Cross Complementing 1) encoding gene is located 1,200 bp from U1 (position 3,270,470 to 3,272,519 in Scaffold-11). Data correspond to gDNA amplification relative to amplification of the housekeeping gene elongation factor 1 (ELF1). The Y-axis was transformed using the square root function for better data visualization. Statistical test results are available in Table B in S2 Dataset. (B) Read depth of HdIV loci compared to the rest of the wasp genome. Read depth values per analyzed region (see Materials and methods) are presented for each locus type (proviral segments and IVSPERs) at pupal stage 1 (hyaline pupa) and pupal stage 3 (pigmented pupa). The read depths per HdIV locus (HdIV) are compared to the read depth per random genome regions outside of HdIV loci (wasp). Note that the read depth value for random wasp regions is lower for DNA samples collected from stage 3 versus stage 1 pupae. This difference is attributed to the higher proportion of reads mapping to HdIV regions among the total number of reads in stage 3 compared to stage 1. The significance levels are indicated as follows: ns = non-significant, **p<0.01, and ***p<0.001. Statistical test results are available at https://github.com/flegeai/EVE_amplification/blob/main/notebooks/Count_tables.ipynb.
Fig 3.
DNA amplification in pupal stage 3 was assessed by mapping genomic DNA Illumina reads against the 12 large H. didymator genome scaffolds. In each scaffold, red bars indicate amplified loci, with the intensity of red corresponding to increased values of the counts per million (CPM) ratio between pupal stage 3 and pupal stage 1. The positions of IVSPERs and isolated IV replication genes are indicated by purple squares, while proviral segments are indicated by green circles. For selected HdIV loci, amplification curves (representing the ratio of the CPM values calculated for 10 bp intervals between pupal stage 3 and pupal stage 1) are shown in boxes. Amplification curves for all of the annotated HdIV loci are shown in S1 Fig. Each HdIV locus is indicated in red while 10,000 bp of flanking sequence on each side of the locus is also shown. For proviral segments, loci are defined as the sequence delimited by two direct repeats; IVSPERs are defined as the region between the start and stop codon of the first and last coding sequences in the cluster; isolated IV replication genes are defined by their coding sequence.
Fig 4.
HdIV amplified regions in Scaffold-11.
(A) Detail of the amplified region at the Hd11 locus. Hd11 (red bar) represents two overlapping segments Hd11-1 (defined by left and right DRs, DR1L and DR1R respectively, blue bar) and Hd11-2 (defined by DR2L and DR2R, orange bar). (B) Detail of two other amplified regions containing IVSPERs and HdIV proviral loci. In (B), amplification curves of IVSPERs are highlighted in yellow. In (A) and (B), amplification curves represent the ratio of the CPM values (calculated for 10 bp intervals) obtained in pupal stage 3 compared to pupal stage 1. For each locus, amplification values at the summit of the peaks (bold type) and at the start and end positions of HdIV segments are indicated with arrows. Each amplification curve figure was generated by Integrated Genome Viewer (IGV) [31].
Table 1.
HdIV loci amplified in calyx cells from stage 3 pupae identified by read mapping and/or the MACS2 algorithm.
For each scaffold, the position and size of the HdIV loci are indicated. Loci newly identified in the present work are marked with asterisks. Corresponding amplified regions (i.e., the peak predicted by the MACS2 algorithm) are provided for each locus or groups of loci. Start and end positions delimiting the HdIV loci and the amplified regions detected by MACS2 are indicated. The distance between the start or the end of the amplified region and the locus is presented. For each HdIV locus and amplified region detected by MACS2, read depth values are provided for calyx cell samples collected from stage 1 or stage 3 pupae. Read depth is based on the length of the HdIV locus or the amplified region. ND indicates amplified regions not detected by MACS2.
Fig 5.
U16 proteins were identified in the campoplegine Hyposoter didymator [12], Campoletis sonorensis [16], and Bathyplectes anurus [18], and in two banchine wasps Glypta fumiferanae [13] and Lissonota sp. [34]. (A) Phylogenetic tree of U16 proteins inferred using a maximum-likelihood method. Branch supports are labeled near the nodes. (B) Identity heat map of U16 proteins. (C) Alignment of U16 proteins. The predicted PriCT-2 domain is indicated by the red outline. In panels (B) and (C), increased blue intensity depicts a stronger degree of identity between the proteins. Abbreviations: Hd, H. didymator; Cs, C. sonorensis; Ba, B. anurus; Gf, G. fumiferanae; Lsp, Lissonota sp.
Fig 6.
(A) RT-qPCR data showing relative expression of U16 in dsGFP (control) and dsU16 injected females. ** p<0.01. Images of ovaries dissected from newly emerged adult females that were injected with dsGFP (left) or dsU16 (right). Note the blue color in the oviduct of the dsGFP control indicating the presence of HdIV virions. (B) Schematics and electron micrographs showing that (a) calyx cell nuclei (N) from females treated with dsGFP-injected contain subvirions (V) while (b) calyx cell from a dsU16-injected wasps do not. This results in no accumulation of virions in the calyx lumen as illustrated in the schematic images. Abbreviations: CL, calyx lumen; Cyt, cytoplasm. Scale bars = 5 μm, zooms = 1 μm.
Fig 7.
Impact of U16 RNAi knockdown on DNA proviral amplification.
(A) Comparative distribution of read depths in dsGFP- and dsU16-injected females. Read depth values in three biological replicates are given for HdIV loci (V) and random genomic regions outside of HdIV loci (W) with equal size distribution. IVSPERs and IV replication genes loci are shown in the left panel, while proviral segment loci are shown in the right panel. (B) Read depth values for IVSPERs and IV replication genes residing outside an IVSPER in three biological replicates treated with dsU16- or dsGFP. Names of HdIV loci and the scaffold (Scaf-) they are located are indicated. (C) Read depth values for proviral segment loci in the three biological replicates of the dsU16 and dsGFP samples. For better visualization, only the scaffold (Scaf-) on which the proviral segments are located is indicated. The list of the proviral segment loci within each scaffold is available in Table 1. The y-axis was transformed by the log function for better data visualization. In (A), significance levels are indicated as follows: **p<0.01, and ***p<0.001. In (B) and (C), all differences between dsU16 and dsGFP samples are statistically significant at p<0.05. Statistical test results are available at https://github.com/flegeai/EVE_amplification/blob/main/notebooks/Count_tables.ipynb.
Fig 8.
Impact of U16 RNAi knockdown on amplification of selected wasp and HdIV genes.
Relative genomic amplification of select HdIV genes in two-day-old females injected with dsGFP or dsU16. The wasp gene XRCC1, located within the amplified region of the IVSPER-1 locus, was incorporated into the analysis. Wasp histone (H1) and ribosomal protein (rpl) genes served as controls. Samples were obtained from calyx cells (where virion are produced) and hind legs (control). Statistical significance levels are denoted as follows: ns = non-significant, *p<0.05, **p<0.01, and ***p<0.001. Statistical test results are available in Table D in S2 Dataset. The y-axis values were transformed using the square root function for better data visualization.
Fig 9.
Impact of U16 RNAi knockdown on HdIV replication gene expression and proviral segment amplification.
(A) Relative expression of nine IVSPER genes in 2-day-old adult females injected with dsGFP (control) or dsU16. (B) Relative DNA amplification of the integrated linear (proviral) and circularized (episomal) forms of viral segment Hd29 in 2-day-old adult females injected with dsGFP (control) or dsU16. The left panel illustrates the position of primer pairs designed to selectively amplify the proviral form (Proviral Left and Right, indicated by red and black arrows), the circularized form (Episomal, red arrows), or both (Proviral + Episomal, brown arrows). Note that the primers designed to amplify circular molecules may also anneal to concatemers generated during segment amplification and processing. The right panel presents the relative amplification of each form using DNA from dsGFP- and dsU16-injected females. In both (A) and (B), significance levels are indicated as follows: ns = non-significant, *p<0.005, **p<0.01, and ***p<0.001. Statistical test results are available in Tables F (A) and H (B) in S2 Dataset. The y-axis values were transformed using the square root function for better data visualization.