Fig 1.
Phylogenetic analysis of the R. prolixus viruses.
(A) Schematic of an ovariole in R. prolixus. Mitotically active trophocytes (i.e. nurse cells) are harbored in Zone 1 of the tropharium, while polyploid trophocytes are present in Zone 2 and 3. The egg chamber in meroistic telotrophic ovaries is formed by the oocyte surrounded by somatic epithelial cells. Trophic cords connect the tropharium to the growing egg chamber and provide transport routes for nutrients and other molecules. (B) Phylogenetic tree of the RpVs constructed with RdRp sequences using Neighbor-Joining method with 1000 bootstrap replicates. Bootstrap values are displayed in tree branches. The RpVs are grouped in three families: Iflaviridae, Permutotetraviridae and Solemoviridae. (C) Genome organization of the RpV1 (MZ328304) and RpV2 (MZ328305) iflaviruses compared to the closest relatives Nesidiocoris tenuis iflavirus 1 and Slow bee paralysis virus respectively. RpV1 displays the 1nt frameshift typical of iflaviruses, that was not detected in the N. tenuis’ virus. (D) Diagram of the RpV3 (MZ328306), RpV4 (MZ328307) and RpV7 (MZ328310) and their closest relative Hubei permutotetra-like virus 8. (E) Genome organization of RpV5 (MZ328308), RpV6 (MZ328309) and Atrato Sobemo-like virus 5. rhv-like = Picornavirus/Calicivirus coat protein, CRPV_capsid = Cricket Paralysis Virus capsid protein, RdRp = RNA-dependent RNA Polymerase, RT_like = Reverse Transcriptase_like.
Fig 2.
Stage-specific quantification of the RpVs during R. prolixus development.
(A) Normalized RNA-Seq reads for each virus in previtellogenic stages (PVS) of oogenesis. (B) Normalized RNA-Seq reads for each virus in unfertilized mature eggs (Egg) dissected from the abdomen of adult females. Biological replicates for both stages of oogenesis are scaled by counts per million (CPM). The endogenous Rp-rp49 gene is also displayed to underscore the abundance of the RpVs in ovarian tissues. Read coverage over the RpV1 (C), RpV3 (D) and RpV4 (E) viral genomes. Red lines indicate the organization of the genome for each virus. (F) Quantification of the RpVs in previtellogenic stages (PVS), mature eggs (Egg), embryos and 1st instar nymphs. RpV2 genome copies were below qRT-PCR detection limits and were not included in the box plot. Y-axis displays the number of viral genome copies per microgram of total RNA (Log10). (G) Quantification of RpV1 in different tissues of R. prolixus using available RNA-Seq datasets [46]. Y-axis displays the Counts per Million (CPM).
Fig 3.
vsiRNA profiling during R. prolixus oogenesis.
(A) Length distribution of the RpVs small RNAs in previtellogenic stages (PVS) of oogenesis. (B) Length distribution of the viral small RNAs in mature eggs (Egg). In both stages of R. prolixus oogenesis, a peak at 22nt is readily detectable for all the RpVs, except for RpV2 and TrV, which did not generate vsiRNA reads above background levels. RpV2 was lost from our insectarium by the time that the small RNA datasets were produced. TrV was never detected in our colony and was used as control for the background small RNA levels. (C-H) vsiRNA profiling along the RpV1, RpV4 and RpV6 genomes and length distribution of the sense and antisense vsiRNAs. Sense and antisense vsiRNAs detected in previtellogenic stages of oogenesis (dark blue = sense, red = antisense) and mature chorionated eggs (light blue = sense, orange = antisense) are displayed for each virus. (I-J) Ping-pong signal for the RpV1 and RpV6 viruses in PVS (white bars) and Egg (grey bars). The red box highlights the position of the typical 10nt overlap between sense and antisense small RNA pairs.
Fig 4.
Analysis of the vsiRNA pools shared by the RpVs.
(A) Venn diagram showing the vsiRNAs expressed in PVS and Egg stages that are shared by the permutotetraviruses. (B) Venn diagram showing the vsiRNAs expressed in PVS and Egg stages that are shared by the sobemoviruses. Only vsiRNAs with perfect match to the viral genomes were considered in these analyses. (C) Small RNA-Seq coverage of the vsiRNAs simultaneously shared by the permutotetraviruses RpV3, RpV4 and RpV7. The coverage along the consensus genome (see Methods) is displayed to highlight the accumulation of the shared vsiRNAs in the viral 3’UTR. Similar results were obtained for the RpV4 and RpV7 genomes (S7 Fig). (D) Small RNA-Seq coverage of the vsiRNAs common to the sobemoviruses RpV5 and RpV6. Sense and antisense shared vsiRNAs detected in previtellogenic stages of oogenesis (dark blue = sense, red = antisense) and mature chorionated eggs (light blue = sense, orange = antisense) are displayed.
Table 1.
Oligonucleotides used in this study.