Fig 1.
SANTV and LEBV delay and reduce C. briggsae progeny production in laboratory conditions.
(A) The Santeuil virus (SANTV, strain JUv1264) was initially found in the C. briggsae JU1264 strain, isolated from a snail. The Le Blanc virus (LEBV, strain JUv1498) was found in C. briggsae JU1498 from a rotting peach (Félix et al. 2011 [10]; Franz et al. 2012 [11]). (B-E) The JU1264 strain was bleach-treated and infected by SANTV, LEBV or both. (B) Representative examples of infected animals at 120 hrs, showing a paler coloration of the gut, compared to an uninfected C. briggsae adults. Bars: 0.25 mm. (C) The viruses shorten the lifespan of C. briggsae JU1264 (logrank test, p = 5.44 × 10−9, p = 5.21 × 10−10, p = 1.08 x 10−7, for mock vs LEBV, SANTV and both, respectively). (D) Total brood size (left) and number of embryos laid over time (right) in the same experiment. Compared to mock infection, the total brood size is smaller for LEBV infection and co-infection (Wilcoxon rank sum test with continuity correction, p = 0.04 and p = 0.01, respectively). JU1264 animals show a significant delay in progeny production when infected with either virus (linear model, p = 2.2 × 10−16). ***: p<0.001,**: p<0.01,*: p<0.05. Panels C-D correspond to a single experiment (see Methods).
Fig 2.
Variation in sensitivity to SANTV and LEBV of C.
briggsae wild isolates (A) Experimental infection protocol, starting from a bleached C. briggsae culture. The transfer by chunking a piece of agar is indicated by beige rectangles cut out from a plate. (B) Sensitivity of C. briggsae wild isolates to SANTV and LEBV. The two viruses were inoculated in parallel. This graph represents the percentage of infected hosts as assayed by FISH for the corresponding virus. Dots are replicates within a block, with 100 animals scored per replicate (see S4 Table for the detailed results and S2 Fig and Methods for the experimental design). Experimental blocks are represented by colors and the bar indicates the grand mean of the blocks. The strains on the x axis are ordered by their rank of LEBV sensitivity. (C) Two-dimensional plot displaying the grand mean of panel B for SANTV and LEBV. The dots represent individual strains that are colored by categories. Their sensitivity to each virus is coded as a binary trait and the combination color-coded. The variance among replicates was considered, which explains that strains with similar positions on this plot are differently colored, one being labeled "Unclear" (see S4 Table for the categories). The strains show a significant correlation between their sensitivity to SANTV and LEBV (regression line in dark grey, with the 95% confidence interval in light grey). CI: Confidence interval. In addition, this plot highlights the specificity of infection for some strains, such as HK104, located far outside this diagonal.
Fig 3.
Relationship between C. briggsae virus sensitivity, geographic origin and genetic relatedness.
(A) World map showing the geographical distribution of the C. briggsae strains tested in Fig 2. Their sensitivity to both viruses is color-coded as in Fig 2C. In determining specific resistance, a low level of viral replication was considered as indicating some sensitivity, thus neglecting for the sake of simplicity possible quantitative variation in sensitivity between the two viruses. For example, JU1564 and JU1907 could be included as specifically resistant to SANTV. The map base was generated using the R software and the world map data of the ggplot2 package [76]. (B) Genetic relationship represented by a haplotype network between the C. briggsae isolates, based on available genome sequences at CaeNDR. * indicate strains used in further studies.
Fig 4.
Differential pattern of small RNAs against SANTV and LEBV.
(A) Proportions of 16–33 nucleotide reads mapping to viral genomes in infection experiments performed in parallel in three C. briggsae strains and a C. elegans strain. The letters M and D refer to mono- and co-infections with SANTV-JUv1264 and LEBV-JUv1498 viruses. The C. elegans N2 infection is used as a control for reads from the viral inoculum. Uninfected C. briggsae AF16 animals were used as a negative control. The host strain names are color coded as in Fig 3, and viruses are color coded as in Fig 1. (B) Differential pattern of small RNAs mapping to the two RNA molecules of the two viruses in two C. briggsae hosts, JU1264 and HK104. The stack bar charts show the distribution in length and 5′ nucleotide of small RNAs mapping onto each viral RNA. Negative values correspond to antisense small RNAs. The percentage of small RNAs mapping to the RNA1 and RNA2 molecules normalized to SANTV RNA2 length were computed for each infection condition and indicated on the bottom right of each graph. See S4 and S5 Tables for detailed counts and S6 Fig for mapping along the viral genomes.
Fig 5.
A major locus on chromosome IV underlies the variation in SANTV infection rate between C. briggsae AF16 and HK104.
(A) Distribution of the proportion of infected animals after exposure to SANTV in Advanced Recombinant Inbred Lines (RILs) between AF16 and HK104. The mean phenotypes of the parents are shown above the graph. See S6 Table for detailed data. (B) Quantitative genetic mapping of the proportion of infected animals after infection with SANTV. Green line: phenotype coded as a quantitative trait. Orange line: phenotype coded as binary. The genetic map with the markers along the six chromosomes is shown on the x axis. The black horizontal line denotes the p<0.05 significance threshold calculated using 10,000 permutations of the data. The QTL peak on chromosome IV is at 77.2 cM. (C) LOD score grid for the two-QTL analysis, represented with a color scale from blue = 0 to red = 10. The upper left triangle corresponds to the additive model and the lower right triangle to the full model. Both analyses point to the same significant regions, i.e. a main locus on chromosome IV and a second one on the right tip of chromosome III, with a LOD score of 10.5, above the threshold calculated by simulations (see Methods).
Fig 6.
Confirmation of the AF16 x HK104 QTLs on chromosomes IV and III using Near Isogenic Lines (NILs).
The genotypes of the lines are shown schematically below the graph, with orange representing the AF16 background (resistant) and green the HK104 background (sensitive to SANTV). The parental backgrounds are highlighted by a light grey rectangle. The color of the star sign at the QTL position on chromosome IV represents the inferred allelic state in the corresponding line. The QTL on chromosome III is represented by a double arrow. "B": background (the other chromosomes). Positions of recombination breakpoints are in megabases (Mb). Detailed genotypes can be found in S10 Table. The infections by SANTV were performed on two different days, with two replicates each day and 100 individuals per replicate. Bars represent the standard deviation among replicates. The significance p values were obtained in a generalized linear model (glm) taking independent experimental blocks and infection replicates into account, testing NILs against their relevant background parent. The p values using the two strains testing for the QTL on chromosome IV and those using the two-QTL strain JU2832 are corrected for multiple testing.
Fig 7.
A major locus on chromosome II underlies the variation in LEBV infection rate between C. briggsae JU1498 and HK104.
(A) Distribution of the proportion of infected animals after exposure to LEBV in Recombinant Inbred Lines (RILs) between JU1498 and HK104 (S8 Table for detailed data). (B) Bulk sequencing of pools of resistant and sensitive lines. The top plot shows the HK104 allele frequency of each pool along the C. briggsae genome (Cb4 assembly) (S9 Table for detailed data). The bottom plots show the LOD scores along the physical map of the six chromosomes. The dotted lines represent the threshold of significance at p<0.01.
Fig 8.
Confirmation and fine mapping of the JU1498 x HK104 locus using Near Isogenic Lines (NILs).
(A) Reciprocal introgressions of the right end of chromosome II. Infections by LEBV (black) and SANTV (grey) were performed in duplicates. n = 100 animals, except for SANTV infection of JU3241 (details in S10 Table). The color of the star (*) at the QTL position represents the inferred state in the corresponding line. (B). Assays of further recombinants of the chromosome II QTL, showing the mean of three infection replicates. Detailed genotypes and scorings can be found in S10 Table. Positions of breakpoints in Mb are indicated in relevant cases. ***: p<0.001 comparing JU4034 with its parent strain HK104 pairwise using a generalized linear model.
Fig 9.
Schematic summary of the findings.
The transcriptional result in C. briggsae JU1264 are from Chen et al. (2017) [23].