Fig 1.
Parasitic wasp ribosomes are depurinated during host protection.
Levels of intact and depurinated wasp ribosomes are significantly decreased and increased, respectively, in Spiroplasma-positive flies, compared to Spiroplasma-negative flies (A). Results of t-tests for intact ribosomes: D. melanogaster + L. heterotoma, p < 0.001; D. melanogaster + L. boulardi, p = 0.028; D. neotestacea + L. heterotoma, p = 0.010, and for depurinated ribosomes, all fly-wasp pairs, p < 0.001; n = 8, 8, 6, and 5 flies per treatment, for D. melanogaster S+, S-, and D. neotestacea S+, S-, respectively. Jitter points are the mean of two technical replicates per wasp-infested larva. Outcomes of wasp exposure for fly and wasp survival were measured as emerged adults (B). Each strain of Spiroplasma kills Leptopilina. L. heterotoma and L. boulardi emergence is significantly reduced in Spiroplasma-positive flies relative to Spiroplasma-negative controls (t-tests, L. heterotoma in D. melanogaster, p < .001; L. boulardi in D. melanogaster, p = .003; L. heterotoma in D. neotestacea, p < .001). Y-axes are labeled with total number of adult emergents per dish and error bars show the standard deviation of the mean number of adult emergents across replicate dishes (sample sizes: L. heterotoma in D. melanogaster, 60 larvae, 4 dishes; L. boulardi in D. melanogaster, 40 larvae, 2 dishes; L. heterotoma in D. neotestacea, 40 larvae, 2 vials).
Fig 2.
Hallmark of RIP attack in wasps begins soon after hatching.
Abundance of depurinated ribosomes across a timecourse of L. heterotoma infestation of D. melanogaster in the presence and absence of sMel (n = 6 fly larvae or pupae per timepoint). Jitter points are the mean of two technical replicates per wasp-infested larva. Depurinated L. heterotoma ribosomes are detected 48 hours after oviposition in sMel-positive D. melanogaster, with increasing levels at subsequent timepoints. For all significant comparisons, p < .001 (t-tests). Legends above boxplots indicate parasitic wasp developmental stage at each time point indicated.
Fig 3.
Depurination of Drosophila ribosomes is minimal and does not explain host mortality during parasitic wasp infestation.
Levels of intact Drosophila ribosomes are not significantly decreased in Spiroplasma-positive flies (blue), compared to Spiroplasma-negative flies (gold) (A,C,E; p = .348, p = .026, p = .693 for L. heterotoma-infested D. melanogaster, L. boulardi-infested D. melanogaster and L. heterotoma-infested D. neotestacea, respectively). Depurinated Drosophila ribosomes are significantly more abundant in Spiroplasma-positive (blue) compared to Spiroplasma-negative flies (gold; B,D,F; p < .001); however, there is no difference in abundance of depurinated Drosophila ribosomes between wasp-infested (blue) and uninfested hosts (pink) regardless of fly survival outcome following defense. Jitter points are the mean of two technical replicates per larva. (G) Levels of depurinated Drosophila ribosomes are plotted for samples of host hemolymph, hemocytes, bled larvae and unbled controls. Jitter points are the mean of two technical replicates per pool of eight larvae. The proportion of depurinated host and infesting wasp ribosomes from larval samples collected in a separate experiment are shown for comparison. Post hoc significance test results are shown above boxplots (Tukey tests).
Fig 4.
Diversity of ribosome-inactivating proteins in Spiroplasma.
A maximum likelihood phylogram of Spiroplasma ribosome-inactivating proteins (RIPs), along with a diagram showing putative domains. E. coli Shiga toxin is also included. Sequences from defensive Spiroplasmas of Drosophila are shown in red. RIP number designations correspond with the numbering system used for RIP transcripts elsewhere in the manuscript. Branches are labeled with approximate likelihood-ratio test scores of .75 or higher.
Fig 5.
The ectoparasitic wasp Pachycrepoideus vindemmiae successfully develops in Spiroplasma-positive Drosophila melanogaster and does not show evidence of RIP attack.
Pachycrepoideus vindemmiae (Pv) successfully parasitizes Spiroplasma-positive D. melanogaster (A). Neither fly nor wasp emergence was significantly affected by the presence of Spiroplasma (p ≥ .5). The proportion of depurinated ribosomes is much less than what is seen in Leptopilina species (p < .001; B). There is no difference in the level of intact P. vindemmiae ribosomes in sMel-positive flies compared to levels in sMel-negative flies (p = .778). Levels of depurinated ribosomes in sMel-positive flies are modestly, albeit significantly, greater than in sMel-negative flies (p = .004; C). Twelve sMel-positive and nine sMel-negative fly pupae were tested for RIP activity and jitter points are the mean of two technical replicates per wasp-infested larva. Significant comparisons from Tukey post hoc tests are labeled above boxplots.