Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster

Abstract

Although host-parasitoid interactions are becoming well characterized at the organismal and cellular levels, much remains to be understood of the molecular bases for the host immune response and the parasitoids' ability to defeat this immune response. Leptopilina boulardi and L. heterotoma, two closely related, highly infectious natural parasitoids of Drosophila melanogaster, appear to use very different infection strategies at the cellular level. Here, we further characterize cellular level differences in the infection characteristics of these two wasp species using newly derived, virulent inbred strains, and then use whole genome microarrays to compare the transcriptional response of Drosophila to each. While flies attacked by the melanogaster group specialist L. boulardi (strain Lb17) up-regulate numerous genes encoding proteolytic enzymes, components of the Toll and JAK/STAT pathways, and the melanization cascade as part of a combined cellular and humoral innate immune response, flies attacked by the generalist L. heterotoma (strain Lh14) do not appear to initiate an immune transcriptional response at the time points post-infection we assayed, perhaps due to the rapid venom-mediated lysis of host hemocytes (blood cells). Thus, the specialist parasitoid appears to invoke a full-blown immune response in the host, but suppresses and/or evades downstream components of this response. Given that activation of the host immune response likely depletes the energetic resources of the host, the specialist's infection strategy seems relatively disadvantageous. However, we uncover the mechanism for one potentially important fitness tradeoff of the generalist's highly immune suppressive infection strategy.

Figure 1. Phylogenetic Comparison of Lb17 and Lh14 to Other Characterized Strains

Parsimony trees with bootstrap scores (circled) made from ribosomal RNA ITS2 sequences show the close relationship between (A) Lb17 and (B) Lh14 with other strains of these species [37].

Figure 2. Lb17 VLPs

TEM micrograph of the rounded, vesicle-filled VLPs from the lumen of the long gland reservoir of Lb17 shows that Lb17 VLPs are similar to those from other virulent L. boulardi strains [46].

Figure 3. Infectivity of Lb17 and Lh14 on Multiple Drosophila Species

Percentage of fly larvae that were successfully parasitized by the wasps (i.e., the percentage of fly larvae from which a wasp eventually hatched). Asterisks indicate that a significant proportion (>5%) of the fly larvae melanotically encapsulated wasp eggs or larvae. The Drosophila phylogeny is a consensus of multiple studies [73,113]; branch lengths are approximate.

Figure 4. Melanotic Encapsulation of Lb17 Eggs by D. yakuba

(A, C) White light pictures of melanized wasp eggs dissected from D. yakuba larvae. (B, D) Fluorescence pictures of the same melanized wasp eggs. (B) Hoechst nuclear stain—note abundance of cells encapsulating melanized egg. (D) Rhodamine-phalloidin actin cytoskeleton stain—note that encapsulating cells have flattened (lamellocyte) morphology.

Figure 5. Different Phenotypic Effects of Lb17 and Lh14 Venom

(A) Percentage of Drosophila lamellocytes lysed (bipolar) after incubation with wasp venom in vitro (means and standard errors shown). (B) hop^Tum-l larvae form melanotic tumors (black arrows), even when infected with either Lb17 (C, E, F) or Lh14 (D, G, H). Though dominant larvae of neither wasp species are encapsulated (white arrows), melanization of supernumerary wasp larvae (white arrowheads) occurs only in Lb17 attacked Drosophila.

Figure 6. Regulatory Changes in Canonical Immune Pathways after Lb17 Attack

Genes in red are significantly overexpressed and in blue underexpressed by Lb17 attacked flies in at least one of three treatment comparisons (Lb17 versus control, Lb17 + Lh14 versus control, or Lb17 versus Lh14) at one of the two early time points post infection (2–5 or 9–12 h). Dashed arrows represent presumed pathway interactions. Horizontal lines represent cell and nuclear membranes; genes inside the nucleus are targets of the upstream pathway. The main Toll pathway AMP Drosomycin was more than 4-fold up-regulated after Lb17 attack, but this change was not significant due to high variance among control replicates. Of the 33 overexpressed genes represented in this figure, only two, PGRP-SA and PGRP-SD, were also overexpressed in the Lh14 versus control comparison (relative to 21 in the Lb17 versus control comparison).

Figure 7. Confirmation of Microarray Results at the Protein Level

(A) PO enzyme activity (standardized by total protein levels) in Drosophila larvae 24 h after wasp attack. Means and upper/lower 95% confidence intervals are represented by horizontal lines. (B) Fat body expression of Drosomycin-GFP 24 h after wasp attack.