Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite

Abstract

Background: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects.

Results: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body.

Conclusions: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

Keywords: Drosophila melanogaster; Immune recognition; Parasitoid wasps.

Fig. 1

The effect of parasitoid wasp exposure on lamellocyte differentiation and the melanization of oil droplets. A Concentration of lamellocytes in the hemolymph of unchallenged larvae and larvae 48 h postinjection with oil or oil + wasp homogenate. The data points are independent measurements of hemolymph pooled from 8 to 10 larvae. B Oil droplets injected into larvae are either melanized (arrow) or not. Melanization of the cuticle resulting from injection wounding is often visible (arrowhead). C Proportion of larvae with melanized oil droplets 48 h after different immune challenges. Different letters represent treatments with statistically significant differences (Tukey’s honest significant difference test, p < 0.01)

Fig. 2

Transcriptional response of humoral immune genes to wasp exposure larvae were injected with oil, wasp + oil, or unchallenged. RNA from the fat body was sequenced 24 h post treatment. A The number of genes with significant changes in expression compared with unchallenged conditions. B The expression of 29 genes with significant changes in expression after immune challenge. C Expression of selected genes in the fat body measured by quantitative PCR 24 h after injection with oil droplets or wasp + oil droplets. For each treatment, 4 pools of 5–7 larvae were used to extract RNA

Fig. 3

Transcriptional response of hemocytes to wasp exposure larvae were injected with oil, wasp + oil, or unchallenged. RNA from the hemocytes was sequenced 24 h post treatment. A The number of genes with significant changes in expression compared with unchallenged conditions. B Changes in gene expression induced by injection of wasp homogenate (x-axis) and by injection of oil (y-axis). Because both treatments cause injury, genes solely regulated by injury will be close to the dashed diagonal 1:1 line. Genes specifically activated by wasp PAMPs will be on the x-axis. Relative expression is represented as log₂ (fold change). C Inferred proportion of immature (LAM1 and LAM2) and mature (LAM3) lamellocytes estimated from the RNA-seq data using digital cytometry. Each point is an independent sample, and the bars are the mean

Fig. 4

The effect of injecting homogenates of different insects on the melanization of oil droplets. The bar chart shows the proportion of oil droplets that were melanized with 95% binomial confidence intervals. The red line is the rate at which oil droplets were melanized without any insect homogenate (N = 241). The red bars are parasitoids of D. melanogaster. The tree [35] is colored according to estimated melanization rates from a phylogenetic mixed model in which the parasitoid status was included as a fixed effect. Sample sizes are given beside the bars. The silhouettes represent different insect orders (from top to bottom: Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, Dermaptera, Hemiptera, Odonata)

Fig. 5

Model of immune activation by parasitoid wasps