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. 2005;6(11):R94.
doi: 10.1186/gb-2005-6-11-r94. Epub 2005 Oct 31.

Genome-wide gene expression in response to parasitoid attack in Drosophila

Bregje Wertheim  1 , Alex R Kraaijeveld, Eugene Schuster, Eric Blanc, Meirion Hopkins, Scott D Pletcher, Michael R Strand, Linda Partridge, H Charles J Godfray
Affiliations

Genome-wide gene expression in response to parasitoid attack in Drosophila

Bregje Wertheim et al. Genome Biol. 2005.

Abstract

Background: Parasitoids are insect parasites whose larvae develop in the bodies of other insects. The main immune defense against parasitoids is encapsulation of the foreign body by blood cells, which subsequently often melanize. The capsule sequesters and kills the parasite. The molecular processes involved are still poorly understood, especially compared with insect humoral immunity.

Results: We explored the transcriptional response to parasitoid attack in Drosophila larvae at nine time points following parasitism, hybridizing five biologic replicates per time point to whole-genome microarrays for both parasitized and control larvae. We found significantly different expression profiles for 159 probe sets (representing genes), and we classified them into 16 clusters based on patterns of co-expression. A series of functional annotations were nonrandomly associated with different clusters, including several involving immunity and related functions. We also identified nonrandom associations of transcription factor binding sites for three main regulators of innate immune responses (GATA/srp-like, NF-kappaB/Rel-like and Stat), as well as a novel putative binding site for an unknown transcription factor. The appearance or absence of candidate genes previously associated with insect immunity in our differentially expressed gene set was surveyed.

Conclusion: Most genes that exhibited altered expression following parasitoid attack differed from those induced during antimicrobial immune responses, and had not previously been associated with defense. Applying bioinformatic techniques contributed toward a description of the encapsulation response as an integrated system, identifying putative regulators of co-expressed and functionally related genes. Genome-wide studies such as ours are a powerful first approach to investigating novel genes involved in invertebrate immunity.

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Figures

Figure 1
Figure 1
The Drosophila immune response after attack by parasitoids. (a) The parasitoid Asobara tabida stabs a second instar Drosophila melanogaster larvae with her ovipositor and inserts a single egg. (b) The parasitoid egg is susceptible to nonself recognition by membrane-bound and noncellular pattern recognition proteins in the larval hemolymph. (c) Hemocyte proliferation and differentiation is triggered, and the blood cells aggregate around the parasitoid egg. (d) The hemocytes form a multilayered capsule around the parasitoid egg and melanin is deposited on the capsule. (e) The parasitoid egg dies when it becomes fully melanized.
Figure 2
Figure 2
Venn diagrams of genes that changed expression after parasitoid attack and known immunity genes. The differentially expressed genes after parasitoid attack differed largely from those with a GeneOntology (GO) annotation for immunity or defense (GO database September 2004; the GO codes are also shown in the figure). Some of the probe sets in our set matched to multiple genes (see additional data files), thus reporting on the expression of potentially all of these genes. We included the multiple gene annotations per probe set to define our set of differentially expressed genes for the comparisons.
Figure 3
Figure 3
Gene expression levels and distribution of regulatory motifs for the genes differentially expressed after parasitoid attack. (a) Expression levels for genes (rows) at different sample time points (columns: 1-9 parasitized larvae; 10-18 unparasitized larvae). The expression levels are given as multiples of the median for that gene, using a color code illustrated at top right. At the left the dendrogram produced by the clustering algorithm is shown, with the 16 clusters discussed in the text depicted in different colors (with their code numbers; the final column on the right shows the clusters again using the same color key). (b) The distribution of putative regulatory motifs in the -1,000 to +50 base pair upstream regions of the genes. The colors indicate the number and strength of the matches for each motif (see code on upper right, in which a score of 0 is equivalent to no matches, 10 is equivalent to one strong or two weak matches, and 20 is equivalent to multiple strong matches).
Figure 4
Figure 4
Gene expression profiles and functional annotations for the eight largest clusters of co-expressed genes. On the left-hand side the average expression levels for the genes in the eight clusters are shown (log2-transformed expression values, divided by the median for that gene across all time points and treatments). Dashed lines represent parasitized and unbroken lines represent unparasitized larvae, and the bars indicate standard errors. Functional annotations associated with clusters are shown along the top, and colors in the matrix indicate the strength of association (yellow = Ease scores (see text) <0.05; red = after Bonferroni correction at P < 0.05; grey = at least one gene with this annotation). The full annotation for all probe sets is provided in Additional data file 1.
Figure 5
Figure 5
Expression profiles of genes from pathways and processes known to be involved in immunity. Each graph depicts the log2 expression values for a single gene at different time points (in hours) after parasitoid attack. The blue circles and red triangles show the individual replicates of the control and parasitized larvae, respectively. The lines denote the average expression at each time point. See text for a discussion of the selected genes.
Figure 6
Figure 6
Overview and summary of our findings. The two left-hand columns show the time elapsed since parasitoid attack and a diagrammatic summary of major cellular and metabolic consequences of parasitism. The three right hand columns show the results of this study and the gene clusters that we hypothesize are associated with the different processes sketched on the far left. These three columns show the following: over-represented transcription factor binding motifs arranged by cluster (with code number) ordered by their time of maximum expression; average expression profiles of genes in these clusters (parasitized larvae in red, unparasitized larvae in blue) with marked temporal restricted expression; and functional annotations associated with genes in these clusters, in the same order as in the first of the three columns. A group of genes with relatively constant levels of reduced expression in parasitized larvae is shown separately at the bottom.
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