Influence of Wolbachia on host gene expression in an obligatory symbiosis

Abstract

Background: Wolbachia are intracellular bacteria known to be facultative reproductive parasites of numerous arthropod hosts. Apart from these reproductive manipulations, recent findings indicate that Wolbachia may also modify the host's physiology, notably its immune function. In the parasitoid wasp, Asobara tabida, Wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. The absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of Wolbachia in the wasp could also be responsible for this dependence. In order to decipher the general mechanisms underlying host-Wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals.

Results: As no genetic data were available on A. tabida, we constructed several Expressed Sequence Tags (EST) libraries, and obtained 12,551 unigenes from this species. Gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction (SSH). As pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death (PCD) and immunity (broad sense) was analyzed by quantitative RT-PCR. We showed that Wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation. We also showed that Wolbachia may interact with immune gene expression to ensure its persistence within the host.

Conclusions: This study allowed us to constitute the first major dataset of the transcriptome of A. tabida, a species that is a model system for both host/Wolbachia and host/parasitoid interactions. More specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of Wolbachia should also be investigated beyond reproductive manipulations.

Figure 1

Sequence treatment (A) and functional annotation procedure (B).

Figure 2

Characteristics of the EST libraries A. Summary of the different EST libraries from Asobara tabida, used to build a transcriptomic map, but also to address the question of the effect of symbiosis and bacterial challenge (b. ch.) on host gene expression. cDNA libraries were sequenced with or without normalization (Norm. or Non norm., respectively). Suppression Subtractive Hybridizations (SSHs) were performed with or without the Mirror Orientation Selection procedure (MOS). The influence of ovarian phenotype was addressed using two different populations known to exhibit extreme phenotypes after Wolbachia removal: females from the Pi3 strain (Pierrefeu, France) do not produce any eggs, while females from the NA strain (Saanich, Canada) produce a few eggs that fail to develop normally. Immune challenge was performed by injecting 1.8x10⁵ Salmonella typhimurium in aposymbiotic females, and RNA was extracted 3h, 6h and 12h after challenge. Abbreviations stand for: DPOv: Distal Part of the Ovaries (e.g. without the eggs), Ov: Ovaries, F: Females, S: Symbiotic, A: Aposymbiotic, C: immune Challenge, NC: No immune Challenge. ESTs: Expressed Sequenced Tags, mito: mitochondrial genes, rRNA: ribosomal RNA, UG: number of unigenes found after a clustering/assembly. B. Abundance classes of ESTs and Unigenes. C. Unigene occurrences among the EST libraries. The horizontal axis represents the different EST libraries. The occurrence of unigenes within the libraries is shown on the vertical axis. A horizontal reading of the graph indicates the percentage of unigenes shared by several EST libraries. D. Gene Ontology (GO) annotation results for High Scoring Pair (HSP) coverage of 0%. GO annotation was first carried out using the Score Function (SF) of the Blast2go software. The GO terms selected by the annotation step were then merged with Interproscan predictions (SF+IPR). Finally, the annex augmentation was run (SF+IPR+ANNEX). E. Annotation distribution of GO terms.

Figure 3

Differential expression of candidate genes in response to Wolbachia infection, depending on tissue and population. The Pi3 strain exhibits a strong ovarian phenotype after Wolbachia removal (no eggs in the ovaries), while the NA strain produces a few eggs that fail to develop normally. Quantitative RT-PCR was performed either in males or in ovaries (whole ovaries for the NA strain, and a distal part of the ovaries (DPOv) for the Pi3 strain). Details of the expression patterns are given in Additional file 3. The ratios between the average expression under aposymbiotic and symbiotic conditions are given. Genes over-expressed in symbiotic individuals are highlighted in blue; whereas those over-expressed in aposymbiotic individuals are highlighted in orange (Wilcoxon’s test on expression data, p-values adjusted using FDR’s correction). A dash indicates that there is no expression in the given tissue. Genes have been ordered within signaling pathways, and from the receptors to the effectors in immune pathways. Asterisks are assigned to pleiotropic genes implicated in several biological functions. PGRP: PeptidoGlycan Recognition Protein, SPE: Spätzle-Processing Enzyme, IAP: Inhibitor of APoptosis, TEP: ThiolEster-containing Protein, LCH: Light Chain, HCH: Heavy Chain, GST: Gluthatione-S-Transferase, SOD: SuperOxide Dismutase, HSP: Heat Shock Protein, TCTP: Translationally-Controlled Tumor Protein, ATG: Autophagy-related protein, Sxl: Sex-Lethal, MAPK: MAP kinase.