Identification of the weevil immune genes and their expression in the bacteriome tissue

Abstract

Background: Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome).

Results: To study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome.

Conclusion: The current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.

Figure 1

Schematic representations of putative Sitophilus zeamais proteins with similarity to antibacterial peptides, lysozymes, PGRP and Tollip. For each gene, the whole cDNA sequence was obtained from the corresponding EST by RACE-PCR and was then confirmed by whole cDNA amplification and sequencing. For each cDNA, ORF was predicted using the MacMolly software package. The top scale shows the length of the various domains of the proteins (aa, amino acid). Black regions indicate the predicted signal peptide (TargetP) and gray regions the putative propeptide domain according to the conserved R-x-(K/R)-R motif. mcs: minimal cleavage site corresponding to an R-x-x-R motif with an additional arginine in P6 position, which may enhance cleavage. Regions with similarity to conserved domains detected by InterProScan are indicated as hatched regions with the associated E-value. Coleoptericin, IPR009382; Defensin, IPR001542; Kunitz, IPR002223; Lysozyme, IPR000974; Destabilase, IPR008597; PGRP, IPR002502; C2, IPR000008; CUE, IPR003892. The accession numbers of the complete coding sequences are: INF-18, EU282111; INF-145, EU282117; INF-42, EU282112; INF-163, EU282118; INF-165, EU282113; INF-479, EU282119; INF-217, EU282115; INF-282, EU282114; INF-152, EU282120; INF-441, EU282121; INF-9, EU282122; INF-359, EU282116.

Figure 2

Analysis of immune gene expression in aposymbiotic larvae challenged with Sitophilus zeamais primary endosymbiont. Transcript of genes induced by an E. coli challenge were quantitated by qRT-PCR in untreated aposymbiotic larvae (control) and in larvae six hours after a 69 μl injection of either sterile water (sterile) or SZPE cells (heat-killed or viable) obtained from 50 bacteriomes dissected from symbiotic fourth-instar larvae. ESTs were classified according to sequence similarity as in Table 2. Each bar represents the mean of three independent measurements with standard error.

Figure 3

Analysis of gene expression in the bacteriome tissue. As described in Materials and Methods, transcripts of candidate genes were quantitated by qRT-PCR in whole aposymbiotic fourth-instar larvae (control) and in bacteriomes dissected from symbiotic fourth-instar larvae. ESTs were classified according to sequence similarity as in Table 2. Each bar represents the mean of three independent measurements with standard error. The asterisk represents a significant (p < 0.05) difference between the bacteriome and the control, and a significantly high expression in a bacteriome is indicated by an arrow. ND, non-determined.