Host PGRP gene expression and bacterial release in endosymbiosis of the weevil Sitophilus zeamais

Abstract

Intracellular symbiosis (endosymbiosis) with gram-negative bacteria is common in insects, yet little is known about how the host immune system perceives the endosymbionts and controls their growth and invasion without complete bacterial clearance. In this study, we have explored the expression of a peptidoglycan recognition protein gene of the weevil Sitophilus zeamais (wPGRP); an ortholog in Drosophila (i.e., PGRP-LB) was recently shown to downregulate the Imd pathway (A. Zaidman-Remy, M. Herve, M. Poidevin, S. Pili-Floury, M. S. Kim, D. Blanot, B. H. Oh, R. Ueda, D. Mengin-Lecreulx, and B. Lemaitre, Immunity 24:463-473, 2006). Insect challenges with bacteria have demonstrated that wPGRP is induced by gram-negative bacteria and that the level of induction depends on bacterial growth. Real-time reverse transcription-PCR quantification of the wPGRP gene transcript performed at different points in insect development has shown a high steady-state level in the bacteria-bearing organ (the bacteriome) of larvae and a high level of wPGRP up-regulation in the symbiotic nymphal phase. Concomitantly, during this stage fluorescence in situ hybridization has revealed an endosymbiont release from the host bacteriocytes. Together with the previously described high induction level of endosymbiont virulence genes at the nymphal phase (C. Dale, G. R. Plague, B. Wang, H. Ochman, and N. A. Moran, Proc. Natl. Acad. Sci. USA 99:12397-12402, 2002), these findings indicate that insect mutualistic relationships evolve through an interplay between bacterial virulence and host immune defense and that the host immunity engages the PGRP gene family in that interplay.

FIG. 1.

Bacterial growth in aposymbiotic infected larvae. Larvae were infected with either E. coli or P. aeruginosa. The number of bacteria was measured at 2, 6, 12, and 24 h after infection. Data are expressed as CFU (CFU per larva) and represent the means of two samples of five larvae.

FIG. 2.

Quantification of wPGRP gene transcripts from control and challenged aposymbiotic larvae. (A) Shown are the images of the Northern blot hybridized with wPGRP and with β-actin probes. Cal, calibrator sample; C, unchallenged aposymbiotic larvae; S, mock-infected larvae; E and P are larvae infected with E. coli and P. aeruginosa, respectively; numbers 1, 2, and 3 indicate sample repetitions. Larvae were incubated for 1, 2, 6, and 12 h following the treatment. (B) Levels of wPGRP gene transcripts normalized with the expression of the β-actin gene. Data (expressed in arbitrary units) are means of three independent repetitions normalized with the relative intensity of the β-actin gene. Bars represent the standard deviations. Data were analyzed with ANOVA (see Results).

FIG. 3.

Real-time RT-PCR quantification of wPGRP gene steady-state levels at different points of the weevil life cycle. Total RNA was extracted from bacteriomes (B), oocytes (O), 3-day-old embryos (E), fourth-instar larvae (L), nymphs (N), and 3-week-old adults (A) of symbiotic (black) and aposymbiotic (gray) weevils. Each bar represents the means of three independent measurements with standard deviations after normalization.

FIG. 4.

FISH monitoring of SZPE at different phases of insect development. Shown are DAPI visualizations of whole embryos from S. zeamais at (image 1) 30 min, (image 2) 6 h, (image 3) 3 days, and (image 4) 4 days, respectively. Images 5, 6, and 7 are SZPE magnifications from the areas indicated by the arrows in images 1, 2, and 3, respectively. Images 8 to 13 are rhodamine visualizations of SZPEs in the larval bacteriome intimately attached to the gut (image 8), during the bacteriome dissociation at the first nymphal stages (images 9, 10, and 11), and in the mesenteric caeca bacteriomes of aged nymphs (image 12) and young adults (image 13). AP, anterior pole; PP, posterior pole; hc, head capsule; Lb, larval bacteriome; bm, bacterial migration; ba, bacterial aggregation; g, gut; mb, mesenteric bacteriome; b, bacteriocyte; gl, gut lumen; at, adipocyte tissue. Arrows indicate the position of SZPEs. Scale bar, 10 μm.