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Science 334 (6054): 362-365

Copyright © 2011 by the American Association for the Advancement of Science

Antimicrobial Peptides Keep Insect Endosymbionts Under Control

Frédéric H. Login1,2, Séverine Balmand1,2, Agnès Vallier1,2, Carole Vincent-Monégat1,2, Aurélien Vigneron1,2, Michèle Weiss-Gayet2,3, Didier Rochat4, and Abdelaziz Heddi1,2,*

1 INSA-Lyon, INRA, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, F-69621 Villeurbanne, France.
2 Université de Lyon, F-69003 Lyon, France.
3 Université Lyon 1, CNRS UMR5534, Centre de Génétique et de Physiologie Moléculaire et Cellulaire, F-69622 Villeurbanne, France.
4 INRA, Université Pierre et Marie Curie, UMR1272 Physiologie de l’Insecte Signalisation et Communication, F-78026 Versailles, France.


Figure 1
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Fig. 1. ColA peptide distribution in weevil tissues. (A) Schemes of larva and adult weevils showing bacteriome localizations (in red). (B) (Upper panel) Tissues from aposymbiotic S. zeamais: Left and middle images are cuticle sections stained with a preimmune serum [negative controls can be found in SOM (14)] and with antibody against ColA (anti-ColA), respectively; the right image is a gut section stained with anti-ColA. ColA signals are detected in the fat body, with relatively high intensity under the cuticle and within gut epithelial cells. (Middle and lower panels) Tissues from symbiotic insects stained with anti-ColA. Middle panel: ColA signals can be seen in oocytes and follicular cells (left), in apical bacteriomes of ovaries (middle), and in adult mesenteric caeca (right); lower panel: ColA signals in larval bacteriome (left), within bacteriocytes (middle), and in bacteriome squashes (right) (14). Arrows indicate high ColA signals at the periphery of tissues and show ColA colocalizing with SPE in bacteriocytes and bacteriome squashes. (C). Immunogold staining of SPE with anti-ColA. Bacteriocyte sections are shown with ColA spots inside symbiont cytoplasm and attached to bacterial membranes.

 

Figure 2
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Fig. 2. ColA and ColB activities against bacteria. (A) ColA (solid line) and ColB (dashed line) activities against E. coli (triangles) and M. luteus (squares). ColA and ColB have a similar inhibitory effect (analysis of variance, P =0.76). They show bactericidal activities against M. luteus at low concentrations. For E. coli, low concentrations of ColA and ColB have bacteriostatic activity, and higher concentrations kill this bacterium. (B) Effect of low concentrations of ColA and ColB on E. coli morphology. Bacteria were incubated in LB broth (left, control), in LB with 8 μM ColA (middle), or in LB with 8 μM ColB (right). Cell gigantism is observed with ColA peptide only. (C) Gram staining of endosymbionts from S. oryzae (rod-shaped, left), S. zeamais (spiral, middle), and R. ferrugineus (filamentous, right). (D) Chromosome visualization of E. coli treated with 8 μM ColA (left), SPE (middle), and Nardonella (right). SPE and Nardonella were isolated from larval bacteriomes of S. oryzae and R. ferrugineus, respectively. Chromosome number was highest in Nardonella.

 

Figure 3
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Fig. 3. Effects of colA inhibition with RNAi on SPE size and location. (A) Typical forward scatter–area/side scatter–area plots showing size (x axis) and granularity (y axis) of SPE isolated from larvae injected with dsRNA-gfp (left) and with dsRNA-colA (right). Three SPE populations were defined arbitrarily. Small-sized cells (P1) and intermediate-sized cells (P2) significantly increased with dsRNA-colA treatment, whereas the population of large-sized cells (P3) decreased ({chi}2-test, P < 0.0001; see percentage values). The mean size of P2 and P3 significantly decreased (Mann-Whitney test, P < 0.0001), whereas the mean size of P1 was equal in dsRNA-gfp– and dsRNA-colA–injected larvae (Mann-Whitney test, P = 0.37). (B) FISH visualization of SPE 9 days (upper panels) and 14 days (lower panels) after larvae were injected with dsRNA-gfp (left) and dsRNA-colA (right). colA inhibition resulted in SPE escaping the bacteriome (see arrows). See table S4 for experimental details.

 


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