To detect unigene similarities with other species, several blasts (with high cut-off e-values)
were performed against the following databases: NCBI nr (blastx (release: 1 March 2011); e-value < 5, HSP length > 33aa), Refseq genomic database (blastn, e-value < 10), Unigene division Arthropods (tblastx, #8 Aedes aegypti, #37 Anopheles gambiae, #3 Apis mellifera, #3 Bombyx mori, #53 Drosophila melanogaster, #9 Tribolium castaneum; e-value < 5). Gene Ontology annotation was carried out using blast2go software [45]. In the first step (mapping), a pool of candidate GO terms was obtained for each unigene by retrieving GO terms associated with the hits obtained after a blastx search against NCBI nr. In the second step (annotation), reliable GO terms were selected from the pool of candidate GO terms by applying the Score AC220 cell line Function (FS) of Blast2go with ‘permissive annotation’ parameters (EC-weight=1, e-value-filter=0.1, GO-weight=5, HSP/hit coverage cut-off = 0%). In the third step of the annotation procedure, the pool of GO terms selected during the annotation step was merged with GO terms associated with the Interpro domain (InterproScan predictions based on the longest ORF). Finally, the Annex augmentation step was run to modulate the annotation by adding GO terms derived from implicit relationships between GO terms [46]. Statistical analyses on libraries We have used the randomization
procedure (with 500 random datasets) and the R statistic, described in [47], to detect unigenes whose transcript Tubastatin A in vivo abundance (number of ESTs) in symbiont-free and symbiont-full bacteriome libraries was statistically different (at a FDR of 5.5%). In order to perform a functional enrichment analysis of the unigenes extracted from the SSH, we used the Fatigo web tool [48] against the SO library. Transcriptomic study Sample preparation Transcriptomic analysis was performed on larval bacteriomes, whole symbiotic and aposymbiotic larvae, non-treated, mock-infected (injected with PBS), and injected with 105 E. coli (TOP10, Invitrogen, Cergy-pontoise, France). The E. coli bacterium was used here because it has been shown to efficiently induce
the weevil immune system [6], and this bacterium does not necessitate an L2 safety lab structure for manipulation. Larvae were then maintained at 27.5°C and 70% rh for 3-mercaptopyruvate sulfurtransferase 6 hours. For each modality, 5 samples of 5 selleck chemicals pooled larvae were prepared and then frozen at -80°C. Bacteriomes were dissected from non-treated larvae that have been maintained at 27.5°C and 70% rh for 6 hours. 5 samples of 25 pooled bacteriomes were dissected and then frozen at -80°C until RNA extraction. Total RNA extraction and cDNA synthesis Total RNA from whole larvae was extracted with the TRIzol Reagent (Invitrogen, Cergy-pontoise, France), following the manufacturer’s instructions. RNA was incubated with 1 U/g of RQ1 RNase-Free DNase (Promega, Charbonnières-les-Bains, France) for 30 min, at 37°C.