The analysis produced a total of 79,204 reads with an average length of 320.6 nucleotides that became, after quality filtering and clustering (needed for Ribosomal Database Project

analysis), 75,564 for 97%, 76,724 for 95%, and 73,579 for 90% of similarity (Additional file 2). Reads were assigned to 41 operational taxonomic units (OTUs) at 90% of sequence identity threshold, and to 45 OTUs at 95% and 97% identity threshold, respectively, in order to perform rarefaction analysis. The total number of clusters obtained after filtering was of 2,107 (1,756 singletons) for 97%, 910 (530 singletons) for 95%, and 244 (124 singletons) for 90% of similarity, respectively. The rarefaction curves tended towards saturation at similar numbers of clusters at 97%, 95% and 90% pairwise ID thresholds (Figure 2). Subsequent analysis was, therefore, conducted at 97% ID. Figure 2 Rarefaction curves of OTUs clustered at different % ID in the gut of RPW larvae. Only three phyla ABT-263 account for 98% of the reads: these are Proteobacteria (64.7%), Bacteroidetes (23.6%) and Firmicutes (9.6%); the remaining 2% is represented by Tenericutes (1.4%) Fusobacteria (0.4%) and other

Bacteria (0.2%) (Figure 3a). Proteobacteria are mainly represented by Gammaproteobacteria (96.7%) followed by Betaproteobacteria (2.71%) (Figure 3b). More than 98% of the reads were classified at the family level, with Enterobacteriaceae representing the 61.5% of the assemblage, followed by Porphyromonadaceae (22.1%) and CHIR-99021 ic50 Streptococcaceae (8.9%) (Additional file 3).

More than half of the reads (52.7%) could be classified at the genus level and eight bacterial genera were detected in the larval RPW gut at an abundance ≥1% (Figure 4a). Dysgonomonas sequences account for the 21.8% of the whole sequences and this is the most represented genus in the gut of RPW larvae, Idoxuridine followed by Lactococcus (8.9%) Salmonella (6.8%), Enterobacter (3.8%), Budvicia (2.8%), Entomoplasma (1.4%) Bacteroides (1.3%) and Comamonas (1%). Other twelve genera are represented at a value between 1% and 0.1% (Figure 4b). The phylogenetic tree of 16S rRNA gene amplicons clustered at 97% consensus is shown in the Additional file 4. Figure 3 Relative abundance of a) bacterial Phyla and b) classes of Proteobacteria in the gut of field caught RPW larvae as detected by pyrosequencing. Values ≤ 0.1% are included in “other bacteria” (see Additional file 2). Figure 4 Relative abundance of bacterial genera a) above 1% and b) below 1% in the gut of field caught RPW larvae as detected by pyrosequencing. “Others” indicates 35 genera below 0.1% (see Additional file 2). Diversity of cultivable bacteria Bacterial isolation under aerobic conditions was carried out on three lots of three pooled RPW larval guts (lots A, B, C), all sampled in April 2011. The dilution plate counts on NA gave an average of 1.5 × 107 CFU gut-1, without differences among the three pools.