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  • A higher wood-feeding termite species, N. arborum (Smeathman) was used in this study. The genus Nasutitermes Dudley, 1890 is a key taxon widespread in all tropical areas, with 243 species currently described. Nasutitermes species are wood-feeding termites and their post-embryonic development has been well established. A nest of N. arborum (Termitidae, Nasutitermitinae) was collected in the Republic of the Congo (Brazzaville), in the Mayombe forest within UNESCO-Man and the Biosphere Reserve, in the district of Mvouti (4°14’S, 12°26’E), with the permission of Dieudonné MBOUMBA, the Administrative Head of the district of Mvouti (Congo). This field study did not involve endangered or protected species. The nest was kept in a termitarium at the Institut de Recherche pour le Développement (IRD) in Pointe Noire (Congo). Termites were identified on the basis of morphological criteria and in comparison with laboratory collections by Dr Alain Robert, entomologist and termite expert. The nest was partially broken to collect the eggs, larvae and adult workers. The two larval instars were distinguished using the standard morphometric criteria for the Nasutitermes genus [10,11]. Triplicate samples of 50 eggs, 30 individuals of each larval stage and 25 workers were used. The eggs were surface-sterilized by dipping them twice in 70% ethanol for 1 min, followed by five rinses in sterile water. The whole gut of termite larvae and workers was removed aseptically using fine sterile forceps under a bacteriological hood and pooled for each replicate in 1.5 ml sterile microtubes. Guts and eggs were first crushed using a polypropylene micro pestle in 1.5 ml microtubes. The DNA was then extracted using the DNeasy Blood & Tissue Kit (QIAGEN, QIAGEN GmbH, D.40724 Hilden, Germany) in accordance with manufacturer’s instructions. The final DNA concentration was determined by electrophoresis in a 1% agarose gel, with a quantitative DNA ladder (High DNA Mass Ladder, Invitrogen, 5791 Van Allen Way Carlbad, CA 92008) for comparison. Prior to pyrosequencing analyses, the DNA concentration of the samples was quantified photometrically using a ND–1000 Spectrophotometer (NanoDrop products, Wilmington, USA). Pyrosequencing and processing of 16S rRNA gene sequence: The purified DNA from the triplicates was pooled for pyrosequencing. Aliquots with similar DNA concentrations were sent to the Research and Testing Laboratory (Lubbock, TX, USA) for sequencing. A DNA fragment spanning the V1-V3 variable regions of the 16S rRNA genes was amplified using the eubacterial primers 28F and 519R and pyrosequenced using a Roche 454 FLX pyrosequencer. The resulting sequences were analyzed using Mothur v.1.33.3 [12]. Pyrosequencing reads were processed using the method described by Schloss [13]. Sequencing error was reduced using an implementation of the AmpliconNoise algorithm and low-quality sequences were removed (minimum length 200 bp, allowing 1 mismatch to the barcode, 2 mismatches to the primer, and homopolymers no longer than 8 bp). Sequences were then trimmed to keep only high quality reads (Q ≥ 35). Chimeras were removed using the chimera.uchime mothur command. Singletons were included in the analysis. Sequences were aligned and classified according to the SILVA bacterial SSU reference database v.102 [14]. They were then assigned to genus-level phylotypes using the naive Bayes classifier implemented in Mothur and clustered into operational taxonomic units (OTU) using the average neighbor algorithm and a sequence identity cutoff of 97%. The Greengenes database [15], and the DictDb reference database v.2.3 [6] were used for the taxonomic assignment of OTUs. The shared OTUs (97% sequence identity) between life stages (eggs, L2 larvae and workers) were determined using the Venn diagram package implemented in Mothur. The Venny 2.0.2 software was used to plot resulting data. Relative abundance of each group of OTUs was added manually. Sequences are available in NCBI Sequence Read Archive under BioSample accession ID SAMN03114856 and SAMN03114878 to SAMN03114880 associated with BioProjects PRJNA270400- PRJNA270403. In each pyrotag library, the relative abundance of an OTU is the percentage of reads included in this OTU with respect to the total number of reads. The relative abundance of each taxon in a given sample is the sum of abundances of all the OTUs included. Clone libraries and phylogenetic analysis: The near-full-length 16S rRNA genes were amplified using the 27F/1390R primers [16]. PCR reactions were performed in 25 μL reaction mixtures containing 15.2 μl Taq polymerase Master Mix (Qiagen), 0.25 μM of each primer and 50 ng of template DNA as described by Thongaram et al [16] using a Prime Elite thermal cycler (TECHNE®, Bibby Scientific Limited, Staffordshire, UK). Amplicons were gel-purified using the GFX™ Purification Kit (GE Healthcare, Buckinghamshire, UK) and cloned into the pCR4-TOPO vector using the TOPO TA cloning kit (Invitrogen), following the manufacturer’s instructions. White clones were screened for the presence of the expected insert by PCR amplification using the vector-specific primers M13F-20/M13-26R. For each sample, 70 positive clones were double-sequenced at Beckman Coulter Genomics (Takeley, CM22 6TA Essex United Kingdom). The sequences were quality checked and processed in the same way as for pyrotag sequences. A phylogenetic analysis was performed using OTUs classified within the most representative phylum (Spirochaetes). A nucleotide Blast search was run and the five closest sequences imported for each OTU. Other Spirochaetes 16S rRNA gene sequences were randomly imported. The redundant sequences were removed and the sequences were aligned using SILVA SINA (www.arb-silva.de/aligner/) with the reference dataset SILVA SSU Ref NR [17]. The alignment was improved manually by removing conserved gaps and ambiguously aligned regions. The phylogenetic tree was reconstructed using the Maximum Likelihood method implemented in MEGA v 6.0 [18] and the Tamura-Nei model with 1000 bootstraps.
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