Quantitative PCR reactions were performed in presence of SYBR Green on ABI Prism 7000 gene expression system according to the manufacturers’ instructions (Applied Biosystems, France) using 5-time dilution of each DNA. Bacteria were quantified using specific primers designed to amplified a 16S rDNA 150-bp-length fragment of Blochmannia (16SFor 5′-AGAATTCCAGGTGTAGCGGTG-3′ and 16SRev 5′-TACGGCATGGACTACCAGGG-3′). Ant DNA were quantified using specific primers designed to amplify a 18S rDNA 150-bp-length fragment (18SFor 5′-TTAGAGTGCTTAAAGCAGGC-3′ buy Cilengitide and 18SRev 5′-ACCTCTAACGTCGCAATACG-3′). These primers had been efficiently

used in another study with Blochmannia floridanus [14]. The 18S rRNA ant gene copy number was used so as to normalize each dissected

sample with the same quantity of ant DNA material. This gene was first specifically cloned and sequenced. Then real-time PCR specific primers (18SFor 5′-TTAGAGTGCTTAAAGCAGGC-3′ buy CH5424802 and 18SRev 5′-ACCTCTAACGTCGCAATACG-3′) were design based on the sequence and used to generate by classic PCR a 18S rDNA specific amplicon used to establish a standard curve expressing the Cycle Threshold (Ct) versus the logarithm of the copy number of 18S rDNA purified PCR products. These specific primers were also used to amplify 18S rDNA using DNA extracted from dissected samples. The exact copy number of 18S rDNA was established based on the experimentally obtained Ct value and the standard curve. This value was used to correct the calculated copy number of learn more bacterial 16S rDNA. Fluorescent 4��8C In Situ hybridisation (FISH) Bacteriocyte were visualized by FISH with oligonucleotide probes as previously described in the method topic “”Symbiont identification”". Acknowledgements We thank Danielle Mersch and Stephane Dorsaz from Lausanne University and Abraham Hefetz from Tel-Aviv University for collection of the mated queen ants. Heike Feldhaar and Sascha Stoll from Wurzburg University aided us on FISH and Quantitative PCR techniques. Terezinha Della Lucia and Elisabeth Huguet aided us to improve the writing. The first author was financially supported by grants from the Capes (Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior-Brasil). References 1. Wernegreen JJ: Endosymbiosis: Lessons in conflict resolution. Plos Biol 2004, 2:307–311.CrossRef 2. Feldhaar H, Straka J, Krischke M, Berthold K, Stoll S, Mueller MJ, Gross R: Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia. BMC Biol 2007, 5:48.CrossRefPubMed 3. Dethlefsen L, McFall-Ngai M, Relman DA: An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 2007, 449:811–818.CrossRefPubMed 4. Margulis L, Fester R: Symbiosis as a source of evolutionary innovation-speciation and morphogenesis Cambridge, MIT Press 1991. 5. Moran NA: Symbiosis as an adaptive process and source of phenotypic complexity.