All unique reads were assigned to taxonomic clades by using the RDPclassifier [39,40]. selleck compound The classifier assigns taxonomic ranks as deep as possible with estimated certainty by using a Bayesian approach. We set the threshold to a 50% bootstrap cutoff for ranks to be adapted, which has been shown to assign more than 90% of sequences to genus level with 95% accuracy in most variable regions of the 16S. We restricted our estimations to the genus level to reduce artifical overestimation of taxonomic units, which is stringent below 97% clustering as suggested by Kunin et al. [37]. This ensures that analyses provide accurate profiling of microbial communities [37]. Singletons were excluded from further analyses. Chloroplast reads were considered to be contamination due to nest-building material and the reed themselves and accordingly removed.
Also, pollen was reported to carry occasional plastid genomic DNA [41]. Hierarchic taxonomic assignments for all bacteria on a generic level were displayed and investigated with KronaTools [42]. The other samples processed with the same sequencing chip included 16S microbial samples obtained non-invasively from surfaces of invertebrate animals that were cultured according to Schokraie et al. [43](3) and plants (5). We assumed the origins to be very different and unlikely to share large proportions of the microbiota. Omnipresent species were thus considered as lab contamination and ignored in the following analyses. Raw sequencing data alongside quality information was uploaded to the public database for environmental sequencing data of the European Nucleotide Archive (EMBL-EBI: ENA) and are retainable through the SRA study accession number ERP002613.
Specific bacteria of interest, i.e. pathogens or commensals known to be of importance for bees or other arthropods were identified to species level by BLASTn [44]. These clades specifically screened for were [1,9,45-59]: ? Gut bacteria: Bacillus subtilis (strains C4, G2III and M1), Bartonella, Bifidobacterium, Burkholderia cepacia, Gilliamella, Enterobacter, Gluconobacter, Klebsiella, Lactobacillus, Saccharibacter, Snodgrassella. ? Potential pathogens: Achromobacter, Bacillus cereus, Bacillus thuringiensis, Brevibacillus, Clostridium botulinum, Enterococcus, Melissococcus plutonius, Mesoplasma, Paenibacillus, Photorhabdus luminescens, Pseudomonas entomophila, Pseudomonas protegens, Rickettsiella grylli, Spiroplasma melliferum, Xenorhabdus bovienii, Xenorhabdus nematophila, Stenotrophomonas.
? Non-pathogenic intracellular bacteria: Mycoplasma, Regiella insecticola, Rickettsia, Sulcia, Wolbachia, Zinderia. ? Associated with pollens: Aurobasidium, Bacillus, Rhizopus. For each of these taxa of interest, we prepared a local BLAST database populated with all 16S sequences of this group present at NCBI (accession date 10th February 2013). We aligned all of our RDP Cilengitide clade-classified sequences against the corresponding databases.