sulcatus, O. rugosostriatus, O. salicicola and O. armadillo) collected in the field and kept in the laboratory until egg deposition. During that period
of time weevils were fed with leaves of Prunus sp., Potentilla sp. or Fragaria sp.. Freshly laid weevil eggs (at most 10 days old) were collected and surface sterilized according to the method developed by Hosokawa et al . The eggs AZD6244 were air dried under the clean bench and transferred individually with sterile featherweight forceps in Petri dishes filled with sterile TSA (40,0 g/l DifcoTM Tryptic Soy Agar, pH 7.3 ± 0.2; Voigt Global Distribution Inc, Lawrence, Kansas). In order to enlarge the contact of egg and TSA agar and to check the success of surface sterilisation,
eggs were rolled several times over the agar plate. For further analysis only eggs with no bacterial growth on TSA were included. Eggs were kept usually at 21-24°C until eclosion. Freshly emerged larvae (approximately 24-72 hours old) without egg material were individually collected from the TSA agar plates, and were stored frozen at -80°C until further processing. Total metagenomic DNA (~20-40 ng/µl DNA per larva) was extracted from the complete larvae using the MasterPureTM DNA Purification Kit (Epicentre® Biotechnologies, Madison, Wisconsin). Taxonomic identity of each larva was confirmed according to a Selleckchem Fosbretabulin diagnostic PCR-RFLP pattern of the COII region . For metagenomic analysis seven individuals of each Otiorhynchus species were included. Bacterial 16S rDNA PCR amplification and 454 pyrosequencing Universal bacteria primers (fwd: 5’-MGAGTTTGATCCTGGCTCAG-3’ and rev: 5’-GCTGCCTCCCGTAGGAGT-3’; LGX818 mw ), amplifying an approximately 450 bp fragment of the 16S rDNA, were used in the present study. These primers are covering the V1-V2 regions of the 16S rDNA gene and showed good phylogenetic resolution from phylum
to family level in a recent study by Hamp et al . Primers were modified by the addition of a GS FLX Titanium Key-Primer A and B (A: CGTATCGCCTCCCTCGCGCCA and B: CTATGCGCCTTGCCAGCCCGC), Megestrol Acetate a four-base library “key” sequence (TCAG) and a multiplex identifier (MID) sequence specific to each Otiorhynchus species. The MID sequences (forward/reverse) were as follows for the respective weevil species: O. salicicola (ATCGCG / CGCGAT), O. rugosostriatus (ATAGCC / GGCTAT), O. sulcatus (CCATAG / CTATGG) and O. armadillo (CTTGAG / CTCAAG). PCR reaction mixture consisted of 0.1 µl of Phire® Hot Start II DNA Polymerase (Finnzymes Oy, Espoo, Finland), 0,2 mM dNTPs (Metabion, Martinsried, Germany), 10 pmol primers and 40-80 ng of DNA template in a final volume of 20 µl. The PCR parameters (C1000TM Thermal Cycler, Bio-Rad Laboratories GmbH, München, Germany) were 95°C for 3 min followed by 35 cycles of 93°C for 60 s, 50°C for 60 s and 72°C for 70 s. A final extension step at 72°C for 5 min was added.