Midgut microbiota sequencing of lab reared 5th instar
Briefly, 16S universal Eubacterial primers- 27Fmod (5_-AGRGTTTGATCMTGGCTCAG-3_) and 519R (5_-GTNTTACNGCGGCKGCTG-3_) with a sample specific bar code was used for amplifying the DNA. PCR was performed using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA). The PCR reaction conditions were 94  C for 3 minutes, followed by 28 cycles of 94  C for 30 seconds; 53  C for 40 secods, and 72  C for 1 minute after which a final elongation step at 72  C for 5 minutes was performed. Midgut DNA amplification was followed by purification of amplicon products using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). The amplicons were sequenced using Roche 454 FLX titanium instruments (MR DNA Lab, TX, USA) and reagents and following manufacturer s protocol.