Quantifying Gut Microbiome Changes in Mosquitoes

Mosquitoes were collected 5 day after dsRNA treatment or antibiotic treatment and surface sterilized with 70% ethanol twice and 0.9% NaCl twice. Midguts were dissected and homogenized in 0.9% NaCl. Homogenates were serially diluted and plated on LB agar plates. CFUs were counted 2 days after incubation at 28°C. Total DNA was extracted by the method of Holmes and Bonner as described [64 (link)]. Bacterial density was quantified by qPCR using universal 16S rRNA primers [28 (link)] (S1 Table). Ribosomal gene S7 was used as the internal reference. Significance was determined using the Student’s t-test.
The composition of the gut microbiota in dsRNA treated mosquitoes was analyzed by pyrosequencing that targeted the V3-V4 region of bacterial 16S rRNA [65 ]. 10 midguts of dsRNA treated mosquitoes were pooled for 1 biological replicate. DNA of 3 biological replicates of each treatment were prepared for further sequencing analysis (S1 Text).
For fluorescent in situ hybridization (FISH), abdomens of dsLD treated females 2 day post blood meal were fixed and sectioned as described [66 (link)]. Slides were hybridized with 10ng/μl universal 16S ribosomal RNA probe (5’-GCTGCCTCCCGTAGGAGT-3’) labeled with Alexa Fluor 555 (Life technology). Tissues were visualized using Nikon ECLIPSE IVi microscope connected to a Nikon DIGITAL SIGHT DS-U3 digital camera.