Diverse microbial communities live in the gut regions of animals. The precise ecological and evolutionary circumstances that govern relationships between hosts and their gut communities is unclear. In this study, we hypothesize that host feeding strategy shapes the microbial communities within the gut systems of insects. We collected five insect species from the Sevilleta National Wildlife Refuge that exhibited herbivorous, detritovorous and carnivorous diets. Using gut samples from the insects we measured if and how microbial communities are shaped based on any effect host feeding strategy might have. Preliminary analysis of bacterial communities using 16S rDNA sequences has thus far revealed that the sampled community profiles initially appear to show signs of being determined by host feeding type. Analysis has also shown that sequences from the phyla Firmicutes and Proteobacteria appear to contribute most significantly to the differences between communities of different feeding types. We expect that upon further data recovery, the extent of the effect host feeding type has on the communities will be clarified. Additionally we intend to incorporate bacterial community data from previous studies to further broaden our sample set. We expect our results to further define the ecological circumstances that shape the microbial populations within living systems.
DNA Extraction, PCR Amplification & Sequencing:
Total community DNA was extracted for each intestinal sample by using the phenol-choloroform method with ethanol precipitation. Bacterial 16S rRNA genes were amplified from the community DNA with the bacteria-specific forward primer 8F 5’AGAGTTTGATCCTGGCTCAG 3’ and the reverse primer 1391R 5’ GACGGGCGGTGTGTRCA 3’. PCR amplification was performed in 50 ul reactions containing 5 ul 10X buffer (Promega Buffer B w/ 1.5 mM MgCl2 ), 12.5 mM concentration of each dNTP (BioLine USA Inc.), 20 pmol of each forward and reverse primer, 2.5 U Taq polymerase (Promega, US), and approximately 150 ng of DNA. The PCR thermal cycling program consisted of 30 cycles at 30s at 94oC, 30s at 50oC and 90s at 72oC. Amplified 16S rRNA genes were spin purified using a DNA purification kit (MoBio Laboratories) and cloned using the TOPO TA Cloning Kit (Invitrogen), according to manufacturer’s instructions. Clones were sequenced using 8F primers.
16S rRNA gene sequence data was screened for quality using CodonCode Aligner (CodonCode Corporation). High quality sequences of length greater than 500 bp were then exported for further analysis.
Five coleopterans were chosen based on availability at sampling time (September 13-20 of 2008). Three individuals were collected for each species, all from the Nunn Flats area of the Sevilleta NWR. Coleopterans were chosen based on observed or known feeding habits. Samples were collected alive and placed in plastic bags and stored on ice. Samples were then stored in freezer upon arrival at the lab and processed within 48 hours.
Samples were dissected using sterile technique. A cut was made along the elytra of the insects, and the intestinal tract entirely removed from the sample. Intestinal tracts were stored in sucrose lysis buffer until DNA was extracted from the samples. After intestine sample removal, insects were stored in 95% ethanol and identified and vouchered at the Museum of Southwest Biology at UNM.
Data are accessible from Genbank: