Honeybee gut flora of Devon: Highly specific or variable, a survey at
the individual, hive, site and county level.
Recent decline in honeybee numbers has precipitated in an increased
interest in the microbial communities associated with bee guts. Some
microbes positively affect digestion and immune system function of the
bee, whilst others are pathogens and therefore have a negative effect on
bee health.
Recent work has suggested that honeybees have a highly specific and
non-diverse microbial fauna in their guts. This project will use next
generation sequencing to test this hypothesis in local honeybee hives.
You will be given (dead) bees from a single beehive, and process material
from intestines. You will extract DNA and use PCR to amplify the
prokaryotic and eukaryotic microbial fauna from five individual bees. This
material will be exhaustively sequenced using the in house HiSeq Illumina,
and you will obtain 10-100s of thousands of SSU sequences, which will
give allow you build up the full microbial profile of each individual
processed bee.
Whilst the data will be analysed by students individually, another
component of the project will integrate the data into a larger database to
give a profile of bee gut diversity over a single apiary and across the
county.
Part1: Locate beekeepers at 4 sites and obtain bees.
Beekeepers: Collect 60 bees from four different beehives (on a particular
week).
Students: Assigned a single hive. Consult beekeepers about history of
beehive. Origin of queen and past antimicrobial treatments.
Part 2: Processing of bees.
Students: Remove the guts from frozen bees, retain the thirty bestdissected samples and transfer to individual eppendorfs for later DNA
extraction.
Materials required: Petri dishes. Scalpel, forceps, ethanol, flame, bench
space, eppendorfs, freezer space.
Part 3: Molecular Biology.
Students: Extract DNA from bee guts. Extract DNA from ten bees per hive.
Grind in a 1.5-ml Eppendorf tube in DNA lysis buffer (consisting of 20 mM
Tris–Cl, 2 mM sodium EDTA and 1.2% Triton X-100). Digest with 20 mg/ml
lysozyme and incubated for 30 min at 37°C, followed by
Phenol/chloroform extraction. PCRs will be carried out on five best DNA
samples. 3 PCRs per sample for each primer set.
Materials required: 10 primer sets per students, 2 x tagged Prokaryote
primers SSU-V9 region (Degnan and Ocham 2012) eukaryote SSU-V6
primers (Amaral-Zettler 2009). Phusion taq, Filter tips, Phenol, Micro
pestles.
Part 4: Sequencing
Students: PCR samples standardised for quantity and pooled and
submitted to Konrad for sequencing.
Konrad to carry out sequencing and basic analysis including assembly of
sequences removal of low quality reads and bioinformatic separation of
samples according to tags and remove primers.
Part 5: Bioinformatics
Students: Prepare pie charts of Genus/Family of eukaryotes and
prokaryotes. Use Unifrac to cluster data.
Expected data out:
Students: Eukaryotic and prokaryotic diversity charts from 5 bees from a
single hive.
Overall: Microbial profiles of 50-75 honeybees across three or four sites in
Devon. Should conservatively get 50000 reads for both Euks/Proks per bee.
Reference for methodology:
ISME J. 2012 Jan; 6(1): 183-94. doi: 10.1038/ismej.2011.74. Epub 2011 Jun
16.
Illumina-based analysis of microbial community diversity. Degnan PH,
Ochman H.