Cuticular hydrocarbon extractions and GC analysis

Honey bee CHC extraction

Supplies

1. Hexane GC grade (Fluka brand sold through Sigma Cat# 52767-2.5L)
2. Octadecane (Millipore Sigma CAS  593-45-3)
3. Hexacosane (Millipore Sigma CAS 630-01-3)
4. 6ml screw vial clear 16mm 100pk - (Agilent Crosslab Cat# 9301-1377)
5. screw cap 16mm PTFE/sil septa 100/pk - (Agilent Crosslab Cat# 8010-0101)
6. 2ml screw vial clear w/wr 12mm 100/pk - (Agilent Crosslab Cat# 5182-0715) 
7. screw cap 12mm blue PTFE lined solid top 100/pk - (Agilent Crosslab Cat# 5183-2075)
8. insert 250ul conical w/ft 9mm 100/pk - (Agilent Crosslab Cat# 8010-0131)
9. narrow feather weight forceps - (BioQuip Cat# 4748)

Protocol

1. Collect bees, flash freeze in liquid nitrogen and keep in -80°C freezer.
2. Allow hexane to come to room temperature and prepare the hexane standard by dissolving 10 ng/ul of octadecane and hexacosane in as much hexane that you need for the experiment. Keep extra hexane standard in a -20 C freezer.
3. Prepare as many 6ml vials containing 500ul of hexane as is required for that day’s extractions. Make sure to include mock extraction vials (5-6 per hexane standard), and 3 aliquots per hexane standard vial (put immediately in 2 mL vials). Immediately close vials with screw cap. 
4. Remove 4 bees from the freezer and keep them on dry ice.
5. Quickly remove each bee from their microcentrifuge tube with the feather weight forceps and place into a prepared 6ml vial. If you’re using the whole bee body, you could alternatively dump the bee from the microcentrifuge tubes directly into the hexane vial.
   a.    Immediately cap vials!
   b.    Note: The scissors and the forceps should be cleaned AFTER EVERY USE. Do this by dipping them serially first in GC grade methanol then pass three times through separate washes of GC grade hexane. After each dip, wipe the forceps with a Kimwipe. Different 6ml vials can serve as wash vials. 
6. Immediately cap vials and place on the vortex at speed 1 (lowest speed).
   1. I created a makeshift vial holder for the vortex using an empty tip box stuffed with a Styrofoam Falcon tube holder taped to the vortex platform.
7. Mix for 2min. 
8. After 2min, quickly remove the hexane from the 6 mL vial and transfer to a 2 mL vial (the septa from the 6 mL vials will leach chemicals into the hexane after too long) using a sterile pipette.
9. When all samples are collected, remove a 100ul aliquot from each and pipette it into a new 2ml vial and cap. 
10. Store the remaining hexane extract in the first 2ml vial at -80°C. Keep this sample as a backup in case something goes wrong with the shipping.
11. Continue to store the 100ul in the 2 ml vials at -20°C until ready to be analyzed.
12. If shipping, ship the samples on dry ice. 

Gas Chromatograph Analysis

Samples were analyzed using an Agilent 7890A gas chromatograph system with a flame ionization detector (GC/FID) and PTV injector (cool-on-column mode), and outfitted with a DB-1 20 m x 0.18 mm Agilent 121–1022 fused silica capillary column (Agilent Technologies, IncSanta Clara, CA, USA). 

1. Inject sample volumes of 1.0 μl onto the column. 
2. Apply helium as the carrier gas at a constant flow rate of 1 ml/min. 
3. Analyze the extract with a column temperature profile that begins at 50°C (held for 1 min) and is ramped at 36.6 °C/min to 150°C and then at 5°C/min to 280°C, and hold for 10 min. 
4. Program the injector and FID temperatures to 280°C and 300°C, respectively.
5. Use Agilent OpenLAB CDS (EZChrom Edition) software to calculate the retention time and total area of each peak. 
6. Normalize data to known quantity (ng) of internal standard hexacosane. You should know the amount of internal octadecane standard and should see this amount when you normalize to hexacosane.
7. Calculate the relative proportion of each compound by dividing the normalized ng of each peak by the sum of the total ng of CHCs per sample.
8. Perform statistical analysis, such as Permutation MANOVA, using relative proportion data or raw ng values.