I would like to collect hemolymph (at least 10 microlitres) from worker bumblebees in the field. I tried the protocol by Borsuk et al. 2017 (Borsuk G, Ptaszyńska AA, Olszewski K, Domaciuk M, Krutmuang P, Paleolog J (2017) A New Method for Quick and Easy Hemolymph Collection from Apidae Adults. PLoS ONE 12(1):e0170487. doi:10.1371/journal.pone.0170487): I captured a bumblebee with a hand net, blocked the bee on the edge of a polystyrene piece with my thumb, removed one antenna (then the other to be sure) with tweezers, gently pressed the bee’s abdomen to increase hemolymph pressure… and not a drop came out!
I also tried to collect hemolymph by puncturing the cuticle with a capillary (between the head/ thorax or between the second and third tergites of the abdomen [side sting]), but I could find only small amounts of hemolymph and there is a high risk of contamination (probably connected to a prolonged drought?).
Has anybody tried the method conceived by Borsuk and colleagues?
It is much less invasive than the microcaps method, so it would be better for my type of study.
Do you have any suggestions related to the best method to collect hemolymph from wild bumblebees in field conditions?
you can collect the hemolymph from the joint connection between the cocsa and sternum
I have found some material in the following paper. http://www.mcponline.org/content/5/12/2252.full
"Hemolymph Collection—
Drones, hive workers, worker larvae, and three queens were collected from colonies maintained at the University of British Columbia, Canada. An additional queen was acquired from a feral colony in Vancouver, Canada. Samples were taken in early autumn and spring. Additional worker larvae samples were collected at the Agriculture Canada Research Station in Beaverlodge, Alberta, Canada. For collection from all adult castes, the bee was held between soft forceps, trapping its wings against the thorax, while a glass disposable 5-μl Microcap pipette was inserted dorsally into the bee between the second and third abdominal terga from the thorax. Clear and slightly yellow hemolymph was drawn out by capillary action. If cloudy yellow intestinal contents were taken, the sample was discarded. Hemolymph was dispensed into a 1.5-ml microcentrifuge tube containing 100 μl of PBS with EDTA-free protease inhibitor mixture kept chilled on ice. For collection of larval hemolymph, 5-day-old (estimated) larvae were taken and washed in 10 ml of PBS to reduce contamination from worker jelly. A small incision was then made two-thirds of the way down one side of the larva, and milky white hemolymph was drawn as described above. Inspection of larval hemolymph by light microscopy revealed that the cloudiness of the liquid was due to a high number of hemocytes and prohemocytes. The microcentrifuge tubes were centrifuged at 16,100 × g in a microcentrifuge (Eppendorf) for 10 min at 4 °C to pellet these cells and other debris, which were discarded. For protein identification, hemolymph from three to six workers or drones was pooled, while for quantitation, three samples of two bees from each caste were analyzed independently. Hemolymph from the queens was not pooled at all. The protein concentration of clarified hemolymph was assayed using Coomassie Plus Protein Assay reagent (Pierce) according to the manufacturer’s instructions and stored at −20 °C."
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