Tests were performed between 12:00 and 16:00 hrs. Using forceps,
we transferred flies by their hook and fixed them to a magnetic
clamp, which was then attached to a rack. This treatment established
free movement of the flies’ tarsi and proboscis and was a
modification from a previously described PER assay Scheiner 2014Scheiner 2004, itself derived from similar assays developed for honeybees Jr 1998Scheiner 2014Scheiner 2004, itself derived from similar assays developed for honeybees Jr 1998. The modifications were designed to prevent unnecessary stress and pressure on the
abdomen of the flies. A group of six to eight flies was tested in
parallel. A filter paper soaked with sucrose solution was presented
for 5 s to all six tarsi but not the proboscis. Seven different
concentrations (0, 0.1%, 0.3%, 0.6%, 1%, 3%, and 30%) were presented
in series with an inter-stimulus interval of 80 s. The proboscis
extension response was recorded. Finally, the proboscis was
stimulated by 30% sucrose solution. Flies not responding to this last
stimulation or responding to the first stimulation (water only) were
discarded from the analysis.