Like many insects, honey bee can increase its cold tolerance through freeze avoidance, using antifreeze proteins (AFPs) to lower its supercooling point (SCP). Proline is the most dominant amino acid in honey bee hemolymph, which can be obtained by the insect through feeding. In the current study the antifreeze activity of this amino acid was evaluated on worker honey bees, immediately before the start of cold season. The experiment was established on four treatments including three different concentrations of proline (1%, 3% and 4.35%) diluted in 1:1 water sucrose syrup, and the syrup without proline (control). Newly emerged worker honey bees were fed on the mentioned diets for 2 weeks, under cage condition, and then 20 bees from each treatment (cage) were selected randomly for determination of cold hardiness inside a cooling bath. Using a CHY data logger, equipped with a K100 sensor attached to the bee's gaster, the SCP, the amount of released heat and the rate of this release as measures of insect cold hardiness were recorded. Proline significantly reduced honey bees' SCP. The lowest point, -7.67 ± 0.2646°C, was observed in the concentration of 1% proline. The amount of released heat and the rate of this release were not significantly different across the treatments.
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