Short communication. Impact of the amino acid proline on the cold hardiness of honey bee, Apis mellifera L.

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.

2002). Consequently, growth of the crystal (addition of water molecules to the crystal surfaces) can only occur between the adsorbed AFPs and in high radius of curvature fronts (high surface free energy), rather than the preferred low radius of curvature fronts (low surface free energy). Therefore, according to the Kelvin effect, the temperature must be lowered below the colligative melting point for growth to proceed (Duman et al., 2004).
There are many apiculture industries in Zanjan, Iran, suffering bees cold lose. Despite this, rare works have been done on the improvement of these colonies' cold tolerance by diet manipulation. Knowledge of SCP would indicate the coldest possible temperature extremes that A. mellifera could survive and provide insights about optimum hive utilization in the cold situations. The aims of this study were to determine (i) the supercooling point of A. mellifera, and (ii) the impact of the amino acid, proline on honey bee cold hardiness.
Studies were conducted at University of Zanjan (36.4108N 48.2424E, 1586 m asl) in the f irst days of autumn (immediately before the start of cold season). Three combs containing worker honey bee pupae were selected from a colony reared in this university. To produce cohorts with similar age, the pupae were allowed to hatch and 4 groups of 1-3 days old emerged bees (each group included 50 bees) were separated and put into 4 cages to establish the experiment treatments.
The dimensions of experimental wooden cages, with transparent mesh walls, were 15 cm × 10 cm × 15 cm. Each cage was provided with a vial of water and a piece of wax comb on the underside of the cage lid. The cages were held in the dark in an incubator at 33 ± 2°C and 50 ± 5% RH, during the experiment.
The treatments consisted of three different concentrations of proline (1%, 3% and 4.35%) diluted in 1:1 water sucrose syrup, and the syrup without proline (control), as bees' diet. Honey bees with their diet (in a vial) were put inside the cages, and 15 cm 3 of each food was added daily to its related cage for 14 days. Every day, dead individuals were removed from cages, and at last day (day 14), 20 bees from each cage were captured to assess their cold hardiness according to the method provided by Jones et al. (2008).
The cold hardiness of honey bees were determined in a cooling bath in which temperature was reduced by a rate of 0.5°C min -1 . A CHY data logger, equipped with a K100 sensor (with accuracy of 0.1°C), was used to track temperature changes. The thermal sensor was attached to the gaster of worker honey bee and the insect was put in the cooling bath inside a test tube with 1-cm in diameter. The data logger was interfaced with a computer, and utilizing its software, the SCP, the amount of released heat and the rate of this release as measures of insect cold hardiness were recorded.
The experiment was performed in a completely randomize design (CRD), and the analysis of variance was performed on supercooling point, released heat and the rate of this release. If treatments were significant at p < 0.05, then differences between means were determined using the Turkey's HSD test at 95% confidence level.
Mean SCPs of each treatment are shown in the Table 1. Honey bees' average SCPs in the three prolinecontaining treatments were significantly lower than the amount of control cohort. The lowest point (-7.67 ± 0.2646°C), which was significantly different from the control and the two other treatment cohorts, was observed in 1% proline. Mean SCPs recorded in the concentrations 3% (-6.79 ± 0.1552°C) and 4.35% (-6.85 ± 0.2197°C) were statistically equal. There was no significant difference between the four treatments of experiment in the amount of released heat and the rate of this release.
To our knowledge, this is the first study of SCP in relation with the amino acid proline on honey bee. Supercooling ability can often be attributed to the accumulation of cryoprotectant chemicals and/or the absence on ice nucleating agents (Lee & Denlinger, 1991). In the current study we found that a change in the proline concentration in diet can alter honey bee's SCP, so proline could be considered as an important antifreeze agent in its body.
On the other hand, the lowest mean SCP was observed in the lowest concentration of proline and it was significantly different from the three other means. A possible explanation to this process could be the impact of high level concentrations of proline on the insect feeding rate. Thereupon, it is predicted that a concentration threshold exists, above which the feed rate and so the proline acquisition decrees and below which, by increasing the amino acid in the diet, supercooling point decreases.
It has been demonstrated that proline is the most dominant amino acid in honey bee hemolymph (Mullins, 1985), which the insect can obtain through feeding on pollen, the main proline source for honey bee (Lipp, 1991;Funck et al., 2012;Hossain et al., 2012). Our results with regard to the existence of this amino acid in bees' hemolymph and natural diet warrant further studies on dietary manipulation of the proline concentration in the insect body for a more successful overwintering.