Effect of feeding different levels of the isoleucine amino acid on hemolymph protein concentration and growth of hypopharyngeal glands in worker bees (Apis mellifera)

Introduction: Honey bees need nutrients such as protein, carbohydrates, lipids, and minerals. Among nutrients, protein is of particular importance due to its role in brood rearing, developing hypopharyngeal glands and ovaries, longevity, bee immunity, bee weight, and flight muscle development. The protein stored in the body of bees is vitally important in strengthening the immune system, producing royal jelly, and especially in wintering. The honey bee obtains its protein needs through the digestion of different pollen grains with different amounts of protein in the midgut. Among the amino acids required by honeybees, the most important are leucine, isoleucine, and valine. Pollen quality depends on the supply of amino acids required by honey bees. Most pollen grains are poor and deficient in isoleucine. This study aimed to evaluate the effect of different levels of isoleucine on hemolymph protein and the growth of hypopharyngeal glands in worker beesMaterials and methods: To investigate the effect of different levels of isoleucine on hemolymph protein and the growth of hypopharyngeal glands in worker bees, an experiment was conducted in a completely randomized design with five experimental diets and four replications (cages). 100 honey bees were kept in each cage. Diets included 1 M sucrose syrup (control), and different levels of isoleucine containing 0.5, 1, 1.5, and 2 micrograms per liter of 1 M sucrose syrup. Before starting the experiment, the egg frames in the hives were marked so that the bees were of the same age at the time of the experiment. The cages were placed in an incubator at a temperature of 34°C and a humidity of 60%. Samples were taken to measure hemolymph protein on days 1, 6, 12, and 18 of 10 bees and for growth of hypopharyngeal glands on days 3, 6, 9, 12, and 15 of three bees. The hemolymph protein amount was measured by UV spectrophotometer and Bradford method and the growth of hypopharyngeal glands by the diameter of the ascites.Results and discussion: The results showed that the difference in hemolymph protein and the growth of hypopharyngeal glands of bees was significant in the whole experiment period and at different ages (P<0.01). A comparison of the experimental treatments revealed that the 1.5 μg isoleucine treatment had the highest amount of hemolymph protein among the diets (6.65 μl/μg), and the difference with other treatments was significant (P<0.01). The control treatment had significantly the lowest hemolymph protein among the experimental diets (3.57 μl/μg). The use of high amounts of isoleucine decreased the hemolymph protein. Therefore, the appropriate level of isoleucine in a honey bee diet is 1.5 μg. In all diets, except the control diet, the highest amount of hemolymph protein was observed on the sixth day, but in diet control, the amount of hemolymph protein decreased. Regarding the hypopharyngeal glands, the comparison of the treatments revealed that the 1.5 μg isoleucine treatment had the largest acini diameter (0.083 mm) and its difference with other treatments was significant (P<0.05). The control treatment (0.065 mm) significantly had the lowest acini diameter among the experimental treatments. Comparing the diameter of acini at different ages showed that the largest diameter of acini was observed at three days of age and the smallest diameter was seen at 15 days of age in all treatments. Some of the increase in hemolymph protein is likely due to the consumption of isoleucine, and some of it is due to the increase in the activity of hypopharyngeal glands because the consumption of isoleucine has increased the growth of hypopharyngeal glands.Conclusions: In this experiment, it was found that the hypopharyngeal glands grew to the maximum level, and then, the amount of protein in the hemolymph of bees reached the highest level. Based on the findings of this research, the use of isoleucine in the diet of bees can increase the production of royal jelly and brood rearing due to its influence on the growth of hypopharyngeal glands and the amount of hemolymph protein. The appropriate level of this amino acid in bee nutrition is 1.5 μg per liter of sucrose syrup because consuming more than this optimum level harms hemolymph protein and the growth of hypopharyngeal glands.The difference between hemolymph protein and hypopharyngeal gland growth of bees was significant in the whole experimental period and at different ages (P <0.01). Diets containing 1.5 μg of isoleucine and control diet had the highest (6.65 μl / μg) and the lowest (3.57 μl / μg) of hemolymph protein and the highest (0.083 mm) and lowest (0.65 mm), respectively. There was no significant difference between hemolymph protein in syrup containing 0.5 and 2 micrograms of isoleucine and growth of hypopharyngeal glands in treatments of 0.5, 1 and 2 micrograms of isoleucine. Therefore, the best level of isoleucine used in bees for proper production performance was the feeding of syrup containing 1.5 μg of isoleucine.