Outsourcing glycolysis! Role of proline in honeybee flight

Insect flight is among the most energy demanding behaviours in the animal world. Supposedly in hymenopteran flight the apex of evolutionary optimization of mass based output of mechanical energy of any muscular system has been achieved. It is already well known that in several insect species proline is utilised as an energy carrier to power flight and heating. Proline is oxidised in the flight muscle and free ammonia transaminated to form alanine wich in due course is reconstituted to proline in the fat body. Thus energy derived from carbohydrate, lipid or amino acids is relayed by proline while ammonia accumulation is curtailed. Yet in honeybee workers, who have high levels of free proline and low levels of free alanine in their haemolymph, proline is metabolised vigorously although measurements of the respiratory quotient (RQ=1) indicate carbohydrate as the primary energy source. Some preliminary experiments done by me indicate that radioactive label from injected proline does not shift to alanine yet radioactive label from injected glucose does shift to proline during flight. Those data lead to the formulation of the hypothesis, that in honeybee workers (drones and queen might realize another mechanism) proline is oxidised in the flight muscle and at the same rate synthesised in the fatbody from carbohydrate (stable proline concentrations during sustained flight point to that). Free ammonia, left over from proline oxidation, might not be synthesised to alanine but is shifted in the free form to the fatbody for proline resynthesis. This would relate to a very efficient mechanism of shuffling acetyle units derived from carbohydrate into the Krebs-cycle of the flight muscle, taking strain off the glycolitic capacities of a flight muscle already packed with mitochondria and myofibrils. Rough estimation from my preliminary experiments indicate, that up to 30% of carbohydrate derived acetyle might be transferred through this pathway. In this project the mechanism proposed above will be ascertained in honeybee workers, drones and queen and the proportion of energy relayed by this mechanism will be determined under different aspects (during rest and flight but also in post-emergence development of the flight muscle and during larval development). In future investigations comparative studies of proline relayed energy metabolism in diverse insect species should address evolutionary aspects of this pathway. Towards this end in this project a standardised protocol for screening various species will be devised which should facilitate work under field conditions as well as sending samples per mail.

In honeybee flight the mass based energy turnover in the flight muscle cells is the highest yet reported in any animal tissue. Honeybees survival depends on their ability to perform sustained flight of worker bees in foraging nectar, pollen or water as well as in mating flights of drones and queens and flights in absconding behavior.Although carbohydrate, derived from nectar or honeydew, is well established as the primary energy source in honeybee flight, high levels of the amino acid proline in honeybee blood and tissue and limitations of glycolytic capacities in the flight muscle cells gave rise to the suspicion, that a secondary, intermediate, pathway of energy transfer to the flight muscle mitochondria could exist.The amino acid proline is known as an energy source in some insect species but has not yet been shown to contribute to sustained flight. Glycolysis is a metabolic pathway in the cells to process carbohydrate in a way, that the product can be utilized by the mitochondria to store energy in the form of ATP. Proline, after some transformation, can be utilized in the mitochondria directly, circumnavigating glycolysis in the cells.Our experiments were carried out by injecting small amounts of proline, which was marked with radioactive carbon 14, into honeybees and following the fate of the radioactive label throughout the internal metabolism of the bees. Flight behavior was simulated by fixing the bees on the arms of a roundabout, where bees flew for up to two hours and the flight parameters were registered. What we found was, depending on flight duration, the radioactive label was decreasing . This indicates that carbon 14 was exhaled as proline was consumed in respiration, the process leading to energy storage in ATP. Yet, the overall content of proline in the bees did not decrease! Obviously, as carbon 14 marked proline was consumed, proline containing no additional carbon 14 was synthesized in the bee from non- radioactive sugar which was fed to the bees before flight. This, in short, means, that about 20% of the flight energy derived from carbohydrate does not go directly to glycolysis in the flight muscle but is (presumably in the fat body in the abdomen) transformed in a continuous cycle to proline, which is transferred through the bloodstream to the flight muscle. There proline in transformed into a molecule, ketoglutarate, which can be directly introduced into the mitochondrial pathways. As yet we have shown that this mechanism is present in honeybee workers and drones and the full capacity of this pathway is reached only as honeybees mature to perform sustained flight.We suggest that this partial outsourcing of glycolysis from flight muscle to fat body is a further puzzle piece in understanding honeybee flight muscle performance.