Honeybee resistance against Pl

Paenibacillus larvae (PI) is a bee-pathogen which causes considerable damage every year in beekeeping world- wide. In Austria and in most European countries, a medical treatment of honey bee colonies with conservative antibiotics is not permitted. Breeding of bees for resistance or a method for treatment with bee-generated substances is a desirable goal. At the Institut for Zoology, University of Graz, a substance was found in larvae as well as in adult bees which inhibits the growth of Pl in vitro. At present this substance is available in an unpurified fraction. The target of this project is the purification of the substance or the group of substances, respectively, their analyses (metabolic profiling) and the description of the respective genes (genomics). These results should enable the classification of the substance and their integration into the family of protein-type antibiotics. At the same time, insight should be gained into the evolutionary development of immunity of insects in general and particularly the immunity of colony forming insects.

This is the final report of the successfully accomplished project: "Honeybee's Resistance against Paenibacillus larvae (Pl)", which took place at the Institute of Zoology at the University of Graz. The goal of the project was to elucidate the mechanism of adult honeybees resistance against the spore-forming bacterium Paenibacillus larvae that is responsible for the devastating honeybee disease American foulbrood (AFB). During the course of the project it could be demonstrated that a phospholipid (lecithin) in the midgut of the adult honeybee is responsible for an antibacterial activity against this bacterium. This compound, lyso-phosphatidylcholine (LPC) is contained to c. 1% in the midgut of honeybees where it prevents Pl from proliferation and also kills vegetative forms that germinate from the spores of Pl. Its activity in vitro was found to be in the micro molar range both against Pl and also Melissococcus plutonius, the causative agent of European foulbrood (EFB). The finding of an antibacterially acting phospholipid came surprising since to-date antimicrobial principles isolated from honeybees exclusively belong to proteinous compounds. Hence our strategy was initially targeted towards the workup of peptides and proteins. The step leading to the elucidation of LPC included in vitro assays used for bioactivity-guided fractionation by HPLC of midgut extracts. The positive influence of LPC against other bee diseases, such as EFB, was proven. To prove the principle of LPCs activity against Pl, experiments with honeybee larvae using artificial larval rearing were carried out. In this in vivo assay in an in vitro environment, larvae, which were artificially infected with Pl spores and treated with LPC, showed a significantly reduced mortality. After LPC was identified and its yet undescribed activity of LPC against gram-positive sporulating bacteria was sufficiently proven, a European patent application claiming LPC to be useful in the treatment of honeybee brood diseases was filed in May 2013. After a positive evaluation of the novelty of this finding, the application was kept up and is currently processed by the European Patent Office. The findings of this project could therefore be of crucial importance in the development of therapeutic agents against bee diseases.