Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.