Stonebrood Disease-Histomorphological Changes in Honey Bee Larvae (Apis mellifera) Experimentally Infected with Aspergillus flavus.

Stonebrood (Aspergillus sp.) is a rare, poorly described disease of the Western honey bee (Apis mellifera) that can affect adult bees and brood. This study describes the pathogenesis using artificially reared pathogen-free Apis mellifera larvae, experimentally infected (5 × 102 spores/larva) with Aspergillus flavus. Between days 1 and 5 p.i. (larval age 4 until 8 days), five uninfected control larvae, up to five infected living larvae, and up to five infected dead larvae were examined macroscopically. Subsequently, the larvae were photographed, fixed (4% formaldehyde), and processed for histological examination (hematoxylin-eosin stain, Grocott silvering). Sections were digitized, measured (area, thickness), and statistically analyzed. In total, 19 of the 43 collected infected larvae showed signs of infection (germinating spores/fungal mycelium): dead larvae (from day 2 p.i.) showed clear histological and macroscopic signs of infection, while larvae collected alive (from day 1 p.i.) were only locally affected. Infected larvae were significantly smaller (day 2 p.i.: p < 0.001, 4 p.i.: p < 0.01, 5 p.i.: p < 0.01) than uninfected larvae (control group). Our study shows that the pathogenesis of stonebrood is characterized by a short period between Aspergillus germination and the onset of disease (about one day), and a rapid larval death.