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 x 102 spores/larva) with Aspergillus flavus. Between day 1 and 5 p.i. (larval age 4 until 8 days), 5 uninfected control larvae, up to 5 infected living larvae, and up to 5 infected dead larvae were collected and 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. 19 of 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). The short incubation period and the early death during pathogenesis could explain why the disease often goes unnoticed: the worker bees remove dead larvae when mycelium has not yet infiltrated the brood cell.