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

## TL;DR

This study examines how Aspergillus flavus causes Stonebrood disease in honey bee larvae, showing rapid infection and death.

## Contribution

The study provides the first detailed histomorphological analysis of Stonebrood disease in honey bee larvae.

## Key findings

- 19 out of 43 infected larvae showed signs of Aspergillus infection, including germinating spores or mycelium.
- Infected larvae were significantly smaller than uninfected larvae at multiple time points.
- The disease progresses rapidly, with a short period between spore germination and larval death.

## Abstract

Stonebrood (Aspergillus sp.), a rare disease of the Western honey bee Apis mellifera affecting adult bees and brood, has been described only occasionally. This study analyzes the course of the disease (pathogenesis) using artificially reared pathogen-free Apis mellifera larvae experimentally infected with Aspergillus flavus and examined macroscopically and histologically. In 19 of the 43 larvae taken from the infected group, signs of infection in the form of germinating spores or fungal mycelium were detected. These larvae were significantly smaller than the larvae from the 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.

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.