Summary Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

Summary — This paper reports improvements on a previous technique of ours for producing chalkbrood disease in Apis mellifera under controlled conditions. Mummification reached almost 95% (the previous technique reached 70%) when fifth instar larvae were chilled at 18 °C 24 h before sealing and kept at 25 °C for 6 days after operculation. When the larvae were chilled, but the temperature after operculation was 30 or 35 °C, mummification reached 43.65 and 29%, respectively. Percentages of mummification were lower when chilling prior to sealing cells was not applied: 77.62% (at 25 °C after sealing), 15.31 % (at 30 °C) and 2.22% (at 35 °C). The effect of a high relative humidity (rh) (87%) combined with slight chilling (30 °C) induced a higher percentage of mummification (7.75%) then compared to the same temperature but lower rh (only 0.95% of larvae were mummified at 68% rh).

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Summary — Third instar larvae from a honeybee colony were fed with high doses of spores of Ascosphaera apis, the causative agent of chalkbrood disease. Optimal survival of spores was detected during a short period after sealing the cell and before larval spinning by culture of the gut contents removed from 4 stages of brood development. The inocula (5 x 10 5 spores/larva) did not induce the disease and were not present in the digestive tract before pupation. In a second experiment, third instar larvae, fed with the same amounts of spores as before, received a cooling stress (22 ± 2°C, for 24 h). When chilling was applied 24 h before or after operculation, mummification occurred in the majority of larvae (59.6 and 65.5%, respectively). Chilling of older brood (spinning larvae or pupa) produced a much lower incidence of chalkbrood. This confirms the need for predisposing conditions over a short period of brood development for the development of this disease. chalkbrood / Apis mellifera / Ascosphaera apis / predisposing conditions / stress / brood chilling INTRODUCTION