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).
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
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