Comparative study of three assays to evaluate hygienic behavior in honey bee (Apis mellifera L.) colonies

The discriminatory capacity, cost and practicality of three assays to measure hygienic behavior in 60 honey bee colonies were compared in a first experiment. Capped worker brood of these colonies were killed with an insect pin (P), frozen in a freezer at -18 degrees C (CC) and with liquid nitrogen at -195 degrees C (CN2). The percentage of brood removed by the bees was recorded 24 h after being killed. Since the P assay showed a low discriminatory capacity, an additional experiment with 50 colonies was performed, reducing the evaluation time to 8 h and verifying the influence of body fluids and odors released by the pin-killed brood. Colonies were significantly more hygienic (P < 0.01) with the P assay (88%) than with the CC (69%) and CN2 (65%) assays. There were significant correlations among assays (r > 0.54, P < 0.01) and between replicates within assays (r > 0.37, P < 0.01). Variation among colonies was greater with the CN2 and CC assays than with the P assay. The P assay was the least expensive and easiest to apply (6.59 pesos, 0.58 dollars per colony), but showed the lowest discriminatory capacity, that could not be enhanced even though the time of evaluation was reduced. This could have been caused by the strong stimulus from odors and haemolymph released from the pin-killed brood. The CN2 and the CC assays showed a high discriminatory capacity, but the former was more expensive (14.80 pesos, 1.31 dollars) and the least practical. The CC assay was the second least expensive (10.15 pesos, 0.90 dollars) and practical. The application of either freeze assays is recommended for screening hygienic behavior in honey bees because of their reliability.