1a.Objectives (from AD-416)
Objective 1: Investigate geographical differences in morbidity and mortality from Nosema sp., including interactions with other biotic and abiotic challenges in full colony studies in stationary location in Weslaco, TX.
(This objective is a part of a larger experiment conducted in a number of different locations throughout the United States).
Objective 2: Determine individual bee morbidity and mortality from Nosema sp., including interactions with other biotic and abiotic challenges using caged studies.
Objective 3: Investigate the honey bee defense pathways against microbial pathogens.
Objective 4: Develop diagnostic tools for field and laboratory detection and monitoring of honey bees diseases (Nosema ceranae).
1b.Approach (from AD-416)
A new apiary will be established to conduct Grand Experiment study described in "Biotic threats" section. Thirty honey bee colonies will be maintained at Weslaco, TX, site by the Research Unit personnel. Bee samples will be collected bi-weekly and stored in -80 C; pollen samples will be collected on a monthly basis and shipped to collaborators for pesticide residue analysis.
Cage Experiment: Nosema ceranae infection study will be conducted in Weslaco in collaboration with Dr. Webster, Kentucky State University. Bees will be infected with different biotypes of N. ceranae in cages. Honey bee samples will be collected at the different stages as the disease progresses to determine the differential gene expression in response to infection. Bee samples collected from the cage infection study will be analyzed using quantitative PCR (qRT-PCR) to estimate the differences in the level of bee immune responses between Nosema-infected and healthy bees. Infection of bees with N. cerana will be confirmed using DNA-base method and microscopic observation of the dissected bees.
Dipstick assay: We propose to develop a highly sensitive dipstick test for detection of Nosema infection in bee colonies. The test is based on the antibodies of N. ceranae raised in small animals, and it will be able to detect Nosema antigen in honey bee samples. A dipstick assay will be evaluated and optimized on honey bee samples collected in six different locations in the U.S. Production and marketing of the dipsticks is not included in the scope of this study.
We developed the bioassay methodology for infecting honey bees in small cages using Nosema spores. This new methodology ensures that mortality of caged bees is not associated with the stress due to the handling of newly emerged bees and/or rearing conditions of bees in cages. Following our new infection protocol, we have determined the level of bee mortality for different species of Nosema (N. ceranae vs. N. apis) at a range of concentrations from 50 to 50,000 spores per bee. A high dose of N. apis spores resulted in an increased bee mortality of infected bees recorded during the first and second week post inoculation. This is at least a week earlier than the increase of mortality recorded for N. ceranae-treated bees. However, the levels of bee mortality differ significantly when bees are fed an intermediate concentration of spores (500 spores per bee) producing 70% vs. 50% bee mortality in N. apis vs. N. ceranae treatments, respectively. In general, spore production in bees treated with a high dose of either species of Nosema spores resulted in higher mortality and greater spore production independent of Nosema species.