2009 Annual Report
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.
In this study, we monitored co-occurrence of pathogens, mites, and pesticides, and identified the factors associated with colony mortality. In the spring of the first year (February 2009), the Honey Bee Research Unit established a stationary apiary from bee packages that were purchased locally. An apiary location was chosen to provide sufficient natural nectar and pollen flow for the test colonies to be self-sustaining. Ambient temperatures for the entire test period will be recorded by the local weather station, KRGV News Channel 5. In April, these initial colonies were re-queened using Koehnen's Queens, Ordbend, CA, and were maintained according to the confirmed protocol. Assessment of the colony strength was conducted at the start of the study. All 30 colonies were measured for the amount of adult bees and brood area.
The Nosema cage study was conducted at Weslaco, TX. In the first part of the study, caged bees were infected with N. ceranae and N. apis, and bee samples were collected at different time points, post-infection. Five cages of bees were infected with N. ceranae, five with N. apis, and five were non-infected controls. Nosema species were confirmed using the PCR method. The workers were placed in cages (14 × 12 × 16 cm) equipped with small foundations and two inverted scintillation vials containing a sugar solution and distilled water installed on top of the cage. The study was conducted in three independent trials. Sixty bees were fed individually with Nosema spores, marked, and returned to cages containing an additional 40 non-infected and non-marked workers. Bees in cages were incubated at 34 C and 60% RH and were provided with water, sucrose solution, and pollen placed at the bottom of each cage. Samples from each cage were collected at specific time points (0 hr, 24 hr, 48 hr, 72 hr, 7 days, and 2 weeks) to determine early, mid, and late gene expression profiles. For each time point, marked bees were collected in duplicate and stored at -80 C. Samples were sent to a collaborator to determine host genes differentially expressed during the progression of the disease. Molecular mechanisms that protect bees from Nosema invasion will be examined.
We are also developing a robust, specific, and sensitive diagnostic test for the detection of Nosema parasites in honey bee samples. The dipstick test is a "front line" diagnostic based on an antigen capture assay that detects N. ceranae or N. apis spores wall antigen. A new detection device is intended for use with the whole insect and does not require any additional sample preparation, equipment, or specialized skills. Therefore, it could be used in local laboratories and directly in the field. This test will be used by hobbyist and commercial beekeepers to monitor the progression of the disease and to minimize the use of synthetic chemicals in bee colonies.