Introduction:

Background

• Honey bees carry an electrostatic charge as they fly.  This charge is involved in pollen collection and is an important factor in particle adsorption (Fig. 1).
• Bees typically fly up to 4 km from the hive to find food sources, and are known to fly up to 13.7 km from the hive to find food.
• There are an estimated 2.9 million beehives in the US (excluding beekeepers with fewer than 5 hives).
• Beehives are very low maintenance and can be kept almost anywhere.

Proposed applications

• Bacteria, and probably fungi and viruses, can all be assayed with this technology
• Surveillance for biowarfare/ bioterrorism agents
• Surveillance for anti-crop agents
• Surveillance for non-BW crop pathogens (i.e. general agricultural use)
• Survey endemic micro-organisms (background)
• Survey potential environmental indicator micro-organisms

Methods

• Laboratory tests in custom wind tunnel (Fig. 2) with controlled temperature, wind speed, and humidity.
• Aerosol generation (test scenario) - Stained or unstained spores are suspended in water and aerosolized using clean compressed air.
• Bee collection - Bees are collected individually with sterilized forceps and placed into sterile screw-cap test tubes.
• Culture - Bees are sonicated in sterile water and the rinse solution is heated to 70°C to kill vegetative bacteria, then filtered.  The spore-retaining filter is rolled onto an agar culture plate and/or dried and viewed under a microscope.
• PCR - Bees are rinsed in sterile water.  Spore coat disrupted with detergent, DNA precipitated, and PCR performed with spore-specific primers:
• BG36f-TTCAGACTACACTTTCGGGG
• BG266r-GGAAACCCCCTAACACTTAGC

Detection of aerosolized spores

• Bees adsorbed spores from an aerosol cloud (Fig. 3) generated in the SwRI TENT facility
• A mathematical model has been developed for spore adsorption onto bees (Fig. 4) PDF, 258Kb

Detection of surface deposited spores

• Bees adsorb spores from flower surfaces (Table 1).  A patch of 30-50 flowers on an aster plant were dusted with <1 mg spores.  Bees were collected after contacting 1-3 inoculated flowers for 5-30 seconds and then landing on a non-inoculated flower.  Control bees were collected from nearby non-inoculated flowers.

Future development:

Develop molecular methods to survey biological organisms in hive material

• Assay wax, honey, pollen, and propolis fractions for microbial DNA
• Assay for pathogens in endemic areas using local hives

• Automatic bee collection - maximum spore retention but bees sacrificed
• Develop system to collect/concentrate adsorbed materials while excluding bees - bees unharmed but reduced material recovery

References:
Electrostatic charge:

• Measurement of electric charges carried by bees: Evidence of biological variations.  Colin, ME, D Richard, and S Chauzy (1991).  J. Bioelec. 10: 17-32.
• Are electrostatic forces involved in pollen transfer?  Corbet, SA, J Beament, and D Eisikowitch (1982).  Plant, Cell, and Environ. 5: 125-129.
• Surface electric potentials on worker honeybees leaving and entering the hive.  Erickson, EH (1975).  J. Apic. Res. 14: 141-147.
• Bees scavenge airborne bacteria.  Lighthart, B, KR Prier, GM Loper, and JJ Bromenshenk (2000).  Microb. Ecol. 39: 314-321. PDF, 180Kb
• An adsorption model of aerosolized bacterial spores (Bacillus subtilis var. niger) onto free-flying honey bees (Hymenoptera: Apidae) and its validation.  Prier, KR, B Lighthart, and JJ Bromenshenk (2001).  Environ. Entomol. 30:1188-1194. PDF, 258Kb
• Mechanisms of generation and perception of electric fields by honey bees.  Yes'kov, YK, and AM Sapozhnikov (1976).  Biofizika 21: 1124-1130.

• The flight range of the honeybee.  Eckert, JE (1933).  J. Agric. Res. 47: 257-285.
• Foraging range and distribution of honey bees used for carrot and onion pollination.  Gary, NE, PC Witherell, and J Marston (1972). Environ. Entomol. 1: 71-78.

• The value of honey bees as pollinators of U.S. crops in 2000.  Morse, RA, and NW Calderone (2000).  Bee Culture 128: Insert, 15 p.