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Revision as of 00:02, 19 September 2015
Project Description
Bee populations around the world have been declining since the early 1990s. In 2015 alone, US beekeepers reported that 42% of their colonies died within the past year. Honeybee Colony Collapse Disorder (CCD) is a serious problem, given the ecological and economical importance of honeybees. CCD is a phenomenon in which adult worker bees die while away from the colony, leaving behind a non-functioning colony. Worker bees are usually the most severely affected, and bees that leave the hive are often unable to return, eventually resulting in the slow collapse of the hive.
Though the mechanisms by which CCD occurs are likely manifold and remain uncertain, neonicotinoid pesticides have been implicated. Imidacloprid, a neonicotinoid pesticide, is usually applied to plants as a seed coating. On germination and growth, imidacloprid is taken up by the plant and deposited in many plant tissues, poisoning all pests that then feed on the plant.
Death may occur as an acute effect at high doses, but this level of exposure to bees is not common in commercial agriculture. This fact is supported by the lack of bee cadavers surrounding a hive, where contaminated food stores would quickly decimate a population of bees.
Consequently, it is thought that the sublethal effects of imidacloprid are far more likely to lead to CCD. Sublethal chronic effects include a loss of coordination, lethargy, and lowered pathfinding abilities, severely impacting the efficacy of bee driven pollination. Imidacloprid is a neurotoxin that binds irreversibly to acetylcholine receptors in the central nervous system, eventually leading to paralysis and death.
We hypothesize that a strain of honeybee intestinal bacterium can be engineered to degrade imidacloprid, a widely-used neonicotinoid. In doing so, honeybees harboring the engineered bacterium will become resistant to common field doses of imidacloprid allowing for its sustained use while reducing the risk of CCD.
Gilliamella apicola is a bacterium native to the midgut of the bee. By engineering Gilliamella to metabolize imidacloprid into oxidizable organic compounds we can create a strain of Gilliamella capable of conferring resistance to imidacloprid. While the exact imidacloprid degradation pathway is unknown, an early step for degradation of neonicotinoids in vivo and in the environment involves the production of 6-chloronicotinic acid (6-CNA). Though 6-CNA requires a very high dose to induce acute toxicity, it still induces sublethal effects.
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In order to create our pro-bee-otic, bacteria specific to Apis mellifera were chosen: the β-proteobacteria, Snodgrassella alvi, and the γ-proteobacteria, Gilliamella apicola. By using microaerophilic bacteria that are unique to the honey bee gut, a specificity is acquired and the chances of pests acquiring the engineered imidacloprid resistant strains are decreased. However, due to the small amount of existing literature on G. apicola and S. alvi, an aspect of the project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.
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Imidacloprid is a neonicotinoid commonly used in pesticides around the world. Studies have shown that the use of this pesticide has adverse, and commonly fatal, effects on insects. Unfortunately, honeybees are also affected by imidacloprid. As such, imidacloprid has been cited as one of many factors contributing to colony collapse disorder (CCD). Previous work has been done to characterize enzymes capable of modifying imidacloprid into less toxic products. Out of all cited enzymes, three candidate enzymes were chosen due to their ability to modify imidacloprid and confer partial resistance to their hosts. Experiments were conducted to create bacterial strains containing the enzymes and to test their ability to modify imidacloprid into less toxic products.
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6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid modification that is both toxic to bees and a persistent environmental contaminant. Our degradation pathway allows for the formation of a krebs cycle intermediate, fumaric acid which can be utilized in central metabolism. To potentially aid in the increased survival of honey bees from the effects of imidacloprid, our probeeotic must be capable of not only modifying imidacloprid, but also detoxifying the 6-CNA breakdown product. This is achieved by degradation by cch2 and the nic cluster. cch2 has been isolated from Bradyrhizobiaceae SG-6C and the nic cluster has been isolated from Pseudomonas putida KT2440.
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In order to test the viability of our pro-bee-otic, experiments on honeybees gut colonization with E. coli containing pesticide-degrading genes were designed and conducted. First, the bee gut colonization was verified after feeding bees a sucrose-water solution supplemented with E. coli. Second, experiments testing colonization of the bee gut with E. coli, harboring pesticide-degradating genes were performed. Bee gut colonization experiments were designed to mimic the real-life situation of how the pro-bee-otic would be delivered to the bees through a sucrose-water solution in the hive environment.
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UBC iGEM 2015