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|Session Title: Aging and Stress Session Type: Poster|
|Session Location: Pauley Pavilion Session Time: Sunday - Tuesday|
|Poster Board Number: 408B Presentation Time: MON, June 27, 2005, 1:30-3:00PM|
|Keywords: KW65 - Stress response|
A novel method of quantifying C. elegans feeding. Windy
A. Boyd, Sandra J. McBride, Jonathan H. Freedman. Nicholas School of
the Environment and Earth Sciences, Duke University, Durham,
Feeding is a complex behavior controlled by an integrated and well-regulated neuromuscular system. C. elegans feed via constant pharyngeal pumping as they move throughout their environment. Coordinated contractions of two bulbs pull bacteria into the pharynx, and the ground bacteria are then passed into the intestine. Exposure to certain chemicals is known to decrease feeding in C. elegans (Avery and Horvitz 1990. The Journal of Experimental Zoology 253:263-270; Boyd WA et al. 2003. Environmental Toxicology and Chemistry 22:3049-3055). However, previous assays involved microscopic examination of individual worms and counting the number of particles (either iron particles or fluorescent beads) each worm ingested. Here we present a new method that compares the relative feeding levels of control worms to those exposed to increasing concentrations of toxicants. In most cases, concentration-dependent decreases in feeding, as measured by decreased mean red fluorescence, were observed after chemical exposures.
Gravid, adult hermaphrodites were dispensed into 96-well plates using a Complex Object Parametric Analyzer and Sorter (COPAS) BIOSORT and were exposed to various concentrations of toxicants for 24 h. After this exposure, 0.5- m red fluorescent beads were added to each well at a constant concentration and worms were allowed to feed normally for 15 min. Sodium azide was then added to anesthetize worms. Samples were aspirated from the plates with the COPAS REFLX, which measured the length, optical density, and red fluorescence of individual worms.
To test the sensitivity our feeding assay, we quantified the feeding of several strains of Eat mutants that exhibit abnormal pharyngeal pumping (Avery 1993. Genetics 133:897-917). Because the strength of the Eat phenotype varied, not all of the mutants fed less than the wild-type N2 strain. However, eat-8 exhibited a strong Eat phenotype and fed less than N2s. At this point, eat-7, 8, and 17 have been tested and Eat mutant testing is ongoing.
Chlorpyrifos, an organophosphate pesticide known to be a potent neurotoxicant, was used as a model toxicant to investigate C. elegans feeding after chemical exposures. Interestingly, worms tended to feed more at lower chlorpyrifos concentrations (not statistically significant) with decreased feeding as concentrations increased. Data from chlorpyrifos and other chemicals will be presented.
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