A high-throughput, in vivo assay that quantitatively measures the activity of the C. elegans pharynx is presented. This assay was validated using strains of C. elegans that have mutations that affect pharyngeal function, chemicals known to modulate feeding behavior, and environmentally relevant toxicants. In all cases, the results from this study are consistent with previous observations.
There is good agreement between the rankings of the Eat phenotypes measured using the current assay, compared to previously published observations (). There are, however, exceptions that may be attributed to differences in experimental procedure and interpretation. The original descriptions of the Eat phenotypes were based on direct observations of pharyngeal pumping 
. These observations utilized a small sample size and were subjective. In contrast, the current methodology examines hundreds of nematodes and is quantitative. In addition, the relative strength of a phenotype may not be directly related to the amount of food consumed. For example, eat-17(ad707)
was previously described as a strong Eat phenotype, but shows one of the highest feeding levels in the current assay. Higher feeding levels would be predicted based on the observation that eat-17(ad707)
nematodes “stuff their pharynxes” with bacteria. In contrast, egl-19(ad695)
is described as a weak relaxation-deficient Eat, but fell in the middle of the feeding levels; possibly because the terminal bulb contractions are not timed properly with those in the corpus preventing transport of microspheres into the intestine.
In examining the applicability of this assay in testing pharmaceutical agents, the effects of two drugs, arecoline and serotonin, known to alter pharyngeal activity in C. elegans
were examined. The presence of food is known to stimulate pumping whereas serotonin can stimulate pumping, even in the absence of food 
. In contrast, arecoline inhibits pumping, independent of the presence of food 
. Consistent with these observations, exposure to the muscarinic agonist, arecoline, resulted in a concentration-dependent inhibition of feeding, independent of the starvation status while serotonin stimulated feeding in starved nematodes, but inhibited feeding in fed animals (). Serotonin increased feeding level in a manner similar to previous reports, in which pumping increased from 22 pumps/min in starved control nematodes to 267 pumps/min in nematodes treated with 10 mg/ml (~25 mM) serotonin 
. The effective concentrations for serotonin activity differed slightly between the two studies. This can be attributed to the method of exposure; where previous exposures were on agar plates and the current via liquid medium. In the absence of drugs, we observed that fed nematodes ingested more microspheres than starved nematodes (). These observations were consistent with those previously reported, in that well-fed nematodes pump regularly when bacteria are present, while starved animals pump slowly and irregularly in the absence of food 
In previous toxicological studies, feeding has been estimated from the amount of bacteria or particles removed from liquid media 
. To test the applicability of the new feeding assay in chemical screening, two environmental toxicants chlorpyrifos and cadmium were examined. Chlorpyrifos inhibits acetyl cholinesterase activity, blocking neuronal activity and paralyzing the animal. C. elegans
express several acetyl cholinesterase genes that are potential targets of chlorpyrifos 
. Although the effects of chlorpyrifos on C. elegans
feeding have not been evaluated, other organophosphate pesticides have been shown to inhibit nematode movement 
values ranging from 1.0 to 2.2 µM for feeding inhibition by chlorpyrifos in the current study () are similar to the EC50
of 5 µM previously reported for movement 
Cadmium is not generally considered to be a neurotoxicant, but has been shown to cause decreased motor activity in animals 
. Cadmium has been shown to decrease feeding in C. elegans
when measured as changes in optical density, bead ingestion, or red acrylic paint uptake 
. An EC50
for feeding inhibition of 128 µM cadmium was found in a previous study by measuring changes in the optical density of the exposure medium 
. This is in good agreement with the calculated EC50
s of 260, 262, and 324 µM cadmium determined using the current assay (). In addition, as previously observed, cadmium concentrations above 200 µM do not fully inhibit feeding, but instead resulted in minimal feeding levels 
An application of the microsphere feeding assay that was not explored in this study is the use of multiple fluorescents probes. The COPAS Biosort is able to simultaneously measure two of three available fluorescence emission wavelengths: 500–520, 535–555, or 600–620 nm 
. Therefore, the accumulation of “red-labeled” fluorescence microspheres can be quantified while at the same time measuring the expression of a green fluorescence protein probe without interference. Routinely a myo-2
::GFP expressing transgenic strain of C. elegans
is used in these assays. If the use of red fluorescent markers were necessary, other fluorescent microspheres are available with various excitation and emission characteristics.
The C. elegans
fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable in large scale genetic or RNAi screens 
used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems including mammalian cardiac models.