Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay

Abstract

Background: Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Methodology/principal findings: Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50) of 2 microM.

Conclusions/significance: 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 to 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.

Figure 1. Separation of adult C. elegans from their offspring and corresponding adult feeding.

Extinction (optical density; log₁₀ scale) versus time of flight (length; log₁₀ scale) for (panel A) wild-type and (panel B) eat-2(ad465) nematodes, showing the results of the clustering algorithm to separate either wild-type (blue) or eat-2 (red) adults from larvae (green). In panel C the relationship between fluorescence (accumulated microspheres) versus time of flight for the adult wild-type (blue) and eat-2 (red) nematodes is shown. Each point corresponds to a single nematode.

Figure 2. Comparison of wild-type and eat mutant C. elegans feeding.

Boxplots of normalized fluorescence for 19 strains of adult nematodes aggregated over all experiments. Normalization of fluorescence measurements consisted of size-adjustment, square-root transformation, and centering and scaling to the respective day's wild-type measurements. The upper and lower limits of each box give the 25th and 75th percentiles of the data respectively, and the line inside the box gives the median. Whiskers of the boxplot extend to 1.5 times the interquartile range, with extreme measurements indicated by horizontal lines. Sample sizes are given in parentheses above each boxplot.

Figure 3. Effects of neuroactive compounds and food availability on wild-type C. elegans feeding.

Size-adjusted fluorescence values for arecoline (left panel) and serotonin (right panel) for one representative experiment in the absence (black) or presence (gray) of bacteria. Each boxplot represents between 250–300 nematodes.

Figure 4. Effect of chlorpyrifos and cadmium on C. elegans feeding.

Fitted concentration-response curves of single representative experiments based on observed mean size-adjusted fluorescence measurements as a percent of the control for two toxicants, cadmium (triangles, top axis) and chlorpyrifos (circles, bottom axis). Each point represents approximately 120 nematodes on average, with counts ranging from 104 to 141.