Caenorhabditis elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. To enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates at a single worm resolution, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 unrestrained crawling worms simultaneously and extract locomotion and feeding behaviors. We demonstrate the tool’s robustness and high-throughput capabilities by measuring feeding in different use-case scenarios, such as through development, with genetic and chemical perturbations that result in faster and slower pumping, and in the presence or absence of food. Finally, we demonstrate that our tool is capable of long-term imaging by showing behavioral dynamics of mating animals and worms with different genetic backgrounds. The low-resolution fluorescence microscopes required are readily available in C. elegans laboratories, and in combination with our python-based analysis workflow makes this methodology easily accessible. PharaGlow therefore enables the observation and analysis of the temporal dynamics of feeding and locomotory behaviors with high-throughput and precision in a user-friendly system.

No abstract

C. elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. Therefore, to enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 freely moving worms simultaneously and extract locomotion and feeding behaviors. Our tool reliably detects pharyngeal pumping in adult worms with a maximum deviation of 5% in the number of pumps compared to an expert annotator. We demonstrate the tool's robustness and high-throughput capabilities by measuring feeding in different use-case scenarios. This includes tracing pharyngeal dynamics during development, revealing their highly conserved nature throughout all life cycle stages. We also observed pumping after food deprivation, corroborating previous studies in which starvation time strongly influences pumping. Finally, we further validated our behavioral tracker by exploring two previously characterized pumping defective mutants: unc-31 and eat-18. Remarkably, our analysis of eat-18 mutants identified unreported defects in pumping and overall locomotion regulation, highlighting the potential of this toolkit. Pharaglow therefore enables the observation and analysis of the temporal dynamics of food intake with high-throughput and precision in a user-friendly system.