(A) Effect of naloxone on pumping. Worms genetically engineered to lack ASI neurons were not affected. ASI neurons were genetically ablated by the recCaspase method (Chelur and Chalfie, 2007; Beverly et al., 2011) (ASI−) ***Different from 0 mM, p < 0.001; N.S. not significantly different from 0 mM (two-way ANOVA, concentration effect). The effect of naloxone on wild-type is significantly different from its effect on npr-17, p < 0.001 (two-way ANOVA, genotype × concentration interaction). (B) npr-17 expression in ASI neurons is required for opioid-mediated feeding control. npr-17 mutants were not affected by naloxone, but this phenotype was rescued with by expression of npr-17 under control of the ASI-specific promoter gpa-4. ***Different from 0 mM, p < 0.001, N.S., not significantly different from 0 mM (two-way ANOVA, concentration effect). The interaction between genotype and concentration is significant at p < 0.001 for Pges-1::npr-17 rescued vs WT, but not Pnpr-17::npr-17 or Pgpa-4::npr-17 rescued. The interaction between genotype and concentration is significant at p < 0.001 for Pnpr-17::npr-17 and Pgpa-4::npr-17 rescued vs npr-17, but not Pges-1::npr-17 rescued. (C) Chronic effect of naloxone on pumping. Worms grown to adulthood with or without 10 mM naloxone plate were then tested with or without 10 mM naloxone. ***p < 0.001; N.S. not significantly different. The stimulatory effect of taking naloxone away from worms grown in the presence of naloxone is significant at p < 0.05. (ANOVA + Tukey tests).