The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) ␣-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family. Ivermectin is used to treat numerous parasitic infections of humans, pets, and livestock (1). Treatment with ivermectin is the cornerstone of efforts to eradicate river blindness (onchocerciasis). However, reports of resistance to ivermectin in nematodes are increasingly common (2-4). Ivermectin also kills the nematode Caenorhabditis elegans at therapeutic concentrations, making C. elegans a useful model system in which to examine mechanisms of ivermectin toxicity and resistance. Ivermectin activates glutamate-gated chloride channels (GluCls) that contain ␣-type channel subunits (5-7). In C. elegans, ␣-type subunits are encoded by a family of genes including: glc-1 (encoding GLC-1͞GluCl␣1), avr-15 (encoding AVR-15͞GluCl␣2), and possibly other uncharacterized genes found in the genome sequence (5-8). Severe loss-of-function mutations in glc-1 or avr-15 do not make worms resistant to ivermectin (6, 7), either because GluCls are not physiologically important targets of ivermectin, or because multiple GluCl genes contribute independently to ivermectin sensitivity. To clarify the role of the GluCls in the nematocidal effects of ivermectin, we had screened for ivermectin-resistant mutants (6). Here we analyze the effects of these and other, previously characterized mutations on ivermectin sensitivity. We show that simultaneous mutation of three genes encoding GluCl ␣-type subunits confers high-level resistance to ivermectin. Our results suggest that the ability of ivermectin to target several members of a multigene family may decrease the rate at which resistance evolves. MethodsGenetics. Unless otherwise indicated the mutant alleles used were: avr-14(ad1302), avr-15(ad1051), gcl-1(pk54::Tc1), unc-7(e5), unc-9(e101), osm-1(ad1307), osm-5(ad1308), dyf-11(ad1303), and che-3(ad1306). avr-15(ad1051), glc-1(pk54::Tc1), unc-7(e5), and unc-9(e101) appear to be molecular nulls (refs. 6,7,and 18; T. Starich, personal communication). Ivermectin-resistant mutants were isolated in a screen for ivermectin resistance in an avr-15(ad1051) background by using the mutagen ethyl methanesulfonate as described (6). All strains were outcrossed twice with N2 ...

Animals have evolved diverse behaviors that serve the purpose of finding food in the environment. We investigated the food seeking strategy of the soil bacteriaeating nematode Caenorhabditis elegans. C. elegans bacterial food varies in quality: some species are easy to eat and support worm growth well, while others do not. We show that worms exhibit dietary choice: they hunt for high quality food and leave hard-to-eat bacteria. This food seeking behavior is enhanced in animals that have already experienced good food. When hunting for good food, worms alternate between two modes of locomotion, known as dwelling: movement with frequent stops and reversals; and roaming: straight rapid movement. On good food, roaming is very rare, while on bad food it is common. Using laser ablations and mutant analysis, we show that the AIY neurons serve to extend roaming periods, and are essential for efficient food seeking.Supplementary material available online at

Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

A guanylyl cyclase (GC-D) was recently shown to be expressed in a subclass of neurons within the neuroepithelim of the rat, but given that only a single cyclase was discovered, whether it represents an odorant͞pheromone receptor as has been suggested for the large family of seventransmembrane receptors remains unclear. Through cloning and expression of cDNA we now demonstrate that at least 29 genomic or cDNA sequences found in Caenorhabditis elegans represent guanylyl cyclases. Many of the membrane forms retain cysteine residues conserved within the extracellular, ligand-binding domain of known cyclase receptors. Of eight orphan cyclase receptor::GFP (green f luroescence protein) fusion constructs for which signals were obtained, all were expressed in specific sensory neurons. Furthermore, a cyclase͞GFP fusion protein (GCY-10͞GFP) was principally expressed in the sensory cilium, suggesting these cyclases function as primary chemosensory receptors. For the first time, we also found that chemosensory neurons (ASE), known to be bilaterally symmetric, demonstrate absolute right or left sidedness with respect to the expression of three different cyclases. Thus, the guanylyl cyclases represent an unexpectedly large and new family of sensory neuron receptors that may complement the 7-transmembrane family of odorant͞ pheromone receptors.

The Caenorhabditis elegans gene eat-4 affects multiple glutamatergic neurotransmission pathways. We find that eat-4 encodes a protein similar in sequence to a mammalian brainspecific sodium-dependent inorganic phosphate cotransporter I (BNPI). Like BNPI in the rat CNS, eat-4 is expressed predominantly in a specific subset of neurons, including several proposed to be glutamatergic. Loss-of-function mutations in eat-4 cause defective glutamatergic chemical transmission but appear to have little effect on other functions of neurons. Our data suggest that phosphate ions imported into glutamatergic neurons through transporters such as EAT-4 and BNPI are required specifically for glutamatergic neurotransmission.

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Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.