Rebecca A. Butcher

A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans

Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual’s own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviours. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behaviour. Anatomical gap junctions connect RMG to several classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviours by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.

The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

The ≈80-kb pksX gene cluster in Bacillus subtilis encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of B. subtilis, we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an α-hydroxy carboxylic acid to a ω-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an (ω-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaenes structure has enabled us to annotate the pksX gene cluster and should facilitate the study of bacillaenes biosynthesis as well as its biological role in B. subtilis.

Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes

Evolution can follow predictable genetic trajectories, indicating that discrete environmental shifts can select for reproducible genetic changes. Conspecific individuals are an important feature of an animal’s environment, and a potential source of selective pressures. Here we show that adaptation of two Caenorhabditis species to growth at high density, a feature common to domestic environments, occurs by reproducible genetic changes to pheromone receptor genes. Chemical communication through pheromones that accumulate during high-density growth causes young nematode larvae to enter the long-lived but non-reproductive dauer stage. Two strains of Caenorhabditis elegans grown at high density have independently acquired multigenic resistance to pheromone-induced dauer formation. In each strain, resistance to the pheromone ascaroside C3 results from a deletion that disrupts the adjacent chemoreceptor genes serpentine receptor class g (srg)-36 and -37. Through misexpression experiments, we show that these genes encode redundant G-protein-coupled receptors for ascaroside C3. Multigenic resistance to dauer formation has also arisen in high-density cultures of a different nematode species, Caenorhabditis briggsae, resulting in part from deletion of an srg gene paralogous to srg-36 and srg-37. These results demonstrate rapid remodelling of the chemoreceptor repertoire as an adaptation to specific environments, and indicate that parallel changes to a common genetic substrate can affect life-history traits across species.

Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans.

Life Histories to Suit Nematode worms can profoundly manipulate their life histories in several ways. For example, Caenorhabditis elegans has two genders: males and hermaphrodites. Some clues for the evolution of this peculiar mating system have been revealed by Baldi et al. (p. 1002), who turned females of a related species, Caenorhabditis remanei, into hermaphrodites by modifying a gene involved in making sperm and another gene required for activating the spermatids. In most animals, the germ line is fully established during adulthood and a reproductive period is determined, at least in part, by aging of the germ line and the viability of oocytes. The reproductive longevity of hermaphrodite C. elegans can be increased at least 15-fold by starvation. Angelo and Van Gilst (p. 954, published online 27 August; see the Perspective by Ogawa and Sommer) found that in starved worms, the germline component of the reproductive system is actively killed, with the exception of a small set of preserved stem cells. When the worms are able to feed again, these cells regenerate into an entirely new and functional germ line. But this is not all. Kim et al. (p. 994, published online 1 October; see the Perspective by Ogawa and Sommer) show that subsets of the complex mixture of structurally related molecules in dauer pheromone act via distinct G protein–coupled receptors either to initiate longterm effects on development and physiology by modulating the neuroendocrine axis, or to trigger short-term acute effects on behavior by altering neuronal responses. Chemical signals that determine alternative nematode developmental programs act via two G protein–coupled receptors. Intraspecific chemical communication is mediated by signals called pheromones. Caenorhabditis elegans secretes a mixture of small molecules (collectively termed dauer pheromone) that regulates entry into the alternate dauer larval stage and also modulates adult behavior via as yet unknown receptors. Here, we identify two heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) that mediate dauer formation in response to a subset of dauer pheromone components. The SRBC-64 and SRBC-66 GPCRs are members of the large Caenorhabditis-specific SRBC subfamily and are expressed in the ASK chemosensory neurons, which are required for pheromone-induced dauer formation. Expression of both, but not each receptor alone, confers pheromone-mediated effects on heterologous cells. Identification of dauer pheromone receptors will allow a better understanding of the signaling cascades that transduce the context-dependent effects of ecologically important chemical signals.

Biosynthesis of the Caenorhabditis elegans dauer pheromone

To sense its population density and to trigger entry into the stress-resistant dauer larval stage, Caenorhabditis elegans uses the dauer pheromone, which consists of ascaroside derivatives with short, fatty acid-like side chains. Although the dauer pheromone has been studied for 25 years, its biosynthesis is completely uncharacterized. The daf-22 mutant is the only known mutant defective in dauer pheromone production. Here, we show that daf-22 encodes a homolog of human sterol carrier protein SCPx, which catalyzes the final step in peroxisomal fatty acid β-oxidation. We also show that dhs-28, which encodes a homolog of the human d-bifunctional protein that acts just upstream of SCPx, is also required for pheromone production. Long-term daf-22 and dhs-28 cultures develop dauer-inducing activity by accumulating less active, long-chain fatty acid ascaroside derivatives. Thus, daf-22 and dhs-28 are required for the biosynthesis of the short-chain fatty acid-derived side chains of the dauer pheromone and link dauer pheromone production to metabolic state.

A potent dauer pheromone component in Caenorhabditis elegans that acts synergistically with other components

In the model organism Caenorhabditis elegans, the dauer pheromone is the primary cue for entry into the developmentally arrested, dauer larval stage. The dauer is specialized for survival under harsh environmental conditions and is considered “nonaging” because larvae that exit dauer have a normal life span. C. elegans constitutively secretes the dauer pheromone into its environment, enabling it to sense its population density. Several components of the dauer pheromone have been identified as derivatives of the dideoxy sugar ascarylose, but additional unidentified components of the dauer pheromone contribute to its activity. Here, we show that an ascaroside with a 3-hydroxypropionate side chain is a highly potent component of the dauer pheromone that acts synergistically with previously identified components. Furthermore, we show that the active dauer pheromone components that are produced by C. elegans vary depending on cultivation conditions. Identifying the active components of the dauer pheromone, the conditions under which they are produced, and their mechanisms of action will greatly extend our understanding of how chemosensory cues from the environment can influence such fundamental processes as development, metabolism, and aging in nematodes and in higher organisms.

Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans

Too Close for Comfort Pheromones are often used for sexual communications in animals, but they can also serve as a measure of population density. Now, Yamada et al. (p. 1647) have found that population density in the nematode worm Caenorhabditis elegans regulates plasticity of olfactory behavior, in which attraction to an odorant decreases after prolonged exposure. Using two rounds of genetic screens, a peptide named SNET-1 and a homolog of a mammalian transmembrane peptidase neprilysin were found to mediate pheromonal regulation. This regulation of olfactory behavior may serve to coordinate the behavior of individual animals in relation to the status of the whole population. A nematode odor response is regulated by population density through dauer pheromone, a neuropeptide, and neprilysin peptidase. Population density–dependent dispersal is a well-characterized strategy of animal behavior in which dispersal rate increases when population density is higher. Caenorhabditis elegans shows positive chemotaxis to a set of odorants, but the chemotaxis switches from attraction to dispersal after prolonged exposure to the odorants. We show here that this plasticity of olfactory behavior is dependent on population density and that this regulation is mediated by pheromonal signaling. We show that a peptide, suppressor of NEP-2 (SNET-1), negatively regulates olfactory plasticity and that its expression is down-regulated by the pheromone. NEP-2, a homolog of the extracellular peptidase neprilysin, antagonizes SNET-1, and this function is essential for olfactory plasticity. These results suggest that population density information is transmitted through the external pheromone and endogenous peptide signaling to modulate chemotactic behavior.

A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles that seek out and infect insect hosts. Upon sensing internal host cues, the infective juveniles regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of infective juveniles. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a small-molecule pheromone to control infective juvenile development. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species. An understanding of chemical signaling in parasitic nematodes may enable the development of chemical tools to control these species.

A Small Molecule Suppressor of FK506 that Targets the Mitochondria and Modulates Ionic Balance in Saccharomyces cerevisiae

FK506 inhibits the evolutionarily conserved, Ca(2+)-dependent phosphatase calcineurin, which in yeast is essential for growth during sodium stress. We undertook a chemical genetic modifier screen to identify small molecules that suppress the ability of FK506 to inhibit yeast growth in high NaCl. One of these small molecule suppressors, SFK1 (suppressor of FK506 1), causes a mitochondrially induced death in low salt, concomitant with the release of reactive oxygen species. Biochemically, SFK1 interacts with Por1p, a channel protein in the outer mitochondrial membrane, suggesting that SFK1 interacts with the mitochondria directly. A genome-wide screen of yeast deletion strains for hypersensitivity to SFK1 yielded several strains with impaired mitochondrial function, as well as several with reduced sodium tolerance. Our data link ionic balance to mitochondrial function and suggest a role for calcineurin in mediating this signaling network.

Balancing selection shapes density-dependent foraging behaviour

The optimal foraging strategy in a given environment depends on the number of competing individuals and their behavioural strategies. Little is known about the genes and neural circuits that integrate social information into foraging decisions. Here we show that ascaroside pheromones, small glycolipids that signal population density, suppress exploratory foraging in Caenorhabditis elegans, and that heritable variation in this behaviour generates alternative foraging strategies. We find that natural C. elegans isolates differ in their sensitivity to the potent ascaroside icas#9 (IC-asc-C5). A quantitative trait locus (QTL) regulating icas#9 sensitivity includes srx-43, a G-protein-coupled icas#9 receptor that acts in the ASI class of sensory neurons to suppress exploration. Two ancient haplotypes associated with this QTL confer competitive growth advantages that depend on ascaroside secretion, its detection by srx-43 and the distribution of food. These results suggest that balancing selection at the srx-43 locus generates alternative density-dependent behaviours, fulfilling a prediction of foraging game theory.