A Keller-Segel model for C elegans L1 aggregation.

We describe a mathematical model for the aggregation of starved first-stage C elegans larvae (L1s). We propose that starved L1s produce and respond chemotactically to two labile diffusible chemical signals, a short-range attractant and a longer range repellent. This model takes the mathematical form of three coupled partial differential equations, one that describes the movement of the worms and one for each of the chemical signals. Numerical solution of these equations produced a pattern of aggregates that resembled that of worm aggregates observed in experiments. We also describe the identification of a sensory receptor gene, srh-2, whose expression is induced under conditions that promote L1 aggregation. Worms whose srh-2 gene has been knocked out form irregularly shaped aggregates. Our model suggests this phenotype may be explained by the mutant worms slowing their movement more quickly than the wild type.

The FMRFamide Neuropeptide FLP-20 Acts as a Systemic Signal for Starvation Responses in Caenorhabditis elegans.

Most animals face frequent periods of starvation throughout their entire life and thus need to appropriately adjust their behavior and metabolism during starvation for their survival. Such adaptive responses are regulated by a complex set of systemic signals, including hormones and neuropeptides. While much progress has been made in identifying pathways that regulate nutrient-excessive states, it is still incompletely understood how animals systemically signal their nutrient-deficient states. Here, we showed that the FMRFamide neuropeptide FLP-20 modulates a systemic starvation response in Caenorhabditis elegans. We found that mutation of flp-20 rescued the starvation hypersensitivity of the G protein ß-subunit gpb-2 mutants by suppressing excessive autophagy. FLP-20 acted in AIB neurons, where the metabotropic glutamate receptor MGL-2 also functions to modulate a systemic starvation response. Furthermore, FLP-20 modulated starvation-induced fat degradation in a manner dependent on the receptor-type guanylate cyclase GCY-28. Collectively, our results reveal a circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

Modelling the ballistic-to-diffusive transition in nematode motility reveals variation in exploratory behaviour across species.

A quantitative understanding of organism-level behaviour requires predictive models that can capture the richness of behavioural phenotypes, yet are simple enough to connect with underlying mechanistic processes. Here, we investigate the motile behaviour of nematodes at the level of their translational motion on surfaces driven by undulatory propulsion. We broadly sample the nematode behavioural repertoire by measuring motile trajectories of the canonical laboratory strain Caenorhabditis elegans N2 as well as wild strains and distant species. We focus on trajectory dynamics over time scales spanning the transition from ballistic (straight) to diffusive (random) movement and find that salient features of the motility statistics are captured by a random walk model with independent dynamics in the speed, bearing and reversal events. We show that the model parameters vary among species in a correlated, low-dimensional manner suggestive of a common mode of behavioural control and a trade-off between exploration and exploitation. The distribution of phenotypes along this primary mode of variation reveals that not only the mean but also the variance varies considerably across strains, suggesting that these nematode lineages employ contrasting 'bet-hedging' strategies for foraging.

Starvation-induced collective behavior in C. elegans.

We describe a new type of collective behavior in C. elegans nematodes, aggregation of starved L1 larvae. Shortly after hatching in the absence of food, L1 larvae arrest their development and disperse in search for food. In contrast, after two or more days without food, the worms change their behavior--they start to aggregate. The aggregation requires a small amount of ethanol or acetate in the environment. In the case of ethanol, it has to be metabolized, which requires functional alcohol dehydrogenase sodh-1. The resulting acetate is used in de novo fatty acid synthesis, and some of the newly made fatty acids are then derivatized to glycerophosphoethanolamides and released into the surrounding medium. We examined several other Caenorhabditis species and found an apparent correlation between propensity of starved L1s to aggregate and density dependence of their survival in starvation. Aggregation locally concentrates worms and may help the larvae to survive long starvation. This work demonstrates how presence of ethanol or acetate, relatively abundant small molecules in the environment, induces collective behavior in C. elegans associated with different survival strategies.

An opioid-like system regulating feeding behavior in C. elegans.

Neuropeptides are essential for the regulation of appetite. Here we show that neuropeptides could regulate feeding in mutants that lack neurotransmission from the motor neurons that stimulate feeding muscles. We identified nlp-24 by an RNAi screen of 115 neuropeptide genes, testing whether they affected growth. NLP-24 peptides have a conserved YGGXX sequence, similar to mammalian opioid neuropeptides. In addition, morphine and naloxone respectively stimulated and inhibited feeding in starved worms, but not in worms lacking NPR-17, which encodes a protein with sequence similarity to opioid receptors. Opioid agonists activated heterologously expressed NPR-17, as did at least one NLP-24 peptide. Worms lacking the ASI neurons, which express npr-17, did not response to naloxone. Thus, we suggest that Caenorhabditis elegans has an endogenous opioid system that acts through NPR-17, and that opioids regulate feeding via ASI neurons. Together, these results suggest C. elegans may be the first genetically tractable invertebrate opioid model.

Regulation of synaptic transmission at the Caenorhabditis elegans M4 neuromuscular junction by an antagonistic relationship between two calcium channels.

In wild-type Caenorhabditis elegans, the synapse from motor neuron M4 to pharyngeal terminal bulb (TB) muscles is silent, and the muscles are instead excited by gap junction connections from adjacent muscles. An eat-5 innexin mutant lacking this electrical connection has few TB contractions and is unable to grow well on certain foods. We showed previously that this defect can be overcome by activation of the M4 → TB synapse. To identify genes that negatively regulate synaptic transmission, we isolated new suppressors of eat-5. To our surprise, these suppressors included null mutations in NPQR-type calcium channel subunit genes unc-2 and unc-36. Our results are consistent with the hypothesis that Ca(2+) entry through the NPQR-type channel inhibits synaptic transmission by activating the calcium-activated K(+) channel SLO-1, thus antagonizing the EGL-19 L-type calcium channel.

A model of the effect of uncertainty on the C elegans L2/L2d decision.

At the end of the first larval stage, the C elegans larva chooses between two developmental pathways, an L2 committed to reproductive development and an L2d, which has the option of undergoing reproductive development or entering the dauer diapause. I develop a quantitative model of this choice using mathematical tools developed for pricing financial options. The model predicts that the optimal decision must take into account not only the expected potential for reproductive growth, but also the uncertainty in that expected potential. Because the L2d has more flexibility than the L2, it is favored in unpredictable environments. I estimate that the ability to take uncertainty into account may increase reproductive value by as much as 5%, and discuss possible experimental tests for this ability.

Density dependence in Caenorhabditis larval starvation.

Availability of food is often a limiting factor in nature. Periods of food abundance are followed by times of famine, often in unpredictable patterns. Reliable information about the environment is a critical ingredient of successful survival strategy. One way to improve accuracy is to integrate information communicated by other organisms. To test whether such exchange of information may play a role in determining starvation survival strategies, we studied starvation of L1 larvae in C. elegans and other Caenorhabditis species. We found that some species in genus Caenorhabditis, including C. elegans, survive longer when starved at higher densities, while for others survival is independent of the density. The density effect is mediated by chemical signal(s) that worms release during starvation. This starvation survival signal is independent of ascarosides, a class of small molecules widely used in chemical communication of C. elegans and other nematodes.

The pharynx of the nematode C. elegans: A model system for the study of motor control.

Motor control is a complex process that requires interplay among the nervous system, muscles and environment. The simple anatomy, well-characterized muscle movements and ample resources for molecular and cellular dissection make the pharynx of the nematode C. elegans an attractive model system for the study of motor control. The C. elegans pharynx shows two clear muscle movements that are essential for food intake, pharyngeal pumping and isthmus peristalsis. Here, we review our recent findings on the mechanism by which food activates the feeding motions. To understand this process, we characterized the behavior of the feeding motions in response to serotonin, an endogenous pharyngeal pumping activator whose action is triggered by food. We found that: (1) the timing of onset and frequencies of the two feeding motions are distinct; (2) isthmus peristalsis is selectively coupled to the preceding pump; (3) like food, serotonin activates isthmus peristalsis as well as pharyngeal pumping. By genetic analysis, we showed that two separate neural pathways activate the two feeding motions explaining the differences between the two feeding motions. We also proposed a model that explains how the two feeding motions are separately controlled, yet coupled by the interaction between the nervous system and the muscles in the pharynx. Finally, we briefly discuss future approaches to further understand the mechanism that couples the two feeding motions in C. elegans and to possibly understand evolution of motor control in the pharynx by expanding findings in C. elegans to other nematode species.

The jaw of the worm: GTPase-activating protein EAT-17 regulates grinder formation in Caenorhabditis elegans.

Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.

Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in Caenorhabditis elegans.

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

The geometry of locomotive behavioral states in C. elegans.

We develop a new hidden Markov model-based method to analyze C elegans locomotive behavior and use this method to quantitatively characterize behavioral states. In agreement with previous work, we find states corresponding to roaming, dwelling, and quiescence. However, we also find evidence for a continuum of intermediate states. We suggest that roaming, dwelling, and quiescence may best be thought of as extremes which, mixed in any proportion, define the locomotive repertoire of C elegans foraging and feeding behavior.

Recognition of familiar food activates feeding via an endocrine serotonin signal in Caenorhabditis elegans.

Familiarity discrimination has a significant impact on the pattern of food intake across species. However, the mechanism by which the recognition memory controls feeding is unclear. Here, we show that the nematode Caenorhabditis elegans forms a memory of particular foods after experience and displays behavioral plasticity, increasing the feeding response when they subsequently recognize the familiar food. We found that recognition of familiar food activates the pair of ADF chemosensory neurons, which subsequently increase serotonin release. The released serotonin activates the feeding response mainly by acting humorally and directly activates SER-7, a type 7 serotonin receptor, in MC motor neurons in the feeding organ. Our data suggest that worms sense the taste and/or smell of novel bacteria, which overrides the stimulatory effect of familiar bacteria on feeding by suppressing the activity of ADF or its upstream neurons. Our study provides insight into the mechanism by which familiarity discrimination alters behavior.DOI:http://dx.doi.org/10.7554/eLife.00329.001.

NSBP-1 mediates the effects of cholesterol on insulin/IGF-1 signaling in Caenorhabditis elegans.

Nematode sterol-binding protein 1 (NSBP-1) is a homolog of nucleosome assembly protein 1 in mammals that is expressed widely in Caenorhabditis elegans. NSBP-1 mutants are biologically lethal, demonstrating the significance of the gene in growth and development. We investigated how cholesterol influences the insulin signaling pathway through this novel sterol-binding protein in C. elegans. Here we report that NSBP-1 influences many biological processes mediated by insulin signaling, such as longevity, dauer formation, fat storage, and resistance to oxidative stress. We found that NSBP-1 is phosphorylated by AKT-1 downstream of insulin signaling. In the absence of insulin signaling, NSBP-1 is translocated to the nucleus and binds to DAF-16, a FOXO transcription factor, in a cholesterol-dependent manner. Moreover, NSBP-1 and DAF-16 regulate a common set of genes that can directly modulate fat storage, longevity, and resistance to stress. Together, our results present a new steroid-binding molecule that can connect sterol signaling to insulin signaling through direct interaction with FOXO.

C. elegans feeding.

C. elegans feeding depends on the action of the pharynx, a neuromuscular pump that joins the mouth to the intestine. The pharyngeal muscle captures food-bacteria-and transports it back to the intestine. It accomplishes this through a combination of two motions, pumping and isthmus peristalsis. Pumping, the most visible and best understood of the two, is a cycle of contraction and relaxation that sucks in liquid from the surrounding environment along with suspended particles, then expels the liquid, trapping the particles. Pharyngeal muscle is capable of pumping without nervous system input, but during normal rapid feeding its timing is controlled by two pharyngeal motor neuron types. Isthmus peristalsis, a posterior moving wave of contraction of the muscle of the posterior isthmus, depends on a third motor neuron type. Feeding motions are regulated by the presence and quality of food in the worm's environment. Some types of bacteria are better at supporting growth than others. Given a choice, worms are capable of identifying and seeking out higher-quality food. Food availability and quality also affect behavior in other ways. For instance, given all the high-quality food they can eat, worms eventually become satiated, stop eating and moving, and become quiescent.

Serotonin activates overall feeding by activating two separate neural pathways in Caenorhabditis elegans.

Food intake in the nematode Caenorhabditis elegans requires two distinct feeding motions, pharyngeal pumping and isthmus peristalsis. Bacteria, the natural food of C. elegans, activate both feeding motions (Croll, 1978; Horvitz et al., 1982; Chiang et al., 2006). The mechanisms by which bacteria activate the feeding motions are largely unknown. To understand the process, we studied how serotonin, an endogenous pharyngeal pumping activator whose action is triggered by bacteria, activates feeding motions. Here, we show that serotonin, like bacteria, activates overall feeding by activating isthmus peristalsis as well as pharyngeal pumping. During active feeding, the frequencies and the timing of onset of the two motions were distinct, but each isthmus peristalsis was coupled to the preceding pump. We found that serotonin activates the two feeding motions mainly by activating two separate neural pathways in response to bacteria. For activating pumping, the SER-7 serotonin receptor in the MC motor neurons in the feeding organ activated cholinergic transmission from MC to the pharyngeal muscles by activating the Gsα signaling pathway. For activating isthmus peristalsis, SER-7 in the M4 (and possibly M2) motor neuron in the feeding organ activated the G(12)α signaling pathway in a cell-autonomous manner, which presumably activates neurotransmission from M4 to the pharyngeal muscles. Based on our results and previous calcium imaging of pharyngeal muscles (Shimozono et al., 2004), we propose a model that explains how the two feeding motions are separately regulated yet coupled. The feeding organ may have evolved this way to support efficient feeding.

Laser microsurgery in Caenorhabditis elegans.

Laser killing of cell nuclei has long been a powerful means of examining the roles of individual cells in C. elegans. Advances in genetics, laser technology, and imaging have further expanded the capabilities and usefulness of laser surgery. Here, we review the implementation and application of currently used methods for target edoptical disruption in C. elegans.

Appetite Control: worm's-eye-view.

Food is important to any animal, and a large part of the behavioral repertoire is concerned with ensuring adequate nutrition. Two main nutritional sensations, hunger and satiety, produce opposite behaviors. Hungry animals seek food, increase exploratory behavior and continue feeding once they encounter food. Satiated animals decrease exploratory behavior, take rest, and stop feeding. The signals of hunger or satiety and their effects on physiology and behavior will depend not only on the animal's current nutritional status but also on its experience and the environment in which the animal evolved. In our novel, nutritionally rich environment, improper control of appetite contributes to diseases from anorexia to the current epidemic of obesity. Despite extraordinary recent advances, genetic contribution to appetite control is still poorly understood partly due to lack of simple genetic model systems. In this review, we will discuss current understanding of molecular and cellular mechanisms by which animals regulate food intake depending on their nutritional status. Then, focusing on relatively less known muscarinic and cGMP signals, we will discuss how the molecular and behavioral aspects of hunger and satiety are conserved in a simple invertebrate model system, C. elegans so as for us to use it to understand the genetics of appetite control.

Hox and a newly identified E2F co-repress cell death in Caenorhabditis elegans.

The development of an organism depends on individual cells receiving and executing their specific fates, although how this process is regulated remains largely unknown. Here, we identify a mechanism by which a specific cell fate, apoptosis, is determined through the cooperative efforts of Hox and E2F proteins. E2F transcription factors are critical, conserved regulators of the cell cycle and apoptosis. However, little is known about the two most recently discovered mammalian E2Fs-E2F7 and E2F8. In the nematode Caenorhabditis elegans, we identify a novel E2F7/8 homolog, EFL-3, and show that EFL-3 functions cooperatively with LIN-39, providing the first example in which these two major developmental pathways-E2F and Hox-are able to directly regulate the same target gene. Our studies demonstrate that LIN-39 and EFL-3 function in a cell type-specific context to regulate transcription of the egl-1 BH3-only cell death gene and to determine cell fate during development.

Caenorhabditis elegans behavioral genetics: where are the knobs?

Thousands of behavioral mutants of Caenorhabditis elegans have been studied. I suggest a set of criteria by which some genes important in the evolution of behavior might be recognized, and identify neuropeptide signaling pathways as candidates.