Chemical basis of microbiome preference in the nematode C. elegans.

Animals are exposed to many microbes in their environment, some of which have been shown to colonize various tissues including the intestine. The composition of the intestinal microbiota affects many aspects of the host's physiology and health. Despite this, very little is known about whether host behavior contributes to the colonization. We approach this question in the nematode C. elegans, which feeds on bacteria and also harbors an intestinal microbiome. We examined the behavior of C. elegans towards CeMbio, a simplified microbiome consisting of twelve strains that represent the bacteria found in the animal's natural environment. We observed that C. elegans raised on E. coli shows a strong preference for three members of CeMbio (Lelliottia amnigena JUb66, Enterobacter hormaechei CEent1, and Pantoea nemavictus BIGb0393) compared to E. coli. Previously, these three bacterial strains have been shown to support faster C. elegans development time than E. coli OP50 and are low colonizers compared to eight other members of CeMbio. We then used gas chromatography coupled to mass spectrometry to identify that these three bacteria release isoamyl alcohol, a previously described C. elegans chemoattractant. We suggest that C. elegans seeks bacteria that release isoamyl alcohol and support faster growth.

The nematode worm C. elegans chooses between bacterial foods as if maximizing economic utility.

In value-based decision making, options are selected according to subjective values assigned by the individual to available goods and actions. Despite the importance of this faculty of the mind, the neural mechanisms of value assignments, and how choices are directed by them, remain obscure. To investigate this problem, we used a classic measure of utility maximization, the Generalized Axiom of Revealed Preference, to quantify internal consistency of food preferences in Caenorhabditis elegans, a nematode worm with a nervous system of only 302 neurons. Using a novel combination of microfluidics and electrophysiology, we found that C. elegans food choices fulfill the necessary and sufficient conditions for utility maximization, indicating that nematodes behave as if they maintain, and attempt to maximize, an underlying representation of subjective value. Food choices are well-fit by a utility function widely used to model human consumers. Moreover, as in many other animals, subjective values in C. elegans are learned, a process we find requires intact dopamine signaling. Differential responses of identified chemosensory neurons to foods with distinct growth potentials are amplified by prior consumption of these foods, suggesting that these neurons may be part of a value-assignment system. The demonstration of utility maximization in an organism with a very small nervous system sets a new lower bound on the computational requirements for utility maximization and offers the prospect of an essentially complete explanation of value-based decision making at single neuron resolution in this organism.

The Basics of Setting up Successful Teaching Labs and Short-Term Projects with C. elegans.

Caenorhabditis elegans is an excellent organism for teaching or doing short-term research projects because of the many freely-available comprehensive resources describing its genome and biology, its short-generation time and the ease of working with it in the lab. However, it can be daunting to begin a short-term project or a teaching lab with an organism with which one has little or no experience. Therefore, in this article, we will discuss the minimal equipment, reagents, and protocols needed to begin doing experiments with C. elegans as well as inclusive teaching practices. We will also describe a teaching laboratory for introductory or advanced neuroscience undergraduate courses where students learn to understand the relationship between genetic mutations and behavior through using chemotaxis assays. In addition, instructions for students for the lab module are provided in Subheading "Example of Teaching Lab Module for Students".

Using Optogenetics to Understand Neuronal Mechanisms Underlying Behavior in C. elegans.

Approaches for inhibiting and activating neurons are essential for understanding how neurons and neuronal circuits produce behavior. Optogenetics is a recently-developed technique which uses light to manipulate neuronal activity with temporal precision in behaving animals and is widely-used by neuroscience researchers. Optogenetics is also an excellent method to incorporate into an undergraduate neuroscience laboratory module because students can learn to conduct and analyze quantitative behavioral assays, reinforce their understanding of synaptic transmission, and investigate the genetic and neuronal basis of behavior. Here, we describe a module in which students use light to activate serotonergic neurons expressing the light-activated ion channel channelrhodopsin in wildtype and mutant Caenorhabditis elegans, and observe and analyze the effects on the movement behavior of the nematode. The methods described here provide a foundation which students can use to design and conduct additional experiments that may have never been done before. Thus, this laboratory module provides an opportunity for students to learn a state-of-the-art neuroscience technique, think about neuroscience on genetic, cellular and behavioral levels, and to develop an independent research project.

Identification of attractive odorants released by preferred bacterial food found in the natural habitats of C. elegans.

Food choice is critical for survival because organisms must choose food that is edible and nutritious and avoid pathogenic food. Many organisms, including the nematode C. elegans, use olfaction to detect and distinguish among food sources. C. elegans exhibits innate preferences for the odors of different bacterial species. However, little is known about the preferences of C. elegans for bacterial strains isolated from their natural environment as well as the attractive volatile compounds released by preferred natural bacteria isolates. We tested food odor preferences of C. elegans for non-pathogenic bacteria found in their natural habitats. We found that C. elegans showed a preference for the odor of six of the eight tested bacterial isolates over its standard food source, E. coli HB101. Using solid-phase microextraction and gas chromatography coupled with mass spectrometry, we found that four of six attractive bacterial isolates (Alcaligenes sp. JUb4, Providenica sp. JUb5, Providencia sp. JUb39, and Flavobacteria sp. JUb43) released isoamyl alcohol, a well-studied C. elegans attractant, while both non-attractive isolates (Raoultella sp. JUb38 and Acinetobacter sp. JUb68) released very low or non-detectable amounts of isoamyl alcohol. In conclusion, we find that isoamyl alcohol is likely an ethologically relevant odor that is released by some attractive bacterial isolates in the natural environment of C. elegans.

Identification of Odor Blend Used by Caenorhabditis elegans for Pathogen Recognition.

Animals have evolved specialized pathways to detect appropriate food sources and avoid harmful ones. Caenorhabditis elegans can distinguish among the odors of various species of bacteria, its major food source, but little is known about what specific chemical cue or combination of chemical cues C. elegans uses to detect and recognize different microbes. Here, we examine the strong innate attraction of C. elegans for the odor of the pathogenic bacterium, Serratia marcescens. This initial attraction likely facilitates ingestion and infection of the C. elegans host. Using solid-phase microextraction and gas chromatography coupled with mass spectrometry, we identify 5 volatile odors released by S. marcescens and identify those that are attractive to C. elegans. We use genetic methods to show that the amphid chemosensory neuron, AWCON, senses both S. marcescens-released 2-butanone and acetone and drives attraction to S. marcescens. In C. elegans, pairing a single odorant with food deprivation results in a reduced attractive response for that specific odor. We find that pairing the natural odor of S. marcescens with food deprivation results in a reduced attraction for the natural odor of S. marcescens and a similar reduced attraction for the synthetic blend of acetone and 2-butanone. This result indicates that only 2 odorants represent the more complex odor bouquet of S. marcescens. Although bacterial-released volatiles have long been known to be attractive to C. elegans, this study defines for the first time specific volatile cues that represent a particular bacterium to C. elegans.

Multigenic natural variation underlies Caenorhabditis elegans olfactory preference for the bacterial pathogen Serratia marcescens.

The nematode Caenorhabditis elegans can use olfaction to discriminate among different kinds of bacteria, its major food source. We asked how natural genetic variation contributes to choice behavior, focusing on differences in olfactory preference behavior between two wild-type C. elegans strains. The laboratory strain N2 strongly prefers the odor of Serratia marcescens, a soil bacterium that is pathogenic to C. elegans, to the odor of Escherichia coli, a commonly used laboratory food source. The divergent Hawaiian strain CB4856 has a weaker attraction to Serratia than the N2 strain, and this behavioral difference has a complex genetic basis. At least three quantitative trait loci (QTLs) from the CB4856 Hawaii strain (HW) with large effect sizes lead to reduced Serratia preference when introgressed into an N2 genetic background. These loci interact and have epistatic interactions with at least two antagonistic QTLs from HW that increase Serratia preference. The complex genetic architecture of this C. elegans trait is reminiscent of the architecture of mammalian metabolic and behavioral traits.

Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent.

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton-KHC complexes, and mitochondria are present in klc (-/-) photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH(2) terminus-splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain-independent, anterograde transport of mitochondria.