Carbon dioxide (CO2) is a byproduct of oxidative metabolism that can be sensed by different species including mammals, insects, and nematodes and can lead to both physiological and behavioral responses. In the free-living nematode Caenorhabditis elegans the behavioral response can be either avoidance or neutral to CO2, indicating that the behavior is flexible. In this thesis, I investigated the neural basis of behavioral flexibility. We found that the CO2 circuit can be modulated by diverse sensory neurons that respond to ambient oxygen (O2), temperature, and food odor. Additionally, we identified two interneurons downstream of the CO2-sensing BAG neurons, AIY and RIG, that have opposing roles in CO2-evoked responses. A decrease in AIY activity and an increase in RIG activity promote appropriate avoidance behavior to CO2 levels. Our results show that just like thermosensory and oxygen circuits, the CO2 circuit can exhibit a network of multisensory integration to enable animals to generate flexible behaviors to changing environments.

The valence of sensory stimuli (i.e. a measure of attractiveness or aversiveness that an animal attaches to a stimulus) is flexible and determined by a combination of factors including environment, behavioral state, and experience. Together, these factors prime the nervous system in order to drive appropriate behaviors. In this dissertation I investigate how environmental context and behavioral state regulate the valence of the chemosensory cue carbon dioxide (CO2) in the free-living nematode Caenorhabditis elegans. CO2 can signify the presence of food, conspecifics, and predators. When raised in standard laboratory conditions C. elegans avoids CO2, but this response is flexible. Whereas oxygen, salt, and food all promote CO2 avoidance, food-deprivation shifts CO2-response valence from avoidance to attraction. We have identified two sets of neurons, RIG and AIY, that contribute to the starvation-dependent shift. We have demonstrated that dopamine signaling enhances the CO2-evoked responses in both of these neurons in order to drive avoidance in fed worms. All animals must make ecologically relevant decisions to survive, and our results provide fundamental insights into how neural circuits are dynamically sculpted by internal state and context to drive behavior.

Adaptability is essential to organisms’ fitness and survival. Evolutionary success depends on access to an array of behavioral choices in the face of changing environmental conditions. To navigate complex landscapes, organisms can interpret the significance of sensory stimuli, and assign context-appropriate valence, by integrating factors such as cues from their internal and external environments, and memories of previously experienced conditions, to dynamically shape neural circuits and generate ethologically relevant behaviors. In this thesis, I explore the cellular and molecular mechanisms that shape the carbon dioxide (CO2) circuit in the free-living nematode, Caenorhabditis elegans. CO2 is a complex sensory cue that can signify the presence of fruitful or dangerous surroundings. As a result, C. elegans can display a variety of different behaviors in response to CO2, from robust attraction to robust avoidance. Although sensory signaling of the CO2-responsive BAG neurons has been extensively characterized, how BAG communicates with postsynaptic interneurons, and how the CO2 signal is propagated through the nervous system to generate a context-appropriate behavior is unknown. First, we have found that neuromodulatory state and environmental oxygen (O2) levels converge on the CO2 circuit via the URX sensory neurons. The lab-derived N2 C. elegans strain expresses high levels of NPR-1 neuropeptide receptor, which inhibits URX and results in CO2 avoidance, regardless of environmental O2. In the C. elegans wild isolate “Hawaii”, loss of npr-1 leads to modulation of URX by environmental O2, and results in CO2 avoidance at low O2, and loss of CO2-evoked behavior at high O2. Second, we present a new circuit motif that demonstrates how divergent responses to a single sensory input, CO2, can arise from an identical set of sensory and interneuron connections. We show that C. elegans exhibit an experience-dependent behavioral valence switch in response to CO2. While animals raised at ambient CO2 are repelled by CO2, animals raised in a high CO2 environment are attracted to CO2. Whether CO2 is attractive or repulsive is determined by the coordinated activity of specialized valence-encoding interneurons, AIY, RIG, and RIA, whose responses are subject to context-dependent modulation. An additional interneuron pair, AIZ, regulates behavioral sensitivity regardless of valence. Glutamatergic and neuropeptidergic signaling mediate both CO2 avoidance and attraction, and different neuropeptides play distinct roles in regulating valence and sensitivity. Our results elucidate a microcircuit motif whereby a fixed set of neurons are leveraged to generate alternative outputs in response to a single chemosensory input.

Skin-penetrating parasitic nematodes infect approximately 1 billion people and are endemic in many developing regions worldwide. Nematode infections can lead to severe intestinal distress, anemia, stunted growth and cognitive impairment in children, and in some cases death. Skin penetrating nematodes live in the environment as developmentally arrested third stage infective larvae (iL3s) that actively search for mammals to infect, a process called host seeking. Despite the pervasive nature of these infections, very little is known about cellular and molecular mechanism that promote host-seeking behavior. In this dissertation I explore the behavioral strategies, and underlying chemosensory mechanisms, that allow skin penetrating iL3s to detect hosts in the environment. I also characterize how iL3s use sensory mechanisms to resume their development upon entering the host. We focused on the human parasitic threadworm Strongyloides stercoralis as a model for understanding chemosensory-driven behaviors in skin-penetrating nematodes. First, we investigated the host-seeking behaviors of S. stercoralis iL3s and compared its behavior to that of other parasitic nematodes. S. stercoralis iL3s are especially active in the environment, are stimulated by elevated temperatures approximating that of the human body, and also navigate toward volatile odorants emitted from human skin and sweat. The odorant response profile of S. stercoralis iL3s was distinct from that of other parasitic nematodes that infect other mammals, indicating that olfaction may play a critical role in host specificity. We observed that the host seeking behaviors of S. stercoralis iL3s are dramatically different from those of passively ingested parasitic nematode species. Thus, parasitic nematodes with different infection modes may identify and navigate to hosts in distinct ways, and in turn may require distinct environmental control measures. To address the molecular mechanisms of host seeking behavior, we leveraged the unique genetic tractability of S. stercoralis to develop CRISPR-Cas9-mediated targeted mutagenesis in parasitic nematodes. As a proof-of-concept for CRISPR-Cas9 feasibility in S. stercoralis we targeted the twitchin gene Ss-unc-22 and generated iL3s with severe motility defects, the first mutant phenotype resulting from targeted mutagenesis observed in any parasitic nematode species. Our CRISPR-Cas9 technique was then applied to disrupt S. stercoralis genes with roles in chemosensation. We targeted the Ss tax-4 gene, a subunit of a cyclic nucleotide-gated ion channel, since its ortholog in the model free-living nematode Caenorhabditis elegans is known to be required for sensory-driven behaviors. Ss-tax-4 iL3s were deficient in thermosensory driven host seeking and could not positively thermotaxis in a temperature gradient, a robust behavior observed in wild-type iL3s. Ss-tax-4 iL3s were also unable to navigate towards host-emitted 3-methyl-1-butanol (isoamyl alcohol), indicating that olfactory driven host-seeking behaviors were also disrupted in the absence of functional chemosensory pathways. Next, we asked if chemosensory mechanisms were also necessary for iL3s to successfully establish an infection in the host. To address this question, we subjected S. stercoralis iL3s to host-like conditions in vitro and assessed iL3 activation, an initial developmental step inside the host where iL3s resume feeding behavior. iL3s consistently activated in the presence of 37C and 5% CO2 environments, but removal of either stimulus eliminated activation. Ss-tax-4 iL3 were unable to activate in the presence of heat and CO2, indicating that sensory function is also critical for in-host development. Our results suggest that two novel avenues for reducing the disease burden caused by skin penetrating nematodes may be to interfere with chemosensory-driven host seeking in the environment, or to inhibit sensory-driven development inside the host. Our results also lay the foundation for investigating the sensory neural circuits underlying the parasite-specific behaviors of an important group of helminths.