I.  Host-seeking behaviors of gastrointestinal parasitic nematodes
Gastrointestinal parasitic nematodes cause extensive morbidity and economic loss worldwide, particularly in low-resource settings. These parasites have an infective larval stage that actively searches for hosts to infect using a wide variety of host-emitted sensory cues. However, the host-seeking behaviors of the infective larvae remain poorly understood. We are interested in the behavioral responses of infective larvae to host-emitted sensory cues, and the neural circuits and signaling pathways that underlie them. We investigate host-seeking behavior in a wide variety of gastrointestinal nematodes, including some species that infect by skin penetration and other species that infect by passive ingestion. For mechanistic studies of host seeking, we use the human-parasitic nematode Strongyloides stercoralis and the closely related rat-parasitic nematode Strongyloides ratti, since these species are amenable to molecular genetic analysis. Our research will provide insight into the neural basis of parasitism and parasitic behaviors, and may enable the development of new strategies for combating harmful nematode infections.

Odor-driven host seeking by S. stercoralis
S. stercoralis is a skin-penetrating worm that infects approximately 30-100 million people worldwide.  S. stercoralis is found in tropical and subtropical regions throughout the world, 

S. stercoralis

Photo by M. Castelletto.
including the United States, and is estimated to infect 30-100 million people worldwide. S. stercoralis infection can cause chronic gastrointestinal distress, and can be fatal for immunosuppressed individuals. S. stercoralis infective larvae actively seek out human hosts, but little is known about their host-seeking behavior. We have shown that S. stercoralis infective larvae are highly motile relative to other parasitic worms, use a cruising strategy to actively search for hosts, and are attracted to a diverse array of human skin and sweat odorants. Many of the odorants that attract S. stercoralis are also mosquito attractants, suggesting that human-parasitic nematodes and mosquitoes respond to some of the same human-emitted olfactory cues. A comparison of olfactory behavior in S. stercoralis and six other nematode species revealed that parasitic nematodes show species-specific olfactory preferences. Moreover, olfactory preferences reflect host specificity rather than genetic relatedness, suggesting an important role for olfaction in host finding and host selection (Castelletto et al., 2014). We are now elucidating the neural circuitry that underlies odor-driven host seeking in S. stercoralis. By comparing olfactory neural circuit function in S. stercoralis and C. elegans, we hope to gain insight into how parasitic nervous systems have evolved to support parasite-specific behavioral repertoires. We are also investigating the responses of S. stercoralis to other host-emitted sensory cues, including heat.

Odor-driven host seeking by H. polygyrus
We also recently examined the olfactory behaviors of the passively ingested nematode Heligmosomoides polygyrus. Passively ingested nematodes infect when they are swallowed by a host, and therefore they were not thought to engage in host seeking. We found that H. polygyrus infective larvae are in fact robustly attracted to host-emitted odorants, suggesting that like skin-penetrating nematodes, passively ingested nematodes use olfactory cues to migrate toward hosts. Odor-driven host-seeking by passively ingested nematodes may enable the infective larvae to position themselves in the environment in the vicinity of potential hosts, where they are more likely to be ingested. In addition, we found that the olfactory responses of H. polygyrus infective larvae are highly flexible: some odorants, including carbon dioxide (CO2), can be either attractive or repulsive depending on the environmental conditions previously experienced by the infective larvae. Similar olfactory plasticity was observed in the passively ingested ruminant parasite Haemonchus contortus, but not the skin-penetrating parasites S. stercoralis and Ancylostoma ceylanicum, suggesting that it may be a general feature of passively ingested nematode behavior. Experience-dependent olfactory plasticity may enable infective larvae to switch between dispersal behavior and host-seeking behavior (Ruiz et al., 2017).

II.  Tools for studying the sensory neurobiology of parasitic nematodes
Another major focus of the lab is on developing tools and approaches for studying the neural basis of host seeking in parasitic nematodes. Toward this end, we recently developed a method for generating targeted gene disruptions in S. stercoralis and S. ratti using the CRISPR-Cas9 system. As a proof-of-concept, we disrupted the S. stercoralis unc-22 gene, which encodes the large muscle protein twitchin. We found that S. stercoralis and S. ratti unc-22 mutant infective larvae showed severely impaired swimming and crawling behaviors. Importantly, homozygous mutant infective larvae can be generated in the first generation, making it possible to study homozygous recessive phenotypes without the need for labor-intensive host passage (Gang et al., 2017). We are now using this approach to target a number of different genes that may be required for sensory-driven host seeking. We are also developing other tools for studying neural circuit function in parasitic nematodes.

III.  The neural basis of CO2-response valence in C. elegans

Carbon dioxide avoidance by C. elegans‎

CO2 is a critical host cue for many parasites, including many parasitic nematodes.  It is also an important sensory cue for many free-living animals, as it can signal the presence of food, mates, predators, or pathogens.  We are investigating the neural basis of CO2 response in the free-living nematode C. elegans. We previously showed that C. elegans adults are repelled by CO2. For example, exposing the head of a forward-moving worm to CO2 causes the worm to reverse. We identified a pair of sensory neurons, the BAG neurons, that are required for CO2-avoidance behavior.  We also showed that CO2 avoidance requires the receptor guanylate cyclase GCY-9, a putative CO2 receptor (Hallem et al., 2011a). In addition, we found that BAG neurons also mediate CO2 response in parasitic nematodes, indicating that the neural basis of CO2 response is at least partly conserved across nematode species (Hallem et al., Curr Biol 2011b).

More recently, we used the CO2 response of C. elegans as a model system for investigating the neural mechanisms that determine chemosensory valence, i.e. whether a chemosensory stimulus is attractive or repulsive. We found that CO2 is repulsive for C. elegans adults raised at ambient CO2 (~0.038% CO2), but attractive for C. elegans adults raised at high CO2 (2.5% CO2). Both CO2 repulsion and CO2 attraction are mediated by a single microcircuit consisting of the BAG sensory neurons and the downstream RIG, RIA, AIY, and AIZ interneurons. The activity of the RIG, RIA, and AIY interneurons is subject to experience-dependent modulation, enabling them to mediate responses of opposite valence. By contrast, the AIZ interneurons mediate behavioral sensitivity to CO2 regardless of response valence. While chemosensory valence is often determined by whether appetitive or aversive interneuron populations are activated, our results illustrate an alternative mechanism of valence encoding in which the same interneurons mediate both attraction and aversion through modulation of sensory neuron to interneuron synapses (Guillermin et al., 2017). We have shown that many parasitic nematodes also exhibit flexible responses to CO2 (Lee et al., 2016; Ruiz et al., 2017), and we are currently investigating whether similar circuit mechanisms operate in parasitic nematodes to regulate the response to this important host cue. 



Research in the Hallem lab is currently supported by the NIH, NSF, MacArthur Foundation, Burroughs-Wellcome Fund, and HHMI.