Overview
Our research investigates the sensory behaviors of gastrointestinal parasitic nematodes. Gastrointestinal parasitic nematodes infect over one billion people worldwide, particularly in low-resource settings. Some of these parasites actively invade hosts by skin penetration, while others infect by passive ingestion when they are swallowed with food or water contaminated with infested feces. The majority of our research focuses on the skin-penetrating human-parasitic threadworm Strongyloides stercoralis. S. stercoralis is endemic in tropical and subtropical regions throughout the world, and is estimated to infect 370 million people worldwide. Infection with S. stercoralis and other gastrointestinal nematodes can cause chronic gastrointestinal distress as well as stunted growth and long-term cognitive impairment in children. S. stercoralis infection can also be fatal for immunosuppressed individuals. Current drugs used to treat nematode infections are inadequate: some are toxic, drug resistance is a growing concern, and reinfection rates are high. A better understanding of the basic biology of these parasites could enable the development of new approaches to nematode control.

The overarching goal of our research is to understand how parasitic nematodes use sensory cues to find and infect hosts. We aim to understand host-seeking and host-infection behaviors at the levels of genes, neural circuits, and behaviors. For comparative studies of host-seeking and host-infection behaviors, we use a diverse group of parasitic nematode species. For mechanistic studies, we use S. stercoralis and the closely related rat-parasitic nematode Strongyloides ratti, since these species are uniquely amenable to genetic engineering techniques. We also use the free-living nematode Caenorhabditis elegans as a comparative model for the parasitic nematodes. Our research is illuminating how parasitic worms use sensory cues to locate hosts to infect, and how sensory neural circuits of parasitic animals differ from those of free-living animals to enable parasitic behaviors. A few of the major findings from our recent research are highlighted below.

Odor-driven host seeking by S. stercoralis
S. stercoralis and other skin-penetrating nematodes have an infective larval stage that inhabits the soil and 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 showed that S. stercoralis infective larvae (see photo at right) 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 the specific adaptations of the S. stercoralis olfactory system that control odor-driven parasitic behaviors. We are also now investigating the role of olfactory and gustatory cues in driving skin penetration, development inside the host, and intra-host navigation.

Odor-driven host seeking by H. polygyrus
We 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).

Temperature-driven behaviors of S. stercoralis
We conducted an in-depth analysis of the temperature-driven behaviors of parasitic nematodes, with a primary focus on S. stercoralis. We found that S. stercoralis and other mammalian-parasitic nematodes respond
robustly to thermal gradients. Like C. elegans, parasitic infective larvae show both positive and negative thermotaxis, and the switch between these behaviors is regulated by recent cultivation temperature. When engaging in positive thermotaxis, infective larvae migrate toward mammalian body temperature (see video to the right). While positive thermotaxis contributes to host seeking, negative thermotaxis may drive environmental navigation. Both behaviors likely enable infective larvae to optimize host finding on a diurnal temperature cycle. To begin to address the role of multisensory integration during host seeking, we then investigated the relationship between chemosensory cues and thermosensory cues. We found that at temperatures well below host body temperature, infective larvae will bypass an attractive odorant to travel up a thermal gradient. However, at temperatures near host body temperature, the infective larvae will migrate less far up a thermal gradient when an attractive host odorant is present. Our results suggest that thermotaxis drives long-range navigation toward hosts, while chemosensation is used at closer range to distinguish hosts from non-hosts (Bryant et al., 2017). Finally, we showed that targeted mutagenesis of the S. stercoralis tax-4 gene prevents positive thermotaxis, providing the first insights into the molecular basis of host seeking. We are now investigating the neural circuits that drive thermosensory behaviors in S. stercoralis. We are also further investigating how infective larvae integrate thermosensory and chemosensory cues during host seeking.

Development of a CRISPR/Cas9 system for S. stercoralis and S. ratti
Another major focus of our research is on the development of new tools and approaches for studying the molecular and cellular mechanisms that drive host seeking and infectivity in parasitic nematodes. Our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, prior to this study, it had not been possible to generate targeted gene disruptions resulting in mutant phenotypes in any parasitic nematode species. We developed a method for generating CRISPR/Cas9-mediated targeted gene disruptions in S. stercoralis and S. ratti. Importantly, we were able to obtain homozygous gene disruptions resulting in recessive mutant phenotypes. Our results demonstrated for the first time that the CRISPR/Cas9 system can be used to generate mutant phenotypes in parasitic nematodes (Gang et al., 2017). We are now using this system to disrupt a number of different genes to identify signaling pathways that drive host seeking and infectivity in parasitic nematodes. We are also further optimizing the CRISPR/Cas9 system for S. stercoralis and S. ratti. At the same time, we are developing other tools for studying neural circuit function in parasitic nematodes.

The neural basis of carbon dioxide response in C. elegans
Carbon dioxide (CO2) is a critical host cue for many parasites, including many parasitic nematodes. We found that many parasitic nematodes show flexible responses to CO2 such that CO2 can be either attractive or
repulsive depending on the age or prior experience of the worm 
(Lee et al., 2016Ruiz et al., 2017). To begin to understand how parasitic nematodes generate flexible responses to CO2, we turned to the model free-living nematode C. elegansWe found that C. elegans also generates flexible responses to CO2: animals cultivated at ambient CO2 (~0.04%) are repelled by CO2 (see video to the right), while animals cultivated at high CO2 (2.5%) are attracted to CO2. We then used this system to investigate the neural mechanisms that determine CO2 response valence. We found that CO2 response is mediated by the same sensory neuron and a single pathway of downstream interneurons regardless of valence. The CO2-evoked activity of these interneurons is subject to experience-dependent modulation, enabling them to drive opposite behavioral responses to CO2. Across phyla, chemosensory valence is typically determined by whether attraction-promoting or aversion-promoting interneuron populations are activated. Our results reveal an alternative mechanism of valence determination in which the same interneurons contribute to both attractive and aversive responses through modulation of sensory-neuron-to-interneuron synapses (Guillermin et al., 2017). 

CO2 response valence in C. elegans is regulated not only by ambient CO2 levels but also by hunger state. We found that CO2 response valence shifts from aversion to attraction during starvation. This change is regulated by biogenic amine signaling: dopamine promotes CO2 repulsion in well-fed animals, whereas octopamine promotes CO2 attraction in starved animals. Biogenic amines also regulate the temporal dynamics of the shift from aversion to attraction such that animals lacking octopamine show a delayed shift to attraction. Biogenic amine signaling regulates CO2 response valence by modulating the CO2-evoked activity of two pairs of interneurons in the CO2 circuit. Our results illuminate a new role for biogenic amine signaling in regulating chemosensory valence as a function of hunger state (Rengarajan et al., 2019). We are now investigating whether similar molecular and cellular mechanisms operate in parasitic worms to regulate CO2 response valence.

Research in the Hallem lab is currently supported by the NIH (1R01AI136976 and 1R01DC017959), MacArthur Foundation, Burroughs-Wellcome Fund, and HHMI.

Last updated 8-14-2019.