generating-transgenics-knockouts-strongyloides-species

Generating Transgenics and Knockouts in <em>Strongyloides</em> Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

University of California, Los Angeles

Over one billion people worldwide are infected with parasitic nematodes. Many parasitic nematodes actively search for hosts to infect using volatile chemical cues, so understanding the olfactory signals that drive host seeking may elucidate new pathways for preventing infections. The free-living nematode Caenorhabditis elegans is a powerful model for parasitic nematodes: because sensory neuroanatomy is conserved across nematode species, an understanding of the microcircuits that mediate olfaction in C. elegans may inform studies of olfaction in parasitic nematodes. Here we review circuit mechanisms that allow C. elegans to respond to odorants, gases, and pheromones. We also highlight work on the olfactory behaviors of parasitic nematodes that lays the groundwork for future studies of their olfactory microcircuits.

Olfactory circuits and behaviors of nematodes.

Many parasitic nematodes have an environmental infective stage that searches for hosts. Olfaction plays an important role in this process, with nematodes navigating their environment using host-emitted and environmental olfactory cues. The interactions between parasitic nematodes and their hosts are also influenced by the olfactory behaviors of the host, since host olfactory preferences drive behaviors that may facilitate or impede parasitic infection. However, how olfaction shapes parasite-host interactions is poorly understood. Here we investigated this question using the insect-parasitic nematode Howardula aoronymphium and its host, the mushroom fly Drosophila falleni. We found that both H. aoronymphium and D. falleni are attracted to mushroom odor and a subset of mushroom-derived odorants, but they have divergent olfactory preferences that are tuned to different mushroom odorants despite their shared mushroom environment. H. aoronymphium and D. falleni respond more narrowly to odorants than Caenorhabditis elegans and Drosophila melanogaster, consistent with their more specialized niches. Infection of D. falleni with H. aoronymphium alters its olfactory preferences, rendering it more narrowly tuned to mushroom odor. Our results establish H. aoronymphium-D. falleni as a model system for studying olfaction in the context of parasite-host interactions.

Olfactory Preferences of the Parasitic Nematode Howardula aoronymphium and its Insect Host Drosophila falleni.

Many chemosensory stimuli evoke innate behavioral responses that can be either appetitive or aversive, depending on an animal's age, prior experience, nutritional status, and environment [1-9]. However, the circuit mechanisms that enable these valence changes are poorly understood. Here, we show that Caenorhabditis elegans can alternate between attractive or aversive responses to carbon dioxide (CO), depending on its recently experienced CO environment. Both responses are mediated by a single pathway of interneurons. The CO-evoked activity of these interneurons is subject to extreme experience-dependent modulation, enabling them to drive opposite behavioral responses to CO. Other interneurons in the circuit regulate behavioral sensitivity to CO independent of valence. A combinatorial code of neuropeptides acts on the circuit to regulate both valence and sensitivity. Chemosensory valence-encoding interneurons exist across phyla, and valence is typically determined by whether appetitive or aversive 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. This circuit design represents a previously unrecognized mechanism for generating rapid changes in innate chemosensory valence.

A Single Set of Interneurons Drives Opposite Behaviors in C. elegans.

How sexually dimorphic behaviors are represented in the brain is a long-standing question. Two new studies in C. elegans uncover novel molecular mechanisms that allow neurons shared by opposite sexes to generate distinct sex-specific behaviors.

Sexual Dimorphisms: How Sex-Shared Neurons Generate Sex-Specific Behaviors.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are a major source of neglected tropical disease [1-6]. Their life cycle includes an infective third-larval (iL3) stage that searches for hosts to infect in a poorly understood process that involves both thermal and olfactory cues. Here, we investigate the temperature-driven behaviors of skin-penetrating iL3s, including the human-parasitic threadworm Strongyloides stercoralis and the human-parasitic hookworm Ancylostoma ceylanicum. We show that human-parasitic iL3s respond robustly to thermal gradients. Like the free-living nematode Caenorhabditis elegans, human-parasitic iL3s show both positive and negative thermotaxis, and the switch between them is regulated by recent cultivation temperature [7]. When engaging in positive thermotaxis, iL3s migrate toward temperatures approximating mammalian body temperature. Exposing iL3s to a new cultivation temperature alters the thermal switch point between positive and negative thermotaxis within hours, similar to the timescale of thermal plasticity in C. elegans [7]. Thermal plasticity in iL3s may enable them to optimize host finding on a diurnal temperature cycle. We show that temperature-driven responses can be dominant in multisensory contexts such that, when thermal drive is strong, iL3s preferentially engage in temperature-driven behaviors despite the presence of an attractive host odorant. Finally, targeted mutagenesis of the S. stercoralis tax-4 homolog abolishes heat seeking, providing the first evidence that parasitic host-seeking behaviors are generated through an adaptation of sensory cascades that drive environmental navigation in C. elegans [7-10]. Together, our results provide insight into the behavioral strategies and molecular mechanisms that allow skin-penetrating nematodes to target humans.

A Critical Role for Thermosensation in Host Seeking by Skin-Penetrating Nematodes.

Parasitic helminth infections are the most common source of neglected tropical disease among impoverished global communities. Many helminths infect their hosts via an active, sensory-driven process in which environmentally motile infective larvae position themselves near potential hosts. For these helminths, host seeking and host invasion can be divided into several discrete behaviors that are regulated by both host-emitted and environmental sensory cues, including heat. Thermosensation is a critical sensory modality for helminths that infect warm-blooded hosts, driving multiple behaviors necessary for host seeking and host invasion. Furthermore, thermosensory cues influence the host-seeking behaviors of both helminths that parasitize endothermic hosts and helminths that parasitize insect hosts. Here, we discuss the role of thermosensation in guiding the host-seeking and host-infection behaviors of a diverse group of helminths, including mammalian-parasitic nematodes, entomopathogenic nematodes, and schistosomes. We also discuss the neural circuitry and molecular pathways that underlie thermosensory responses in these species.

Temperature-dependent behaviors of parasitic helminths.

Infection with gastrointestinal parasitic nematodes is a major cause of chronic morbidity and economic burden around the world, particularly in low-resource settings. Some parasitic nematode species, including the human-parasitic threadworm Strongyloides stercoralis and human-parasitic hookworms in the genera Ancylostoma and Necator, feature a soil-dwelling infective larval stage that seeks out hosts for infection using a variety of host-emitted sensory cues. Here, we review our current understanding of the behavioral responses of soil-dwelling infective larvae to host-emitted sensory cues, and the molecular and cellular mechanisms that mediate these responses. We also discuss the development of methods for transgenesis and CRISPR/Cas9-mediated targeted mutagenesis in Strongyloides stercoralis and the closely related rat parasite Strongyloides ratti. These methods have established S. stercoralis and S. ratti as genetic model systems for gastrointestinal parasitic nematodes and are enabling more detailed investigations into the neural mechanisms that underlie the sensory-driven behaviors of this medically and economically important class of parasites.

Terror in the dirt: Sensory determinants of host seeking in soil-transmitted mammalian-parasitic nematodes.

Hunger affects the behavioral choices of all animals, and many chemosensory stimuli can be either attractive or repulsive depending on an animal's hunger state. Although hunger-induced behavioral changes are well documented, the molecular and cellular mechanisms by which hunger modulates neural circuit function to generate changes in chemosensory valence are poorly understood. Here, we use the CO response of the free-living nematode  to elucidate how hunger alters valence. We show that CO response valence shifts from aversion to attraction during starvation, a change that is mediated by two pairs of interneurons in the CO circuit, AIY and RIG. The transition from aversion to attraction is regulated by biogenic amine signaling. Dopamine promotes CO repulsion in well-fed animals, whereas octopamine promotes CO 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 CO response valence by modulating the CO-evoked activity of AIY and RIG. Our results illuminate a new role for biogenic amine signaling in regulating chemosensory valence as a function of hunger state.

Feeding state sculpts a circuit for sensory valence in .

Inter-individual variability in behavioral flexibility is widespread throughout the animal kingdom, but its underlying mechanisms remain poorly understood. In this issue of Neuron, Beets et al. (2020) provide novel insights into the genetic basis of inter-individual differences in behavioral flexibility using the model nematode C. elegans.

Decoding Inter-individual Variability in Experience-Dependent Behavioral Plasticity.

Human-parasitic nematodes infect over a quarter of the world's population and are a major cause of morbidity in low-resource settings. Currently available treatments have not been sufficient to eliminate infections in endemic areas, and drug resistance is an increasing concern, making new treatment options a priority. The development of new treatments requires an improved understanding of the basic biology of these nematodes. Specifically, a better understanding of parasitic nematode development, reproduction and behavior may yield novel drug targets or new opportunities for intervention such as repellents or traps. Until recently, our ability to study parasitic nematode biology was limited because few tools were available for their genetic manipulation. This is now changing as a result of recent advances in the large-scale sequencing of nematode genomes and the development of new techniques for their genetic manipulation. Notably, skin-penetrating gastrointestinal nematodes in the genus  are now amenable to transgenesis, RNAi and CRISPR/Cas9-mediated targeted mutagenesis, positioning the  species as model parasitic nematode systems. A number of other mammalian-parasitic nematodes, including the giant roundworm  and the tissue-dwelling filarial nematode , are also now amenable to transgenesis and/or RNAi in some contexts. Using these tools, recent studies of  species have already provided insight into the molecular pathways that control the developmental decision to form infective larvae and that drive the host-seeking behaviors of infective larvae. Ultimately, a mechanistic understanding of these processes could lead to the development of new avenues for nematode control.

Recent advances in functional genomics for parasitic nematodes of mammals.

Approximately 800 million people worldwide are infected with one or more species of skin-penetrating nematodes. These parasites persist in the environment as developmentally arrested third-stage infective larvae (iL3s) that navigate toward host-emitted cues, contact host skin, and penetrate the skin. iL3s then reinitiate development inside the host in response to sensory cues, a process called activation. Here, we investigate how chemosensation drives host seeking and activation in skin-penetrating nematodes. We show that the olfactory preferences of iL3s are categorically different from those of free-living adults, which may restrict host seeking to iL3s. The human-parasitic threadworm  and hookworm  have highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the  gene abolishes iL3 attraction to a host-emitted odorant and prevents activation. Our results suggest an important role for chemosensation in iL3 host seeking and infectivity and provide insight into the molecular mechanisms that underlie these processes.

Elissa A. Hallem

Department of Microbiology,
Immunology,
and Molecular Genetics,
Molecular Biology Institute,
Department of Microbiology, Immunology, and Molecular Genetics

University of California, Los Angeles

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

Elissa A. Hallem

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Immunology,and Molecular Genetics,Molecular Biology Institute,Department of Microbiology, Immunology, and Molecular Genetics

University of California, Los Angeles

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

Department of Microbiology,
Immunology,
and Molecular Genetics,
Molecular Biology Institute,
Department of Microbiology, Immunology, and Molecular Genetics