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The skin-penetrating gastrointestinal parasitic nematode Strongyloides stercoralis causes strongyloidiasis, which is a neglected tropical disease that is associated with severe chronic illness and fatalities. Unlike other human-infective nematodes, S. stercoralis cycles through a single free-living generation and thus serves as a genetically tractable model organism for understanding the mechanisms that enable parasitism. Techniques such as CRISPR/Cas9-mediated mutagenesis and transgenesis are now routinely performed in S. stercoralis by introducing exogenous DNA into free-living adults and then screening their F1 progeny for transgenic or mutant larvae. However, transgenesis in S. stercoralis has been severely hindered by the inability to establish stable transgenic lines that can be propagated for multiple generations through a host; to date, studies of transgenic S. stercoralis have been limited to heterogeneous populations of transgenic F1 larvae. Here, we develop an efficient pipeline for the generation of stable transgenic lines in S. stercoralis. We also show that this approach can be used to efficiently generate stable transgenic lines in the rat-infective nematode Strongyloides ratti. The ability to generate stable transgenic lines circumvents the limitations of working with heterogeneous F1 populations, such as variable transgene expression and the inability to generate transgenics of all life stages. Our transgenesis approach will enable novel lines of inquiry into parasite biology, such as transgene-based comparisons between free-living and parasitic generations.

Strongyloides stercoralis, commonly known as the human threadworm, is a skin-penetrating gastrointestinal parasitic nematode that infects hundreds of millions of people worldwide. Like other Strongyloides species, S. stercoralis is capable of cycling through a single free-living generation. Although S. stercoralis and the free-living nematode Caenorhabditis elegans are evolutionarily distant, the free-living adults of S. stercoralis are similar enough in size and morphology to C. elegans adults that techniques for generating transgenics and knockouts in C. elegans have been successfully adapted for use in S. stercoralis. High-quality genomic and transcriptomic data are also available for S. stercoralis. Thus, one can use a burgeoning array of functional genomic tools in S. stercoralis to probe questions about parasitic nematode development, physiology, and behavior. Knowledge gained from S. stercoralis will inform studies of other parasitic nematodes such as hookworms that are not yet amenable to genetic manipulation. This review describes the basic anatomy of S. stercoralis.

An animals preference for many chemosensory cues remains constant despite dramatic changes in the animals internal state. The mechanisms that maintain chemosensory preference across different physiological contexts remain poorly understood. We previously showed that distinct patterns of neural activity and motor output are evoked by carbon dioxide (CO2) in starved adults vs dauers of Caenorhabditis elegans, despite the two life stages displaying the same preference (attraction) for CO2. However, how the distinct CO2-evoked neural dynamics and motor patterns contribute to CO2 attraction at the two life stages remained unclear. Here, using a CO2 chemotaxis assay, we show that different interneurons are employed to drive CO2 attraction at the two life stages. We also investigate the molecular mechanisms that mediate CO2 attraction in dauers vs adults. We show that insulin signaling promotes CO2 attraction in dauers but not starved adults and that different combinations of neurotransmitters and neuropeptides are used for CO2 attraction at the two life stages. Our findings provide new insight into the distinct molecular and cellular mechanisms used by C. elegans at two different life stages to generate attractive behavioral responses to CO2.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue Strongyloides: omics to worm-free populations.

Skin-penetrating nematodes, including the human threadworm Strongyloides stercoralis and hookworms in the genera Necator and Ancylostoma, are gastrointestinal parasites that are a major cause of neglected tropical disease in low-resource settings worldwide. These parasites infect hosts as soil-dwelling infective larvae that navigate towards hosts using host-emitted sensory cues such as odorants and body heat. Upon host contact, they invade the host by penetrating through the skin. The process of skin penetration is critical for successful parasitism but remains poorly understood and understudied. Here, we review current knowledge of skin-penetration behaviour and its underlying mechanisms in the human parasite S. stercoralis, the closely related rat parasite Strongyloides ratti, and other skin-penetrating nematodes such as hookworms. We also highlight important directions for future investigations into this underexplored process and discuss how recent advances in molecular genetic and genomic tools for Strongyloides species will enable mechanistic investigations of skin penetration and other essential parasitic behaviours in future studies. This article is part of the Theo Murphy meeting issue Strongyloides: omics to worm-free populations.

Many chemosensory cues evoke responses of the same valence under widely varying physiological conditions. It remains unclear whether similar or distinct neural mechanisms are involved in the detection and processing of such chemosensory cues across contexts. We show that in Caenorhabditis elegans, a chemosensory cue is processed by distinct neural mechanisms at two different life stages that share the same valence state. Both starved adults and dauer larvae are attracted to carbon dioxide (CO2), but CO2 evokes different patterns of neural activity and different motor outputs at the two life stages. Moreover, the same interneuron within the CO2 microcircuit plays a different role in driving CO2-evoked motor output at the two life stages. The dauer-specific patterns of CO2-evoked activity in this interneuron require a dauer-specific gap junction complex and insulin signaling. Our results demonstrate that functionally distinct microcircuits are engaged in response to a chemosensory cue that triggers the same valence state at different life stages, revealing an unexpected complexity to chemosensory processing.

Migrations performed by helminths are impressive and diverse, and accumulating evidence shows that many are controlled by sophisticated sensory programs. The migrations of vector-borne helminths are particularly complex, requiring precise, stage-specific regulation. We review the contrasting states of knowledge on snail-borne schistosomes and mosquito-borne filarial nematodes. Rich observational data exist for the chemosensory behaviors of schistosomes, while the molecular sensory pathways in nematodes are well described. Recent investigations on the molecular mechanisms of sensation in schistosomes and filarial nematodes have revealed some features conserved within their respective phyla, but adaptations correlated with parasitism are pronounced. Technological developments are likely to extend these advances, and we forecast how these technologies may be applied.

The oft-neglected human-parasitic threadworm, Strongyloides stercoralis, infects roughly eight percent of the global population, placing disproportionate medical and economic burden upon marginalized communities. While current chemotherapies treat strongyloidiasis, disease recrudescence and the looming threat of anthelminthic resistance necessitate novel strategies for nematode control. Throughout its life cycle, S. stercoralis relies upon sensory cues to aid in environmental navigation and coordinate developmental progression. Odorants, tastants, gases, and temperature have been shown to shape parasite behaviors that drive host seeking and infectivity; however, many of these sensory behaviors remain poorly understood, and their underlying molecular and neural mechanisms are largely uncharacterized. Disruption of sensory circuits essential to parasitism presents a promising strategy for future interventions. In this review, we describe our current understanding of sensory behaviors - namely olfactory, gustatory, gas sensing, and thermosensory behaviors - in Strongyloides spp. We also highlight the ever-growing cache of genetic tools optimized for use in Strongyloides that have facilitated these findings, including transgenesis, CRISPR/Cas9-mediated mutagenesis, RNAi, chemogenetic neuronal silencing, and the use of fluorescent biosensors to measure neuronal activity. Bolstered by these tools, we are poised to enter an era of rapid discovery in Strongyloides sensory neurobiology, which has the potential to shape pioneering advances in the prevention and treatment of strongyloidiasis.

Soil-transmitted parasitic nematodes infect over one billion people and cause devastating morbidity worldwide. Many of these parasites have infective larvae that locate hosts using thermal cues. Here, we identify the thermosensory neurons of the human threadworm Strongyloides stercoralis and show that they display unique functional adaptations that enable the precise encoding of temperatures up to human body temperature. We demonstrate that experience-dependent thermal plasticity regulates the dynamic range of these neurons while preserving their ability to encode host-relevant temperatures. We describe a novel behavior in which infective larvae spontaneously reverse attraction to heat sources at sub-body temperatures and show that this behavior is mediated by rapid adaptation of the thermosensory neurons. Finally, we identify thermoreceptors that confer parasite-specific sensitivity to body heat. Our results pinpoint the parasite-specific neural adaptations that enable parasitic nematodes to target humans and provide the foundation for drug development to prevent human infection.

BackgroundSkin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points during their life cycle, Strongyloides species encounter a wide variety of host-associated and environmental bacteria. However, the microbiome associated with Strongyloides species, and the behavioral and physiological interactions between Strongyloides species and bacteria, remain unclear.ResultsWe first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have an associated microbiome consisting of specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to a narrow range of environmental bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, and thereby greatly decreases parasite viability.ConclusionsSkin-penetrating nematodes encounter bacteria from various ecological niches throughout their life cycle. Our results demonstrate that bacteria function as key chemosensory cues for directing parasite movement in a life-stage-specific manner. Some bacterial genera may form essential associations with the nematodes, while others are detrimental and serve as a potential source of novel nematicides.

Advances in genomics techniques are expanding the range of nematode species that are amenable to transgenesis. Due to divergent codon usage biases across species, codon optimization is often a critical step for the successful expression of exogenous transgenes in nematodes. Platforms for generating DNA sequences codon-optimized for the free-living model nematode Caenorhabditis elegans are broadly available. However, until now such tools did not exist for non-Caenorhabditis nematodes. We therefore developed the Wild Worm Codon Adapter, a tool for rapid transgene codon optimization for expression in non-Caenorhabditis nematodes. The app includes built-in optimization for parasitic nematodes in the Strongyloides, Nippostrongylus, and Brugia genera as well as the predatory nematode Pristionchus pacificus. The app also supports custom optimization for any species using user-provided optimization rules. In addition, the app supports automated insertion of synthetic or native introns, as well as the analysis of codon bias in transgene and native sequences. Here, we describe this web-based tool and demonstrate how it may be used to analyze genome-wide codon bias in Strongyloides species.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

Soil-transmitted gastrointestinal parasitic nematodes infect approximately 1 billion people worldwide, predominantly in low-resource communities. Skin-penetrating gastrointestinal nematodes in the genus Strongyloides are emerging as model systems for mechanistic studies of soil-transmitted helminths due to the growing availability of functional genomics tools for these species. To facilitate future genomics studies of Strongyloides species, we have designed a web-based application, the Strongyloides RNA-seq Browser, that provides an open source, user-friendly portal for accessing and analyzing Strongyloides genomic expression data. Specifically, the Strongyloides RNA-seq Browser takes advantage of alignment-free read mapping tools and R-based transcriptomics tools to re-analyze publicly available RNA sequencing datasets from four Strongyloides species: Strongyloides stercoralis, Strongyloides ratti, Strongyloides papillosus, and Strongyloides venezuelensis. This application permits on-demand exploration and quantification of gene expression across life stages without requiring previous coding experience. Here, we describe this interactive application and demonstrate how it may be used by nematode researchers to conduct a standard set of bioinformatics queries.

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S. stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S. ratti is closely related to S. stercoralis and is often used as a laboratory model for S. stercoralis. Both S. stercoralis and S. ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique. Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

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 Strongyloides stercoralis and hookworm Ancylostoma ceylanicum have highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the S. stercoralis tax-4 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.

Carbon dioxide (CO2) is an important sensory cue for many animals, including both parasitic and free-living nematodes. Many nematodes show context-dependent, experience-dependent and/or life-stage-dependent behavioural responses to CO2, suggesting that CO2 plays crucial roles throughout the nematode life cycle in multiple ethological contexts. Nematodes also show a wide range of physiological responses to CO2. Here, we review the diverse responses of parasitic and free-living nematodes to CO2. We also discuss the molecular, cellular and neural circuit mechanisms that mediate CO2 detection in nematodes, and that drive context-dependent and experience-dependent responses of nematodes to CO2.

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 CO2 response of the free-living nematode Caenorhabditis elegans to elucidate how hunger alters valence. We show that CO2 response valence shifts from aversion to attraction during starvation, a change that is mediated by two pairs of interneurons in the CO2 circuit, AIY and RIG. The transition from aversion to attraction 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 AIY and RIG. Our results illuminate a new role for biogenic amine signaling in regulating chemosensory valence as a function of hunger state.

Parasitic nematodes infect over 2 billion individuals worldwide, primarily in low-resource areas, and are responsible for several chronic and potentially deadly diseases. Throughout their life cycle, these parasites are thought to use astacin metalloproteases, a subfamily of zinc-containing metalloendopeptidases, for processes such as skin penetration, molting, and tissue migration. Here, we review the known functions of astacins in human-infective, soil-transmitted parasitic nematodes - including the hookworms Necator americanus and Ancylostoma duodenale, the threadworm Strongyloides stercoralis, the giant roundworm Ascaris lumbricoides, and the whipworm Trichuris trichiura - as well as the human-infective, vector-borne filarial nematodes Wuchereria bancrofti, Onchocerca volvulus, and Brugia malayi. We also review astacin function in parasitic nematodes that infect other mammalian hosts and discuss the potential of astacins as anthelmintic drug targets. Finally, we highlight the molecular and genetic tools that are now available for further exploration of astacin function and discuss how a better understanding of astacin function in human-parasitic nematodes could lead to new avenues for nematode control and drug therapies. [ABSTRACT FROM AUTHOR]

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.

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.

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.

Parasitic nematodes of humans and livestock cause extensive disease and economic loss worldwide. Many parasitic nematodes infect hosts as third-stage larvae, called iL3s. iL3s vary in their infection route: some infect by skin penetration, others by passive ingestion. Skin-penetrating iL3s actively search for hosts using host-emitted olfactory cues, but the extent to which passively ingested iL3s respond to olfactory cues was largely unknown. Here, we examined the olfactory behaviors of the passively ingested murine gastrointestinal parasite Heligmosomoides polygyrus. H. polygyrus iL3s were thought to reside primarily on mouse feces, and infect when mice consume feces containing iL3s. However, iL3s can also adhere to mouse fur and infect orally during grooming. Here, we show that H. polygyrus iL3s are highly active and show robust attraction to host feces. Despite their attraction to feces, many iL3s migrate off feces to engage in environmental navigation. In addition, H. polygyrus iL3s are attracted to mammalian skin odorants, suggesting that they migrate toward hosts. The olfactory preferences of H. polygyrus are flexible: some odorants are repulsive for iL3s maintained on feces but attractive for iL3s maintained off feces. Experience-dependent modulation of olfactory behavior occurs over the course of days and is mediated by environmental carbon dioxide (CO2) levels. Similar experience-dependent olfactory plasticity occurs in the passively ingested ruminant-parasitic nematode Haemonchus contortus, a major veterinary parasite. Our results suggest that passively ingested iL3s migrate off their original fecal source and actively navigate toward hosts or new host fecal sources using olfactory cues. Olfactory plasticity may be a mechanism that enables iL3s to switch from dispersal behavior to host-seeking behavior. Together, our results demonstrate that passively ingested nematodes do not remain inactive waiting to be swallowed, but rather display complex sensory-driven behaviors to position themselves for host ingestion. Disrupting these behaviors may be a new avenue for preventing infections.

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

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 (CO2), depending on its recently experienced CO2 environment. Both responses are mediated by a single pathway of interneurons. The CO2-evoked activity of these interneurons is subject to extreme experience-dependent modulation, enabling them to drive opposite behavioral responses to CO2. Other interneurons in the circuit regulate behavioral sensitivity to CO2 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.

Members of the gammaproteobacterial Photorhabdus genus share mutualistic relationships with Heterorhabditis nematodes, and the pairs infect a wide swath of insect larvae. Photorhabdus species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl-trans-stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from Photorhabdus luminescens that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.

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.

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.

BackgroundEntomopathogenic nematodes (EPNs) are lethal parasites of insects that are of interest as biocontrol agents for insect pests and disease vectors. Although EPNs have been successfully commercialized for pest control, their efficacy in the field is often inconsistent for reasons that remain elusive. EPN infective juveniles (IJs) actively search for hosts to infect using a diverse array of host-emitted odorants. Here we investigate whether their host-seeking behavior is subject to context-dependent modulation.ResultsWe find that EPN IJs exhibit extreme plasticity of olfactory behavior as a function of cultivation temperature. Many odorants that are attractive for IJs grown at lower temperatures are repulsive for IJs grown at higher temperatures and vice versa. Temperature-induced changes in olfactory preferences occur gradually over the course of days to weeks and are reversible. Similar changes in olfactory behavior occur in some EPNs as a function of IJ age. EPNs also show temperature-dependent changes in their host-seeking strategy: IJs cultured at lower temperatures appear to more actively cruise for hosts than IJs cultured at higher temperatures. Furthermore, we find that the skin-penetrating rat parasite Strongyloides ratti also shows temperature-dependent changes in olfactory behavior, demonstrating that such changes occur in mammalian-parasitic nematodes.ConclusionsIJs are developmentally arrested and long-lived, often surviving in the environment through multiple seasonal temperature changes. Temperature-dependent modulation of behavior may enable IJs to optimize host seeking in response to changing environmental conditions, and may play a previously unrecognized role in shaping the interactions of both beneficial and harmful parasitic nematodes with their hosts.

The phylum Nematoda comprises a diverse group of roundworms that includes parasites of vertebrates, invertebrates, and plants. Human-parasitic nematodes infect more than one billion people worldwide and cause some of the most common neglected tropical diseases, particularly in low-resource countries [1]. Parasitic nematodes of livestock and crops result in billions of dollars in losses each year [1]. Many nematode infections are treatable with low-cost anthelmintic drugs, but repeated infections are common in endemic areas and drug resistance is a growing concern with increasing therapeutic and agricultural administration [1]. Many parasitic nematodes have an environmental infective larval stage that engages in host seeking, a process whereby the infective larvae use sensory cues to search for hosts. Host seeking is a complex behavior that involves multiple sensory modalities, including olfaction, gustation, thermosensation, and humidity sensation. As the initial step of the parasite-host interaction, host seeking could be a powerful target for preventative intervention. However, host-seeking behavior remains poorly understood. Here we review what is currently known about the host-seeking behaviors of different parasitic nematodes, including insect-parasitic nematodes, mammalian-parasitic nematodes, and plant-parasitic nematodes. We also discuss the neural bases of these behaviors.

Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are lethal parasites of insects that are of interest as models for understanding parasite-host interactions and as biocontrol agents for insect pests. EPNs harbor a bacterial endosymbiont in their gut that assists in insect killing. EPNs are capable of infecting and killing a wide range of insects, yet how the nematodes and their bacterial endosymbionts interact with the insect immune system is poorly understood. Here, we develop a versatile model system for understanding the insect immune response to parasitic nematode infection that consists of seven species of EPNs as model parasites and five species of Drosophila fruit flies as model hosts. We show that the EPN Steinernema carpocapsae, which is widely used for insect control, is capable of infecting and killing D. melanogaster larvae. S. carpocapsae is associated with the bacterium Xenorhabdus nematophila, and we show that X. nematophila induces expression of a subset of antimicrobial peptide genes and suppresses the melanization response to the nematode. We further show that EPNs vary in their virulence toward D. melanogaster and that Drosophila species vary in their susceptibilities to EPN infection. Differences in virulence among different EPN-host combinations result from differences in both rates of infection and rates of postinfection survival. Our results establish a powerful model system for understanding mechanisms of host-parasite interactions and the insect immune response to parasitic nematode infection.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are responsible for some of the most common neglected tropical diseases. The infective larvae of skin-penetrating nematodes are thought to search for hosts using sensory cues, yet their host-seeking behavior is poorly understood. We conducted an in-depth analysis of host seeking in the skin-penetrating human parasite Strongyloides stercoralis, and compared its behavior to that of other parasitic nematodes. We found that Str. stercoralis is highly mobile relative to other parasitic nematodes and uses a cruising strategy for finding hosts. Str. stercoralis shows robust attraction to a diverse array of human skin and sweat odorants, most of which are known mosquito attractants. Olfactory preferences of Str. stercoralis vary across life stages, suggesting a mechanism by which host seeking is limited to infective larvae. A comparison of odor-driven behavior in Str. stercoralis and six other nematode species revealed that parasite olfactory preferences reflect host specificity rather than phylogeny, suggesting an important role for olfaction in host selection. Our results may enable the development of new strategies for combating harmful nematode infections.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'. [ABSTRACT FROM AUTHOR]

Advances in the functional genomics and bioinformatics toolkits for Strongyloides species have positioned these species as genetically tractable model systems for gastrointestinal parasitic nematodes. As community interest in mechanistic studies of Strongyloides species continues to grow, publicly accessible reference genomes and associated genome annotations are critical resources for researchers. Genome annotations for multiple Strongyloides species are broadly available via the WormBase and WormBase ParaSite online repositories. However, a recent phylogenetic analysis of the receptor-type guanylate cyclase (rGC) gene family in two Strongyloides species highlights the potential for errors in a large percentage of current Strongyloides gene models. Here, we present three examples of gene annotation updates within the Strongyloides rGC gene family; each example illustrates a type of error that may occur frequently within the annotation data for Strongyloides genomes. We also extend our analysis to 405 previously curated Strongyloides genes to confirm that gene model errors are found at high rates across gene families. Finally, we introduce a standard manual curation workflow for assessing gene annotation quality and generating corrections, and we discuss how it may be used to facilitate community-driven curation of parasitic nematode biodata. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'. [ABSTRACT FROM AUTHOR]