Targeted mutagenesis in a human-parasitic nematode.

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

Morphogenesis of Strongyloides stercoralis Infective Larvae Requires the DAF-16 Ortholog FKTF-1

Based on metabolic and morphological similarities between infective third-stage larvae of parasitic nematodes and dauer larvae of Caenorhabditis elegans, it is hypothesized that similar genetic mechanisms control the development of these forms. In the parasite Strongyloides stercoralis, FKTF-1 is an ortholog of DAF-16, a forkhead transcription factor that regulates dauer larval development in C. elegans. Using transgenesis, we investigated the role of FKTF-1 in S. stercoralis' infective larval development. In first-stage larvae, GFP-tagged recombinant FKTF-1b localizes to the pharynx and hypodermis, tissues remodeled in infective larvae. Activating and inactivating mutations at predicted AKT phosphorylation sites on FKTF-1b give constitutive cytoplasmic and nuclear localization of the protein, respectively, indicating that its post-translational regulation is similar to other FOXO-class transcription factors. Mutant constructs designed to interfere with endogenous FKTF-1b function altered the intestinal and pharyngeal development of the larvae and resulted in some transgenic larvae failing to arrest in the infective stage. Our findings indicate that FKTF-1b is required for proper morphogenesis of S. stercoralis infective larvae and support the overall hypothesis of similar regulation of dauer development in C. elegans and the formation of infective larvae in parasitic nematodes

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection.

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

Experience-dependent olfactory behaviors of the parasitic nematode Heligmosomoides polygyrus.

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

Experience-dependent olfactory behaviors of the parasitic nematode <i>Heligmosomoides polygyrus</i>

<div><p>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 <i>Heligmosomoides polygyrus</i>. <i>H</i>. <i>polygyrus</i> 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 <i>H</i>. <i>polygyrus</i> 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, <i>H</i>. <i>polygyrus</i> iL3s are attracted to mammalian skin odorants, suggesting that they migrate toward hosts. The olfactory preferences of <i>H</i>. <i>polygyrus</i> 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 <i>Haemonchus contortus</i>, 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.</p></div

<i>H</i>. <i>polygyrus</i> iL3s are attracted to host feces.

<p><b>A</b>. <i>H</i>. <i>polygyrus</i> iL3s were attracted to mouse feces. By contrast, <i>S</i>. <i>ratti</i> and <i>S</i>. <i>stercoralis</i> iL3s were not attracted to the feces of their hosts (rat and dog, respectively). ***<i>p</i><0.001, Kruskal-Wallis test with Dunn’s post-test. n = 12–14 trials for each species. Data for <i>S</i>. <i>ratti</i> and <i>S</i>. <i>stercoralis</i> are from Castelletto <i>et al</i>., 2014 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006709#ppat.1006709.ref018" target="_blank">18</a>]. <b>B-C</b>. <i>H</i>. <i>polygyrus</i> iL3s respond differently to feces from different animals (<b>B</b>), and prefer mouse feces to gerbil or rabbit feces (<b>C</b>). Labels above and below each box in <b>C</b> indicate the opposing cues in the fecal preference assay. **<i>p</i><0.01, ***<i>p</i><0.001, Kruskal-Wallis test with Dunn’s post-test. n = 10–14 trials per condition. <b>D</b>. <i>H</i>. <i>polygyrus</i> iL3s prefer fresh mouse feces to aged mouse feces, but cannot distinguish mouse feces from infected animals versus uninfected animals. *<i>p</i><0.05, ***<i>p</i><0.001, Kruskal-Wallis test with Dunn’s post-test. n = 11–14 trials per condition. <b>E</b>. In a short-term dispersal assay, <i>H</i>. <i>polygyrus</i> iL3s leave feces to engage in host seeking. iL3s were placed on fresh mouse feces and allowed to crawl freely for 1 hour. The number of iL3s either on the feces, in zone 1, or in zone 2 (right) was then quantified. Approximately half of the iL3 population migrated off of the feces. n = 11 trials, with 15–40 iL3s per trial. <b>F</b>. In a long-term dispersal assay, nearly all <i>H</i>. <i>polygyrus</i> iL3s eventually left their original fecal pellet to engage in host seeking. The cumulative percentage of nematodes that had migrated off of their original fecal pellet was quantified each day over the course of 10 days. n = 13 trials. For all graphs, lines indicate medians and interquartile ranges.</p

Navigational strategies of <i>H</i>. <i>polygyrus</i> in comparison to those of <i>S</i>. <i>ratti</i> and <i>S</i>. <i>stercoralis</i>.

<p><b>A</b>. Dispersal behavior across species. iL3s were placed at the center of an agar plate and allowed to crawl freely for 1 hour in the absence of applied sensory stimuli. The percentage of iL3s in the outer zone, defined as the region of the plate outside a 4-cm-diameter circle (right), was determined. <i>H</i>. <i>polygyrus</i> and <i>S</i>. <i>ratti</i> iL3s dispersed to a similar extent, while <i>S</i>. <i>stercoralis</i> iL3s dispersed to a greater extent. ***<i>p</i><0.001, Kruskal-Wallis test with Dunn’s post-test. n = 9–11 trials for each species and condition. <b>B</b>. Crawling speed across species. <i>H</i>. <i>polygyrus</i> iL3s crawled more slowly than <i>S</i>. <i>ratti</i> and <i>S</i>. <i>stercoralis</i> iL3s. ***<i>p</i><0.001, one-way ANOVA with Holm-Sidak’s post-test. n = 23–31 iL3s per species. For <b>A</b>-<b>B</b>, graphs show medians and interquartile ranges. <b>C</b>. Nictation frequencies were similar across species (<i>p</i> = 0.65, chi-square test). n = 22–70 iL3s per species. Data for <i>S</i>. <i>ratti</i> and <i>S</i>. <i>stercoralis</i> are from Castelletto <i>et al</i>., 2014 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006709#ppat.1006709.ref018" target="_blank">18</a>].</p