A sensory code for host seeking in parasitic nematodes

Abstract

Parasitic nematode species often display highly specialized host-seeking behaviors that reflect their specific host preferences. Many such behaviors are triggered by host odors, but little is known about either the specific olfactory cues that trigger these behaviors or the underlying neural circuits. Heterorhabditis bacteriophora and Steinernema carpocapsae are phylogenetically distant insect-parasitic nematodes whose host-seeking and host-invasion behavior resembles that of some devastating human- and plant-parasitic nematodes. We compare the olfactory responses of Heterorhabditis and Steinernema infective juveniles (IJs) to those of Caenorhabditis elegans dauers, which are analogous life stages. The broad host range of these parasites results from their ability to respond to the universally produced signal carbon dioxide (CO(2)), as well as a wide array of odors, including host-specific odors that we identified using thermal desorption-gas chromatography-mass spectroscopy. We find that CO(2) is attractive for the parasitic IJs and C. elegans dauers despite being repulsive for C. elegans adults, and we identify a sensory neuron that mediates CO(2) response in both parasitic and free-living species, regardless of whether CO(2) is attractive or repulsive. The parasites' odor response profiles are more similar to each other than to that of C. elegans despite their greater phylogenetic distance, likely reflecting evolutionary convergence to insect parasitism.

Figure 1. Life cycles of insect-parasitic nematodes

A–B. Photomicrographs of an H. bacteriophora (A) and an S. carpocapsae (B) infective juvenile (IJ). Both species harbor a bacterial symbiont – H. bacteriophora harbors Photorhabdus luminescens and S. carpocapsae harbors Xenorhabdus nematophila – in the gut during the IJ stage. Nomarski images are overlaid with epifluorescence images; bacterial symbiont is labeled with GFP. In both cases, the anterior end of the worm is at the top. C. The life cycle of insect-parasitic nematodes. The IJ stage is a developmentally-arrested third larval stage, and is the only free-living stage. IJs infect insect larvae by entering through a natural body opening, although H. bacteriophora can also penetrate directly through the larval cuticle. Following infection, IJs expel their symbiotic bacteria into the host, where it plays a critical role in overcoming the host immune system [6, 7]. The nematodes develop and reproduce inside the insect cadaver until the food is depleted, at which point new IJs form and disperse into the soil in search of new hosts [46]. D. Jumping by S. carpocapsae. Still images of a jumping IJ. A standing IJ (0.0 s) curls (1.4 s) into a lariat structure (2.0 s) and propels itself into the air (2.3 s). Jumping was observed on an agar surface sprinkled with sand. Red arrows indicate the jumping IJ; time is recorded in the lower right. A single jump can propel the nematode nine body lengths in distance and seven body lengths in height, and can be elicited by chemosensory and mechanical stimuli [47]. E–G. Representative photomicrographs illustrating the insect-parasitic lifestyle. E. A Steinernematid IJ jumped onto and attached to a katydid antenna. Arrowhead indicates attached IJ. F. A cricket (Acheta domesticus) cadaver infected with Steinernematids. Adult nematodes are visible beneath the cuticle throughout the cadaver; some of the most prominent nematodes are indicated by the arrowhead. G. IJs emerging from a depleted waxworm (Galleria mellonella) cadaver. Arrowhead indicates a clump of IJs; arrow indicates a single IJ. See also Figure S1 and Move S1.

Figure 2. BAG neurons are required for CO₂ response in free-living and parasitic nematodes

A. Parasitic IJs and C. elegans dauers are attracted to CO₂ in a chemotaxis assay (Figure S2A). n = 5–6 trials for each species. B. CO₂ induces jumping by S. carpocapsae in a jumping assay (Figure S2B). n = 4–11 trials. C–E. BAG neurons are required for CO₂ attraction in H. bacteriophora and S. carpocapsae IJs, and C. elegans dauers. n = 12–34 worms for each treatment (C–D) or n = 18–29 trials (E). The assay in E was a 10 min. assay, since the difference between wild-type and BAG- animals was apparent after only 10 min. F. BAG neurons are required for CO₂-evoked jumping by S. carpocapsae IJs. n = 10–18 worms for each treatment. ***, P<0.001; *, P<0.05, Fisher’s exact test (C, D, F) or unpaired t test (E). Error bars represent SEM. For C, D, and F, y-axis values represent the percentage of worms that yielded a positive behavioral response; error bars are not present because each worm was scored once individually. AWC chemosensory neurons were ablated as a control. 10% CO₂ was used for all experiments. See also Figure S2 and Movie S2.

Figure 3. BAG neurons are required for some but not all host-seeking behaviors

A. Volatiles released by live waxworms (Galleria mellonella), crickets (Acheta domesticus), mealworms (Tenebrio molitor), and superworms (Zophobas morio) attract the parasitic IJs but not C. elegans dauers. n = 6–27 trials. B. Insect volatiles also stimulate jumping by S. carpocapsae. n = 3–11 trials. **, P<0.01, one-way ANOVA with Dunnett’s post-test. For A–B, error bars represent SEM. C. BAG neurons are required for chemotaxis toward waxworms in H. bacteriophora. n = 10–38 worms for each treatment. **, P<0.01, Fisher’s exact test. D. BAG neurons are not required for jumping evoked by waxworm odors in S. carpocapsae. n = 20–39 worms for each treatment. No significant differences were observed between treatment groups. For C–D, values shown represent the percentage of worms that yielded a positive behavioral response; error bars are not present because each worm was scored once individually. AWC or ASI chemosensory neurons were ablated as controls. E–F. Attraction of H. bacteriophora (E) and S. carpocapsae (F) to G. mellonella is eliminated and A. domesticus is reduced when CO₂ is chemically removed from host headspace using soda lime. n = 6–14 trials for each treatment. ***, P<0.001; **, P<0.01; *, P<0.05, Mann-Whitney or unpaired t test (host vs. host + soda lime). See also Figure S3.

Figure 4. Odor response profiles of free-living and parasitic nematodes

A. Odor response profiles of C. elegans dauers, H. bacteriophora IJs, and S. carpocapsae IJs. n = 5–33 trials for each odorant. B. A comparison of odorant-evoked chemotaxis and jumping by S. carpocapsae. Both the chemotaxis index (C.I.) and the jumping index (J.I.) range from −1 to +1, with −1 indicating perfect repulsion and +1 indicating perfect attraction (Figures S2B and S4A). n = 5–8 trials for chemotaxis and 3–10 trials for jumping. Data for chemotaxis is from A. For A and B, response magnitudes are color-coded according to the scale shown to the right of each heat map, and odorants are ordered based on hierarchical cluster analysis. Host odorants identified by TD-GC-MS of insect headspace are highlighted in red. C. The odor response profiles of H. bacteriophora and S. carpocapsae are more similar to each other than to that of C. elegans, despite the fact that H. bacteriophora and C. elegans are more closely related phylogenetically. Left, behavioral dendrogram of olfactory responses across species. Behavioral distance is based on the Euclidian distances between species based on their odor response profiles. Right, phylogenetic neighbor-joining tree. Branch lengths in the phylogenetic tree are proportional to genetic distances between taxa; scale bar represents 0.02 nucleotide substitutions per site. See also Figure S4 and Table 1.