Signaling by AWC Olfactory Neurons Is Necessary for Caenorhabditis elegans' Response to Prenol, an Odor Associated with Nematode-Infected Insects.

Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.

EPNs Exhibit Repulsion to Prenol in Pluronic Gel Assays.

Entomopathogenic nematodes (EPNs) are lethal parasites of insects that have become valuable in biological control and as a model system for studying host-parasite interactions, behavioral ecology, neurobiology, and genomics, among other fields. Their ability to locate hosts is paramount to successful infection and host seeking has been extensively studied in many species in the lab. Here, we explored the usefulness of pluronic gel as a medium to assess EPN host seeking in the lab by characterizing the response of Steinernema carpocapsae, S. feltiae, S. glaseri, S. riobrave, Heterorhabditis bacteriophora, and H. indica to the odor prenol. We found that the infective juveniles (IJs) of these species were repelled by prenol in pluronic gel. We then evaluated how storing the IJs of S. carpocapsae, S. feltiae, and S. glaseri for different amounts of time affected their behavioral responses to prenol. The response of S. carpocapsae was significantly affected by the storage time, while the responses of S. feltiae and S. glaseri were unaffected. Our data support the notion that pluronic gel is a useful medium for studying EPN behavior and that the response of S. carpocapsae to informative odors is significantly affected by long-term storage.

Touch-stimulation increases host-seeking behavior in Steinernema Carpocapsae.

Previous research demonstrated that Steinernema carpocapsae infective juveniles (IJs) exposed to a host cuticle were more attracted toward certain host-associated volatile odors. We wanted to test the specificity of attraction that results from exposure to host cuticle. Host recognition behavior was analyzed after stimulating IJs by allowing them to physically interact with Galleria mellonella cuticles. The subsequent behavioral response and the proportion of the population participating in chemotaxis to multiple host odors were measured. We found that exposure to host cuticles resulted in a significantly higher percentage of the population participating in host-seeking behavior, with threefold more nematodes participating in chemotaxis. We tested whether exposure to live or dead host cuticle resulted in a different response and found that a higher percentage of IJs exposed to a live host cuticle participated in chemotaxis than IJs exposed to a dead host cuticle, but that IJs exposed to a dead host demonstrated significantly higher participation than was observed for non-stimulated IJs. To test whether the increase in IJ participation in host-seeking behaviors after exposure to a live host cuticle was specific, we exposed stimulated IJs to a known repulsive odor, a neutral odor, and two predicted attractants. We found that stimulation of IJs through physical contact with a host cuticle induces a specific enhancement of host-seeking behavior to host-specific odors rather than a general increased chemotactic response to all volatile stimuli. However, the nematodes displayed an enhanced response to multiple host-specific odors. Future work should focus on the mechanism through which contact with live host cuticle stimulates increased behavioral response.Previous research demonstrated that Steinernema carpocapsae infective juveniles (IJs) exposed to a host cuticle were more attracted toward certain host-associated volatile odors. We wanted to test the specificity of attraction that results from exposure to host cuticle. Host recognition behavior was analyzed after stimulating IJs by allowing them to physically interact with Galleria mellonella cuticles. The subsequent behavioral response and the proportion of the population participating in chemotaxis to multiple host odors were measured. We found that exposure to host cuticles resulted in a significantly higher percentage of the population participating in host-seeking behavior, with threefold more nematodes participating in chemotaxis. We tested whether exposure to live or dead host cuticle resulted in a different response and found that a higher percentage of IJs exposed to a live host cuticle participated in chemotaxis than IJs exposed to a dead host cuticle, but that IJs exposed to a dead host demonstrated significantly higher participation than was observed for non-stimulated IJs. To test whether the increase in IJ participation in host-seeking behaviors after exposure to a live host cuticle was specific, we exposed stimulated IJs to a known repulsive odor, a neutral odor, and two predicted attractants. We found that stimulation of IJs through physical contact with a host cuticle induces a specific enhancement of host-seeking behavior to host-specific odors rather than a general increased chemotactic response to all volatile stimuli. However, the nematodes displayed an enhanced response to multiple host-specific odors. Future work should focus on the mechanism through which contact with live host cuticle stimulates increased behavioral response.

Differentiating between scavengers and entomopathogenic nematodes: Which is Oscheius chongmingensis?

Entomopathogenic nematodes (EPNs) continue to be explored for their potential usefulness in biological control and pest management programs. As more insect-associated species of nematodes are discovered and described, it is possible that scavengers and kleptoparasites may be mischaracterized as EPNs. If a nematode species is truly an entomopathogen it should display similar infectivity, as well as behaviors and preferences, to those of established EPN species, such as Steinernema carpocapsae. In this study we evaluated dauers of the putative EPN species Oscheius chongmingensis. We examined virulence, odor preferences as a measure of host-seeking behavior, and features of its bacterial symbiont Serratia nematodiphila. We determined that O. chongmingensis behaves more like a scavenger than an EPN. Not only did O. chongmingensis exhibit very poor pathogenicity in Galleria mellonella (wax moth larvae), it also displayed odor (host-seeking) preferences that are contrary to the well-known EPN S. carpocapsae. We also found that the bacterial symbiont of O. chongmingensis was antagonistic to S. carpocapsae; S. carpocapsae IJs were unable to develop when S. nematodiphila was a primary food source. We conclude that there is insufficient evidence to support the characterization of O. chongmingensis as an EPN; and based on the attributes of its preferences for already-infected or deceased hosts, suggest that this nematode is a scavenger, which may be on an evolutionary trajectory leading to an entomopathogenic lifestyle.

Dispersal and Repulsion of Entomopathogenic Nematodes to Prenol.

Chemosensory cues are crucial for entomopathogenic nematodes (EPNs)-a guild of insect-killing parasitic nematodes that are used as biological control agents against a variety of agricultural pests. Dispersal is an essential element of the EPN life cycle in which newly developed infective juveniles (IJs) emerge and migrate away from a resource-depleted insect cadaver in order to search for new hosts. Emergence and dispersal are complex processes that involve biotic and abiotic factors, however, the elements that result in EPN dispersal behaviors have not been well-studied. Prenol is a simple isoprenoid and a natural alcohol found in association with EPN-infected, resource-depleted insect cadavers, and this odorant has been speculated to play a role in dispersal behavior in EPNs. This hypothesis was tested by evaluating the behavioral responses of five different species of EPNs to prenol both as a distal-chemotactic cue and as a dispersal cue. The results indicate that prenol acted as a repulsive agent for all five species tested, while only two species responded to prenol as a dispersal cue.

Host seeking parasitic nematodes use specific odors to assess host resources.

Entomopathogenic nematodes (EPNs) are insect parasites used as biological control agents. Free-living infective juveniles (IJs) of EPNs employ host-seeking behaviors to locate suitable hosts for infection. We found that EPNs can differentiate between naïve and infected hosts, and that host attractiveness changes over time in a species-specific manner. We used solid-phase microextraction and gas chromatography/mass spectrometry to identify volatile chemical cues that may relay information about a potential host's infection status and resource availability. Among the chemicals identified from the headspace of infected hosts, 3-Methyl-2-buten-1-ol (prenol) and 3-Hydroxy-2-butanone (AMC) were selected for further behavioral assays due to their temporal correlation with the behavioral changes of IJs towards the infected hosts. Both compounds were repulsive to IJs of Steinernema glaseri and S. riobrave in a dose-dependent manner when applied on an agar substrate. Furthermore, the repulsive effects of prenol were maintained when co-presented with the uninfected host odors, overriding attraction to uninfected hosts. Prenol was attractive to dauers of some free-living nematodes and insect larvae. These data suggest that host-associated chemical cues may have several implications in EPN biology, not only as signals for avoidance and dispersal of conspecifics, but also as attractants for new potential hosts.

Genomics of Entomopathogenic Nematodes and Implications for Pest Control.

Entomopathogenic nematodes (EPNs) have been used in biological control but improvement is needed to realize their full potential for broader application in agriculture. Some improvements have been gained through selective breeding and the isolation of additional species and populations. Having genomic sequences for at least six EPNs opens the possibility of genetic improvement, either by facilitating the selection of candidate genes for hypothesis-driven studies of gene-trait relations or by genomics-assisted breeding for desirable traits. However, the genomic data will be of limited use without a more mechanistic understanding of the genes underlying traits that are important for biological control. Additionally, molecular tools are required to fully translate the genomic resources into further functional studies and better biological control.