A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

The entomopathogenic nematode Heterorhabditis bacteriophora is a lethal parasite of insects that engages in a symbiotic relationship with a bacterial partner, Photorhabdus luminescens, which it uses to kill insect hosts.1-3 Small-molecule cues are critical mediators of the interactions between nematode, bacterium, and insect in this tripartite system. In the soil, the nematode survives as a developmentally arrested infective juvenile (IJ) that seeks out an insect host based on chemical cues that the insect produces.4 Once the nematode enters the insect host, it responds to chemical cues in the insect’s hemolymph by regurgitating the symbiotic bacteria that it stores in its gut.2 The bacterial pathogens multiply inside the insect, disabling the insect’s immune system and producing antibiotics that prevent other microorganisms from proliferating on the insect’s carcass.5 In addition, the bacteria also produce small-molecule signals that induce the IJs to develop into adults and transition into the reproductive phase of their life cycle.6-9 Ultimately, once the insect has been consumed by the bacteria and nematodes feeding off of it, IJs accumulate inside the insect and then disperse to seek out new insect prey. Initially, after IJs infect an insect host and recover to the adult stage, the adults lay eggs that develop through four larval stages (J1-J4) to the next generation of adults (Figure 1A). After one or two rounds of egg laying, however, the adults begin to retain eggs inside their body cavity.1 After these retained eggs hatch, the resulting juveniles digest maternal tissues and develop into IJs. This process, known as endotokia matricida, leads to maternal death, followed by emergence of the IJs. Low food availability within the maternal uterus is thought to trigger egg retention and IJ formation.10 Endotokia matricida may facilitate transmission of P. luminescens to the gut of the IJs before they leave the insect host.1 Although IJs can form via endotokia matricida at both low and high nematode densities, at low nematode densities the IJs will recover to the J4 stage, but at high nematode densities the IJs will accumulate.1 Figure 1 The life cycles of H. bacteriophora and C. elegans. (A) Inside an insect, H. bacteriophora undergoes reproductive growth, progressing through four larval stages (J1–J4) to the adult stage. IJs form in large numbers via endotokia matricida and ... The IJ stage of entomopathogenic nematodes is similar to the dauer stage of Caenorhabditis elegans (Figure 1B), in that it is developmentally arrested, non-feeding, and specialized for dispersal. The IJ stage of H. bacteriophora enables the worm to survive in the environment as it seeks out a new insect host. Similarly, the dauer stage of C. elegans enables the worm to survive in the environment as it seeks out a bacteria-rich food source, such as a rotting piece of fruit.11 In C. elegans, dauer development is controlled by the dauer pheromone, which the nematode secretes into its environment during reproductive growth and uses to sense its population density. The active components of the dauer pheromone, which include several derivatives of the 3,6-dideoxysugar ascarylose with different fatty acid-like side chains (Figure 1C), promote dauer formation and inhibit dauer recovery.12-15 The dauer pheromone targets G protein-coupled receptors on exposed chemosensory neurons in the head of the worm and suppresses signaling by the insulin / insulin-like growth factor-1 (IGF-1) and TGFβ neuroendocrine pathways.16-18 Unlike the free-living C. elegans, H. bacteriophora is an obligate parasite in nature and has many parasitic adaptations, including a buccal tooth to slice into hosts.3 H. bacteriophora is closely related to the strongylids, vertebrate parasites which include human hookworms.19 The IJ larval stage is similar to the infective L3 (iL3) larval stage of animal-parasitic nematodes in that it is required for successful infection and in that it resumes reproductive development in response to host cues.20-22 Furthermore, there is conservation in the neurons and signaling pathways that control IJ recovery in H. bacteriophora23 and iL3 recovery in animal-parasitic nematodes22, 24-27. Importantly, H. bacteriophora can be cultivated in the laboratory outside of its host on a lawn of bacterial symbionts, making it much easier to manipulate than an animal-parasitic nematode. The genome sequence of H. bacteriophora, as well as that of P. luminescens, is publicly available, and RNA interference, which has been an important technique for examining gene function in C. elegans, can also be used to study signaling pathways in H. bacteriophora.3, 28 Here, we show that H. bacteriophora secretes a pheromone that prevents IJ recovery to the J4 stage. Using activity-guided fractionation and NMR-based structure elucidation, we identify the chemical structure of the pheromone as a structurally novel ascaroside. Other ascarosides, including the dauer pheromone ascarosides from C. elegans, have much less activity in H. bacteriophora, suggesting that there is a high degree of structural specificity. Once the nematodes reach a high density inside an insect host, the IJ pheromone may facilitate the accumulation of IJs by preventing IJ recovery.