Interspecies relationships of wild amoebae and bacteria with C. elegans create environments propitious for multigenerational diapause.

The molecular and physical communication within the microworld supports the entire web of life as we know it. How organisms such as bacteria, amoeba and nematodes -all superabundant-interact to sustain their niche, however, is not known, especially how their associations generate and affect behavior of animals in fluctuating environments. To have a frame to study interactions between microbe and animal, we collected soil from a temperate semi-arid climate and isolated the culturable genus of bacteria Comamonas, Stenotrophomonas, Chryseobacterium and Rhodococcus and the amoeba Tetramitus. This ensemble was then fed in long-term experiments to the nematode C. elegans to study developmental rate, diapause entry, fertility, feeding behavior and neuronal integrity. We observed that the ensemble is long lasting and induces animals to diapause after a few generations under conditions that are not canonically pathogenic. We called this phenomenon Dauer Formation in Natural Ensembles (DaFNE). DaFNE requires the communication between live bacteria and the nematode intestine, suggesting the existence of a bidirectional interaction in the holobiont. While all bacteria from the ensemble colonize the intestine of the nematodes, Comamonas is the most represented and Rhodococcus the scarcest. The amoeba Tetramitus can be ingested by C. elegans, but it is not part of its microbiota. DaFNE depends on pheromone and nematode quorum, but high temperature in the homeostatic range, triggers diapause with fewer numbers. DaFNE increases as generations pass and is also remembered transgenerationally. The RNA interference (RNAi) pathway is needed for initiation of DaFNE, indicating the communication via RNA is crucial to execute bacterially induced behaviors in natural environments. SignificanceMicrobes have an overwhelming influence over the animals they live with, modulating development and decision making. Microscopic nematodes are the most abundant multicellular animals in the biosphere, suggesting they possess well-rehearsed successful relationships with their associated microbiota. Little is known about the modulation of nematode behavior in complex ecosystems with multiple organisms interacting. We use bacteria and amoeba from a natural ecosystem and introduce the pioneer nematode C. elegans to study behavioral parameters in long lasting experiments. The most striking response of nematodes to this natural environment is the commitment to diapause of a significant portion of the population. We call this form of hibernation Dauer Formation in Natural Ensembles or DaFNE. We propose that animals in nature may hibernate frequently, as a result of the communication with their natural biota. We find that DaFNE requires pheromone production in nematodes and also the RNA interference pathway, suggesting the RNA repertoire of both entities may be at play. Higher temperatures in the optimal range for nematode growth, require much less nematode quorum for DaFNE, indicating that a non-noxious increase in temperature favors diapause in natural environments. Nematodes respond to each bacterium in different ways when grown in monocultures and in the ensemble. This suggests that the abundance of specific species in nature may shift behavioral preferences and outputs in microscopic animals. We also show that the amoeba Tetramitus can be ingested by worms, demonstrating that C. elegans is a broader microbivore. Like worms, amoebae display specific responses to bacteria and add variability to behaviors elicited by nematodes. Finally, bacteria in the ensemble unlike in monocultures, are not exhausted during the length of the experiments even in the presence of bacterivore nematodes and amoebae.