We showed that the development of nematodes on bacterial pathogens increases the lifespan of adult nematodes exposed to the same or different pathogens, and increases resistance of nematodes to heat shock. It is important to note that such increased robustness of nematodes developed on pathogens is not due to the culling of weak individuals from the population, as the same fraction of nematode larvae reached adulthood following development on pathogenic and innocuous strains. In addition, there was no difference in the lifespan of adult nematodes on the innocuous OP50 strain after they developed on OP50 or on the pathogenic 536 strain, which indicates that development on a pathogen does not damage larvae.
Hosts can defend themselves from pathogens using a variety of strategies . The avoidance strategy minimizes the risk of infection with pathogen and economizes the nematode’s resources, which otherwise would be used for the activation of its defenses . The avoidance strategy requires that host first detects pathogens. In nature, C. elegans is predominantly found in decaying organic matter where it feeds on microorganisms, which can be good or harmful food . C. elegans possesses a complex chemosensory neuronal system that allows discrimination between these two groups of microorganisms based on the detection of a number of volatile odorant and water-soluble compounds [36–38]. For example, nematodes are repulsed by a cyclic lipodepsipentapeptide produced by Serratia marcescens, a bacterium that is harmful to nematodes . C. elegans also has the capacity to identify pathogens and harmful xenobiotics by monitoring its essential cellular activities, such as protein translation or oxidative respiration. Perturbations of these functions induce aversion behavior and defense mechanisms . We did not study the molecular basis of the discrimination between different bacterial strains by nematodes. However, the fact that the observed hormetic effect is not bacterial species specific suggests that it is more likely induced by the nematode surveillance of its own essential cellular functions, rather than by recognition of the specific compounds produced by bacteria.
Nematodes also have a capacity to memorize, into adulthood, environmental cues encountered during early life . Can avoidance behavior based on memory of past conditions contribute to the longer lifespan of nematodes developed on a pathogen and exposed to the same pathogen as adults, compared to nematodes developed on an innocuous strain and exposed to pathogens as adults? This is probably not the case in our study because independently of the developmental conditions, nematodes have the same amount of the 536 pathogenic bacteria in their intestine, which indicates that 536/536 nematodes do not live longer than OP50/536 nematodes because they avoid pathogens and eat less. The fact that the intestinal pathogen burden is independent of developmental conditions also suggests that, in our case, the longer lifespan of nematodes developed on, and then exposed to, pathogens as adults were not the result of enhanced capacity to kill bacteria, but likely due to the increased tolerance to the damages inflicted by pathogenic bacteria. Tolerance is defined as the ability to limit negative impact on hosts by increasing cellular repair and maintenance capacity, without affecting pathogen burden [33, 41]. Increased investment in repair and maintenance function often comes with a cost for the host. In our study there is no evidence of such cost as, nematodes developed on pathogen and then transferred to non-stressful conditions, i.e., innocuous bacterial strain, have lifespans identical to the lifespan of nematodes exposed to the innocuous bacterial strain throughout life. However, it cannot be excluded that the cost of tolerance becomes evident under experimental conditions that are closer to those found in natural environments.
From a molecular perspective, increased tolerance to pathogens of the nematodes developed on pathogens could be, at least partially, due to the induction of the heat shock response. We observed that adult nematodes developed on pathogens induce higher levels of expression of the hsp-16.2 gene than nematodes developed on an innocuous strain. The hsp-16.2 gene expression was shown to be regulated by DAF-16 and HSF-1 transcription factors in the N2 nematodes [42–44]. It was previously observed that the expression level of hsp-16.2 in individual nematodes exposed to a mild heat shock was predictive both of thermotolerance and lifespan [32, 45]. Overproduction of HSP-16.2 also suppresses beta-amyloid peptide toxicity . There are many other examples of the correlation between increased stress resistance and longevity in nematodes . For example, the DAF-2/DAF-16 insulin-like pathway mediates stress resistance, longevity and pathogen resistance in C. elegans. This supports our hypothesis that the nematode’s response to developmental conditions observed in this study is genetically controlled by the DAF-2/DAF-16 insulin-like pathway. However, it was reported that daf-16 and dbl-1 mutants have an increased amount of live bacteria in their intestine compared to N2 nematodes when maintained on E. coli OP50 . On the contrary, a pmk-1 mutant has similar amount of live bacteria in its intestine when compared to that of N2 nematodes. Thus, the absence of increased survival for daf-16 and dbl-1 mutants could also be due to an increased intestinal bacterial load following development on the pathogen, counterbalancing the beneficial effect of early pathogen exposure. Further investigation of these signaling pathways will be necessary to determine the direct or indirect involvement of these pathways in the control of nematode response to developmental exposure to bacterial pathogens.
Observed effect of developmental conditions on the adult lifespan and stress resistance could be described as hormetic. Hormetic response in C. elegans, i.e., increased stress resistance and/or increased lifespan, was shown to be induced with sub-lethal abiotic stresses, like oxidative and thermal stress [47, 50]. However, all forms of stress, e.g. UV or ionizing radiation, do not produce hormetic effects in C. elegans. Our study shows that some biotic factors, i.e., bacterial pathogens, can be added to the list of known hormetic stressors. One possible explanation for the hormetic effects of exposure to low levels of stress is that the level of response to stress is more than necessary to repair the damage caused, thus increasing the nematode defense capacity and allowing tolerance to future stresses. A particularity of our study, contrary to other above-mentioned studies of the hormetic effect of different stressors on C. elegans, is that we induced hormetic effect by exposing nematodes to bacterial pathogens during development as stressors. We hypothesize that hormetic effects of early exposure to bacteria are common in nature as they were observed in a variety of organisms. For example, priming of innate immunity by pathogens has been reported for invertebrates such as C. elegans[52, 53] and insects . In some cases, immune priming can even be trans-generational, as observed for the mealworm beetle Tenebrio molitor. It was shown that the presence of bacteria during the first week of the Drosophila melanogaster adult life increases the flies lifespan . The early life interactions between human host and bacteria, which largely occur through the colonization of the newborn intestine, are also increasingly recognized as being critical for the maturation of human immune system as well as for the metabolic homeostasis of the host . Newborn babies whose intestinal colonization was modified by the mode of delivery (natural vs. Cesarean section) or by antibiotic treatments, show a delay in immune response maturation . Early exposure of children, up to five years of age, to non-septic bacteria-rich environments, has a significant protective effect against the onset of allergies . Epidemiologic studies also show an increased occurrence of autoimmune diseases in human populations in “clean” environments . These observations constitute the basis of the “hygiene hypothesis,” according to which the lack of exposure to microbes due to high hygienic conditions commonly found in the Western world prevents correct maturation of the immune system and predisposes individuals to allergies and other immune diseases. These observations also correspond to the programming concept, which refers to stimuli that during critical periods of development may “program” the long-term structure or function of an organism . Our data establish that, at least in the C. elegans model, intentional administration of particular bacterial strains during early life could indeed modulate disease susceptibility during adulthood.
Our study strongly suggests that variations induced during development are maintained in genetically identical adult nematodes, as it was previously reported for the nematodes that transiently passed through the stress-induced dauer larval stage . In C. elegans, the molecular basis of such life-long memory of modifications of developmental transcription profiles is mediated by histone modifications . Whether histone modifications are also responsible for the phenotypes we observed remains to be determined.
Although natural selection might favor improved sensing and response mechanisms, adaptation could also result in the emergence of more sophisticated response strategies. Developmental plasticity allows for the production of phenotypically diverse offspring in populations of genetically identical individuals, which increases the probability of survival in changing and challenging environments. The evolution of the ability to mount such predictive developmental modifications depends on a number of features, such as the cost of developmental plasticity, ability to correctly detect and interpret environmental cues that depends on the frequency by which various environmental conditions are encountered, and finally on the long term fitness advantage provided. Therefore, as described for E. coli plus Saccharomyces cerevisiae, and C. elegans, the observed phenomenon could extend beyond merely sensing and responding immediately to a given stimulus and could contribute to a predictive response strategy that uses the appearance of a stimulus as a cue that future conditions might be stressful.