Multicellular organisms have evolve powerful controlling mechanisms to cope with diverse perturbations in order to assure phenotypically quasi-invariant developmental outcome 1 . However, development can be influenced by environmental stimuli, e.g., temperature, nutrients and oxygen availability, and the presence of predators or parasites. The exposure to stresses during development can be deleterious or beneficial to the adults. For example, changes during development influence adulthood disease propensity in humans 2 . The adult Caenorhabditis elegans that transiently passed through the stress-induced dauer larval stage exhibit an extended lifespan compared to animals with normal development 3 . Environmental conditions, i.e., temperature, and oxygen and carbon dioxide concentrations 4 5 , were shown to direct the development of nematode Strongyloides ratti to distinct parasitic or free-living adults whose aging rates can differ by 30-fold 6 . Biotic factors can also induce dramatic developmental alterations. For example, in response to waterborne cues from predators, some Daphnia species grow protective helmets 7 . Microorganisms, which are a major environmental component, also have an important impact on development. The effect of microorganisms on development was established by comparing axenic animals against animals colonized with microbes. In fruit fly and zebrafish, axenic development can result in slower development, reduced fecundity, and/or early death 8 9 . Axenic C. elegans have a slower development, reduced fecundity, and increased lifespan 10 . In mammals, microorganisms can affect the maturation of the mucosal immune system 11 , modulate the proliferation and the differentiation of intestinal epithelia 12 , regulate angiogenesis 13 , and play a key role in the extraction and the processing of nutrients 14 .

Microorganisms are classified as commensals when they are innocuous and/or beneficial for the host, and pathogens when they are harmful to the host 15 . It should be noted that pathogens most frequently do not kill their hosts, but decrease host fitness and increase host susceptibility to environmental challenges. Adult hosts mount different responses to various types of microorganisms, which have a profound effect on the hosts themselves. While it is clear that presence or absence of microorganisms can have an important impact on development, how exposure to different types of microorganism, i.e. innocuous and pathogenic, during development affects adult animals health, stress resistance and longevity is unknown. Thus, in this study we examined the consequences of exposure of C. elegans nematodes during development to different bacterial strains. C. elegans was chosen because it is a well-studied animal model for aging, development, and host-pathogen interactions, and isogenic populations can be generated and maintained. The bacterium Escherichia coli was chosen because it is a well-studied model organism and a species with a wide virulence spectrum, ranging from innocuous to highly pathogenic in diverse hosts. It is also of particular importance that the standard laboratory food for C. elegans is one E. coli strain, OP50 16 17 . Survival/longevity of nematodes fed with OP50 is considered as the baseline for all C. elegans studies. E. coli strains are among the first colonizers of the intestinal tract of newborn warm-blooded animals. E. coli strains are mainly harmless commensals, but some strains are able to cause intestinal and extra-intestinal diseases, which by their frequency and potential severity cause considerable morbidity and significant health-care costs 18 . We previously demonstrated that E. coli pathogenic strains can significantly reduce the lifespan of the adult C. elegans 19 20 , and that E. coli pathogenicity factors are responsible for the observed lifespan reduction.

For this study, C. elegans nematodes were fed on different bacterial strains during development and then with the same or different bacteria as adults. We found that nematodes developed on a pathogen lived longer when exposed to pathogens as adults than nematodes that were developed on an innocuous strain. Some of the findings observed with the pathogenic E. coli were confirmed using a second pathogen, a Gram-positive Enterococcus faecalis. The observed increase in nematode lifespans is controlled by the DBL-1 transforming growth factor beta-like, DAF-2/DAF-16 insulin-like signaling, and partially by the PMK-1 p38 MAP kinase pathways. Developmental exposure to one pathogen increases resistance of adult nematodes to the same and other pathogens, as well as to heat shock. Hence, development on pathogens has a hormetic effect on adult nematodes. Hormesis is a response of organisms to low doses of a damaging environmental factor resulting in resistance to higher doses of the same, but also to other stressful environmental factors 21 22 . The possible consequences of the observed hormetic effect in natural environments are also discussed.

For this study, we used the reference OP50 and pathogenic 536 E. coli strains, as well as Enterococcus faecalis OG1RF strain. The innocuity of the OP50 strain was attested by observation that the mean lifespan of the C. elegans fer-15 nematodes was the same on the live and UV-killed OP50 bacteria 20 . fer-15 is a derivative of the C. elegans N2 strain, which we used in the present study. Strain 536 is an uropathogenic E. coli isolated from a patient with acute pyelonephritis 23 . Pathogenicity of the 536 strain was previously demonstrated in the mice sepsis model and in C. elegans 19 . Adult fer-15 nematodes exposed to strain 536 have a 50% shorter mean lifespan compared to nematodes exposed to the OP50 strain 19 . Deletion of pathogenicity islands II and III significantly reduces the pathogenicity of strain 536 in C. elegans fer-15 19 , while it does not reduce the bacterial growth rates nor resistance to biologically relevant stressors 24 . This shows that strain 536 reduces C. elegans lifespan because of its pathogenicity factors and not because it does not provide an adequate food source.

N2 nematodes were developed on one bacterial strain and late L4 stage nematodes were subsequently maintained on the same strain or transferred to a different one. For this reason, the designation of each experimental condition contains: (i) the name of the strain on which larvae were developed and (ii) the name of the strain to which adult nematodes were exposed. For example, OP50/536 indicates that nematodes were developed on OP50 and then transferred onto strain 536. All experiments were performed at 25°C.

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 33 . 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 34 . 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 35 . 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 37 38 . For example, nematodes are repulsed by a cyclic lipodepsipentapeptide produced by Serratia marcescens, a bacterium that is harmful to nematodes 39 . 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 37 . 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 40 . 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 43 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 46 . There are many other examples of the correlation between increased stress resistance and longevity in nematodes 47 . For example, the DAF-2/DAF-16 insulin-like pathway mediates stress resistance, longevity and pathogen resistance in C. elegans 48 . 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 49 . 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 51 . 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 54 . In some cases, immune priming can even be trans-generational, as observed for the mealworm beetle Tenebrio molitor 55 . It was shown that the presence of bacteria during the first week of the Drosophila melanogaster adult life increases the flies lifespan 56 . 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 57 . 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 58 . 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 59 . Epidemiologic studies also show an increased occurrence of autoimmune diseases in human populations in “clean” environments 60 . 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 61 . 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 3 . In C. elegans, the molecular basis of such life-long memory of modifications of developmental transcription profiles is mediated by histone modifications 3 . 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 62 , and C. elegans 52 , 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.

Our study shows that development on pathogens has a hormetic effect on adult nematodes, as it results in increased resistance to different pathogens and to heat shock. Such developmental plasticity of C. elegans nematodes, which are self-fertilizing homozygous animals producing offspring with negligible genetic variation, could increase the probability of survival in changing environments.

The authors declare that they have no competing interests.

ML, TM, XM and IM designed experiments, analyzed and interpreted data, and drafted the manuscript. ML, TM, XM and DFA performed experiments. All authors read and approved the final manuscript.