Studies by a Princeton University-led team have shown how the tiny roundworm Caenorhabditis elegans can transmit complex learned avoidance behaviors to its offspring, giving the young an inherited mechanism for surviving dangerous conditions. The research, reported in Cell, showed how C. elegans worms that become ill from ingesting pathogenic Pseudomonas aeruginosa (known as P14) quickly learned to avoid the bacterium, and could also pass this aversive behavior on to their offspring through a process known as transgenerational epigenetic inheritance (TEI), but only for four generations. Princeton researchers Rebecca Moore, PhD, Rachel Kaletsky, PhD, and Coleen Murphy, PhD, report their findings in a paper titled, “Piwi/PRG-1 Argonaute and TGF-β Mediate Transgenerational Learned Pathogenic Avoidance.”
Some of an organism’s features are determined wholly by its genes, which are inherited from the parents and passed down through the germline—eggs and sperm. Other characteristics are influenced by both the DNA variations hard-wired into the organism’s genetic code, and by environmental factors, such as nutrition, environmental stress, or temperature, which can affect the expression levels of genes. C. elegans, for example, will adapt to conditions of starvation or heat stress by varying the expression of multiple genes. Such epigenetic alterations can be made at the level of the genome by mechanisms including changes to DNA packing and so accessibility, or by destroying RNA transcripts that act to translate gene sequences into protein templates. If epigenetic alterations are made at the level of the germline they can be passed down to future generations through the process of TEI.
Research has already shown that C. elegans’ adaptations to heat stress or starvation can be passed down for multiple generations but these are physiological responses. As the authors wrote, “ … it is not known whether complex neurological behaviors, such as learning and memory, can be inherited across generations.” To investigate this more closely the Princeton team focused on the ability of C. elegans to learn which bacterial food sources are unsafe. The roundworm must be able to distinguish between nutritious and harmful bacteria in its natural environment. In fact, about 30% of the organism’s natural microbial environment is composed of Pseudomonas species, the authors noted, some of which are pathogenic. Although P. aeruginosa P14 will kill C. elegans within hours or days of exposure, the roundworm is initially attracted to P14, but then quickly learns to avoid the bacterium. “In their natural environment, worms come into contact with many different bacterial species,” said Murphy, who is a professor in Princeton’s department of molecular biology and the Lewis-Sigler Institute for Integrative Genomics. “Some of these are nutritious food sources, while others will infect and kill them. Worms are initially attracted to the pathogen Pseudomonas aeruginosa, but upon infection, they learn to avoid it. Otherwise they will die within a few days.”
The investigators’ studies showed that this learned avoidance behavior can be passed from parent C. elegans to offspring, but only if the adult worms had actually been infected by the P14 pathogen. Exposure to odors released by the pathogen wasn’t enough. “ … pathogenic learning and transgenerational inheritance of pathogen avoidance require both physical contact and infection with PA14,” the investigators wrote. Further experiments indicated that the ability to pass down P14 avoidance behavior to progeny could be effected through eggs or sperm.
Interestingly, TEI of pathogen avoidance persisted through four generations of offspring, and no further. Fifth generation progeny reverted to naïve behavior, and rather than avoiding the P14 P. aeruginosa pathogen, were attracted to the bacterium.
The studies also showed that TEI of avoidance behavior was specific to P14, rather than operating as a universal response to pathogen exposure. C. elegans worms could also learn to avoid another, but less common pathogenic bacterium, Serratia marcescens, but couldn’t pass this avoidance behavior down to their offspring.
Through further experiments the investigators discovered that elevated expression of the TGF-β ligand daf -7 in the P14-exposed worms was required for their progeny to inherit pathogen aversion. They found that daf-7 expression in a type of sensory neuron known as ASI neurons correlated strongly with the inherited avoidance behavior. “The process of inheriting this learned avoidance [also] requires the activity of small RNAs called piRNA,” Murphy said.
piRNAs have been implicated in other TEI pathways in C. elegans, where they are thought to silence gene expression and indirectly regulate DNA packing. The researchers found that the piRNA-associated protein PRG-1, while not necessary for the C. elegans adults to learn avoidance of P. aeruginosa, was required for increased daf-7 expression in progeny, and for their inherited avoidance behavior. Whether piRNAs and PRG-1 operate primarily in the adult, the progeny, or in both to promote inheritance of avoidance behavior isn’t understood. Importantly, though, expression of daf-7 stayed high in the ASI neurons of progeny for four generations, then dropped back to basal levels in the fifth generation, which is when the inherited avoidance behavior also disappeared.
So, if avoidance behavior gives offspring such survival advantage, “ … why is this behavior not hard-wired, and what is the advantage of losing the TEI avoidance behavior after several generations?” the authors questioned. Given that the Pseudomonas genus, which includes several nonpathogenic species, makes up perhaps a third of C. elegans’ natural environment, the authors queried whether it might, in fact, be beneficial for the progeny of PA14-exposed parents to reacquire PA14 attraction later at a later stage.
To try and answer this, Moore et al., studied behavioral responses to PA14 grown at different temperatures, which affect bacterial virulence. They discovered that while PA14 is pathogenic to C. elegans when grown at relatively high temperatures of 25˚C, when that bacterium was grown at lower temperatures it was safer to eat, and could possibly represent a valuable food source. In effect, “forgetting” the learned avoidance after a few generations may be necessary to allow the worms to become reattracted to nutritious, non-pathogenic Pseudomonas when environmental conditions change, the team concluded. “… naïve avoidance and indefinite transgenerational inheritance of avoidance of all Pseudomonas species might be a poor long-term strategy that could deprive animals of adequate nutrition.”