Investigating the Heritability of a Learned Pathogen Avoidance Behavior in Caenorhabditis elegans
Authors: Meissner Laura E , Manilla Gabriella A , Brown Taylor G , Youngman Elaine M 
Published: November 2023

In defiance of the laws of traditional Mendelian inheritance, multigeneration epigenetic inheritance of changes in gene expression in response to the experience of parental generations has been widely observed in both plants and animals. The roundworm Caenorhabditis elegans (C. elegans) has emerged as a premier model system for the study of transgenerational epigenetic inheritance, and heritable, epigenetic effects on gene expression have been seen in response to a variety of environmental conditions. It has been hypothesized that worms might be particularly adept at epigenetic inheritance of acquired traits due to their abundant use of small RNA silencing pathways, which have been shown to mediate gene silencing phenomena that can be epigenetically inherited for tens of generations. Here we investigate whether a well‐studied learned behavior in the worm can be inherited, and whether small RNA‐mediated gene silencing plays a role in mediating learningC. elegans that have been infected by the pathogenic soil bacterium Pseudomonas aeruginosa (P. aeruginosa, strain PA14) learn to avoid this food source through a mechanism involving alterations in serotonin expression in specific chemosensory neurons. In this associative learning regimen, worms are trained on plates that contain both pathogenic PA14 and nonpathogenic Escherichia coli (E. coli). At adulthood, worms are assayed for their preference between PA14 and E. coli using a standard behavioral choice assay. Similar to what has been seen previously, we find that only 29% of trained worms choose PA14 over E. coli, whereas naïve worms (which have never been exposed to PA14) choose PA14 37% of the time (p<.01, one‐tailed t test). To determine whether this learned avoidance behavior can be inherited by subsequent generations, the F1 progeny of trained worms were raised on plates that contained only E. coli and tested in the choice assay at adulthood. 41% of these worms were found in the PA14 spot, which was not significantly different from naïve worms (p=0.21), suggesting that learned pathogen avoidance cannot be epigenetically inherited. Some previously reported cases of siRNA‐mediated epigenetic inheritance in worms required that animals be exposed to the initial silencing trigger for several generations before epigenetic inheritance was established. To ask whether multi‐generation infection to pathogen could result in inheritance of avoidance behavior, synchronized populations of worms were continuously grown in the presence of pathogen for 3, 5, and 7 generations. When we performed learning assays on F1 progeny of the third, fifth, and seventh generation trained worms, we again saw a lack of inheritance of pathogen avoidance. To test the hypothesis that siRNAs play a role in the pathogen avoidance behavior, we are performing learning assays on strains of C. elegans containing mutations that disrupt siRNA pathways. In addition, we are using immunofluorescence for serotonin and mRNA‐seq for genome‐wide analysis to ask whether sub‐phenotypic changes in gene expression can be inherited even in the absence of inheritance of the avoidance behavior. Although other studies have examined epigenetic inheritance of learned behaviors or changes in gene expression, few studies have examined both. Taken together, our experiments will address this important gap between gene expression and phenotype.