Absence of Evidence is Not Evidence of Absence: The many flaws in the case against transgenerational epigenetic inheritance of pathogen avoidance in C. elegans

After examining the data and methods presented in Gainey, et al., bioRxiv, 20241 we conclude that the authors did not use an experimental paradigm that would have allowed them to replicate our results on transgenerational epigenetic inheritance (TEI) of learned avoidance that we regularly observe2-5. That is, we agree with the authors that their experiments show no evidence of TEI. However, there are substantial differences in their execution of every step of the work that also make it impossible for the authors to claim that they are replicating our experiments or protocols. Based on these differences, we do not believe there is an issue of "robustness" and "reliability" in our TEI findings, but rather that Hunter and colleagues have in fact not tested the central condition of TEI - that is, small RNA production by bacteria and subsequent uptake by C. elegans - nor have they carried out proper behavioral and imaging assays to assess this behavior. Our subsequent work shows that indeed this example of transgenerational epigenetic inheritance is not just observed in laboratory settings with PA14, but is also induced by wild strains of Pseudomonas, exhibiting its robustness. Just as we offered advice and training to Hunter and colleagues, we are happy to advise anyone who wishes to learn this assay. We have now performed the experiments in the absence of azide, a deviation from our protocol that Hunter and colleagues deliberately made, and found that this omission may account for most if not all of the differences from our results. It is disingenuous for the authors to have presented their work as if they have used our protocol, given the fact that they chose to not use the same conditions for the most basic assay used in the work, the chemotaxis assay, which would have been necessary to replicate in order to reproduce our work. Therefore, Gainey et al.s claims that our protocol or results are "irreproducible" are not supported by their evidence.