Independent Validation of Transgenerational Inheritance of Learned Pathogen Avoidance in Caenorhabditis elegans

Reviewer #1 (Public review):

Summary:

The manuscript addresses the discordant reports of the Murphy (Moore et al., 2019; Kaletsky et al., 2020; Sengupta et al., 2024) and Hunter (Gainey et al., 2025) groups on the existence (or robustness) of transgenerational epigenetic inheritance (TEI) controlling learned avoidance of C. elegans to Pseudomonas aeruginosa. Several papers from Colleen Murphy's group describe and characterize C. elegans transgenerational inheritance of avoidance behaviour. In the hands of the Murphy group, the learned avoidance is maintained for up to four generations, however, Gainey et al. (2025) reported an inability to observe inheritance of learned avoidance beyond the F1 generation. Of note, Gainey et al used a modified assay to measure avoidance, rather than the standard assay used by the Murphy lab. A response from the Murphy group suggested that procedural differences explained the inability of Gainey et al.(2025) to observe TEI. They found two sources of variability that could explain the discrepancy between studies: the modified avoidance assay and bacterial growth conditions (Kaletsky et al., 2025). The standard avoidance assay uses azide as a paralytic to capture worms in their initial decision, while the assay used by the Hunter group does not capture the worm's initial decision but rather uses cold to capture the location of the population at one point in time.

In this short report, Akinosho, Alexander, and colleagues provide independent validation of transgenerational epigenetic inheritance (TEI) of learned avoidance to P. aeruginosa as described by the Murphy group by demonstrating learned avoidance in the F2 generation. These experiments used the protocol described by the Murphy group, demonstrating reproducibility and robustness.

Strengths:

Despite the extensive analyses carried out by the Murphy lab, doubt may remain for those who have not read the publications or for those who are unfamiliar with the data, which is why this report from the Vidal-Gadea group is so important. The observation that learned avoidance was maintained in the F2 generation provides independent confirmation of transgenerational inheritance that is consistent with reports from the Murphy group. It is of note that Akinosho, Alexander et al. used the standard avoidance assay that incorporates azide, and followed the protocol described by the Murphy lab, demonstrating that the data from the Moore and Kaletsky publications are reproducible, in contrast to what has been asserted by the Hunter group.

Weaknesses:

While I would have liked to see a confirmation of the daf-7::GFP data in F2, and perhaps inheritance of avoidance beyond F2, the premise of the manuscript is that they have independently verified the transgenerational inheritance of learned avoidance as described by the Murphy lab, and this bar has been met.

Reviewer #2 (Public review):

Summary:

The manuscript "Independent validation of transgenerational inheritance of learned pathogen avoidance in C. elegans" by Akinosho and Vidal-Gadea offers evidence that learned avoidance of the pathogen PA14 can be inherited for at least two generations. In spite of initial preference for the pathogen when exposed in a 'training session', 24 hours of feeding on this pathogen evoked avoidance. The data are robust, replicated in 4 trials, and the authors note that diminished avoidance is inherited in generations F1 and F2.

Strengths:

These results contrast with those reported by Gainey et al, who only observed intergenerational inheritance for a single generation. Although the authors' study does not explain why Gainey et el fail to reproduce the Murphy lab results, one possibility is that a difference in a media ingredient could be responsible.

Weaknesses:

The authors do not list the sources of their media ingredients, which might be important with regard to reproducibility.

Reviewer #3 (Public review):

Summary

This short paper aims to provide an independent validation of the transgenerational inheritance of learned behaviour (avoidance) that has been published by the Murphy lab. The robustness of the phenotype has been questioned by the Hunter lab. In this paper, the authors present one figure showing that transgenerational inheritance can be replicated in their hands. Overall, it helps to shed some light on a controversial topic.

Strengths

The authors clearly outline their methods, particularly regarding the choice of assay, so that attempting to reproduce the results should be straightforward. It is nice to see these results repeated in an independent laboratory.

Weaknesses

Previous reports on this topic have provided raw data, which is helpful when assessing sample sizes. The authors provided a spreadsheet containing the choice assay results for individual assays, but not the raw data. In the methods, it is stated that F2 animals were produced from F1 animals by bleaching, but there are many more F2 assays than F1. Were multiple F2 assays performed on the offspring from one F1 plate? If so, they do not represent independent assays.

I think that the introduction somewhat overstates their findings - do they really "address potential methodological variations that might influence results"? This makes it sound as though they test different conditions, whereas they only use one assay setup throughout.