Genome-wide association and environmental suppression of the mortal germline phenotype of wild C. elegans

  1. Lise Frézal
  2. Marie Saglio
  3. Gaotian Zhang
  4. Luke Noble
  5. Aurélien Richaud
  6. Marie-Anne Félix

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Abstract

The animal germline lineage needs to be maintained along generations. However, some Caenorhabditis elegans wild isolates display a mortal germline phenotype, whereby the lineage becomes sterile after several generations at 25°C. We used a genome-wide association approach to study the genetic basis for this phenotype in C. elegans populations. We detected a significant peak on chromosome III around 5 Mb, which was confirmed using introgression lines. These results indicate that a seemingly deleterious genotype is maintained at intermediate frequency in the species. Environmental rescue is a likely explanation and we indeed find that naturally associated bacteria and microsporidia suppressed the phenotype. The tested bacteria also suppressed the temperature-sensitive mortal germline phenotype of mutants in small RNA inheritance ( nrde-2 ) and histone modifications ( set-2 ). Even Escherichia coli strains of the K-12 lineage suppressed the phenotype compared to B strains. By shifting a strain cultured on E. coli K-12 back to E. coli B, we found that C. elegans can keep over several generations the memory of the suppressing conditions. Thus, the mortal germline phenotype of wild C. elegans is lin part revealed by laboratory conditions and may represent variation in epigenetic inheritance and environmental interactions. This study also points to the importance of non-genetic memory in the face of environmental variation.

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    Reply to the reviewers

    We thank the reviewers for their time, the positive reviews and the useful comments. We answer below and explain the changes made to the manuscript. The comments of the reviewers are in italics.

    Reviewer #1

    1. 'For GWAS, the strains that were fertile after 20 generations were considered non-Mrt.' One aspect of Fig 1D that could be clarified are the dots at generation 21. If these represent strains that were always fertile at generation 21, then perhaps give these a different color to indicate that sterility was never observed?

    Response: This is a good idea. We added colors in Figure 1, which makes it clearer.

    We also provide a different color for surviving replicates in all relevant figures.

    1. 'The mean Mrt values of strains ranged from sterile at 3 generations to fertile after 20 generations at 25°C, with a skewed distribution toward high values (Figure 1B).' Based on Table S2, part of the explanation for this skewed distribution in later generations is that some strains became sterile rapidly for some blocks, whereas the same strain did not become sterile in other blocks. For example, JU1200, JU360, PB303. I suggest providing a second color for Fig. 1D for strains that sometimes displayed sterility and sometimes did not.

    __Response: __We now colored the isolates that never became sterile, with the same color code as in panel B. Because we stopped the scoring at G20 and code fertility at G20 as '21', those with a mean below 21 show some sterility in at least one case.

    Because the number of generations at which we stopped the phenotyping (20) is arbitrary, the fact a line stayed fertile at 20 generations in one replicate is not very meaningful, especially considering that the number of replicates is not the same for all strains. The key point of the variance graph is to show that the strains with the most variance are those with high but

    For those that were sometimes fertile and sometimes sterile, I suggest creating a graph in Figure 1 that shows generations at sterility or lack of sterility, color coded by block. This will allow the significance of strains with high generation Mrt values to be better appreciated for readers who do not look at the supplementary table.

    __Response: __Yes, we added this graph in Figure S1. This is indeed useful.

    1. The GWAS section could benefit from a simple explanation of the premise of GWAS for non-specialist readers.

    __Response: __Yes, we added: "A genome-wide association study (GWAS) is a genetic mapping that uses the natural diversity of a panel of organisms of a given species to test for statistical independence between the allelic state of polymorphic markers and the phenotype of interest (Andersen and Rockman 2022). A statistical association between the marker and the phenotype indicates that a polymorphism tightly linked to the marker in the data (i.e. in linkage disequilibrium with it) causes the variation in phenotype. For statistical reasons, GWAS can only detect polymorphisms that are at intermediate frequencies in the panel, i.e. cases where both alleles occur at frequencies higher than 5%. We only used such polymorphisms in the GWAS (see Methods)."

    And further down:

    "To diminish the multiple testing burden, the initial analysis in Figure 1E used a restricted set of markers, after pruning those that were in high linkage to each other."

    1. One problem might be that the Mrt phenotype is widespread among wild strains. To the authors' credit, they consider results observed in different laboratories as valid, even when the results do not agree. If the Mrt phenotype is influenced by the environment, then some laboratory environments might result in 'false negative' Mrt results that could be ignored in favor of positive results from another lab that appear strong. Might focusing on strains with a set of strong positive results from one lab allow the authors to draw stronger GWAS conclusions?
    1. The authors' perform GWAS based on the variance of the Mrt phenotype data. Would the GWAS data be more illuminating if the authors only considered strains that become sterile fairly rapidly, within 10 generations. The authors might then have a second category that included strains that become sterile from generation 11-20. If the genetic basis for the Mrt phenotypes is the same, then GWAS of strains that become sterile in less than 10 generations might yield similar peaks as GWAS for strains that become sterile between generations 11-20.

    __Response: __These two comments are strongly related so we answer them together. Note that the GWAS is not mapping the variance values but the Mrt values themselves.

    We actually initially only used block 1 (a single replicate, all strains performed in parallel in our laboratory) and also detected the chromosome III association using a categorical variable (threshold at 11), but decided to show the results with all data to maximize power, taking into account the generation value and block effects.

    We investigated other ways to code the data (e.g. categorically) and removing the strains of the most variable middle category, as proposed by the reviewer. This changed the *p *values and the rank of the markers on chromosome III but not the overall result.

    In summary, we did a variety of tests, which pointed to chromosome III, a region that was validated using crosses (Figure 2).

    Note that in the revision, we updated the GWAS plot and fine mapping table as we noticed a few problems in our previous mapping. 1) We removed 3 isolates that were classified in Lee et al. 2021 as divergent. 2) We included strains that had been lost in the pipeline because their names did not match CeNDR isotypes. This increased the significance of the chromosome III peak.

    __Response: __There was no comment 6.

    1. 'We did not investigate whether a second locus present in JU775 on the right arm of Chr III might have a lesser effect.'

    __Response: __We are not sure what the reviewer meant. Considering the difficulties with the stronger effect locus, we did not try to study loci with a weaker effect.

    1. It might be interesting to test the memory of growth on beneficial bacteria on JU4134, which had a Mrt phenotype that was strongly suppressed by the beneficial bacteria.

    __Response: __We agree that testing other strains would be useful but given the duration of such experiments (30 generations and two weeks of preparation before), we respectfully decline to perform this experiment that does not seem strictly necessary.

    1. The Mrt phenotype of mutants in small RNA inheritance and histone modifying enzymes 'appears however distinct from that of the prg-1/piwi mutant (for which the cause of sterility is debated), especially the latter does not show temperature dependence and is suppressed by starvation.' While it is true that the cause of sterility is debated for the prg-1/piwi mutant, this mutant is defective for small RNA silencing and likely has parallels with some defects in histone modifying enzymes. Anecdotal reports suggest that starvation might affect the Mrt phenotype or longevity of histone modifying enzyme mutants. Moreover, the cause of sterility is not clear for small RNA inheritance and histone modifying enzyme mutants. It is fair to say that the distinction between temperature-sensitivity or lack of temperature sensitivity of small RNA mutants is not understood. Could the authors please comment here about whether any of the wild strains display sterility at 20°C.

    __Response: __The temperature-dependence of the wild isolates is progressive between 20-25°C. We previously showed that strains with a very strong Mrt phenotype, such as QX1211, can display sterility at 20°C (Figure 1B in Frézal et al. 2018). However, its Mrt phenotype is still temperature-dependent as the sterility occurs much earlier at 25°C.

    1. If intracellular bacteria are simply somatic, then how is it that they are transmitted to progeny. If they are released into the environment and then consumed by hatched larvae, this is soma-to-soma transmission.

    __Response: __These microsporidia (which are eukaryotes related to fungi) are indeed transmitted horizontally. To make this clear, we added: "colonizing its intestinal cells and being transmitted horizontally via defecation and ingestion of spores". The soma-to-germline interaction concerns the effect of microsporidia on germline maintenance.

    Minor:

    1. 'We measured the mortal germline (Mrt) phenotype'. Mortal Germline (Mrt)

    __Response: __It is unclear as to whether phenotypes start with a capital letter when they are in full words. We did write phenotypes in previous works with a capital letter but have changed because *C. elegans *nomenclature rules (https://cgc.umn.edu/nomenclature) suggest that they should not: "Phenotypic characteristics can be described in words, e.g., dumpy animals or uncoordinated animals." For the mortal germline phenotype in particular, we find several ways to write it in articles (with 0, 1 or 2 capital letters, including the three reviewers). We are happy to change it if required.

    Reviewer #2

    Major comments: The authors claimed that the variants causing Mrt exist at intermediate frequency in the natural population but the evidence supporting this claim is rather limited.

    __Response: __Thank you for this comment as it helped us clarify the manuscript.

    To better explain the notion of intermediate frequency in the GWAS, we added an explanation of the principle of the GWAS (see above) and again in the Discussion: "The intermediate frequency of the candidate alleles derives from the GWAS approach, which cannot detect rare alleles, such as set-24, that are present in a single strain of the dataset."

    We also illustrated the frequency by adding a plot (Fig. 1F) showing the association of the most associated candidate SNP, with a visual depiction of the frequency. We further added in Results: "For SNPs with a high significance (p-4) in the fine mapping, the frequency of the Mrt associated allele was comprised between 21 and 41% in our GWAS strain set (Table S3); as an example, the Mrt allele of the associated SNP shown in Figure 1F (III:4677491) displayed a frequency of 29% in the restricted strain set. Over the global wild strain set with genotypes at CeNDR in 2020, these numbers are 17-58% and 39%, respectively. "

    To strengthen the claim, the authors should examine the distribution and frequency (perhaps coupled with phylogenetic analysis) of the Ch III haplotype in the wild isolates. The authors should also examine the GWAS peak for the signature of balancing selection (e.g., dN/dS ratio).

    __Response: __Thank you for this comment. The different associated SNPs in Table S3 differ in their allele frequency (Table S3), hence they belong to different haplotypes. We added a supplementary Figure S2 with an analysis of the haplotype structure. Those at a low frequency (around 20%) belong to the same haplotype (e.g. JU775 and MY10) but some associated alleles are present in more haplotypes (40-50%), such as JU1793. Even if we neglect recombination, the history of mutations in the region is complex and there is not a single associated haplotype. We now show the genotypes of these different haplotypes at all SNPs in Table S3. We also added Table S4 that shows the co-occurrence of relevant haplotypes in local populations.

    Concerning tests of balancing selection, without knowing the causal polymorphism and linked haplotype, this is far reaching. We only feel confident to say that the causal polymorphism(s) is present at a significant frequency. We added however the fact that irrespective of which polymorphisms are causal, both alleles were found to coexist locally.

    Results: relevant text was added at the end of the GWAS section.

    Discussion: "The co-occurrence of relevant chromosome III haplotypes on multiple continents and in local populations (Table S4) is suggestive of balancing selection; however, a linked locus other than that causing the Mrt phenotype may be involved."

    Does JU775 carry polymorphisms in genes that are known to be involved in Mrt? These genes may genetically interact with the Ch III variant, as suggested by the partial penetrant phenotypes of the introgressed lines. It would be helpful to have a table summarize the variation in these genes.

    __Response: __It is difficult to deduce much from a genomic variant analysis, so we refrain from showing tables of polymorphisms beyond that used for the fine GWAS mapping in Table S3. For example, a non-synonymous SNP may or may not alter protein activity and cis-regulatory elements are difficult to assess. Moreover, an obviously null allele may be compensated by another polymorphism in the background. The JU775 alleles and bam files are publically available from CeNDR (Erik Andersen's lab): https://caendr.org/data/data-release/c-elegans/latest

    It is curious to me that for experiments with HT115, the expression of the RNAi vectors was induced with IPTG. Is this step necessary? It is known that even the backbone of L4440 could trigger a non-specific RNAi response (PMID: 30838421). I wonder if activating exogenous RNAi response is required for Mrt rescue.

    __Response: __Indeed: this experiment was initially aimed at testing RNAi sensitivity of JU775, thus IPTG was added on the plate (Figure 7, panel B). We therefore repeated the memory experiment with OP50 and without IPTG, with a similar result (Figure 7, panel A).

    In figure 7, it appears that the worms transferred from MG1655/HT115 to OP50 showed an even stronger rescue (higher Mrt value) than the ones constantly on MG1655/HT115. This suggests to me that fluctuations in food composition may strongly affect epigenetic inheritance. Please clarify as this is very interesting, if true.

    __Response: __Note: This answers the comment above (IPTG is not required).

    We indeed noticed this strong rescue but do not wish to make a point as we did no attempt to reproduce this result in the exact same conditions. The experiment in panel B does not show this effect.

    Optional - Numerous studies have shown that SKN-1 regulates metabolism in response to food composition and availability (PMID: 23040073). Additionally, some recent studies have indicated a role of SKN-1 in epigenetic inheritance triggered by exogenous RNAi. In particular, SKN-1 promotes stress-induced epigenetic resetting (PMID: 33729152). I wonder if SKN-1 modulates Mrt based on bacterial diet.

    __Response: __We tested* skn-1b/c *hypomorphic and gain-of-function mutants in the N2 background on E. coli OP50 and did not see an effect of the skn-1 allele.

    Minor comments Line 47: typo "...they defined..."

    __Response: __We did mean "thus defined".

    Line 100-101: weird sentence structure. Please consider rephrasing.

    __Response: __We simplified to "a wild C. elegans strain can keep the memory of its culture on a suppressing bacterial strain."

    Line 138-139: I don't quite understand what "intermediate-frequency chromosome III alleles" means here. Some SNPs were found in Ch III 4-6Mb? Please expand.

    __Response: __We rephrased to: "because this isolate carries the chromosome III alleles associated in the GWAS analysis with the Mrt phenotype (Table S3)."

    Line 213 - it was unclear to me why the assay was performed at 23C instead of 25C. I later learned in the method section that microsporidia cannot be cultured at 25C. I think it will be helpful to add that information when microsporidia is introduced to improve clarity.

    __Response: __We added: " We used a temperature of 23°C because these microsporidia kill C. elegans too rapidly at 25°C."

    Reviewer #3.

    Minor points

    1. Could the authors please define "experimental blocks"

    __Response: __We added the following sentence in Results: "Each Mrt assay started at a certain date constitutes an experimental block."

    1. Legend to supplementary snp table should be completed: define AF, impact, modifier, moderate, AA1, AA2...

    __Response: __This is added in the first sheet of the table. We also simplified the table and removed some of these columns.

    1. Please define "intermediate-frequency allele"

    __Response: __We added in Results: "GWAS can only detect polymorphisms that are at intermediate frequencies in the panel, i.e. cases where both alleles occur at frequencies higher than 5%." We also added below: " "For SNPs with a high significance (p-4) in the fine mapping, the frequency of the Mrt associated allele was comprised between 21 and 41% in our GWAS strain set (Table S3); as an example, the Mrt allele of the associated SNP shown in Figure 1F (III:4677491) displayed a frequency of 29% in the restricted strain set."

    1. Figure 7 legend: Authors should be more specific in describing the figure: After 10 (A panel), 13 or 20 generations (B panel) on the K-12 strain... What is E. coli OP50 start 'G10'? the 15° stock?

    __Response: __We changed to: " After 10 (A panel), 13 or 20 generations (B panel) on the K-12 strain" and added some details in:

    "A control from a 15°C culture maintained without starvation ("15°C stock") was bleached in parallel (labeled "E. coli OP50 start "G10" " in the graph of panel A)."

    Optional: Did the authors attempt to rescue the Mrt phenotype with individual metabolites (eg Vit B12...)? These are not straight forward experiments and most likely part of a future study.

    __Response: __We indeed tested several metabolites that are known to differ in* C. elegans *raised on *E. coli OP50 versus K-12 strains for their effect on the Mrt phenotype. None was able to rescue the mortal germline phenotype. However, especially in these long multigenerational experiments, it is difficult to know whether the metabolites are stable. We monitored vitamin B12 activity by using an acdh-1::GFP reporter that is known to be repressed by vitamin B12 - so we are confident of this negative result, which we now show in Figure S4. As cell wall lipopolysaccharide (LPS) differ between E. coli *K-12 and B strains, we also tested the *E. coli *LPS mutants, which had no eff

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    Referee #3

    Evidence, reproducibility and clarity

    • The nematode C. elegans is at the forefront of research on transgenerational epigenetic inheritance. In this work the authors studied the effects of natural genetic variations on multigenerational inheritance, using the temperature-sensitive Mortal germline phenotype (Mrt) as a paradigm in C. elegans. In ts Mrt mutants, animals become progressively sterile at 25{degree sign}C (stressful temperature) over subsequent generations and, importantly, this phenotype is reversible. The present study originated from the authors' previous observation that multiple C. elegans wild isolates display a ts-Mrt phenotype when cultured in the lab, raising the question of whether this intrinsically deleterious phenotype may be suppressed in the wild, and how natural genetic variation affects this phenotype.

    • By comparing 132 wild isolates of C. elegans, the authors found a wide distribution in ts-Mrt phenotypes ranging from 3 to 20 generations to reach sterility at 25{degree sign}C. The variance among a restrictive set of 115 replicates was low for strong Mrt values and high at intermediate trait values. Given this distribution, the authors analyzed the data using generalized linear mixed models. This reviewer is unable to evaluate the appropriateness of these models. They then performed GWAS mapping combined with analysis of introgression lines and identified a QTL on chromosome III between 4.66 and 6 .49Mb that includes a number of potentially interesting candidates that were not further analyzed in this work.

    • Because the authors noticed that the Mrt phenotype commonly appears after bleaching the culture, a treatment that kills associated microbes, they then tested the impact of naturally associated microbes on the Mrt phenotype. They found that freshly isolated strains such as JU3224 could be propagated for more than 20 generations at 25{degree sign}C with their associated microbes, while after bleaching on OP50 (bacteria commonly used in lab culture) they developed a Mrt phenotype at 25{degree sign}. They then fed the isolates with naturally associated bacteria isolated in the lab-either their own or from other isolates. Reassociation of single bacterial clones, or a mix of these, fully or partially rescued the Mrt phenotype. Importantly, bacteria isolated from one strain was able to rescue the Mrt of another strain, suggesting common mechanisms of action in rescuing the Mrt phenotype. Surprisingly Microsporidia, usually detrimental to C. elegans, also rescued the Mrt phenotype. These results show that infection of somatic tissues can influence the germline.

    • ts Mrt mutations so far identified affect nuclear small RNA pathways, small RNA amplification and histone modifications in the germline. The authors further show that the Mrt phenotype of laboratory mutants in small RNA inheritance or chromatin factors such as the set-2 histone methyltrasferase is also suppressed by culture on bacteria other than E. coli OP50.

    • Finally, the authors tested whether animals have a memory of their past bacterial environment by shifting animals of the C. elegans JU775 strain that had been cultured for several generations at 25{degree sign}C on an E. coli K-12 strain (on which their Mrt phenotype was suppressed) to the laboratory E. coli OP50, which usually reveals the Mrt phenotype. Lines that were propagated for 10-20 generations at 25{degree sign}C on an E. coli K-12 strain (MG1655 or HT115) showed a rescued phenotype when transferred back on OP50, consistent with a multigenerational memory of the bacterial environment.

    • All experiments are well executed, clearly presented and of the highest standard.

    Significance

    C. elegans is an excellent model system to study transgenerational inheritance. However, most studies on epigenetic inheritance in this system are carried out under standard laboratory conditions, and the phenotypes followed often not very robust (stress resistance, longevity..) raising questions as to their interpretation. This work is an important contribution to the field because it reveals how a widely studied phenotype (the Mrt phenotype) relates to natural isolates. The results reported demonstrate a clear link between the environment and the multigenerational transmission of non-genetic information. They also raise interesting questions on the ability of a species to transiently provide environmental cues to a variable number of generations. Finally, these results offer hints that that the Mrt phenotype may result from inherited metabolic changes, as observed using other experimental paradigms in C. elegans, including starvation. This work will therefore be of interest to a wide audience interested in epigenetic inheritance and the environment, soma-germline communication, and host pathogen interactions.

    Minor points

    1. Could the authors please define "experimental blocks"

    2. Legend to supplementary snp table should be completed: define AF, impact, modifier, moderate, AA1, AA2...

    3. Please define "intermediate-frequency allele"

    4. Figure 7 legend: Authors should be more specific in describing the figure: After 10 (A panel), 13 or 20 generations (B panel) on the K-12 strain... What is E. coli OP50 start 'G10'? the 15{degree sign} stock?

    Optional:

    Did the authors attempt to rescue the Mrt phenotype with individual metabolites (eg Vit B12...)? These are not straight forward experiments and most likely part of a future study.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Frézal et al. reported novel interactions between microbes and C. elegans in the regulation of epigenetic inheritance. By screening for 132 isotypes, the authors found natural genetic variants that contribute to the mortal germline (Mrt) phenotype. The authors further found that naturally associated gut bacteria, microsporidia, and E. coli K12 could rescue Mrt phenotype in wild isolates as well as in epigenetic mutants. Finally, the authors showed that the epigenetic memory of bacterial environment could propagate transgenerationally. I find this paper highly intriguing as it provides valuable insights into the impact of the environment on epigenetic inheritance and its effects on evolution within ecologically relevant contexts.

    Major comments:

    • The authors claimed that the variants causing Mrt exist at intermediate frequency in the natural population but the evidence supporting this claim is rather limited. To strengthen the claim, the authors should examine the distribution and frequency (perhaps coupled with phylogenetic analysis) of the Ch III haplotype in the wild isolates. The authors should also examine the GWAS peak for the signature of balancing selection (e.g., dN/dS ratio).

    • Does JU775 carry polymorphisms in genes that are known to be involved in Mrt? These genes may genetically interact with the Ch III variant, as suggested by the partial penetrant phenotypes of the introgressed lines. It would be helpful to have a table summarize the variation in these genes.
      It is curious to me that for experiments with HT115, the expression of the RNAi vectors was induced with IPTG. Is this step necessary? It is known that even the backbone of L4440 could trigger a non-specific RNAi response (PMID: 30838421). I wonder if activating exogenous RNAi response is required for Mrt rescue.

    • In figure 7, it appears that the worms transferred from MG1655/HT115 to OP50 showed an even stronger rescue (higher Mrt value) than the ones constantly on MG1655/HT115. This suggests to me that fluctuations in food composition may strongly affect epigenetic inheritance. Please clarify as this is very interesting, if true.

    • Optional - Numerous studies have shown that SKN-1 regulates metabolism in response to food composition and availability (PMID: 23040073). Additionally, some recent studies have indicated a role of SKN-1 in epigenetic inheritance triggered by exogenous RNAi. In particular, SKN-1 promotes stress-induced epigenetic resetting (PMID: 33729152). I wonder if SKN-1 modulates Mrt based on bacterial diet.

    Minor comments:

    • Line 47: typo "...they defined..."

    • Line 100-101: weird sentence structure. Please consider rephrasing.

    • Line 138-139: I don't quite understand what "intermediate-frequency chromosome III alleles" means here. Some SNPs were found in Ch III 4-6Mb? Please expand.

    • Line 213 - it was unclear to me why the assay was performed at 23C instead of 25C. I later learned in the method section that microsporidia cannot be cultured at 25C. I think it will be helpful to add that information when microsporidia is introduced to improve clarity.

    Significance

    This study beautifully demonstrates how diet composition affects epigenetic inheritance. This study is rigorous (replicated by 3 different labs) and the data is solid. Using natural wild isolates and naturally associated microbes, the authors described how diet composition affects germline mortality and epigenetic inheritance. Interestingly, the authors showed that Mrt phenotype might be the result of standard lab cultivation conditions and it was masked when the worms were fed on naturally associated bacteria and microsporidia. Overall, the findings are very interesting and novel. While mechanistic insights are currently lacking, it is outside the scope of this paper. This paper provides an interesting paradigm to study how genetic and environmental variation influence epigenetic inheritance and evolution. I believe this paper will be of great interest to audiences across many fields of biology, including quantitative biology, evo-devo, ecology, and genetics and epigenetics.

    My field of expertise: C. elegans biology, epigenetic inheritance, genetics.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Frezal, Felix and colleagues study 132 wild isolates of the C. elegans species and demonstrate that the majority of these strains will become sterile within 20 generations if grown at 25oC. This is a very thorough analysis of a multigenerational trait that the authors show is commonly found in wild C. elegans strains. The authors use GWAS to identify a peak on chromosome III that is enriched in strains that become sterile at 25oC. Consistently genetic crosses place this segment of chromosome III from a Mrt wild strain into the N2 background resulted in a strong Mrt phenotype. The authors noticed that bleaching of wild C. elegans strains to remove associated bacteria promoted the Mrt phenotype. Remarkably, the authors show that growth of bleached wild strains on bacteria isolated from the wild strains prior to bleaching is sufficient to suppress the Mrt phenotype. These results were obtained with two wild isolates and with multiple species of wild bacteria, strongly supporting the authors' conclusions. The authors also show that independent types of intracellular bacteria that infect the intestine can partially suppress the Mrt phenotypes. The authors also show partial to strong rescue of temperature-sensitive epigenetic mutants set-2, set-24 and nrde-2 by wild bacteria. Remarkably, the authors demonstrate that growth of the introgressed JU775 strain on a N2 background can be grown on suppressor bacteria for 10 to 20 generations, then bleached and placed on OP50, then there is a multigenerational memory of the suppressor bacteria. This intriguing result is consistent with bacteria having an epigenetic effect on C. elegans Mrt phenotypes, which are themselves in some cases caused by epigenetic defects.

    Comments for the authors:

    1. 'For GWAS, the strains that were fertile after 20 generations were considered non-Mrt.'

    One aspect of Fig 1D that could be clarified are the dots at generation 21. If these represent strains that were always fertile at generation 21, then perhaps give these a different color to indicate that sterility was never observed?

    1. 'The mean Mrt values of strains ranged from sterile at 3 generations to fertile after 20 generations at 25oC, with a skewed distribution toward high values (Figure 1B).'

    Based on Table S2, part of the explanation for this skewed distribution in later generations is that some strains became sterile rapidly for some blocks, whereas the same strain did not become sterile in other blocks. For example, JU1200, JU360, PB303. I suggest providing a second color for Fig. 1D for strains that sometimes displayed sterility and sometimes did not.

    For those that were sometimes fertile and sometimes sterile, I suggest creating a graph in Figure 1 that shows generations at sterility or lack of sterility, color coded by block. This will allow the significance of strains with high generation Mrt values to be better appreciated for readers who do not look at the supplementary table.

    1. The GWAS section could benefit from a simple explanation of the premise of GWAS for non-specialist readers.

    2. One problem might be that the Mrt phenotype is widespread among wild strains. To the authors' credit, they consider results observed in different laboratories as valid, even when the results do not agree. If the Mrt phenotype is influenced by the environment, then some laboratory environments might result in 'false negative' Mrt results that could be ignored in favor of positive results from another lab that appear strong. Might focusing on strains with a set of strong positive results from one lab allow the authors to draw stronger GWAS conclusions?

    3. The authors' perform GWAS based on the variance of the Mrt phenotype data. Would the GWAS data be more illuminating if the authors only considered strains that become sterile fairly rapidly, within 10 generations. The authors might then have a second category that included strains that become sterile from generation 11-20. If the genetic basis for the Mrt phenotypes is the same, then GWAS of strains that become sterile in less than 10 generations might yield similar peaks as GWAS for strains that become sterile between generations 11-20.

    4. 'We did not investigate whether a second locus present in JU775 on the right arm of Chr III might have a lesser effect.'

    5. It might be interesting to test the memory of growth on beneficial bacteria on JU4134, which had a Mrt phenotype that was strongly suppressed by the beneficial bacteria.

    6. The Mrt phenotype of mutants in small RNA inheritance and histone modifying enzymes 'appears however distinct from that of the prg-1/piwi mutant (for which the cause of sterility is debated), especially the latter does not show temperature dependence and is suppressed by starvation.'

    While it is true that the cause of sterility is debated for the prg-1/piwi mutant, this mutant is defective for small RNA silencing and likely has parallels with some defects in histone modifying enzymes. Anecdotal reports suggest that starvation might affect the Mrt phenotype or longevity of histone modifying enzyme mutants. Moreover, the cause of sterility is not clear for small RNA inheritance and histone modifying enzyme mutants. It is fair to say that the distinction between temperature-sensitivity or lack of temperature sensitivity of small RNA mutants is not understood. Could the authors please comment here about whether any of the wild strains display sterility at 20oC.

    1. If intracellular bacteria are simply somatic, then how is it that they are transmitted to progeny. If they are released into the environment and then consumed by hatched larvae, this is soma-to-soma transmission.

    Minor comments:

    1. 'We measured the mortal germline (Mrt) phenotype'. Mortal Germline (Mrt)

    Significance

    All in all, this is an interesting and well-written manuscript that represents a considerable amount of work and demonstrates that a temperature-sensitive multigenerational sterility phenotype is widespread among wild C. elegans strains. This Mrt phenotype is modulated by the food they consume or by intracellular bacterial parasites that reside in somatic intestinal cells. This may mean that the intestine is a major modulator of the Mrt phenotype, which may be a consequence of lab culture conditions and may not occur for wild strains in the wild. Nevertheless, the phenotype or phenotypes are intriguing and likely relevant to natural variation.

    The limitations of this manuscript include a lack of understanding of the precise genes involved or if small RNAs or metabolites from bacteria are involved. But this manuscript represents an enormous effort and raises many interesting points that will be addressed in future efforts.

  5. Version published to 10.1101/2023.05.17.540956v1 on bioRxiv