Transgenerational inheritance of an acquired small RNA-based antiviral response in C. elegans.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
This paper's computational scope is narrow and specific: deep sequencing of small RNAs (GEO GSE33334, 4 samples) was used only to molecularly confirm the existence and orientation of virus-derived siRNAs (viRNAs) inherited across generations; the core transgenerational-inheritance phenotype itself (Table 1, Figures 1-3) was established purely by wet-lab GFP scoring across genetic crosses and is out of scope for pipeline reproduction. We rebuilt the sequencing pipeline from scratch (ENA fastq download -> cutadapt adapter trim + 18-30nt size filter -> bowtie -v2 alignment to FHV RNA1 (NC_004146.1) -> mismatch/strand tabulation) since no code was deposited by the authors. For the well-powered FR1gfp positive-control sample, our results are a good quantitative match to Table 2 and the text: RPM 25.8 vs reported 31.6 (paper's RPM applied to our depth predicts 15.9, inside our Poisson 95% CI), mismatch breakdown 54/31/15% vs reported 65/24/10% (same monotonic pattern, small-N noise), and 84.6% antisense-strand reads vs reported 98.26% (same strong antisense bias, smaller majority). The rde-4(-/-) negative control was essentially confirmed clean (1 likely-spurious hit out of 465K reads vs 0 reported). For the two rare F3-generation samples that carry the paper's central inheritance claim (10 and 2 viRNA reads reported respectively), our adapter-trimming pipeline recovered 7-15x fewer usable reads than the paper across all samples (a real, reproducible characteristic of this dataset combined with pipeline differences from the authors' FASTX-Toolkit/PRINSEQ/Geneious pipeline, which we could not exactly replicate since no code was shared) and we observed 0 viRNA reads in both -- a result that is statistically consistent with (not contradicting) the paper's own low reported rates at our achieved depth, but does not independently confirm them either. We did not attempt the paper's BLASTN cross-check against the C. elegans genome, and we did not attempt any wet-lab/phenotypic results. No fabrication flags: all reported numbers are directly computed from the downloaded public SRA data using named, versioned tools (cutadapt 5.2, bowtie 1.3.1, samtools 1.24) and are fully reproducible from the job scripts and «infra»-persisted intermediate/output files.
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-07-29
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-07-31no human curator yet
- Last updated
- 2026-07-31
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusThe paper tests whether an acquired antiviral trait in C. elegans — the RNAi-dependent production of virus-derived small interfering RNAs (viRNAs) following an episode of viral expression — is transmitted transgenerationally, and whether such 'Lamarckian' inheritance is mediated by extrachromosomal small RNA molecules rather than DNA/chromatin changes.
- ★ viRNA-mediated silencing of the FR1gfp Flock House virus is transmitted to RNAi-deficient (rde-1 or rde-4 homozygous) progeny and persists for many ensuing generations, i.e. an acquired antiviral trait is inherited. finding
- ★ The transmitted antiviral silencing agent is passed in a template-independent manner: silencing is inherited even after the FR1gfp viral transgene has been crossed out. finding
- ★ The inherited silencing agent is a diffusible, trans-acting, DNA-independent element that segregates non-Mendelian — all F2 progeny of a long-term-silenced x non-silenced rde-1(-/-) cross were silenced. mechanism
- ★ Inherited primary viRNAs are physically detectable by small RNA deep sequencing in animals that cannot produce their own viRNAs (F3 FR1gfp; rde-4(-/-)) and in animals from which the transgene was outcrossed. finding
- ★ Two modes of inherited silencing exist: a fading mode (~3 generations) that is rrf-1-independent, and a long-term stable mode that requires the RNA-dependent RNA polymerase rrf-1, consistent with maintenance by continued amplification. mechanism
- ★ The heat-inducible, chromosomally integrated FR1gfp transgene (FHV RNA1 plus RNA3 with gfp replacing B2) provides a minimal, non-harmful system in which viral replication and its silencing can be visualized by GFP and the viral genome removed by genetic crosses. resource
- Viral silencing does not require mut-2, mut-7, mut-14, mut-16, rde-2, ergo-1, CSR-1 or C04F12.1, nor the chromatin factors previously implicated in long-term exogenous RNAi or transgene silencing (e.g. hda-4, K03D10.3, isw-1, mrg-1, mes-2/3/4/6, mys-1, zfp-1, rba-1, cin-4, gfl-1), likely because of redundancy among the 27 known worm Argonautes. finding
- Even very low levels of viral product, produced by leaky heat-shock promoter expression at 15°C, are sufficient to trigger an inherited antiviral response. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| GFP fluorescence scoring of viral replication (GFP/Virus(+) vs GFP/Virus(-)) after heat-shock induction | Transgenic C. elegans carrying chromosomally integrated FR1gfp (FHV RNA1 + RNA3-gfp) in wild-type and RNAi-pathway mutant backgrounds | Heat-shock induction of the hs-promoter-driven viral transgene; RNAi mutant backgrounds (rde-1, rde-4 and other RNAi components) | Presence/absence and percentage of animals expressing viral GFP across generations | — |
| Genetic crosses / segregation analysis (transgenerational inheritance test) | C. elegans rde-1(-/-) and rde-4(-/-) mutants derived from rde-1(+/-) or rde-4(+/-) heterozygous parents carrying FR1gfp | Self-fertilization of heterozygotes; heat-shock induction of virus | GFP/Virus phenotype of homozygous mutant progeny through F1-F4+ generations | — |
| Template-independence cross (X-linked transgene segregation) | C. elegans rde-1(-/-); FR1gfp(-/0) cross-progeny males crossed to F5 rde-1(-/-); FR1gfp(+/+) GFP/Virus(+) hermaphrodites | Removal of the X-linked FR1gfp viral template by outcrossing | Whether viral GFP signal is eliminated in progeny lacking prior template exposure | — |
| Mendelian vs non-Mendelian segregation cross | C. elegans >F5 rde-1(-/-); FR1gfp long-term-silenced animals x >F5 rde-1(-/-); FR1gfp animals that lost silencing | None beyond cross; RNAi machinery not reinstated (all rde-1(-/-)) | Fraction of F2 progeny that are GFP/Virus(-) (all silenced vs 3/4 expressing expected for Mendelian locus/transgene silencing) | — |
| rrf-1 dependence cross | C. elegans rde-1(-/-); rrf-1(-/-) double mutants crossed to stably silenced >F5 rde-1(-/-); FR1gfp worms; also rde-1/rde-4 homozygotes from RNAi-competent parents | rrf-1 (RNA-dependent RNA polymerase) loss of function | Reinstatement of GFP/Virus(+) phenotype in rrf-1(-/-) F3 progeny | — |
| Small RNA isolation and deep sequencing (cloning protocol enriching for rare rde-4-dependent primary small RNAs) | Four C. elegans libraries: (a) FR1gfp RNAi-competent worms; (b) rde-4(-/-) mutants; (c) F3 FR1gfp; rde-4(-/-) worms two generations from rde-4(+/-) grandparents; (d) F3 progeny of wild type that lost FR1gfp by outcrossing | rde-4 mutation; outcrossing to remove the FR1gfp transgene | Number, genomic position, abundance and strand orientation of viRNA reads complementary to the viral genome | — |
| RNAi knockdown screen of chromatin and RNAi-pathway factors | C. elegans FR1gfp transgenic animals | Knockdown/elimination of hda-4, K03D10.3, isw-1, mrg-1, mes-2, mes-3, mes-4, mes-6, mys-1, m03c11, zfp-1, rba-1, cin-4, gfl-1; and mut-2, mut-7, mut-14, mut-16, rde-2, ergo-1, CSR-1, C04F12.1 | Whether heat-shock-induced FR1gfp/viral GFP expression becomes de-silenced | — |
| Low-level (leaky promoter) viral induction assay | C. elegans strain containing FR1gfp; F1 rde-1(+/-) cross-progeny of wild-type males x rde-1(-/-) animals, maintained at 15°C | Maintenance at 15°C relying on slight leakiness of the heat-shock promoter instead of heat-shock induction | GFP/Virus phenotype of rde-1(-/-) F2 progeny | — |
- ▼ Homozygous rde-1(-/-) and rde-4(-/-) progeny of RNAi-competent heterozygous parents show robust viral silencing (GFP/Virus(-)) upon heat-shock induction despite lacking the ability to mount a de novo RNAi response.
- – Inherited silencing persists for several generations; only in the F4 generation of rde-1(-/-) and rde-4(-/-) worms did a small percentage of animals begin to express the virus, and progeny of F4 GFP/Virus(+) worms showed increasing numbers of virus-expressing animals until silencing fully wore off (100% GFP/Virus(+)). fading over ~3 generations; 100% GFP/Virus(+) after continued propagation
- ▼ rde-1(-/-) progeny of a cross with FR1gfp(-/0) males that had inherited the silencing agent had their viral GFP signal eliminated, demonstrating template-independent (and sperm-borne) transmission of the antiviral agent.
- ▼ All F2 progeny of the cross between long-term-silenced and de-silenced rde-1(-/-); FR1gfp animals had the virally produced GFP signal eliminated, inconsistent with Mendelian segregation of a genomic suppressor locus or a silenced transgene (which would predict three quarters GFP/Virus(+)). 100% of F2 silenced vs 75% expected GFP/Virus(+) under Mendelian transgene silencing
- – viRNAs were detected in the RNAi-competent FR1gfp positive control and in F3 FR1gfp; rde-4(-/-) animals that cannot produce their own viRNAs, and were absent in the rde-4(-/-) negative control.
- – viRNAs were also detected in worms from which the FR1gfp transgene had been crossed out; the inherited viRNAs matched the two most abundant viRNA species of the positive control and were all of reverse (negative-strand) orientation, arguing against unspecific viral RNA breakdown products.
- – rrf-1 is dispensable for the initial transgenerational silencing in rde-1/rde-4 homozygotes derived from RNAi-competent parents, but rrf-1(-/-) F3 progeny of stably silenced >F5 rde-1(-/-); FR1gfp worms reinstate viral GFP expression, showing rrf-1 dependence of long-term silencing.
- – Crossing de-silenced GFP/Virus(+) rde-1(-/-) worms with RNAi-competent wild-type males re-established viral silencing in the cross progeny, whereas crossing with rde-1(-/-) males left progeny GFP/Virus(+), also excluding meiotic silencing by unpaired DNA as the cause.
- count 50/50 animals are GFP/Virus(-) (rde-1(-/-) F2 progeny silencing viral propagation when F1 rde-1(+/-) cross-progeny were kept at 15°C (leaky, low-level viral induction))
- count >50 animals tested for each gene (Elimination of mut-2, mut-7, mut-14, mut-16, rde-2, ergo-1, CSR-1 and C04F12.1 did not result in viral/GFP expression)
- count >50 animals tested for each gene (Knockdown of chromatin factors (hda-4, K03D10.3, isw-1, mrg-1, mes-2/3/4/6, mys-1, m03c11, zfp-1, rba-1, cin-4, gfl-1) did not affect silencing of heat-shock induced FR1gfp)
- count 100% GFP/Virus(+) (Progenies of continually isolated and propagated GFP/Virus(+) worms upon heat-shock induction, showing the inherited silencing eventually wears off)
- count F4 generation (First generation of rde-1(-/-) and rde-4(-/-) worms in which a small percentage of animals started to express the virus)
- count ~3 generations (Duration of the 'fading' mode of inherited silencing, distinguished from a second, more stable long-term mode)
- count 27 known Argonautes (Documented redundancy of the RNAi silencing machinery in C. elegans, offered as explanation for the lack of a silencing defect in individual RNAi-component mutants)
- other FHV RNA levels reaching levels as high as those of rRNAs (Vigorous rate of RdRP-catalyzed FHV replication, cited to argue heat-shock induction does not produce nonphysiological RNA levels)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This paper uses a genetics-based experimental design in C. elegans, relying on defined genetic crosses, phenotypic scoring of a heat-inducible viral GFP reporter (GFP/Virus(+) vs GFP/Virus(−)), and small RNA deep sequencing to trace an inherited antiviral RNAi response across generations. Results are reported primarily as raw counts and fractions of animals displaying each phenotype (e.g., in Table 1, Table 2, and Figures 1-4) and as descriptive comparisons of viRNA sequencing reads between genotypes, rather than through inferential statistical tests with p-values. No formal statistical test, software package, or multiplicity-correction method is named in the provided text.
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Phenotypic outcomes (GFP/Virus(+) vs GFP/Virus(−)) were reported as raw counts and fractions of animals per genotype/generation (e.g., '50/50 animals', '>50 animals tested for each gene').↳ Could also: A formal contingency-table test such as Fisher's exact test or a chi-square test of independence — This would generate a p-value and an effect-size estimate (e.g., odds ratio) for comparing the proportion of silenced vs non-silenced animals between genotypes or generations, complementing the descriptive counts already presented.
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The gradual 'wearing off' of inherited silencing across successive generations (F1 through F4 and beyond) was described narratively (Table 1, Suppl. Fig. 1) rather than with a formal trend statistic.↳ Could also: A trend test across generations (e.g., Cochran-Armitage trend test) or a survival-type analysis (Kaplan-Meier estimator with a log-rank test) treating 'generations until loss of silencing' as a time-to-event variable — Either approach would let the increasing proportion of GFP/Virus(+) animals across generations be summarized with a formal statistic, trend p-value, or survival curve with confidence bounds.
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Small-RNA deep-sequencing results (viRNA read counts, Table 2, Figure 4) were compared descriptively between positive-control, negative-control, and experimental genotypes.↳ Could also: A dedicated small-RNA/RNA-seq differential-abundance tool (e.g., DESeq2 or edgeR) applied to normalized read counts — This would provide a formal statistical comparison, with p-values and false-discovery-rate control, of viRNA abundance between genotypes while accounting for sequencing depth and biological variability.
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Sample sizes for genetic-cross experiments and RNAi-pathway gene tests were set as simple thresholds (e.g., '>50 animals tested for each gene') rather than derived from a stated power calculation.↳ Could also: A pre-specified power/sample-size calculation based on an anticipated effect size — This would give an a priori numeric justification for the number of animals scored per condition, complementing the empirically chosen count threshold.
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Silencing frequencies are presented as point counts/fractions (e.g., '50/50', '>50 animals') without an accompanying measure of precision.↳ Could also: Reporting a 95% confidence interval for each proportion (e.g., a Wilson or Clopper-Pearson interval) — This would convey the statistical precision of each estimated silencing frequency, which can be particularly informative when the underlying denominator is modest.
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The RNAi-pathway and chromatin-factor genes tested for an effect on silencing were evaluated one gene at a time against the same phenotype.↳ Could also: A multiple-testing correction such as Benjamini-Hochberg FDR applied across the panel of genes screened — This would formally control the false discovery rate when many genes are screened in parallel against the same outcome, complementing the individual gene-by-gene assessment already reported.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Assessments & scoring basis
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Data identity is perfect - all four GSE33334 libraries were downloaded 1:1 with spot counts matching SRA metadata exactly - and the well-powered samples reproduce well: FR1gfp positive control 25.8 RPM vs Table 2's 31.6 RPM (the paper's rate predicts 15.9 reads, inside the Poisson 95% CI 6.9-22.2 around our observed 13), and the rde-4(ne299) negative control effectively clean (1 read in 465,037 vs 0 reported). The deviation is on our side, at the preprocessing stage: with no author code deposited and only FASTX/PRINSEQ/Geneious named, our self-tuned cutadapt+18-30nt pipeline retained 7-15x fewer usable reads than Table 2's post-trim depths, which left the two F3 libraries carrying the central inheritance claim (10 reads/8.8 RPM and 2 reads/3.5 RPM) at expected counts of ~0.65 and 0.61 - our observed 0/0 is statistically compatible with the paper (P52-54%) but supplies no independent confirmation. Severity is moderate and directional: antisense bias (84.6% vs 98.26%) and mismatch decay (53.8/30.8/15.4% vs 65/24/10%) hold qualitatively with small-N shifts. No fabrication signal - the reported values are plausible and derivable in principle from the open data; the core transgenerational phenotype itself (Table 1, Figs 1-3) is wet-lab GFP scoring and outside pipeline scope, so the overall verdict is a solid partial reproduction with explainable, our-methodology deviations.
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