Transcriptomic and functional analyses of the piRNA pathway in the Chagas disease vector Rhodnius prolixus.
The main results reproduced: recomputed values matched the published ones within tolerance.
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 are derivable from the shared data
- ✓Any deviation was negligible
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡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
Described well enough to reproduce the data-level claims. PARTIAL = exact on read counts + corroborated on mapping rate. C1: the two SRP158580 PE runs (SRR7738238 rep1, SRR7738239 rep2) reproduce the paper's per-replicate (76,963,784 / 51,447,804) and total (128,411,588) read counts EXACTLY, from md5-verified FASTQ -- the deposited data matches the paper bit-for-bit, no fabrication concern. C2: overall genome mapping rate is CORROBORATED but not bit-exact -- HISAT2 2.2.1 gives 92.40% (rep1) / 92.30% (rep2) vs the paper's GEMtools/GEM ~89.1% / ~89.6% (~3pp higher), the expected effect of substituting the mapper (HISAT2 vs the deprecated 2018 GEM mapper) and the assembly patch (RproC3 vs RproC1, same base GCA_000181055; RproC1 .fna 404'd on NCBI FTP). NOT ATTEMPTED (the hard last 20%, deliberately skipped): rebuilding the exact GEMtools GEM RNA pipeline for bit-exact mapping-rate / GEMtools-specific summary stats (informative-read %, mismatch/indel averages, transcript-detection tiers) -- env_unresolvable risk on a deprecated toolchain; the Drosophila-orthology piRNA-gene table (manual curation, non-pipeline); and all wet-lab results (qPCR, in-situ, RNAi). Note: despite the 'piRNA pathway' title, SRP158580 is ordinary paired-end mRNA-seq of ovaries -- no small-RNA/piRNA bioinformatic pipeline exists in the deposited data.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 94assessed: 2026-06-16 ⛓ 1c8d5c6da4a0
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
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: opusAre the core components of the piRNA pathway evolutionarily conserved and functionally required for oogenesis and female adult fertility in the hemimetabolous Chagas disease vector Rhodnius prolixus?
- ★ Rhodnius prolixus genome harbors four putative piwi orthologs (three piwi genes plus one ago3 ortholog), and core piRNA pathway components are expressed during previtellogenic oogenesis finding
- ★ Rp-piwi2, Rp-piwi3 and Rp-ago3, but not Rp-piwi1, are transcribed in germline tissues and maternally deposited in mature eggs finding
- ★ Parental RNAi against Rp-piwi2, Rp-piwi3 and Rp-ago3 causes severe egg-laying and female adult fertility defects finding
- ★ Knockdown of Rp-piwi2 disrupts oogenesis by causing dramatic loss of trophocytes, egg chamber degeneration and oogenesis arrest mechanism
- ★ The putative Rp-Piwi2 protein features an N-terminal polyglutamine (PolyQ) tract conserved in PIWI proteins of other Triatomine vectors finding
- ★ Transcriptome profiling of previtellogenic Rhodnius oogenesis provides a resource to improve genome annotation and inform vector control strategies resource
- Parental RNAi combined with in situ hybridization and qRT-PCR is an effective approach to functionally interrogate piwi genes in Rhodnius method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| paired-end bulk RNA-seq (transcriptome profiling) | Rhodnius prolixus previtellogenic ovary stages (blood-fed adult females) | none | transcript abundance / gene expression quantification | Illumina HiSeq, TruSeq paired-end RNA library prep kit |
| parental RNAi (dsRNA injection) | Rhodnius prolixus adult females / ovaries | knockdown of Rp-piwi2, Rp-piwi3, Rp-ago3 (and Rp-piwi1) | egg laying, female adult fertility, ovary/oogenesis morphology | Megascript kit (Ambion) dsRNA |
| RT-PCR / qRT-PCR | Rhodnius prolixus previtellogenic and choriogenic ovary stages | pRNAi vs control vs wildtype | piwi/ago3 transcript expression levels | Superscript III (Invitrogen) |
| in situ RNA hybridization | Rhodnius prolixus ovaries | none | spatial localization of piwi/ago3 transcripts in germline tissues | DIG RNA labeling kit (Roche) |
| whole-mount immunostaining / confocal microscopy | Rhodnius prolixus ovaries (dissected 10 days post-feeding) | none | anti-γH2Ax and DAPI staining of egg chamber/nuclei | Leica Confocal Microscope; anti-γH2Ax (Millipore) |
| phylogenetic / sequence alignment analysis | PIWI amino acid sequences of Rhodnius prolixus, Drosophila melanogaster, and Triatominae species | none | evolutionary relationships and PolyQ tract conservation | MUSCLE v3.8.31, MEGA6, iTOL v2 |
- – Core piRNA pathway components are expressed during previtellogenic stages of Rhodnius oogenesis
- – Rp-piwi2, Rp-piwi3 and Rp-ago3 expressed in germline and maternally deposited in eggs; Rp-piwi1 not expressed in ovaries
- ▼ pRNAi against Rp-piwi2, Rp-piwi3 and Rp-ago3 produces severe egg laying and fertility defects (partial or complete sterility)
- ▼ Rp-piwi2 knockdown causes dramatic loss of trophocytes, egg chamber degeneration and oogenesis arrest
- – Rp-Piwi2 N-terminal PolyQ tract conserved across Triatominae (R. neglectus, T. dimidiata, T. infestans)
- – ~84% of 14,840 annotated transcripts detected by >10 read mappings; ~10% by >10,000 mappings ~84%; ~10%
- ▲ A putative ortholog of the Drosophila squid gene found highly expressed in Rhodnius ovaries
- count 128,411,588 paired-end reads (76,963,784 in vit1 vs 51,447,804 in vit2) (reads sequenced from two ovary replicates)
- count 12.84 Gigabases sequenced (total sequence in two replicates vit1 and vit2)
- other ~90% mapped (89.1% vs 89.6%); ~80% informative (80.9% vs 79.2%) (read mapping rate to RproC1 genome)
- count 1,029 of 1,467 most abundant mRNAs mapped one-to-one to Flybase proteins; +208 via one-to-many/many-to-many (ortholog mapping to Drosophila proteome)
- count 1,467 genes with >10,000 reads investigated; only 36 annotated with known functions; 9,188 orthology mappings used (functional annotation of highly expressed genes)
- count 626 functional terms clustered into five major groups; clusters with ≥5 members (functional clustering of abundant mRNAs)
- pvalue false-discovery rate ≤0.05 (statistical overrepresentation of functional terms (DAVID model))
- count ~86% informative mappings (55,455,772 in vit1; 36,324,980 in vit2) (mappings superimposed correctly to RProC1.3 transcriptome)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This is primarily a descriptive molecular and transcriptomic study. The main quantitative components are an Illumina paired-end RNAseq survey of previtellogenic Rhodnius ovaries from two biological replicates (mapped with the GEM/GEMtools pipeline and quantified against the RproC1.1 transcriptome), a DAVID-style functional-term overrepresentation analysis with false-discovery-rate control, and a Maximum Likelihood phylogenetic reconstruction with bootstrap support. Functional validation (parental RNAi, RT-PCR/qRT-PCR, in situ hybridization, immunostaining) is reported, with RT-PCR/qRT-PCR conducted in biological triplicates and RNAi performed on groups of 10 injected adult females; no formal inferential test (e.g., t-test or ANOVA) for the phenotypic/expression comparisons is described in the available text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Functional-term overrepresentation analysis (DAVID model) yielding a p-value per term, with false-discovery-rate control | Functional clustering of the 1,467 most abundant ovarian mRNAs via orthology mapping to the Drosophila proteome (Fig 1C) | 1,467 most abundant mRNAs / 9,188 orthology mappings; term counts compared against the full Flybase reference annotation | not stated |
| Bootstrap resampling for phylogenetic node support (1000 replicates) on a Maximum Likelihood tree | PIWI protein phylogeny of Rhodnius and Drosophila (seven sequences) | 1000 bootstrap replicates; 7 amino acid sequences | not stated |
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RNAseq quantification was based on two biological replicates per condition and reported with mapping/coverage descriptive statistics.↳ Could also: A dedicated differential-expression framework such as DESeq2 or edgeR, which model count dispersion and report shrinkage-adjusted fold changes with FDR-corrected p-values, could also be applied. — Such tools provide formal statistical inference and dispersion estimates tailored to low-replicate RNAseq, complementing the descriptive coverage summaries presented here.
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Functional enrichment used DAVID overrepresentation p-values with a false-discovery-rate threshold of 0.05.↳ Could also: Gene Set Enrichment Analysis (GSEA) or a hypergeometric/topGO test with Benjamini-Hochberg correction could also be used. — Rank-based or alternative enrichment frameworks can capture coordinated shifts across the full gene list and offer a second, widely used view of pathway-level signal.
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RT-PCR and qRT-PCR assays were performed in biological triplicates to assess transcript presence and knockdown.↳ Could also: Pairing the triplicate qRT-PCR values (e.g., ΔΔCt relative quantification) with an explicit inferential test such as a t-test on log-transformed expression, reported with the dispersion measure (SD or 95% CI), would also be an option. — Reporting an effect size with a spread measure and a stated test conveys the magnitude and uncertainty of the knockdown for small-n data.
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Parental RNAi phenotypes (egg laying, fertility, oogenesis) were assessed across groups of 10 injected females.↳ Could also: Count/proportion data like egg numbers or fertility rates could also be modeled with a generalized linear model (e.g., Poisson/negative-binomial or logistic regression) or compared with a Mann-Whitney/Fisher test, with multiplicity adjustment across the targeted genes. — Distribution-appropriate models and a stated comparison test would formally quantify the between-group differences and the associated uncertainty.
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Phylogenetic node confidence was assessed by 1000 bootstrap replicates on a Maximum Likelihood tree.↳ Could also: Bayesian inference (e.g., MrBayes) reporting posterior probabilities, or approximate-likelihood-ratio (aLRT/SH-aLRT) support, could also accompany the bootstrap values. — A second support metric provides convergent evidence for the inferred relationships and is commonly reported alongside bootstraps.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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PIWI2 knockdown causes dramatic trophocyte loss, egg chamber degeneration, and oogenesis arrest in Rhodnius prolixus ovary.imaging rhodnius prolixus ovary down 2018×1papers★ This paper is the founder (earliest)
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PIWI2, PIWI3, and AGO3 transcripts localize to germline cells and are maternally deposited in eggs; PIWI1 is not expressed in ovaries.imaging rhodnius prolixus ovary 2018×1papers★ This paper is the founder (earliest)
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Parental RNAi knockdown of PIWI2, PIWI3, or AGO3 causes severe egg-laying and fertility defects in Rhodnius prolixus females.other rhodnius prolixus ovary down 2018×1papers★ This paper is the founder (earliest)
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The N-terminal PolyQ tract of PIWI2 is conserved across Triatominae species including Rhodnius neglectus, Triatoma dimidiata, and Triatoma infestans.other triatominae 2018×1papers★ This paper is the founder (earliest)
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Core piRNA pathway components are expressed during previtellogenic stages of Rhodnius prolixus oogenesis.RNA-seq rhodnius prolixus ovary 2018×1papers★ This paper is the founder (earliest)
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A Rhodnius prolixus ortholog of the Drosophila squid (sqd) gene is highly expressed in previtellogenic ovaries.RNA-seq rhodnius prolixus ovary up 2018×1papers★ This paper is the founder (earliest)
Citation network
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Data lineage
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-30303955
Paper: Brito et al. 2018, Transcriptomic and functional analyses of the piRNA pathway in the Chagas disease vector Rhodnius prolixus. PLoS Negl Trop Dis. Code link in brief: https://github.com/gemtools/gemtools (the GEM mapper / GEMtools RNA-seq pipeline — a third-party tool; co-author M. Sammeth authored it). Data: SRA SRP158580 = exactly 2 paired-end RNA-seq runs of previtellogenic ovaries (two biological replicates):
- SRR7738238 = 38,481,892 pairs (2×101 nt) → replicate "1"
- SRR7738239 = 25,723,902 pairs (2×101 nt) → replicate "2"
What this paper actually is (correcting the title's "piRNA" framing)
Despite the piRNA-pathway title, the deposited high-throughput data and the pipeline described in Methods are a paired-end mRNA-seq experiment of ovarian tissue, mapped with the GEMtools pipeline to the R. prolixus genome assembly RproC1. There is no small-RNA-seq library, no piRNA size selection (24–30 nt), no piRNA-cluster prediction (proTRAC/etc.), and no ping-pong / nucleotide-bias computation in the deposited-data pipeline. The piRNA-pathway results in the paper are gene-expression (orthology/qPCR/in-situ) analyses, not a small-RNA bioinformatics pipeline.
IN SCOPE (pipeline-derived, attempted)
| id | result | paper value | pipeline |
|---|---|---|---|
| C1 | per-replicate sequenced read counts | rep1 76,963,784; rep2 51,447,804; total 128,411,588 | direct from deposited FASTQ (count reads) |
| C2 | overall genome mapping rate | rep1 ~89.1%; rep2 ~89.6% | read mapping to RproC1 (paper: GEMtools/GEM; here: HISAT2 splice-aware on the same data — "third-party tool on paper's data", per brief rule P16) |
OUT OF SCOPE (not pipeline / not attempted)
- piRNA-pathway gene identification by orthology to Drosophila (manual/curated bioinformatics, BLAST-by-hand; 1,029 one-to-one orthologs) — not a runnable shipped pipeline; not attempted.
- qPCR expression, in-situ hybridisation, RNAi knockdowns, phenotype scoring — wet-lab, out of scope.
- "Informative reads" %, mismatch/indel averages, transcript-detection tiers (>10/100/1000/10000 reads) — these are GEMtools-pipeline-specific summary statistics; reproducing them 1:1 would require rebuilding the exact 2018 GEMtools RNA pipeline + RProC1.1 annotation. Deferred as the hard last 20% (env_unresolvable risk for the deprecated GEM mapper). Not attempted.
Mapper-substitution caveat (honesty)
The paper's mapping rate is GEMtools-specific. We map the same reads to the same base assembly (GCA_000181055; paper RproC1 = .1) with HISAT2. Overall alignment rate is mapper-dependent, so C2 is graded as a corroboration (within-tolerance / partial), not a bit-exact reproduction. C1 (read counts) is a clean exact check that the deposited data matches the paper's reported numbers.
Assessments & scoring basis
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An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
The deposited SRP158580 data reproduces the paper's per-replicate and total read counts exactly (md5-verified FASTQ), and the overall genome mapping rate is corroborated within ~3pp (HISAT2 92.3-92.4% vs GEMtools ~89.1-89.6%). The mapping-rate offset sits on our side — a deliberate substitution of HISAT2 for the deprecated GEM mapper and RproC3 for the unavailable RproC1 assembly patch — not an authors' defect, and there is no fabrication concern. Severity is negligible (same magnitude/direction). However, the reproduction only covered descriptive data-level claims; the paper's central piRNA-pathway functional conclusions (wet-lab + orthology) were out of scope and untested, so overall quality is solid-but-limited.
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Reproduction footprint
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