A single-cell survey of Drosophila blood.
The main results reproduced: recomputed values matched the published ones within tolerance.
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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.
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- Reproduced
- 2026-06-19
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no human curator yet
- Last updated
- 2026-07-29
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Deep full-text extraction
Model: opusHow diverse is the Drosophila larval hemocyte (blood cell) repertoire at the molecular level, and can single-cell RNA sequencing across steady-state and inflammatory conditions resolve mature hemocyte types into distinct subtypes, intermediate states, and activated states to reveal new immune functions?
- ★ scRNA-seq of Drosophila larval hemocytes across unwounded, wounded, and wasp-infested conditions resolves 17 clusters spanning plasmatocytes, crystal cells, lamellocytes, and a non-hemocyte population. finding
- ★ Plasmatocytes are resolved into multiple states defined by cell cycle (PM2), immune activation/antimicrobial peptide expression (PM3-7), metabolism, and several minor subpopulations (PM8-12). finding
- ★ Crystal cells and lamellocytes each split into two clusters representing a mature state and an immature/intermediate state (CC1 immature vs CC2 mature; LM1 intermediate vs LM2 mature). finding
- ★ Rare subsets of crystal cells express the FGF ligand branchless (bnl) and rare lamellocytes express the FGF receptor breathless (btl), revealing FGF signaling components within hemocytes. finding
- ★ FGF signaling (bnl/btl) mediates inter-hemocyte crosstalk required for effective immune responses against parasitoid wasp eggs in vivo. mechanism
- ★ A novel Metchnikowin-like antimicrobial peptide gene (CG43236/Mtk-like, Mtkl) is identified enriched in plasmatocyte cluster PM7. finding
- ★ A searchable Drosophila blood scRNA-seq web portal provides a resource of hemocyte gene expression profiles across conditions. resource
- Harmony batch correction integrated into Seurat was used to merge inDrops, 10X Chromium, and Drop-seq datasets and remove batch effects. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| single-cell RNA sequencing (inDrops) | Drosophila melanogaster larval hemocytes | wounding / wasp infestation / unwounded control | per-cell gene expression (UMIs, transcriptomes), cell clustering | inDrops |
| single-cell RNA sequencing (10X Chromium) | Drosophila melanogaster larval hemocytes | wounding / wasp infestation / unwounded control | per-cell gene expression / cell clustering | 10X Chromium |
| single-cell RNA sequencing (Drop-seq) | Drosophila melanogaster larval hemocytes | wounding / wasp infestation / unwounded control | per-cell gene expression / cell clustering | Drop-seq |
| quantitative real-time PCR (qRT-PCR) | Drosophila larval sessile and circulating hemocytes (unwounded) | none (steady state) | expression of plasmatocyte cluster marker genes in sessile vs circulating compartments | — |
| bulk RNA-seq comparison (pseudobulk vs published bulk) | Drosophila larval whole hemocytes and lz+ crystal cells | none | Spearman correlation of pseudobulk scRNA-seq vs published bulk RNA-seq | — |
| in vivo functional immune assay | Drosophila larvae | parasitoid wasp (Leptopilina boulardi) infestation; FGF (bnl/btl) manipulation | immune response against wasp eggs | — |
- – 19,458 hemocytes profiled across conditions yielding 17 clusters after Harmony integration 19,458 cells; 17 clusters
- – PM6 cecropin-enriched cluster proportion matches previously observed fraction of cecropin-expressing hemocytes upon infection ~5.5% (vs prior ~5-10%)
- ▲ PM AMP clusters (PM6, PM7) and lamellocyte LM2 emerge mainly upon wounding or wasp infestation
- ▼ Crystal cell clusters CC1 and CC2 are underrepresented in wasp inf. 24 hr, consistent with crystal cell loss after oviposition
- – Pseudobulk scRNA-seq correlates strongly with published bulk RNA-seq for whole hemocytes and crystal cells, indicating high data quality Spearman r ~0.79
- – A non-hemocyte cluster lacks pan-hemocyte markers He and srp ~0.2% of cells
- ▲ Novel Mtk-like AMP (Mtkl/CG43236) identified among top expressed genes of cluster PM7
- count 19,458 cells (total hemocytes profiled across all conditions/platforms)
- count median 1010 genes per cell (median genes detected per cell across conditions)
- count median 2883 UMIs per cell (median unique molecular identifiers per cell)
- correlation Spearman ~0.79 (pseudobulk scRNA-seq vs published bulk RNA-seq for hemocytes and crystal cells)
- count 17 clusters (total clusters identified after Harmony batch correction)
- other ~5.5% (proportion of cecropin-enriched PM6 cluster)
- other ~90-95% (plasmatocyte fraction of total hemocytes)
- other ~0.2% (fraction of profiled cells in non-hemocyte cluster)
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.
The study is a descriptive single-cell RNA-sequencing survey of Drosophila hemocytes across unwounded, wounded, and wasp-infested larvae, profiling 19,458 cells with 3–4 replicates per condition on three platforms (inDrops, 10X Chromium, Drop-seq). Data sets were merged, batch-corrected with Harmony within the Seurat pipeline, and unsupervised clustering identified 17 clusters annotated by known and de novo marker genes (ranked by average log fold-change). Validation relied on Spearman correlation of pseudobulk scRNA-seq against published bulk RNA-seq, plus qRT-PCR of selected markers; results were reported largely as cluster identities, marker dot/heat maps, and cell-fraction changes rather than formal hypothesis tests.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Spearman rank correlation | pseudobulk scRNA-seq vs published bulk RNA-seq for all hemocytes and for crystal cells (Figure 1—figure supplement 2A,B) | — | not stated |
| Cluster marker / differential expression ranking by average log fold-change (avg_logFC) | top genes per cluster, dot plot (Figure 1D) and marker tables (Supplementary file 2) | — | not stated |
| qRT-PCR expression comparison (sessile vs circulating hemocytes) | plasmatocyte marker genes in unwounded larvae (Figure 1—figure supplement 1G) | — | na |
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Pseudobulk-to-bulk concordance was quantified with a Spearman correlation coefficient (~0.79).↳ Could also: A Pearson correlation on log-transformed counts, or a concordance correlation coefficient, could also be reported, optionally with a confidence interval. — Pearson on log-scale captures linear agreement and a confidence interval would also convey the precision of the correlation estimate; reporting both gives a fuller picture of agreement.
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Cluster marker genes were prioritized by average log fold-change.↳ Could also: Adjusted p-values from a differential-expression test (e.g. Wilcoxon rank-sum with Benjamini-Hochberg FDR, or model-based methods such as MAST or DESeq2 on pseudobulk) could also accompany the fold-change ranking. — Pairing effect size with an FDR-controlled significance value would also communicate statistical confidence in each marker alongside its magnitude.
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Cell-fraction changes across conditions were summarized as proportions/percentages per cluster (Figure 2D).↳ Could also: A compositional-analysis framework (e.g. scCODA, or a Dirichlet-multinomial / proportion test across replicates) could also be applied. — Such approaches would also incorporate replicate-to-replicate variability and the compositional nature of the data when describing fraction shifts.
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Batch effects across condition, replicate, and technology were addressed with Harmony.↳ Could also: Alternative integration methods such as Seurat CCA/RPCA anchors, scVI, or fastMNN could also be used. — Comparing integration methods would also let one describe how robust the cluster structure is to the choice of batch-correction algorithm.
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Marker expression in sessile vs circulating compartments was assessed by qRT-PCR (Figure 1—figure supplement 1G).↳ Could also: Replicate-level qRT-PCR values could also be summarized with SD or a 95% CI and compared with a t-test or Mann-Whitney U, depending on n and distribution. — Reporting spread and an accompanying test would also convey the variability and the strength of the sessile-vs-circulating difference.
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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AMP-enriched plasmatocyte clusters (PM6, PM7) and lamellocyte LM2 are induced upon wounding or wasp infestationscRNA-seq drosophila larval hemocytes up 2020×1papers★ This paper is the founder (earliest)
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a cecropin-enriched plasmatocyte cluster (PM6) comprises a fraction matching previously observed cecropin-expressing hemocytes upon infectionscRNA-seq drosophila larval hemocytes up 2020×1papers★ This paper is the founder (earliest)
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crystal cell clusters are underrepresented after wasp infestation, consistent with crystal cell loss following ovipositionscRNA-seq drosophila larval hemocytes down 2020×1papers★ This paper is the founder (earliest)
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a non-hemocyte cluster lacks the pan-hemocyte markers Hml (He) and srpscRNA-seq drosophila larval hemocytes none 2020×1papers★ This paper is the founder (earliest)
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a novel Metchnikowin-like AMP Mtkl (CG43236) is highly expressed in plasmatocyte cluster PM7scRNA-seq drosophila larval hemocytes up 2020×1papers★ This paper is the founder (earliest)
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pseudobulk scRNA-seq correlates strongly with published bulk RNA-seq for whole hemocytes and crystal cellsscRNA-seq drosophila larval hemocytes none 2020×1papers★ This paper is the founder (earliest)
Citation network
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Data lineage
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
All examined headline values reproduce from the authors' own deposited GSE146596 metadata: cell count (19,458) and cluster count (17) are exact, and median genes/UMIs differ only by 1 (rounding/even-N median, ~0.1%). No value was found non-derivable from the shipped data — no fabrication signal. The main scope caveat is on our side, not the authors': this is an internal-consistency audit of the deposited output vs the manuscript, not a from-FASTQ re-derivation, and cell-type identities (C3) plus independent re-clustering (C7) remain manual/pending.
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