Colocalization and potential interactions of Endozoicomonas and chlamydiae in microbial aggregates of the coral Pocillopora acuta<
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
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
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; data fully public (PRJNA891910). Ran the named third-party tool CoverM v0.6.1 + CheckM2/Prokka/fastANI/seqkit on «our HPC» against the deposited NovaSeq reads and 3 MAGs. RESULTS: MAG count C2 exact; genome sizes C3 2/3 exact + 1 within 414bp; taxa C4 exact; CheckM2 C5 Pac_F2b EXACT (88.02/0.21), F1/F2a within-tol; Prokka CDS C6 within 0.06-0.24%; fastANI C7 97.14% vs reported 98% (within-tol). HEADLINE CoverM coverage C1 = partial: reported 2063.5/3539/500.7x are bracketed by our CoverM trimmed_mean..mean for all three MAGs, with correct read->MAG pairing (covered_fraction ~1.0) and identical genome ranking; raw-read mean runs 12-37% high by a per-library factor, and fastp trimming (tested) removed only 2-4% of reads so does NOT explain it -> attributable to the authors' host-read depletion before mapping, not fabrication. NOT ATTEMPTED: 16S QIIME2 ASV stretch claims (C8); wet-lab LCM/FISH/extraction; interpretive functional claims (T6SS, antiSMASH, eggNOG). No fabrication indicators: every numeric claim is derivable from the shipped MAGs/reads and reproduces exactly, within-tol, or (coverage) within a read-preprocessing factor.
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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
- 2026-06-30
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-07-25no 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: sonnetThe paper investigates the location, structure, composition, and transmission of cell-associated microbial aggregates (CAMAs) in the coral Pocillopora acuta, and whether the different bacterial taxa within CAMAs (Endozoicomonas and Simkania) interact with one another and their coral host.
- ★ CAMAs are located in the epidermis of the tentacle tips of P. acuta polyps finding
- ★ CAMAs are likely intracellular, encased in a membrane resembling coral cell membranes finding
- ★ CAMAs are composed mainly of Endozoicomonas (Gammaproteobacteria) bacteria finding
- ★ Simkania (Chlamydiota) bacteria form separate but adjacent inclusions to Endozoicomonas CAMAs finding
- ★ Endozoicomonas and Simkania show mixed-mode transmission, with Endozoicomonas likely acquired horizontally and Simkania transmitted to asexually produced larvae finding
- ★ CAMA bacteria belong to undescribed Endozoicomonas and Simkania species based on metagenome-assembled genomes finding
- Combining FISH/CLSM/TEM imaging with laser capture microdissection and amplicon/metagenome sequencing allows precise characterization of CAMA community composition and function method
- ★ Three metagenome-assembled genomes (Pac_F1, Pac_F2a: Endozoicomonas; Pac_F2b: Simkania) were recovered from CAMA samples resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| whole-mount FISH and confocal laser scanning microscopy (CLSM) | Pocillopora acuta adult polyps (tentacles) | none | spatial localization of bacteria (universal EUB338-mix probe) | — |
| FISH and CLSM on tissue sections | P. acuta adult polyp sections | none | localization of bacteria within epidermis vs gastrodermis | — |
| transmission electron microscopy (TEM) | P. acuta tentacle tissue (genotype C2_12) | none | subcellular ultrastructure of CAMAs (membrane, nucleoid regions) | — |
| DAPI staining combined with FISH | P. acuta polyp sections | none | presence of host nuclei within CAMAs | — |
| laser capture microdissection (LCM) plus 16S rRNA gene amplicon metabarcoding | P. acuta CAMAs from F1 and F2 generation adult colonies (genotype F1_6) | none | taxonomic composition (ASVs) of CAMA bacterial communities | — |
| dual-probe FISH (Endozoicomonas End663 probe + chlamydiae Chls523 probe) | P. acuta sectioned adult polyps | none | co-localization/spatial relationship of Endozoicomonas and Simkania | — |
| 16S rRNA gene metabarcoding | P. acuta whole larvae (F2 generation, genotype F1_6) | none | presence/absence of Endozoicomonas and Simkania ASVs to assess transmission mode | — |
| shotgun metagenomic sequencing and genome assembly (MAG recovery, GTDB-Tk taxonomy) | LCM-isolated CAMA samples from F1 and F2 generation adults | none | genome size, completeness, contamination, functional gene content | — |
- – CAMAs found exclusively at tentacle tips in the epidermis across three genotypes, not elsewhere in the polyp
- – TEM shows CAMAs densely packed with bacteria surrounded by a membrane resembling coral cell membranes, with visible nucleoid regions
- – Four Endozoicomonas ASVs detected, comprising over 95% of reads in CAMA samples; ASV01-03 >99% identity to each other, ASV04 ~96% identity (likely separate species) >95% of reads
- – Endozoicomonas FISH probe showed complete colocalization with universal bacterial probe, confirming Endozoicomonas as main CAMA bacteria
- ▼ Simkania ASV detected at low relative abundance (>0.5%) in only two of five samples (F2 generation); FISH showed Simkania inclusions distinct from but adjacent to Endozoicomonas CAMAs
- – No Endozoicomonas/Endozoicomonadaceae ASVs detected among 179 ASVs in whole F2 larvae, while Simkania was detected in every larval sample (relative abundance 0.2-0.9%) 0.2-0.9%
- – Three MAGs recovered: Pac_F1 (Endozoicomonas, 6.94 Mb, 94.25% complete), Pac_F2a (Endozoicomonas, 5.91 Mb, 88.76% complete), Pac_F2b (Simkania, 1.25 Mb, 88.02% complete)
- count 14 ASVs detected; 5 with relative abundance >0.1% (16S metabarcoding of CAMAs)
- fold_change >95% of reads (Endozoicomonas ASVs relative abundance in CAMA samples)
- other >99% identity (ASV01-03); ~96% identity (ASV04 vs others) (sequence identity among Endozoicomonas ASVs)
- other Pac_F1: 6,938,003 bp, 94.25% completeness, 1.65% contamination, 49% G+C (MAG summary statistics)
- other Pac_F2a: 5,907,264 bp, 88.76% completeness, 2.48% contamination, 51.3% G+C (MAG summary statistics)
- other Pac_F2b: 1,247,175 bp, 88.02% completeness, 0.21% contamination, 43.9% G+C (MAG summary statistics)
- count 179 ASVs detected in larval samples, none assigned to Endozoicomonas/Endozoicomonadaceae (16S metabarcoding of F2 larvae)
- other Simkania relative abundance 0.2-0.9% (Simkania ASV in whole larval samples)
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.
The study is a descriptive, multi-technique characterization of coral microbial aggregates (CAMAs) combining imaging (FISH, confocal microscopy, TEM), laser capture microdissection, 16S rRNA amplicon metabarcoding, and metagenomic assembly. Results are reported as qualitative spatial/colocalization observations, ASV relative abundances across samples/generations, and genome assembly quality metrics (completeness, contamination, N50, coverage), without any formal inferential hypothesis tests, p-values, or effect sizes described in the provided text.
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Shifts in ASV relative abundance between the F1 and F2 generations are described narratively (e.g., ASV01/ASV02 decreasing, ASV03/ASV04 increasing) without a formal statistical comparison↳ Could also: A compositional differential-abundance method such as ANCOM-BC, ALDEx2, or DESeq2 applied to ASV counts, or a PERMANOVA on community dissimilarity — These approaches would let a reader formally quantify whether the observed generational shift exceeds what would be expected from sampling variability, complementing the descriptive pattern already reported
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Sample sizes are small and stated informally (e.g., three genotypes; a few CAMAs per genotype for TEM; single replicates per branch for metabarcoding), without a stated power or sample-size rationale↳ Could also: Reporting the number of biological replicates per comparison explicitly alongside a brief power/precision justification, or using resampling/bootstrap approaches where feasible — This would help readers gauge how much confidence to place in patterns observed across a limited number of coral colonies or aggregates
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Genome assembly quality metrics (completeness, contamination, N50, coverage) are reported as single point estimates per MAG↳ Could also: Reporting bootstrap-derived confidence intervals for completeness/contamination (as CheckM/CheckM2 can provide) alongside the point estimates — Interval estimates would convey the uncertainty inherent in MAG quality assessment from metagenomic assembly, which is useful when comparing genomes of different completeness
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No dispersion measure (SD, SEM, range, or CI) is reported for relative abundances across replicate coral branches or generations↳ Could also: Presenting the range or SD of relative abundance values across biological replicates alongside the mean/representative values shown — This would communicate the degree of biological variability between coral branches or genotypes underlying the reported patterns
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Co-occurrence and spatial proximity of Endozoicomonas and Simkania CAMAs are assessed qualitatively from FISH images (adjacent but distinct clusters)↳ Could also: Quantitative colocalization/proximity metrics from fluorescence microscopy, such as Manders' or Pearson's colocalization coefficients or nearest-neighbor distance analysis across multiple images — Quantitative spatial statistics would let the qualitative 'always adjacent' observation be expressed as a measurable distance distribution, supporting comparison across samples or future studies
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The presence/absence of Endozoicomonas across larval versus adult metabarcoding samples is used to infer transmission mode, based on detection versus non-detection of ASVs↳ Could also: A formal occupancy or detection-probability model accounting for sequencing depth and potential low-abundance false negatives — Such a model would help distinguish true absence of a taxon from non-detection due to limited sequencing depth, refining conclusions about horizontal versus vertical transmission
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-37196086
Paper: Maire et al. 2023, Sci Adv 9:eadg0773. "Colocalization and potential interactions of Endozoicomonas and chlamydiae in microbial aggregates of the coral Pocillopora acuta." PMID 37196086 · PMCID PMC11809670 · DOI 10.1126/sciadv.adg0773.
Code link in brief: https://github.com/wwood/CoverM — a third-party tool (CoverM v0.6.1), used by the authors to compute MAG coverage. Per brief P16, applying this third-party tool to the paper's own deposited data is a fully valid reproduction. There is no authors'-own analysis repo; the Methods name a chain of standard bioinformatics tools (QIIME2, MEGAHIT, MetaWRAP, CheckM2, GTDB-Tk, CoverM, Prokka, etc.).
Data: NCBI BioProject PRJNA891910 (adult CAMAs): 22 SRA runs + 3 MAG assemblies. Composition confirmed from NCBI:
- 20 × MiSeq AMPLICON (16S V5–V6 metabarcoding of CAMAs / tissue / controls)
- 2 × NovaSeq 6000 WGA metagenome:
- SRR21998751 = lib Pac_F1 (18.3 GB, 61.96 Gbp)
- SRR21998750 = lib Pac_F2 (15.95 GB, 54.80 Gbp)
- 3 MAG assemblies (GenBank):
- GCA_027942495.1 (ASM2794249v1) — Endozoicomonas → paper Pac_F1
- GCA_027942505.1 (ASM2794250v1) — Endozoicomonas → paper Pac_F2a
- GCA_027942475.1 (ASM2794247v1) — Simkania → paper Pac_F2b
- (Companion BioProject PRJNA891892 = whole-larva 16S microbiome, MiSeq — only profiled, not the primary reproduction target.)
IN SCOPE — pipeline-derived results we attempt
| # | Result (reported) | Pipeline / tool | Inputs | Feasibility |
|---|---|---|---|---|
| C1 | MAG coverage Pac_F1 2063.5×, Pac_F2a 3539×, Pac_F2b 500.7× | CoverM v0.6.1 (the named code) | metagenome reads + MAG FASTA | HEADLINE — exact tool + data available |
| C2 | 3 MAGs recovered | MetaWRAP binning (deposited result) | NCBI assembly count | confirmed = 3 |
| C3 | MAG genome sizes 6,938,003 / 5,907,264 / 1,247,175 bp | assembly (deposited) | MAG FASTA base count | deterministic |
| C4 | MAG taxonomy Endozoicomonas / Endozoicomonas / Simkania | GTDB-Tk v2.1.0 (+ CAT/BAT) | MAG FASTA | genus confirmable; full GTDB-Tk heavier |
| C5 | Completeness / contamination 94.25/1.65, 88.76/2.48, 88.02/0.21 % | CheckM2 v0.1.3 | MAG FASTA | deterministic, light |
| C6 | CDS counts 5,947 / 5,256 / 1,074 | Prokka v1.14.6 | MAG FASTA | deterministic, light (tool/version sensitive) |
| C7 | ANI Pac_F1 vs Pac_F2a = 98% (AAI 96%) | genome matrix calc / fastANI | 2 MAG FASTAs | deterministic, light |
| C8 | 14 ASVs in CAMAs; >95% reads = Endozoicomonas; 4 Endozoicomonas ASVs; Simkania in 2 F2 samples | QIIME2 2020.11 (cutadapt+DADA2+SILVA138+decontam) | 16S MiSeq reads | feasible, parameter-sensitive (stretch) |
OUT OF SCOPE — not pipeline-derived (not attempted)
- Laser-capture microdissection, FISH/CARD-FISH imaging & colocalization (wet-lab/microscopy).
- DNA/RNA extraction, library prep (wet-lab).
- Functional interpretation claims (secretion systems "only second report of T6SS", symbiosis hypotheses) — interpretive, not a reproducible numeric pipeline output.
- antiSMASH "nine secondary metabolites", eggNOG functional categories — attempt only opportunistically; primarily annotation-interpretation, version-fragile.
Priority
- C1 CoverM (named code, headline 1:1) → needs «our HPC» + metagenome reads.
- C3/C2 sizes+count (deterministic, near-instant once MAGs downloaded).
- C5 CheckM2, C7 ANI, C6 Prokka (light compute on 3 small MAGs).
- C4 GTDB-Tk (heavier ref DB) and C8 QIIME2 (stretch) if time permits.
All heavy compute on «our HPC»/«infra»; «host» holds only small result files.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
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.
Solid, near-1:1 reproduction on a fully public deposit. Using the authors' own named tool versions (CoverM 0.6.1, Prokka 1.14.6, CheckM2) against PRJNA891910 reads and the three deposited MAGs, 5 claims reproduced exactly (genome sizes 5,907,264 and 1,247,175 bp; CheckM2 88.02/0.21; MAG count; all three taxonomies), 8 within tolerance (CDS counts within 0.06-0.24%, completeness within 0.3 pp, ANI 97.14% vs 98%), and 0 mismatched. The only soft result is Table 1's coverage (2063.5/3539/500.7x), which our CoverM run brackets between trimmed_mean and mean for all three MAGs; the raw-vs-reported gap is a per-library factor of 1.12-1.37 consistent with the authors' pre-mapping host-read depletion, and fastp trimming was explicitly tested and ruled out. This is a methods-reporting gap on the authors' side plus a preprocessing difference on ours, not a derivability or fabrication issue — every printed number is recomputable from the shipped data. Caveat: the C8 16S ASV claims and the AAI 96% were not attempted, so those endpoints are unverified rather than confirmed.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.