Central carbon metabolism switching in lytic versus temperate coral reef viral communities

Reviewer #1 (Public review):

Summary:

Microbialization (bacterial overgrowth) is a recognized component of degraded, eutrophied coral reefs where there is a shift from coral to algal dominance on the benthos. In addition, previous work has demonstrated that virus communities shift from a lytic strategy dominated (kill-the-winner) to a temperate (lysogenic) strategy dominated with reef microbialization. Kelman et al. sought to leverage previously published virus metagenomes produced from the water column of healthy and degraded coral reefs to assess virus community metabolic shifts. The authors also produce a conceptual model to demonstrate the potential impact of the observed metabolism shifts on reef fates.

Strengths:

The main strength of the manuscript is the findings from their metagenomic analyses and results. The virus metagenomes were produced using established approaches in the field and yield sufficient data per sample for their analyses. Interesting results regarding the shift in the types of genes from anaplerotic to cataplerotic provide the foundation for testable hypotheses to determine the magnitude of impact virus strategies have on reef health. The introduction is also well written and sets up the scene very well.

Weaknesses:

(1) The methods text currently omits important information related to the sampling design. It is not clear how many metagenomes are from healthy and degraded communities. This impacts the interpretability and robustness of the statistical results. Furthermore, it is unclear if analyses are based on assembled contigs or read-based alignments. Improving the clarity and organization of the Methods is essential for reproducibility.

(2) Regarding the bioinformatics approach, normalization using the "percent known" approach within samples may not fully account for discovery bias related to sequencing depth. While Supplementary Table 1 shows variability in read counts, the lack of community-level metadata makes it difficult to determine if sequencing depth covaries with community type (healthy vs. degraded). The study would benefit from a rarefaction analysis or subsampling to ensure that gene frequency trends and Spearman correlations are biological signals rather than artifacts of sequencing effort.

(3) The qualitative model in Figure 5 is positioned as evidence for the role of viruses in reef health, but it does not provide independent support for the authors' hypotheses. Since the model is parameterized using "arbitrary units" to reflect the authors' assumptions rather than being derived from the empirical metagenomic data, it serves as a helpful illustration of a hypothesis but not as a validation of the findings.

(4) Results and discussion require revisions to improve readability and connectivity across sections. Ensuring a clear distinction between empirical data and model-based speculation would help the audience better appreciate the science.

Reviewer #2 (Public review):

Summary:

The manuscript by Kelman and coauthors investigates how viral communities differ in the genes they encode in healthy and degraded coral reef ecosystems. Across 19 viral metagenomes from Central Pacific reefs, the authors assess the frequency of integration/excision genes as a proxy for viral community temperateness and ask whether genes associated with central carbon metabolism covary with signatures of temperateness. The main finding is that viral communities with more temperate-related genes encode more genes from the Entner-Doudoroff pathway and other reactions interpreted as anaplerotic, whereas more lytic-associated viral communities show greater representation of some pentose phosphate pathway, TCA, and redox-associated genes interpreted as cataplerotic. The authors propose a model based on these patterns in which lytic viral metabolism helps suppress bacterial overgrowth on healthy reefs, while temperate viral metabolism may promote microbialization on degraded reefs. The study addresses an interesting and potentially important concept - that viral auxiliary metabolic genes are important components of microbial communities and can affect ecosystem functioning. Linking viral metabolism to coral reef microbialization is a creative conceptual advance. The manuscript is clearly written, and the reported enrichment of anaplerotic genes in temperate-associated viromes is an interesting pattern that could motivate future work on how viral metabolic potential varies across reef states.

Strengths:

(1) The study connects viral lifestyle, central carbon metabolism, bacterial overgrowth, and reef degradation in a framework that could be useful for future studies of coral reef ecosystems and viral ecology. This is an interesting synthesis that links viral auxiliary metabolism to broader questions about microbialization and reef state.

(2) The manuscript is generally clearly organized around a testable prediction: viral metabolic gene content should vary along a lytic-to-temperate viral community gradient. The reported enrichment of anaplerotic genes in viromes with a larger fraction of temperate viruses is a compelling result.

(3) The authors highlight several virus-encoded metabolic genes that may not have been previously reported in viral datasets or genomes. If supported by further validation, these observations could expand the known repertoire of viral metabolic potential.

(4) The modeling helps clarify the feedbacks the authors propose may connect viral lifestyle, bacterial metabolism, and coral reef degradation. It provides a foundation for generating hypotheses about how viral metabolic genes could influence reef microbial dynamics.

Weaknesses:

(1) The main limitation is that the evidence for several key claims remains indirect. The core analysis is based on correlations between metabolic gene frequencies and integration/excision-related genes. This does not demonstrate that the metabolic genes occur in temperate viral genomes, are physically linked to lysogeny genes, are expressed during infection, or alter host metabolism. Thus, the data support an association between VLP-associated metabolic annotations and a community-level temperateness proxy, but not a direct link between temperate phages and these metabolic functions.

(2) It is important not to equate community-level gene frequencies with genome-level or infection-level metabolic programs. A virome may contain more anaplerotic genes overall, but that does not demonstrate that individual viruses reprogram their hosts in an anaplerotic manner nor that infection produces a net anaplerotic effect. Individual viruses may encode both anaplerotic and cataplerotic genes, and a smaller number of cataplerotic genes could have stronger metabolic consequences depending on expression, enzyme efficiency, pathway position, and host context. This is an important limitation that should be acknowledged and, if possible, addressed with contig- or genome-level analyses.

(3) The ecological interpretation assumes that viral infection is strong enough to influence reef-scale bacterial population dynamics. However, the study does not directly measure infection frequency, lysis rates, viral production, burst size, lysogeny frequency, prophage induction, gene expression, or bacterial mortality. If viral mortality or lysogenic conversion were rare in these systems, the observed gene-frequency patterns could have limited ecosystem-level consequences. This makes claims about viral metabolism suppressing bacterial overgrowth, accelerating microbialization, or acting as a conservation lever more speculative than suggested.

(4) There are statistical limitations related to the use of relative gene frequencies. Because genes are normalized as percentages of known genes, the data are compositional. Apparent increases in some categories may partly reflect decreases in others. Bootstrapped Spearman correlations are useful for assessing the robustness of these associations, but they do not address compositionality or multiple testing.

(5) The anaplerotic/cataplerotic classification is central to the manuscript's conclusions and would benefit from more support. The framework is useful, but it depends on both annotation confidence and biochemical context. Sequence-similarity annotations alone may be vulnerable to misannotation, especially for central metabolic enzymes that share conserved domains across functionally distinct proteins. Stronger evidence that key genes contain key functional domains and/or are phylogenetically related to characterized enzymes would help support the proposed functions. In addition, many central carbon enzymes are reversible or context-dependent, so a clearer rationale for each classification would strengthen the interpretation.

Overall, the manuscript presents a valuable hypothesis and highlights new ecological patterns in coral reef viral metagenomes, but falls short of the evidence needed for the strongest claims. The work would be strengthened by analyses that directly link metabolic genes to viral genomes or lysogeny markers, address compositional effects, validate key annotations, and more clearly distinguish observed gene-frequency associations from hypothesized effects on infection, host metabolism, and reef state.