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Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper

• This study demonstrates that the marine cyanobacterium Prochlorococcus MED4 employs a light-dependent, predator-inducible synthesis of aldehydes (primarily formaldehyde) as a specific anti-predation defense mechanism against the ciliate Uronema marinum. The findings reveal important mechanistic insights into prey-predator interactions in marine ecosystems and their implications for global biogeochemical cycling.

Major comments - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• Experimental Design: The study employs a well-designed multi-pronged experimental approach. The initial grazing assays screen multiple cyanobacterial and protist strains, followed by mechanistic characterization using targeted approaches. The experimental model organisms are appropriately selected and cultured under controlled conditions with specified light regimens and growth phases.

• Analytical Rigor: The biochemical identification of aldehydes via GC-MS analysis is robust, showing 3.4- and 14.0-fold increases in formaldehyde and acetaldehyde concentrations in co-cultures with U. marinum. The toxicity assessment using LC50 determinations provides quantitative support for the defense mechanism.

• Mechanistic Insights: The investigation of phagolysosomal acidification using LysoTracker Red DND-26 provides elegant evidence for differential digestion efficiency between U. marinum and other protists, supporting the proposed intracellular aldehyde accumulation mechanism.

Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• Potential Limitations

• The study primarily focuses on Prochlorococcus MED4; generalizability to other Prochlorococcus ecotypes (e.g., NATL2A) requires verification

• The ecological relevance of laboratory-determined LC50 values to field conditions remains unclear

• The specificity of this defense to U. marinum is demonstrated, but interaction with other marine predators warrants broader investigation

Clarifications Needed

Light-dependence mechanism: While transcription of mdh and queD genes decreased 19-26% under dark conditions, the regulatory mechanism linking light sensing to aldehyde biosynthesis pathway activation requires further elaboration

Specificity mechanism: The paper establishes that the defense is specific to U. marinum, but the underlying determinants (differential formaldehyde sensitivity vs. digestive efficiency) deserve a clearer mechanistic distinction

Growth phase effects: The investigation of prey growth phase effects on grazing resistance is mentioned, but results and implications are not fully presented in the provided sections

Presentation of Data

The figure captions and supplementary figure references (S5A, S5B-C, S5D, S5E-H) indicate comprehensive supporting data. The schematic diagram (Figure 3G) effectively illustrates the proposed defense mechanism.

Comments on reporting - information on the statistical analyses or availability of data.

• Statistical Analyses

• The document indicates the use of one-way ANOVA followed by Tukey's HSD post hoc test with a significance threshold of p < 0.05. However:

• Sample size (replication numbers) should be explicitly stated in the main text for all experiments

• Error bars represent standard deviations of triplicate experiments, but whether all experiments met this standard is unclear

• The specific statistical tests applied to transcriptomics data, toxicity data, and survival curves require clarification

Data Availability

The manuscript indicates support funding from the Southern Marine Science and Engineering Guangdong Laboratory and the National Natural Science Foundation of China. A statement regarding data and code availability in public repositories should be included.

Methodological Details

The materials and methods section provides comprehensive details on strains used, culture conditions, and grazing assay parameters

Suggestions for future studies

• Ecological validation: Conduct mesocosm or field experiments to test whether observed defense mechanisms operate under natural marine conditions and predation pressures

• Evolutionary perspective: Investigate the evolutionary history of aldehyde biosynthesis genes across Prochlorococcus lineages and their phylogenetic relationship with defense mechanisms in other cyanobacteria

• Genomic survey: Expand analysis to additional Prochlorococcus ecotypes and assess the prevalence and variation of mdh and queD genes encoding aldehyde-synthesizing enzymes

• Compound specificity: Investigate whether other aldehydes or volatile organic compounds beyond formaldehyde and acetaldehyde contribute to the defense phenotype

• Host-pathogen dynamics: Examine the applied potential for biocontrol of U. marinum infections in marine fish aquaculture, as mentioned in the significance statement

• Metabolic costs: Quantify the metabolic burden and fitness costs of maintaining light-dependent aldehyde synthesis under varying nutrient and energy conditions

Conflicts of interests of reviewers

• The authors declare no competing interests

• The author utilized an AI Tool for the purpose of enhancing the readability and language of the review.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they used generative AI to come up with new ideas for their review.