Hepcidin from the Chinese Spiny Frog (Quasipaa spinosa) Integrates Membrane-Disruptive Antibacterial Activity with Macrophage-Mediated Protection Against Elizabethkingia miricola

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review report

 

This manuscript presents the first functional characterization of a hepcidin homolog from the Chinese spiny frog Quasipaa spinosa (QsHep), emphasizing its roles in antibacterial defense and macrophage activation. The authors combine molecular cloning, expression profiling, synthetic peptide assays, macrophage functional tests, and an in vivo infection model. Overall, the study is well-framed within the context of hepcidin’s dual role in vertebrate immunity and iron regulation. The experiments are logically organized, and the results collectively point to QsHep as an antimicrobial and immunomodulatory peptide with potential application in amphibian aquaculture. The manuscript is generally well-written, logically structured, and the findings are clearly presented. However, there are some methodological issues that need to be addressed to strengthen the manuscript.

 

1. The frogs’ sex, developmental stage, and health screening criteria were not reported, all of which may influence immune responses.
2. Water quality parameters (pH, ammonia, nitrite, dissolved oxygen, photoperiod) were not described, limiting replicability.
3. It is unclear whether the transcriptome dataset was validated independently (e.g., through RACE-PCR, genome alignment).
4. No information on open-reading frame confirmation, sequence verification, or sequencing depth/quality.
5. No description of how bacterial strains were maintained, prepared, or standardized before microdilution.
6. How bacterial pellets were washed or normalized before treatment?
7. How LDH from bacteria (not eukaryotic cells) was validated for assay compatibility?

One-way ANOVA is used, but: Many datasets involve multiple factors (e.g., dose × stimulation), making ANOVA insufficient. No post hoc tests (Tukey, Dunnett) are reported. The statement “means ± SEM” is acceptable, but exact biological replicates per assay are missing

Author Response

Comment 1. The frogs’ sex, developmental stage, and health screening criteria were not reported, all of which may influence immune responses.

Response 1: We appreciate this important comment. We have now clarified the sex, developmental stage and health-screening criteria of the experimental animals in the Materials and Methods. Specifically, all experiments were conducted with sexually mature, post-metamorphic Chinese spiny frogs of both sexes. These details have been added to Section 2.1 (Animals), and cross-referenced in Sections 2.3, 2.7 and 2.12.

Comment 2. Water quality parameters (pH, ammonia, nitrite, dissolved oxygen, photoperiod) were not described, limiting replicability.

Response 2: We thank the reviewer for pointing out this omission. In the revised manuscript, we have now described the husbandry conditions and key water-quality parameters, including pH, temperature, dissolved oxygen, nitrogenous waste control, and photoperiod, to improve transparency and replicability. These details have been added to the Materials and Methods section.

Comment 3. It is unclear whether the transcriptome dataset was validated independently (e.g., through RACE-PCR, genome alignment).

Response 3: The liver transcriptome dataset of Chinese spiny frog used in this study was generated in our previous work, which analyzed the liver immune response to Proteus mirabilis infection (Liu et al., 2024, Open Life Sci 19:20221003). In that study, raw reads were subjected to standard quality control and de novo assembly, and assembly quality and annotation statistics were reported in detail. Moreover, the expression patterns of several representative differentially expressed genes were independently validated by quantitative real-time PCR, confirming the reliability of the RNA-seq dataset. In the present work, this validated liver transcriptome was re-used to identify the full-length QsHep sequence and to evaluate its transcriptional regulation.

Comment 4. No information on open-reading frame confirmation, sequence verification, or sequencing depth/quality.

Response 4: We thank the reviewer for this helpful suggestion. In the revised manuscript, we now state in Section 2.2 that the open reading frame (ORF) of QsHep was predicted using ORF Finder and that the cDNA sequence was confirmed by Sanger sequencing. We also clarify that the liver transcriptome dataset (PRJNA1012438) used to obtain the QsHep cDNA was generated and independently validated in our previous study, where sequencing depth and quality metrics were reported in detail (Liu et al., 2024, Open Life Sci 19:20221003).

Comment 5. No description of how bacterial strains were maintained, prepared, or standardized before microdilution.

Response 5: We thank the reviewer for this useful comment. In the revised Methods, we now describe that all bacterial strains were stored at −80°C in glycerol stocks, subcultured on agar plates, and then grown in broth to mid-log phase before testing. The bacterial suspension was adjusted to a 0.5 McFarland standard and further diluted to yield a final inoculum of approximately 1 × 10⁵ CFU/mL in each well for the broth microdilution assay.

Comment 6. How bacterial pellets were washed or normalized before treatment?

Response 6: We thank the reviewer for this helpful comment. In the revised Methods, we now clarify that after culture, bacteria were harvested by centrifugation, washed twice with sterile PBS, and resuspended to the same optical density (OD600) before peptide treatment so that all bacterial pellets were normalized to a comparable cell density.

Comment 7. How LDH from bacteria (not eukaryotic cells) was validated for assay compatibility?

Response 7: We appreciate this important comment. Lactate dehydrogenase (LDH) is a ubiquitous cytosolic enzyme present in virtually all living cells, including bacteria, and the colorimetric LDH kit we used detects enzymatic activity rather than species-specific epitopes. To ensure assay compatibility with our bacterial strains, we included wells in which bacteria were fully lysed by detergent; these lysates produced strong LDH signals above medium background and were used as 100% release controls. We have added a sentence in the LDH assay subsection of the Methods to clarify this validation.

Comment 8. One-way ANOVA is used, but: Many datasets involve multiple factors (e.g., dose × stimulation), making ANOVA insufficient. No post hoc tests (Tukey, Dunnett) are reported. The statement “means ± SEM” is acceptable, but exact biological replicates per assay are missing.

Response 8: We thank the reviewer for this thoughtful statistical comment. In the revised manuscript, we have clarified both our study design and the statistical analyses. All experiments in this study were designed to address a single experimental factor at a time, namely the effect of different concentrations of QsHep under a given condition. Accordingly, datasets involving more than two groups within a single factor were analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons test, whereas comparisons between only two groups were analyzed by unpaired Student’s t-tests. In addition, we now explicitly state in the Statistical analysis subsection that all data are presented as means ± SEM and that “n” refers to biological replicates (independent cell preparations or individual frogs), with the exact n indicated in each figure legend. Regarding the respiratory burst assay, our experimental question was limited to two pre-specified comparisons: QsHep versus BSA in unstimulated cells, and QsHep + PMA versus BSA + PMA in PMA-stimulated cells. We did not aim to test a full dose × stimulation interaction for this assay. Accordingly, these data were analyzed using one-way ANOVA within each stimulation condition rather than a two-way ANOVA, and this has now been clarified in the Statistical analysis section. We hope that these clarifications address the reviewer’s concerns regarding the suitability of ANOVA, the post hoc tests used, and the reporting of biological replicates.

Reviewer 2 Report

Comments and Suggestions for Authors

The article studies QsHep, an amphibian hepicidin that combines membrane-disruptive antibacterial activity with the activation of macrophage effector functions and confers significant protection against bacterial infection in vivo.  

The experimental design is sound, and the presentation is straigthforward. 

This reviewer would have liked to see more emphasis be given to possible utilisation of these compounds, in a prophylactic approach. The authors are urged to include a discussion on that 

Author Response

Comment 1. This reviewer would have liked to see more emphasis be given to possible utilisation of these compounds, in a prophylactic approach. The authors are urged to include a discussion on that. 

Response 1: We thank the reviewer for this constructive suggestion. In the revised Discussion, we have now added a dedicated paragraph highlighting the potential prophylactic applications of QsHep. Based on our in vitro antimicrobial and immunomodulatory data, and on published work showing that hepcidin peptides can be administered orally or as feed additives to enhance innate immunity, improve growth performance, and increase disease resistance in farmed fish, we propose that QsHep or QsHep-derived analogues could be developed as prophylactic immunostimulants in Chinese spiny frog culture (e.g., via in-feed supplementation or immersion during high-risk periods). We also emphasize that future in vivo studies will be required to optimize dose, route, and timing, and to assess long-term safety and efficacy before QsHep-based prophylactic strategies can be implemented in practice.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no further comments.