Linalool’s Multifaceted Antimicrobial Potential: Unveiling its Antimicrobial Efficacy and Immunomodulatory Role Against Saprolegnia parasitica

Reviewer #1 (Public review):

Summary:

The work seeks to investigate the efficacy of linalool as a natural alternative for combating Saprolegnia parasitica infections, which would provide great benefit to aquaculture. This paper shows the effect of linalool in vitro using a variety of techniques including changes in S. parasitica membrane integrity following linalool exposure and alterations in cell metabolism and ribosome function. Additionally, this work goes on to show that prophylactic and concurrent treatment of linalool at the time of S. parasitica infection can improve survival and tissue damage in vivo in their grass carp infection model. The conclusions of the paper are partially supported by the data, cleaning up, clarifying, and elaborating on some aspects of this work is necessary.

(1) Adding microscopy of the untreated group to compare Figure 2A with would further strengthen the findings here.

(2) Quantification of immune infiltration and histological scoring of kidney, liver, and spleen in the various treatment groups would increase the impact of Figure 4.

(3) The data in Figure 6 I is not sufficiently convincing as being significant.

(4) Comparisons of the global transcriptomic analysis of the untreated group to the PC, LP, and LT groups would strengthen the author's claims about the immunological and transcriptomic changes caused by linalool and provide a true baseline.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to delineate the antimicrobial activity of linalool and tried to investigate the mode of action of linalool against S. parasitica infection. One of the main focuses of this work was to identify the in vitro and in vivo mechanisms associated with the protective role of linalool against S. parasitica infection.

Strengths:

(1) The authors have used a variety of techniques to prove their hypothesis.

(2) An adequate number of replicates were used in their studies.

(3) Their findings showed a protective role of linalool against oomycetes and makes it an attractive future antibiotic in the aquaculture industry.

Weaknesses:

There are several weaknesses in this manuscript.

(1) The authors have taken for granted that the readers already know the experiments/assays used in the manuscript. There was not enough explanation for the figures as well as figure legends.

(2) The authors missed adding the serial numbers to the references.

(3) The introduction section does not provide adequate rationale for their work, rather it is focused more on the assays done.

(4) Full forms are missing in many places (both in the text and figure legends), also the resolution of the figures is not good. In some figures, the font size is too small.

(5) There is much mislabeling of the figure panels in the main text. A detailed explanation of why and how they did the experiments and how the results were interpreted is missing.

(6) There is not enough experimental data to support their hypothesis on the mechanism of action of linalool. Most of the data comes from pathway analysis, and experimental validation is missing.

Overall, the conclusions drawn by the authors are partially justified by the data. Importantly, this paper has discovered the novelty of the compound linalool as a potent antimicrobial agent and might open up future possibilities to use this compound in the aquaculture industry.

Author response:

Reviewer #1:

(1) Adding microscopy of the untreated group to compare Figure 2A with would further strengthen the findings here.

Thank you for your comments on our manuscript. We will carefully revise this part. Actually, we used a time-lapse method to capture images at 0 minute before any drugs were added. We will change '0 min' to 'untreated,' which will further strengthen our findings.

(2) Quantification of immune infiltration and histological scoring of kidney, liver, and spleen in the various treatment groups would increase the impact of Figure 4.

Thank you for your comments on our manuscript. To further strengthen Figure 4, we will use quantification of immune infiltration and histological scoring of the kidney, liver, and spleen in different groups. Additionally, we will use ImageJ software for molecular immunohistochemistry and determine the ratio of normal to abnormal cells, providing more comprehensive insights into the effects of the treatments.

(3) The data in Figure 6 I is not sufficiently convincing as being significant.

Thank you for your comments on our manuscript. Previous researches have shown that antibiotics and other drugs can cause alterations in gut microbiota. Therefore, we plan to study the effects of linalool on gut microbiota. The results of this part were mostly built on gut microbiota sequencing and correlation analysis, we have tried several times to isolate vital microbes from the gut, but this is a very challenging work and the results were not good. Thus, in this study, we just predicted the effects of linalool on gut microbiota. In the future, we will continue to delve into interesting aspects of how linalool affects gut microbiota.

(4) Comparisons of the global transcriptomic analysis of the untreated group to the PC, LP, and LT groups would strengthen the author's claims about the immunological and transcriptomic changes caused by linalool and provide a true baseline.

Thank you for your comments on our manuscript. We will compare the global transcriptomic analysis of the untreated group with the PC, LP, and LT groups to strengthen the claims about the immunological and transcriptomic changes induced by linalool, thereby providing a true baseline.

Reviewer #2:

(1) The authors have taken for granted that the readers already know the experiments/assays used in the manuscript. There was not enough explanation for the figures as well as figure legends.

Thank you for your comments on our manuscript. We will provide more detailed explanations of the experiments and assays used in the manuscript, as well as enhance the descriptions in the figure legends, to ensure that readers have a clear understanding of the figures and context.

(2) The authors missed adding the serial numbers to the references.

Thank you for your comments on our manuscript. We will add serial numbers to the references to ensure proper citation and improve the clarity of our manuscript.

(3) The introduction section does not provide adequate rationale for their work, rather it is focused more on the assays done.

Thank you for your comments on our manuscript. We will add a section to the introduction that provides a rationale for our work, specifically focusing on the impact of plant extract on immunoregulation.

(4) Full forms are missing in many places (both in the text and figure legends), also the resolution of the figures is not good. In some figures, the font size is too small.

Thank you for your comments on our manuscript. We will ensure that all abbreviations are expanded where necessary, both in the text and figure legends. Additionally, we will improve the resolution of the figures and increase the font size where needed to enhance clarity.

(5) There is much mislabeling of the figure panels in the main text. A detailed explanation of why and how they did the experiments and how the results were interpreted is missing.

Thank you for your comments on our manuscript. We will improve the labeling of the figure panels, provide detailed explanations of the experimental methods, including their rationale and interpretation, and clarify the connections between the methods.

(6) There is not enough experimental data to support their hypothesis on the mechanism of action of linalool. Most of the data comes from pathway analysis, and experimental validation is missing.

Thank you for your comments on our manuscript. We have tried our best to link transcriptomic data, pathway analysis, experimental validation. We carried out many experiments to substantiate the changes inferred from the transcriptomic data as SEM, TEM, CLSM, molecular docking, RT-qPCR, histopathological examinations. The detailed information is listed as follows. (1) As shown in Figure 2, we combined the transcriptomic data related to membrane and organelle with SEM, TEM, and CLSM images. After deep analysis of these data and observation together, we illustrated that cell membrane may be a potential target for linalool. (2) As shown in Figure 3, we carried out molecular docking to explore the specific binding protein of linalool with ribosome which were screen out as potential target of linalool by transcriptomic data. (3) As shown in Figure 5, transcriptomic data illustrated that linalool enhanced the host complement and coagulation system. To substantiate these changes, we carried out RT-qPCR to detect those important immune-related gene expressions, and found that RT-qPCR analysis results were consistent with the expression trend of transcriptome analysis genes. (4) As shown in Figure 4 and 5, transcriptomics data revealed that linalool promoted wound healing tissue repair, and phagocytosis (Figure. 5E). To ensure these, we carried out histopathological examinations, and found that linalool alleviated tissue damage caused by S. parasitica infection on the dorsal surface of grass carp and enhancing the healing capacity (Figure. 4G). But we know the antimicrobial mechanism of linalool need further investigation, we will conduct more experiments to explore the antimicrobial mechanism of action of linalool in the future.