Terpenoid Mixtures as Repellents Against the American Cockroach: Their Synergy and Low Toxicity Against Non-Target Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To be accept this manuscript must to evaluate Cymbopogon citratus, Cinnamomum verum and Illicium verum essential oils and demostrated the presence of the compunds in the essential oils refered and then resubmit this paper.

 

 

Comments for author File: Comments.pdf

Author Response

Please see the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a well-conceived and timely study investigating the synergistic repellent effects of binary terpenoid mixtures against Periplaneta americana and their safety for non-target organisms. The topic is highly relevant given the growing need for effective, eco-friendly alternatives to synthetic insecticides like DEET. However, the manuscript requires significant revisions to enhance clarity, methodological detail, statistical reporting, and the depth of discussion before it can be considered for publication. The following major points and minor suggestions should be addressed.

1 Cockroach Rearing: Provide more specific details. What was the photoperiod and relative humidity during rearing? How many adults were used in total for bioassays, and how were they randomized?

2 Chemicals: Clarify the solvent used for preparing DEET and terpenoid solutions. Was it also 70% ethanol? Specify this for all treatments and controls.

3 Bioanalysis of Repellent Activity: The formula for Repellent Rate (%RR) is unconventional (A-B/A+B). The standard is typically (Number in control - Number in treatment) / (Total) * 100, or a similar variant. Justify or clarify this formula. Furthermore, describe the physical setup (Figure 2) in the text: dimensions of the plastic box, size of treated/untreated zones, how the filter paper was treated, and how cockroaches were introduced and counted.

4 Toxicity Bioassays: For the guppy bioassay, specify the water quality parameters (e.g., pH, dissolved oxygen). For the earthworm bioassay, state the incubation temperature. For both, explicitly mention the number of individuals per replicate and the total number of replicates per concentration.

5 Statistical Analysis: The description is confusing. It states "simple regression assessed the repellent efficacy..." followed by an almost identical sentence. Clarify the statistical models used. Specifically, how were RC values (RC25, RC50, RC90) and their confidence intervals derived? Probit analysis is mentioned; confirm this was used for RC values and LT50 values. For ANOVA, always report the degrees of freedom (df) and F-value alongside the p-value in the results text or tables, not just in figure captions.

6 Figures 3 & 4: The figures are extremely dense and difficult to interpret. Consider simplifying them. For instance, Figure 3 could be moved to supplementary material, and Figure 4 could focus on key concentrations (e.g., low and high) or the most effective mixture. Ensure all axis labels are fully visible.

7 Synergy Calculations (SV & IV): The formulas (3) and (4) in the text appear to be identical, which is incorrect. One should calculate SV and the other IV. Please verify and correct these formulas. The current description of SV ("if SV is less than 1, a synergistic impact is present; if SV is more than 1, no synergy is present") contradicts the bar graph in Figure 7 where all SV values are positive and much greater than 1, yet are described as "strong synergy." This is a critical confusion that must be resolved. Define and apply the synergy metrics (SV, IV, or others like Co-toxicity Coefficient) correctly and consistently with established literature.

8 Units Consistency: There is a concerning inconsistency in units. The text and tables switch between μl/cm³ (e.g., page 4, concentrations) and μl/m³ (e.g., page 1, Abstract; Table 1). μl/cm³ is equivalent to 1,000,000 μl/m³, which is a massive discrepancy. This must be checked and corrected throughout the manuscript. Likely, the intended unit for the repellent chamber is μl/cm² (volume per area of filter paper) or the chamber concentration is in μl/l air or μg/cm³. Please standardize this precisely.

9 Table 1 & 2: In Table 1, add a column for the slope ± SE from the probit analysis. The Chi-square values seem very high for some mixtures (e.g., 138.6); comment on the goodness-of-fit. In Tables 2 and 3, the mortality rate for DEET is listed as "100a" on some days. Use a consistent format (e.g., 100.0 ± 0.0a).

10 Deepen the discussion on the mechanistic basis of the synergy. The paragraph on page 14 touches on mechanisms but remains speculative. Elaborate on how the specific combination of geranial (an acyclic monoterpene aldehyde) and trans-cinnamaldehyde (a phenylpropenal) might interact at the molecular level (e.g., simultaneous targeting of acetylcholinesterase and octopamine receptors, or disruption of different neuronal pathways). Cite relevant studies on the mode of action of these compounds.

10 Compare and contrast with existing literature more critically. The discussion rightly cites supportive studies but should also address any conflicting results or explain why the observed synergy is particularly strong compared to other terpenoid combinations.

11 Address the DEET resistance point more substantively. The statement that cockroaches "might be resistant to DEET" is raised but not explored. If this is a hypothesis from your data, discuss it in the context of known resistance mechanisms. If not, rephrase to state that your natural mixtures simply outperform DEET under these test conditions.

Author Response

Please see the attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

My comments had been solved

Author Response

Please see the attached file.

Author Response File: Author Response.pdf