Formicidal Potential of Thymol Derivatives: Adverse Effects on the Survival and Behavior of Acromyrmex balzani

Round 1

Reviewer 1 Report

The manuscript analyzes the effect of new molecules as insecticides for the leaf-cutting ant Acromyrmex balzani. The authors modified the monoterpene thymol molecule by reactions of substitution and esterification and tested the new molecules by analyzing the lethality and the changes in the behavior of the ants at different concentrations. They also compared the results with a commonly used pesticide, deltamethrin. They found the molecule thymyl chloroacetate can have lethal effects on the ants, and might be used in controlling it.

The manuscript is overall well-written, the sampling effort and the experimental apparatus are more than satisfactory, and the results can have interesting practical perspectives. I have only some doubts about the statistical analyses and the ecological consequences of the compound. Here below point-by-point comments;

Lines 111-112: Taking a look at Fig. 3, it appears that it is not completely true that acetone does not interfere with the survival of the ants. KM curves show that almost 20% of the ants died at the end of the experiment in controls, which are made by acetone only, if I correctly understood. I believe that you have to report your preliminary analyses—at least in a supplementary file—and discuss that acetone has a lower impact with respect to the other compounds, but it is not zero, and it may play a role in the functionality of the pesticide. It is probable that the effects of the tested molecule and acetone add up. Please consider this point and explain it in the discussion and method sections.

Lines 223-230: The ANOVA design is not specified. Looking at the results, it seems that you used both factors, different molecules, and different doses, as orthogonal factors, and you have tested the interaction between them. In some cases, interaction resulted to be significant. In those cases, I am not sure you can test single factors and then perform Dunnett comparisons. You may check this recent article dealing with this point (10.1371/journal.pone.0271668). Moreover, you also say ‘To verify differences between doses within treatments, ANOVA was used followed by paired t-test at P < 0.05’, but if you test it with the ANOVA, why do this again? If you do it, you treat the data similarly to a nested design, that you should have performed with the ANOVA, and you probably had to apply a correction (e.g. Bonferroni). I suggest clarifying and better justifying your choices for using this test.

Furthermore, you claim that all these variables are normally distributed, but in Fig. 8 is reported the boxplot (why it is differently drawn? Please use the same representation of the others) of the ‘meander behavior’, which appears to be not normally distributed. Probably data should be transformed, but you have to check both normality and homoscedasticity before using parametric ANOVA (as you correctly did in previous analyses).

Line 340: Is it the mean of the total distance covered? SE are the error bars, I guess, not the circle. Please rephrase.

All figure captions: A. balzani is not in italics, and the name of the test is always called Dunnest, instead of Dunnett.

Lines 405-406: change ‘research has’ with ‘researches have’.

Lines 421-422: I can’t find a link between the effect of microbes and the effects on ants. If the compound has a detrimental effect on microbes, the fact that it has or does not have an effect on ants is completely disconnected. Please better specify what did you mean or delete the previous sentence.

Line 425: You probably meant ‘Cl atoms’, not ‘Cl molecules’.

From line 444: It is not necessary recalling figure numbers in the discussion, it is useless and distractive.

As a final comment, I wanted to read something about the possible environmental effects of these new molecules, and the ecological consequences of their use. Are they specific for this ant species? They can kill all insects or not? Are they harmless for other animals, humans included? And what about plants? How long is the permanence in the environment? If you don’t have responses to these questions, it is difficult to say ‘to be used in the management of A. balzani’, because the harmful effects might be higher than the benefits. You probably need to add a part in the discussion section where you say that these points have not already been assessed and that this is the first step toward the use of the new compounds in controlling this species.

Author Response

We are grateful to the referee for the comments and have provided a detailed list of the referee's comments along with our action on them in an attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Botanical insecticides are an indispensable candidate for the safety of pest control of humans and the environment in modern agriculture. In this manuscript, the author tested the bioactivity of the monoterpene thymol and its derivatives on the behavior of the leaf-cutting ant Acromyrmex balzani. The manuscript is of good quality with a clear structure, data analysis, high results, and deep discussion. I have only shown some minor suggestions for the manuscript.

Line 103, the mean size of the ant as well as the sample number should be provided.

Line 172. How do these behaviors (cleaning, antennation, avoidance, aggression of leaf-cutting ants) be classified? Do you describe it or show your references?

Line 246, the scientific name of species should be in italics. The author may omit it in the following figures, and please correct them.

Author Response

We are grateful to the referee for the comments and have provided a detailed list of the referee's comments along with our action on them in an attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript reported the toxicity of the monoterpene thymol and its derivatives, as well as the sublethal effects of these compounds on the behavior of the leaf-cutting ant Acromyrmex balzani. A. balzani is an important pest of crops, posing a potential threat to the safety production of agricultural industry. The experiment is well designed and innovative in this field. The introduction provide sufficient background and include all relevant references. The methods are adequately described. I think the paper can be accepted in present form. 

(1) Introduction

Supplement the damage and economic losses caused by Acromyrmex balzani on agricultural and forestry crops.

(2)Discussion

Complete the current status of thymol derivatives used to control pests in your country. Is the control of worker ants effective in controlling the damage and spread of leaf-cutting ant colonies?

Author Response

We are grateful to the referee for the comments and have provided a detailed list of the referee's comments along with our action on them in an attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors responded to my comments point-by-point and adjusted the text accordingly. I believe that now the paper is suitable for publication.