-->PONE-D-25-51282-->-->Predatory activity and nematocidal compounds released into liquid culture filtrates as attack strategies of a Mexican strain of Arthrobotrys oligospora against Haemonchus contortus infective larvae-->-->PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: General comment:

This manuscript presents solid, innovative, and highly interesting research. However, regarding the in vitro anthelmintic evaluation data of the fungus and its filtrate, it is necessary to clarify whether the assumptions underlying the statistical analysis were tested to ensure that the data met these requirements. Otherwise, the data should be transformed or analyzed using nonparametric methods to enhance the scientific rigor and validity of the conclusions.

Specific comments:

Key words: Please, instead the words “Haemonchus contortus” and “nematocidal activity” choose other two words that are not contained in your title.

Introduction:

In line 57, source 11 most not be included here because this article talks about nematophagous fungi extracts and not about plant extracts. Therefore, you can include this source in the part of the sentence where you talk about nematophagous fungi.

In line 66, I think it is important to mention the specific place in Mexico (city) where the fungi of the study were collected.

Figures:

Figure 1. Could you please substitute the figures A, B and C with other figures with better resolution?

Material and methods

All trade names must have the "®" mark, especially in the methodology section (lines 97, 110, etc.).

In line 132 please add any description of this H. contortus strain and a reference.

In line 139, it is important to express that the protocol was also “approved” and not only supervised by the Committee of Care and Use of Experimental Animals.

In line 144, please add the plate measurements.

In line 153, please add the reference Abbott formula.

Statistical design

Was it evaluated whether the data complied with the assumptions of the analysis of variance (homoscedasticity)? If yes, please add this information to this section, because this kind of data is frequently not normal, making it necessary to transform the data or use a non-parametric analysis.

In line 204, please put H. contortus in italics.

In the section Zymography assays, please add the ® mark to all the commercial names. Please check this in all the manuscript.

Results

Would it be possible to include images in Figures 2 and 4 with better resolution? Additionally, it would be interesting to include a scale showing the length in the images.

In table 1, This range corresponds to the minimum and maximum values observed or measured standard deviation.

In line 281, used italics for scientific names. Please ensure that all scientific names are consistently italicized throughout the manuscript.

In table 3, Since different letters were not observed in the means, there was no significant difference between the control and the treated group, right?

In table 4, I think it would be interesting to make statistical comparisons between the means of the different filtrate concentrations, and with the negative and positive controls used, could you add these results to this table?

Microscopic analysis:

These chances were also observed and lower concentration or not? were observed to a lesser degree? Please specify this in your results.

In line 132 the information cannot be read.

The information in the title of Figure 6 is very extensive. Is it possible to summarize it without omitting the essential information?

Discussion:

In line 364, add a reference to support your statement.

In line 387, please include against “infective larvae” of …the gastrointestinal parasitic nematode H. contortus, ….

Table 8 is not cited or mentioned in the discussion section and should be properly integrated into the text. In general, the number of tables in the discussion could be reduced, keeping only those essential to support or strengthen your arguments.

Do you think that the microscopic changes observed in the intestinal cells of the infective larvae could be due to the direct penetration of some of the metabolites through the intact cuticle and membranes, or rather to the prior enzymatic degradation of the cuticle by these proteolytic enzymes? Alternatively, do you think that the ingestion of these metabolites and fungal enzymes might also have contributed to the observed changes in the intestinal cells of the larvae? I believe it would be interesting to address this point in your discussion.

In line 510, it is important to indicate that the Internal Committee for the Care and Use of Experimental Animals (CICUAE-FESC) not only supervised but also approved the study.

Reviewer #2: 131 It does not specify whether the lamb was previously dewormed (anthelminthic treatment) or if it was evidenced to be free of gastrointestinal parasites.

143 What was the variability (standard deviation) of the mean values of recovered larvae with or without the action of the predatory fungus?

163 't'-test for small samples (discrimination by least significant difference?) please clarify, because in line 198, mean separation with Tukey's test was used for lethal activity. Was this design only for lethal activity or also for predatory activity?

254 What was the variability of the mean values (±s)?

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General Comment: A very interesting study with several components, the results are promising regarding the biological control of gastrointestinal parasitism and contributing to the control of anthelmintic resistance. However, because this is an in vitro study with interesting results, the true in vivo activity of the fungus remains unknown.

As part of a doctoral thesis, it would be interesting to know if any phase or sequence for in vivo animal trials is being considered. It has been observed that many very promising in vitro results have not yielded the same outcome when applied in vivo, much less in field activity under real-world conditions. So far, the gap in the transition from in vitro to in vivo, with the same or better results has not been overcome. There are many more variables, but an approximation would be interesting to see.

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Reviewer #1: No

Reviewer #2: No

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Predatory activity and nematocidal compounds released into liquid culture filtrates as attack strategies of a Mexican strain of Arthrobotrys oligospora against Haemonchus contortus infective larvae

PONE-D-25-51282R1

Dear Dr. Higuera-Piedrahita,

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Kind regards,

Wesley Lyeverton Correia Ribeiro, Ph.D.

Academic Editor

PLOS One

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