Effects of Bt-Cry1Ah1 Transgenic Poplar on Target and Non-Target Pests and Their Parasitic Natural Enemy in Field and Laboratory Trials

Round 1

Reviewer 1 Report

This study is the result of considerable research effort.  The authors created a transgenic poplar producing Bt toxins, then measured survival of several insects on the plants.  This was followed by a survival test with a common non-target insect.  This is an exceptionally complete series of experiments.  Unfortunately, there are several problems that need to be addressed before the paper should be considered for publication.

 

Major concerns

 

1. A major problem is that Duncan’s Multiple Range Test (DMRT) was used to compare differences between means following an ANOVA. This is not an appropriate test for this type of study. DMRT was designed to be a VERY liberal test to find any possible difference in resistance between plant lines to insects or pathogens.  This test has not been used extensively for biological studies (outside of plant resistance) for probably 20 years.  Most authors now use Tukeys Honestly Significant Different test, an LSD test, or something similar.  Many journals specifically prohibit the use of DMRT test except for plant resistance studies.  The data need to be reanalyzed using an appropriate test.

 

2. Presentation of statistical differences in Tables and Figures needs to be corrected. For example, for Table 1, the presentation of the significance results for “18d” and “Pupation rate” is not appropriate for most journals. The letters should follow the increasing mortality (or the decreasing pupation rate) in alphabetical order. This same pattern should be followed for all columns in all tables. Similarly, the statistical separation presented in figure 4 should be corrected.  The lowest value (the ck) was given an ‘a’ value, and the next highest was given a ‘b’ value.  This pattern should be followed until the highest value is given an ‘e’.  However, currently, the ‘c’ value is the highest of all, and the other letters indicate lower values.  I suggest the authors examine some recent papers in Forests to see how the significance results are normally presented.  As written, the statistical analysis is confusing.

 

3. Figure 5: The authors state that ”Vertical bars represent the means ± standard deviation (SD; n = 3). *, p < 0.05; **, p < 0.01”. I assume the asterisks indicate a significant difference from the CK, but I am not sure.  Was an ANOVA conducted for each plant accession versus the CK separately?  Unfortunately, I could not figure out what was done. I am also not sure what can be done for the tests that had only 3 insects as replicates.  This is a very small number, but the authors were able to statistically detect differences. I suspect some reviewers might have a concern that the authors did not test 45 insects independently (which is what gave them over 40 degrees of freedom).  The insects were examined in 3 containers with groups of 5 insects. The authors should probably consult a statistician to see if their apriori decision to use individual insects as replicates is a[appropriate given the experimental design.

 

 

Minor problems

4. Line 120: “Pupae masses of H. cunea,…”, should be just ‘Pupae’

 

5. Line 122: what does “porecultured” mean?Perhaps this could be explained in the paper. Also, “matted” should be ‘mated’

 

6. Line 160: The authors state that “Pupae were parasitized in finger tubes…”. What is a finger tube?

 

7. Line 163: What constitutes a “well-parasitized” pupae? Understanding what this means would be useful for someone repeating any of the work.

 

8. Figure 1: I did not find figure 1 to be particularly useful. In fact, these pictures raised more questions. For example, in Figure 1. The “E” panel includes a picture of leaves being cleaned (this was not described in the paper), and the ‘c’ photo of panel “E” is described as “catching insects”, when it is a picture of leaves jammed into small containers. Perhaps I am misunderstanding what was intended. 

 

9. Line 254” The authors state: “Although the difference was not significant, emergence time in the treatment was slightly longer than that in the control (Fig. 8A). On line 258, the authors note: “On average, the weight of pupae fed on non-transgenic poplar was higher than that of pupae fed on transgenic poplar, but this difference was not significant (Fig. 8C)”. Describing trends or patterns in a non-significant data set is almost never justified. I suggest that the authors simply state the differences were not significant, but leave out the ‘trends’ comments.

 

10. Line 284: the statement that “Moreover, H. cunea larvae have a high tolerance for starvation” would benefit a reference, since starvation effects were not evaluated in this study.

 

11. Line 298: The authors statement that “According to current findings, it is unlikely that Bt poplar is toxic to non-target insects” is probably overstated. The statement could be true, but the authors tested only one non-target insect species.  Extrapolating this test to include all non-target insects on poplar is probably not justified.

 

 

 

Author Response

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Reviewer 2 Report

The paper explains the interest of Bacillus thurigiensis as biological control agent pointing out the reasons that make this bacterium interesting in poplar forest. The paper describes clearly how to use this potentiality by means of  genetic engineering technology.

Material and Methods are very clear and allows readers to replicate experiments.

The use of schemas and the large amount of bibliography consulted makes this paper very comprehensible and grounded.However as this is a polemic subject and although the authors refer this controversy, I suggest a more carefully redaction in line 311 because nobody knows what could happen in the future using this approach.

In line 339 I suggest remove the point and continue the sentence with the word “and”.

Author Response

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Reviewer 3 Report

Dear all,

A well performed and analyzed experimental presentation. English language needs a considerable improvement.

Author Response

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