Egg Deposition of Micromelalopha sieversi (Staudinger) on Clones of Populus from Section Aigeiros Induces Resistance in Neighboring Plants
Round 1
Reviewer 1 Report
The paper is very interesting and results are valuable. However I have serious doubts about the methodology used;
The first major question is related to the distance between the “oviposited” plants and the “neighbouring” plants. From what I understood the oviposition bioassay 1 involves groups of 4 plants placed inside a cage with 50 cm distance between them in square design (considering the cage size). After is stated that the neighboring plants are placed also 50 cm distance, in a different cage, but with a square placement only 2 trees will be at the stated distance from the nearest oviposited plants and 100 cm from the far pair of oviposited plants, while the other 2 neighbor plants will be at 100 cm and 150 cm distance. The results are presented as means and standard deviations of the number of eggs laid for the groups of 4 plants (figure 2) and apparently those means were used for the statistical analysis. Since the conditions are different for the pairs of plants (pair closer and pair more away), first the variability between the 4 plants in each group should be presented and accounted along the statistical analyzes. For all the previous, the statement in line 295 of Discussion needs to be validated.
ANOVA is not the most adequate method since data used for the statistical analysis were means.
Suggestion: compare within groups variability and if there’s no difference between the 4 trees each tested group (to be presented in the paper), then use the sum of eggs for the ANOVA tests.
Apparently the authors have no idea of the distance that the volatiles have effect, since in the conclusions they say that the optimal distance at which the volatile compounds operate needs to be evaluated, so the variable distance between plants in the methodology may be enough for the neighboring plants may not be in the same experimental conditions and all data needs to be reviewed and regrouped.
The second major question is related to the oviposition counting; In lines 100-101 the authors say “after egg masses appeared on oviposited plants, 20 pairs of moths were placed in the cages of neighboring and control plants” and the data used for analysis were the counting of eggs after 72h of oviposition (line 102). Volatiles were collected after 24 h, 48 h and 72 h (line 109). If the 72 h of egg period finish is the same as the last volatile collection, then the amount of volatiles accumulated in the different replicates could be very different depending on the egg laying activity of the moths, at the collection times, although “the number of eggs laid… was basically unchanged after the first discovery” (lines 160-161). The cumulated curve of the number of eggs laid along time in each treatment should be present to clarify and a statistical analysis also is needed to support the previous statement.
Apparently in line 305 references are not correctly cited; should be: “elm trees (13), and pine needles (16)…”
Author Response
Response to Reviewer 1 Comments
Point 1: The first major question is related to the distance between the “oviposited” plants and the “neighbouring” plants. From what I understood the oviposition bioassay 1 involves groups of 4 plants placed inside a cage with 50 cm distance between them in square design (considering the cage size). After is stated that the neighboring plants are placed also 50 cm distance, in a different cage, but with a square placement only 2 trees will be at the stated distance from the nearest oviposited plants and 100 cm from the far pair of oviposited plants, while the other 2 neighbor plants will be at 100 cm and 150 cm distance. The results are presented as means and standard deviations of the number of eggs laid for the groups of 4 plants (figure 2) and apparently those means were used for the statistical analysis. Since the conditions are different for the pairs of plants (pair closer and pair more away), first the variability between the 4 plants in each group should be presented and accounted along the statistical analyzes. For all the previous, the statement in line 295 of Discussion needs to be validated.
Response 1: ANOVA was performed for the 4 oviposited plants, neighboring plants, and control plants at different positions (1, 2, 3, 4) in each cage, respectively. The results showed that there was no significant difference in the number of eggs on oviposited plants (F(3,12) = 0.069, P = 0.975), neighboring plants (F(3,12) = 0.653, P = 0.596), and control plants (F(3,12) = 0.644, P = 0.601) at different positions of ‘108’ (Figure 3-A). There was also no significant difference in the number of eggs on oviposited plants (F(3,12) = 0.215, P = 0.884), neighboring plants (F(3,12) = 0.203, P = 0.892), and control plants (F(3,12) = 0.206, P = 0.890) at different positions of ‘111’ (Figure 3-B). The method has been supplemented in the paper. The statement in line 295 of Discussion has been modified.
Figure 3-A. Number of eggs on oviposited plants, neighboring plants, and control plants at different positions on ‘108’
Figure 3-B. Number of eggs on oviposited plants, neighboring plants, and control plants at different positions on ‘111’
ANOVA is not the most adequate method since data used for the statistical analysis were means.
Suggestion: compare within groups variability and if there’s no difference between the 4 trees each tested group (to be presented in the paper), then use the sum of eggs for the ANOVA tests.
Response 1: There was no difference in the number of eggs on oviposited plants, neighboring plants, and control plants at different positions on ‘108’ and ‘111’ , respectively (to be supplemented in the paper). Therefore, the four plants in each cage be counted as a whole. So use the sum of eggs for the ANOVA tests. The corresponding contents has been modified.
Apparently the authors have no idea of the distance that the volatiles have effect, since in the conclusions they say that the optimal distance at which the volatile compounds operate needs to be evaluated, so the variable distance between plants in the methodology may be enough for the neighboring plants may not be in the same experimental conditions and all data needs to be reviewed and regrouped.
Response 1: In this experiment, four oviposited plants were surrounded by a square area with a side length of 50 cm. Four neighboring plants were surrounded by the same area. The two areas were 50 cm apart. The neighboring plants clearly produced induced resistance. In the conclusions, there is a sentence that ‘the optimal distance at which the volatile compounds operate…’, this statement is not accurate enough. It should be replaced by ‘In this experiment, four oviposited plants of the two Aigeiros clones were surrounded by a square area with a side length of 50 cm, respectively. Four neighboring plants were surrounded by the same area. The two areas were 50 cm apart. The neighboring plants produces clear induced resistance’. The corresponding contents has been modified.
The second major question is related to the oviposition counting; In lines 100-101 the authors say “after egg masses appeared on oviposited plants, 20 pairs of moths were placed in the cages of neighboring and control plants” and the data used for analysis were the counting of eggs after 72h of oviposition (line 102). Volatiles were collected after 24 h, 48 h and 72 h (line 109). If the 72 h of egg period finish is the same as the last volatile collection, then the amount of volatiles accumulated in the different replicates could be very different depending on the egg laying activity of the moths, at the collection times, although “the number of eggs laid… was basically unchanged after the first discovery” (lines 160-161). The cumulated curve of the number of eggs laid along time in each treatment should be present to clarify and a statistical analysis also is needed to support the previous statement.
Response 2: It was observed that most of the 20 pairs of male and female mated and laid eggs on the day of emergence. Later, occasionally individual plants will appear little scattered egg grains. The data of three treatments at three time periods were supplemented. The results showed that there was no significant difference in the number of eggs on oviposited plants (F(2,9) = 0.000, P = 1.000), neighboring plants (F(2,9) = 0.010, P = 0.990), and control plants (F(2,9) = 0.000, P = 1.000) of ‘108’ at 24h, 48h, and 72h after oviposition (Figure 2-A). There was also no significant difference in the number of eggs on oviposited plants (F(2,9) = 0.002, P = 0.998), neighboring plants (F(2,9) = 0.010, P = 0.990), and control plants (F(2,9) = 0.007, P = 0.993) of ‘111’ at 24h, 48h, and 72h after oviposition (Figure 2-B). Therefore, the data of three treatments used for the statistical analysis was the number of eggs at 72 h after oviposition.
Under the condition that the number of eggs is basically unchanged, the change of volatiles is mainly affected by the degree of eggs maturation (from immature to mature).
Figure 2-A. Number of eggs on oviposited plants, neighboring plants, and control plants of ‘108’ at 24 h, 48 h and 72 h after oviposition
Figure 2-B. Number of eggs on oviposited plants, neighboring plants, and control plants of ‘111’ at 24 h, 48 h and 72 h after oviposition
Apparently in line 305 references are not correctly cited; should be: “elm trees (13), and pine needles (16)…”
Response 2: It has been modified.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Guo et al. investigated the effect of oviposition of the moth Micromelalopha sieversi (Staudinger) on poplar leaves on neighboring plants. The results suggest that the infected plants communicate with, up then, uninfected plants which result thereafter in a lower susceptibility of the neighboring plant. Guo et al. identified several volatile compounds secreted after oviposition. These compounds are perceived by the moths and treatment of plants with some of these compounds reduced the number of eggs. The presented results are a nice addition to the literature.
Comments:
*) l.16: replace “having” by “being”
*) l.75: delete “are”
*) Tables 1 and 2 have some formatting errors. Several line breaks are not appropriate. The lines indicating the significance should show only letters, no numbers.
*) l.247-249: “ There was no significant difference in the response of mated females to the EAG with different concentrations of styrene […]. Mated females exhibited the largest response to styrene at 10 ng/μl.” -> the two sentences contradict each other. Either there is a difference or not.
*) l.270: add “of” to read “number of eggs”
*) l.286: “These results indicate…” better “These results suggest…”. There might be many different other reasons, which have not been investigated.
*) At some positions, there appear to be changes in the text style, e.g. from l.301 to l.302.
*) l.306: “on” -> “of” or “from” to read “eggs from M.”
Author Response
Response to Reviewer 2 Comments
*) l.16: replace “having” by “being”
Response 1: It has been modified.
*) l.75: delete “are”
Response 2: It has been modified.
*) Tables 1 and 2 have some formatting errors. Several line breaks are not appropriate. The lines indicating the significance should show only letters, no numbers.
Response 3: It has been modified.
*) l.247-249: “ There was no significant difference in the response of mated females to the EAG with different concentrations of styrene […]. Mated females exhibited the largest response to styrene at 10 ng/μl.” -> the two sentences contradict each other. Either there is a difference or not.
Response 4: It has been modified.
*) l.270: add “of” to read “number of eggs”
Response 5: It has been modified.
*) l.286: “These results indicate…” better “These results suggest…”. There might be many different other reasons, which have not been investigated.
Response 6: It has been modified.
*) At some positions, there appear to be changes in the text style, e.g. from l.301 to l.302.
Response 7: It has been modified.
*) l.306: “on” -> “of” or “from” to read “eggs from M.”
Response 8: It has been modified.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Thank you for the corrections and now only two more questions need to be ammended:
First: the ANOVA tests applied which results are shown in lines 200-210 are not adequate, since the number of trees in each treatment is small. Therefore equivalent non-parametric statistic tests should be applied instead. Like in all other situations along the paper the assumptions for ANOVA should be checked and if some are not fulfilled (such as: small number of samples, normal distribution of the data, homogeneity of variances, etc.) then non-parametric statistics should be used instead.
Second: rephrase sentence in Line 211: “Therefore, for further statistical analysis the four plants groups in each cage were considered as a replicate”.
Author Response
First: the ANOVA tests applied which results are shown in lines 200-210 are not adequate, since the number of trees in each treatment is small. Therefore equivalent non-parametric statistic tests should be applied instead. Like in all other situations along the paper the assumptions for ANOVA should be checked and if some are not fulfilled (such as: small number of samples, normal distribution of the data, homogeneity of variances, etc.) then non-parametric statistics should be used instead.
Response 1: The Kruskal-Wallis test of non-parametric test was performed for the 4 oviposited plants, neighboring plants, and control plants at different positions (1, 2, 3, 4) in each cage, respectively (in lines 200-210). The results showed that there was no significant difference in the number of eggs on oviposited plants (P = 0.936), neighboring plants (P = 0.755), and control plants (P = 0.637) at different positions in each cage on ‘108’. There was also no significant difference in the number of eggs on oviposited plants (P = 0.985), neighboring plants (P = 0.798), and control plants (P = 0.839) at different positions in each cage on ‘111’. The corresponding contents has been modified and supplemented in the method.
The data in all other situations have been checked and met the condition of ANOVA, which can be analyzed by ANOVA.
Second: rephrase sentence in Line 211: “Therefore, for further statistical analysis the four plants groups in each cage were considered as a replicate”.
Response 2: This sentence has been added to the result.
Author Response File: Author Response.pdf
