A Laser Irradiation Method for Controlling Pieris rapae Larvae
Round 1
Reviewer 1 Report
The study focuses on the lethal and antifeedant effect of laser irradiation on Pieris rapae and measures the efficacy of selected factors on the two variables: mortality and antifeedant effect, at different working parameters. Various parameter combinations are evaluated, and the optimal combination is identified by performing an ‘optimization analysis’ using ‘surface analysis’ (by which a quadratic regression model is obtained for each variable) and multiple verification tests. Lastly, using the identified ‘optimal’ combination, different larval instars are tested. Overall, the topic is relevant, and the manuscript is well organized. However, it needs extensive revision for language and appropriate wording.
General comments:
The study heavily relies on data analysis and statistical procedures. In my view, the study primarily aims at validating a specific procedure to evaluate the efficacy of laser irradiation on selected ‘indicators’ and analyze the results, using Pieris rapae as an insect model rather than evaluating laser as a control tool for this pest. I suggest the suitability of these procedures and the wording of the statistical analysis section is revised by a statistician/mathematician. Some information could probably be removed for conciseness.
To this reviewer, the results are presented in a way somewhat confusing and difficult to read. It may help to present the results as ‘experiment 1’, ‘experiment 2’, etc. Plus, it may be worth trying to be more explicit as to why ‘optimization and validation’ tests are needed or the benefits of presenting data as surface plots.
Specific comments:
In my opinion, the title is misleading for the reasons mentioned above. In line 25: briefly define ‘antifeedant percentage’. In line 61, avoid saying that the authors ‘tried to use’. Lines 63-64: change the wording, something like ‘…can kill arthropod pests, including eggs, nymphs and adults’. The sentence in line 67 does not make sense. Re-word the paragraph in lines 69-72, it does not make much sense either. Lines 72-74 seem to contradict what is mentioned in lines 69-71. Re-word lines 78-80: ‘laser irradiation is recommended as a pest control tool’? In the Introduction, remove the last paragraph.
In 2.1, it is not clear whether the larvae were reared on cabbage or broccoli, or if cabbage was used only to ‘lure’ the larvae to the petri dishes. In line 96: you can just say ‘therefore, fourth instar larvae were used’. In line 102, explain ‘an Arduino UNO’. In line 124, explain or remove ‘..according to the test results’. In line 126: ‘..instar larva were used’. The larvae were tested, not ‘performed’. The experiment is performed.
In line 129: how were the ‘target values’ (5, 0.01, 2, middle) determined? Be more explicit on where the ‘eigenvalues’ come from. In line 132: which ‘cross marks’? Test process: I would combine steps 3 and 4. Line 138, ‘after’ I think fits better than ‘within’ 24 hours. In line 141: better say ‘tests’, not ‘experiments’. In lines 167-168 and line 172: explain how the ranges of the four parameters were selected; for example for laser power(W), from 1 - 10 to 1.5 - 7.5 (five to three levels). Not sure ‘evaluation indicators’ is the appropriate term, maybe ‘dependent variables’?
In line 188: laser may have an antifeedant effect rather than ‘activity’ which to me sounds more like as a result of a biotic factor. Also, in line 196: to estimate instead of to ‘identify’. In line 200: the best working parameter combination instead of combination of ‘all the factors’. In line 201: explain ‘membership degrees’.
In line 202: why is improving the antifeedant effect the priority? In other words, if you can kill the larva, why is it more important to suppress its feeding? By killing the larva you can prevent them from becoming adults and reproduce. In line 204: ‘According to previous studies..’.
In line 212: remove ‘..in the paper’. In the Statistical Analysis, remove the first paragraph, maybe move somewhere above. Lines 232-233: remove the sentence, it has been already mentioned in the Methods. In line 246: remove ‘A’. In line 249: ‘..after 24 h (stats), 48 h (stats), and 72 h (stats) (…) were detected’. In lines 303-304: ‘Therefore, only results measured after 24 h were used’. In line 308: ‘the test results are shown’. In line 315: the larvae were collected.
Paragraph from line 308-317 belongs to ‘Methods’. footnote #2 in Table 2: why are only codes -1 and 1 mentioned? In line 329: explain ‘manually optimized’. In line 336: what about BC? In lines 356-357: I assume the difference between the predicted and the adjusted R was less than 0.2? In line 413: how is ‘desirability’ calculated and why is it important? Lines 394-395 repeat same information as in 215-216.
Title in Table 4 is supposed to reflect what is shown in the table, not how data were obtained. Footnote #2: 7.07 mm2, 6.158 mm2, and 4.908 mm2. Footnote #6: If this refers to the column then the number should be placed in the column, not in the row. Line 419: does ‘in actual situations’ refer specifically to a field setting?
Start the discussion with a concise, clear statement about the main contribution of the study. Any conclusion drawn from the first experiment? Line 473: ‘In this study, (…) by laser irradiation is proposed’. Line 474: By testing (…) and studing. Line 475: It has been mentioned that there is no interaction between experimental factors (lines 142-143). This first paragraph needs to be re-worded. Line 480: I think there is no need to include the comprehensive score here. Line 485: the optimal combination of parameters had a higher lethal and antifeedant effect on young larvae. Any unexpected results? To include some citations would be beneficial for the discussion.
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer:
Thank you very much for your comments and suggestion. Since the cover letter contains figures and equations, the file is uploaded as an attachment. Please see the attachment.
Thank you and best regards.
Yours sincerely,
Yajun Li
Author Response File: 
Author Response.docx
Reviewer 2 Report
The work addresses the possibility of the Laser Irradiation Method in Controlling Pieris rapae Larvae
The introduction is clear, sufficiently developed and with relevant references.
The methodology is clearly presented, and sufficient numbers of larvae and replicates per treatment were used.
The results are presented in a very exhaustive way.
The discussion should be further developed, and the results that were obtained should be compared to other works in the area, even if of other species.
It could have addressed the difficulties in the field, as most larvae will be hidden inside the plants or under the leaves. Will the control
method work under these conditions?
Author Response
Dear Reviewer:
Thank you very much for your comments concerning our manuscript entitled ‘A Novel Laser Irradiation Method Exhibits High Efficiency at Controlling Pieris rapae Larvae’ (ID: applsci-1373124). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval.
The main corrections in the manuscript and the responds to the comments are as following
1. Response to comment: (The discussion should be further developed)
Response: In paragraphs 2 and 3 of the Discussion, the laser irradiation method was compared and discussed with the pesticides, plant extracts and other physical control methods. The contents are as follows:
It has been shown that many insecticides, such as 1.8% avermectin, 5% chlorobenzamide, 6% spinetoram and some plant extracts [43-47], have been commonly used to control P. rapae larvae in recent years. Most of these insecticides exhibited a pest reduction rate of less than 80% within 3 days after treatment, and the insecticidal effectiveness de-creased over time [47]. Moreover, among the 74 plant extracts tested, the most effective plant extract caused an antifeedant percentage of 96.10% after 48 hours [43]. After 24 hours, the laser-based method used in this research showed the same insecticidal effectiveness on larvae as traditional chemical control for 3 days, and the increase in the antifeedant percentage after 48 hours reached 99.27%. Thus, the laser-based method for controlling larvae exhibits the merits of avoiding the development of resistance and effectively reducing the harm of pesticide residues in the environment to human beings.
At present, the frequency vibration insecticidal lamp is widely used in physical control methods [48]. The lamp uses the phototaxis of pests to lure pests, and then kill pests through the power grid. According to the investigation of Ye et al. [49], the lamp can trap and kill 30 species of pests in 5 orders, 17 families, and can trap and kill pests in main orders and families in vegetable fields. However, the lamp has the same killing ability for beneficial insects and non-target insects in the control area, threatening the natural eco-logical balance [50]. On the other hand, the above methods have a certain trapping and killing effect on adults such as moths, but have no effect on larvae directly endangering vegetable crops.
2. It could have addressed the difficulties in the field, as most larvae will be hidden inside the plants or under the leaves. Will the control method work under these conditions?
Response: In the preliminary experiment, we investigated the living habits of P. rapae. The study found that the P. rapae do not hidden inside the plant or under the leaves all the time. In the morning and evening, more than 75% of the 3rd-5th instar P. rapae appeared on the leaf surface. And the larvae mainly located on the petiole, vein and undeveloped new leaves of the outer leaves. P. rapae can be clearly observed from directly above the vegetables. The total amount of food eaten by the 1st- to 3rd-instar P. rapae larvae accounted for only 2.53% of the total food consumption. Even if it is not immediately irradiated by laser, it will not have a serious impact on the growth of host plants. Therefore, the method of laser irradiation to control P. rapae proposed in this study can overcome the situation that the larvae stay inside the plant or under the leaves.
The above are my corrections in the paper and the responds to the comments. Once again, thank you very much for your comments and suggestion. If you have any queries, please don’t hesitate to contact me at the address below.
Thank you and best regards.
Yours sincerely,
Yajun Li
