Use of Essential Oils and α-Pinene as Insecticides against Sitophilus zeamais and Their Effects on Maize Seed Germination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study demonstrates the toxic and repellent effects of essential oils from four different species, as well as alpha-pinene, on Sitophilus zeamais under storage conditions. Additionally, it evaluates the impact on corn seed germination. The study is well-designed, with a clear methodology and correct statistical analyses. The discussion is thoroughly addressed; however, some points require further refinement.

P3 L106 “Combine 500 g of crushed resin with 3 liters of distilled water in a round-bottom 106 flask.” Sentence is not appropriate. Instead: “Five hundred grams of crushed resin were combined with 3 liters of distilled water in a round-bottom 106 flask.” 

 

P3 L 144. After this process, in each repetition, 12 unsexed adults of S. zeamais with 0–15 days old.” Verb is missing

 

P4 L165 “For each repetition, 20 individuals, aged 0–15 days, were placed in these containers without sex.

“…. Without sex discrimination”

 

P4 L172 “In the fumigation test, for each treatment, there are four repetitions.Do not use the verb in present tense. In the fumigation test, for each treatment, four repetitions were conducted.

 

P4 L178-180 Sentence is confuse.  Instead: “For the persistence test, 500 g of BR-106 corn seeds were allocated for each product. From this, 400 g were used, divided into four 100 g samples, and each sample was individually treated and homogenized as described earlier in the contact test. The treated samples were then stored in a transparent zip lock bag, wrapped in aluminum foil, and kept under refrigeration at 10°C until the experiment was set up ”

 

P5L203-206 Replace “you” by “ti”

 

P5 L242. “The composition of the oils varied among themselves, and the major compounds were identified.” The use of the term “themselves” is not appropriate, instead: "The composition of the oils varied among the different species, and the major compounds were identified."

Section 3.2 from Results: "What is the purpose of reporting the LC15 and LC30 values?"

 

Figure 1 is missing

 

P8 L300 “therefore, α-pinene is responsible for the mortality caused by J. communis (Table 3).”

The original affirmative statement seems too strong. A more measured suggestion could be that minor compounds in the oil may interact antagonistically with alpha-pinene, leading to an increase in the lethal concentration of the essential oil."

P8 L306: “Persistence of the insecticidal effect of essential oils and α-pinene on stored seeds” Why .only Gaultheria procumbens and Juniperus communis essential oils were selected?

 

Table 5. Fresh matter is not a reliable parameter to evaluate the toxicity effect of the essential oils. Instead, replace by Dry matter (table 6).

 

Table 5 and Table 6 are related and redundant. Germination speed index (Table 5), average germination time and Average germination speed are parameters related and redundant as they are based on number of seeds that germinated in time ‘i´. Remove table 6, it is enough to report the germination speed index of table 5 and report the average germination time in the text as an average value since no significant differences were observed. Do not report AGS.

 

P11 L384. The phrase 'which was able to stop their enzymes from working' is unclear and lacks context. It should be specified what is being referred to, such as the particular substance or condition that inhibits enzyme activity. Which enzymes?

 

P11 L393. There was a variation in the inclination of the products, ranging from 2.57 to 11.33, indicating some toxicological heterogeneity among the products tested. What is this referred to? It is unclear redaction. What are 2.57 and 11.33 values? When you say products, do you refer to essential oils? It is not appropriate word to name the essential oils.

 

P12L428. “which is a possible justification for α-pinene being more toxic to S. zeamais than to S.  zeamais. The essential oil of J. communis.” Check the redaction. The concepts here are confusing.

Comments on the Quality of English Language

The English is clear and well-constructed.

Author Response

Comments 1: P3 L106. “Combine 500 g of crushed resin with 3 liters of distilled water in a round-bottom 106 flask.” Sentence is not appropriate. Instead: “Five hundred grams of crushed resin were combined with 3 liters of distilled water in a round-bottom 106 flask.” 
Response: The suggestion was accepted and modified in the final manuscript.

Comment 2: P3 L 144. After this process, in each repetition, 12 unsexed adults of S. zeamais with 0–15 days old.” Verb is missing
Response: The suggestion was accepted and "were placed" was added in the final manuscript. 

Comment 3 P4 L165. “For each repetition, 20 individuals, aged 0–15 days, were placed in these containers without sex. “…. Without sex discrimination”
Response: The suggestion was accepted and "discrimination" was added in the final manuscript. 

Comment 4 P4 L172. “In the fumigation test, for each treatment, there are four repetitions.Do not use the verb in present tense. In the fumigation test, for each treatment, four repetitions were conducted.
Response: The suggestion was accepted and modified in the final manuscript.

Comment 5 P4 L178-180. Sentence is confuse.  Instead: “For the persistence test, 500 g of BR-106 corn seeds were allocated for each product. From this, 400 g were used, divided into four 100 g samples, and each sample was individually treated and homogenized as described earlier in the contact test. The treated samples were then stored in a transparent zip lock bag, wrapped in aluminum foil, and kept under refrigeration at 10°C until the experiment was set up ”
Response: The suggestion was accepted and modified in the final manuscript.

Comment 6 P5L203-206. Replace “you” by “ti” 

Response: The suggestion was accepted and modified in the final manuscript.

Comment 7 P5 L242. “The composition of the oils varied among themselves, and the major compounds were identified.” The use of the term “themselves” is not appropriate, instead: "The composition of the oils varied among the different species, and the major compounds were identified."

Response: The suggestion was accepted and modified in the final manuscript.

• Section 3.2 from Results: "What is the purpose of reporting the LC15 and LC30 values?"

Response: The CL15 and CL30 values of the essential oils were reported as they were used in the repellency assessment. This parameter, being a sublethal effect (aiming not to kill the insect, but to repel it), is tested at low concentrations.

Comment 8.  Figure 1 is missing

Response: The figure was added in the final manuscript.

Comment 9 P8 L300. “therefore, α-pinene is responsible for the mortality caused by J. communis (Table 3). ”The original affirmative statement seems too strong. A more measured suggestion could be that minor compounds in the oil may interact antagonistically with alpha-pinene, leading to an increase in the lethal concentration of the essential oil."

Response: The suggestion was accepted and modified in the final manuscript.

Comment 10 P8 L306: “Persistence of the insecticidal effect of essential oils and α-pinene on stored seeds” Why only Gaultheria procumbens and Juniperus communis essential oils were selected?

Response: Gaultheria procumbens and Juniperus communis essential oils were selected due to their high toxicity and the availability of these oils in larger volumes.

Comment 11: Table 5. Fresh matter is not a reliable parameter to evaluate the toxicity effect of the essential oils. Instead, replace by Dry matter (table 6).

Response: The dry and fresh mass analyzed aims to observe the physiological qualities of seeds and the impact of oils on the seeds, rather than the toxicity of the oils. It should be noted that this is a standard test for the physiological quality of seeds, as established by the Rules for Seed Testing (RAS), a Brazilian regulation that follows international standards. The authors considered both legal and scientific criteria in the planning of the experiments and indicate the continuity of the data.

- Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes / Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. – Brasília : Mapa/ACS, 2009. 399 p.

- DADLANI, M.; YADAVA, D.K. (Editors). Seed Science and Technology, 2023. Springer Singapore. DOI: https://doi.org/10.1007/978-981-19-5888-5

Comment 12: Table 5 and Table 6 are related and redundant. Germination speed index (Table 5), average germination time and Average germination speed are parameters related and redundant as they are based on number of seeds that germinated in time ‘i´. Remove table 6, it is enough to report the germination speed index of table 5 and report the average germination time in the text as an average value since no significant differences were observed. Do not report AGS.

Response: The authors followed legislative and scientific criteria for the preparation of the analyses, as previously mentioned, and we believe that an excess of information is not a flaw but rather fundamental to emphasize understanding. Considering that the request is specific to only one reviewer as opposed to the others, we have opted to keep the data as expressed in the manuscript. We appreciate the reviewer’s suggestion but have chosen not to comply with it.

Comment 13 P11 L384. The phrase 'which was able to stop their enzymes from working' is unclear and lacks context. It should be specified what is being referred to, such as the particular substance or condition that inhibits enzyme activity. Which enzymes?

Response: The reviewed frase was added:
"Isolated compounds and essential oils from plants of botanical families similar to those studied here have been reported to be toxic to several insect pests. For instance, essential oil from Gaultheria procumbens demonstrated toxicity against stored grain pests, such as Sitophilus oryzae L. and Rhyzopertha dominica Fabr., by inhibiting acetylcholinesterase (AChE) activity. Kiran and Prakash (2015) [17] found that the in vivo inhibition of AChE activity in these pests ranged between 6.12% and 27.50% when exposed to lethal and sublethal doses of this essential oil."

Comment 14 P11 L393. There was a variation in the inclination of the products, ranging from 2.57 to 11.33, indicating some toxicological heterogeneity among the products tested. What is this referred to? It is unclear redaction. What are 2.57 and 11.33 values? When you say products, do you refer to essential oils? It is not appropriate word to name the essential oils.

Response: The reviewed frase was added:

"There was a variation in the slope values of the toxicity curves for the essential oils, ranging from 2.57 to 11.33, indicating a degree of toxicological heterogeneity among the oils tested."

Comment 15 P12L428. “which is a possible justification for α-pinene being more toxic to S. zeamais than to S.  zeamais. The essential oil of J. communis.” Check the redaction. The concepts here are confusing.

Response: The redaction was correct as follows: "which is a possible justification for α-pinene being more toxic to S. zeamais than the essential oil of J. communis." and a possible justification for α-pinene being more toxic to S. zeamais compared to the essential oil of J. communis could be the higher concentration or potency of α-pinene in its pure form, leading to increased toxicity against S. zeamais when used independently.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

You have prepared a very interesting paper titled "Use of essential oils and α-pinene as insecticides on Sitophilus zeamais and their effects on maize seed germination." In my opinion, the work is well thought out, well written, and conforms to the IMRAD standards. You have prepared the discussion of the results in a particularly engaging manner, which presents an interesting dialogue between your findings and the information obtained by other authors.

Please respond to the following questions. Your responses will be very valuable in enhancing the overall understanding of the work.

1. How do the authors intend to account for biodiversity and potential differences in the chemical composition of the discussed essential oils in the context of their effectiveness against S. zeamais? Have possible differences in effectiveness based on the source of plant material been considered?

2. What are the specific health and environmental implications associated with the use of natural products like essential oils compared to traditional pesticides?

3. How was it ensured that the seed storage conditions were appropriate for the tests, and how was variability in these conditions controlled during the experiments? Did the authors consider any potential sources of systematic errors in their experiments?

4. The discussion frequently raises the topic of variability in the composition of essential oils. Do the authors plan to examine different sources of plants or cultivation conditions to assess how these factors affect the effectiveness of natural insecticides? What are the implications of this for future research?

5. When discussing the mechanisms of action of essential oils on pests, the authors mention the inhibition of acetylcholinesterase activity. Have the authors investigated this mechanism using more detailed experiments, such as enzyme activity assays or molecular modeling, to better understand the specific interactions between the chemical compounds and the pests?

6. What specific steps can be taken to implement the findings of this study in agricultural practice? Are there guidelines for the application of these essential oils in commercial conditions, as well as assessments of their long-term effects on pollinators and other beneficial organisms?

Author Response

Comment 1 - How do the authors intend to account for biodiversity and potential differences in the chemical composition of the discussed essential oils in the context of their effectiveness against S. zeamais? Have possible differences in effectiveness based on the source of plant material been considered?
Response:  The chemical composition of essential oils is very diverse. The biodiversity, especially in relation to different botanical families and plant species, can influence Through a screening of various plants, those with the highest potential for alternative control of stored grain insects can be identified. Different sources of essential oils can exhibit variable effectiveness against S. zeamais due to the diversity of compounds present and their synergistic interactions. Therefore, the origin of plant material is a crucial consideration, and future studies should explore how different species and cultivation conditions affect chemical composition and, consequently, the oils' effectiveness.


Comment 2: What are the specific health and environmental implications associated with the use of natural products like essential oils compared to traditional pesticides?
Response: Chemical pesticides pose significant health risks to applicators, potentially causing acute or chronic intoxication. For products like phosphine, there are additional environmental concerns related to the disposal of used tablets, which can leave toxic residues. In contrast, essential oils, as natural products, present lower toxicity risks for both applicators and the environment. However, their safe and effective application requires further study to ensure they can be a viable and sustainable alternative to synthetic pesticides.

Comment 3: How was it ensured that the seed storage conditions were appropriate for the tests, and how was variability in these conditions controlled during the experiments? Did the authors consider any potential sources of systematic errors in their experiments?

Response: All tests were conducted in the same laboratory under standardized conditions. Furthermore, all evaluations were performed by a single researcher, the first author, which minimizes potential variability in data collection. Therefore, the possibility of systematic error was reduced. The methodology used is well-established in the field, supported by various published references, and previously validated by the research group in renowned scientific journals.

Comment 4: The discussion frequently raises the topic of variability in the composition of essential oils. Do the authors plan to examine different sources of plants or cultivation conditions to assess how these factors affect the effectiveness of natural insecticides? What are the implications of this for future research?

Response:The chemical composition of essential oils can vary depending on several factors, including the time of collection, plant age, plant part used for extraction, and management practices. This chemical variability influences the toxicity of the oils, not only concerning the major compounds but also their synergistic interactions with minor components. In the future, essential oils offer an excellent opportunity for managing resistance associated with synthetic pesticides, as they often have multiple modes of action. However, large-scale application requires the development of nanoemulsion techniques, formulations that prevent phytotoxicity, and the selection of plant materials that are more resistant to light and potentially have longer residual effects.

Comment 5: When discussing the mechanisms of action of essential oils on pests, the authors mention the inhibition of acetylcholinesterase activity. Have the authors investigated this mechanism using more detailed experiments, such as enzyme activity assays or molecular modeling, to better understand the specific interactions between the chemical compounds and the pests?

Response: This study did not include detailed experiments on the mechanisms of action, such as acetylcholinesterase inhibition or analyses using molecular markers. However, other studies in the literature have explored enzymatic inhibition by essential oils. Future research could include these approaches to deepen our understanding of the mode of action of these oils against S. zeamais.

Comment 6: What specific steps can be taken to implement the findings of this study in agricultural practice? Are there guidelines for the application of these essential oils in commercial conditions, as well as assessments of their long-term effects on pollinators and other beneficial organisms?

Response: Basic research like the present study is essential to provide practical information on formulations for large-scale applications. To implement these findings in the field, it is necessary to investigate the toxicity of the essential oils at different doses and the susceptibility of target pests, such as S. zeamais. However, there is currently a lack of studies on the effects of these oils on pollinators. Regarding natural enemies, such as predators and parasitoids, studies have indicated that most essential oils are selective, thereby preserving these beneficial organisms. Therefore, management strategies should include clear guidelines for the safe and sustainable application of these products in commercial conditions.

Reviewer 3 Report

Comments and Suggestions for Authors

The comments are in the manuscript.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

*The questions/suggestions from Reviewer 3 were addressed in the same PDF file where the comments were made.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Well-done study using several essential oils tested as potential botanical insecticides for pest control of stored corn seeds, a lot of experiments using wide range of concentrations, besides acute toxicity also repellent effects, persistence and fumigation effects were studied. In addition, phytotoxicity affecting seed germination was assessed and chemical analysis of essential oils was performed. The study brings a lot of new results which have application potential. Manuscript is well written, methods are sound nevertheless I recommend to revise it - see suggestions below.

Specific comments (numbers indicate line in ms):

Title: consider to write ... insecticides against Sitophilus ... or ... insecticides for Sitophilus zeamais control ... Also, remove quotation marks.

Abstract

Some modification can be done to improve the clarity.

22-23 part on germination can be left for next sentence where it is germination is repeated.

23-24 the effects like toxicity should be specified it is against pest so that readers do not think it is phytotoxicity

31-32 but how about other compounds/oils? From this sentence it could be understood that others did affect germination.

Keywords: perhaps "phytotoxicity" and/or "storage pest" could be added

Introduction: I believe it should be enhanced/more elaborated, e.g. some details on phytotoxicity. Moreover, I miss any information on safety of α-pinene and other compounds for human.

52-55 One-sentence paragraphs do not look nice, try to merge them or modify the text.

Material and Methods

91-92 this sentence is repetition of the previous one. please delete it

95-100 Because full scientific names with authors were already written in Introduction, there is no need to repeat them, just use abbreviated form (e.g. J. communis)

110-111

116 delete quotation mark

124-127 change bold text to normal

143 specify model/manufacturer of micropippetor (or micropipette?)

145 seems incomplete sentence (I guess ...adults ... were introduced ...)

156 the same problem: some text is missing

166 what does it mean "without sex"?

167 what type of paper? brand or manufacturer?

170-174 was there any control, i.e. the same design but without any compound applied?

206 check description of variables used in equation

228-229 ANOVA requires normal distribution which in not in case of % of mortality so data should be either normalized first (e.g. by arcsin sqrt transformation) or GLM should be used

Results

266 in addition to table, figures with concentration-response curves and original data would be appreciated. The same for fumigation toxicity in next section

Table 4 - it would be better seen as graph, means +- SE

Table 6 - testemunha = control?

Discussion - it is rather long (> 4 pages) but considering the amount of results it can be justified

388 species names in italics

434 this short sentence is unclear

579-589 the last paragraph should be placed as Conclusions section

 

Comments on the Quality of English Language

English seems to be fine, only few mistakes/incomplete sentences etc.

Author Response

Comment 1: Title: consider to write ... insecticides against Sitophilus ... or ... insecticides for Sitophilus zeamais control ... Also, remove quotation marks.

Response: The quotation marks were removed, and "on" was replaced by "Against" in the title.

Comment 2: 22-23 part on germination can be left for next sentence where it is germination is repeated.

Response: For better reading flow, the following part was removed: "as well as to evaluate the possible effects on the germination of the seeds." 

Comment 3: 23-24 the effects like toxicity should be specified it is against pest so that readers do not think it is phytotoxicity.

Response: As 'contact toxicity' is mentioned in the text, the authors believe that the expression is clear enough given the context.

Comment 4: 31-32 but how about other compounds/oils? From this sentence it could be understood that others did affect germination.
Response: The germination test was not conducted with all the oils and compounds, only with those of higher toxicity (higher LC₅₀ and LC₉₅ in the contact test and oil availability). This information was added to the materials and methods section.

Comment 5: Keywords: perhaps "phytotoxicity" and/or "storage pest" could be added.

Response: The keyword "Storage pest" was added in the manuscript.

Comment 6: Introduction: I believe it should be enhanced/more elaborated, e.g. some details on phytotoxicity. Moreover, I miss any information on safety of α-pinene and other compounds for human.

Comment 7: 52-55 One-sentence paragraphs do not look nice, try to merge them or modify the text.
Response:  The paragraphs was rephrased as follows: 

"The plant species that produce essential oils are distributed throughout the world and in several plant families and has a toxicity potential for insects [12–16]. For example, essential oils from plant species Gaultheria procumbens L. (Ericaceae) [17–18], Juniperus communis L. (Cupressaceae) [19], and the genus Protium (Burseraceae) [20] showed toxicity to pests that attack stored products in previous studies, as well as the compound α-pinene [21–23]."

Comment 8: 91-92 this sentence is repetition of the previous one. please delete it
Response: The sentence was deleted. 

Comment 9: 95-100 Because full scientific names with authors were already written in Introduction, there is no need to repeat them, just use abbreviated form (e.g. J. communis)

Response: The scientific names were corrected as required in the final manuscript.

Comment 10: 110-111

Response: ?

Comment 11: 116 delete quotation mark
Response: The quotation mark was removed. 

Comment 12: 124-127 change bold text to normal
Response: The bold texts were changed to normal. 

Comment 13: 143 specify model/manufacturer of micropippetor (or micropipette?)
Response: The manufacturer was cited in the final manuscript. 

Comment 14:  145 seems incomplete sentence (I guess ...adults ... were introduced ...)
Response: It was rephrased in the final manuscript. 

Comment 15: 156 the same problem: some text is missing
Response: It was rephrased in the final manuscript. 

Comment 16: 166 what does it mean "without sex"?
Response: It was changed for "unsexed" as the others. 

Comment 17: 167 what type of paper? brand or manufacturer?
Response: It was used filter-paper. This information was added in the manuscript. 

Comment 18: 170-174 was there any control, i.e. the same design but without any compound applied?
Response: Yes, there was. The following sentence was added: "There was a control treatment (no oil application)"

Comment 19: 206 check description of variables used in equation
Response: They were checked ans corrected in the manuscript. 

Comment 20: 228-229 ANOVA requires normal distribution which in not in case of % of mortality so data should be either normalized first (e.g. by arcsin sqrt transformation) or GLM should be used
Response: Yes, the data used has previously undergone the normality test. It was highlighted in the final manuscript. 

Results

Comment 21: 266 in addition to table, figures with concentration-response curves and original data would be appreciated. The same for fumigation toxicity in next section
Response: The submitted data supports the research and as only one reviewer requested it, we found it unnecessary to include it and did not comply with this request.

Comment 22: Table 4 - it would be better seen as graph, means +- SE
Response: As it is just a suggestion, we thought it best not to accept it and the data remains represented in table form.

Comment 23: Table 6 - testemunha = control?
Response: "testemunha" was replaced by "control"

Discussion - it is rather long (> 4 pages) but considering the amount of results it can be justified

Comment 24: 388 species names in italics
Response: They were all corrected. 

Comment 25: 434 this short sentence is unclear
Response: It was changed by "related to the methodology of application used" for a better understanding

Comment 26: 579-589 the last paragraph should be placed as Conclusions section
Response: The "conclusions section" was added in the final manuscript wih this last paragraph

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper has been substantially improved. Please correct "pineno" to "pinene" in Figure 1.