Biological and Physiological Changes in Spodoptera frugiperda Larvae Induced by Non-Consumptive Effects of the Predator Harmonia axyridis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript is detailed and provides enough insight into the authors' study. 

The conclusion, however, vaguely concludes the study and instead introduces new concepts making it more a part of the discussion -needing more elaboration.

The increase in ROS appears specific to earlier instars, not just to the general larvae. Saying so seems misleading.

The authors raise an interesting point with "...adjust of metabolic pathways", however, there is no earlier discussion before the conclusion. It would be better to discuss this in the discussion not just place it in the conclusion. similarly for the "redistribution of resources". 

Energy cost/storage was not addressed in the manuscript. Based on Figure 4 the "Non-consumptive effect" present seems to be related to feeding habits in the presence of the predator. Did the authors confirm feeding in their experiments?  Changes in feeding habits would also influence other factors such as those mentioned in Figure 2. 

Author Response

Dear Sir/Madam,

Thank you for the response received from the Reviewers on our manuscript. Please now find enclosed a revised version of the manuscript. We fully appreciate all the constructive comments received from the Reviewers. We have taken on board and revised the manuscript carefully according to the comments and suggestions received. We believe the revision has greatly added to the quality of the manuscript.

The Reviewer comments are now listed below with our response given to each one.

I trust this revised manuscript now proves satisfactory. All co-authors are in agreement with the revised version.

Yours sincerely,

 

Major points:

Manuscript is detailed and provides enough insight into the authors' study. 

The conclusion, however, vaguely concludes the study and instead introduces new concepts making it more a part of the discussion -needing more elaboration.The increase in ROS appears specific to earlier instars, not just to the general larvae. Saying so seems misleading.The authors raise an interesting point with "...adjust of metabolic pathways", however, there is no earlier discussion before the conclusion. It would be better to discuss this in the discussion not just place it in the conclusion. similarly for the "redistribution of resources". 

Thank you for your valuable feedback regarding the conclusion section of our manuscript. We have carefully revised the conclusion to address your concerns. The updated conclusion now provides a clearer summary of the study's findings without introducing new concepts. We have also deleted the discussion of specific points, such as the increase in ROS, adjustments to metabolic pathways and redistribution of resources. Please see line 372-391.

Energy cost/storage was not addressed in the manuscript. Based on Figure 4 the "Non-consumptive effect" present seems to be related to feeding habits in the presence of the predator. Did the authors confirm feeding in their experiments? Changes in feeding habits would also influence other factors such as those mentioned in Figure 2. 

Thank the reviewer for your comments. About “energy cost/storage”, when insect activity increases or they experience stress, carbohydrates are rapidly consumed to provide the required energy. And fat substances are the primary form of long-term energy storage in insects. Fats are stored in fat bodies, providing a prolonged energy supply for the insect. The changes in carbohydrates and fat substances within insects can indeed reflect their energy cost and storage. About feeding, we confirm feeding in our experiments. We provided enough maize leaves for the Armyworm and replaced them regularly, and the feeding behavior of Armyworm did not change significantly.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, I have reviewed the manuscript "Survival strategies of Spodoptera frugiperda larvae under non-consumptive effects of the predator Harmonia axyridis."

The authors must address some doubts, suggestions, and observations that were found.

In the Introduction section

Lines 50-55 are repeated later; it is recommended that lines 76-79 be deleted at first mention.

Line 56 includes taxonomic authority, order, and family of Spodoptera frugiperda.

Line 74, full name, includes taxonomic authority, order, and family of H. axyridis.

In the Materials and Methods section

Lines 82, What were the stages of development of Spodoptera frugiperda that were collected?

Lines 82-83 include geographic coordinates.

Lines 83-88: There is confusion. On line 83, they mention that the individuals were raised under laboratory conditions, and on line 86, they mention that they were raised under greenhouse conditions. Please clarify.

Lines 89-91. What is the origin of Harmonia axyridis? Was it collected? Did some institutions donate them? Was its taxonomic identification required?

Line 89. under laboratory conditions? Include temperature, relative humidity, and photoperiod. Did you use a growth chamber?

Also confusing is that the predator was raised with Acyrthosiphon pisum. However, the authors also mention that H. axyridis was fed with S. frugiperda larvae for three generations before starting the experiment. Really, How were S. frugiperda larvae raised?

Line 98. The authors mention "young maize leaves." They refer to "young maize leaves" as the first leaf that appears, the second leaf that appears, the third leaf that appears, etcetera. Please be more precise.

Line 99. How do they determine the age of H. axyridis? How did they know that the age of H. axyridis ranged between 3 and 5 days? Was there any methodology to determine the age of H. axyridis?

Line 98 and 111. Include the agronomic issues of how corn was grown, fertilization, irrigation, frequency of application of fertilization, and irrigation. Variety, line of corn was used? Under what conditions (greenhouse or field) was the corn crop used to feed the different stages of development of S. frugiperda carried out? Include all variables since they evaluate the non-consumptive effects on nutrients in S. frugiperda larvae. Because the food quality influences the development of fall armyworm larvae.

Line 112. Spodoptera frugiperda larvae were exposed to the predator H. axyridis for 2 to 3 days. They were 24 hours apart; this difference may have stressed the S. frugiperda larvae that were exposed to the predator for three days more than those that were exposed for two days.

Why that exposure interval?

Line 112. Between 2 to 3 days, S. frugiperda larvae were exposed to the predator H. axyridis. What did the predator consume during those days? How many days does H. axyridis live without feeding?

Line 121. Was the collection random?

The experimental design is unclear in subsections 2.2 and 2.3 and was reflected in subsection 2.6 (Data Analysis). The experimental design must have contained a control group (without the presence of the predator) of the same developmental stage of the treatment (with the presence of the predator) and then apply a Student's t-test to make the comparison to observe the effect of the predator on each developmental stage (first, second, and third) of the fall armyworm larvae: for example treatment: first instar in the presence of the predator; control group: first instar without the presence of the predator, and so on until the third instar.

In the ANOVA, the authors compare the three instars against a control group. However, they do not specifically mention the developmental stage of fall armyworms. Furthermore, and most importantly for me, this comparison is erroneous. This comparison cannot be analyzed because a single statistical test includes the three instars and the control group, that is, third vs. control, first vs. control, and second vs. control, and it is the same control group = it is the same group of individuals. The correct thing is that each treatment (whether first, second, or third instar in the presence of the predator) was compared with a control (first, second, or third instar without the presence of the predator), and then the Student's t-test.

 

Therefore, mentioning that there are or are not differences in the developmental duration of larvae, pupation rate, developmental duration of pupa, abnormality rate, pupal mass, and emergence rate of the first, second, and third instar of S. frugiperda with the presence or not of the predator may be wrong. Therefore, the results presented may be biased towards reality and the conclusions.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Dear Sir/Madam,

Thank you for the response received from the Reviewers on our manuscript. Please now find enclosed a revised version of the manuscript. We fully appreciate all the constructive comments received from the Reviewers. We have taken on board and revised the manuscript carefully according to the comments and suggestions received. We believe the revision has greatly added to the quality of the manuscript.

The Reviewer comments are now listed below with our response given to each one.

I trust this revised manuscript now proves satisfactory. All co-authors are in agreement with the revised version.

Yours sincerely,

 

Major points:

Dear Authors, I have reviewed the manuscript "Survival strategies of Spodoptera frugiperda larvae under non-consumptive effects of the predator Harmonia axyridis."

The authors must address some doubts, suggestions, and observations that were found.

In the Introduction section

Lines 50-55 are repeated later; it is recommended that lines 76-79 be deleted at first mention.

Done. We have deleted it. Please see line 83-95.

Line 56 includes taxonomic authority, order, and family of Spodoptera frugiperda.

Done. We added taxonomic authority, order, and family of Spodoptera frugiperda at first mention. Please see line 59.

Line 74, full name, includes taxonomic authority, order, and family of H. axyridis.

Done. We added taxonomic authority, order, and family of H. axyridis at first mention. Please see line 58-59.

In the Materials and Methods section

Lines 82, What were the stages of development of Spodoptera frugiperda that were collected?

Done. Spodoptera frugiperda adult individuals were collected. Please see line 104.

Lines 82-83 include geographic coordinates.

Done. We added geographic coordinates. Please see line 105.

Lines 83-88: There is confusion. On line 83, they mention that the individuals were raised under laboratory conditions, and on line 86, they mention that they were raised under greenhouse conditions. Please clarify.

Done. We have revised details. Please see line 109.

Lines 89-91. What is the origin of Harmonia axyridis? Was it collected? Did some institutions donate them? Was its taxonomic identification required?

Harmonia Axyridis adult individuals were collected in 2018 from lettuce in farm of South China Agricultural University (SCAU). we added details in line 111-112. The identification of H. axyridis can be made based on external appearance.

Line 89. under laboratory conditions? Include temperature, relative humidity, and photoperiod. Did you use a growth chamber?

Our study used Harmonia axyridis reared in a specialized laboratory with controlled lighting, temperature, and humidity tailored for this species. Please see line 113-114.

Also confusing is that the predator was raised with Acyrthosiphon pisum. However, the authors also mention that H. axyridis was fed with S. frugiperda larvae for three generations before starting the experiment. Really, How were S. frugiperda larvae raised?

The predator was reared on pea aphids Acyrthosiphon pisum as food to maintain the colony. After Spodoptera frugiperda adults lay eggs, the eggs are allowed to hatch into larvae. Rearing Spodoptera frugiperda larvae alone in predator-free petri dishes. Once hatched, the larvae are fed fresh corn leaves, which are their natural host plant. In addition, before starting the experiment, it’s necessary to switch the beetles’ food from aphids to 1st-3rd instar S.frugiperda and feed them three generations to acclimate to their prey. Please see line 112-116.

Line 98. The authors mention "young maize leaves." They refer to "young maize leaves" as the first leaf that appears, the second leaf that appears, the third leaf that appears, etcetera. Please be more precise.

Done. Please see line 123-124.

Line 99. How do they determine the age of H. axyridis? How did they know that the age of H. axyridis ranged between 3 and 5 days? Was there any methodology to determine the age of H. axyridis?

Maintain detailed records of rearing conditions, including the date of eclosion, and monitor the beetles regularly to track their developmental milestones. Ladybugs that are 3-5 days post-eclosion can be used for the experiment.

Line 98 and 111. Include the agronomic issues of how corn was grown, fertilization, irrigation, frequency of application of fertilization, and irrigation. Variety, line of corn was used? Under what conditions (greenhouse or field) was the corn crop used to feed the different stages of development of S. frugiperda carried out? Include all variables since they evaluate the non-consumptive effects on nutrients in S. frugiperda larvae. Because the food quality influences the development of fall armyworm larvae.

We apologize for that we overlooked this important information. We planted maize seeds (Zhongnong Sweet Maize 488) in plastic pots (28 cm in diameter, 20 cm in height) filled with a soil-sand mixture (10% sand, 5% clay, and 85% peat). The plants were cultivated in greenhouse, free from arthropod infestations, and maintained under natural temperature and light conditions. The plants were regularly watered and used for the experiment once they developed 6-8 fully expanded leaves.We have added details. Please see line 98-103. Meanwhile, when the maize plants develop 6-8 fully expanded leaves, we cut the 2nd and 3rd leaves and use them to feed Spodoptera frugiperda under laboratory conditions.

Line 112. Spodoptera frugiperda larvae were exposed to the predator H. axyridis for 2 to 3 days. They were 24 hours apart; this difference may have stressed the S. frugiperda larvae that were exposed to the predator for three days more than those that were exposed for two days. Why that exposure interval?

The exposure duration was determined based on the developmental stage of the larvae. The stress exposure period varied because it was linked to the larvae's progression to the next developmental stage, which is confirmed by measuring cephalic capsule width ( Santos et al. 2003). Since the developmental times for each larval stage (1st, 2nd, and 3rd instars) differ, ranging from 2 to 3 days, the exposure duration varied accordingly.Therefore, the described exposure period of 2-3 days accounts for the variability in developmental time across different larval stages. This approach ensured that the exposure duration was relevant to the larvae's developmental progress and provided a more accurate assessment of the effects of predation stress.

Line 112. Between 2 to 3 days, S. frugiperda larvae were exposed to the predator H. axyridis. What did the predator consume during those days? How many days does H. axyridis live without feeding?

In our study, no food was given to H. axyridis during the exposure. In general, ladybirds can live without food for about one weeks.

Line 121. Was the collection random?

Yes. We have revised this. Please see line 148-149.

The experimental design is unclear in subsections 2.2 and 2.3 and was reflected in subsection 2.6 (Data Analysis). The experimental design must have contained a control group (without the presence of the predator) of the same developmental stage of the treatment (with the presence of the predator) and then apply a Student's t-test to make the comparison to observe the effect of the predator on each developmental stage (first, second, and third) of the fall armyworm larvae: for example treatment: first instar in the presence of the predator; control group: first instar without the presence of the predator, and so on until the third instar.

In the ANOVA, the authors compare the three instars against a control group. However, they do not specifically mention the developmental stage of fall armyworms. Furthermore, and most importantly for me, this comparison is erroneous. This comparison cannot be analyzed because a single statistical test includes the three instars and the control group, that is, third vs. control, first vs. control, and second vs. control, and it is the same control group = it is the same group of individuals. The correct thing is that each treatment (whether first, second, or third instar in the presence of the predator) was compared with a control (first, second, or third instar without the presence of the predator), and then the Student's t-test.

Therefore, mentioning that there are or are not differences in the developmental duration of larvae, pupation rate, developmental duration of pupa, abnormality rate, pupal mass, and emergence rate of the first, second, and third instar of S. frugiperda with the presence or not of the predator may be wrong. Therefore, the results presented may be biased towards reality and the conclusions.

We understand the reviewers' concerns regarding the control group setup. In biological experiments, the control group for first-instar stress treatment typically corresponds to first-instar no-stress treatment (and also includes second and third instars with no stress). For second-instar stress treatment, the control group corresponds to second-instar no-stress treatment (and also includes first and third instars with no stress). For third-instar stress treatment, the control group corresponds to third-instar no-stress treatment (and also includes first and second instars with no stress). All three control groups are no-stress treatments for all developmental stages (first, second, and third instars) because all experiments are conducted under the same conditions. To simplify the experimental design and reduce resource consumption, we used a unified control group as the comparison baseline for all stress treatment groups. We believe that, given that all experimental groups are under the same conditions, a single control group can reasonably provide the needed baseline comparison. Relevant literature also indicates that using a single control group in similar experimental designs is a common and reasonable practice,such as Fan et al.( 2021) Non-consumptive effects of Encarsia formosa on the reproduction and metabolism of the whitefly Bemisia tabaci, Biocontrol.

Furthermore, in this part of the experiment, after the stress treatment, the individuals continued to develop until pupation and eventual adult emergence. We also aimed to explore at which instar stage the fall armyworm is most affected. Therefore, in the data analysis, we used ANOVA to compare differences among the four groups. In sections 2.4 and 2.5, due to the need for immediate sampling after treatment, we set up respective control groups and used T-tests for data analysis.

We believe this design effectively evaluates the impact of stress on different instar stages of the fall armyworm while ensuring scientific rigor and reliability of the results.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The proper hypothesis may be given in the introduction. Why the study has been planned, whether the predator is significantly more active in the maize ecosystem. If yes, the study results will be interesting. Unrelated discussions might be removed. 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Good

Author Response

Dear Sir/Madam,

Thank you for the response received from the Reviewers on our manuscript. Please now find enclosed a revised version of the manuscript. We fully appreciate all the constructive comments received from the Reviewers. We have taken on board and revised the manuscript carefully according to the comments and suggestions received. We believe the revision has greatly added to the quality of the manuscript.

The Reviewer comments are now listed below with our response given to each one.

I trust this revised manuscript now proves satisfactory. All co-authors are in agreement with the revised version.

Yours sincerely,

 

Major points:

The proper hypothesis may be given in the introduction. Why the study has been planned, whether the predator is significantly more active in the maize ecosystem. If yes, the study results will be interesting. Unrelated discussions might be removed.

Done. We have added proper hypothesis in the introduction. Please see line 83-95. Many studies have shown that non-consumptive effects can be directly applied in biological control. Therefore, we designed relevant experiments to explore whether stress of Harmonia axyridis can have a negative impact on Armyworm, providing a basis for developing new biological control methods. Harmonia axyridis is significantly more active in the maize ecosystem, which can prey on aphids, mealybugs, whiteflies, mites, and young larvae of lepidoptera. However, our experiments were conducted in the laboratory, and no field trials have been performed. Therefore, the non-consumptive effects in the field still need further investigation.    

Line 60 Kindly verify the statement, at what stage the infestation results in crop failure

Done. Please see line 69.

Line 69 Give the common name and authority of the predator

Done. Please see line 58-59.

Line 89. Harmonia axyridis revise to H. axyridis 

Done. Please see line 111.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript provides valuable insights into the non-consumptive effects of Harmonia axyridis on Spodoptera frugiperda larvae, contributing to our understanding of pest management strategies. The study is well-structured and the experimental design is sound, offering robust data. However, the manuscript could benefit from clearer presentation of results and a stronger connection between findings and practical implications for agricultural pest control. Enhancing the discussion with more comparisons to existing research and addressing potential limitations would also strengthen the manuscript. Overall, with some revisions, this study has strong potential for publication in the journal "Agriculture."

Title

• The title is clear and informative but consider specifying the type of non-consumptive effects to make it more precise (e.g., "Behavioural and physiological non-consumptive effects...").

Abstract

• The abstract is concise and effectively summarizes the study. However, the readability could be improved by breaking down long sentences.
• The abstract lack the methodology.

Introduction

• The introduction provides a good background but could benefit from more detailed in expanding on the novelty of your study by comparing it more explicitly with existing research on non-consumptive effects.

•  

Materials and Methods

• The methodology is well-detailed, but it might be useful to include more information on the statistical methods used. Specify the rationale behind choosing certain tests and any assumptions that were made.

Discussion

• The discussion effectively links the findings to broader ecological and pest management contexts. However, it could be strengthened by further comparing your results with those of similar studies.
• The section on predator detection mechanisms could be expanded by discussing potential applications or future research directions, particularly on how these mechanisms could be leveraged in integrated pest management.

Conclusion

• The conclusion is succinct but could benefit from a stronger emphasis on the practical implications of the findings for agricultural practices.
• Consider adding a sentence or two about the limitations of the study and potential areas for future research.

Please, refer to the attached for other comments.

Comments for author File: Comments.pdf

Author Response

Dear Sir/Madam,

Thank you for the response received from the Reviewers on our manuscript. Please now find enclosed a revised version of the manuscript. We fully appreciate all the constructive comments received from the Reviewers. We have taken on board and revised the manuscript carefully according to the comments and suggestions received. We believe the revision has greatly added to the quality of the manuscript.

The Reviewer comments are now listed below with our response given to each one.

I trust this revised manuscript now proves satisfactory. All co-authors are in agreement with the revised version.

Yours sincerely,

Major points:

The manuscript provides valuable insights into the non-consumptive effects of Harmonia axyridis on Spodoptera frugiperda larvae, contributing to our understanding of pest management strategies. The study is well-structured and the experimental design is sound, offering robust data. However, the manuscript could benefit from clearer presentation of results and a stronger connection between findings and practical implications for agricultural pest control. Enhancing the discussion with more comparisons to existing research and addressing potential limitations would also strengthen the manuscript. Overall, with some revisions, this study has strong potential for publication in the journal "Agriculture."

Title

The title is clear and informative but consider specifying the type of non-consumptive effects to make it more precise (e.g., "Behavioural and physiological non-consumptive effects...").

We revised title to “Biological and Physiological Changes in Spodoptera frugiperda larvae under non-consumptive effects of the predator Harmonia axyridis”. Please see line 2-4.

Abstract

The abstract is concise and effectively summarizes the study. However, the readability could be improved by breaking down long sentences.

The abstract lack the methodology.

Done. We have broken down long sentence and added the methodology, please see line 13-31.

Introduction

The introduction provides a good background but could benefit from more detailed in expanding on the novelty of your study by comparing it more explicitly with existing research on non-consumptive effects.

Done. We have expanded on the novelty of our study by comparing it more explicitly with existing research on non-consumptive effects. Please see line 56-65.

Why those instars been chosen?

Harmonia axyridis primarily feeds on 1st-3rd instar larvae and does not feed on 4th-6th instar larvae. The 4th-6th instar larvae may exhibit aggressive behavior towards Harmonia axyridis. Therefore, 1st-3rd instar larvae were selected for the experiment.

Materials and Methods

The methodology is well-detailed, but it might be useful to include more information on the statistical methods used. Specify the rationale behind choosing certain tests and any assumptions that were made.

What about night for this every 3h?

Done. We have added details on the statistical methods, please see line 204-219. And we have added the rationale, please see line 159-174, 184-202.

To ensure a continuous supply of food and avoid any potential shortages, pieces of maize leaves were added to the larvae's habitat every 3 hours during the day. For the nighttime period, we maintained the same feeding schedule by having the leaves added every 3 hours as well, ensuring that the larvae had a constant food supply throughout the entire 24-hour cycle.

Discussion

The discussion effectively links the findings to broader ecological and pest management contexts. However, it could be strengthened by further comparing your results with those of similar studies.

The section on predator detection mechanisms could be expanded by discussing potential applications or future research directions, particularly on how these mechanisms could be leveraged in integrated pest management.

Done. We have compared our results with those of similar studies. Please see line 296-299, 310-311, 328. We have expanded discussing potential applications or future research directions. Please see line 336-342.

Conclusion

The conclusion is succinct but could benefit from a stronger emphasis on the practical implications of the findings for agricultural practices.

Consider adding a sentence or two about the limitations of the study and potential areas for future research.

Please, refer to the attached for other comments.

Done. We have discussed practical implications of the findings for agricultural practices, please see line 382-386. and we have added limitations, please see line 386-391.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I have reviewed the article again, along with the responses to my observations in the first review. The authors have justified and substantially improved the document.