Predatory Potential of Nymphal Odonates on Aedes aegypti Developing in Freshwater and Brackish Water Habitats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is a nice manuscript using aquatic insects against Aedes larval stages. In the fight against chemical insecticides, everything is valid to protect the environment. I see a well-designed study, strong treatment repetitions, the correct molecular identifications of immature nymphs, etc.

However, most similar studies are very successful but conducted under lab conditions. Open field operations demand important data, primarily related to mass-rearing, and many details about the sustainability of the predators in the artificial Aedes aegypti containers in household backyards.

In lines #347-352, I suggest adding comments about failures and successes during the mass-rearing of other Odonata species worldwide. It will be useful for young Investigators to consider this for future research projects. 

A typical question also needs to be addressed. What will the predator eat after killing all Aedes larvae in the water FW or BW container? Is there an alternative and available species there? In lines #233-248, the authors describe PR and PI as less than 50% in most tested species. A logical question for readers would be: Did they find or need to eat another organism? Anyway, I am sure that supplementary comments will enrich the paper.

Regarding water salinity, I suggest supplementing it with some limnological information, such as shade, pH, and conductivity. Indeed, it will help better describe the prey and predator environments in Sri Lanka.

Author Response

RESPONSE TO REVIEWERS’ COMMENTS_ Reviewer 1 (insects-3097398)

Thank you for overall comments and we have addressed all the comments.

Comment 1:

However, most similar studies are very successful but conducted under lab conditions. Open field operations demand important data, primarily related to mass-rearing, and many details about the sustainability of the predators in the artificial Aedes aegypti containers in household backyards.

Response 1: Thank you for pointing this out. We agree with this comment. We have addressed this point in the revised manuscript in line # 385-391.

Comment 2:

In lines #347-352, I suggest adding comments about failures and successes during the mass-rearing of other Odonata species worldwide. It will be useful for young Investigators to consider this for future research projects.

Response 2:

We agreed with the suggested review. This has been addressed in line # 385-400.

Comment 3:

A typical question also needs to be addressed. What will the predator eat after killing all Aedes larvae in the water FW or BW container? Is there an alternative and available species there?

Response 3:

Thank you for pointing out this. There are alternative foods for the predators present in the mesocosm setup. The microbiota present in the natural pond water will be the alternative food for the odonates. We did mention this in the line # 139.

Comment 4:

In lines #233-248, the authors describe PR and PI as less than 50% in most tested species. A logical question for readers would be: Did they find or need to eat another organism? Anyway, I am sure that supplementary comments will enrich the paper.

Response 4:

Thank you.

The statement was unfortunately misread. What mentioned was “on average, predatory rate of the tested predators were above 50% for all the species with one exception”.  To describe clearly, we have now described predatory impact as percentage consumption per hour.

Comment 5:

Regarding water salinity, I suggest supplementing it with some limnological information, such as shade, pH, and conductivity. Indeed, it will help better describe the prey and predator environments in Sri Lanka.

Response 5: Thank you for pointing out this. We agree with the suggestion. Only water salinity was considered as to evaluate the development of odonates in different salinity conditions. We agree with your suggestion and these factors will definitely be considered for wider research on the biological control of dengue vectors in the future.

Reviewer 2 Report

Comments and Suggestions for Authors

Arthiyan et al. report the results of a laboratory study on odonate nymph predation of Aedes larvae. The manuscript is fairly clearly written, but the presentation of the Results and the statistical inferences require improvement.
I have written comments and numbered points on a scanned copy of the manuscript.
NUMBERED POINTS (see scanned file)
1. significantly higher than what? Do you mean "the highest"?
2. Do not use keywords that are already in the title.
3. Please provide information on the diet used to rear larvae and how adults from the colony were blood-fed.
4. It was unclear whether you are reporting 98% similarity with database sequences or 98% agreement between sequence based identifications and morphological identifications.
5. Matured is a strange term to use with nymphs (implies sexual maturity). Do you mean late instar or final instar?
6. Could you provide us with some details on the "naturally occurring biota" that odonates were consuming?
7. The number of abbreviations increases with the use of these terms. I suggest using the full words in the text rather than abbreviations for improved clarity.
8. a) I was uncertain about the 3-way ANOVA. How did you check for equality of variances prior to ANOVA?
b) Were the data normal?
c) How did you compare means?
d) You need to make it clear that species, salinity and time were the three factors that you used in the ANOVA analysis.
9. Are the ± values SE? or SD?
10. Please provide units for the predatory impact response . Is this percentage of larvae consumed/hour [line 189]? If so, how are values so large 23% of larvae consumed per hour over 24 h is >>100% consumption! Are these per day values? Unclear.
11. Table 3.
a) This does not appear to be a three-way ANOVA - you only compare the FW/BW effects (all non-significant). I expected to see 3-way comparisons shown by different letters after numerical values to compare species, time and salinity. Table S1 does not do much to help.
b) What about all the interactions? The purpose of 3-way ANOVA is arguably to reveal important interaction effects, isn't it?
12. Your results suggest that odonates could contribute to the biological control of mosquito vectors. It would therefore be wise to conserve their populations. However, vector control activities may often disrupt the aquatic habitats in which mosquitoes develop. Are their any studies on the effect of vector control practices on odonate populations or odonate diversity?
13. You may wish to conclude that the laboratory findings require validation in field studies on mosquito oviposition sites.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

RESPONSE TO REVIEWERS’ COMMENTS_ Reviewer 2 (insects-3097398)

Thank you for overall comments and corrections in the manuscript. We have accepted almost all corrections indicated in the manuscript. Also, we have attended to the suggestions, and adjusted the text according to the scanned review document of our article sent by the reviewer.

Comment 1:

significantly higher than what? Do you mean "the highest"?

Response 1: Thank you for highlighting this. This has been addressed in line # 29

Comment 2:

Do not use keywords that are already in the title.

Response 2: Agreed. This has been addressed in the line # 34 and 35.

Comment 3:

Please provide information on the diet used to rear larvae and how adults from the colony were blood-fed.

Response 3: This has been addressed in line # 85-87.

Comment 4:

It was unclear whether you are reporting 98% similarity with database sequences or 98% agreement between sequence-based identifications and morphological identifications.

Response 4: We report the percentage similarity of the DNA sequence of each species with the database sequences. It is addressed in line # 121-122.

Comment 5:

Matured is a strange term to use with nymphs (implies sexual maturity). Do you mean late instar or final instar?

Response 5: This is reworded as late instar in line # 146.

Comment 6:

Could you provide us with some details on the "naturally occurring biota" that odonates were consuming?

Response 6: This has been addressed in line # 141.

Comment 7:

The number of abbreviations increases with the use of these terms. I suggest using the full words in the text rather than abbreviations for improved clarity.

Response 7: Agreed, and this has been addressed

Comment 8:

1. a) I was uncertain about the 3-way ANOVA. How did you check for equality of variances prior to ANOVA?
2. b) Were the data normal?
3. c) How did you compare means?
4. d) You need to make it clear that species, salinity and time were the three factors that you used in the ANOVA analysis.

Response 8:

Thank you for pointing out this. There was a mistake during this analysis, as the normality of the data was not determined at the initial analysis. Now, the data was reanalyzed and found to be not normality distributed. Appropriate non-parametric tests were done and the statistical results have been revised.

This has been addressed in line #198-204, and table 3 was revised.

Comment 9:

Are the ± values SE? or SD?

Response 9: This has been addressed in line #225-226, and revised in table 1

Comment 10:

Please provide units for the predatory impact response. Is this percentage of larvae consumed/hour [line 189]? If so, how are values so large 23% of larvae consumed per hour over 24 h is>>100% consumption! Are these per day values? Unclear

Response 10: The unit of predatory impact is percent/h; percentage of larvae consumed per hour. The units of the PI were incorporated in the text in line # 194, and 257-263.

The interpretation is that if the value is 23 percent/h, the predator will consume 23% of the available prey per hour.

Comment 11

Table 3.

1. a) This does not appear to be a three-way ANOVA - you only compare the FW/BW effects (all non-significant). I expected to see3-way comparisons shown by different letters after numerical values to compare species, time and salinity. Table S1 does not do much to help.
2. b) What about all the interactions? The purpose of 3-way ANOVA is arguably to reveal important interaction effects, isn't it?

Response 11: Agreed, and this has been addressed in line # 298-299 as indicated as response to the comment 8. Table 3 has been revised based on the new revised reanalysis. We have removed the table S1.

Comment 12:

Your results suggest that odonates could contribute to the biological control of mosquito vectors. It would therefore be wise to conserve their populations. However, vector control activities may often disrupt the aquatic habitats in which mosquitoes develop. Are their any studies on the effect of vector control practices on odonate populations or odonate diversity?

Response 12: We agreed with this important comment. But we are of the view that this can be addressed by a separate study. Also, we could not come across any reports relevant to this.

Comment 13:

You may wish to conclude that the laboratory findings require validation in field studies on mosquito oviposition sites.

Response 13: We agreed with the suggestion, and it was incorporated in the text in line # 406-411.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have improved their manuscript, but there is one issue that I still do not understand. Lines 254-260: the authors state that the predatory impact was 23% at 24 h (line 257) but as predatory impact is percentage of larval prey consumer PER HOUR how can values like 23% at 24 h be possible? according to this logic 23% per hour x 24 h would be = 552%. The same applies to 18% (line 259) which would be 432% at 24 h and also in line 260.

This needs to be clarified.

I suspect that the authors have miscalculated predatory impact or are mis-reporting this value.

Comments on the Quality of English Language

Minor editing.

Author Response

Comment 1:

The authors have improved their manuscript, but there is one issue that I still do not understand. Lines 254-260: the authors state that the predatory impact was 23% at 24 h (line 257) but as predatory impact is percentage of larval prey consumer PER HOUR how can values like 23% at 24 h be possible? according to this logic 23% per hour x 24 h would be = 552%. The same applies to 18% (line 259) which would be 432% at 24 h and also in line 260.

This needs to be clarified.

I suspect that the authors have miscalculated predatory impact or are mis-reporting this value.

Response 1: Thank you for highlighting this again.

We appreciate the comment.

We regret for our miscalculation of predatory impact (PI). The PI has been recalculated as number of larvae consumed by a predator per hour (line #191-194). Other calculation errors have also been attended and indicated in track changes mode in line # 185, 243, 246-247, 254-260,265-266, 272, 273, and the Table 2 has been revised.