Review Reports - Larvicidal Effect of Vitex ovata Thunb. (Lamiales: Lamiaceae) Leaf Extract towards Aedes (Stegomyia) aegypti (Linnaeus, 1762) (Diptera: Culicidae)

Round 1

Reviewer 1 Report

Line 34: aegypti; also, are author's names required after first mention of a taxon?

Line 61: Disagreement - plants are or a plant is, but not "plant are".

Line 68: Not yet available?

Line 72: italics.

Line 75: percentage was or percentages were, not "percentages was".

Line 76: except

Line 80: italics

Line 83: italics

Line 84: different letters, not different alphabets. Different alphabets would be Latin, Greek, and Cyrillic, for example.

Also line 84: row or column?

Line 88: delete "looked"

Line 99: aegypti

Line 108: Vitex is a genus, not a family.

Line 110: use a comma after schiliebeni

Line 111: spelling of aegypti, and use a comma.

Line 121: darkening was - disagreement between subject and verb.

Line 130: aegypti

Line 135 and elsewhere: leaf extract rather than leaves extract.

Line 142: it would be better to write that the washing was done to remove soil. Soil is scientific; dirt is housekeeping.

Line 157 & line 166: ground cat food

Table 1: is the second column the mean number of dead larvae over four replicates? This is not explicitly stated.

Line 174 & 175: Percent data are not distributed normally and need to be arcsine transformed prior to ANOVA. Was this done?

Author Response

Reviewer 1 Comments	Author's Response
Line 34: aegypti; are author's names required after first mention?	It was corrected with the author's name.
Line 61: Disagreement - plants are or a plant is, but not "plant are".	It was corrected accordingly.
Line 68: Not yet available?	It was corrected accordingly.
Line 72; 80 & 83: italics.	All was corrected accordingly.
Line 75: percentage was or percentages were, not "percentages was".	It was corrected accordingly.
Line 76: except	It was corrected accordingly.
Line 84: different letters, not different alphabets. Row or column.	It was corrected accordingly.
Line 88: delete "looked".	It was corrected accordingly.
Line 99 & 130: aegypti	All was corrected accordingly.
Line 108: Vitex is a genus, not a family.	It was corrected accordingly.
Line 111: spelling of aegypti, and use a comma.	It was corrected accordingly.
Line 121: darkening was - disagreement between subject and verb.	It was corrected accordingly.
Line 135 and elsewhere: leaf extract rather than leaves extract.	It was corrected accordingly.
Line 142: it would be better to write "soil" instead of dirt.	It was corrected accordingly.
Line 157 & 166: ground cat food.	All was corrected accordingly.
Table 1: is the 2nd column the mean number of dead larvae of 4 replicates?	Based on 3 replicates. The text was added in L78-79. The method was corrected on L183.
Line 174 & 175: Percent data are not distributed normally and need to be arcsine transformed prior to ANOVA. Was this done?	Thank you for the reminder. The data were now transformed. The result has been added with new ANOVA output.

Reviewer 2 Report

Overall, this was an interesting and well-presented study by Aziz et al. While simple in design, the methodology was sound and the results were clearly described, in regards to the larvicidal effect of V. ovata crude leaf extracts on Aedes aegypti larvae.

I have only a few suggestions for the authors, mostly pertaining to the discussion:

It was unclear to me whether the authors were proposing moving forward with further testing of crude V. ovata leaf extracts, or whether they thought identification of the active larvicidal component (within the crude extract) was the critical next step. I think this should be clarified. Also, potentially moving to small-scale field trials was mentioned in the abstract but not discussed anywhere else in the text. Perhaps it is too early to move to field trials given these initial results, especially if the authors first want to identify the bio-active component of the V. ovata crude leaf extract.
I would be interested to see more discussion around the feasibility of applying 5-10g/L of crude leaf extract to larval breeding sites. This seems like a large quantity to be adding per L, so I would welcome further discussion around this point. For instance, how much live plant matter is needed to generate 5-10g of extract? Would large scale application at this concentration be logistically feasible?
Throughout, the authors mention that using plant-derived bio-larvicides would be more environmentally safe than applying synthetic chemicals. I would be interested in the authors discussing whether there are any environmental risks of adding high concentrations of plant extracts to larval breeding sites. For instance, could highly concentrated V. ovata extracts impact other insect species? What about toxicity to mammals? Will this be tested in the future?
Are the authors proposing V. ovata extracts be used as a bio-larvicide only in Malaysia or also in other locations? This wasn’t clear to me.
I would suggest additional experiments in the future, testing the larvicidal effect of this V. ovata extract on other/earlier larval instars.

For the results section, I have the following two comments:

Lines 76-78: Since there was no statistical difference in the mortality between 5000mg/L and 10000mg/L, I think this sentence should read, “The two highest concentrations tested, 5000mg/L and 10000mg/L, had the highest larvicidal activities with 76% and 85% mortality within 24hr, respectively (stats showing no difference in mortality between the two concentrations).”
Can the authors confirm that the images in Figure 2 are both live larvae?

Author Response

Reviewer 2 Comments	Author's Response
Whether proposing moving forward with further testing of crude V. ovata leaf extracts, or identification of the active larvicidal component (within the crude extract)? Clarify.	L133-144: We proposed further testing the crude extract by using different solvents and identifying the phytochemicals with larvicidal properties.
Discussion: potentially moving to small-scale field trials.	L145-153: Before moving to field trials, other toxicological studies have to be conducted.
Discussion: how much live plant matter is needed to generate 5-10g of extract? Would large scale application at this concentration be logistically feasible?	L145-153: We think if V. ovata can be further proven with good larvicidal properties, non-toxic to other organisms and environmentally safe, it is suitable for most countries with this plant's presence. Vitex. ovata can grow rapidly and is considered an invasive coastal species.
Discussion: any environmental risks of adding high concentrations of plant extracts to larval breeding sites. For instance, could highly concentrated V. ovata extracts impact other insect species? What about toxicity to mammals? Will this be tested in the future?	L145-153: As discussed above, further testing has to be conducted to determine the toxicity effect on other non-target organisms.
Discussion: can V. ovata extracts be used as a bio-larvicide only in Malaysia or also in other locations?	L145-153: The crude extract can be used as a potential larvicide in other locations, not only Malaysia, as long as the plant source is available.
Lines 76-78: Since there was no statistical difference in the mortality between 5000mg/L and 10000mg/L, I think this sentence should read, "The two highest concentrations tested, 5000mg/L and 10000mg/L, had the highest larvicidal activities with 76% and 85% mortality within 24hr, respectively (stats showing no difference in mortality between the two concentrations)."	The text has been changed according to the suggestion.
Can the authors confirm that the images in Figure 2 are both live larvae?	No. Both larvae were dead when the pictures were taken. The control larva was killed (using a freezer) for capturing the photo.

Round 2

Reviewer 1 Report

Title: Leaf extract, not leaves extract!

Lines 110 - 118: Check journal style. Do you need to include authors after all of these species names?

Lines 198 - 200: "An acceptable insecticide does not need to cause high mortality on target organisms but should be eco-friendly." If the insecticide does not cause high mortality to the target organisms, what good is it? This sentence essentially says that something that causes 1% mortality is acceptable as a pesticide. I don't think that's right.

Author Response

Reviewer 1 Comments	Author's Response
Title: Leaf extract, not leaves extract!	Corrected.
Lines 110 - 118: Check journal style. Do you need to include authors after all of these species names?	We included all the authors name after the scientific names of the species listed on first mentioned.
Lines 198 - 200: "An acceptable insecticide does not need to cause high mortality on target organisms but should be eco-friendly." If the insecticide does not cause high mortality to the target organisms, what good is it? This sentence essentially says that something that causes 1% mortality is acceptable as a pesticide. I don't think that's right.	Apology on the misleading statement. The sentence was amended.

Author Response File: Author Response.docx