Sublethal Pyriproxyfen Exposure Alters Anopheles arabiensis Fitness and Pyrethroid Susceptibility Without Trans-Generational Carry-Over
, Halfan Ngowo 1,2 and Dickson Wilson Lwetoijera 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIt is suggested that the significance of the values ​​obtained in Table 1 be further explained, as well as the different concentrations and comparisons of permethrin and deltamethrin.
The same applies to Table 2. Information not described in the text.
The same applies to Table 3. Information not described in the text.
The same applies to Table 4. Information not described in the text.
The same applies to Table 5. Information not described in the text.
The conclusions section does not include any values ​​from the results obtained in the experiment.
In general, the discussion section is consistent with the experiment. However, for the results section, it is suggested that alternative graphs be used to represent means and standard deviations to integrate the results. I believe this section can be improved.
Valuable information as an alternative control strategy.
Comments on the Quality of English LanguageA second review is required.
Author Response
Comments 1. It is suggested that the significance of the values ​​obtained in Table 1 be further explained, as well as the different concentrations and comparisons of permethrin and deltamethrin.
Response 1. The suggested changes have already been incorporated in the revised manuscript. Line 223-240, page 6. (Result section).
Comment 2. The same applies to Table 2. Information not described in the text.
Response 2. The suggested changes have already been incorporated in the revised manuscript. Line 245-260, page 7. (Result section).
Comment 3. The same applies to Table 3. Information not described in the text.
Response 3. The suggested changes have already been incorporated in the revised manuscript. Line 267-276, page 9. (Result section).
Comment 4. The same applies to Table 4. Information not described in the text.
Response 4. The suggested changes have already been incorporated in the revised manuscript. Line 283-293, page 10. (Result section).
Comment 5. The same applies to Table 5. Information not described in the text.
Response 5. The suggested changes have already been incorporated in the revised manuscript. Line 301-311, page 12. (Result section).
Comment 6. The conclusions section does not include any values ​​from the results obtained in the experiment.
Response 6. The suggested changes have already been incorporated in the revised manuscript. Line 419-421, page 15. (Conclusions section)
Comment 7. In general, the discussion section is consistent with the experiment. However, for the results section, it is suggested that alternative graphs be used to represent means and standard deviations to integrate the results. I believe this section can be improved.
Response 7. The suggested changes have already been incorporated in the revised manuscript. (Line 279, Figure 2, page 10), (Line 297, Figure 3, page 11) and (Line 316, Figure 4, page 13).
Reviewer 2 Report
Comments and Suggestions for AuthorsComments to the Authors
Dear Authors, the article is certainly interesting because it faces important issues related to the use of appropriated pesticides against mosquito vectors. The article is generally well written but a few revisions regarding language are necessary. The statistical analysis is also fine and results are well described. Specific comments to the Manuscript are listed below but a few major issues must be highlighted. In my opinion, using a further non-resistant population as control should have been opportune before drawing conclusions. In addition, the rationale behind testing the effects of the treatment across generations is not clear to me and the Authors do not furnish any reference to studies highlighting this kind of phenomenon. Also, the fact that the exposure to PPF can increase pyrethroid resistance should be anticipated in the introduction to allow the reader to better understand the study. Overall these issues do not consent the article to be published in present form but they can be addressed through revisions.
Line 17: “substance”
COMMENT: please use a more specific term (as an example: pesticide, chemical, insect growth regulator)
Line 21: “this effect did not continue in later generations”
COMMENT: please use a clearer sentence (as an example: “this effect is not transmissible to the next generation” or “the progeny of the treated females are not affected”
Line 28: “a juvenile hormone analogue” “promising chemistry”
COMMENT: please add a comma after analogue and revise “promising chemistry”. I suppose the Authors meant “promising chemical” or “ promising pesticide”
Line 30: “of sublethal PPF exposure”
COMMENT: please revise to “Subletal PPF doses”
Line 32: “Laboratory-reared, pyrethroid-resistant mosquito larvae”
COMMENT: why did the Authors use a pyrethroid-resistant population? Is that the only available in the area? However, this could lead to misleading conclusions. A non-resistant population used as control should have been opportune. Can the Authors conclude that the treatment increases further the resistance? This should be clear.
COMMENT: the rationale behind testing the effects of the pyriproxyfen on 2nd and 3rd generation is not clear. Did the Authors suppose this IGR could pass to the progeny in some way? Please explain
Table 1: “Summary of 24-Mortality”
COMMENT: please, clearly explain what does “24-mortality” stand for
Table 2: “Summary of knockdown 60-mininutes”
COMMENT: also in this case the sentence is not immediately clear to the readed and “mininutes” must be revised
Lines 275-276: Previous studies have examined the combined effects of PPF and pyrethroids to understand its interactions with conventional vector control tools like LLINs and IRS (22,23,35–39).
COMMENT: I suggest moving this sentence to the introduction to allow the reader to better understand the rationale behind the study
Line 278: “pyrethroid susceptibility across three generations”
COMMENT: it is not clear to me why effects have been studied across three generation, nor the Authors report previous studies highlighting this phenomenon…
Lines 370-371: “This study demonstrates temporary effects of sublethal dose of PPF exposure on An. arabiensis susceptibility to pyrethroids and fitness across multiple generations
COMMENT: this sentence is not enough clear to me. Furthermore, effects across generations have not been demonstrated
Comments on the Quality of English LanguageEnglish is generally fine but there are a few sentences to be revised especially in simple summary and abstract
Author Response
Comment 1. Line 17: “substance”
COMMENT: please use a more specific term (as an example: pesticide, chemical, insect growth regulator)
Response 1. The suggested changes have already been incorporated in the revised manuscript. Line 17, page 1. (Simple summary section).
Comment 2. Line 21: “this effect did not continue in later generations”
COMMENT: please use a clearer sentence (as an example: “this effect is not transmissible to the next generation” or “the progeny of the treated females are not affected”
Response 2. The suggested changes have already been incorporated in the revised manuscript. Line 21, page 1. (Simple summary section).
Comment 3. Line 28: “a juvenile hormone analogue” “promising chemistry”
COMMENT: please add a comma after analogue and revise “promising chemistry”. I suppose the Authors meant “promising chemical” or “promising pesticide”
Response 3. The suggested changes have already been incorporated in the revised manuscript. Line 28, page 1. (Abstract section).
Comment 4. Line 30: “of sublethal PPF exposure”
COMMENT: please revise to “Sublethal PPF doses”
Response 4. The suggested changes have already been incorporated in the revised manuscript. Line 30, page 1. (Abstract section).
Comment 5. Line 32: “Laboratory-reared, pyrethroid-resistant mosquito larvae”
COMMENT: why did the Authors use a pyrethroid-resistant population? Is that the only available in the area? However, this could lead to misleading conclusions. A non-resistant population used as control should have been opportune. Can the Authors conclude that the treatment increases further the resistance? This should be clear.
Response 5. We thank the reviewer for this important point. We used a pyrethroid-resistant An. arabiensis population because previous evidence suggests that amplification of resistance following sublethal PPF exposure is most likely to occur in mosquitoes that already exhibit baseline resistance, as reported by Opiyo et al. (2021). Additionally, our study was conducted in south-eastern Tanzania (Kilombero, Morogoro), an area where pyrethroid resistance has been well documented. For example, Matowo et al. (2017) and Urio et al. (2022) reported high levels of pyrethroid resistance in local Anopheles populations driven by both agricultural and public-health insecticide pressure. Using this resistant colony therefore allowed us to examine whether PPF exposure could further enhance resistance within a population already under strong selective pressure. Importantly, both the PPF-exposed and non-exposed control groups originated from the same resistant population, and WHO susceptibility tests were performed on both groups to directly compare any amplification of resistance attributable to PPF exposure. This design ensures that any observed differences arise from PPF treatment rather than inherent population differences, thus supporting the validity of our conclusions regarding potential resistance intensification.
Comment 6. Table 1: “Summary of 24-Mortality”
COMMENT: please, clearly explain what does “24-mortality” stand for
Response 6. The suggested changes have already been incorporated in the revised manuscript. Line 242, page 6. (Results section).
Comment 7. Table 2: “Summary of knockdown 60-mininutes”
COMMENT: also, in this case the sentence is not immediately clear to the readed and “mininutes” must be revised.
Response 7. The suggested changes have already been incorporated in the revised manuscript. Line 261, page 7. (Results section)
Comment 8. Lines 275-276: Previous studies have examined the combined effects of PPF and pyrethroids to understand its interactions with conventional vector control tools like LLINs and IRS (22,23,35–39).
COMMENT: I suggest moving this sentence to the introduction to allow the reader to better understand the rationale behind the study.
Response 8. The suggested changes have already been incorporated in the revised manuscript. Line 101, page 3. (Introduction section).
Comment 9.
Line 278: “pyrethroid susceptibility across three generations”
COMMENT: it is not clear to me why effects have been studied across three generation, nor the Authors report previous studies highlighting this phenomenon…
Response 9. We thank the reviewer for raising this important point. The decision to examine the effects of pyriproxyfen (PPF) across three generations was driven by the need to address existing knowledge gaps regarding its potential long-term influence on pyrethroid resistance. Although PPF has been widely studied and shown to be effective in both larviciding and as an additive in bed nets, recent evidence such as the findings by Opiyo et al. (2021) indicates that sublethal exposure may temporarily elevate pyrethroid resistance in malaria vectors. These observations highlighted the possibility that PPF could induce physiological or metabolic adjustments that persist beyond the directly exposed generation.
To investigate whether such effects could extend to the progeny, we assessed susceptibility and key fitness traits over three generations. Our results indicate that any PPF-related changes were restricted to the first generation, with subsequent generations showing parameters comparable to controls. Thus, evaluating multiple generations allowed us to determine that these effects are transient and not heritable.
Comment 10. Lines 370-371: “This study demonstrates temporary effects of sublethal dose of PPF exposure on An. arabiensis susceptibility to pyrethroids and fitness across multiple generations
COMMENT: this sentence is not enough clear to me. Furthermore, effects across generations have not been demonstrated
Response 10. The suggested changes have already been incorporated in the revised manuscript. Line 419-421, page 15. (Conclusion section).
