A Deltamethrin Crystal Polymorph for More Effective Malaria Control

A deltamethrin crystal polymorph for more effective malaria control

Jingxiang Yanga,b, Bryan Erriaha,b, Chunhua T. Hua,b, Ethan Reitera,b, Xiaolong Zhua,b, Vilmalí López-Mejíasc,d, Isis Paola Carmona-Sepúlvedac,d, Michael D. Warda,b,1, and Bart Kahra,b,1

aDepartment of Chemistry, New York University, New York, NY 10003; bMolecular Design Institute, New York University, New York, NY 10003; cDepartment of Chemistry, University of Puerto Rico, Río Piedras, San Juan, PR 00931; and dCrystallization Design Institute, Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926

Edited by Lia Addadi, Weizmann Institute of Science, Rehovot, Israel, and approved August 28, 2020 (received for review June 26, 2020) Pyrethroid contact insecticides are mainstays of malaria control, form II, produced by cooling melts of the commercial form I but their efficacies are declining due to widespread insecticide from ca. 110 °C to room temperature. This polymorph is ap- resistance in Anopheles mosquito populations, a major public proximately 12 times faster acting in laboratory assays against health challenge. Several strategies have been proposed to over- disease vectors than the commercially available crystals of form I come this challenge, including insecticides with new modes of ac- that are used in the field. Epidemiological modeling suggests tion. New insecticides, however, can be expensive to implement in that substitution of DM form II for commercial form I would low-income countries. Here, we report a simple and inexpensive reduce malaria transmission. The use of more-active crystal method to improve the efficacy of deltamethrin, the most active polymorphs is a simple and powerful strategy for improving the and most commonly used pyrethroid, by more than 10 times against efficacy of existing compounds for malaria control, obviating the Anopheles mosquitoes. Upon heating for only a few minutes, the limitations associated with the introduction of new compositions commercially available deltamethrin crystals, form I, melt and crys- of matter. Using polymorphs of existing insecticides circumvents tallize upon cooling into a polymorph, form II, which is much faster the need for developing new compounds, thereby mitigating the acting against fruit flies and mosquitoes. Epidemiological modeling cost of new manufacturing processes and regulatory testing. suggests that the use of form II in indoor residual spraying in place of form I would significantly suppress malaria transmission, even in Materials and Methods the presence of high levels of resistance. The simple preparation of Details of the materials and methods are included in SI Appendix. These CHEMISTRY form II, coupled with its kinetic stability and markedly higher effi- include methods for preparing and/or distinguishing DM forms I and II as cacy, argues that form II can provide a powerful, timely, and afford- single crystals (SI Appendix, Table S1), in thin films and in commercial dusts. able malaria control solution for low-income countries that are Methods are provided for determining average velocity and lethality of DM losing protection in the face of worldwide pyrethroid resistance. exposed to Drosophila raised locally as well as susceptible female Anopheles quadrimaculatus and Aedes aegypti purchased as adults. The model of malaria transmission (14) is described briefly in Lethality Comparison of deltamethrin | polymorphism | malaria | epidemiological modeling | Polymorphs in addition to SI Appendix. Methods of chemical analysis are mosquito

more fully described in SI Appendix, including descriptions of optical mi- SUSTAINABILITY SCIENCE croscopy, Raman spectroscopy, and X-ray crystallography. ombatting malaria, a disease afflicting >200 million people Cannually, requires effective insecticides as part of integrated Significance vector management control strategies (1). Interventions, including indoor residual spraying (IRS) of insecticides and The use of deltamethrin, a crystalline contact insecticide and a insecticide-treated bed nets (ITNs), are estimated to have re- leading tool in combatting malaria vectors, faces an uncertain duced malaria cases in Africa by 80% between 2000 and 2015 future, threatened by developing resistance of mosquitoes. A (2). Pyrethroids are the most widely used insecticides for malaria more active crystalline polymorph of deltamethrin, discovered control today because of their high insect lethality and low here, speeds the knockdown of susceptible mosquitoes by a mammalian toxicity (3). Among pyrethroids, deltamethrin (DM, factor of up to 12 compared with the currently used crystalline (S)-cyano(3-phenoxyphenyl)methyl(1R,3R)-3-(2,2-dibromo- form. The faster-acting deltamethrin polymorph is predicted to vinyl)-2,2-dimethylcyclopropane-1-carboxylate; Fig. 1A) is one of suppress malaria transmission and associated human mortality the foremost insecticides for IRS, providing malaria control for while reducing environmental exposure because less agent is millions (3, 4). Unfortunately, resistance to pyrethroids has be- required to achieve the same effect. The outstanding perfor- come widespread in Anopheles mosquito populations, rapidly mance of form II promises increased serviceable use of delta- reducing the efficacy of DM and threatening the substantial methrin crystals for indoor residual spraying. Metastable forms progress in malaria control during the 21st century (5, 6). of contact insecticides should be considered generally for Consequently, new insecticides and mosquito-targeted antima- public health applications. larials have been proposed (7, 8). The development, evaluation, and introduction of new chemical agents for malaria control, how- Author contributions: J.Y., X.Z., M.D.W., and B.K. designed research; J.Y., B.E., C.T.H., E.R., ever, requires substantial investment and evaluation, and it incurs X.Z., V.L.-M., and I.P.C.-S. performed research; J.Y., B.E., X.Z., M.D.W., and B.K. analyzed data; and J.Y., M.D.W., and B.K. wrote the paper. potential risks. Improving the effectiveness of compounds currently in Competing interest statement: New York University has applied for a patent on the use of use is preferable (9). Such improvements are needed as urgently as form II of deltamethrin with J.Y., X.Z., M.D.W., and B.K. as inventors, to encourage de- ever during the global COVID-19 crisis (10). The number of deaths velopment for malaria prophylaxis. from malaria in Africa this year is projected to double as a result of This article is a PNAS Direct Submission. supply chain limitations due to coronavirus-related disruptions (11). Published under the PNAS license. More effective interventions are needed even more. 1To whom correspondence may be addressed. Email: [email protected] or bart.kahr@ DM is a contact insecticide; its active form for IRS is crys- nyu.edu. talline (Fig. 1B) (13). As such, its activity depends upon the in- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ terface between the crystals and whole organisms. Herein, we doi:10.1073/pnas.2013390117/-/DCSupplemental. describe the discovery of a more lethal DM crystal polymorph,

www.pnas.org/cgi/doi/10.1073/pnas.2013390117 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. (A) Molecular structure of DM. (B) Microcrystals in suspension concentrate (Suspend SC, DM concentration: 5%), a formulation used for IRS. (C and D) Melt grown DM (C) form I and (D) form II spherulites observed between crossed polarizers. PXRD confirmed that the fibers in the spherulite were oriented along the crystallographic <010> direction. (E) PXRD data for form I (blue) and form II (orange) from their respective films. (F and G) Single crystal structures of forms (F) I (12) and (G) II determined at 100 K.

Results and Discussion widespread application of DM, only one crystal structure, mea- Polymorph of DM. Female mosquitoes resting on DM-treated sured at room temperature, has been reported (in 1975), desig- walls contact the surfaces of DM crystals (13), absorbing the nated here as form I (Cambridge Structural Database reference toxin through their tarsi. Despite the considerable study and code PXBVCP10) (12). The structure of DM form I was

Fig. 2. Lethality comparison of DM forms I and II against D. melanogaster.(A) Experimental setup for lethality comparisons using crystalline films of DM form I or II covered with microislands of PEG (sprayed on films to reduce their activity so that the KTs could be compared more readily), schematically illustrated by the blue dots in the side view and revealed in scanning electron micrographs. At higher magnification (10-mm scale bar), form II crystallites were observed between PEG islands. (B) The motions of D. melanogaster exposed to crystalline films. The dots represent the average speed of all fruit flies in 1 min,

smoothed (solid line) by a Savitzky−Golay filter. (C) KTs for D. melanogaster exposed to crystalline films. KT50 values are indicated, as in F.(D) Setup for lethality comparisons of dusts. Form I dust was converted to form II dust by heating, either using a microwave oven or convective heating (SI Appendix, Fig. S5). (E) Motions of D. melanogaster exposed to dusts. (F) KTs for D. melanogaster exposed to dusts.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.2013390117 Yang et al. Downloaded by guest on September 24, 2021 redetermined at 100 K (Fig. 1F and SI Appendix, Fig. S1C and supine—for the PEG-treated crystalline films were 400 min for Table S1). form I compared with 61 min for form II (Fig. 2C), corre- Commercial DM (form I) confined between glass slides was sponding to a ratio of 6.6 for Drosophila, undoubtedly a kinetic melted at ca. 110 °C to 120 °C (Tm = 99 °C), either by direct difference associated with uptake of DM molecules from the conduction heating or with a conventional microwave oven, and crystal surface through the insect tarsi. then cooled to 25 °C. This protocol resulted in the growth of Some of the most widely used formulations of DM are so- polycrystalline spherulites (15) consisting of fine fibrils (Fig. 1D). called dusts, microcrystals dispersed on inert carriers (e.g., On rare occasions, fields of chaotic polycrystalline textures also chalk and silica). Commercial DM dust (D-fence Dust; 0.05 wt% were observed (Fig. 1C). Powder X-ray microdiffraction (PXRD) DM) was heated for 5 min above the melting point of DM form I and micro-Raman spectroscopy confirmed that the chaotic tex- either in a microwave oven or in a 150 °C oil bath. Although the tures were DM (form I), whereas the spherulites were a poly- DM polymorphs could not be identified at the low 0.05 wt % morph, herein designated as form II (Fig. 1E and SI Appendix, Fig. loading, the lethality assays described below for heat-treated DM S1 A and B). The single crystal structure of form II was deter- dust parallel the results for the crystalline films of forms I and II, mined at 100 K (Fig. 1G and SI Appendix,Fig.S1D and Table S1). corroborating the conversion of form I in the commercial dust to The melting point of form II (68 °C) is much lower than form I, form II. Commercial and heat-treated dusts are denoted as form suggesting form II is metastable. Unconfined films of form II I and form II dusts, respectively. Female fruit flies were exposed nucleate form I within 3 h, but the transformation is slow (SI to 2.0 mg of forms I and II dusts (equivalent to 1 μgofDM Appendix,Fig.S2). However, crystalline DM form II confined microcrystals each) (Fig. 2D). The fruit flies exposed to form II between the glass slides is stable for at least 12 mo at 25 °C and at were immediately hyperactive (Fig. 2E), while the onset of hy- least 6 mo at 40 °C. peractivity was 40 min for form I. The KT50 values for forms I and II were 113 and 20 min, respectively (Fig. 2F), a ratio of 5.7, Lethality Comparison of Polymorphs. A comparison of the lethality comparable to the corresponding DM films described above. of DM forms I and II was difficult for neat films owing to their Both microwave and convective heating produced equivalent exceptionally short knockdown times (KTs, the time required for results (SI Appendix, Fig. S5). The remarkably stability of form II a fly to become immobile in a supine position); insects were in dusts can be attributed to the low probability of the nucleation knocked down within minutes of exposure to either form. of form I in minute crystals of form II. Therefore, films of DM form I or II were partially covered with Female A. quadrimaculatus and A. aegypti mosquitoes exposed

nebulized, micrometer-size droplets of polyethylene glycol to 2.0 mg of either DM form I or form II dust also were more CHEMISTRY (PEG) to reduce the surface area for contact of the insect tarsi susceptible to form II, as indicated by the onset of hyperactivity with DM, thus bringing the KTs within a more conveniently (Fig. 3 A and C and SI Appendix, Fig. S6A). The KT50 values for measured range (Fig. 2A and SI Appendix, Fig. S3). Female fruit Aedes exposed to forms I and II dust were 192 and 21 min, re- flies (Drosophila melanogaster), an accepted model for testing spectively (Fig. 3B), a ratio of 9.1. The corresponding KT50 insecticide lethality against mosquitoes (16), were exposed to values for Anopheles mosquitoes were 282 and 24 min, respec- these films (Fig. 2A), following a procedure similar to that pre- tively (Fig. 3D), a ratio of 11.8. Anopheles mosquitoes exposed to viously reported (17–19). Fly hyperactivity, the first symptom of DM form II were virtually inactive after the hyperactivity peak poisoning (20), and KTs were analyzed by measurement of the (SI Appendix, Fig. S6). SUSTAINABILITY SCIENCE average speed of fly motion, using a video camera and a custom MATLAB program (SI Appendix, Fig. S4). Peak hyperactivity Impact on Current Malaria Control Intervention. A model of malaria occurred after 160 min of exposure to form I compared with transmission (SI Appendix, Fig. S7) previously developed by 40 min for form II (Fig. 2B). The KT50 values—the knockdown Catteruccia, Childs, and coworkers was employed using a vari- times required for 50% of the insects to become immobile and able to account for the lethality difference between DM forms

Fig. 3. Lethality of DM crystalline forms I and II against A. aegypti and A. quadrimaculatus mosquitoes. (A) The motions of A. aegypti mosquitoes exposed to dusts. The dots represent the average speed of all mosquitoes in 1 min. Solid lines are smoothed trend lines (as in C), See also SI Appendix, Fig. S6.

(B) Comparison of A. aegypti KTs for forms I and II dusts. KT50 values are indicated, as in D.(C) Motions of A. quadrimaculatus mosquitoes exposed to dusts. (D) Comparison of A. quadrimaculatus KTs using dusts.

Yang et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 24, 2021 I and II (8, 14). The mosquito population dynamics and human 3 years, when the mosquito population and human prevalence malaria infection prevalence were incorporated into this model converged to 370 and 45%, respectively. During the following to evaluate the potential consequences of the application of form 3 years, the simulation was performed under the condition that II in IRS. We assumed that the mosquito population is ho- DM form I was used via IRS. Due to high levels of resistance, mogenous, and 1 unit of form II was equal to 12 units of form I. however, the introduction of form I only marginally reduced The introduction of form II was predicted, by modeling, to in- both the population of infected mosquitoes (298) and the human crease the effectiveness of IRS under a broad range of human infection prevalence (40%). Starting from the seventh year, DM infection prevalence, intervention coverage, and insecticide re- form II was substituted for DM form I. Both the population of sistance conditions within the model (Fig. 4A and SI Appendix, infected mosquitoes and the human infection prevalence expe- Figs. S8 and S9). This suggests that application of form II may rienced a significant drop and converged to 18 and 4%, respec- suppress malaria in endemic regions where formulations of form tively. This decrease, arising from the introduction of DM II in I suffer from pyrethroid resistance. simulation, would translate to suppression of malaria transmis- We then modeled the effect of switching from form I to form sion in hot spots where resistance to pyrethroids is as high as II on the infectious mosquito population and human malaria 70%. A simulation performed with a lower initial infection infection dynamics. The simulation was performed for hypo- prevalence (20%) indicated that the switch from form I to form thetical regions (marked in Fig. 4A as red squares) characterized II would decrease the human infection prevalence from 18 to by high resistance (70%), moderate infection prevalence (45%), 0.3% at even higher resistance (80%) and lower IRS coverage and high IRS coverage (75%) (Fig. 4B). The simulation was (60%) (SI Appendix, Fig. S8). We emphasize in Fig. 4A that the performed in the absence of insecticide intervention in the first gains in effectiveness of form II from IRS can be relatively small,

Fig. 4. Simulation of malaria transmission dynamics. (A) Effectiveness of IRS for inhibition of malaria transmission, using the same doses of DM form I (Left) or II (Right) at varying levels of coverage and insecticide resistance with an initial human infection prevalence of 45%. Insecticide resistance corresponds to the percentage of mosquitoes that survive after contacting the applied dose of form I. Coverage corresponds to the probability of a mosquito contacting the insecticide during a single resting episode. (B) Predicted reduction in population of infected mosquitoes and human prevalence upon application of DM form I and then switching to form II (coverage = 75%; resistance = 70%; infection prevalence = 45%, denoted in Fig. 4A as red squares). Both the population of infected mosquitoes and human disease prevalence dropped precipitously.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.2013390117 Yang et al. Downloaded by guest on September 24, 2021 even if the coverage is 100%, for mosquito population with significantly improved by applying form II during IRS. The extremely high-level resistance. simple preparation of form II by cooling from the melt, coupled DM form II, because it is less thermodynamically stable than with its kinetic stability and markedly greater efficacy, argues form I, favors uptake through dissolution of DM molecules by that DM form II can serve as a powerful, timely, and affordable secretions or detachment from underlying crystal planes. Even tool for controlling malaria and other infectious diseases for low- for insects with cuticle thickness resistance (21), metabolic re- income countries that are losing protection in the face of sistance (overexpressed detoxification enzymes) (22), knock- worldwide pyrethroid resistance. down resistance (23), and the newly discovered chemosensory The Anopheles species that transmit malaria in sub-Saharan protein SAP2 binding resistance (24), lethal doses may still reach Africa belong to the gambiae and funestus species. Our obser- the sodium ion channel target site, due to the significantly in- vations of the activities of DM forms I and II are consistent creased uptake of DM molecules from form II. DM form II dust across three insect species, D. melanogaster, A. aegypti, and A. continues to exhibit greater lethality than form I dust 3 mo after quadrimaculatus. We anticipate that the DM forms would have heating (SI Appendix, Fig. S10), consistent with the kinetic sta- comparable relative activities against susceptible gambiae and bility of form II crystals. As recommended by the World Health funestus. Notably, the differences in activities have been mea- Organization (WHO), DM must be sprayed every 3 mo to 6 mo sured for common insecticides against the sibling species of one to maintain its residual effect (13). The 3-mo durability of DM Anopheles organism, and DM was the least variable (ca. ±10%) form II formulations, combined with its higher activity, promises (33). Nevertheless, the assays described here should be per- significant benefits for field applications. formed for the Anopheles species most relevant in Africa—in Since the more active form II can be produced by simply addition to the six major Anopheles vectors that are common in melting form I, which is currently in use, the cost of its imple- South Asia—by scientists who maintain these species. Both mentation would be low compared with the development of al- susceptible and pyrethroid-resistant organisms should likewise be ternative interventions. IRS alternatives to pyrethroids, although compared, as the experimental results above do not address recommended for insecticide resistance management (25), have whether form II will exhibit the same increase in efficacy against proven expensive for scale-up (5). The use of DM form II in IRS, insecticide-resistant mosquito species. however, could be integrated into the extensive manufacturing Polymorph discovery promises to improve the performance of and distribution pipelines of DM that are already in operation in other contact insecticides (17–19), as less thermodynamically malaria-endemic regions. Form II may even be less costly overall stable crystal forms are expected to surrender their molecules to than form I because less compound would be needed to achieve the desired outcome, allowing for greater IRS coverage with insects more readily. Greater attention to the crystal phases of CHEMISTRY limited funding. Moreover, the overall environmental exposure contact insecticides, both existing compounds and new ones, can would be reduced. provide an opportunity to prevent vector-borne disease while The WHO recently recommended the use of the synergist reducing risks to the environment. piperonyl butoxide (PBO) for coincorporation in ITNs (26, 27). Code Availability. The customized program used in this study for PBO doubles the speed of action of DM against Anopheles by neutralizing metabolic pyrethroid resistance (28, 29). ITNs with insect lethality measurement can be obtained from GitHub at the DM and PBO have improved the control of malaria transmission following URLs: https://github.com/Jingxiang-Yang/Deltamethrin- in areas where pyrethroid resistance is prevalent (30). The ad- Average-Velocity-Calculation and https://github.com/Jingxiang- SUSTAINABILITY SCIENCE ditional cost of PBO-incorporated ITNs poses a challenge for Yang/Deltamethrin-Polymorph-Malaria. The code also is avail- resource-poor countries, however (31, 32). DM form II, which able upon requests to the corresponding authors. improves knockdown speeds 12-fold, promises to be beneficial Data Availability. even in the absence of additives, while mitigating insecticide Crystal structure data reported in this paper resistance wherever DM is used in its crystalline form, as in IRS. have been deposited at the Cambridge Crystallographic Data Centre with accession numbers 1985206 (form I) and 1985207 Conclusion (form II). A crystalline polymorph of DM, form II, is 12 times faster acting ACKNOWLEDGMENTS. This work was supported primarily by the Materials against disease-carrying mosquitoes than DM form I when used Research Science and Engineering Center program of the NSF under Award as microcrystals embedded in commonly sprayed dusts. Epide- DMR-1420073. The X-ray facility was supported partially by the NSF under miological modeling suggests that malaria control could be Award CRIF/CHE-0840277.

1. World Health Organization, World Malaria Report 2019, (World Health Organization, 13. World Health Organization, Indoor Residual Spraying: An Operational Manual for 2019). Indoor Residual Spraying (IRS) for Malaria Transmission Control and Elimination, 2. S. Bhatt et al., The effect of malaria control on Plasmodium falciparum in Africa (World Health Organization, 2015). between 2000 and 2015. Nature 526, 207–211 (2015). 14. L.-M. Childs et al., Disrupting mosquito reproduction and parasite development for 3. J.-E. Casida, Michael Elliott’s billion dollar crystals and other discoveries in insecticide malaria control. PLoS Pathog. 12, e1006060 (2016). chemistry. Pest Manag. Sci. 66, 1163–1170 (2010). 15. A.-G. Shtukenberg, Y.-O. Punin, E. Gunn, B. Kahr, Spherulites. Chem. Rev. 112, 4. M. Elliott, A.-W. Farnham, N.-F. Janes, P.-H. Needham, D.-A. Pulman, Synthetic in- 1805–1838 (2012). secticide with a new order of activity. Nature 248, 710–711 (1974). 16. D. Schneider, Using Drosophila as a model insect. Nat. Rev. Genet. 1, 218–226 (2000). 5. World Health Organization, World Malaria Report 2018, (World Health Organization, 2018). 17. J. Yang et al., DDT polymorphism and the lethality of crystal forms. Angew. Chem. Int. 6. K.-H. Toé et al., Increased pyrethroid resistance in malaria vectors and decreased bed Ed. Engl. 56, 10165–10169 (2017). net effectiveness, Burkina Faso. Emerg. Infect. Dis. 20, 1691–1696 (2014). 18. J. Yang et al., Inverse correlation between lethality and thermodynamic stability of 7. J. Hemingway, The way forward for vector control. Science 358, 998–999 (2017). contact insecticide polymorphs. Cryst. Growth Des. 19, 1839–1844 (2019). 8. D.-G. Paton et al., Exposing Anopheles mosquitoes to antimalarials blocks Plasmo- 19. X. Zhu et al., Manipulating solid forms of contact insecticides for infectious disease dium parasite transmission. Nature 567, 239–243 (2019). prevention. J. Am. Chem. Soc. 141, 16858–16864 (2019). 9. K. Kupferschmidt, Pick your poison. Science 354, 171–173 (2016). 20. R.-A. Alzogaray, A. Fontán, E.-N. Zerba, Evaluation of hyperactivity produced by py- 10. J. Wang et al., Preparedness is essential for malaria-endemic regions during the rethroid treatment on third instar nymphs of Triatoma infestans (Hemiptera: Redu- COVID-19 pandemic. Lancet 395, 1094–1096 (2020). viidae). Arch. Insect Biochem. Physiol. 35, 323–333 (1997). 11. World Health Organization, The Potential Impact of Health Service Disruptions on the 21. V. Balabanidou et al., Cytochrome P450 associated with insecticide resistance cata- Burden of Malaria: A Modelling Analysis for Countries in Sub-Saharan Africa, (World lyzes cuticular hydrocarbon production in Anopheles gambiae. Proc. Natl. Acad. Sci. Health Organization, 2020). U. S. A. 113, 9268–9273 (2016). 12. J.-D. Owen, Absolute configuration of the most potent isomer of the pyrethroid insecticide 22. B.-J. Stevenson et al., Cytochrome P450 6M2 from the malaria vector Anopheles α-cyano-3-phenoxybenzyl-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate by gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed. crystal structure analysis. J. Chem. Soc. Perkin Trans. 1 1975,1865–1868 (1975). Insect Biochem. Mol. Biol. 41, 492–502 (2011).

Yang et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 24, 2021 23. D. Martinez-Torres et al., Molecular characterization of pyrethroid knockdown re- 29. F. Darriet, F. Chandre, Combining piperonyl butoxide and dinotefuran restores the sistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol. Biol. 7, efficacy of deltamethrin mosquito nets against resistant Anopheles gambiae (Diptera: 179–184 (1998). Culicidae). J. Med. Entomol. 48, 952–955 (2011). 24. V.-A. Ingham et al., A sensory appendage protein protects malaria vectors from py- 30. N. Protopopoff et al., Effectiveness of a long-lasting piperonyl butoxide-treated in- rethroids. Nature 577, 376–380 (2020). secticidal net and indoor residual spray interventions, separately and together, 25. World Health Organization, Global Plan for Insecticide Resistance Management in against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised Malaria Vectors, (World Health Organization, 2012). controlled, two-by-two factorial design trial. Lancet 391, 1577–1588 (2018). 26. V. Corbel et al., Field efficacy of a new mosaic long-lasting mosquito net (PermaNet 31. G.-F. Killeen, H. Ranson, Insecticide-resistant malaria vectors must be tackled. Lancet 3.0) against pyrethroid-resistant malaria vectors: A multi centre study in Western and 391, 1551–1552 (2018). Central Africa. Malar. J. 9, 113 (2010). 32. J. Sachs, P. Malaney, The economic and social burden of malaria. Nature 415, 680–685 27. World Health Organization, Conditions for deployment of mosquito nets treated (2002). with a pyrethroid and piperonyl butoxide (World Health Organization, 2017). https:// 33. S.-N. Surendran, P.-J. Jude, T.-C. Weerarathne, S.-H.-P. Parakrama Karunaratne, apps.who.int/iris/bitstream/handle/10665/258939/WHO-HTM-GMP-2017.17-eng.pdf? R. Ramasamy, Variations in susceptibility to common insecticides and resistance sequence=5. Accessed 15 September 2020. mechanisms among morphologically identified sibling species of the malaria vector 28. D. Gylnne-Jones, PBO—The Insecticide Synergist, (Academic, 1998). Anopheles subpictus in Sri Lanka. Parasit. Vectors 5, 34 (2012).

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.2013390117 Yang et al. Downloaded by guest on September 24, 2021