Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia

Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia Monenus Geleta, Eba Alemayehu Simma, Delenasaw Yewhalaw This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7704930/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Malaria control in Ethiopia relies mainly on case management with ACT and vector control interventions, notably long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). Anopheles arabiensis is the predominant vector, while the invasive An. stephensi is rapidly spreading across multiple regions, intensifying challenges for malaria elimination. Insecticide resistance further complicates control efforts. Methods This study assessed the susceptibility of An. gambiae s.l. and An. stephensi to commonly used and new public health insecticides at two sites: Awash Sabhat Kilo, where An. stephensi is established, and an area dominated by An. gambiae s.l. WHO tube bioassays tested pirimiphos-methyl (0.25%), propoxur (0.1%), bendiocarb (0.1%), deltamethrin (0.25%), alpha-cypermethrin (0.05%), and clothianidin (2%). CDC bottle bioassays examined susceptibility to pirimiphos-methyl, deltamethrin, chlorfenapyr, clothianidin, and broflanilide. Molecular identification and allele-specific PCR targeted resistant alleles, particularly mutations in the voltage-gated sodium channel (VGSC) gene. Data were analyzed with SPSS v20. Results Results showed An. gambiae s.l. populations were susceptible to propoxur and pirimiphos-methyl but resistant to bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. An. stephensi displayed broader resistance, including to pirimiphos-methyl, propoxur, bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. Both species remained fully susceptible to chlorfenapyr, clothianidin (at higher doses), and broflanilide across tested concentrations. Resistance to deltamethrin and pirimiphos-methyl was dose-dependent. Molecular identification confirmed An. arabiensis as the major species within the An. gambiae complex. Knockdown resistance ( kdr ) mutations were detected in An. arabiensis with a genotypic frequency of 22.8%. Conclusion These findings highlight the serious threat insecticide resistance poses to Ethiopia’s malaria control efforts. The persistence of resistance in both An. arabiensis and An. stephensi raises concerns about the long-term sustainability of insecticide-based strategies and underscores the need for alternative or integrated approaches. Broflanilide insecticide resistance Anopheles stephensi Ethiopia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 BACKGROUND Mosquitoes as a large group of arthropods that play an important role in the transmission of many diseases to humans such as malaria, filariasis, yellow fever, dengue fever [ 1 ]. In Ethiopia, malaria remains a major public health concern with millions of cases and thousands of deaths reported annually [ 2 ]. Unlike most of the African continent, malaria is caused by infection with Plasmodium vivax or P. falciparum . Efforts to control the transmission of malaria currently target An. arabiensis , the primary malaria vector in Ethiopia, as well the secondary vectors An. funestus , An. pharoensis , and An. nili [ 3 ]. Anopheles stephensi is an important vector that plays a major role in transmitting malaria in urban areas and is an established vector in many parts of India. The first report of An. stephensi in the Horn of Africa was from Djibouti in 2013[ 4 ] and this mosquito was first detected in the Somali Regional State of Ethiopia in 2016 [ 5 ] and subsequently been confirmed to have a broad distribution in Northeast and eastern Ethiopia [ 6 ] and distribution in south Ethiopia [ 7 ]. Transmission of malaria can be reduced by adopting vector control measures such as indoor residual spraying (IRS) with insecticides, larval control measures, and personal protection measures. The combination of tools and methods used to combat malaria now includes insect nets treated with long-lasting insecticides (LLIT) and artemisinin-based combination therapy, supported by IRS of insecticide and intermittent preventive treatment during pregnancy [ 8 ]. Chemical control had been the main method for combating the adult stage of malaria vector since the eradication era. Insecticide application for adult mosquito control started with organochlorines (DDT, dieldrin, and BHC) during the 1960s, followed by organophosphates (malathion and pirimiphos-methyl) for 2 decades from 1966 and continued with the carbamate, propoxur during 1977–1990, and then with pyrethroids (lambda-cyhalothrin/deltamethrin). Temephos, chlorpyriphos-methyl, and pirimiphos-methyl were used for larviciding from 1970 to 1992. In Guinea An. gambiae s.l. was resistant to pyrethroid, alpha cypermethrin, lambdacyhalothrin and DDT but susceptible to deltamethrin [ 9 ]. In Togo An. gambiae s.l . found resistant against bendiocarb, deltamethrin and propoxur [ 10 ]. In Ethiopia An. gambiae s.l. was resistant to two groups of pyrethroid insecticides (deltamethrin and alpha-cypermethrin), but susceptible to pirimiphos-methyl, propoxur and bendiocarb [ 11 ]. Resistance of An. stephensi to DDT, dieldrin, and malathion was reported for the first time in 1957, 1960, and 1976, respectively [ 11 ]. Anopheles stephensi in Ethiopia was highly resistant to DDT, malathion, pirimiphos-methyl, bendiocarb, propoxur, deltamethrin, and permethrin [ 12 ]. In Awash Sebat Kilo Afar region (2019, 2020) the resistance intensity of An. stephensi to alpha-cypermethrin, deltamethrin and permethrin was assessed through exposure to 1×, 5× and 10× the diagnostic dose [ 13 ]. The various mechanisms, including metabolic resistance and site insensitivity can cause insecticide resistance [ 14 ]. Metabolic resistance mechanism is based on the enzyme systems which all mosquitoes possess to help them to detoxify naturally occurring insecticides. Three categories of enzymes, namely esterases, P450s and glutathione-S-transferases are known to confer resistance to insecticides in insect pest such as malaria vectors. In Ethiopia An. arabiensis very high frequency of the West African kdr allele (L1014F), was observed with higher kdr allele indicating that target site resistance mechanism might contribute for the observed high level of pyrethroids (deltamethrin and alpha-cypermethrin) resistance in the population[ 11 ]. In Afghanistan An. stephensi , general esterases (GES), glutathione S -transferases (GSTs), cytochrome P450s and insensitive acetylcholinesterase (AChEs) were implicated in insecticide resistance [ 15 ]. General esterases are involved in OPs resistance in An. stephensi from Pakistan [ 16 ]. WHO standard insecticide susceptibility bioassays have been performed on An. stephensi from Afghanistan showing resistance to organochlorines, carbamates and pyrethroid insecticides [ 17 ]. Anopheles stephensi from India had increased activities of esterases and GSTs associated with deltamethrin and permethrin resistance [ 18 ]. Insecticides generally act at a specific site within the insect, typically within the nervous system (e.g. OP, carbamate, DDT and pyrethroids insecticides ) . Knockdown resistance ( kdr ) mutation is widespread in Anopheles species in Africa especially An. gambiae [ 17 ]. Originally, the L1014F mutation ( kdr west ) was detected in 2000; a second kdr mutation ( kdr east ) was detected in Kenyan An. gambiae [ 17 ]. The first report of a kdr L1014F resistance mechanism in An. stephens i was in the DUB-S strain in 2003 [ 17 ]. The presence of kdr east in An. stephensi from India was reported for the first time by Singh et al. [ 15 ]. Currently, most resistance monitoring is dependent on bioassays, using fixed insecticide concentrations and exposure times, and the data is reported as percentage mortality and/or Knock down (KD) effect. WHO recommends that insecticide susceptibility status of malaria vectors should be monitored annually [ 8 ]. When insecticide resistance is detected, its intensity and the biochemical and molecular mechanisms should also be investigated [ 8 ]. Accurate information on the underlying resistance mechanisms in An. stephensi is needed for proper management of insecticide resistance and a better management of malaria through vector control interventions. Therefore, the aim of this study was to determine the insecticide resistance status of An. gambiae s.l. and An. stephens i to different classes of insecticides and characterize molecular resistance mechanisms in An. gambiae s.l. METHODS Study area The research took place in Awash Sabhat kilo and Jimma towns. Awash Sabhat kilo is 220 km southeast of Addis Ababa in the Afar region (Fig. 1 ). The town is located at 8.989149" N, 40.164715" E, and is 916 meters above sea level. It has about 24,700 residents and experiences a semi-arid climate with main rains in July-August and brief rains in April/May. Temperatures average 25.8°C, ranging from 17.3°C to 33.6°C. The Awash River Valley is Ethiopia's most irrigated region, with extensive farming. The area has year-round malaria, with 536 cases per 1000 people in 2019[ 19 ]. In 2018, the presence of An. stephensi mosquitoes was reported in the town [ 8 ]. Jimma town is 353km southwest of Addis Ababa, at 7°41' N and 36° 50'E, with an elevation of 1780 meters. The area, known as Woyna Daga, has good conditions for farming and living. The climate is warm, with temperatures between 14°C and 30°C. Yearly rainfall is between 1138mm and 1690mm, with most rain falling from June to August and the least in December and January. The heavy rainfall makes this highland region one of Ethiopia's most fertile areas, perfect for farming. The Bacho bore Kebele area has many mosquito breeding sites and regular malaria cases. Both P. falciparum and P. vivax malaria types are common here, with An. arabiensis mosquitoes being the main carriers. Mosquito collection and identification From May 2022 to October 2024, mosquito larvae and pupae were collected in Jimma town and Awash Sebhat Kilo, Ethiopia. An. arabiensis mosquito was obtained from Jimma University’s disease research center, while An. gambiae s.l was collected from natural habitats such as swamps, rice fields, puddles, ditches, tree holes, and water containers in the Jimma Bacho bore area. An. stephensi mosquito was sampled from human-made sites, including wells, tanks, coolers, gutters, and construction sites in Awash Subhat Kilo. For each site, researchers recorded coordinates, water type (permanent/temporary), source, collection type, sun exposure, vegetation, and water quality. Larvae were placed in labeled plastic containers and transported to the insectary, where they were reared to adults with yeast powder and sail farm food under controlled conditions (25 ± 2°C, 75 ± 10% RH, 12:12 h light–dark cycle). Pupae were transferred to emergence cages, and adults were held safely to prevent contamination and ant damage. Mosquito species were identified morphologically [ 20 ]. Insecticide Susceptibility Tests WHO Tube test The WHO tube test method was used to assess mosquito susceptibility [ 21 ]. Female mosquitoes of An. gambiae s.l., An. stephensi , and laboratory-reared An. arabiensis (3–5 days old, non-blood-fed) were tested. For each species, 100 wild mosquitoes were used, divided into four groups for exposure to different insecticides, alongside two control groups. The insecticides tested were pirimiphos-methyl (0.25%), propoxur (0.1%), bendiocarb (0.1%), deltamethrin (0.05%), alpha-cypermethrin (0.05%), and clothianidin (2%). Tests were conducted according to WHO guidelines (2016). After exposure, mosquitoes were transferred to holding tubes, provided with 10% sugar solution, and maintained for 24 hours at 27 ± 2°C and 80 ± 10% relative humidity. Mortality was then recorded. All dead and surviving specimens were preserved individually in Eppendorf tubes over silica gel for subsequent molecular assays. Resistance status was classified according to WHO criteria: 98–100% mortality indicates susceptibility, 90–97% suggests possible resistance requiring confirmation, and < 90% indicates resistance [ 21 ]. CDC bottle bioassays The CDC bottle bioassay was conducted with broflanilide (various concentrations), chlorfenapyr (100 µg/bottle), and clothianidin (10 µg/bottle) following standard protocols [ 22 ]. Chlorfenapyr was mixed with acetone, and clothianidin with acetone + MERO (800 ppm). Bottles and caps were coated with 1 ml of solution, rolled until dry, and stored in the dark. Control bottles received acetone or acetone + MERO only. For broflanilide, 1 mg of technical-grade insecticide was dissolved in 2 ml of acetone + MERO (800 ppm) to prepare a 500 µg/ml stock. Comparator insecticides, deltamethrin and pirimiphos-methyl, were prepared using acetone alone. Subsequently, 1mg of technical-grade broflanilide was added and fully dissolved to produce a 500 µg/ml broflanilide stock solution. Serial dilutions were obtained by mixing 1 ml of stock with 9 ml of acetone + MERO, yielding 50 µg/ml; the same procedure was used for deltamethrin and pirimiphos-methyl [ 22 ]. Wheaton bottles were arranged by concentration. In total, 2,400 non-blood-fed female mosquitoes ( An. gambiae s.l., An. stephensi , and lab-reared An. arabiensis ), aged 3–5 days, were tested. For each dose, 25 mosquitoes were introduced (four replicates); controls included two bottles with 25 mosquitoes each. Mortality was recorded every 15 min over 60 min (Fig. 2 ). Molecular identification of An. gambiae s.l and An. stephensi , and detection of target site mutations DNA Extraction and Species Identification Whole-genome DNA was extracted using DNAzol reagent (MRCgene, USA) following standard protocols [ 23 ]. Individual mosquitoes were processed: 50 survivors and 10 dead from deltamethrin exposure, plus 10 controls. Each mosquito was homogenized in 100 µl DNAzol, incubated for 2–3 min, and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was transferred to a new 1.5 ml tube, mixed with 200 µl 100% ethanol, and centrifuged again (13,000 rpm, 5 min, 4°C). The pellet was washed with 100 µl 70% ethanol, air-dried, and re-suspended in 100 µl TE buffer. Extracted DNA was used for species identification and resistance genotyping. Species identification was performed using PCR following Safi et al. (2019) [ 24 ]. In total, 120 female An. gambiae s.l. and 39 An. stephensi were tested. For An. gambiae s.l., samples included 50 survivors, 10 dead, and 10 controls from deltamethrin assays. For An. stephensi , samples included 30 survivors, 5 dead, and 4 controls. PCR assays were conducted at Jimma University’s Tropical and Infectious Diseases Research Centre. For An. gambiae s.l., species-specific primers (AR, AG, QD, and UN) were used. PCR reactions contained MgCl₂, Tris-HCl, KCl, Triton X-100, dNTPs, and SilverStar DNA polymerase. Products were visualized on agarose gels stained with ethidium bromide. For An. stephens i, ITS2 primers were used to amplify a 400–500 bp fragment. Reactions included forward and reverse primers, PCR water, master mix, and DNA template. PCR cycling conditions were 95°C (denaturation), 50°C (annealing), and 72°C (extension), and products were resolved on 2% agarose gels. Detection of kdr Mutation Knockdown resistance ( kdr ) mutations were screened using allele-specific PCR following established protocols. After WHO tube tests, 58 female An. arabiensis (dead and surviving) were analyzed for kdr and ace-1 mutations. The assay employed allele-specific PCR targeting the L1014F mutation. Data analysis Knockdown was recorded every 10 min for 1 h, and mortality was assessed after 24 h for all treatments and controls. When control mortality ranged from 5–20%, data were corrected using Abbott’s formula. Mortality confidence limits (5%) and lethal times (LT₅₀, LT₉₀ with 95% CIs) were estimated using the Finney method. Insecticide results were graphed in Excel. For molecular analysis, the frequency of kdr mutations in wild An. arabiensis (dead and surviving) was determined, and genotype distributions were assessed with Hardy–Weinberg software [ 25 ]. RESULTS Overall, 6,150 female Anopheles mosquitoes were reared from immature stages collected in two Ethiopian sites: Bacho Bore Kebele, Jimma town (Oromia region), and Awash Sebat Kilo town (Afar region) (Table 1 ). An. gambiae s.l was primarily associated with natural water bodies and showed seasonal breeding patterns, whereas An. stephensi preferred artificial containers. Table 1 Characterization of Anopheles mosquitoes breeding habitats in Jimma and Awash 7 kilo towns, Ethiopia S.N Breeding site An. gambiae s.l. An. stephensi 1 Geographic location Jimma Town (Bacho Bore) Awash 7 kilo town, Afar 2 Type of breeding site temporary Permanent or semi-permanent 3 Origin of the water rain man-made 4 Nature of the water collection Puddle, ditch Ditch, container, over tank 5 Exposure to sunlight shaded, sunlit or partial light shaded, sunlit or partial light 6 Presence of vegetation emergent, submerged, floating floating 7 Characteristics of the water Clear, turbid clear WHO Tube Test result A total of 900 An. gambiae s.l mosquitoes were tested for insecticide resistance, with 600 exposed to insecticides and 300 serving as controls. The insecticides tested were pirimiphos-methyl (0.25%), bendiocarb (0.1%), deltamethrin (0.05%), alpha-cypermethrin (0.05%), clothianidin (2%), and propoxur (0.1%). According to WHO criteria, An. gambiae s.l. from Jimma were resistant to deltamethrin, alpha-cypermethrin, bendiocarb, and clothianidin, with mortality rates below the 90% threshold: 18% (deltamethrin), 43% (alpha-cypermethrin), 63% (bendiocarb), and 73% (clothianidin). The mosquitoes were fully susceptible to propoxur and pirimiphos-methyl (Fig. 3 ). Mosquitoes from Awash 7 Kilo Town exhibited high resistance to most tested insecticides. After 24 hours, mortality rates were: clothianidin 97%, propoxur 31%, pirimiphos-methyl 23%, alpha-cypermethrin 9%, deltamethrin 8%, and bendiocarb 7%. As effective insecticides are expected to achieve ≥ 90% mortality, only clothianidin approached efficacy, though early resistance signs were observed. Overall, these mosquitoes were substantially more resistant than An. gambiae s.l (Fig. 4 ). CDC bottle bioassays result A total of 100 adult female An. gambiae s.l and An. stephensi were exposed to chlorfenapyr (100 µg/ml), resulting in 100% mortality, indicating full susceptibility. Complete susceptibility was also observed for An. stephensi at 10 µg/ml and An. gambiae s.l at 4 µg/ml of clothianidin (Fig. 5 ). Although WHO tube tests suggested resistance to clothianidin in these populations, CDC bottle assays confirmed full vulnerability. For broflanilide, 700 adult females of each species were tested across concentrations of 50, 25, 12.5, 6.25, 3.125, 1.562, and 0.78 µg/ml. An. arabiensis were fully susceptible at all concentrations (Fig. 6 ). An. gambiae s.l. exhibited 100% mortality within 24 hours at all doses (Fig. 7 ), while An. stephensi showed complete mortality at higher concentrations (50–3.125 µg/ml) and 99% mortality at the two lowest doses (1.563 and 0.78 µg/ml) (Fig. 8 ). Across species, higher concentrations of broflanilide consistently produced greater mortality, with An. arabiensis being the most susceptible, and An. gambiae s.l. and An. stephensi showing similar susceptibility patterns. A total of 700 adult females of each mosquito species were tested against pirimiphos-methyl (80, 40, 20, 10, 5, 2.5, and 1.25 µg/ml) and deltamethrin (50, 25, 12.5, 6.25, 3.125, 1.562, and 0.78 µg/ml). For pirimiphos-methyl, An. arabiensis were fully susceptible at all concentrations, while An. gambiae s.l. showed complete mortality at the four highest concentrations (80–10 µg/ml), susceptibility at 5 µg/ml, and resistance at the two lowest concentrations (2.5–1.25 µg/ml). An. stephensi were fully susceptible at 80 and 40 µg/ml, possibly susceptible at 20 µg/ml, and resistant at the remaining lower concentrations (10–1.25 µg/ml) (Figs. 9 – 11 ). For deltamethrin An. arabiensis were fully susceptible at all tested concentrations, with 100% mortality at 48 hours (Fig. 12 ). An. gambiae s.l. were susceptible at the three highest concentrations (50–12.5 µg/ml) and possibly resistant at the lower four concentrations (6.25–0.78 µg/ml) (Fig. 13 ). An. stephensi were susceptible at 50 and 25 µg/ml, while all lower concentrations (12.5–0.78 µg/ml) showed resistance (Fig. 14 ). Molecular identification of An. gambiae s.l. and An. stephensi using PCR Of the An. gambiae s.l samples, 117 (97.5%) were successfully amplified and all were identified as An. arabiensis . For An. stephensi , 39 samples were tested, of which 16 (41.0%) were confirmed as An. stephensi , while 23 (59.0%) failed to amplify. Detecting L1014F Kdr alleles in An.arabiensis The L1014F kdr (West African kdr) mutation was detected in An. arabiensis from Bacho Bore kebele, with a frequency of 22.8% (13 heterozygotes and no homozygotes). Among 57 specimens analyzed, 13 (22.8%) were heterozygous (RS), and no homozygous resistant (RR) individuals were observed. The mutation frequencies are summarized in Table 2 . Table 2 Summary of L1014F kdr genotype frequency in survived and dead An. arabiensis exposed to pyrethroids in Bacho bore kebeles, Jimma town Ethiopia. An.arabiensis Number tested RS RR SS Kdr allele frequency Dead 8 0 0 8 0 Alive 49 13 0 36 0.26 total 57 13 0 44 0.26 RR: homozygous resistances: RS: heterozygous resistance and SS: homozygous susceptible DISCUSSION This study evaluated the susceptibility of two major mosquito species in Ethiopia, An. gambiae s.l. and An. stephensi , to various insecticides. Mosquito resistance is influenced by multiple factors, including genetics, physiology, behavior, environment, and frequency of insecticide use [ 26 ]. An. gambiae from Bacho Bore, Jimma, was fully susceptible to the organophosphate pirimiphos-methyl, consistent with findings from Ethiopia [ 11 ], Sudan [ 27 ], Burkina Faso [ 28 ], and Ghana [ 29 ], but differing from reports of high resistance in western Kenya [ 30 ]. The same population was fully susceptible to propoxur but showed partial resistance to bendiocarb, similar to observations in Lake Tana, Ethiopia, and Faranah, Guinea [ 31 , 9 ], while studies from southeastern Benin reported full susceptibility [ 9 ]. Resistance to pyrethroids (deltamethrin and alpha-cypermethrin) was observed, consistent with findings from Gambella, Ethiopia [ 11 ]. Anopheles gambiae s.l. showed variable susceptibility to neonicotinoids, including clothianidin, indicating that resistance patterns differ by insecticide class and region. This underscores the need for regular monitoring of insecticide effectiveness in malaria vector control. An. stephensi populations in Awash Sebhat Kilo (Afar Region) and Kebri Dehar (Somali Region) demonstrated resistance to multiple insecticides, including pyrethroids, bendiocarb, propoxur, and pirimiphos-methyl, with some tolerance to clothianidin [ 6 , 32 ]. Compared with An. gambiae s.l. , An. stephensi displayed higher resistance, posing a significant challenge to current malaria control strategies. CDC bottle bioassays revealed that both An. gambiae and An. stephensi were fully susceptible to clothianidin (10 µg/ml), chlorfenapyr (100 µg/ml), and broflanilide (50–0.78 µg/ml), although the lowest broflanilide concentrations were slightly less effective against An. stephensi (Figs. 5 – 8 , 10 – 14 ). Pirimiphos-methyl and deltamethrin also showed high efficacy at higher concentrations. Differences between CDC bottle and WHO tube tests were noted: clothianidin resistance was better detected using WHO paper tests, while CDC assays allowed evaluation of higher insecticide doses, effectively overcoming resistance in local populations. These results highlight the importance of testing methodology and dose optimization in monitoring mosquito susceptibility. Molecular analysis of An. arabiensis from Bacho Bore revealed the presence of the West African kdr mutation (L1014F) at low frequency (22.8% heterozygotes), with no homozygous resistant individuals and no detection of the East African kdr (L1014S) mutation (Table 2 ). The low frequency of kdr mutations, despite high phenotypic resistance to deltamethrin, suggests other mechanisms contribute to resistance in this population. Molecular testing of An. stephensi could not be performed due to material constraints. Overall, these findings underscore the urgent need for continued monitoring of insecticide resistance across regions and mosquito species in Ethiopia. Future research should expand geographic coverage, examine additional resistance mechanisms, and test various insecticide doses using CDC bottle assays. Conclusion This study highlights the high level of insecticide resistance in mosquito populations in Ethiopia. In Awash Subhat Kilo Town (Afar Region), An. stephensi exhibited resistance to most tested insecticides, while An. gambiae remained susceptible only to pirimiphos-methyl and propoxur. An. stephensi was susceptible to chlorfenapyr and broflanilide but showed resistance to clothianidin. An. gambiae s.l populations were still susceptible to clothianidin, broflanilide, and chlorfenapyr. The widespread resistance of An. stephensi is concerning and underscores the need for urgent interventions. Further research is required to understand the mechanisms of resistance and develop more effective control strategies. Ongoing monitoring of mosquito susceptibility is essential to guide insecticide selection and ensure the success of malaria control programs. Abbreviations AChE Acethylcholinesterase CDC Center for Disease Control DDT Dichloro-diphenyl-trichloroethane GST Glutathione-S-transferases IRS Indoor residual spraying KDR Knockdown resistance LLIN Long-lasting insecticidal net PCR Polymerase chain reaction RPM Revolution per minute VGSC Voltage gatedsodium channel WHO World Health Organization Declarations Ethics approval and consent to participate Consent for publication Competing interests: The authors declare that they have no competing interests. Funding: No funding was obtained for this research. Author Contribution EAS and DY conceived and designed the study. MG conducted the field and the laboratory work and analyzed the data. MG and EAS drafted the manuscript, and DY critically reviewed it. All authors read approved the final version of the manuscript. Acknowledgement The authors acknowledge the crucial support from the Department of Biology and the Tropical and Infectious Disease Research Center (TIDRC) at Jimma University, which provided both financial and logistical resources for the research. The authors also express gratitude to the authorities and communities of Bocho Bore Kebele in Jimma town, Oromia region, and Awash 7 Kilo town in the Afar region, for their invaluable cooperation. This assistance was fundamental to the successful execution of the fieldwork and the collection of essential data for the study. Data Availability All data generated are included in this manuscript. References Matovu HA, Muyanja CK, & S, B. (2015). The Proximate and Chemical Composition of Improved . 15 No. 5 , 10474–10490. Tesfaye, S., & Yesuf, A. (2024). 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F. de S., Hien, A. S., Kaboré, D. A., Kientega, M., Ouédraogo, A. G., Pennetier, C., Koffi, A. A., Moiroux, N., & Dabiré, R. K. (2021). Insecticide resistance status of malaria vectors Anopheles gambiae (s.l.) of southwest Burkina Faso and residual efficacy of indoor residual spraying with microencapsulated pirimiphos-methyl insecticide. Parasites and Vectors , 14 (1), 1–9. https://doi.org/10.1186/s13071-020-04563-8 Baffour-Awuah, S., Annan, A. A., Maiga-Ascofare, O., Dieudonné, S. D., Adjei-Kusi, P., Owusu-Dabo, E., & Obiri-Danso, K. (2016). Insecticide resistance in malaria vectors in Kumasi, Ghana. Parasites and Vectors , 9 (1), 1–8. https://doi.org/10.1186/s13071-016-1923-5 Kitungulu, N., Guyah, B., Webale, M., Shaviya, N., Machani, M., Mulama, D., & Ndenga, B. (2022). Resistance of Anopheles gambiae sensu lato to Pirimiphos-methyl Insecticide in Kakamega County, Highlands of Western Kenya. African Health Sciences , 22 (1), 589–597. https://doi.org/10.4314/ahs.v22i1.68 Kendie, F. A., Wale, M., Nibret, E., & Ameha, Z. (2023). Insecticide susceptibility status of Anopheles gambiae (s.l. ) in and surrounding areas of Lake Tana, northwest Ethiopia. Tropical Medicine and Health , 51 (1). https://doi.org/10.1186/s41182-023-00497-w Yared, S., Gebressielasie, A., Damodaran, L., Bonnell, V., Lopez, K., Janies, D., & Carter, T. E. (2020). Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia. Malaria Journal , 19 (1), 1–7. https://doi.org/10.1186/s12936-020-03252-2 Tchouakui, M., Assatse, T., Tazokong, H. R., Oruni, A., Menze, B. D., Nguiffo-Nguete, D., Mugenzi, L. M. J., Kayondo, J., Watsenga, F., Mzilahowa, T., Osae, M., & Wondji, C. S. (2023). Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa. Scientific Reports , 13 (1), 1–10. https://doi.org/10.1038/s41598-023-29605-w Additional Declarations No competing interests reported. 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2","display":"","copyAsset":false,"role":"figure","size":358509,"visible":true,"origin":"","legend":"\u003cp\u003eSerial dilution of broflanilide, deltamethrin and pirimiphos-methyl insecticide\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/65fb25dd21f81f127a763b08.png"},{"id":93538756,"identity":"4e49b7b7-cee1-4508-b0f1-78c89b3eea80","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38288,"visible":true,"origin":"","legend":"\u003cp\u003eMean mortality rates of \u003cem\u003eAn.gambiae \u003c/em\u003es.l\u003cem\u003e \u003c/em\u003eusing WHO tube test (Pirimiphos methyl(0.25%) propoxur(0.1%), Clothianidin(2%), Bendiocarb(0.1%), Alpha cypermethrin (0.05%) and Deltamethrin (0.25%) \u003cem\u003e(solid line shows cutoff point as per WHO, 2016).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/889cc5242bc98762c520226e.png"},{"id":93539573,"identity":"0c5d7592-8ac7-409b-bb7c-ddd78e7951a3","added_by":"auto","created_at":"2025-10-15 02:19:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39876,"visible":true,"origin":"","legend":"\u003cp\u003eMean mortality rate of \u003cem\u003eAn. stephensi \u003c/em\u003eusing\u003cem\u003e \u003c/em\u003eWHO tube test \u003cem\u003e(solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/f9c0cf1a9506ff465dc5e087.png"},{"id":93538767,"identity":"c14669ad-b3f8-4d2f-b38d-d5804e95782a","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38054,"visible":true,"origin":"","legend":"\u003cp\u003eMean mortality rates of \u003cem\u003eAn.gambiae\u003c/em\u003e s.l., \u003cem\u003eAn. arabiensis\u003c/em\u003e strain and \u003cem\u003eAn. stephensi\u003c/em\u003e 24 exposed to Chlorfenapyr and Clothianidin using CDC bottle bioassay \u003cem\u003e(solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/2a9588b7c7bdefe10103bccd.png"},{"id":93538759,"identity":"99cf8c03-e791-4bbb-885b-ba527f111617","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49383,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of strain of \u003cem\u003eAn.arabiensis\u003c/em\u003eby broflanilide\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/899ea2d1648ffe88873d02b7.png"},{"id":93538763,"identity":"5d96701f-5a6f-4c77-88ce-4a64a80582b8","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44300,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003eAn.gambiae \u003c/em\u003es.l.by broflanilide\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/fa0409fa7c7bcfafd68dc5eb.png"},{"id":93539576,"identity":"6fccedd0-288b-44a8-bcbf-ffb4fee92dcf","added_by":"auto","created_at":"2025-10-15 02:19:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":67644,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003eAn.stephensi\u003c/em\u003eby broflanilide\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/50917e6b363b2aff214000bd.png"},{"id":93538772,"identity":"484cf227-3d8f-496f-a67b-e1842890b34e","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":48581,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of strain of \u003cem\u003eAn.arabiensis\u003c/em\u003eby pirimiphos methyl\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/fd949acd43e10a5afe57f25f.png"},{"id":93538765,"identity":"fb5a21dc-98ee-4192-994e-0ba5ab003cce","added_by":"auto","created_at":"2025-10-15 02:11:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":50272,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003eAn. gambiae\u003c/em\u003es.l. by pirimiphos methyl\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/0802a4c6a3acac337d9e519e.png"},{"id":93538775,"identity":"d4a40a85-16c6-4e1b-a88a-0d66c9f482dc","added_by":"auto","created_at":"2025-10-15 02:11:59","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":49400,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003eAn. stephensi\u003c/em\u003e by Pirimiphos methyl\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/5950bb643f3ea475f3abcc51.png"},{"id":93539579,"identity":"7acccbd0-12f7-4bb0-9fee-9e4dbf60fac9","added_by":"auto","created_at":"2025-10-15 02:19:59","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":72513,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of strain of \u003cem\u003eAn.arabiensis\u003c/em\u003eby deltamethrin\u003cem\u003e(solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/977582f6100c1e08d912f901.png"},{"id":93538783,"identity":"23726e65-57cb-40bd-9540-17d791eb3640","added_by":"auto","created_at":"2025-10-15 02:11:59","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":56974,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003eAn. gambiae\u003c/em\u003es.l by deltamethrin\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/ffc9dd26a8a18c88408ffa1e.png"},{"id":93538777,"identity":"156da851-6a1e-4068-96c1-bc37709a021c","added_by":"auto","created_at":"2025-10-15 02:11:59","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":58363,"visible":true,"origin":"","legend":"\u003cp\u003eWHO bottle bioassay mortality of \u003cem\u003e\u003cstrong\u003eAn. stephensi\u003c/strong\u003e\u003c/em\u003eby deltamethrin\u003cem\u003e (solid line shows cutoff point as per WHO, 2016)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/6dfef1c74408f5bdd904d7e0.png"},{"id":98628603,"identity":"2e5c15a8-4904-4277-9e26-f3aa94755463","added_by":"auto","created_at":"2025-12-19 17:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2202973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7704930/v1/afe9ebde-3ae9-48c4-8a89-56d078347e34.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eMosquitoes as a large group of arthropods that play an important role in the transmission of many diseases to humans such as malaria, filariasis, yellow fever, dengue fever [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Ethiopia, malaria remains a major public health concern with millions of cases and thousands of deaths reported annually [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Unlike most of the African continent, malaria is caused by infection with \u003cem\u003ePlasmodium vivax\u003c/em\u003e or \u003cem\u003eP. falciparum\u003c/em\u003e. Efforts to control the transmission of malaria currently target \u003cem\u003eAn. arabiensis\u003c/em\u003e, the primary malaria vector in Ethiopia, as well the secondary vectors \u003cem\u003eAn. funestus\u003c/em\u003e, \u003cem\u003eAn. pharoensis\u003c/em\u003e, and \u003cem\u003eAn. nili\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eAnopheles stephensi\u003c/em\u003e is an important vector that plays a major role in transmitting malaria in urban areas and is an established vector in many parts of India. The first report of \u003cem\u003eAn. stephensi\u003c/em\u003e in the Horn of Africa was from Djibouti in 2013[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and this mosquito was first detected in the Somali Regional State of Ethiopia in 2016 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and subsequently been confirmed to have a broad distribution in Northeast and eastern Ethiopia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and distribution in south Ethiopia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTransmission of malaria can be reduced by adopting vector control measures such as indoor residual spraying (IRS) with insecticides, larval control measures, and personal protection measures. The combination of tools and methods used to combat malaria now includes insect nets treated with long-lasting insecticides (LLIT) and artemisinin-based combination therapy, supported by IRS of insecticide and intermittent preventive treatment during pregnancy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChemical control had been the main method for combating the adult stage of malaria vector since the eradication era. Insecticide application for adult mosquito control started with organochlorines (DDT, dieldrin, and BHC) during the 1960s, followed by organophosphates (malathion and pirimiphos-methyl) for 2 decades from 1966 and continued with the carbamate, propoxur during 1977\u0026ndash;1990, and then with pyrethroids (lambda-cyhalothrin/deltamethrin). Temephos, chlorpyriphos-methyl, and pirimiphos-methyl were used for larviciding from 1970 to 1992. In Guinea \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e was resistant to pyrethroid, alpha cypermethrin, lambdacyhalothrin and DDT but susceptible to deltamethrin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In Togo \u003cem\u003eAn. gambiae s.l\u003c/em\u003e. found resistant against bendiocarb, deltamethrin and propoxur [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In Ethiopia \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e was resistant to two groups of pyrethroid insecticides (deltamethrin and alpha-cypermethrin), but susceptible to pirimiphos-methyl, propoxur and bendiocarb [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Resistance of \u003cem\u003eAn. stephensi\u003c/em\u003e to DDT, dieldrin, and malathion was reported for the first time in 1957, 1960, and 1976, respectively [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Ethiopia was highly resistant to DDT, malathion, pirimiphos-methyl, bendiocarb, propoxur, deltamethrin, and permethrin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In Awash Sebat Kilo Afar region (2019, 2020) the resistance intensity of \u003cem\u003eAn. stephensi\u003c/em\u003e to alpha-cypermethrin, deltamethrin and permethrin was assessed through exposure to 1\u0026times;, 5\u0026times; and 10\u0026times; the diagnostic dose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe various mechanisms, including metabolic resistance and site insensitivity can cause insecticide resistance [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Metabolic resistance mechanism is based on the enzyme systems which all mosquitoes possess to help them to detoxify naturally occurring insecticides. Three categories of enzymes, namely esterases, P450s and glutathione-S-transferases are known to confer resistance to insecticides in insect pest such as malaria vectors. In Ethiopia \u003cem\u003eAn. arabiensis\u003c/em\u003e very high frequency of the West African \u003cem\u003ekdr\u003c/em\u003e allele (L1014F), was observed with higher \u003cem\u003ekdr\u003c/em\u003e allele indicating that target site resistance mechanism might contribute for the observed high level of pyrethroids (deltamethrin and alpha-cypermethrin) resistance in the population[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In Afghanistan \u003cem\u003eAn. stephensi\u003c/em\u003e, general esterases (GES), glutathione \u003cem\u003eS\u003c/em\u003e-transferases (GSTs), cytochrome P450s and insensitive acetylcholinesterase (AChEs) were implicated in insecticide resistance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. General esterases are involved in OPs resistance in \u003cem\u003eAn. stephensi\u003c/em\u003e from Pakistan [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. WHO standard insecticide susceptibility bioassays have been performed on \u003cem\u003eAn. stephensi\u003c/em\u003e from Afghanistan showing resistance to organochlorines, carbamates and pyrethroid insecticides [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eAnopheles stephensi\u003c/em\u003e from India had increased activities of esterases and GSTs associated with deltamethrin and permethrin resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Insecticides generally act at a specific site within the insect, typically within the nervous system (e.g. OP, carbamate, DDT and pyrethroids insecticides\u003cb\u003e)\u003c/b\u003e. Knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutation is widespread in \u003cem\u003eAnopheles\u003c/em\u003e species in Africa especially \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Originally, the L1014F mutation \u003cb\u003e(\u003c/b\u003e\u003cem\u003ekdr west\u003c/em\u003e\u003cb\u003e)\u003c/b\u003e was detected in 2000; a second \u003cem\u003ekdr\u003c/em\u003e mutation \u003cb\u003e(\u003c/b\u003e\u003cem\u003ekdr east\u003c/em\u003e\u003cb\u003e)\u003c/b\u003e was detected in Kenyan \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The first report of a \u003cem\u003ekdr\u003c/em\u003e L1014F resistance mechanism in \u003cem\u003eAn. stephens\u003c/em\u003ei was in the DUB-S strain in 2003 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The presence of \u003cem\u003ekdr east\u003c/em\u003e in \u003cem\u003eAn. stephensi\u003c/em\u003e from India was reported for the first time by Singh \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrently, most resistance monitoring is dependent on bioassays, using fixed insecticide\u003c/p\u003e\u003cp\u003econcentrations and exposure times, and the data is reported as percentage mortality and/or Knock down (KD) effect.\u003c/p\u003e\u003cp\u003eWHO recommends that insecticide susceptibility status of malaria vectors should be monitored annually [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. When insecticide resistance is detected, its intensity and the biochemical and molecular mechanisms should also be investigated [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Accurate information on the underlying resistance mechanisms in \u003cem\u003eAn. stephensi\u003c/em\u003e is needed for proper management of insecticide resistance and a better management of malaria through vector control interventions. Therefore, the aim of this study was to determine the insecticide resistance status of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. and \u003cem\u003eAn. stephens\u003c/em\u003ei to different classes of insecticides and characterize molecular resistance mechanisms in \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy area\u003c/h2\u003e\u003cp\u003eThe research took place in Awash Sabhat kilo and Jimma towns. Awash Sabhat kilo is 220 km southeast of Addis Ababa in the Afar region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The town is located at 8.989149\" N, 40.164715\" E, and is 916 meters above sea level. It has about 24,700 residents and experiences a semi-arid climate with main rains in July-August and brief rains in April/May. Temperatures average 25.8\u0026deg;C, ranging from 17.3\u0026deg;C to 33.6\u0026deg;C. The Awash River Valley is Ethiopia's most irrigated region, with extensive farming. The area has year-round malaria, with 536 cases per 1000 people in 2019[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In 2018, the presence of \u003cem\u003eAn. stephensi\u003c/em\u003e mosquitoes was reported in the town [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eJimma town is 353km southwest of Addis Ababa, at 7\u0026deg;41' N and 36\u0026deg; 50'E, with an elevation of 1780 meters. The area, known as Woyna Daga, has good conditions for farming and living. The climate is warm, with temperatures between 14\u0026deg;C and 30\u0026deg;C. Yearly rainfall is between 1138mm and 1690mm, with most rain falling from June to August and the least in December and January. The heavy rainfall makes this highland region one of Ethiopia's most fertile areas, perfect for farming. The Bacho bore Kebele area has many mosquito breeding sites and regular malaria cases. Both \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e malaria types are common here, with \u003cem\u003eAn. arabiensis\u003c/em\u003e mosquitoes being the main carriers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMosquito collection and identification\u003c/h3\u003e\n\u003cp\u003eFrom May 2022 to October 2024, mosquito larvae and pupae were collected in Jimma town and Awash Sebhat Kilo, Ethiopia. \u003cem\u003eAn. arabiensis mosquito\u003c/em\u003e was obtained from Jimma University\u0026rsquo;s disease research center, while \u003cem\u003eAn. gambiae\u003c/em\u003e s.l was collected from natural habitats such as swamps, rice fields, puddles, ditches, tree holes, and water containers in the Jimma Bacho bore area. \u003cem\u003eAn. stephensi\u003c/em\u003e mosquito was sampled from human-made sites, including wells, tanks, coolers, gutters, and construction sites in Awash Subhat Kilo. For each site, researchers recorded coordinates, water type (permanent/temporary), source, collection type, sun exposure, vegetation, and water quality.\u003c/p\u003e\u003cp\u003eLarvae were placed in labeled plastic containers and transported to the insectary, where they were reared to adults with yeast powder and sail farm food under controlled conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 75\u0026thinsp;\u0026plusmn;\u0026thinsp;10% RH, 12:12 h light\u0026ndash;dark cycle). Pupae were transferred to emergence cages, and adults were held safely to prevent contamination and ant damage. Mosquito species were identified morphologically [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eInsecticide Susceptibility Tests\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eWHO Tube test\u003c/h2\u003e\u003cp\u003eThe WHO tube test method was used to assess mosquito susceptibility [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Female mosquitoes of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., \u003cem\u003eAn. stephensi\u003c/em\u003e, and laboratory-reared \u003cem\u003eAn. arabiensis\u003c/em\u003e (3\u0026ndash;5 days old, non-blood-fed) were tested. For each species, 100 wild mosquitoes were used, divided into four groups for exposure to different insecticides, alongside two control groups. The insecticides tested were pirimiphos-methyl (0.25%), propoxur (0.1%), bendiocarb (0.1%), deltamethrin (0.05%), alpha-cypermethrin (0.05%), and clothianidin (2%). Tests were conducted according to WHO guidelines (2016). After exposure, mosquitoes were transferred to holding tubes, provided with 10% sugar solution, and maintained for 24 hours at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 80\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity. Mortality was then recorded.\u003c/p\u003e\u003cp\u003eAll dead and surviving specimens were preserved individually in Eppendorf tubes over silica gel for subsequent molecular assays. Resistance status was classified according to WHO criteria: 98\u0026ndash;100% mortality indicates susceptibility, 90\u0026ndash;97% suggests possible resistance requiring confirmation, and \u0026lt;\u0026thinsp;90% indicates resistance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCDC bottle bioassays\u003c/h3\u003e\n\u003cp\u003eThe CDC bottle bioassay was conducted with broflanilide (various concentrations), chlorfenapyr (100 \u0026micro;g/bottle), and clothianidin (10 \u0026micro;g/bottle) following standard protocols [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Chlorfenapyr was mixed with acetone, and clothianidin with acetone\u0026thinsp;+\u0026thinsp;MERO (800 ppm). Bottles and caps were coated with 1 ml of solution, rolled until dry, and stored in the dark. Control bottles received acetone or acetone\u0026thinsp;+\u0026thinsp;MERO only.\u003c/p\u003e\u003cp\u003eFor broflanilide, 1 mg of technical-grade insecticide was dissolved in 2 ml of acetone\u0026thinsp;+\u0026thinsp;MERO (800 ppm) to prepare a 500 \u0026micro;g/ml stock. Comparator insecticides, deltamethrin and pirimiphos-methyl, were prepared using acetone alone. Subsequently, 1mg of technical-grade broflanilide was added and fully dissolved to produce a 500 \u0026micro;g/ml broflanilide stock solution. Serial dilutions were obtained by mixing 1 ml of stock with 9 ml of acetone\u0026thinsp;+\u0026thinsp;MERO, yielding 50 \u0026micro;g/ml; the same procedure was used for deltamethrin and pirimiphos-methyl [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWheaton bottles were arranged by concentration. In total, 2,400 non-blood-fed female mosquitoes (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l., \u003cem\u003eAn. stephensi\u003c/em\u003e, and lab-reared \u003cem\u003eAn. arabiensis\u003c/em\u003e), aged 3\u0026ndash;5 days, were tested. For each dose, 25 mosquitoes were introduced (four replicates); controls included two bottles with 25 mosquitoes each. Mortality was recorded every 15 min over 60 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular identification of\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l and\u003c/b\u003e \u003cb\u003eAn. stephensi\u003c/b\u003e, \u003cb\u003eand detection of target site mutations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA Extraction and Species Identification\u003c/p\u003e\u003cp\u003eWhole-genome DNA was extracted using DNAzol reagent (MRCgene, USA) following standard protocols [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Individual mosquitoes were processed: 50 survivors and 10 dead from deltamethrin exposure, plus 10 controls. Each mosquito was homogenized in 100 \u0026micro;l DNAzol, incubated for 2\u0026ndash;3 min, and centrifuged at 13,000 rpm for 20 min at 4\u0026deg;C. The supernatant was transferred to a new 1.5 ml tube, mixed with 200 \u0026micro;l 100% ethanol, and centrifuged again (13,000 rpm, 5 min, 4\u0026deg;C). The pellet was washed with 100 \u0026micro;l 70% ethanol, air-dried, and re-suspended in 100 \u0026micro;l TE buffer. Extracted DNA was used for species identification and resistance genotyping.\u003c/p\u003e\u003cp\u003eSpecies identification was performed using PCR following Safi et al. (2019) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In total, 120 female \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. and 39 \u003cem\u003eAn. stephensi\u003c/em\u003e were tested. For \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., samples included 50 survivors, 10 dead, and 10 controls from deltamethrin assays. For \u003cem\u003eAn. stephensi\u003c/em\u003e, samples included 30 survivors, 5 dead, and 4 controls.\u003c/p\u003e\u003cp\u003ePCR assays were conducted at Jimma University\u0026rsquo;s Tropical and Infectious Diseases Research Centre. For \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., species-specific primers (AR, AG, QD, and UN) were used. PCR reactions contained MgCl₂, Tris-HCl, KCl, Triton X-100, dNTPs, and SilverStar DNA polymerase. Products were visualized on agarose gels stained with ethidium bromide. For \u003cem\u003eAn. stephens\u003c/em\u003ei, ITS2 primers were used to amplify a 400\u0026ndash;500 bp fragment. Reactions included forward and reverse primers, PCR water, master mix, and DNA template. PCR cycling conditions were 95\u0026deg;C (denaturation), 50\u0026deg;C (annealing), and 72\u0026deg;C (extension), and products were resolved on 2% agarose gels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003ekdr\u003c/b\u003e \u003cb\u003eMutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eKnockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations were screened using allele-specific PCR following established protocols. After WHO tube tests, 58 female \u003cem\u003eAn. arabiensis\u003c/em\u003e (dead and surviving) were analyzed for \u003cem\u003ekdr\u003c/em\u003e and ace-1 mutations. The assay employed allele-specific PCR targeting the L1014F mutation.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eKnockdown was recorded every 10 min for 1 h, and mortality was assessed after 24 h for all treatments and controls. When control mortality ranged from 5\u0026ndash;20%, data were corrected using Abbott\u0026rsquo;s formula. Mortality confidence limits (5%) and lethal times (LT₅₀, LT₉₀ with 95% CIs) were estimated using the Finney method. Insecticide results were graphed in Excel. For molecular analysis, the frequency of \u003cem\u003ekdr\u003c/em\u003e mutations in wild \u003cem\u003eAn. arabiensis\u003c/em\u003e (dead and surviving) was determined, and genotype distributions were assessed with Hardy\u0026ndash;Weinberg software [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOverall, 6,150 female Anopheles mosquitoes were reared from immature stages collected in two Ethiopian sites: Bacho Bore Kebele, Jimma town (Oromia region), and Awash Sebat Kilo town (Afar region) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eAn. gambiae\u003c/em\u003e s.l was primarily associated with natural water bodies and showed seasonal breeding patterns, whereas \u003cem\u003eAn. stephensi\u003c/em\u003e preferred artificial containers.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacterization of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes breeding habitats in Jimma and Awash 7 kilo towns, Ethiopia\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS.N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eBreeding site\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eAn. stephensi\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGeographic location\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJimma Town (Bacho Bore)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAwash 7 kilo town, Afar\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eType of breeding site\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etemporary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePermanent or semi-permanent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOrigin of the water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eman-made\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNature of the water collection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePuddle, ditch\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDitch, container, over tank\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExposure to sunlight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eshaded, sunlit or partial light\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eshaded, sunlit or partial light\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresence of vegetation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eemergent, submerged, floating\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003efloating\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCharacteristics of the water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClear, turbid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eclear\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eWHO Tube Test result\u003c/h3\u003e\n\u003cp\u003eA total of 900 \u003cem\u003eAn. gambiae\u003c/em\u003e s.l mosquitoes were tested for insecticide resistance, with 600 exposed to insecticides and 300 serving as controls. The insecticides tested were pirimiphos-methyl (0.25%), bendiocarb (0.1%), deltamethrin (0.05%), alpha-cypermethrin (0.05%), clothianidin (2%), and propoxur (0.1%).\u003c/p\u003e\u003cp\u003eAccording to WHO criteria, \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. from Jimma were resistant to deltamethrin, alpha-cypermethrin, bendiocarb, and clothianidin, with mortality rates below the 90% threshold: 18% (deltamethrin), 43% (alpha-cypermethrin), 63% (bendiocarb), and 73% (clothianidin). The mosquitoes were fully susceptible to propoxur and pirimiphos-methyl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMosquitoes from Awash 7 Kilo Town exhibited high resistance to most tested insecticides. After 24 hours, mortality rates were: clothianidin 97%, propoxur 31%, pirimiphos-methyl 23%, alpha-cypermethrin 9%, deltamethrin 8%, and bendiocarb 7%. As effective insecticides are expected to achieve\u0026thinsp;\u0026ge;\u0026thinsp;90% mortality, only clothianidin approached efficacy, though early resistance signs were observed. Overall, these mosquitoes were substantially more resistant than \u003cem\u003eAn. gambiae\u003c/em\u003e s.l (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCDC bottle bioassays result\u003c/h2\u003e\u003cp\u003eA total of 100 adult female \u003cem\u003eAn. gambiae\u003c/em\u003e s.l and \u003cem\u003eAn. stephensi\u003c/em\u003e were exposed to chlorfenapyr (100 \u0026micro;g/ml), resulting in 100% mortality, indicating full susceptibility. Complete susceptibility was also observed for \u003cem\u003eAn. stephensi\u003c/em\u003e at 10 \u0026micro;g/ml and \u003cem\u003eAn. gambiae\u003c/em\u003e s.l at 4 \u0026micro;g/ml of clothianidin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although WHO tube tests suggested resistance to clothianidin in these populations, CDC bottle assays confirmed full vulnerability.\u003c/p\u003e\u003cp\u003eFor broflanilide, 700 adult females of each species were tested across concentrations of 50, 25, 12.5, 6.25, 3.125, 1.562, and 0.78 \u0026micro;g/ml. \u003cem\u003eAn. arabiensis\u003c/em\u003e were fully susceptible at all concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. exhibited 100% mortality within 24 hours at all doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), while \u003cem\u003eAn. stephensi\u003c/em\u003e showed complete mortality at higher concentrations (50\u0026ndash;3.125 \u0026micro;g/ml) and 99% mortality at the two lowest doses (1.563 and 0.78 \u0026micro;g/ml) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAcross species, higher concentrations of broflanilide consistently produced greater mortality, with \u003cem\u003eAn. arabiensis\u003c/em\u003e being the most susceptible, and \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. and \u003cem\u003eAn. stephensi\u003c/em\u003e showing similar susceptibility patterns.\u003c/p\u003e\u003cp\u003eA total of 700 adult females of each mosquito species were tested against pirimiphos-methyl (80, 40, 20, 10, 5, 2.5, and 1.25 \u0026micro;g/ml) and deltamethrin (50, 25, 12.5, 6.25, 3.125, 1.562, and 0.78 \u0026micro;g/ml). For pirimiphos-methyl, \u003cem\u003eAn. arabiensis\u003c/em\u003e were fully susceptible at all concentrations, while \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. showed complete mortality at the four highest concentrations (80\u0026ndash;10 \u0026micro;g/ml), susceptibility at 5 \u0026micro;g/ml, and resistance at the two lowest concentrations (2.5\u0026ndash;1.25 \u0026micro;g/ml). \u003cem\u003eAn. stephensi\u003c/em\u003e were fully susceptible at 80 and 40 \u0026micro;g/ml, possibly susceptible at 20 \u0026micro;g/ml, and resistant at the remaining lower concentrations (10\u0026ndash;1.25 \u0026micro;g/ml) (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor deltamethrin \u003cem\u003eAn. arabiensis\u003c/em\u003e were fully susceptible at all tested concentrations, with 100% mortality at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e\u003cem\u003e). An. gambiae\u003c/em\u003e s.l. were susceptible at the three highest concentrations (50\u0026ndash;12.5 \u0026micro;g/ml) and possibly resistant at the lower four concentrations (6.25\u0026ndash;0.78 \u0026micro;g/ml) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e). An. stephensi were susceptible at 50 and 25 \u0026micro;g/ml, while all lower concentrations (12.5\u0026ndash;0.78 \u0026micro;g/ml) showed resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular identification of\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l. and\u003c/b\u003e \u003cb\u003eAn. stephensi\u003c/b\u003e \u003cb\u003eusing PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOf the \u003cem\u003eAn. gambiae\u003c/em\u003e s.l samples, 117 (97.5%) were successfully amplified and all were identified as \u003cem\u003eAn. arabiensis\u003c/em\u003e. For \u003cem\u003eAn. stephensi\u003c/em\u003e, 39 samples were tested, of which 16 (41.0%) were confirmed as \u003cem\u003eAn. stephensi\u003c/em\u003e, while 23 (59.0%) failed to amplify.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetecting L1014F\u003c/b\u003e \u003cb\u003eKdr\u003c/b\u003e \u003cb\u003ealleles in\u003c/b\u003e \u003cb\u003eAn.arabiensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe L1014F \u003cem\u003ekdr\u003c/em\u003e (West African kdr) mutation was detected in \u003cem\u003eAn. arabiensis\u003c/em\u003e from Bacho Bore kebele, with a frequency of 22.8% (13 heterozygotes and no homozygotes). Among 57 specimens analyzed, 13 (22.8%) were heterozygous (RS), and no homozygous resistant (RR) individuals were observed. The mutation frequencies are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of L1014F \u003cem\u003ekdr\u003c/em\u003e genotype frequency in survived and dead \u003cem\u003eAn. arabiensis\u003c/em\u003e exposed to pyrethroids in Bacho bore kebeles, Jimma town Ethiopia.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn.arabiensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber tested\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eKdr\u003c/em\u003e allele frequency\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDead\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRR: homozygous resistances: RS: heterozygous resistance and SS: homozygous susceptible\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study evaluated the susceptibility of two major mosquito species in Ethiopia, \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e and \u003cem\u003eAn. stephensi\u003c/em\u003e, to various insecticides. Mosquito resistance is influenced by multiple factors, including genetics, physiology, behavior, environment, and frequency of insecticide use [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eAn. gambiae\u003c/em\u003e from Bacho Bore, Jimma, was fully susceptible to the organophosphate pirimiphos-methyl, consistent with findings from Ethiopia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Sudan [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], Burkina Faso [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Ghana [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but differing from reports of high resistance in western Kenya [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The same population was fully susceptible to propoxur but showed partial resistance to bendiocarb, similar to observations in Lake Tana, Ethiopia, and Faranah, Guinea [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while studies from southeastern Benin reported full susceptibility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Resistance to pyrethroids (deltamethrin and alpha-cypermethrin) was observed, consistent with findings from Gambella, Ethiopia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles gambiae s.l.\u003c/em\u003e showed variable susceptibility to neonicotinoids, including clothianidin, indicating that resistance patterns differ by insecticide class and region. This underscores the need for regular monitoring of insecticide effectiveness in malaria vector control. \u003cem\u003eAn. stephensi\u003c/em\u003e populations in Awash Sebhat Kilo (Afar Region) and Kebri Dehar (Somali Region) demonstrated resistance to multiple insecticides, including pyrethroids, bendiocarb, propoxur, and pirimiphos-methyl, with some tolerance to clothianidin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Compared with \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e, \u003cem\u003eAn. stephensi\u003c/em\u003e displayed higher resistance, posing a significant challenge to current malaria control strategies.\u003c/p\u003e\u003cp\u003eCDC bottle bioassays revealed that both \u003cem\u003eAn. gambiae\u003c/em\u003e and \u003cem\u003eAn. stephensi\u003c/em\u003e were fully susceptible to clothianidin (10 \u0026micro;g/ml), chlorfenapyr (100 \u0026micro;g/ml), and broflanilide (50\u0026ndash;0.78 \u0026micro;g/ml), although the lowest broflanilide concentrations were slightly less effective against \u003cem\u003eAn. stephensi\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e). Pirimiphos-methyl and deltamethrin also showed high efficacy at higher concentrations. Differences between CDC bottle and WHO tube tests were noted: clothianidin resistance was better detected using WHO paper tests, while CDC assays allowed evaluation of higher insecticide doses, effectively overcoming resistance in local populations. These results highlight the importance of testing methodology and dose optimization in monitoring mosquito susceptibility.\u003c/p\u003e\u003cp\u003eMolecular analysis of \u003cem\u003eAn. arabiensis\u003c/em\u003e from Bacho Bore revealed the presence of the West African \u003cem\u003ekdr\u003c/em\u003e mutation (L1014F) at low frequency (22.8% heterozygotes), with no homozygous resistant individuals and no detection of the East African \u003cem\u003ekdr\u003c/em\u003e (L1014S) mutation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The low frequency of \u003cem\u003ekdr\u003c/em\u003e mutations, despite high phenotypic resistance to deltamethrin, suggests other mechanisms contribute to resistance in this population. Molecular testing of \u003cem\u003eAn. stephensi\u003c/em\u003e could not be performed due to material constraints.\u003c/p\u003e\u003cp\u003eOverall, these findings underscore the urgent need for continued monitoring of insecticide resistance across regions and mosquito species in Ethiopia. Future research should expand geographic coverage, examine additional resistance mechanisms, and test various insecticide doses using CDC bottle assays.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the high level of insecticide resistance in mosquito populations in Ethiopia. In Awash Subhat Kilo Town (Afar Region), \u003cem\u003eAn. stephensi\u003c/em\u003e exhibited resistance to most tested insecticides, while \u003cem\u003eAn. gambiae\u003c/em\u003e remained susceptible only to pirimiphos-methyl and propoxur. \u003cem\u003eAn. stephensi\u003c/em\u003e was susceptible to chlorfenapyr and broflanilide but showed resistance to clothianidin. \u003cem\u003eAn. gambiae\u003c/em\u003e s.l populations were still susceptible to clothianidin, broflanilide, and chlorfenapyr. The widespread resistance of \u003cem\u003eAn. stephensi\u003c/em\u003e is concerning and underscores the need for urgent interventions. Further research is required to understand the mechanisms of resistance and develop more effective control strategies. Ongoing monitoring of mosquito susceptibility is essential to guide insecticide selection and ensure the success of malaria control programs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAChE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAcethylcholinesterase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCenter for Disease Control\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDDT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDichloro-diphenyl-trichloroethane\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutathione-S-transferases\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIRS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndoor residual spraying\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKnockdown resistance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLLIN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLong-lasting insecticidal net\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePolymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRPM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRevolution per minute\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVGSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVoltage gatedsodium channel\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWorld Health Organization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e\u003cb\u003eConsent for publication\u003c/b\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNo funding was obtained for this research.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEAS and DY conceived and designed the study. MG conducted the field and the laboratory work and analyzed the data. MG and EAS drafted the manuscript, and DY critically reviewed it. All authors read approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the crucial support from the Department of Biology and the Tropical and Infectious Disease Research Center (TIDRC) at Jimma University, which provided both financial and logistical resources for the research. The authors also express gratitude to the authorities and communities of Bocho Bore Kebele in Jimma town, Oromia region, and Awash 7 Kilo town in the Afar region, for their invaluable cooperation. This assistance was fundamental to the successful execution of the fieldwork and the collection of essential data for the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated are included in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMatovu HA, Muyanja CK, \u0026amp; S, B. (2015). The\u003cem\u003e Proximate and Chemical Composition of Improved\u003c/em\u003e. \u003cem\u003e15 No. 5\u003c/em\u003e, 10474\u0026ndash;10490.\u003c/li\u003e\n\u003cli\u003eTesfaye, S., \u0026amp; Yesuf, A. (2024). Trend analysis of malaria surveillance data in West Wallaga, West Oromia, Ethiopia: a framework for planning and elimination. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1), 1\u0026ndash;8. https://doi.org/10.1186/s12936-024-04874-6\u003c/li\u003e\n\u003cli\u003eEligo, N., Wegayehu, T., Pareyn, M., Tamiru, G., Lindtj\u0026oslash;rn, B., \u0026amp; Massebo, F. (2024). \u003cem\u003eAnopheles arabiensis\u003c/em\u003e continues to be the primary vector of Plasmodium falciparum after decades of malaria control in southwestern Ethiopia. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1), 1\u0026ndash;9. https://doi.org/10.1186/s12936-024-04840-2\u003c/li\u003e\n\u003cli\u003eFaulde, M. K., Rueda, L. M., \u0026amp; Khaireh, B. A. (2014). First record of the Asian malaria vector \u003cem\u003eAnopheles stephensi\u003c/em\u003e and its possible role in the resurgence of malaria in Djibouti, Horn of Africa. \u003cem\u003eActa Tropica\u003c/em\u003e, \u003cem\u003e139\u003c/em\u003e, 39\u0026ndash;43. https://doi.org/https://doi.org/10.1016/j.actatropica.2014.06.016\u003c/li\u003e\n\u003cli\u003eCarter, T. E., Gebresilassie, A., Hansel, S., Damodaran, L., Montgomery, C., Bonnell, V., Lopez, K., Janies, D., \u0026amp; Yared, S. (2022). Analysis of the Knockdown Resistance Locus (\u003cem\u003ekdr\u003c/em\u003e) in \u003cem\u003eAnopheles stephensi\u003c/em\u003e, \u003cem\u003eAn. arabiensis\u003c/em\u003e, and \u003cem\u003eCulex pipiens s.l. \u003c/em\u003efor Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia. \u003cem\u003eAmerican Journal of Tropical Medicine and Hygiene\u003c/em\u003e, \u003cem\u003e106\u003c/em\u003e(2), 632\u0026ndash;638. https://doi.org/10.4269/ajtmh.20-1357\u003c/li\u003e\n\u003cli\u003eBalkew, M., Mumba, P., Yohannes, G., Abiy, E., Getachew, D., Yared, S., Worku, A., Gebresilassie, A., Tadesse, F. G., Gadisa, E., Esayas, E., Ashine, T., Yewhalaw, D., Chibsa, S., Teka, H., Murphy, M., Yoshimizu, M., Dengela, D., Zohdy, S., \u0026amp; Irish, S. (2021). An update on the distribution, bionomics, and insecticide susceptibility of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Ethiopia, 2018\u0026ndash;2020. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(1), 1\u0026ndash;13. https://doi.org/10.1186/s12936-021-03801-3\u003c/li\u003e\n\u003cli\u003eHawaria, D., Kibret, S., Zhong, D., Lee, M. C., Lelisa, K., Bekele, B., Birhanu, M., Mengesha, M., Solomon, H., Yewhalaw, D., \u0026amp; Yan, G. (2023). First report of \u003cem\u003eAnopheles stephensi \u003c/em\u003efrom southern Ethiopia. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(1), 1\u0026ndash;8. https://doi.org/10.1186/s12936-023-04813-x\u003c/li\u003e\n\u003cli\u003eWHO. (2018). \u003cem\u003eGlobal report on insecticide resistance in malaria vectors: 2010\u0026ndash;2016. Geneva: World Health Organization\u003c/em\u003e. https://apps.who.int/iris/bitstream/handle/10665/272533/9789241514057-eng.pdf\u003c/li\u003e\n\u003cli\u003eSovi, A., Keita, C., Sinaba, Y., Dicko, A., Traore, I., Cisse, M. B. M., Koita, O., Dengela, D., Flatley, C., Bankineza, E., Mihigo, J., Belemvire, A., Carlson, J., Fornadel, C., \u0026amp; Oxborough, R. M. (2020). \u003cem\u003eAnopheles gambiae\u003c/em\u003e (s.l.) exhibit high intensity pyrethroid resistance throughout Southern and Central Mali (2016-2018): PBO or next generation LLINs may provide greater control. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 1\u0026ndash;16. https://doi.org/10.1186/s13071-020-04100-7\u003c/li\u003e\n\u003cli\u003eApetogbo, Y., Ahadji-Dabla, K. M., Soma, D. D., Amoudji, A. D., Koffi, E., Akagankou, K. I., Bamogo, R., Ngaffo, K. L., Maiga, S., Atcha‑Oubou, R. T., Dorkenoo, A. 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Pyrethroid susceptibility \u0026amp; enzyme activity in two malaria vectors, \u003cem\u003eAnopheles stephensi\u003c/em\u003e (Liston) \u0026amp; A. culicifacies (Giles) from Mysore, India. \u003cem\u003eIndian Journal of Medical Research\u003c/em\u003e, \u003cem\u003e117\u003c/em\u003e(JAN.), 30\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eMehlhorn, H. (2016). \u003cem\u003eAnopheles stephensi\u003c/em\u003e. \u003cem\u003eEncyclopedia of Parasitology\u003c/em\u003e, 145\u0026ndash;146. https://doi.org/10.1007/978-3-662-43978-4_4931\u003c/li\u003e\n\u003cli\u003eBalkew, M., Mumba, P., Dengela, D., Yohannes, G., Getachew, D., Yared, S., Chibsa, S., Murphy, M., George, K., Lopez, K., Janies, D., Choi, S. H., Spear, J., Irish, S. R., \u0026amp; Carter, T. E. (2020). Geographical distribution of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in eastern Ethiopia. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 1\u0026ndash;8. https://doi.org/10.1186/s13071-020-3904-y\u003c/li\u003e\n\u003cli\u003eA\u0026iuml;zoun, N., Oss\u0026egrave;, R., Azondekon, R., Alia, R., Oussou, O., Gnanguenon, V., Aikpon, R., Padonou, G. G., \u0026amp; Akogb\u0026eacute;to, M. (2013). Comparison of the standard WHO susceptibility tests and the CDC bottle bioassay for the determination of insecticide susceptibility in malaria vectors and their correlation with biochemical and molecular biology assays in Benin, West Africa. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(1). https://doi.org/10.1186/1756-3305-6-147\u003c/li\u003e\n\u003cli\u003eDenlinger, D. S., Creswell, J. A., Anderson, J. L., Reese, C. K., \u0026amp; Bernhardt, S. A. (2016). Diagnostic doses and times for Phlebotomus papatasi and Lutzomyia longipalpis sand flies (Diptera: Psychodidae: Phlebotominae) using the CDC bottle bioassay to assess insecticide resistance. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 1\u0026ndash;11. https://doi.org/10.1186/s13071-016-1496-3\u003c/li\u003e\n\u003cli\u003eFanello, C., Santolamazza, F., \u0026amp; Della Torre, A. (2002). Simultaneous identification of species and molecular forms of the \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex by PCR‐RFLP. \u003cem\u003eMedical and veterinary entomology\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(4), 461-464\u003c/li\u003e\n\u003cli\u003eStica, C., Jeffries, C. L., Irish, S. R., Barry, Y., Camara, D., Yansane, I., Kristan, M., Walker, T., \u0026amp; Messenger, L. A. (2019). Characterizing the molecular and metabolic mechanisms of insecticide resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e in Faranah, Guinea. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(1), 1\u0026ndash;15. https://doi.org/10.1186/s12936-019-2875-y\u003c/li\u003e\n\u003cli\u003eMartin, E. R., Lai, E. H., Gilbert, J. R., Rogala, A. R., Afshari, A. J., Riley, J., Finch, K. L., Stevens, J. F., Livak, K. J., Slotterbeck, B. D., Slifer, S. H., Warren, L. L., Conneally, P. M., Schmechel, D. E., Purvis, I., Pericak-Vance, M. A., Roses, A. D., \u0026amp; Vance, J. M. (2000). SNPing away at complex diseases: Analysis of single-nucleotide polymorphisms around APOE in alzheimer disease. \u003cem\u003eAmerican Journal of Human Genetics\u003c/em\u003e, \u003cem\u003e67\u003c/em\u003e(2), 383\u0026ndash;394. https://doi.org/10.1086/303003\u003c/li\u003e\n\u003cli\u003eZhu, F., Lavine, L., O\u0026rsquo;Neal, S., Lavine, M., Foss, C., \u0026amp; Walsh, D. (2016). Insecticide resistance and management strategies in urban ecosystems. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(1), 1\u0026ndash;26. https://doi.org/10.3390/insects7010002\u003c/li\u003e\n\u003cli\u003eKorti, M. Y., Ageep, T. B., Adam, A. I., Shitta, K. B., Hassan, A. A., Algadam, A. A., Baleela, R. M., Saad, H. A., \u0026amp; Abuelmaali, S. A. (2021). Status of insecticide susceptibility in \u003cem\u003eAnopheles arabiensis \u003c/em\u003eand detection of the knockdown resistance mutation (\u003cem\u003ekdr\u003c/em\u003e) concerning agricultural practices from Northern Sudan state, Sudan. \u003cem\u003eJournal of Genetic Engineering and Biotechnology\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(1), 1\u0026ndash;9. https://doi.org/10.1186/s43141-021-00142-1\u003c/li\u003e\n\u003cli\u003eSoma, D. D., Zogo, B., Hien, D. F. de S., Hien, A. S., Kabor\u0026eacute;, D. A., Kientega, M., Ou\u0026eacute;draogo, A. G., Pennetier, C., Koffi, A. A., Moiroux, N., \u0026amp; Dabir\u0026eacute;, R. K. (2021). Insecticide resistance status of malaria vectors \u003cem\u003eAnopheles gambiae\u003c/em\u003e (s.l.) of southwest Burkina Faso and residual efficacy of indoor residual spraying with microencapsulated pirimiphos-methyl insecticide. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(1), 1\u0026ndash;9. https://doi.org/10.1186/s13071-020-04563-8\u003c/li\u003e\n\u003cli\u003eBaffour-Awuah, S., Annan, A. A., Maiga-Ascofare, O., Dieudonn\u0026eacute;, S. D., Adjei-Kusi, P., Owusu-Dabo, E., \u0026amp; Obiri-Danso, K. (2016). Insecticide resistance in malaria vectors in Kumasi, Ghana. \u003cem\u003eParasites and Vectors\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 1\u0026ndash;8. https://doi.org/10.1186/s13071-016-1923-5\u003c/li\u003e\n\u003cli\u003eKitungulu, N., Guyah, B., Webale, M., Shaviya, N., Machani, M., Mulama, D., \u0026amp; Ndenga, B. (2022). Resistance of \u003cem\u003eAnopheles gambiae sensu lato\u003c/em\u003e to Pirimiphos-methyl Insecticide in Kakamega County, Highlands of Western Kenya. \u003cem\u003eAfrican Health Sciences\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(1), 589\u0026ndash;597. https://doi.org/10.4314/ahs.v22i1.68\u003c/li\u003e\n\u003cli\u003eKendie, F. A., Wale, M., Nibret, E., \u0026amp; Ameha, Z. (2023). Insecticide susceptibility status of \u003cem\u003eAnopheles gambiae (s.l.\u003c/em\u003e) in and surrounding areas of Lake Tana, northwest Ethiopia. \u003cem\u003eTropical Medicine and Health\u003c/em\u003e, \u003cem\u003e51\u003c/em\u003e(1). https://doi.org/10.1186/s41182-023-00497-w\u003c/li\u003e\n\u003cli\u003eYared, S., Gebressielasie, A., Damodaran, L., Bonnell, V., Lopez, K., Janies, D., \u0026amp; Carter, T. E. (2020). Insecticide resistance in \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Somali Region, eastern Ethiopia. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(1), 1\u0026ndash;7. https://doi.org/10.1186/s12936-020-03252-2\u003c/li\u003e\n\u003cli\u003eTchouakui, M., Assatse, T., Tazokong, H. R., Oruni, A., Menze, B. D., Nguiffo-Nguete, D., Mugenzi, L. M. J., Kayondo, J., Watsenga, F., Mzilahowa, T., Osae, M., \u0026amp; Wondji, C. S. (2023). Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in \u003cem\u003eAnopheles funestus\u003c/em\u003e across Africa. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 1\u0026ndash;10. https://doi.org/10.1038/s41598-023-29605-w\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Broflanilide, insecticide resistance, Anopheles stephensi, Ethiopia","lastPublishedDoi":"10.21203/rs.3.rs-7704930/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7704930/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMalaria control in Ethiopia relies mainly on case management with ACT and vector control interventions, notably long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). \u003cem\u003eAnopheles arabiensis\u003c/em\u003e is the predominant vector, while the invasive \u003cem\u003eAn. stephensi\u003c/em\u003e is rapidly spreading across multiple regions, intensifying challenges for malaria elimination. Insecticide resistance further complicates control efforts.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis study assessed the susceptibility of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. and \u003cem\u003eAn. stephensi\u003c/em\u003e to commonly used and new public health insecticides at two sites: Awash Sabhat Kilo, where \u003cem\u003eAn. stephensi\u003c/em\u003e is established, and an area dominated by \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. WHO tube bioassays tested pirimiphos-methyl (0.25%), propoxur (0.1%), bendiocarb (0.1%), deltamethrin (0.25%), alpha-cypermethrin (0.05%), and clothianidin (2%). CDC bottle bioassays examined susceptibility to pirimiphos-methyl, deltamethrin, chlorfenapyr, clothianidin, and broflanilide. Molecular identification and allele-specific PCR targeted resistant alleles, particularly mutations in the voltage-gated sodium channel (VGSC) gene. Data were analyzed with SPSS v20.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eResults showed \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. populations were susceptible to propoxur and pirimiphos-methyl but resistant to bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. \u003cem\u003eAn. stephensi\u003c/em\u003e displayed broader resistance, including to pirimiphos-methyl, propoxur, bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. Both species remained fully susceptible to chlorfenapyr, clothianidin (at higher doses), and broflanilide across tested concentrations. Resistance to deltamethrin and pirimiphos-methyl was dose-dependent. Molecular identification confirmed \u003cem\u003eAn. arabiensis\u003c/em\u003e as the major species within the \u003cem\u003eAn. gambiae\u003c/em\u003e complex. Knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations were detected in \u003cem\u003eAn. arabiensis\u003c/em\u003e with a genotypic frequency of 22.8%.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese findings highlight the serious threat insecticide resistance poses to Ethiopia\u0026rsquo;s malaria control efforts. The persistence of resistance in both \u003cem\u003eAn. arabiensis\u003c/em\u003e and \u003cem\u003eAn. stephensi\u003c/em\u003e raises concerns about the long-term sustainability of insecticide-based strategies and underscores the need for alternative or integrated approaches.\u003c/p\u003e","manuscriptTitle":"Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 02:11:53","doi":"10.21203/rs.3.rs-7704930/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27a0a17e-f708-45ae-8a9c-3537b5992123","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T11:24:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 02:11:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7704930","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7704930","identity":"rs-7704930","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}