Introduction

Malaria is a vector-borne disease caused by five protozoan parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and most recently implicated Plasmodium knowlesi) of the genus Plasmodium and transmitted by Anopheles mosquitoes biting1,2. There are over 460 and 140 described species of the genus Anopheles mosquitoes worldwide and in Africa, respectively. Only an estimated 30 to 40 species are regularly associated with plasmodium transmission to humans worldwide, and of those 140, eight species are known to be efficient vectors of malaria parasites3,4.

Anopheles gambiae complex includes at least eight different vector species, five of which are significant malaria vectors: Anopheles gambiae(s.l), Anopheles coluzzii, Anopheles arabiensis, Anopheles melus, and Anopheles merus 5. Anopheles funestus Giles complex consist of nine species: Anopheles parensis Gillies, Anopheles aruni Sobti, Anopheles confusus Evans and Leeson, Anopheles funestus, Anopheles vaneedeni Gillies and Coetzee, Anopheles rivulorum Leeson, Anopheles fuscivenosus Leeson, Anopheles leesoni Evans, and Anopheles Brucei, two of which, such as Anopheles funestus s.s and Anopheles parensis Gillies, mostly play a critical role in malaria parasite transmission6,7.

Anopheles nili group includes four closely related species: Anopheles nili sensu stricto, Anopheles somalicus, Anopheles carnevalei, and Anopheles ovengensis, of which Anopheles nili sensu stricto is highly anthropophilic and the group’s major malaria vector, and Anopheles stephensi was recently identified as a new malaria vector and widely distributed in Africa8,9. The primary malaria vector in Ethiopia is Anopheles gambae s.l., with secondary vectors including Anopheles pharoensis, Anopheles funestus, Anopheles nili, and the recently discovered Anopheles stephensi10,11.

The public health burden posed by Plasmodium vivax is not considered benign, as it causes severe morbidity and death. Nonetheless, Plasmodium falciparum remains the most serious threat to public health on a global scale, accounting for more than 90% of all malaria deaths in 2018. Children under the age of five are the most vulnerable group, accounting for 67% (272,000) of all malaria deaths worldwide1,12. For this reason WHO has established challenging global technical strategy targets for 2030, including the elimination of malaria in many countries and a reduction in incidence and mortality rates of at least 90%13,14. To achieve these goals, malaria parasites are targeted using anti-malarial drugs and vector control measures through the use of insecticides15.

There are two main methods of malaria vector control: indoor residual spraying (IRS) and insecticide-treated nets (ITNs). Permethrin, etofenprox, bifenthrin, deltamethrin, lambda-cyhalothrin, and alphacypermethrin, DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos-methyl, fenitrothion (organophosphates), and pyriproxyfen are currently available insecticide products for malaria vector control, confined to four chemical classes. Pyrethroid insecticides are the only insecticides approved by the WHO to be used in treated bed nets because of their relatively low human toxicity, excite-repellent properties, rapid rate of knock-down, and killing effects16,17,18. Mosquitoes having resistance to pyrethroid insecticides can be controlled more effectively by using synergist PBO19.

In Ethiopia, IRS was first implemented in 1959 and continues to play an important role in malaria control. Despite the significant role played by insecticides in malaria control in Ethiopia, the disease remains endemic, with populations in some areas remaining at high risk of infection13,20. The emergence of insecticide resistance is one of the challenges to the main malarial controlling approaches21. However, the susceptibility status of malaria vectors to insecticides is largely unknown. Therefore, it is crucial to assess the insecticide susceptibility status of malaria vectors. Hence, the aim of this study was to determine the effectiveness of vector control tools in order to choose the most effective insecticides for vector control in the context of battling malaria.

Methods

Study area

The study was carried out in the Gondar zuria district, Northwest Ethiopia, from March 1, 2022, to August 31, 2022. This district is 45 km away from Gondar, town, and 685 km away from Addis Ababa. It covers an area of 1108.53 km2 and has a population density of 188.4 people per km. Maksegnit town, located at 12 23′ 00’' latitudes and 37 33′ 00’' longitudes, is the capital of the Gondar zuria district and it receives 1047.6 mm of rain annually, with a mean maximum temperature of 27.4 °C, a mean minimum temperature of 14.7 °C, and a relative humidity of 45%22,23. It is malarious with significant malaria transmission for both P. falciparum and P. vivax malaria; Streams and irrigation water serve as permanent Anopheline breeding sites during dry seasons, and it has an altitude ranging between 1750 and 2600 m above sea level. The major malaria transmission season is from September through November, and the minor one is from April to May.

Study design, period, and population

A cross sectional study design was conducted from March 1, 2022, to August 31, 2022 at Gondar zuria district, Northwest Ethiopia. The study populations were adult female Anopheles mosquitoes that fulfilled the inclusion criteria.

Sample size determination

According to WHO guidelines, 120 adult female Anopheles mosquitoes of a given species were required to conduct a single set of WHO insecticide susceptibility test24. Of these, 80 were exposed to the insecticide that was being tested (in four replicates each of around 20 mosquitoes). The remaining 40 mosquitoes serve as “controls” (i.e., two replicates of each of around 20 mosquitoes). A total of 100 adult female Anopheles mosquitoes were required to run a test of the synergist assay.

$${\text{N}} = { 12}0/{\text{test}},{\text{ n}} = {6}00/{5}\;{\text{test}}$$
$${\text{N}} = {1}00,{\text{ n}} = {3}00/{3}\;{\text{test}}$$

The collection of larvae and processing

Larval sampling and rearing procedure

Anopheles mosquitoes larvae were collected using the standard dipping method (dipper) from the breeding site including road puddles, brick pits, marshes, and ditches. The larvae were placed in a plastic tin for transportation to the Maksegnit malaria research center. In the laboratory, the larvae were poured into the larvae tray and debris was removed from the water. Then larvae were placed in the larval trays with their natural water and larvae food.

The pupae were sorted and transferred with pipettes from the enamel trays to beakers with small amounts of water. Each beaker was placed inside a cage with a 10% sugar solution for rearing them in the cage. After two to three days, the pupae emerged to adults, and cages were put in a safe place and protected from contamination25,26. Temperature and relative humidity in testing rooms were within the range of 25 ± 2 °C and 80 ± 10%, respectively.

Insecticide susceptibility test for adult female Anopheles mosquitoes

WHO insecticide susceptibility tests were carried out following the standard World Health Organization27 protocol, using insecticide susceptibility test kits and insecticide-impregnated papers. 120 Anopheles mosquitoes were transferred from cages to six holding tubes with untreated papers for an hour. Batches of 20 mosquitoes in four replicates were exposed to insecticide-impregnated papers with discriminating concentrations of the most commonly used insecticides (permethrin (0.75%), deltamethrin (0.05%), alphacypermethrin (0.05%), pirimiphos-methyl (0.25%), and propoxur (0.1%) for 1 h in WHO test tubes.

The knockdown effects for all tested insecticides were recorded at 10, 15, 20, 30, 40, 50, and 60 min. A control in two replicates (40 female Anopheles mosquitoes were used for each insecticide), each with an equal number of mosquitoes, were exposed to papers impregnated with oil and run concurrently. After that, mosquitoes were placed in holding tubes made of untreated paper, where given 10% sucrose solution by using cotton wool. Finally, 24-h post-exposure mortality was noted, and each Anopheles mosquitoes were identified morphologically using a taxonomic key24. All WHO susceptibility tests were carried out in a room with 25 ± 2 °C temperatures and 80 ± 10% humidity.

$${\text{Observed mortality}} = \frac{{{\text{total }}\;{\text{number}}\;{\text{ of }}\;{\text{died }}\;{\text{mosquitoes}}}}{{{\text{Total}}\;{\text{ tested}}}}*{1}00\%$$

Vector species composition

Anopheles mosquitoes were divided into three groups using a morphological key (Gillies and M. Coetzee,1987) after each test was done: the Anopheles gambiae (s,l,) the Anopheles funestus group, and the Anopheles pharoensis group28.

Synergist-insecticide bioassays

Synergist experiments were carried out using piperonylbutoxide (PBO), an inhibitor of oxidases including P450s, to determine the role of metabolic enzymes in the resistance profile, particularly cytochrome P450s. For these tests, batches of 25 non-exposed three-day-old adult female Anopheles mosquitoes were transferred to four (PBO-only, PBO + pyrethroids, insecticide only, and control labeled) holding tubes with untreated papers for an hour. After an hour, each of the batches in PBO-only and PBO + pyrethroids were transferred to two exposure tubes with 4%PBO-impregnated papers for one hour, and each of the batches of insecticide only, and control tubes were left in their holding tubes. Then, after 50 Anopheles mosquitoes from insecticide only and PBO + pyrethroids were transferred to the two insecticide-treated exposure tubes and exposed for 60 min again. Simultaneously the solvent control test mosquitoes were transferred to the oil-impregnated paper control tube and exposed for an hour. PBO only and control tubes were used as control.

The knockdown numbers for tested insecticides were recorded at 10, 15, 20, 30, 40, 50, and 60 min. Anopheles mosquitoes from insecticide only, control and PBO + pyrethroids tubes were transferred to holding tubes with cotton wool in sugar water after an hour exposure and maintained for 24 h. Mortality was recorded after 24 h and compared to those of insecticides with and without synergist exposure to evaluate the level of susceptibility restoration and the implication of P450s in resistance to the tested insecticide.

Standard definition

The results of the susceptibility tests were evaluated as recommended by WHO criteria: 98–100% mortality indicates susceptibility, 90–97% mortality indicates resistance candidate (more investigation is needed) and less than 90% mortality suggests resistance24.

Data quality assurance

The quality of data was assured by following WHO protocol: insecticide-impregnated papers were used only one times. Sterilized cages and 10% sucrose solution soaked in cotton pads were used. Three-day old adult female Anopheles mosquitoes were used. Temperature and relative humidity were within the range of 25 ± 2 °C and 80 ± 10%, respectively. Finally; each and everything was cross-checked and done carefully.

Data analysis

Data was entered and analyzed using SPSS version 26. The results of the susceptibility tests were evaluated as recommended by WHO criteria24 as follows: 98–100% mortality indicates susceptibility, 90–97% mortality indicates a resistance candidate (more investigation is needed), and less than 90% mortality suggests resistance.

Logit probit analysis was used to determine the KDT 50% and KDT 90% knockdown time of the insecticides (the time taken to knock down 50%, 90%, and 95% of mosquitoes, respectively). One-way ANOVA was used to compare the mean knock down rate of the Anopheles mosquitoes by insecticides to evaluate their effectiveness. An independent sample t-test was used to compare the mean knock down rate of the Anopheles mosquitoes by pyrethroid insecticides with and without PBO.

The Synergist assay was conducted by using PBO following WHO criteria29. Complete restoration of susceptibility following pre-exposure to PBO (i.e., ≥ 98% mean mortality) implies that a monooxygenase-based resistance mechanism fully accounts for the expression of the resistant phenotype in the Anopheles mosquitoes. Partial restoration of susceptibility following preexposure to PBO (i.e., mean mortality in the PBO followed by insecticide samples is greater than mean mortality in the insecticide-only samples but < 98%) implies that a monooxygenase-based resistance mechanism only partially accounts for expression of the resistant phenotype and that other resistance mechanism are likely to be present in the test population. No restoration of susceptibility following pre-exposure to PBO (mean mortality in the PBO followed by insecticide samples is equal to or lower than mean mortality in the insecticide-only samples) implies that the resistance phenotype detected is not based on mono-oxygenase-mediated detoxification.

Results

Anopheles species composition

A total of 900 Anopheles mosquitoes were collected, and three different species were identified: Anopheles gambiae s.l., Anopheles funestus s.l., and Anopheles pharoensis. Anopheles gambiae s.l. was the most abundant malaria vector species with 97.5%, followed by Anopheles funestus and Anopheles phronesis with 1.5% and 0.7%, respectively.

WHO insecticide susceptibility tests

Knockdown time

Permethrin and deltamethrin used 40 min, while propoxur was used 37 min to induce 50%knockdown of Anopheles mosquito, respectively. Pirimiphos-methyl and alphacypermethrin did not show 50% knockdown of Anopheles mosquito after an hour. 50% KDT for permethrin and deltamethrin was high in comparison to propoxur (Table 1). But, at each time interval, the knockdown number of Anopheles mosquitoes increased (Fig. 1).

Table 1 Time to knockdown (in minutes) for 50% of Anopheles mosquitoes in the Gondar Zuria district after one hour of insecticide exposure.
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Fig. 1
figure 1

The knockdown number of Anopheles mosquitoes by each insecticide being tested at each time interval.

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The mean knockdown rate of the Anopheles mosquitoes by insecticides

Mean knock down rate were 22.7% (95% CI 11.97–33.5) for alphacypermethrin, 27.8% (95% CI 8.7–47) for permethrin, 27% (95% CI 7–47) for deltamethrin, 3.3% (95% CI 0.55–7) for pirimiphos-methyl, and 29.7% (95% CI 3.2–56.2) for propoxur. The mean knock down rate of the Anopheles mosquitoes by permethrin and deltamethrin was the same while the mean knock down rate of the Anopheles mosquitoes by propoxur was high, but the mean knock down rate of the Anopheles mosquitoes by pirimiphos-methyl was small after an hour (Fig. 2).

Fig. 2
figure 2

The mean knockdown rate of the Anopheles mosquitoes by each insecticide after an hour.

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However, there was no significant difference between average of the mean knockdown rates of the Anopheles mosquitoes by insecticides that being tested after 1 h (P > 0.092) (Table 2).

Table 2 The mean knockdown rate of the Anopheles mosquitoes after 1 h.
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Phenotypic susceptibility levels of malaria vectors

According to WHO recommendations, phenotypic susceptibility tests on 600 adult female Anopheles mosquitoes were performed to evaluate the resistance profile against five insecticides, namely alphacypermethrin, permethrin, deltamethrin, pirimiphos-methyl, and propoxur. Malaria vectors were resistant to alpha-cypermethrin, permethrin, and deltamethrin, with mortality rates of 67.5%, 88.8%, and 73.75%, respectively. Complete susceptibility (100% death) of the Anopheles mosquitoes was observed with pirimiphos-methyl and propoxur (Table 3). The resistance intensity of alpha-cypermethrin was high when compared with permethrin and deltamethrin (Table 4).

Table 3 Phenotypic susceptibility levels of Anopheles mosquitoes to different classes of insecticides.
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Table 4 Time to knockdown (in minutes) for 50% and 90% of Anopheles mosquitoes from Gondar zuria district following exposure to insecticides only and PBO plus pyrethroids.
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The impact of piperonyl-butoxide in pyrethroid-resistant malaria vectors

The duration required to cause 50% knockdowns of the Anopheles mosquitoes using PBO + deltamethrin, PBO + permethrin, and PBO + alpha-cypermethrin was 22 min (95% CI 18.5–26.2), 29 min (95% CI 23–37), and 17 min (95% CI 13.2–21.4), respectively. When compared to the 50% knockdown time of the Anopheles mosquitoes by pyrethroid insecticides alone, the time required to induce 50% knockdown of the Anopheles mosquitoes by PBO + deltamethrin, PBO + permethrin, and PBO + alphacypermethrin decreased (Table-4). Additionally, PBO + deltamethrin and PBO + alpha-cypermethrin induced 90% knockdown of the Anopheles mosquito in 59 min (95% CI 46.9–86.2).

The mean knockdown rate of the Anopheles mosquitoes was significantly improved by PBO plus alpha-cypermethrin (P < 0.005) when compared to alpha-cypermethrin alone (Table 5). At each time interval, the knockdown number of the Anopheles mosquitoes by PBO + deltamethrin, PBO + permethrin, and PBO + alphacypermethrin was greater than deltamethrin, permethrin, and alphacypermethrin alone (Fig. 3).

Table 5 Mean differences in the knockdown rates of the Anopheles mosquitoes by PBO plus pyrethroid insecticides and pyrethroids only.
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Fig. 3
figure 3

The knockdown number of malaria vectors by insecticides only and PBO + pyrethroids insecticides at each time interval.

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Phenotypic susceptibility levels of malaria vectors to pyrethroids with PBO

Malaria vectors were resistant to alpha-cypermethrin and deltamethrin alone, but the efficacy of alpha-cypermethrin and deltamethrin was fully restored by pre-exposure to PBO from 68 to 100% and 72 to 100%, respectively. Anopheles mosquitoes were resistant to permethrin alone but partially restored by pre-exposure to PBO from 88 to 96% according to WHO guidelines (Fig. 4). Because the mortality rate in the controls and PBO-only was consistently lower than 5%, the mortality rate found in the experimental tests was not adjusted (Table 6).

Fig. 4
figure 4

Mortality rates of the malaria vectors by pyrethroids-only and PBO + pyrethroids.

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Table 6 The PBO restoration of pyrethroids’ effectiveness in pyrethroid-resistant malaria vectors.
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Discussion

For many years, controlling and eradicating malaria has been hampered by insecticide resistance30,31. As a result of Anopheles mosquitoes becoming resistant to the majority of insecticides now used in public health32. For this reason, the World Health Organization27 published a Global Plan for insecticide resistance management in malaria vectors that outlines best-practice strategies (rotations of insecticides, use of interventions in combination, mosaic spraying, and use of mixtures) for preserving or prolonging the effectiveness of the insecticides used for malaria control, based on the best available evidence at the time30,33. However, geographically, there is a poor understanding of insecticide resistance status due to limited evidence or data to decide on appropriate insecticides34,35,36. Hence, this study has been demonstrated for the first time in Gondar zuria woreda regarding the resistance status of malaria vectors to insecticides used for malaria vector control, using standardized WHO protocols to reduce this gap24.

The results of the investigation shown that the Anopheles mosquitoes that transmit malaria parasites in the Gondar zuria woreda were identified along with their composition and density. The Anopheles gambae(s.l) complex was the most abundant malaria vector, followed by the Anopheles funestus group and the Anopheles pharoensis group. This outcome is in line with what Tilahun Adugna and his colleagues reported37. Also, this finding is similar with studies which reported Anopheles gambiae s. l. and Anopheles Pharoensis, which have been identified as primary and secondary vectors in south-western Ethiopia by different researchers, respectively38,39,40,41,42,43,44.

This indicates the presence of a probability to get different anopheles mosquito species from different malaria endemic areas that can be responsible for the transmission of malaria parasites if entomological studies are conducted in different endemic areas.

According to the findings of the investigation, the Gondar zuria woreda malaria vectors were susceptible to carbamate (propoxur) and organophosphate (pirimiphos-methyl) insecticides. However, they were resistant to all of the pyrethroid insecticides tested. Previous findings from Burkina Faso, Uganda, Mali, Rwanda, Kenya, and Tanzania agree with this one from 2005 to 201716,45,46,47,48,49,50,51.

According to Chanyalew T, Balkew M, Yared, and their colleagues, permethrin, alphacypermethrin, and deltamethrin all demonstrate comparable degrees of resistance of this study9,52,53. Additionally, reports from south-central Ethiopia and south-western Ethiopia demonstrated comparable pyrethroid resistance states of this study54,55. It also support by studies whch reports the resistance of deltamethrin and permethrin in Asian countries such as China’s Shandong province, Thailand, and Myanmar56,57. Suspected resistance to deltamethrin is reported from Madagascar and Badakhshan58,59. However, it needs confirmission according to WHO criteria24. But, Susceptible to pyrethroid insecticide is reported by Rana SM and his friends from pakistan60. Marcomba Sand and his colleagues’ report is inline with the report of Rana SM61. Similar findings are reported by Dhiman S and his colleagues62. This may be a product of environmental influences, in contrast to this study findings38.This all indicates the presence of resistance in malaria vectors to pyrethroid insecticides in different malaria-endemic areas.

Additionally, after 60 min, alpha-cypermethrin failed to knock down 50% of the malaria vectors. However, permethrin and deltamethrin KD 50% of the malaria vectors. According to Kinfe and his associate’s reports, permethrin and deltamethrin demonstrate comparable result63. This early detection indicates the low efficacy of alpha-cypermethrin and its higher resistance than permethrin and deltamethrin in malaria vectors. However, there was no significant difference between insecticides when comparing the mean knockdown rates of Anopheles mosquitoes (P > 0.092). This shows the absence of a significant difference regarding the efficacy of pyrethroids against Anopheles mosquitoes.

In this investigation, pirimiphos methyl, an insecticide from the class of organophosphates, was used to assess phenotypic levels of susceptibility. It has been completely (100%) toxic to malaria vectors according to WHO guidelines24. Even while primiphos methyl failed to knock down 50% of malaria vectors after 60 min, it miraculously caused full susceptibility after 24 h. This indicates a pro-insecticide called pirimiphos methyl needs mosquito cytochrome P450 enzymes to be activated in order to cause toxicity64. Hence, it takes a long time to be toxic to malaria vectors, so it should be used considering this. The outcome is in line with reports by Rakotoson JD and Soma DD with their colleagues50,58. Reports from Benin, Zambia, Madagascar, and Ghana are all in agreement with this study result58,65,66,67,68. The susceptibility of malaria vectors to primiphos methyl has also been documented from Gambella and other parts of the country52,69.

Propoxur from the carbamate class was used to evaluate the susceptibility status of malaria vectors. It caused a 50% knockdown of malaria vectors within 37 min. But it couldn’t cause 90% knockdown at 60 min. This report is in agreement with reports from different regions in Ethiopia70. According to WHO standards24, malaria vectors have been verified to be completely susceptible (100%) to propoxur. A report from Nigeria is in line with this71. Similar to this, comparable findings from Ethiopian regions have been reported38,52,63,69. By Alemayehu and his colleagues, the susceptibility of malaria vectors is reported in line with this72. These different comparable results may indicate the effectiveness of propoxur.

The synergistic effect of piperonyl-butoxide (PBO) with pyrethroid insecticides (alpha-cypermethrin, permethrin, and deltamethrin) was observed by exposing Anopheles mosquitoes to synergists before insecticides to check the presence of enzymes that can be controlled by PBO. Permethrin and deltamethrin alone had a knockdown time of 34 and 47 min to knock down 50% of malaria vectors, respectively, but when combined with PBO, the time was lowered to 29 and 22 min to knock down 50% of malaria vectors at 60 min, respectively. After 60 min, among pyrethroid class insecticides that have been assessed, only alpha-cypermethrin was unable to knock down 50% of malaria vectors. However, when they were exposed to PBO prior to insecticides, 50% and 90% knockdown of malaria vectors were observed.

Deltamethrin also could not knock down 90% of malaria vectors after 60 min. But it induced knock down 90% after exposure of Anopheles mosquitoes to PBO, followed by deltamethrin. This report is in line with outcomes from various locations in Africa73,74,75,76,77. The knockdown and mortality rates of the Anopheles mosquitoes have increased by PBO pre-exposure followed by pyrethroid insecticide. It is comparable with other reports that have been mentioned above. The reduction of knockdown time after malaria vectors are exposed to PBO indicates the presence of metabolizing enzymes in the malaria vectors that can metabolize the insecticides.

After 24 h, the mortality rates of malaria vectors by PBO + pyrethroid insecticide and insecticide alone were calculated and compared. Complete restoration to deltamethrin and alpha-cypermethrin-resistant malaria vectors, as well as partial restoration to permethrin-resistant malaria vectors, have been achieved in accordance with WHO guidelines24.

Mortality rates of malaria vectors by deltamethrin, alpha-cypermethrin, and permethrin alone were 72%, 68%, and 88%, respectively, but after being pre-exposed to PBO, mortality rates of malaria vectors were 100%, 100%, and 96%,respectively. This is in line with outcomes from various researches conducted in Africa73,74,75,76,77. This suggests that detoxifying enzymes had a part in the development of resistance. Complete restoration of susceptibility suggests that the resistant phenotype in the malaria vectors is entirely explained by a monooxygenase-based resistance mechanism, whereas partial restoration of susceptibility indicates that additional resistance mechanisms are probably present in the malaria vectors and that a monooxygenase-based resistance mechanism only partially accounts for the manifestation of the resistant phenotype.

An independent sample t-test reveals the absence of a significant difference between the mean knockdown rates of the malaria vectors by permethrin and PBO + permethrin, as well as between deltamethrin and PBO + deltamethrin. However, there was a significant difference between alphacypermethrin and PBO + alphacypermethrin. This implies that the detoxifying enzymes produced by malaria vectors may metabolize alphacypermethrin rather than permethrin and deltamethrin.

The results of this study are not unexpected because several investigations conducted in various locations of Ethiopia have consistently shown the existence of resistance to the pyrethroid insecticides as well as the malaria-causing vectors in this study area is consistent with those identified in different locations of Ethiopia. In general, these data are critical for selecting the most effective insecticides for vector control in order to reduce malaria.

Conclusion

This study demonstrated that malaria vectors are susceptible to pirimiphos-methyl, propoxur, and PBO + pyrethroid insecticides, but resistant to pyrethroids insecticide. Anopheles gambae s.l. Anopheles funestus, and Anopheles pharoensis are malaria vectors in Gondar zuria woreda with Anopheles gambae complex predominating. Thus, the incidence rate of malaria and malaria vectors can be controlled in this area using alphacypermethrin, deltamethrin, and permethrin-impregnated mosquito nets integrated with PBO and/or through indoor residual spraying of sprayable human dwellings with pirimiphos-methyl and propoxur. Based on the findings of this study, we recommend that the use of pyrethroids integrated with bed nets should not provide important protection in the future; rather, it is better to use pyrethroids-impregnated mosquito nets with PBO and/or the use of propoxur and pirimiphos-methyl should continue for effective control of malaria by indoor spraying technique in this area. Since Anopheles mosquitoes could develop various resistant mechanisms with various pathways, more research should be done on molecular confirmation of the presence of the knockdown resistance alleles in this area.