Assessing Insecticide Susceptibility Status of Anopheles Mosquitoes in Gondar Zuria District, Northwest Ethiopia

doi:10.21203/rs.3.rs-3630218/v1

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Assessing Insecticide Susceptibility Status of Anopheles Mosquitoes in Gondar Zuria District, Northwest Ethiopia

https://doi.org/10.21203/rs.3.rs-3630218/v1

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Background

Insecticide-based vector control, which comprises the use of insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS), is the key method of malaria control in Ethiopia. However, it’s effectiveness is threatened as malaria vectors become resistant to insecticides. Assessing the susceptibility status of these Anopheles mosquitoes at different time is important to complement vector control interventions. However, there is no reports on the insecticide susceptibility status of malaria vectors from Gondar zuria woreda, Northwest Ethiopia. Thus, this study investigated the susceptibility status of malaria vectors in Gondar zuria woreda, Northwest Ethiopia against various insecticides recommended for use in ITNs and/or IRS to provide evidence to suggest alternates for malaria vector control tools.

Methods

An entomological study was carried out from March 1, 2022, to August 31, 2022. 900 three-day-old adult female Anopheles mosquitoes from larva collected in the field from Gondar zuria district using dipper were used for bioassay and synergist. They were tested for susceptibility to permethrin (0.75%), deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), and pirimiphos-methyl (0.25%) by using WHO susceptibility test papers. Baches of 20 and 25 adult female Anopheles mosquitoes were used for both bioassay and synergist assay, respectively. 50%KDT and 90%KDT were determined by logit-probit analysis after 1 hour. One-way ANOVA was used to compare the mean knockdown rates of the Anopheles mosquitoes by insecticides after 60 minutes. An independent sample t-test was used to compare the mean knockdown rates of the Anopheles mosquitoes by pyrethroid insecticide with and without PBO after 60 minutes. Mortality rates pos-24 hours were recorded.

Results

A total of 900 female Anopheles mosquitoes were examined, of which 878 (97.5%), 15 (1.5%), and 7 (0.7%) were found to be Anopheles Gambae(s.l), Anopheles funestus group, and Anopheles Pharoensis, respectively. They exhibited susceptibility to pirimiphos-methyl (100%) and propoxur (100%), but resistant to permethrin (88.8%), alphacypermethrin (67.5%), and deltamethrin (73.75). Although permethrin restoration was only 96% in permethrin resistant Anopheles mosquitoes, the efficacy of alphacypermethrin and deltamethrin was totally restored by pre-exposure to PBO.

Conclusion and recommendation

This study demonstrated that malaria vectors are susceptible to pirimiphos-methyl propoxur, and PBO + pyrethroid insecticides but resistant to pyrethroids insecticide-only. It also revealed that Anopheles gambae s.l., Anopheles funestus group, and Anopheles pharoensis group are malaria vectors in Gondar zuria woreda with Anopheles gambae complex predominating. Based on the findings of this study, it is better to use pyrethroids-impregnated mosquito nets with PBO and/or the use of propoxur and primiphos-methyl to control malaria. Further investigations are needed to better understand the molecular basis of pyrethroids insecticide resistant-malaria vectors.

Insecticides

PBO

Malaria vectors

Susceptibility status

Ethiopia

1.1. Background

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 biting (1, 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 parasites(3, 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 transmission(6, 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 Africa(8, 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 stephensi(10, 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 worldwide(1, 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 insecticides(15).

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 effects(16–18).

Mosquitoes having resistance to pyrethroid insecticides can be controlled more effectively by using synergist PBO(19). Piperonyl-butoxide (PBO), inhibits cytochrome P450 monooxygenase enzyme activity, targeting DDT and pyrethroid insecticides. Despite having none of its own pesticidal activity, it enhances the efficacy of insecticides such as pyrethroids and acts as an insecticide synergist by inhibiting the natural defenses of the insects(20).

In Sub-Saharan Africa, an estimated 1.04 billion nets were provided between 2009 and 2016, and 582 million of those were long-lasting insecticidal nets (LLINs). Between 2000 and 2015, these nets are believed to have prevented 663 million clinical cases of malaria in Sub-Saharan Africa, with ITNs accounting for 68% of those instances and indoor residual spraying (IRS) being responsible for 11%. While LLINs are the most commonly used vector control method, IRS protected an estimated 45 million people in Sub-Saharan Africa in 2016(21).

In Ethiopia, IRS was first implemented in 1959 and continues to play an important role in malaria control. LLIN coverage has been scaled up since 2005, with over 64 million nets distributed by 2014, resulting in malaria incidence rates dropped from 43.1 cases per 1000 population annually between 2001 and 2010 to 29 cases per 1000 population annually between 2011 and 2016, and malaria deaths dropped from 2.1 deaths per 100 000 people annually between 2001 and 2010 to 1.1 deaths per 100 000 people annually between 2011 to 2016(10, 11).

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 infection(13, 22). The emergence of insecticide resistance is one of the challenges to the main malarial controlling approaches (21). In most cases, resistance is attributed to two major mechanisms: (i) target-site insensitivity, where mutations in the target site of the insecticide alter binding; and (ii) metabolic-based resistance, where the insecticide is degraded, sequestered, or transported/excreted out of the cell before it can bind to its target. There's also behavioral resistance, which involves behavioral changes in vector behavior as a result of the insecticides' irritating properties, which help the vector evade the insecticides' lethal effects, reduced penetration, in which the exoskeleton is modified in ways that prevent insecticide penetration, and excretion resistance, in which increased excretion helps the vector evade the insecticides' lethal effects(23, 24).

Factors that contribute to the emergence of insecticide resistance include the widespread use of insecticides in agriculture and public health, as well as incorrect insecticide use, which has been linked to the development of insecticide resistance in important malaria vectors(10, 25). 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.

2.1. Description of the 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 latitudes and 37 33 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%(74, 75). 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.

2.2. Study design

An entomological study design was carried out in Gondar zuria district (Maksegnit), Northwest Ethiopia

2.3. Study period

The study was conducted from March 1, 2022, to August 31, 2022.

2.4. Population

2.4.1. Source population

All larval-stage mosquitoes.

2.4.2. Study Population

All adult female Anopheles mosquitoes that fulfilled the inclusion criteria were enrolled in the study.

2.5. Eligibility criteria

2.5.1. Inclusion criteria

Three-day-old laboratory-reared adult female Anopheles mosquitoes were included.

2.5.2. Exclusion Criteria

Culex

Male

Above three-day-old laboratory-reared adult female Anopheles mosquitoes were excluded

2.6. 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 test (76), 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.

N = 120/test, n = 600/5 test

N = 100, n = 300/3 test

2.7. The collection of larvae and processing

2.7.1. 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 contamination(77, 78). Temperature and relative humidity in testing rooms were within the range of 25 ± 2°C and 80 ± 10%, respectively.

2.7.2. Insecticide susceptibility test for adult female Anopheles mosquitoes

WHO insecticide susceptibility tests were carried out following the standard World Health Organization (WHO) 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 minutes. 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-hour post-exposure mortality was noted, and each Anopheles mosquitoes were identified morphologically using a taxonomic key(76). All WHO susceptibility tests were carried out in a room with 25 ± 2°C temperatures and 80 ± 10% humidity.

2.7.3. 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 group(79).

2.8. 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 minutes 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 minutes. 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 hours. Mortality was recorded after 24 hours 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.

2.9. Variables of the study

2.9.1. Dependent variables

Susceptibility status of Anopheles mosquitoes

2.9.2. Independent variables

Permethrin (0.75%)

Deltamethrin (0.05%)

Alpha-cypermethrin (0.05%)

Pirimiphos-methyl (0.25%)

Propoxur (0.1%)

Pyrethroids+(4%PBO)

2.10. 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 resistance(76).

2.11. Data analysis

Data was entered and analyzed using SPSS version 26. The results of the susceptibility tests were evaluated as recommended by WHO criteria(76) 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 criteria(80). 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.

2.12 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.

3.1. 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 (Fig. 1).

3.2. WHO insecticide susceptibility tests

3.2.1. Knockdown time

Permethrin and deltamethrin used 40 minutes, while propoxur was used 37 minutes 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. 2).

3.2.2. 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. 3).

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

Table 2

The mean knockdown rate of the Anopheles mosquitoes after 1 hour
Insecticides	Mean	95% Confidence interval for Mean	p-value
		Lower Bound	Upper Bound
Alphacypermethrin	22.71	11.97	33.46
Permethrin	27.86	8.71	47.01
Deltamethrin	27.43	7.23	47.63
Pirimiphos-methyl	3.29	− .55	7.13
Propoxur	29.71	3.22	56.21
Insecticides	Mean	95% Confidence interval for Mean	p-value

3.2.3. 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 3).

Table 3

Phenotypic susceptibility levels of Anopheles mosquitoes to different classes of insecticides
Insecticides	Total	Type
Pirimiphos-methyl	120	Exposure
Control	80
40	80
0	100%
0	S
Propoxur	120	Exposure
Control	80
R = Resistant, S = Susceptible

3.3. 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 minutes (95% CI = 18.5–26.2), 29 minutes (95% CI = 23–37), and 17 minutes (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 minutes (95% CI = 46.9–86.2).

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.
Total	Type	Number used	50%KDT in minutes	95%CI	90%KDT in minutes	95%CI
100	PBO-Only	25		-	-	-
	PBO + Deltamethrin	25	22	(18.5–26.2)	59	(46.9–86.2)
	Deltamethrin only	25	47	(36.8–76.7)	-	-
	Control	25		-		-
100	PBO-Only	25		-		-
	PBO + Permethrin	25	29	(23–37)	-	-
	Permethrin only	25	34	(28.5–40.2)	-	-

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. 4).

Table 5

Mean differences in the knockdown rates of the Anopheles mosquitoes by PBO plus pyrethroid insecticides and pyrethroids only.
Insecticides	Mean	Standard deviation
Deltamethrin	8.6	4.5
0.089
PBO + Deltamethrin	14.6	7.3
Permethrin	10.57	7
PBO + Permethrin	12	5.6
Alphacypermethrin	7.4	3.2
PBO + Alphacypermethrin	16.3	5.9

3.3.1. 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 68–100% and 72–100%, respectively. Anopheles mosquitoes were resistant to permethrin alone but partially restored by pre-exposure to PBO from 88–96% according to WHO guidelines (Fig. 5). 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).

Table 6

The PBO restoration of pyrethroids' effectiveness in pyrethroid-resistant malaria vectors
Deltamethrin	Total	Type	Number used
	100	PBO-Only	25

For many years, controlling and eradicating malaria has been hampered by insecticide resistance(23, 81). As a result of Anopheles mosquitoes becoming resistant to the majority of insecticides now used in public health(82). For this reason, the World Health Organization (WHO) 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 time(23, 83). However, geographically, there is a poor understanding of insecticide resistance status due to limited evidence or data to decide on appropriate insecticides(35, 84, 85). 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 gap(76).

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 dissecting microscope was used to identify and categorize Anopheles mosquito species to their complex levels based on taxonomic keys. For all tests, 900 Anopheles mosquitoes were required. The Anopheles gambae(s.l) complex was the most abundant malaria vector with 97.5%, followed by the Anopheles funestus group and the Anopheles pharoensis group with 1.5% and 0.7%, respectively (Fig. 1). This outcome is in line with what Tilahun Adugna and his colleagues reported(38). Anopheles gambiae s. l. and Anopheles Pharoensis have been identified as primary and secondary vectors in south-western Ethiopia by different researchers, respectively(39–45).

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 (deltamethrin, permethrin, and alpha-cypermethrin) with none of mortality rates higher than 90% after post-24-hour testing, according to WHO criteria: Mortality rates of 98–100% indicate susceptibility, 90–97% indicate a candidate for resistance (further investigation is needed), and less than 90% indicate resistance(76), as shown in Table-3. Previous findings from Burkina Faso, Uganda, Mali, Rwanda, Kenya, and Tanzania agree with this one from 2005 to 2017 (16, 34, 36, 37, 54–57).

According to Chanyalew T, Balkew M, Yared, and their colleagues, permethrin, alphacypermethrin, and deltamethrin all demonstrate comparable degrees of resistance(9, 59, 60). Additionally, reports from south-central Ethiopia and south-western Ethiopia demonstrated comparable pyrethroid resistance states(61, 62). Resistance to deltamethrin and permethrin has been reported in Asian countries such as China's Shandong province, Thailand, and Myanmar(47, 48).

Suspected resistance to deltamethrin is reported from Madagascar and Badakhshan(49, 50). However, it needs confirmission according to WHO criteria(76). But, Susceptible to pyrethroid insecticide is reported by Rana SM and his friends from pakistan(51). Marcomba Sand and his colleagues' report is inline with the report of Rana SM(52). Similar findings are reported by Dhiman S and his colleagues(53). This may be a product of environmental influences, in contrast to this study findings(43).This all indicates the presence of resistance in malaria vectors to pyrethroid insecticides in different malaria-endemic areas.

Additionally, after 60 minutes, alpha-cypermethrin failed to knock down 50% of the malaria vectors. Nevertheless, permethrin and deltamethrin KD 50% of the malaria vectors, as shown in (Table 1). According to Kinfe and his associate's reports, permethrin and deltamethrin demonstrate comparable result(58). This early detection indicates the low efficacy of alpha-cypermethrin and its higher resistance than permethrin and deltamethrin in malaria vectors, as shown in (Table 1). However, there was no significant difference between insecticides when comparing the mean knockdown rates of Anopheles mosquitoes (P > 0.092), which is shown in (Table-2). 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 guidelines(76). Even while primiphos methyl failed to knock down 50% of malaria vectors after 60 minutes, it miraculously caused full susceptibility after 24 hours, as shown in Table 3. This indicates a pro-insecticide called pirimiphos methyl needs mosquito cytochrome P450 enzymes to be activated in order to cause toxicity(86). 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 colleagues(34, 49). Reports from Benin, Zambia, Madagascar, and Ghana are all in agreement with this study result(49, 67–70). The susceptibility of malaria vectors to primiphos methyl has also been documented from Gambella and other parts of the country(59, 71).

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 minutes. But it couldn’t cause 90% knockdown at 60 minutes. This report is in agreement with reports from different regions in Ethiopia(46). According to WHO standards(76), malaria vectors have been verified to be completely susceptible (100%) to propoxur as shown in (Table-2). A report from Nigeria is in line with this(72). Similar to this, comparable findings from Ethiopian regions have been reported(43, 58, 59, 71). By Alemayehu and his colleagues, the susceptibility of malaria vectors is reported in line with this(73).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 minutes to knock down 50% of malaria vectors, respectively, but when combined with PBO, the time was lowered to 29 and 22 minutes to knock down 50% of malaria vectors at 60 minutes, respectively, as shown in (Table 4). After 60 minutes, 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, as shown in (Table 4).

Deltamethrin also could not knock down 90% of malaria vectors after 60 minutes. But it induced knock down 90% after exposure of Anopheles mosquitoes to PBO, followed by deltamethrin (Table 4). This report is in line with outcomes from various locations in Africa(20, 63–66). 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 hours, 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 guidelines(76).

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%, as shown in (Figure-6). This is in line with outcomes from various research conducted in Africa(20, 63–66). 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, which is shown in (Table 5). This suggests that rather than permethrin and deltamethrin, alphacypermethrin could be metabolized by the detoxifying enzymes that produced by malaria vectors.

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.

This study demonstrated the emergence of pyrethroids insecticide resistance in Anopheles mosquitoes in Gondar zuria districts. However, the study did have certain restrictions. The association between the emergence of the knockdown resistance mutations and the resistance could not be confirmed. Additionally, Anopheles mosquitoes at their species levels were unable to be identified.

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, I 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.

ANOVA: Analysis of Variance

DDT: Dichlorodiphenyltrichloroethane

FMH: Federal Minister of Health

IRS: Indoor Residual Spraying

ITNs: Insecticide Treated nets

LLINs: Long-Lasting Insecticidal nets

KD: Knock Down

PQ: Prequalification

PBO: Piperonylbutoxide

UoG: University of Gondar

WHO: World Health Organization

We declare that this thesis is original and has not been presented or published in this University or institution. And also, all methods were achieved based on appropriate guidelines and regulations.

Ethical Consideration

This study followed the ethical standards of the Declaration of Helsinki. And also, this study was conducted after ethical approval was obtained from research and ethics review committee of the school of biomedical and laboratory sciences.

Consent for publication

Not applicable

Patients or the public involvement

Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.

Availability of data and material

We confirmed that all the data for this manuscript are available, if someone wants to request the data can contact Wagaw Abebe.

Competing interests

The authors declare that they have no competing interests.

Funding

There is no funding in this research.

Author contribution

S. B was conceived, designs the study, interpretation of the data, and wrote original draft of the manuscript. A. J was participated in the design of the study and edited the draft of the manuscript. M. A was participated in the design of the study and edited the draft of the manuscript. D. W was participated in the data collection. W. A was performed data collection and statistical analysis of the data. Finally all the authors were read and approved the final manuscript.

Acknowledgment

We acknowledge our colleagues, Dr. Meshesha Balkew for supplying me necessary materials and his comments and suggestion as well as Mr. Muluken Assefa for his support in the morphological identification of the Anopheles mosquito.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Assessing Insecticide Susceptibility Status of Anopheles Mosquitoes in Gondar Zuria District, Northwest Ethiopia

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Abstract

Figures

1. Introduction

1.1. Background

2. Methodology

2.1. Description of the study area

2.2. Study design

2.3. Study period

2.4. Population

2.4.1. Source population

2.4.2. Study Population

2.5. Eligibility criteria

2.5.1. Inclusion criteria

2.5.2. Exclusion Criteria

2.6. Sample Size determination

2.7. The collection of larvae and processing

2.7.1. Larval sampling and rearing procedure

2.7.2. Insecticide susceptibility test for adult female Anopheles mosquitoes

2.7.3. Vector species composition

2.8. Synergist-insecticide bioassays

2.9. Variables of the study

2.9.1. Dependent variables

2.9.2. Independent variables

2.10. Standard definition

2.11. Data analysis

2.12 Data quality Assurance

3. Results

3.1. Anopheles species composition

3.2. WHO insecticide susceptibility tests

3.2.1. Knockdown time

3.2.2. The mean knockdown rate of the Anopheles mosquitoes by insecticides

3.2.3. Phenotypic susceptibility levels of malaria vectors

3.3. The impact of piperonyl-butoxide in pyrethroid-resistant malaria vectors

3.3.1. Phenotypic susceptibility levels of malaria vectors to pyrethroids with PBO

4. Discussion

5. Limitations

6. Conclusion

7. Recommendations

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

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