J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 196 http://jad.tums.ac.ir Published Online: June 30, 2021 Original Article Current Status of Insecticide Susceptibility in the Principal Malaria Vector, Anopheles gambiae in Three Northern States of Nigeria Mustapha Ahmed Yusuf 1,2; Mohammad Ali Oshaghi1; *Hassan Vatandoost1,3; *Ahmad Ali Hanafi-Bojd1,4; Ahmadali Enayati5; Rabiu Ibrahim Jalo6; Akande Oyebanji Azeez Aliyu Aminu2; Isa Muhammad Daneji2 1Department of Medical Entomology and Vector Control, School of Public Health, International Campus, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical Microbiology and Parasitology, College of Health Sciences, Bayero University, Kano, Nigeria 3Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Zoonoses Research Center, Tehran University of Medical Sciences, Tehran, Iran 5Department of Medical Entomology, School of Public Health and Health Sciences Research Centre, Mazandaran University of Medical Sciences, Sari, Iran 6Department of Community Medicine, College of Health Sciences, Bayero University, Kano, Nigeria *Corresponding authors: Prof Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, Dr Ahmad Ali Hanafi-Bojd, E-mail: aahanafibojd@tums.ac.ir (Received 14 Jul 2020; accepted 01 Jun 2021) Abstract Background: Malaria is a major public health problem in Nigeria with 97% of its population with high morbidity and mortality. Mosquitoes play an important role in the transmission of malaria parasites. This study was conducted to eval- uate the current resistance status of Anopheles gambiae to insecticides. Methods: Larvae of An. gambiae was collected from three zones; A, B and C differentiated on the basis of variation in agricultural ecosystems between August and November, 2018 in the northeast and northwestern parts of Nigeria. They were carefully reared to adult stage and insecticidal susceptibility tests were conducted. Results: The mosquitoes tested showed high levels of resistance to all the insecticides used with the exception of mala- thion. Study zone A, recorded 74% mortality after 24h to deltamethrin compared to 81% from zone B and 82% from zone C, respectively. Mosquitoes from zone B exposed to DDT had the highest level of resistance at 37% compared to 40% and 53% from zones A and C, respectively. Resistant to bendiocarb was also observed, with zone A having the lowest mortality of 44% compared to 48% from zone C and 55% from Zone B, respectively. According to the results of knockdown tests, mosquitoes from Zone A exposed to deltamethrin recorded the lowest knockdown across the study locations while zone B recorded the lowest knockdown for DDT. Conclusion: The results of the study provide an insight into the current status of An. gambiae to four major insecticides in northern Nigeria as guideline for mosquitocontrol. Keywords: Malaria; Anopheles gambiae; Insecticide resistance; Susceptibility; Northern Nigeria Introduction Malaria is a life-threatening parasitic vec- tor-borne disease troubling many countries in the tropical and subtropical regions of the world (1). With over 200 million new cases between 2010 and 2017, Africa still carries the highest burden (92%) worldwide compared to South- East Asia (5%) and the Eastern Mediterranean regions (2%), respectively (2-3). Nigeria, Dem- ocratic Republic of Congo, Mozambique, In- dia and Uganda account for nearly half of all Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com mailto:aahanafibojd@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 197 http://jad.tums.ac.ir Published Online: June 30, 2021 malaria cases worldwide (3). In 2017 alone, there were reported cases of increased malaria transmission (more than half a million cases) from countries with highest burden in Africa (Nigeria, Democratic Republic of Congo and Madagascar) compared with the year 2016 (3). Malaria is a serious problem in Nigeria with approximately 100 million cases and over 200,000 deaths annually (4- 5). It also accounts for nearly 60% of outpatients visits, 30% of under-five hospitalization and contributes to 11% maternal mortality, making Nigeria the country with the highest burden in the world (4-5). The malaria parasite is mainly transmitted in Africa by the Anopheles gambiae s.l. and the major vectors are An. gambiae and An. fu- nestus species complexes (6-9). Anopheles gam- biae forms a species complex comprising of eight morphologically indistinguishable, i.e., identical sibling species in the series Pyretoph- orus in the Anopheles subgenus Cellia across sub-Saharan Africa (6, 10). The individual spe- cies of the complex exhibit similar traits and are closely related, making it very hard to be identified morphologically except for some few larvae and adult females (11). The An. funes- tus complex is comprised of nine sibling spe- cies of which An. funestus sensu stricto (s.s.) is the principal vector with very high density and found across different geographical regions (12). However, the An. gambiae s.l. complex is the most widely distributed Anopheles mos- quitoes in Nigeria (65.2%) followed by the An. funestus (17.3%), respectively (13). The main control measures adopted in Ni- geria for malaria vectors are insecticide-treat- ed nets (ITNs)/long-lasting insecticide-treated nets (LLINs) (14). These methods when proper- ly used against insecticide susceptible mosquito populations contribute significantly in the re- duction of malaria cases (15-16). Other vector control measures used for malaria prevention in Nigeria include personal protective measures such as the use of repellents and house screen- ing (14). There is a serious threat to these con- trol measures as a result of progression of in- secticide resistance to major malaria vectors across malaria endemic countries including Nigeria (14, 17). Insecticide resistance is defined as the abil- ity of some organism to tolerate a specific dose of a toxic substance that will be deadly to oth- er organisms of the same species and from the same environment (18). Over the years, the problem of insecticide resistance is progress- ing and involving more classes of insecticide, and this can significantly affect the strength of vector control programs leading to failure, thus resistance management is designed to delay or prevent insecticide resistance (19). An important part in the resistance management strategies is identifying the resistance and mechanisms in- volved by obtaining the baseline information about the vector susceptibility, detection of re- sistance in the early stage and monitoring its frequency levels over time (20-21). An inte- grated approach where two or more methods are employed in the vector control programs could help in delaying the sustain progression of the resistance (20). A study conducted in 2013 at Bichi (North- ern Nigeria) reported a high resistance of An. gambiae with significant elevation of detoxi- fication enzymes in deltamethrin and bendio- carb resistance strains compared to suscepti- ble species from agricultural and residential ar- eas (22). A similar study from Bichi was con- ducted in 2015 to assess the level of resistance against bendiocarb, permethrin and DDT. Very high resistance to permethrin and DDT was re- ported with less resistant to bendiocarb (14). Resistance to permethrin and DDT exposed An. gambiae s.s across all the geographical zones of Nigeria with the highest level of the re- sistance in the forest savannah, Mosaic and Guinea savanna has been reported (18). The re- sistance profile and kdr mutation of An. gam- biae s.l. populations was also reported from two locations (Auyo and Bunkure) in northern Nigeria (13). Other studies have also reported resistance to commonly used insecticides from http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 198 http://jad.tums.ac.ir Published Online: June 30, 2021 Nigeria (6-7). Also insecticide resistance was reported in this species from the neighboring country Ghana (23-24). Periodic monitoring of susceptibility sta- tus of An. gambiae to insecticides used in public health practice is vital and will guide stake- holders towards the procurement and strategy used in vector control programs. The aim of this study was to examine the current situation of insecticide susceptibility in the principal ma- laria vector, An. gambiae s.l. in northern Nigeria. Materials and Methods Study area The study was conducted across three dif- ferent locations within three states (Gombe, Jigawa and Kano) in northeast and northwest Nigeria from August to November, 2018 (Fig. 1). The locations were designated as study zones A, B and C differentiated by the type of vegetative zone, i.e., A: lies within the Sudan, Northern and Southern Guinea savanna; B: is found within the Sudan, Sahel and Northern Guinea savanna; and C, lies within the Sudan and Northern Guinea savanna (6, 13). Zone A, Yamaltu Deba (10° 13′ 0″ N, 11° 23′ 0″ E) is one of the 11 Local Govern- ment Areas in Gombe State, Nigeria. It has a population of 255,248 and an area of 1,981 km². Gombe State (10° 15′ 0″ N, 11° 10′ 0″ E) is situated in the north-eastern part of Nigeria (25, 26). The state shares common borders with Borno, Yobe, Taraba, Adamawa and Bauchi states. The state has two distinct climates, the dry season (November–March) and the wet sea- son (April–October) with average annual rain- fall of 850mm (27). Zone B, Auyo (12°21′N, 9°59′E) is a lo- cality in Jigawa State, north-western Nigeria within the Sudan and Guinea savannah with pockets of Sahel savannah. The town is known for its irrigation activities in which rice and vegetables are produced. It has a total popula- tion of 132,001 and estimated land mass area of 512km (13, 26). Zone C, Kumbotso (11°53′17′′N, 8°30′10′′E) is situated in Kano state, north-western Nige- ria in the Sudan and Guinea savanna with a population of 409,500 and an area of 158km2 (18, 26). The temperature is generally warm, and the annual rainfall is about 1,300mm between April and September (28). Study Sample Sampling was conducted from different breeding places in the study areas using dip- ping method as described by Habibu, 2017 (14) in order to provide laboratory stock of mos- quitoes. The samples were transferred to the in- sectary at Bayero University, Kano with a rear- ing condition of 28±2 °C temperature, 65±5% relative humidity (RH) and 12:12h D: L. Two to three days old female sugar-fed mosquitoes were used for susceptibility tests (29). The inclusion criteria used for the selec- tion of the sampling areas include: history of pesticide and herbicides use on the land from agriculture activities, vector control and irrigation activity, availability and high den- sity of target species, high intensity of malaria transmission, and paucity of data on suscep- tibility profile of the malaria vector An. gam- biae s.l. Susceptibility test papers The impregnated test papers recommend- ed by WHO used in this study were purchased from the Vector Control Research Unit, School of Biological Sciences, Universiti Sains Ma- laysia 1800 Minden, Penang, Malaysia. WHO susceptibility test The current WHO susceptibility bioassay guideline (21) was followed. At least 120–150 female mosquitoes were aspirated from the mos- quito cage into six holding tubes giving six rep- licate samples of 20–25 mosquitoes per tube. With the mosquitoes in the tubes, the slide unit was immediately closed and the holding tubes set in a vertical position for one hour. Dam- aged mosquitoes were removed at the end of the one hour exposure time. The exposure tubes http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/Borno_State https://en.wikipedia.org/wiki/Yobe_State https://en.wikipedia.org/wiki/Taraba_State https://en.wikipedia.org/wiki/Adamawa_State https://en.wikipedia.org/wiki/Bauchi_State J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 199 http://jad.tums.ac.ir Published Online: June 30, 2021 were lined with a sheet of insecticide-impreg- nated paper, while the plain control tubes were lined with oil-impregnated papers, provided for each group of insecticides by WHO, fastened into position with a copper spring-wire. The plain holding tubes containing the mosquitoes were attached to the vacant position on the slides and with the slide unit opened, the mosquitoes were blown gently into the exposure tubes con- taining the following insecticide treated papers with discriminating dosages: DDT 4%, bendio- carb 0.1%, malathion 5% and deltamethrin 0.05 %. The exposure tubes containing all the mos- quitoes were set aside in a vertical position for one hour after which they were transferred back to the holding tubes by reversing the procedure outlined. Knockdown was recorded on deltame- thrin and DDT from all the study zones. A pad of cotton-wool soaked in sugar solution 5% was placed on the mesh-screen end of the holding tubes and the mosquitoes were kept in the re- covery period for 24 hours. During this time, the holding tubes were kept in the insectary. After the recovery period, the mortality was scored and recorded. A mosquito is consid- ered alive if it is able to fly, regardless of the number of legs remaining (21). Data analysis The 24h mortality was accessed manually, while the susceptibility was defined as: 98–100 % mortality indicates susceptibility, 90–97% mortality requires confirmation of resistance and between 0–89% suggests resistance (21). Microsoft office excel, version 2013 was used to create charts, sort and clean the data. While Statistical Package for the Social Sciences (SPSS) version 16 was used to calculate the 95 % confidence interval and the means of the var- iable using the Student’s t test. Abbott’s for- mula (30) was used to correct for natural mor- tality if the control mortality was between 5 and 20%. The results of the tests with >20% mor- tality in controls were discarded and the test repeated (31). Results A total of 1800 F0 2–3 days old adult fe- male An. gambiae s.l. mosquitoes were used for the bioassays. Anopheles mosquitoes from all the three zones were only susceptible to mal- athion and resistant to DDT, bendiocarb and deltamethrin (Table 1, Fig. 2). A mortality of 74% (95%, CI: 68–79) was recorded in zone A to deltamethrin compared to 81% (CI: 72– 89) from zone B and 82% (CI: 76–87) from zone C (Fig. 2). This study area had a compar- atively lower mortality to deltamethrin, indicat- ing a relatively higher resistance to this insec- ticide. Resistance by An. gambiae to bendiocarb in Nigeria is usually moderate but was found to be very high in all the three locations with zone A having the lowest mortality of 44% (CI: 38–49) compared to 48% (CI: 44–51) from zone C and 55% (CI: 53–56) from zone B, re- spectively (Table 1). The organophosphate, mal- athion was the only insecticide mosquitoes from all the study locations were susceptible to, with 100% mortality both in zones B and C, while zone A recorded 99% (Fig. 2). According to the results of knockdown tests, mosquitoes from Zone A exposed to del- tamethrin recorded the lowest knockdown at 30 minutes 32% (Standard Error, SE= 0.333), compared to 37% (SE= 0.344) and 48% (SE= 0.355) from zones B and C, respectively (Fig. 3). Similarly, at 60 minutes zone A mosquitoes still recorded the lowest knockdown 44% (SE= 0.354) compared to 51% (SE= 0.357) and 56% (SE= 0.355) from zones B and C, respectively (Fig. 3). Comparatively, from the deltamethrin knockdown result observed, zone A recorded the lowest knockdown rate at various time in- tervals shown above, which is in line with the low percentage mortality observed to deltame- thrin after 24h 74% (CI: 68–79) from this study area (Figs. 2, 3). The highest percentage knock- down to deltamethrin was observed with mos- quitoes from zone C, also in keeping with the high percentage mortality to this insecticide http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 200 http://jad.tums.ac.ir Published Online: June 30, 2021 after 24h 82% (CI: 76–87) recorded from this locality (Figs. 2, 3). The highest level of DDT resistance was 37% (CI: 34–39) and seen with mosquitoes from zone B compared to 40% (CI: 37–42) and 53 % (CI: 49–56) from zone A and zone B, re- spectively (Fig. 4). Comparatively, mosquitoes from zone B recorded the lowest mortality to DDT, signifying that they are highly resistant to DDT. Similarly, in confirming the aforemen- tioned statement, these mosquitoes recorded the lowest knockdown of 2% (SE= 0.1) at 30 minutes and 9% (SE= 0.204) at 60 minutes com- pared to 3% (SE= 0.122) and 17% (SE= 0.268) from zone C and 8% (SE= 0.2) and 8% (SE= 0.274) from zone A, respectively (Fig. 4). The high knockdown observed to DDT with mos- quitoes from zone A was in keeping with the high percentage mortality observed with mos- quitoes from this study area to the organochlo- rines (Figs. 2, 4) and was statistically signifi- cant (P< 0.05). Fig. 1. Map showing the geographical locations of the study sites in Nigeria, 2018 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 201 http://jad.tums.ac.ir Published Online: June 30, 2021 Fig. 2. Percentage mortality of Anopheles gambiae s.l. collected from some localities in Nigeria after 24h exposure to insecticides, 2018 Fig. 3. Knockdown profile of Anopheles gambiae s.l. mosquitoes collected from Northern Nigeria exposed to Deltame- thrin 0.05% after 60 minutes, 2018 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 202 http://jad.tums.ac.ir Published Online: June 30, 2021 Table 1. Susceptibility status of Anopheles gambiae s.l. at 95% confidence interval with the mean and standard devia- tion collected from the study zones in Nigeria after 24h exposure to different insecticides, 2018 Mortality (%) Locality Insecticide Exposed Mean ± SD Control 95% confidence interval Resistant Status Zone A Deltamethrin 0.05% 100 74.00±3.13 4.00±0.65 68–79 R DDT 4% 100 53.00± 3.56 0.00±0.00 49–56 R Malathion 5% 100 99.00± 0.71 0.00±0.00 97–100 S Bendiocarb 0.1% 100 44.00± 3.21 0.00±0.00 38–49 R Zone B Deltamethrin 0.05% 100 81.00±2.80 4.00±0.65 72–89 R DDT 4% 100 37.00±3.44 0.00±0.00 34–39 R Malathion 5% 100 100.00±0.71 0.00±0.00 100 S Bendiocarb 0.1% 100 55.00±3.55 0.00±0.00 53–56 R Zone C Deltamethrin 0.05% 100 82.00±2.74 2.00±0.48 76–87 R DDT 4% 100 40.00±3.49 0.00±0.00 37–42 R Malathion 5% 100 100.00±0.71 0.00±0.00 100 S Bendiocarb 0.1% 100 48.00±3.56 0.00±0.00 44–51 R R: Resistant; S: Susceptible Fig. 4. Knockdown profile of Anopheles gambiae s.l. mosquitoes collected from Northern Nigeria exposed to DDT 4% after 60 minutes, 2018 Discussion In the assessment of the resistance level to four insecticides viz; deltamethrin, DDT, mal- athion and bendiocarb by An. gambiae mosquito during the present study, bendiocarb showed http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 203 http://jad.tums.ac.ir Published Online: June 30, 2021 resistance across all the study area, with zone A having the lowest percentage mortality fol- lowed by zone C and B respectively. This find- ing agrees with studies from the north-eastern state of Gombe in the Sudan savanna of Nige- ria (7), where they reported a percentage mor- tality range of 2.3–100%. Similarly, a study from the forest zone vegetation of Kumasi in Ghana reported 38–56% mortality to bendiocarb (24). Another study from the coastal savanna vegeta- tion of Ghana reported a mortality rate of 12.3 % to bendiocarb (23). Most of the studies con- ducted in the Sudan, Guinea, Sahel savanna and humid forest reported moderate resistance to full susceptibility to bendiocarb (13-14). The major challenge arising from this finding is that the initial thought of using carbamate as an alter- native to the rapid spread of pyrethroids re- sistance may no longer hold (23-24, 32). This study also reports resistance to the pyrethroids (deltamethrin) from the study sites with zone A having the lowest percentage mor- tality followed by zone B and C, respectively. This finding is in agreement with studies con- ducted in the northern Guinea savanna of Ni- geria (33) where they reported percentage mor- tality of 83%, and from the Sudan through Guin- ea and some parts of Sahel savanna (13) where 78% mortality to deltamethrin was reported. However, a study conducted in the Sudan sa- vanna disagrees with our finding where a very high resistance of 38% mortality to deltamethrin was published (22). Also, another study from the forest zone vegetation of Kumasi in Ghana reported very high resistance in the range of 15– 46% (24). Recently, high resistance to deltame- thrin in the range of 1–70% from the Sudan and Sahel savanna was reported in Nigeria (6- 7). A rise in pyrethroids resistance by mosqui- toes is becoming a big threat to the vector con- trol strategy in Nigeria, thus necessary and ur- gent steps need to be taken to avoid complete failure of the measures (18). The resistance profile of the mosquitoes from all the three study locations to DDT shows a high level of resistance, with zone B having the highest level followed by C and A. This finding is in agreement with previous studies from Sudan, Guinea and Sahel savanna (6-7, 13-14, 16, 33). Similarly, very high resistance in the range of 12–46% was reported from the forest zone and coastal vegetation of Ghana (23-24). In this study, malathion was the only insecticide found to be very active against the mosquito vector tested across all the study lo- cations and agrees with previous studies from within and outside Nigeria (6, 13, 24, 32, 34). A slow knockdown to deltamethrin, that in- creased with increasing time of exposure was observed from all the zones with mosquitoes from zone A having the lowest knockdown fol- lowed by zone B and then C, respectively. This finding agrees with previous studies reported (35). DDT also recorded very low knockdown from all the study locations. This finding is sim- ilar to previous reports (13, 35). However, a pre- vious study conducted at Bichi in Kano-Nige- ria reported high knockdown to DDT (14). The high level of insecticide resistance by An. gambiae observed may be associated with increased use of pyrethroids treated bed nets (22). This is because farmers in these study lo- cations use a wide range of pesticides and herb- icides to protect their crops, and these pesticides marketed under different trade names belong to all the chemical classes including organo- phosphates, organochlorine, pyrethroids and car- bamates (16). The insecticide resistance observed in this study along with previous reports of re- sistance from these locations involving three ma- jor classes (carbamate, organochlorine, pyre- throids) of insecticides used in public health practice is disturbing and may have a negative effect on the malaria control programme which ultimately can lead to failure of the vector con- trol programme (6-7, 14, 33). Resistance man- agement consisting of all available measures designed to delay or prevent resistance should be implemented in all the malaria endemic zones as soon as possible (18, 24). Some methods that can also limit the progression of insecticides resistance to mosquito vectors of malaria in- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 196–206 M Ahmed Yusuf et al.: Current Status of … 204 http://jad.tums.ac.ir Published Online: June 30, 2021 clude: altering the dose and frequency of pes- ticide application in areas with high seasonal transmission; applying different formulations; avoidance of slow release formulations and iden- tify new pesticides with an alternate mechanism of action (18). Conclusion The results of the study provide an insight into the current status of An. gambiae to four major insecticides in northern Nigeria. This may form a new baseline data for further studies on other classes of insecticides that can be adopt- ed to guide the control of mosquito vectors of malaria in Nigeria. Acknowledgements We are very grateful to Dr Sulaiman S Ib- rahim, Eng MR Abai for all the support and advice given during this work. 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