J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 228 http://jad.tums.ac.ir Published Online: September 30, 2020 Original Article First Report of Target Site Insensitivity in Pyrethroid Resistant Anopheles gambiae from Southern Guinea Savanna, Northern-Nigeria Mustapha Ahmed-Yusuf1,2; *Hassan Vatandoost1,3; *Mohammad Ali Oshaghi1; Ahmad Ali Hanafi-Bojd1,3; Ahmad Ali Enayati4; Rabiu Ibrahim Jalo5 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical Microbiology and Parasitology, College of Health Sciences, Bayero University, Kano, Nigeria 3Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Department of Medical Entomology, School of Public Health and Health Sciences Research Centre, Mazandaran University of Medical Sciences, Sari, Iran 5Department of Community Medicine, Bayero University, Kano, Nigeria *Corresponding authors: Dr Hassan Vatandoost, Email: hvatandoost1@yahoo.com, Dr Mohammad Ali Oshaghi, Email: moshaghi@tums.ac.ir (Received 01 Dec 2019; accepted 11 Jul 2020) Abstract Background: Malaria is a major public health problem and life threatening parasitic vector-borne disease. For the first time, we established and report the molecular mechanism responsible for Anopheles gambiae s.l. resistance to pyre- throids and DDT from Yamaltu Deba, Southern Guinea Savanna, Northern-Nigeria. Methods: The susceptibility profile of An. gambiae s.l. to four insecticides (DDT 4%, bendiocarb 0.1%, malathion 5% and deltamethrin 0.05%) using 2–3 days old females from larvae collected from study area between August and No- vember, 2018 was first established. Genomic DNA was then extracted from 318 mosquitoes using Livak DNA extrac- tion protocol for specie identification and kdr genotyping. The mosquitoes were identified to species level and then 96 genotyped for L1014F and L1014S kdr target site mutations. Results: The mosquitoes were all resistant to DDT, bendiocarb and deltamethrin but fully susceptible to malathion. An. coluzzii was found to be the dominant sibling species (97.8%) followed by An. arabiensis (1.9%) and An. gambiae s.s (0.3%). The frequency of the L1014F kdr mutation was relatively higher (83.3%) than the L1014S (39%) in the three species studied. The L1014F showed a genotypic frequency of 75% resistance (RR), 17% heterozygous (RS) and 8% susceptible (SS) with an allelic frequency of 87% RR and 13% SS while the L1014S showed a genotypic frequency of RR (16%), RS (38%) and SS (46%) with an allelic frequency of 40% RR and 60% SS, respectively. Conclusion: This study reveals that both kdr mutations present simultaneously in Northern-Nigeria, however contribu- tion of L1014F which is common in West Africa was more than twice of L1014S mutation found in East Africa. Keywords: Anopheles gambiae s.l.; Insecticide resistance; Northern Nigeria; Voltage-gated sodium channels (VGSC) Introduction Insecticide resistance is mainly associated with genetic factors that are inherited and can be defined as “the ability in a population to tol- erate doses of insecticide which would prove lethal to the majority of individuals in a normal population of the same species, developed as a result of selection pressure to the insecticide” (1, 2). The resistance is mainly acquired through two methods which include the target site insen- sitivity and metabolic resistance (3). The target site insensitivity is operated through one of the following methods (insensitive acetylcholines- terase (AChE), GABA receptor mutation, or mu- tations in the voltage-gated sodium channel). The nervous system of mosquitoes is been targeted by specific insecticide through which it acts, though sometimes the sensitivity of the site may reduce as a result of mutations leading to Copyright © 2020 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/ http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 229 http://jad.tums.ac.ir Published Online: September 30, 2020 resistance (4). The organophosphate and carba- mates target the acetylcholinesterase (AChE) through carbamoylating the active serine site thereby stopping it from hydrolyzing the ace- tylcholine (5-7). Substitution in the GABA re- ceptor of an Alanine to Serine has been report- ed in Drosophila melanogaster, D. simulans, Aedes aegypti, Anopheles stephensi and An. gambiae as the cause of resistance (8). Re- sistance in the Pyrethroids and DDT is mainly due to mutations in the gene that encodes the voltage-gated sodium channel called the knock- down resistance (9). In the An. gambiae and An. arabiensis mosquitoes, two different knock- down resistance have been reported (10). The leucine-phenylalanine substitution at position 1014 of the sodium channel gene (L1014F), was the first mutation reported from Burkina Faso and Ivory Coast in West Africa (11). While a leucine-serine substitution (L1014S) at the same position was reported as the second mutation from Western Kenya in East Africa (12). A study conducted in Northern Nigeria re- ported high resistance of An. gambiae to per- methrin and DDT with less resistant to bendi- ocarb (13). The resistance profile and kdr mu- tation of An. gambiae s.l. populations was also reported from two locations (Auyo and Bunku- re) in northern Nigeria (14). High presence of An. coluzzii has been reported from previous studies (13-17). Two different studies conduct- ed in Northern Nigeria both reported lower kdr mutations from both resistant and susceptible mosquitoes (18, 19). Similarly, studies conduct- ed in Kenya reported lower kdr mutations to An. gambiae (20, 21). The aim of this study was to investigate spe- cies composition, the insecticide susceptibility status, and to explore type of kdr mutations con- ferring pyrethroids and DDT resistance in mem- bers of the An. gambiae complex from North- ern-Nigeria. Materials and Methods Study Location Yamaltu Deba (10° 13′ 0″ N, 11° 23′ 0″ E) is one of the eleven Local Government Areas in Gombe State, Nigeria (Fig. 1). It has a popu- lation of 255,248, an area of 1,981km² and is located in the north-eastern part of Nigeria, stretching through the Sudan savannah, north- ern and southern guinea savannah (22, 23). Study Sample Dipping method was used to collect larvae samples from different breeding places in the study site as described by (13) in order to pro- vide laboratory stock of mosquitoes. The sam- ples were transported to the insectary at Bayero University Kano with a rearing condition of 28±2 °C temperature, 65±5% relative humidi- ty (RH) and 12:12 hrs D: L. Two to three days old female sugar fed mosquitoes were used for susceptibility tests (24). WHO susceptibility tests Adult susceptibility test was conducted ac- cording to the recent WHO bioassay guideline (25). Twenty five female mosquitoes of 2–3 days old fed on 10% sugar solution, were exposed to malathion 5%, bendiocarb 0.1%, DDT 4.0% and deltamethrin 0.05% impregnated papers for 60 minutes in the standard WHO test kit. Oil-im- pregnated papers were used for the control group. There were four replicates for the treat- ed and two replicates for the control group. At the end of the exposure time, both the treated and control mosquito groups were allowed to recover in holding tubes with cotton pads con- taining 10% sucrose solution on the top for 24 hours and then the number of dead and alive mosquitoes were recorded. A mosquito is con- sidered alive if it is able to fly, regardless of the number of legs remaining. DNA Extraction Genomic DNA was extracted from 318 in- dividual mosquitoes using Livak DNA extrac- tion protocol template preparation kit (26, 27). Specie Identification The mosquitoes were first identified mor- phologically using morphological identification http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 230 http://jad.tums.ac.ir Published Online: September 30, 2020 keys (28, 29). Molecular species identification was performed by PCR-SINE200 technique as previously described (14). Sine PCR reagents were carried out in 15ul master mix contain- ing amplification reaction of 0.51mol of each primer sine 200F and sine 200R, 0.12mM of each dNTP, 0.75mM of MgCl2, 1.5U Taq DNA polymerase, PCR Buffer 10x [200mM Tris HCl (pH 8.4), 500mM KCl], 1.0µl of template DNA extracted from each mosquito. The primer se- quence and thermal cycling conditions are shown in (Table 1). PCR for kdr west (L1014F) and kdr East (L1014S) The amplification protocol used for the de- tection of 1014F and 1014S mutations was per- formed using allele specific PCR in a 12.5µl reaction containing 1µl of template DNA, 1x Qiagen PCR buffer, 0.5mM MgCl2, 0.5nM of each primer, 0.5µM of dNTPs, and 1U of Taq DNA polymerase revised from the protocols previously established by Martinez-Torres et al. (11) and Ranson et al. (30). The primer se- quences and thermal cycling conditions are shown in (Table 2). The primers Agd1, Agd2, Agd4 and Agd5 were used to detect the 1014S mutation whereas primers Agd1, Agd2, Agd3 and Agd4 were used to detect the 1014F mu- tation. Data analysis The 24hrs mortality was accessed manual- ly, while the susceptibility was defined as; 98– 100% mortality indicates susceptibility, 90–97 % mortality requires confirmation of resistance and between 0–89% suggests resistance (25). The Hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic fre- quencies. Microsoft office excel, version 2003 was used to create charts, calculate the stand- ard deviation, sort and clean the data. Abbott’s formula (30) was used to correct for natural mortality, if the control mortality was between 5 and 20%. The results of the tests with >20% mortality in controls, were discarded and the test repeated (25). Results Female mosquitoes exposed to deltamethrin, DDT and bendiocarb showed 74% (95%, CI: 68–79); 53% (CI: 49–56) and 44% (CI: 38– 49) mortalities after 24 hours, respectively (Fig. 2). Whereas, malathion was found to be sus- ceptible (Fig. 2). Molecular specie identification A total of 318 mosquitoes composing of 138 alive (45 exposed to deltamethrin, DDT, bendiocarb and 3 exposed to malathion) and 180 dead (45 exposed to deltamethrin, DDT, bendiocarb and malathion) were identified to specie level. All the dead mosquitoes identi- fied were An. culluzzi, the alive mosquitoes ex- posed to DDT, malathion and bendiocarb also were An. culluzzi while for deltamethrin ex- posed, 53.3% were An. culluzzi, 40% An. ara- biensis and 6.7% An. gambiae s.s (Table 3). Genotyping kdr west (L1014F) A total of 96 mosquitoes: 76 alive (69 An. coluzzii, 6 An. arabiensis, 1 An. gambiae s.s) and 20 dead (13 An. coluzzii, 6 An. arabiensis, 1 An. gambiae s.s) were used for kdr genotyp- ing. The following result was recorded: alive mosquitoes, the only An. gambiae s.s 1/1 (100 %) was homozygote resistant (RR); An. ara- biensis 4/6 (67%) RR, 2/6 (33%) heterozygote resistant (RS); An. coluzzii 55/69 (80%) RR, 9/69 (13%) RS, 5/69 (7%) homozygote sus- ceptible (SS). Dead mosquitoes, the only An. gambiae s.s 1/1 (100%) RR; An. arabiensis 3/6 (50%) RR, 2/6 (33%) RS, 1/6 (17%) SS; An. coluzzii 8/13 (62%) RR, 3/13 (23%) RS, 2/13 (15%) SS (Table 4). Genotypic and allelic frequencies of L1014F The Hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic frequencies. The result was found to be 72 (75%) RR, 16 (17%) RS and 8 (8%) SS; 87% RR and 13% SS (Table 5). http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 231 http://jad.tums.ac.ir Published Online: September 30, 2020 Genotyping kdr west (L1014S) A total of 96 mosquitoes were used, L1014S mutation was only found in 37 (39%) mosquitoes distributed as follows: 24 alive (15 An. coluzzii, 8 An. arabiensis, 1 An. gam- biae s.s) and 13 dead (8 An. coluzzii, 4 An. arabiensis, 1 An. gambiae s.s) were used for kdr genotyping. The following result was rec orded: alive mosquitoes, the only An. gambiae s.s 1/1 (100 %) RS; An. arabiensis 2/8 (33%) RR, 5/8 (56%) RS, 1/8 (11%) SS; An. coluzzii 4/15 (27%) RR, 3/15 (20%) RS, 8/15 (53%) SS. Dead mosquitoes: the only An. gambiae s.s 1/1 (100%) RS; An. arabiensis 3/4 (75%) SS, 1/4 (25%) RS; An. coluzzii 3/8 (38%) RS, 5/8 (62%) SS (Table 4). Genotypic and allelic frequencies (L1014S) The Hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic frequencies. The result was found to be 6 (16 %) RR, 14 (38%) RS and 17 (46%) SS: 40% RR and 60% SS (Table 5). Table 1. (A) PCR primer sequences and (B) thermal cycling conditions used for specie identification of the Anopheles gambiae complex from Yamaltu Deba (Gombe state), Northern Nigeria, 2018 A Primer name sequence (5′ to 3′) Identified species Size of the PCR product (bp) sine200F sine200R TCG-CCT TAG ACC TTG CGT TA CGC TTC AAG AAT TCG AGA TAC An. gambiae s.s. An. coluzzii An. arabiensis 240 470 220 B Step Temperature °C Time Cycle Initial Denaturation 95 5Mins 1 Denaturation Annealing Extension 94 54 72 30Sec 1Min 1Min 35 Final Extension 72 10Mins 1 Table 2. (A) PCR primer sequences and (B) thermal cycling conditions used for detection knockdown resistance muta- tions (L1014F and L1014S) in the Anopheles gambiae complex from Yamaltu Deba (Gombe state), Northern Nigeria, 2018 A Primer name sequence (5′ to 3′) Primer type Combination and Size of PCR product (bp) Agd1 ATAGATTCCCCGACCATG Common forward Agd1+Agd2=293 Agd2 AGACAAGGATGATGAACC Common reverse Agd3 AATTTGCATTACTTACGACA Specific reverse for L1014F Agd1+Agd3=195 Agd4 CTGTAGTGATAGGAAATTTA Specific forward for susceptible L1014L Agd2+Agd4=137 Agd5 ATTTGCATTACTTACGACTG Specific reverse for L1014S Agd1+Agd5=195 B Step Temperature °C Time Cycle Initial Denaturation 95 3Mins 1 Denaturation Annealing Extension 94 60 72 30Sec 1Min 1Min 35 Final Extension 72 5Mins 1 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 232 http://jad.tums.ac.ir Published Online: September 30, 2020 Table 3. Molecular species identification Anopheles gambiae s.l. specimens (A: alive, B: dead) following exposure to the insecticides from Yamaltu Deba (Gombe state), in Northern Nigeria, 2018 A Insecticide No. Exposed An. coluzzii (%) An. arabiensis (%) An. gambiae s.s (%) Deltamethrin 0.05% 45 53.3 40 6.7 DDT 4% 45 100 0 0 Malathion 5% 3 100 0 0 Bendiocarb 0.1% 45 100 0 0 B Insecticide No Exposed An. coluzzii (%) An. arabiensis (%) An. gambiae s.s (%) Deltamethrin 0.05% 45 100 0 0 DDT 4% 45 100 0 0 Malathion 5% 45 100 0 0 Bendiocarb 0.1% 45 100 0 0 Table 4. Genotyping of kdr west (L1014F) and east (L1014S) mutations of Anopheles gambiae s.l. from Northern Ni- geria, 2018 kdr Mutation (L1014F) Alive Dead Species An. gambiae s.s. An. arabiensis An. coluzzii An. gambiae s.s. An. arabiensis An. coluzzii Homozygote resistance 100% 67% 80% 100% 50% 62% Heterozygote resistance 0 33% 13% 0 33% 23% Homozygote susceptible 0 0 7% 0 17% 15% n=1 n=6 n=69 n=1 n=6 n=13 kdr Mutation (L1014S) Alive Dead Species An. gambiae s.s An. arabiensis An. coluzzii An. gambiae s.s An. arabiensis An. coluzzii Homozygote resistance 0 33% 27% 0% 0% 0% Heterozygote resistance 100% 56% 20% 100% 25% 38% Homozygote susceptible 0 11% 53% 0 75% 62% n=1 n=8 n=15 n=1 n=4 n=8 Table 5. The allelic and genotypic frequencies of the L1014F and L1014S, mutations of Anopheles gambiae s.l. from Northern Nigeria, 2018 Genotypic frequencies (L1014F) Homozygote resistance Heterozygote resistance Homozygote susceptible 75% 17% 8.00% n=96 Allelic frequencies (L1014F) Homozygote resistance Homozygote susceptible 87% 13% Genotypic frequencies (L1014S) Homozygote resistance Heterozygote resistance Homozygote susceptible 16% 38% 46% n=96 Allelic frequencies (L1014S) Homozygote resistance Homozygote susceptible 40% 60% http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 233 http://jad.tums.ac.ir Published Online: September 30, 2020 Fig. 1. Map (adapted from google earth) showing the geographical location of the study site, Gombe state (red circle) in Northern Nigeria, 2018 Fig. 2. Susceptibility (24hrs) profile of Anopheles gambiae s.l. to four insecticides from Yamaltu Deba (Gombe state), Northern Nigeria, 2018 Discussion Susceptibility test Bendiocarb showed very high level of re- sistance. This finding agrees with previous study (32), where they reported a percentage mortal- ity range of 2.3–100%. Similarly, a study from Kumasi in Ghana, reported 38–56% mortality to bendiocarb (33). This study reports moderate level of resistance to deltamethrin from the study site. This finding is in agreement with study con- ducted in the northern guinea savanna of Nige- ria (34) where they reported percentage mor- tality of 83%, and from north western part of Nigeria where they reported 78% mortality to http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 234 http://jad.tums.ac.ir Published Online: September 30, 2020 deltamethrin (14). However, a study conducted around the study location disagrees with our finding where they reported a very high re- sistance of 38% mortality to deltamethrin (35). DDT was found to be resistant and this finding is in agreement with previous studies from Su- dan, Guinea and Sahel savanna of Nigeria (13, 14, 19, 30, 36, 37). Malathion was found to be susceptible and agrees with studies from dif- ferent regions within and outside Nigeria (14, 19, 33, 34, 37). Species identification Anopheles coluzzii was found to be the domi- nant sibling species followed by the An. ara- biensis and An. gambiae s.s (Table 3). This is in agreement with a study conducted in north- ern Nigeria, where they reported An. coluzzii as the dominant specie 86.8% followed by An. arabiensis 77% (14). Also, the high presence of An. coluzzii reported is supported by previ- ous studies (13, 15, 17). However, a study con- ducted on molecular identification of An. gam- biae s.l mosquitoes in Kamuli District of Ugan- da, disagrees with our finding where they re- ported 98% of the mosquitoes to be An. gam- biae s.s (38). Another study conducted in Ni- geria by Oyewole and colleagues (2011), re- ported An. gambiae s.s as the dominant spe- cies (74.6%) followed by An. arabiensis 26.4% in contrast to our finding (39). Knockdown resistance (kdr) West (L1014F) and East (L1014S) The kdr mutations were observed in the study location with high frequency of the L1014F in the An. coluzzii species (Table 4). This agrees with study conducted by Ibrahim et al. (2014) where they found the kdr muta- tions in 80.1% of the An. coluzzii and 13.5% in An. arabiensis mosquitoes (12). Oyewole et al. (2011), in their study from south-western Ni- geria reported that 87% of the mosquitoes re- sistant to deltamethrin carried the kdr mutations and 80% of the DDT resistant mosquitoes as well (36). Furthermore, increase L1014F was reported from Ghana by Lynd et al. (2010) Niger, by Czeher et al. (2008) and sharp et al. (2007) from Equatorial Guinea (40-42). A study by Awolola et al. (2003) contradicts our find- ing, where they reported high frequency of L1014F in An. gambiae s.s compared to An. coluzzii (43). Derrick et al. (2011) from Ken- yan, also reported increased presence of kdr in An. gambiae s.s compared to the An. coluzzii. Also, increased presence of L1014S was re- ported by Protopopoff et al. (2008), from Bu- rundi and Verhaeghen et al. (2010), from Ugan- da (44, 45). Genotypic and allelic frequencies This study reports high genotypic frequency particularly in the L1014F kdr compared to the L1014S gene from the study location (Ta- ble 5). This is in agreement with previous study where they reported homozygous resistant of 74.1%, heterozygous resistant of 19.7% and ho- mozygous susceptible of 6.2% for the L1014F in the An. coluzzii. While from the An. ara- biensis; 69.2% were homozygous susceptible, 23.1% heterozygous and 7.7% homozygous re- sistance (14). A study by Habibu and colleagues (2017), contradicts our finding, where they re- ported 65.6% homozygous susceptible, 10% ho- mozygous resistance and 24.4% as heterozy- gous resistance in the L1014S (36). While the L1014F showed 54.4% as homozygous suscep- tible, 21.6% as homozygous resistance and 24% as heterozygous resistance. The L1014S was seen both in the An. coluzzii and An. arabiensis (36). Our study also reports a very high allelic frequency in the L1014F compared to the L1014S (Table 5). This agrees with the results of Habibu et al. (2017) where they reported an allelic frequency of 48.9% and 65.9% in An. gambiae s.s and 20% and 61.8% in An. ara- biensis in the L1014F (13). While the L1014S mutation recorded an allelic frequency of 40% and 55.3% in An. coluzzii; 20% and 30.8% in An. arabiensis. The L1014F has higher asso- ciation with An. coluzzii (36). Studies by Der- rick (2011) and Stump (2004) from Kenyan re- http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 228–238 M Ahmed-Yusuf et al.: First Report of … 235 http://jad.tums.ac.ir Published Online: September 30, 2020 ported lower allelic frequency compared to our findings (20, 21). The high level of insecticide resistance observed may be associated with in- creased use of pyrethroids treated bed nets and carbamate for indoor residual spraying (IRS) in public health and agricultural applications (35, 46). Farmers in the study location use a wide range of pesticides and herbicides to protect their crops and these pesticides marketed un- der different trade names belong to all the chemical classes including organophosphates, organochlorine, pyrethroids and carbamates (36). The high presence of kdr gene seen in this population of mosquitoes could be explained by the increase usage and abuse of insecticides by farmers and the increase coverage of LLIN distribution. Conclusion This study reveals the co-occurrence of L1014F and L1014S mutations with dominance of An. coluzzii and high genotypic and allelic frequencies in the L1014F over L1014S-kdr. Very high level of resistance to DDT, deltame- thrin and bendiocarb was also observed. Acknowledgements The authors wish to thank the sponsors. This research was co-sponsored by International Cam- pus, Tehran University of Medical Science (Pro- ject No. 9513494001) and Institute of Environ- mental Research (Grant. No. 97-02-27-39814), Iran with technical support from Center for In- fectious Diseases Research Bayero University, Kano, Nigeria. The authors declare that they have no competing interests. References 1. 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