Bendiocarb resistance and, kdr associated deltamethrin and DDT resistance in Anopheles gambiae s.l. populations from North Eastern Adamawa State, Nigeria

: Timely and consistent insecticide resistance monitoring efforts are required for early planning of management strategies. Here, we present the first report on bendiocarb, deltamethrin and dichlorodiphenyltrichloroethane (DDT) insecticide resistance in Anopheles gambiae s.l. populations from Adamawa, North Eastern Nigeria. Mosquitoes reared from larval collections were exposed to DDT (4%), bendiocarb (0.1%) and deltamethrin (0.05%) insecticides using standard WHO test kits and protocols. Species-specific Polymerase Chain Reaction (PCR) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assays were used to determine the sibling species composition of the exposed mosquitoes while allele-specific PCR was used for kdr genotyping in the mosquitoes that survived after insecticide exposure. Mosquito populations from all the four study sites in Vimtim, Imburu, Muchala and Bachure were resistant (≤ 87% mortality) to DDT and deltamethrin insecticides. Mosquito populations exposed to bendiocarb showed suspected resistance in Vimtim and Imburu study sites and confirmed resistance in Muchala and Bachure sites respectively. Mosquitoes alive after deltamethrin, DDT and bendiocarb exposures were identified predominantly as An. coluzzii species (78.5 - 92%) compared to An. gambiae s.s (5.3 - 11%) and An. arabiensis (0 - 10.1%). All the 22 kdr positive mosquitoes collected were identified as An. coluzzii. Percentage occurrence of kdr alleles in the mosquito populations alive after deltamethrin and DDT exposures were 30% and 25% respectively. An. coluzzii (≥ 63%) was the predominant species identified in each study site compared to An. gambiae sensu stricto ( s.s ) (≤ 20%) and An. arabiensis (≤ 17.4%) species. The results of kdr associated pyrethroid resistance is a potential threat to the effectiveness of the currently deployed deltamethrin-impregnated bed-net campaigns in Adamawa state. Evidence of bendiocarb carbamate resistance also implies potential ineffectiveness of future bendiocarb Indoor Residual Spraying. The involvement of metabolic resistance mechanisms in the mosquitoes from these study sites should be investigated for proper insecticide resistance management.


INTRODUCTION
Vector control is a major component of the global strategy for malaria prevention, control and elimination. Current best practices regarding malaria vector control involves the use of long-lasting insecticide-treated bed-nets (LLIN) and indoor residual spraying (IRS) of recommended longacting insecticides. In Nigeria, where 25% of the global malaria burden is experienced (WHO, 2019), preliminary trials on improved IRS-based tools such as insecticidetreated durable wall-lining have been conducted in some rural communities (Obembe et al., 2018a;Obembe et al., 2019). However, the main focus of the Nigerian National Malaria strategic Plan (NMEP, 2014) entails the expansion of universal access to insecticide-treated materials through mass distribution of LLINs, significant scaling up of IRS, and expansion of larval source management (larviciding and environmental management). Between May 2009 and November 2013, the Nigerian government, with support from several partners, distributed over 60 million mosquito nets across the country (NMEP, 2014).
Insecticide resistance in the malaria vectors have been attributed to the increased deployment of these insecticidebased tools for malaria vector control (Toé et al., 2014). Indeed, evidence-based concerns are currently being expressed concerning the tendency of insecticide resistance in the malaria vectors to slow, halt or reverse the gains made in malaria vector control (Ochomo et al., 2013;Toé et al., 2014). Therefore, current knowledge on the development of insecticide resistance in the local malaria vector population is required for the design and effective implementation of appropriate control measures. Pyrethroids is still a major class of insecticide recommended by WHO for LLIN impregnation [http://www.who.int/whopes/en/]. Since LLIN serves currently as the primary means of malaria prevention, timely and regular investigations of pyrethroids insecticide resistance and the mechanisms conferring the resistance in local malaria vector populations are imperative for the management of insecticides resistance detected. Resistance to pyrethroids/ DDT is conferred by two main physiological mechanisms including metabolic resistance and target site insensitivity (Hemingway et al., 2004;Karunaratne et al., 2018). Members of the Anopheles gambiae Giles species complex represents the major vectors of malaria in sub-Saharan Africa. According to Coetzee (2020), three of the nine sibling species that make up the members of the Anopheles gambiae Giles species complex currently include An. gambiae arabiensis (Patton, 1905), An. gambiae coluzzii (Coetzee & Wilkerson, 2013) and An. gambiae s.s (Giles, 1902). These morphologically indistinguishable sibling species exhibit diverse ecology and behaviour and their identification is of importance in setting up the most appropriate control interventions. Pyrethroid target site resistance mechanism within these members of the Anopheles gambiae species complex is conferred by knock down resistance (kdr) gene mutations including the substitutions of leucine to phenylalanine (L1014F) identified as kdr-west (Martinez-Torres et al., 1998) and leucine to serine (L1014S) designated kdr-East Africa (Ranson et al., 2000).
Data on malaria vector resistance in Nigeria (Awolola et al., 2005;Oduola et al., 2010;Oduola et al., 2012;Okorie et al., 2015;Adeogun et al., 2017) have mostly been from the South Western part of the Country. Availability of extensive malaria entomological data set in the South West may have contributed to the implementation of control response and the report of lower malaria prevalence rates (32.1%) in this region as compared to those of other zones in the country (NMEP, 2016). According to the malaria prevalence results reported in the latest Nigerian National Malaria Indicator Survey (NMEP, 2016), the North East had the third highest malaria prevalence rates (42.8%) in Nigeria after North West (58.3%) and North Central (50.7%) zones. However, apart from Bauchi (Umar et al. 2014) and Gombe  states, malaria vector resistance data required to guide and subsequently assess the impact of suitable control measures on local vector population is scarcely available in the North East region. In fact, Okorie et al. (2011) earlier reported that the proportion of malaria vector studies carried out between year 2000 and 2010 was lowest in the North East followed by North West, South South, South East and the North Central geo-political zones of Nigeria. Adamawa state currently has the highest malaria prevalence rate (55.5%) among the six states in the North Eastern region of Nigeria (NMEP, 2016). The State represents one of the largest states in Nigeria having flood plains with many rivers passing through especially during the rainy season (Adewumi, 2019). Data on susceptibility/ resistance status of local Anopheles mosquito populations to insecticides such as deltamethrin (the most common insecticide used for LLIN impregnation), carbamate (potential alternative to pyrethroids IRS) and DDT (which could induce cross-resistance to deltamethrin) could serve as useful information for malaria vector control. This study presents a first report on carbamate, deltamethrin and DDT resistance in identified An. gambiae species complex mosquito populations from four study sites in Adamawa state, North Eastern Nigeria.

Study area and mosquito collection
Adamawa State is located in North Eastern Nigeria, and lies between latitude 9º 19' 60.00''N and and longitude 12º 29' 59.99''E. It covers a land area of 38,741 km 2 and shares boundaries with Taraba state in the south and west, Gombe state in its North West, Borno state to the North and Cameroon Republic along its Eastern border (Adebayo and Tukur, 1999). Adamawa has a tropical climate with a mean monthly temperature ranging from 26.7 o C to 27.8 o C and rainfall of 700 mm to 1600 mm (Adebayo and Umar, 1999). The state is blessed with floodplains otherwise known as fadama lands, characterized by the availability and accessibility to both open surface and underground water (Umar et al., 2012). Water as a physical resource is adequate in Adamawa state due to the presence of River Benue and hundreds of other perennial streams and rivers used for fishing and irrigated agriculture in several parts of the state (Adebayo and Umar, 1999). Four study sites from four different Local Government Areas (LGA); Muchala in Gombi LGA (10º33'50"/N, 13º23'00"/E), Vimtim in Mubi-North (10º23'50"/N, 13º21'00"/E), Bachure in Yola North (09º16'50"/N, 12º24'50"/E) and Imburu in Numan LGA (09º30'00"/N, 11.5º20'00"/E) ( Figure 1) were selected from the state based on their proximities to major river tributaries. Anopheles larvae were collected from natural breeding sites such as ground pools, puddles, marshes, swamps and rice fields in each study site from July to October, 2017. Anopheles mosquito larvae and pupae collected together with water from the study sites with the aid of a dipper were transported to the insectary at Adamawa State University where the samples were reared to adulthood. Newly emerged adult Anopheles mosquitoes were maintained on 10% sugar solution.

Insecticide susceptibility assay
World Health Organization (WHO) test papers containing diagnostic concentrations of pyrethroid deltamethrin (0.05%), organochlorine DDT (4%) and carbamate bendiocarb (0.1%) insecticides were used for the tests following WHO standard procedures and test kits (WHO, 2013). Hundred female Anopheles mosquito samples (four replicates of 25 non-blood fed 2 -3 day old) were exposed to the test papers of each insecticide for 1 h. Control experiments were conducted alongside with two replicates of 20 -25 mosquitoes exposed to the control papers. Temperature and relative humidity conditions recorded during the susceptibility tests in the laboratory were 26 o to 29 o C and 74% to 83% respectively. Exposed mosquitoes were transferred into holding tubes, supplied with 10% sugar solution and given a recovery period of 24 h after exposure (WHO, 2013). Dead and alive mosquitoes were separately preserved in Eppendorf tubes (one mosquito per tube) containing silica gel for subsequent molecular analysis at the Molecular Entomology and Vector Control Research Laboratory of The Nigerian Institute of Medical Research.

Identification of sibling species and knockdown resistance (kdr) mutation in Anopheles mosquitoes
Exposed mosquitoes were identified morphologically (Gillies and Coetzee, 1987) and a subset of the identified Anopheles gambiae species complex samples were further characterized into sibling species using PCR (Scott et al., 1993) and PCR-RFLP (Favia et al., 1997) standard procedures. Species specific primers (Primer sequence 5' to 3'): Universal (GTGTGCCCCTTCCTCGATGT), An. gambiae s.s (CTGGTTTGGTCGGCACGTTT), An. merus and An. melas (TGACCAACCCACTCCCTTGA), An. arabiensis (AAGTGTCCTTCTCCATCCTA) and An. quadriannulatus (CAGACCAAGATGGTTAGTAT) designed from the DNA sequence of the intergenic spacer region of An. gambiae s.l. were used for the identification (Scott et al., 1993). Polymerase chain reaction amplification was carried out with an initial denaturation step at 95 o C for 5 min, followed by 30 cycles each consisting of 30 seconds denaturation at 95 o C, 30 seconds annealing at 50 o C and 30 seconds elongation at 72 o C. The final elongation was carried out at 72 o C for 5 minutes. The PCR product was digested using Heamophilus haemolyticus (Hha1) restriction enzyme to further identify the Anopheles gambiae s.s as An. coluzzii or An. gambiae. The digestion was carried out at 37 °C for 6 hours in a thermal cycler (Favia et al., 1997). The PCR products were electrophoresed through ethidium bromide-stained 1.5% agarose gel and visualized under the UV light in a gel documentation machine.
The sibling species characterization was done on all the surviving mosquitoes and a subset of the dead mosquitoes per site in order to determine sibling species composition of Anopheles gambiae s.l. mosquitoes in each of the study sites within the LGAs considered. Sixty nine (53 alive Fourty deltamethrin survivor mosquitoes (10 from each site) and 40 DDT survivor mosquitoes (10 from each site) were genotyped for kdr mutation after Genomic DNA extraction. Genomic DNA extraction was conducted following the protocol of Collins et al. (1987) while kdr-west L1014F mutation genotyping was done using the standard allele specific PCR designed for the West African kdr mutation (Martinez-Torres et al., 1998). The primers; Agd1 (ATAGATTCCCCGACCATG), Agd2 (AGACAAGGATGATGAACC), Agd3 (AATTTGCATTACTTACGACA), and Agd4 (CTGTAGTGATAGGAAATTTA) were used for the kdr genotyping. The PCR conditions consists of 94 °C for 5 min (denaturation phase), 40 cycles of 94 °C for 1 min, 48 °C for 2 min, and 72 °C for 2 min (hybridization and extension phase), and a final extension phase of 72 °C for 10 min (Martinez-Torres et al., 1998). The PCR-Kdr products were run on 2% agarose gel and photographed under UV light in a gel documentation machine.

Data analysis
Proportions of mosquitoes dead after the 24 h postexposure period was used to calculate percentage mortality. Susceptibility or resistance status of exposed mosquito populations were determined using WHO (2013) criteria as follows: 98% to 100% mosquito mortality indicated susceptibility, 90 -97% signified suspected or moderate resistance while less than 90% mosquito mortality implied confirmed resistance. None of the control mortalities was up to 5%. There was no need to correct insecticide induced percentage mortalities with Abbott's formula.

Ethical statement
The study was conducted after obtaining certified ethical approval S/MOH/81/T.II/330 from the State Ministry of Health Yola, Adamawa State, Nigeria.

Mosquito mortality rates and insecticide susceptibility status
Susceptibility status of Anopheles gambiae complex mosquito populations from the four different communities in Adamawa State is presented in Table 1. Mortalities from exposures to bendiocarb (83 -92%) was higher than deltamethrin (75 -87%) and DDT (77 -83%) for each mosquito population except in Bachure community where lower mortality from bendiocarb (83%) was observed compared to deltamethrin (87%).

Sibling species composition of Anopheles gambiae complex from the study locations
The results of sibling species composition of An. gambiae complex mosquitoes collected in all the communities are detailed in Tables 2 and 3. A large proportion of deltamethrin (88.1%) and DDT (78.5%) surviving mosquitoes were identified as An. coluzzii species. Similarly, 65.2% and 76.2% of the deltamethrin and DDT susceptible mosquitoes were identified as An. coluzzii (Table 2). Conversely, the proportion of bendiocarb susceptible mosquitoes identified as An. coluzzii was very low (8.3%) compared to the bendiocarb resistant (92%) An. coluzzii (Table 2). Altogether, 85.4% of all the 205 resistant mosquitoes and 57.1% of the 112 dead/susceptible mosquitoes analyzed were An. coluzzii (Table 2). However, lower proportions 5.3%, 11.4% and 8% of An. gambiae s.s mosquitoes survived deltamethrin, DDT and bendiocarb exposures respectively compared to respective higher proportions of An. gambiae s.s 34.8%, 23.8% and 62.5% that were susceptible to the same insecticides. Consequently, there were higher proportions (36.6%) of susceptible An. gambiae s.s mosquitoes compared to resistant (8.3%) ones ( Table   2). Proportions of susceptible and resistant mosquitoes identified as An. arabiensis were the same (6.3%). All the 22 kdr positive An. gambiae s.l mosquitoes collected were identified as An. coluzzii (Table 3). Overall, samples of Anopheles arabiensis species were not found in Vimtim and Muchala communities. Likewise, the Anopheles arabiensis species had the lowest occurrence in both Bachure (4.6%) and Imburu (17.4%) communities (

Occurrence of the kdr alleles in resistant Anopheles populations
The three kdr genotypes were identified by the characteristic 293bp band common to both susceptible and resistant specimens, a 137bp band associated with susceptible (SS) specimens and a 195bp kdr band (R). Presence of all three bands in a single specimen indicates heterozygosity (RS) (Figure 4).
Occurrences of kdr L1014F alleles in the DDT and deltamethrin survivor mosquito populations were low (<50%). Percentage occurrences of knock down resistant alleles (RR+RS) among the mosquito populations alive after deltamethrin and DDT exposures were 30% and 25% respectively (Table 4). Taken together, 27.5% (22/80) of both DDT and deltamethrin survivor mosquito samples combined were identified as carrying the kdr (RR+RS) alleles which confers target site resistance on the mosquitoes (Table 4).

DISCUSSION
This study presents the first report on insecticide resistance status and kdr L1014F resistance mechanism in An. gambiae complex mosquito populations from Adamawa State, North eastern Nigeria. The DDT, deltamethrin and bendiocarb resistance results obtained are an important contribution to the current malaria vector surveillance and resistance monitoring efforts of the National Malaria Elimination Programme. Specifically, confirmed deltamethrin and suspected bendiocarb resistance similar to those obtained in Vimtim and Imburu sites in this study have been reported in Bauchi State, Northeastern Nigeria (Umar et al., 2014). Equally, confirmed DDT, deltamethrin and bendiocarb resistance observed in Muchala and Bachure have also been noted in Lagos (Oduola et al., 2012) southwest and Kano (Abdu et al., 2017;Ibrahim et al., 2019;Ononamadu et al., 2020) Northwest Nigeria. Deltamethrin and bendiocarb resistance observed in this study could be attributed to intense use of insecticide-based tools such as LLIN for malaria vector control coupled with insecticide deployment in irrigated cereal and vegetable cultivation carried out by farmers in these areas. Such agricultural insecticide linked Anopheles mosquito resistance to carbamate have been reported in other areas in Africa (Yadouleton et al., 2010).
All the kdr positive mosquitoes analyzed were identified as An. coluzzii. This could have contributed to the higher proportions of deltamethrin and DDT resistant An. coluzzii mosquitoes found compared to the An. gambiae s.s species. In contrast, higher proportions of deltamethrin and DDT susceptible An. gambiae s.s mosquitoes compared to resistant ones may be associated with the nonidentification of kdr positive mosquitoes among the An. gambiae s.s species. The fact that 92% of the bendiocarb resistant mosquitoes and a large proportion (≥ 65.2%) of deltamethrin and DDT exposed mosquitoes (susceptible or resistant) were identified as An. coluzzii showed the preponderance of this species in the study sites. Exception to the preponderance of An. coluzzii species (8.3%) was only found among the bendiocarb susceptible mosquitoes. This observation, coupled with high percentage (92%) of An. coluzzii species found among the bendiocarb surviving mosquitoes, call for the investigation of carbamate resistance mechanisms in the An. coluzzii mosquitoes available in these study sites.
Molecular and biochemical studies are required to identify the active mechanisms responsible for confirmed bendiocarb carbamate resistance observed in this study so as to guide the implementation of effective management decisions. Such studies should also investigate the possible operation of additional metabolic resistance mechanisms in the deltamethrin and DDT resistant mosquito populations. Evidence of suspected and confirmed bendiocarb carbamate resistance implies that the Indoor Residual Spraying of bendiocarb carbamate insecticide may not be an effective addition to the current mosquito net campaign intervention in Adamawa State.
The low kdr frequencies in the DDT (25%) and deltamethrin (30%) resistant mosquitoes in this study was associated with 77 -83% DDT and 75 -87% deltamethrin induced mortalities obtained. Comparable trend of low kdr frequencies (19 -25.6%) associated with 81% deltamethrin induced An. gambiae s.l mosquito mortality have been reported in Southwest Nigeria (Okorie et al., 2015). Nevertheless, higher kdr frequencies 49 -95.8% conferring lower DDT (0.83 -56.9%) and deltamethrin (1 -63%) induced An. gambiae s.l mortalities have been reported in some other parts of Nigeria (Abdu et al., 2017;Ibrahim et al., 2019;Ononamadu et al., 2020). Some of these studies implicated metabolic resistance as an additional resistance mechanism conferring phenotypic An. gambiae s.l resistance to deltamethrin and DDT. Alone, the presence of kdr mechanism was initially associated with low level of phenotypic pyrethroids resistance having little or no operational effect on the efficacy of LLIN (Darriet et al., 2000;Asidi et al., 2005). The complacency and increased insecticide selection pressure that followed the earlier observed low level of kdr resistance led to the evolution of more potent resistance mechanisms yielding mosquitoes that are now insensitive to standard LLIN and IRS pyrethroids formulations (Ochomo et al., 2013;Toé et al., 2014). In Jigawa State, Northwestern Nigeria, initial moderate An. coluzzii deltamethrin resistance (78.4% mortality) noted between 2009 and 2011 (Ibrahim et al., 2014) had escalated (1% mortality) in the recent studies conducted in 2017 (Ibrahim et al., 2019). To avoid this situation in Adamawa state, continuous resistance monitoring and management efforts should be made to reduce and possibly reverse the moderate level of resistance observed in this study.
Confirmation of deltamethrin resistance in the Anopheles gambiae s.l populations from all the study sites in this study should be of particular concern especially since 7 out of the 13 WHO recommended LLIN brands available are deltamethrin impregnated (WHO, 2012). Four are alphacypermethrin impregnated while the remaining 2 brands are permethrin infused (WHO, 2012). Anopheles gambiae s.l mosquito populations from Bauchi, another state in north eastern Nigeria, were resistant to other pyrethroids insecticides but susceptible to alphacypermethrin. Further studies on the susceptibility status of Anopheles mosquitoes in Adamawa state to other pyrethroids such as alphacypermethrin should be conducted. Also, the involvement of metabolic mechanisms should be investigated for proper management of the resistance detected in Adamawa state as the wait for novel interventions continues.
Anopheles arabiensis species is known to prefer arid conditions available in the savannah areas of Nigeria (Onyabe and Conn, 2001). The predominance of An. gambiae s.s (Awolola et al., 2005;Obembe et al., 2018b;Oduola et al., 2019) or An. arabiensis (Onyabe and Conn, 2001;Yoriyo et al., 2014;Oduola et al., 2016) mosquito species have been reported in some savannah areas of Nigeria. Adamawa State also belongs to the Savanna area of Nigeria. However, the result of this study in Adamawa shows the prevalence of An. coluzzii species probably because of the peculiarity of Adamawa State as a flood plain area with several perennial streams and rivers (Adebayo and Umar, 1999) providing suitable relatively permanent breeding sites preferred by the predominant An. coluzzii mosquito species (Djègbè et al., 2018). Apart from investigations on the involvement of metabolic resistance mechanisms, further studies on continuous monitoring of insecticide resistance in these areas should consider kdr analysis for all the resistant samples to generate a more robust data on this aspect. Development of kdr type mutations other than L1014F and their contribution towards pyrethroid and DDT resistance should also be investigated. Results from these studies will assist in the implementation of suitable resistance management strategies required to ensure effective insecticide-based malaria vector control.

CONCLUSIONS
Knock down resistance (kdr L1014F) associated deltamethrin pyrethroids and DDT organochlorine resistance were detected for the first time and identified as a potential threat to the current deployment of deltamethrin impregnated bed-nets through mass LLIN campaigns in Adamawa state, Nigeria. Evidences of suspected and confirmed bendiocarb carbamate resistance were also found implying the potential ineffectiveness of the implementation of bendiocarb IRS as an addition to the current LLIN campaign interventions in the State. The predominance of indoor-loving An. coluzzii mosquito sibling species in this study is predictive of the suitability of LLIN as an appropriate malaria vector control strategy in the study area. However, further studies on the involvement of metabolic resistance mechanisms that could contribute to the ineffectiveness of such deployed LLIN should be conducted for the implementation of effective resistance management strategies. Susceptibility of these mosquito populations to other promising pyrethroids insecticides such as alphacypermethrin should be tested to determine the possibility of switching to the use of bed-nets impregnated with such alternative.