J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 50 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article Widespread Report of Multiple Insecticide Resistance in Anopheles gambiae s.l. Mosquitoes in Eight Communities in Southern Gombe, North-Eastern Nigeria *Adedayo Olatunbosun-Oduola1; Ezra Abba2; Olukayode Adelaja1; Adeolu Taiwo-Ande1; Kennedy Poloma-Yoriyo2; Taiwo Samson-Awolola3 1Department of Zoology, University of Ilorin, Ilorin, Kwara State, Nigeria 2Department of Biological Sciences, Faculty of Science, Gombe State University PMB 127, Gombe, Nigeria 3Public Health Division and Epidemiology, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria (Received 20 Mar 2018; accepted 8 Jan 2019) Abstract Background: Timely entomological and insecticide resistance monitoring is a key to generating relevant data for vector management. We investigated the insecticide susceptibility status of Anopheles gambiae s.l. in eight rural farming communities in Southern Gombe, Nigeria. Methods: Overall, 3–5 days-old adult female Anopheles mosquitoes reared from field-collected immature stages between September and November, 2014 were exposed to the diagnostic doses of pyrethroids, organophosphate and carbamate insecticides using the Center for Disease Control Bottle bioassay. The observatory knockdown time from exposure to each insecticide was recorded up to two hours. The dead mosquitoes were then identified morphological- ly and by molecular assays. Results: Mortality results showed resistance in An. gambiae s.l. populations to bendiocarb (2.3–100%), deltamethrin (39–70%), pirimiphos-methyl (65–95%), dichloro-diphenyl-trichloroethane (0–38.1%), permethrin (0–46.3%) and lambda-cyhalothrin (42.5–86.4%). The few cases of full susceptibility were observed from lamdacyhalothrin exposed population of An. gambiae s.l. in Banbam and Pantami respectively. An. gambiae 177 (45%) was significantly higher (P< 0.05) than An. arabiensis 64 (16.3%), An. coluzzii 34 (8.7%) and An. gambiae/An. coluzzii hybrid 78 (19.8%). Conclusion: A strong evidence of widespread resistance in the major malaria vector species in Southern Gombe to all common classes of insecticides is a justification for the State Malaria Elimination Programme to consciously con- sider incorporating insecticide resistance management strategies into control programs in order to sustain the future of current control interventions. Keywords: Multiple; Insecticide resistance; Anopheles gambiae; Gombe south; Nigeria Introduction Malaria is a major public health burden in Nigeria with over 90% of her 167 million peo- ple at constant risk. Nigeria accounts for 29% of the global malaria burden and together with the Democratic Republic of Congo contribute up to 40% of the global burden for malaria (1). Anopheles gambiae s.l. have been reported to be the most widespread mosquitoes respon- sible for malaria transmission in all the eco- logical zones (1). Controlling these vectors pose major challenges as the An. gambiae s.l. mosquito belongs to a complex with each playing different role in transmission and ex- hibiting varying insecticide susceptibility status across the several regions in Nigeria. In Nigeria, vector control strategy remains one of the frontline and effective tools for con- trolling malaria and other insect-borne diseases. The strategy relies heavily on the use of in- secticides from just four available classes of insecticides. In the face of increasing reports of insecticide resistance, sustained efficacy of *Corresponding author: Dr Adedayo Olatunbosun Oduola, E-mail: dayooduola@yahoo.co.uk J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 51 http://jad.tums.ac.ir Published Online: April 27, 2019 these chemicals is desired. Resistance to at least one or more classes of insecticide have as- sumed a geographical scale in more than 50 countries (2). This is a major threat to pyre- throids which is the only class of insecticide approved for use in long lasting insecticide nets) (LLINs as compared to Indoor Residu- al Spray (IRS) which utilizes all the four clas- ses (3). The delicate gains made in the global reductions of malaria deaths since 2010 due to increased funding and concerted efforts to attain control is threatened by emerging re- sistance to insecticides in Anopheles mosqui- toes (2). Insecticide resistance data exist in Nigeria; however, most of these data are not consist- ently monitored for all communities in Nige- ria. Despite an increase in the reporting of in- secticide resistance in southern (4-9) and North- ern Nigeria (10-11), only very little information on insecticide resistance exist for Gombe state. This suggests a major setback for vector con- trol and weak existing capacity for monitoring insecticide resistance. Where these are avail- able, they are not consistently generated to guide implementation of vector control. Sim- ilarly, there is also limited information on the insecticide susceptibility status of members of the major malaria vector species: An. gam- biae s.l. to available classes of insecticides used in malaria vector control in the Southern Gombe State. This study therefore provides information meeting the demand of The Ni- gerian National Malaria Strategic plan to gen- erate surveillance data that can be used to in- form policy. This study for the first time provides such information on the susceptibility status and identity of the major malaria vectors in south- ern Gombe to different available classes of in- secticides used in vector control. Materials and Methods Study Area The study was carried out in the four Local Government Areas of southern part of Gom- be state, 10°17`N, 11°10`E. Gombe is a state in North-Eastern Nigeria, with its capital at Gombe. The state has an area of 20,265km2 and a population of around 1.8 million. The state is characterized by two distinct seasons, which are dry season (Nov to Mar) and wet season (Apr to Oct). The vegetation of Gombe state can be described as Sudan Savanna with open grassland which dries up during the dry season. Gombe State shares boundaries with Yobe State to the North, Adamawa and Taraba States to the South, Borno State to the East, and Bauchi State to the West. The people of Gom- be south are mainly farmers. They produce both food and cash crops. Among its food crops are yam, cassava, maize, millet, sorghum, cowpea, tomato, groundnut, while cotton are produced for cash. Indigenes also keep cattle, goats, sheep, horses, and donkeys and practice the traditional crafts of weaving and dyeing cotton The study was carried out in 8 farming rural communities namely: Kalorgu (N09°49′34.9″, E01117′27.1″), Ture (N09° 49′09.8″, E011° 22′ 44.2″), Bambam (N09° 42′22.7″, E011°32′ 23.5″), Pantami (N09° 41′35.4″, E011°28′ 23.4″), Pokolin (N09°52′39.8″, E011°12′57.5″), Zazzagawa (N09°53′20.1″, E011°12′34.7″), Filiya (N09°34′38.5″, E011°06′52.4″), Diga (N09°39′41.5″, E11⁰07′10.5″) (Fig. 1). Collection of Anopheles Larvae Third to fourth instar larvae and pupae of Anopheles mosquito larvae were collected from breeding sites in the eight communities from four Local Government Areas (Billiri, Kaltun- go, Balanga and Shomgom) of Gombe South, Nigeria. Anopheline larvae were collected from their natural breeding sites (rice farms, small pools, puddles, and potholes) using the dip- ping method during the months of September to Nov 2014. Coordinates of the study sites were established using The Global Position- ing System (GPS). The immature Anopheles mosquitoes col- lected were transported to the Gombe state J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 52 http://jad.tums.ac.ir Published Online: April 27, 2019 malaria control insectary where they were reared to adults. The emerging adult mosqui- toes were placed in the adult mosquito cages and fed with 10% sugar solution soaked in cotton wool. Separation of Female Anopheles Mosquitoes and Morphological Identification of the Mosquitoes The adult female Anopheles used for the test were separated from the males. Using mor- phological characters (12-13) the adult female mosquitoes were identified under a dissecting microscope. The identification focused on dark spots at the upper margins of the wings which is common to all Anopheles. The palpis are elongated and segmented into three. Speckles on the legs, third pre-apical dark area on vein 1 with a pale interruption and tersi 1–4 with conspicuous pale bands are morphological fea- tures for the identification of An. gambiae. CDC Bottle Bioassay Method Ten to fifteen unfed female Anopheles mos- quitoes of 3–5 days old were introduced into four 250ml Wheaton bottles coated with Tech- nical grade insecticide and one control bottle coated with acetone. These were provided by Center for Disease Control (CDC), Atlanta Georgia as described by the Guideline for Eval- uating Insecticide Resistance in Vectors Us- ing the CDC Bottle Bioassay). The numbers of dead and live mosquitoes were monitored at different time intervals (0, 15, 30, 35, 40, 45, 60, 75, 90, 105, 120min). This allowed the de- termination of the total percent mortality against time for all replicates. The tested mos- quito samples were stored in eppendorf tubes containing silica gel for further analysis. PCR Identification of Members of the Anopheles gambiae complex Mosquitoes identified as An. gambiae com- plex was subjected to species-specific poly- merase chain reaction assays to identify mem- bers of the members. The molecular identifi- cation method was based on specific DNA nucleotide sequences in the intergenic spaces of the ribosomal DNA (14). Further analysis to identify the An. gambiae (formerly ‘S’ form) and An. coluzzii (formerly ‘M’ form) was de- termined by incubating amplified material with HhaI restriction enzymes at 37 degrees over a period of 3h to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzyme HhaI produced patterns of DNA bands which differentiated An. gambiae from An. coluzzii (15). Data Analysis The percentage mortality of the mosqui- toes exposed to each of the insecticides was calculated as the proportion of mosquitoes that died at the diagnostic time for each of the in- secticides. Correction with Abbott’s formula was not necessary as control mortalities was less than 5% throughout the test. WHO rec- ommendations for assessing the significance of detected resistance was used. ccording to the criteria, ≥98% mortality at the recommend- ed diagnostic time indicates susceptibility, ≤97% mortality indicates resistance (16). Data were entered into SPSS ver. 17 software (Chi- cago, IL, USA) and species composition in the study communities were analyzed for signif- icant differences using analysis of variance. A general linear model procedure (t-test in- dependent means at P< 0.05 significance) was also used to compare mortality in A gambiae populations exposed to two different insecti- cides within the same (deltamethrin/ lambda- cyhalothrin, deltamethrin/ permethrin, and lamdacyhalothrin/ permethrin) and different classes (pyrethroids and DDT, Organophos- phate and Carbamate) but having the same target site. Ethical Approval This study received ethical approval from University of Ilorin Ethical Research Com- mittee. J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 53 http://jad.tums.ac.ir Published Online: April 27, 2019 Results Insecticide susceptibility tests Three batches of adult female Anopheles mosquitoes each containing 331, 333 and 345 An. gambiae s.l. mosquitoes were exposed to deltamethrin, lambda cyhalothrin and perme- thrin insecticides (Table 1). There was re- sistance (0–86.4%) in all the study communities to the three different pyrethroids tested except in Bambam community where full suscepti- bility (100%) of the An. gambiae s.l. to lamdacyhalthrin was observed (Table 1). In the same manner another batch of 342, 324 and 344 Anopheles mosquitoes exposed to bendi- ocarb, pirimiphos-methyl and DDT showed re- sistance (0–95%) in all the study communities (Table 2). Except in Bambam and Pantanmi where full susceptibility (100%) to bendiocarb was observed (Fig. 2). The resistance profile of the An. gambiae population showed that sus- ceptibility (percentage mortality ≥97%) were found only in Bambam and Pantanmi com- munities. A GLM procedure to compare mortality in An. gambiae s.l. populations exposed to two different insecticides showed no significant difference between mortality of An. gambiae populations exposed to lambdacyhalothrin and deltamethrin (t=-1.58906, P=0.1344). However, mortalities were significantly higher in both lambda cyhalothrin (t= 5.1234, P= 0.0002) and deltamethrin (t= 5.09965, P= 0.0002) exposed populations of An. gambiae s.l. mosquitoes compared to permethrin. Lambda cyhalothrin and deltamethrin insecticides produced the highest mortality in the population of An. gam- biae in most of the study sites compared to per- methrin. In comparing mortalities in An. gambi- ae populations exposed to two different classes of insecticides (organochlorine/ pyrethroids and bendiocarb/ organophosphate), mortality rate was significantly higher in An. gambiae popu- lations exposed to deltamethrin (t= -0.55542, P=0.587372) and lamdacyhalothrin (t=5.07487, P= 0.000169) compared to DDT. In contrast, there was no significant difference between mortalities observed from An. gambiae s.l. populations exposed to DDT and permethrin (t= -0.55542, P= 0.587372). Similarly, a com- parison of differences in mortality rates be- tween An. gambiae s.l. populations exposed to bendiocarb (carbamate) and pirimiphos- methyl (organophosphate) in all the study sites were also not significant (t= -0.84482, P= 0 .412419). Species composition of members of Anoph- eles gambiae s.l. Out of the 2019 An. gambiae s.l. mosqui- toes subjected to the bioassay, three hundred An. gambiae s.l. mosquitoes selected across the study communities were assayed to detect sib- ling species. Anopheles gambiae (formerly re- ferred to as ‘S’ molecular form) and An. coluzzii (formerly referred to as ‘M’ form), An. ara- biensis and hybrid (An. gambiae/ An. coluzzii) were all found across the study communities (Table 3). Anopheles gambiae (formerly S- form) was the significantly dominant species across the study communities constituting 177 (45%) of the total samples analyzed (Table 3). Anopheles arabiensis accounted for 64 (16.3 %) of the total samples scored in the assay. On the other hand An. coluzzii (M-form) consti- tuted 34 (8.7%). The mosquitoes unaccounted for constituted about 40 (10.2%) and these were samples either misidentified samples or sam- ples with poorly preserved DNA (Table 3). Outside the 40 (10.2%) samples not suc- cessfully amplified, analysis of variance to test difference in species composition of members of the An. gambiae s.l. was significant (P= 0.011369). Results showed that An. gambiae was significantly higher compared to An. coluzzii (P= 0.006433.) and A. arabiensis (P= 0.024755). There was no significant difference between An. gambiae and the hybrid species (P= 0.103996) (Fig. 2). J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 54 http://jad.tums.ac.ir Published Online: April 27, 2019 Table 1. Susceptibility status of Anopheles gambiae s.l. mosquitoes to pyrethroids insecticides Study Sites Deltamethrin Lambda-cyhalothrin Permethrin Number Assayed (331) Mortality N (%) Status Number Assayed (333) Mortality N (%) Status Number Assayed (345) Mortality N (%) Status Kalorgu 43 21 (48.8) R 41 25 (61) R 44 6 (13.6) R Ture 44 25 (56.8) R 43 30 (70) R 41 19 (46.3) R Bambam 40 28 (70) R 40 40 (100) S 47 0 (0) R Pantami 40 24 (58.5) R 44 38 (86.4) R 44 2 (4.5) R Pokolin 41 16 (39) R 41 21 (51.2) R 40 13 (32.5) R Zazzagawa 42 26 (61.9) R 40 17 (42.5) R 44 7 (15.5) R Filiya 40 21 (52.5) R 40 17 (42.5) R 45 13 (28.9) R Diga 41 16 (39) R 44 35 (79.5) R 40 5 (12.5) R N= number of mortality, R=Resistant, S=Susceptible, Numbers in brackets represent percentages No mortality was observed in the control experiment set up for the insecticides in each study sit Table 2. Susceptibility status of Anopheles gambiae s.l. mosquitoes to bendiocarb, pirimiphos-methyl and DDT insecticides Study Sites Bendiocarb DDT Pirimiphos-Methyl Number Assayed 342) Mortality N (%) Status Number Assayed (344) Mortality N (%) Status Number Assayed (324) Mortality N (%) Status Kalorgu 46 40 (87) R 45 16 (35.6) R 40 38 (95) R Ture 41 31 (75.6) R 46 13 (28.3) R 40 34 (85) R Bambam 41 41 (100) S 42 7 (16.7) R 43 31 (72.1) R Pantami 43 43 (100) S 40 10 (25) R 40 27 (67.5) R Pokolin 42 12 (28.6) R 41 0 (0) R 40 26 (65) R Zazzagawa 44 1 (2.2) R 44 8 (18.2) R 41 27 (65.9) R Filiya 44 29 (65.9) R 44 10 (22.7) R 40 30 (75) R Diga 40 24 (60) R 42 16 (38.1) R 40 32 (80) R N= number of mortality, R= Resistant, S= Susceptible, Numbers in brackets represent percentages No mortality was observed in the control experiment set up for the insecticides in each study site Table 3. Distribution and composition of members of Anopheles gambiae s.l. in the study areas Study Sites Number Assayed (N) Species composition A. arabiensis An. gambiae (S-form) An. coluzzii (M-form) Hybrid No amplification (NA) Kalorgu 70 13(18.6) 6(8.6) 2(2.9) 41(58.6) 8(11.4) Ture 52 13(18.6) 22(42.3) 2(3.8) 10(19.2) 5(9.6) Bambam 21 6(28.6) 10(47.6) 0(0) 3(14.3) 2(9.5) Pantami 63 7(11.1) 34(54) 6(9.5) 7(11.1) 9(14.2) Pokolin 58 7(12.1) 34(58.6) 7(12.1) 5(8.6) 5(8.6) Zazzagawa 74 8(10.8) 49(66.2) 11(14.9) 6(8.1) 0(0) Filiya 15 1(6.6) 10(66.7) 3(20) 0(0) 1(6.6) Diga 40 9(22.5) 12(30) 3(7.5) 6(15) 10(25) Total 393 64(16.3) 177(45) 34(8.7) 78(19.8) 40(10.2) N= Total number of An. gambiae s.l., NA= No amplification, Numbers in brackets represent percentage J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 55 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 1. Map of Gombe South, Nigeria showing the study area and sampling localities Fig. 2. Insecticide susceptibility/resistance profile of Anopheles gambiae s.l. mosquitoes in eight selected communities in Southern Gombe Nigeria. Error bars represent variability in the data All charts below the red line indicate that population of An. gambiae in the study communities that are resistant to the classes of insecticides J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 56 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 3. Composition of members of the Anopheles gambiae s.l. and hybrid (A gambiae/ An. coluzzii species in Southern Gombe Discussion Resistance of An. gambiae s.l. mosquitoes to all the four major classes of insecticides were observed in all the study communities in south- ern Gombe. This widespread observation of re- sistance in the major vector An. gambiae s.l. and factors responsible for this upsurge in these communities is not known and should be in- vestigated. However, the absence of contin- uous monitoring data in these communities may have denied vector control managers the opportunity to have detected this trend earlier. Resistance of An. gambiae s.l. to several classes of insecticides is not entirely new in Nigeria (4, 17). Permethrin, bendiocarb and DDT resistance were have been reported in An. coluzzii mosquitoes in Kano, Northern Nigeria (11). Summary of insecticide resistance cases and number of countries reporting this have constantly been on the rise (2). However, a consideration of factors (usage of ITN, IRS, agricultural pesticides) previously reported se- lecting for insecticide resistance (17, 18) pro- vided a reasonable clue of what could be the driver of insecticide resistance in Southern Gombe. Utilization of insecticide nets by persons in Gombe is 34%, which is just below the na- tional average of 37% (1). Gombe state ranked 8th out of the 38 states with the highest per- centage of households with at least one ITN (1). There is a significant usage of Insecticide treated nets in Gombe state in Nigeria hence, these insecticide based tools may have con- tributed to the insecticide pressure in select- ing for resistance in the Anopheles gambiae population. On the other hand, the use of IRS may not have added significantly to this pre- sent scale of insecticide resistance because cov- erage of IRS in all the states in the 6 geopo- litical zones of Nigeria is just 2.5% (1). As such, there seem to be some other major con- tributors to the multiple insecticide pressure oc- casioned in these localities. A literature search for recent update on pesticide utilization in Gombe state yielded a positive result on what may have been the source of these insecticide pressures. WAAP (19) reported the widespread cultivation of hor- ticultural crops on which considerable amount of pesticides of different classes are used in Gombe state. Hence, by inference, agricultural use of insecticides therefore may have con- J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 57 http://jad.tums.ac.ir Published Online: April 27, 2019 tributed to the current modification of mosquito susceptibility to the pesticides in Southern Gombe. The status of pyrethroid resistance status of An. gambiae s.l. population in all the study sites is worrisome considering that the major malaria vector control tools in Ni- geria rely on this class of insecticides (20). However, the disparities in mortality rates ex- pressed by the Anopheles population to each of the pyrethroid insecticides indicate that rate of resistance development vary with different insecticides in each location and population. For example, while higher mortality rates (51.2–100%) to lamdacyhalothrin was observed in the Anopheles. populations at all the study sites, lower mortality rates were observed in populations exposed to permethrin (0–46.3%) and deltamethrin (39–70%). This variation is an important indication of the need to introduce insecticide resistance management strategies to slow down the rate at which these mosquitoes develop resistance to the pyrethroids insecticides. This will invari- ably help in prolonging the shelf life of the few available classes of insecticides on which ITNs and IRS rely upon. Recent report from an ag- ricultural setting in Jigawa state: another north- ern state in Nigeria showed multiple resistances to a pyrethroid, carbamate and an organochlo- rine (10). Similar evidences of full suscepti- bility to lambda-cyhalothrin as reported in Bambam study community have also been re- ported in Kenya (21), Sudan (22), Tanzania (23) and Nigeria (24). In observing the pattern of resistance in the vectors, full susceptibility to bendiocarb was observed in, Bambam and Pantami which are both neighbhouring com- munities. This suggests the focal nature of the in- secticide resistance status of Anopheles pop- ulations in each of these communities. While bendiocarb resistance was observed in Anoph- eles population in 6 out of the 8 communities, resistance to pirimiphos-methyl was observed in the An. gambiae populations from all the communities tested. Resistance to pirimiphos- methyl (an organophosphate) in Gombe is of great concern to vector control manager be- cause it is not currently used in any of the current vector control tools. However, the wide- spread resistance in Anopheles population in 8 communities to pirimiphos-methyl (organ- ophosphate) and the co-resistance to bendio- carb (a carbamate with same target site with organophosphate) point towards other sources as these have not been used for vector control. In contrast, findings from Auyo, a communi- ty in Jigawa, Nigeria, Kouande and Tangui- eta in Atacora communities, in Republic of Benin, the An. gambiae populations were susceptible to pirimiphos-methyl (10, 25). In this study, all the Anopheles populations were resistant to deltamethrin exposure in all the study communities. Similar findings of del- tamethrin resistance have been reported in Benin, West Africa (26, 27). Contrary to the findings of this study, in Khartoum city of Sudan, high susceptibility to deltamethrin was reported (22). In this study, similar lower mortality of Anopheles mosqui- toes from all the study communities to per- methrin have been reported severally in Ni- geria: Oyo, Lagos, Niger and Bauchi states (4, 24). Similar evidence of resistance to perme- thrin have also been reported in Atacora, Benin (27). Evidence of DDT resistance was high in this present study and this was not surpris- ing considering that both pyrethroids and DDT (organochlorine) share the same target site. High resistance to one class may have led to cross-resistance in the other class. Insignifi- cant differences in the mortality rates from permethrin and DDT mortality in the Anoph- eles population also showed similar patterns of resistance. A molecular analysis would have provided additional information on probable existence of similar resistance mechanisms in the pop- ulations; however, logistics was a major lim- itation to this study. Several studies in Nigeria have reported high levels of permethrin- DDT resistance in An. gambiae and An. coluzzii in J Arthropod-Borne Dis, March 2019, 13(1): 50–61 A Oduola et al.: Widespread Report of … 58 http://jad.tums.ac.ir Published Online: April 27, 2019 Nigeria (4, 6, 8 and 24). Evidence of resistance to pirimiphos-methyl (mortality rate between 65–95%) was observed in the An. gambiae populations in all the study communities. In comparing mortalities in An. gambiae popu- lations exposed to two different classes of in- secticides (organochlorine/ pyrethroids and bendiocarb/ organophosphate), showed there is significant differences in mortality rates of populations exposed to deltamethrin and lamb- da cyhalothrin (class II pyrethroids). Deltame- thrin and lambda cyhalothrin are still effec- tive compared with permethrin (a class I py- rethroids). Similarly, a comparison of differ- ences in mortality rates between An. gambiae s.l. populations exposed to bendiocarb (car- bamate) and Pirimiphos-methyl (organophos- phate) in all the study sites were also not sig- nificant suggesting possible shared resistance mechanisms. This however requires laboratory validation. Identification of the Anopheles species col- lected in this study showed the predominance of An. gambiae s.l. as the major malaria vectors in all the study communities. The An. gam- biae complex includes sibling species that are the most important vectors of malaria in sub- saharan Africa (28). Of these species An. gam- biae (formerly S-form), An. coluzzii (former- ly M-form) A. arabiensis and hybrid species (An. gambiae/ An. coluzzii) were found to be sympatric in all the study communities. In this study, An. gambiae (formerly S form) was the predominant species over An. arabiensis, An. coluzzii and the hybrid species identified. The sympatric occurrence of An. gambiae, An. coluzzii and An. arabiensis species have been reported in North–Central Nigeria (29). While this study reported, a higher pro- portion (19.8%) of An. gambiae/An. coluzzii hybrids than any previous report in Nigeria (6, 12, 30). Information on the role of these hybrids and their contribution to malaria trans- mission is currently unavailable. In this study, An. gambiae s.s (formerly S form) was found to be the most predominant species. In con- trast, this species was either found to be en- tirely absent or in insignificant proportions where the hybrid occurred with predominant An. coluzzii (6, 10, 12, and 30). This raises questions on the fitness status of these hy- brids compared to the disappearing An. gam- biae when exposed to insecticides. The pre- ponderance of An. gambiae (formerly S form) over An. coluzzii species has always been at- tributed to ecological preference for drier areas of the savannah regions or areas with tempo- rary breeding habitats. The species composi- tion outcome of this study is in agreement with previous nationwide mapping of the distribu- tion of members of the An. gambiae complex in the Sudan savannah where the study com- munities are located (31, 32). Conclusion Monitoring insecticide resistance is an in- tegral part of measures against malaria vec- tors and the resistance management strate- gies depends on the knowledge of the com- position of malaria vectors in an area as well as their resistance profiles. Anopheles gam- biae, An. coluzzii, An. arabiensis, and the hy- brid species identified in all the study com- munities in Southern Gombe were resistant to all the four classes of insecticide approved for vector control. There is an urgent need for the implementation of insecticide resistance management strategies in these localities. Fur- ther studies must also be implemented to as- sess the operative resistance mechanisms in order to inform the choice of alternative vec- tor control interventions to be considered. Acknowledgements The authors wish to thank David Hoel of the Center for Disease Control Atlanta Geor- gia for providing us with the CDC bioassay kits. 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