Susceptibility test of female anopheles mosquitoes to ten insecticides for indoor residual spraying (IRS) baseline data collection in Northeastern Nigeria

Department of Biological Sciences, University of Maiduguri, Maiduguri, Borno State, Nigeria. Department of Crop Protection, University of Maiduguri, Maiduguri, Borno State, Nigeria. Abt Associates/USAID/PMI-AIRS Project, Abuja, Nigeria. Federal Ministry of Health, Abuja, Nigeria. Department of Biology, Abubakar Tafawa Balewa University, Bauchi, Bauchi State, Nigeria. Department of Biology, College of Education, Azare, Azare, Bauchi State, Nigeria. Bauchi State Malaria Control Booster Project, Bauchi, Bauchi State, Nigeria.


INTRODUCTION
WHO current estimates show that malaria mortality rates were reduced by about 42% globally and by 49% in the WHO African Region between 2000 and 2012 and during the same period, malaria incidence rates declined by 25% around the world, and by 31% in the African Region (WHO, 2013a).
In Nigeria, malaria accounts for 60% of outpatient visits to health facilities, 30% of childhood deaths, 25% of death in children under one year and 11% maternal death in addition to about 132 billion naira financial loss in the form of treatment costs, prevention, loss of man-hours, etc in Nigeria (FMoH/ NMCP, 2009). In Nigeria, the economic impact of malaria can be attributed to low gross national income per capital (GNI) of US$260 (FMoH, 2005).
In recent times, IRS is being adopted and scaled up to protect the entire household and community members who possibly have no access to treated bed nets in Africa (Beier et al., 2008).
The Federal Government Policy on Malaria Control in Nigeria focuses on LLINs, IRS, intermittent preventive treatment (IPT) and environmental management (NMCP, 2014). In line with these strategies, the National Malaria Elimination Programme (NMEP) in Nigeria has scaled up indoor residual spraying (IRS) to achieve 85% coverage in 20% of eligible structures in Nigeria in 2014. To achieve these target, IRS activities was progressively expanded in the seven World Bank Supported Malaria Booster States of Bauchi, Gombe, Kano, Jigawa, Rivers, Anambra, Akwa Ibom states, Nigeria from 2009 to 2014 to supplement LLIN and environmental management.
Currently, WHOPES recommends 12 insecticide compounds and formulations, belonging to four chemical classes, for deployment in IRS program (WHO, 2009). The major challenge in use of these insecticides in malarial vector control has been the development of resistance to insecticides among the vector populations. Anopheles mosquitoes resistance to insecticides is spreading rapidly across African countries (Awolola et al., 2002(Awolola et al., , 2005(Awolola et al., , 2007Ndams et al., 2006;Oduola et al., 2010;Ranson et al., 2011;Kabula et al., 2012;Natacha et al, 2013;Ibrahim et al., 2014) and could reduce the impact of malaria prevention interventions using IRS and LLINs, particularly in sub-Sahara Africa (NGuessan et al., 2007;Awolola et al., 2008).
The successful implementation of IRS program partly depends on availability of insecticide(s) susceptible Anopheles mosquitoes in the local environment. Therefore, it is imperative to periodically conduct bioassays tests to assess the susceptibility status of local mosquito species to IRS interventional insecticides. The susceptibility of Anopheles mosquitoes against insecticides was fairly evaluated in southern parts of Nigeria (Olayemi et al., 2011;Oduola et al., 2012) but there was dearth of information in the northern Nigeria (Molta and Ali, 1998;Ndams et al., 2006). No documented evidence on the susceptibility status of Anopheles mosquitoes to guide procurement of IRS insecticide in Northeast Nigeria is available. Therefore, the presents study was conducted to provide baseline data on insecticides susceptibility status of local Anopheles mosquito in Misau, Bauchi State, Nigeria.

Study area and period
The study was conducted in August 2010 in Misau town, Misau L.G.A located at latitude 11.31897 and longitude 10.47587, human population of 263,487 as at the 2006 census with an area of 1,226km 2 . IRS was scaled up in 2009 in the three wards of Misau (Gundari, Kukadi A and Kukadi B) where Lambdacyhalothrin, Deltamethrin and Bifentrin respectively were used. The total coverage for insecticides was 52,000 households.The community has been using LLINs since 2002 till date. The farmers in the suburb cultivate vegetables, rice and wheat on the wetlands where agrochemicals (cypermethrin, lambdacyhalothrin, deltamethrin, dichlovos and primiphos-methyl) are used in pest control. The wetlands also has number of tube bore holes to supplement provision of portable water to Misau community. Pools of standing water from the wetlands and tube bore holes provide active breeding sites for the Anopheles mosquitoes.

Mosquito collection and rearing
The Anopheles species larvae were collected in naturally infested waterbodies in Misau using entomological ladles. When culicine larvae were collected, they were separated from the Anopheline larvae and discarded on the land. The emerging pupae were sucked out of the larval containers using pipette and kept in plastic cups inside a mosquito cage made from five(5) litres white plastic bucket, fastened with cone shape white mosquito netting with its rear end tied in to a knot to prevent escape of emerging adult mosquitoes. The adult that emerged in 1-3 days were reared according to methods of Umar et al. (2008).
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Bioassay techniques
Insecticide susceptibility tests were carried out using the WHO standard procedures and test kits for adult mosquitoes (WHO, 1998). The bioassay was conducted using 2-3 days old, glucosefed but non-blood fed female Anopheles mosquitoes.
For each insecticidal paper and the control, a three replicates of 20 adult female Anopheles mosquitoes were exposed to tubes and allowed to stand for 1 h and numbers of knocked-down mosquitoes were recorded at intervals of 10 min. After the exposure, mosquitoes were then transferred to a recovery tubes and fed with a pad of cotton wool soaked in 10% glucose solution. The holding tubes were kept for 24 h in a secluded, shaded and sterile place. Adult mortality was assessed after 24 h post-exposure by inability to stand upright and walk when probed with glass rod. The dead and survived mosquitoes at the end of experiment were separately kept in labeled 1.5 mL Eppendorf tubes containing silica gel, for species identification. The susceptibility tests were conducted in laboratory under fluctuating temperature (25-33°C) and relative humidity (90-95%).

Identification of Anopheles mosquitoes
Morphological keys of Gillies and DeMeillon (1968) and Gillies and Coetzee (1987) were used in morphological identifications of adult Anopheles mosquitoes.

Data analysis
The knockdown data was subjected to probit analysis using a statistical software (Statsdirect, 2013) to compute the KDT50 and KDT90 (time taken to knockdown 50 and 90% of the exposed mosquitoes) and their 95% confidence intervals. The 24 h mortality was manually assessed. The susceptibility of Anopheles mosquitoes to insecticides was assessed using the current WHO (2013b) criteria: A mortality in the range 98-100% indicates susceptibility and a mortality of less than 98% is suggestive of the existence of resistance. The adult mortality in control experiments were less than 5% and hence were not corrected for (Abbott, 1925).

RESULTS
The results of knockdown assessment of female Anopheles mosquitoes exposed to ten different insecticide impregnated papers is presented in

DISCUSSION
The present study presents for the first time baseline data on the susceptibility status of Anopheles mosquitoes to WHOPES approved IRS insecticides in Misau, Bauchi State, Northeastern Nigeria to guide procurement of IRS insecticides in the state.
The results of knockdown assessment showed that the tested insecticidal papers induced knockdown of adult Anopheles mosquitoes suggesting that knockdown mechanism could be operating in the local Anopheles mosquito populations. This confirm earlier studies which separately indicates the knockdown effects of impregnated papers against Anopheles mosquitoes in Nigeria (Awolola et al., 2005;Oduola et al., 2010;Olayemi et al., 2011;Oyewole et al., 2011;Ibrahim et al., 2014). The knockdown of Anopheles mosquitoes exposed to insecticidal papers indicates the presence of KDR resistance mechanism (Kristan et al., 2003;Awolola et al., 2007;Ibrahim et al., 2014) operating in the populations of Anopheles mosquitoes in Misau.

Insecticide group
Insecticidal paper Concentration (%) The multiple insecticide resistances of Anopheles mosquitoes to the tested pyrethroids, carbamates and organochlorine insecticides may have grave implications for the malaria control programme. It may compromise the efficacy of interventions and potentially lead to the failure of IRS and ITNs based vector control (Awolola et al., 2008).
The resistance of Anopheles mosquitoes to bifenthrin, lamdacyhalothrin and deltamenthrin may be linked to use of these insecticides in 2009 IRS intervention in the communities. It is established that prior exposure of mosquitoes to insecticides may induced selection pressure (Kerah-Hinzoumbé et al., 2008). Pyrethroids based aerosols and coils are used for control of mosquitoes and domestic pests and it might contribute to the development of resistance as reported elsewhere (Kristan et al., 2003). The farmers in the community also use cypermethrin, lambdacyhalothrin, deltamethrin, dichlovos and primiphos-methyl for agricultural crop protection. Previous researchers have reported that exposure of malarial vectors to crop protection insecticides could result in development of insecticide resistance (Etang et al., 2003;Awolola et al., 2007;Müller et al., 2008;Chouaibou et al., 2008;Bigoga et al., 2012;Philbert et al., 2014). LLIN was used in Misau for protection against mosquitoes since 2002 to date and it may induce selections to pyrethroids insecticides. Previous studies revealed that use of LLIN could result in development of insecticide resistance in Anopheles mosquitoes (Kabula et al., 2011).
The morphological analysis of preserved mosquito samples showed populations of A. gambiae, A. funestus and A. nilin were used in the bioassays. A. gambiae is the principal vector of malaria in sub-Saharan Africa (Gillies and Coetzee, 1987;Samdi et al., 2006;Sinka et al., 2010). The fauna of A. gambiae, A funestus and A nili was earlier documented in northern Nigeria (Molineaux and Gramiccia, 1980;Gadzama, 1983;Molta et al., 1999;Samdi et al., 2006;Ahmed, 2013). The A gambiae and A. funestus are major malarial vector in Nigeria (Molineaux and Gramiccia, 1980) and have great implication in malaria transmission in Bauchi State. Therefore, periodic monitoring of insecticides resistance in this mosquito species is imperative to avoid vector control failures.

Conclusion
It is concluded that procurement of IRS insecticide(s) in the state should be guided by the results of the present study until new susceptibility status is established and resistance management strategies should be considered when using the less susceptible insecticides. It is recommended that future studies should focus on investigation on the A. gambiae and A. funestus complexes and elucidations of resistance mechanisms in these mosquito species.