Study area
The study was carried out in three Local Government Areas (LGAs) (Dekina, Ibaji and Omala LGAs) in Kogi East, Kogi State, Nigeria. Kogi East is located in the north-central geopolitical zone of Nigeria which lies in the guinea savannah. The state has two distinct weather; dry season, which lasts from November to March and wet season extends from April to October and annual rainfall ranges from 1016 to 1524 mm. Kogi State has an annual temperature range of 22.8 and 33.2 0C. Lokoja, the state capital is moderately hot throughout the year. The vegetation of the state consists of mixed leguminous (guinea) woodland to savannah forest. Kogi State has wide expanse of fadama in the river basin and long stretches of tropical savannah forest in the Western and Southern belt of the state [30]. The state has a population of projection of more 4,473,500 in 2016 [31] and aside civil service, agriculture is the major occupation of the inhabitants.
Mosquito collection and rearing
Eggs, larvae and pupae of An. gambiae were collected during the wet season from April to October 2017. The immature were collected from 228 natural breeding sites (tyre tracks, rice fields, puddles etc) across the three LGAs using soup ladle, dippers and pipette. These larvae and pupae were transported in containers containing the waters from where they were collected to the insectary of the Department of Biological Sciences, Kogi State University, Ayingba, Kogi State, Nigeria. They were reared to adults under standard protocols as described in Nkya et al. [32]. The larvae were fed daily with a mixture of finely ground fish food and brewer’s yeast, while pupae were transferred into pupae cups and placed in cages. Emerging adults were maintained on 5% sugar solution until they were used for insecticide susceptibility test. Kisumu strain of An. gambiae collected from National Arbovirus and Vector Research Centre, Enugu, Nigeria was used as the reference strain.
Insecticide susceptibility test
Adult susceptibility tube bioassays were carried out with eight insecticides representative of all four classes of insecticides available for use in public health. This was done using WHO established methods [33]. WHO insecticide treated filter papers with diagnostic doses recommended by WHO were used [33]. The insecticides included 4% DDT (organochlorine), alphacypermethrin 0.5%, permethrin 0.5%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, (pyrethroid), bendiocarb 0.1% (carbamate) and pirimiphos-methyl 0.25% (organophosphate). Tube bioassay was conducted using 3 to 5 days old non blood fed adult female mosquitoes.
An adult mosquito was considered alive if it is able to fly, regardless of the number of legs remaining. Any knocked-down mosquitoes, whether or not they have lost legs or wings, were considered moribund and were counted as dead. On completion of the susceptibility test, a subset of mosquitoes from each collection area were properly preserved in RNAlater and SCB solution and send with ice packs using WMX box to the Laboratory of Institute of Molecular Biology and Biotechnology, Heraklion, Greece for molecular analysis. Further investigations were carried out for the identification of resistance mechanisms as noted above if on the diagnostic concentrations a significant number of survivors were found (more than 2%). Further tests were conducted in order to determine the underlying genetic mechanisms responsible for the observed resistance. These investigations included identification of the survivors, and at least 20% of dead mosquitoes, in order to identify in which species the signs of resistance are present. This information was used in assessing the likelihood of cross-resistance between insecticides classes, and also provided valuable information about the potential for spread of resistance in vector populations [33].
Morphological identification
Adult anophelines were identified morphologically with the aid of a light microscope and taxonomic keys of Gillies and De Meillon [34] and Gillies and Coetzee [35].
DNA extraction from single mosquitoes
DNA was extracted from individual mosquitoes preserved in ethanol using the DNAzol™ Reagent (Invitrogen), for isolation of genomic DNA from solid and liquid samples according to the manufacturer’s instructions. The pellet containing DNA was dissolved in 15.0 μL of DEPC treated water.
Total RNA and DNA extraction from mosquito pools
Total RNA and DNA were extracted using a magnetic beads-based approach using the MagaZorb kit (Promega). The quantity and purity of DNA and total RNA were assessed spectrophotometrically (Nanodrop). The quality of RNA was assessed by 1.0% w/v agarose gel electrophoresis.
Specific identification
Each mosquito was identified to species level using the polymerase chain reaction (PCR) assay of Scott et al. [36] and slightly modified by Van Rensburg et al. [37].
Genotyping of mosquito samples and multiplex RT-qPCR for gene expression analysis
Species ID and target site mutation determination were performed using the assays described in the IVCC Vector Population Monitoring Tool (VPMT) Protocol [38] with minor modifications.
Newly developed and integrable to the LabDisk automated diagnostic platform quantitative Reverse Transcription-real-time PCR (qRT-PCR) 3-plex TaqMan® assays were used for the quantification of 7 detoxification genes’ expression (CYP6P3, CYP6M2, CYP9K1, CYP6P4, CYP6Z1, GSTE2, CYP6P1, CYP4G16) using RPS7 for normalization purposes in each assay. Reactions were performed in the Viia7 Real-Time PCR system (Applied Biosystems) using a one-step RT-PCR mastermix supplied by FTD (Fast-track diagnostics, Luxembourg) in a total reaction volume of 10 uL. The thermal cycle parameters were: 50 °C for 15 min, 95 °C for 3 min, and 40 cycles of 95 °C for 3 s and 60 °C for 30 s, presenting a sample to result time of ~75 min. Duplicates of samples were amplified and each run always included a non-template control.
Statistical analysis: Calculation of fold-changes, 95% CIs and statistical significance was performed according to the Pfaffl method [39]. Graphs were constructed with the SigmaPlot software (v12.0).