Species composition and insecticide resistance status of Anopheles gambiae (s.l.) (Culicidae) in Kome, southern Chad and the implications for malaria control
© The Author(s). 2016
Received: 17 March 2016
Accepted: 15 August 2016
Published: 23 August 2016
The development and spread of insecticide resistance among malaria vectors, is a threat to the continued effectiveness of interventions to control and eliminate the disease. The status of insecticide resistance among malaria vector populations at two sites in Kome, southern Chad, was evaluated to inform decisions on vector control.
Mosquito larvae were collected from temporary rain-filled and semi-permanent breeding places at two sites and reared in a laboratory. Emerging Anopheles gambiae (senso lato) (s.l.) adults were morphologically identified, sorted and evaluated for susceptibility to WHOPES recommended insecticides. Standardized biomolecular and biochemical methods were used to determine sibling species and molecular forms: knockdown resistant alleles (kdr-w) for pyrethroids and DDT; acetylcholinesterase-1 resistant alleles for organophosphate and carbamates; biochemical resistance through measurement of the levels of non-specific esterase (α and β), oxidase and glutathione-s-transferases activities.
Anopheles gambiae (s.l.) was the main vector group in the two study sites and comprised of Anopheles gambiae (senso stricto) (s.s.) and An. arabiensis, respectively, at 71 and 29 % in Site A, and 60 and 40 % at Site B. Anopheles gambiae (s.s.) was composed of M (Anopheles coluzzii) and S [nominotypical An. gambiae (s.s.)] molecular forms. Anopheles coluzzii accounted for over 98 % of the sub-group. There was extensive phenotypic resistance to pyrethroids, DDT and carbamates, but full susceptibility to organophosphates. Population-wide frequency of knockdown resistant allele in An. gambiae (s.l.) was 43 homozygous (RR), 19 heterozygous (RS) and 38 % homozygous susceptible (SS). When segregated by species and molecular forms, An. coluzzii had the highest kdr-w frequency of 37.4 homozygous resistant alleles, and 17.5 % heterozygous, with 8.3 % homozygote susceptible alleles. An. gambiae (s.s.) had 1 % homozygous resistant allele. Levels of esterase, oxidase and glutathione-s-transferases were not significantly different compared to fully susceptible laboratory raised An. gambiae (s.s.) Kisumu reference, although few individuals showed significant elevation of esterases (> 0.04 μg/protein), indicating a likely start of biochemical enzyme resistance.
There is an urgent need for action to stop and reverse significant insecticide resistance in the area. A comprehensive entomological surveillance and monitoring program is needed to understand the full extent of resistance to enable realistic insecticide resistance management strategy, and also to track future changes in the vector populations.
There have been dramatic reductions in malaria cases in many places around the world, over the last decade . The development of insecticide resistance by mosquito vectors of malaria, however threatens this achievement, as it could undermine the efficacy of the mostly insecticide-based vector control interventions , including the current main interventions of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). The World Health Organization (WHO) currently recommends six pyrethroids for impregnating nets and 12 insecticides, belonging to four classes of insecticides (pyrethroids, organophosphates, carbamates and organochlorines), for IRS. The insecticides work similarly, by targeting specific sites in the nervous system on insects. Resistance arises when mutation at a target site prevents the insecticide from binding and thereby blocks the action of the insecticide. Knockdown resistance (kdr) results from a mutation in the sodium ion channel target site of DDT and pyrethroids, while target site resistance for carbamate and organophosphates is caused by mutation of the G119S Acetylcholinesterase gene (Ace-1R) [3, 4]. Biochemical or metabolic resistance is conferred by increased production of specific enzymes that metabolize or sequester the insecticide before it exerts toxic effects. Three main enzyme systems are involved in metabolic resistance: esterases, mono-oxygenases and glutathione S-transferases. Different enzymes affect different classes of insecticides.
The West Africa region is noted for elevated insecticide resistance in local mosquito vectors. Target site and metabolic resistance have been demonstrated in parts of Chad and in almost all the bordering countries [5–11]. Chad has adopted the current WHO policy of universal LLIN coverage of all populations at risk of malaria, and has been implementing large scale use of LLINs. As at 2013, over 50 % of the population reported coverage . Very limited application of IRS occur in small locations in the southern part of the country. There are significant gaps in the knowledge on the malaria vectors in the country, and the levels and distribution of insecticide resistance in the vector populations have not been adequately characterized . Although the epidemiological impact of pyrethroid resistance on the efficacy of LLINs and pyrethroid-based IRS, is unclear, limited studies elsewhere in West Africa point to potential reduction in efficacy . Pursuant to the goals of the WHO global action plan for Insecticide resistance management , it is important for Chad to redouble efforts to generate adequate information on the status of insecticide resistance, to enable the development of realistic strategies to stem and reverse the development of insecticide resistance in the country. This study contributes to that effort.
Larval collection sites
Each study site covered about square miles, with the two sites separated by about 8 miles. Temporary rain pools and semi-permanent breeding sites in the two sites, were sampled for mosquito larvae. Sampling took place from 15th October 2013 to 7th November 2013, during the last part of the rainy season. The larvae collected from the two sites were reared separately to adults in the laboratory at temperature of 28 ± 2 °C and relative humidity of 72 ± 5 %. Emerging adult female An. gambiae (s.l.) were morphologically identified using the taxonomic keys of Gillies & Coetzee , sorted out, and fed on 10 % sugar solution. These vectors were subsequently used for the evaluations described in following sections.
Determining phenotypic resistance
The WHO tube method for insecticide susceptibility  was used for determining the phenotypic resistance. Non-blood fed female An. gambiae (s.l.), 2 to 5 days post-emergence, were exposed to WHO diagnostic doses of nine insecticides currently recommended for malaria vector control (deltamethrin 0.05, DDT 4.0, fenithrothion 1.0, lamdacylothrin 0.05, malathion 5, pirimiphos methyl 0.9, propoxur 0.1, bendiocarb 0.1 and permethrin 0.75 %). For each insecticide, a total of 100 An. gambiae (s.l.) were tested, comprising four replicates of 20 mosquitoes per test and a control setup of 20 mosquitoes. Twenty four-hour mortality was scored and susceptibility levels determined using WHO criteria .
Vector species identification
Samples of the dead and surviving morphologically identified An. gambiae (s.l.) from the susceptibility evaluation, were identified into sibling species An. gambiae Giles (senso stricto) and An. arabiensis, using ribosomal DNA-polymerase chain reaction (PCR) . A PCR-RFLP (restriction fragment length polymorphism) procedure described by Fanello et al. , was then used to separate An. gambiae (s.s.), into the M (An. coluzzii) and An. gambiae (s.s.) molecular forms.
Determining target site resistance
The Hot Oligonucleotide Ligation Assay (HOLA) technique described by Lynd et al. , and real time PCR described by Bass et al.  was used to detect the presence of L1014F West Africa knockdown resistance gene (kdr-w) in samples of dead and surviving mosquitoes from the insecticide susceptibility tests. The presence of Ace-1R alleles was identified using the protocol described by Weill et al. .
Biochemical and metabolic enzyme activity
A fresh sample of non-blood fed 1–3 day-old first generation females from wild-caught An. gambiae (s.l.), were selected and killed by storing in a freezer at -20 °C in the field station, then ice-packed and transported to the laboratory and stored at -80 °C for further analysis. Kisumu strain An. gambiae (s.s.) was used as a reference for the assays. The levels of non-specific esterase (α and β), glutathione-s-transferase (GST) and mixed-function oxidase activities in the samples were quantified. Anopheles gambiae (s.s.) Kisumu susceptible strains were used as cut-off point to estimate activity levels (mean + 3 standard deviations, SD). Enzyme assays were performed using the method designed by Brodgon et al. . The concentration of GSTs and acetylcholinesterase produced were calculated following methods described by Hemingway et al. . An extinction coefficient of 4.39 m/M and 13.6 m/M (corrected for a path length of 0.6 cm) was used to convert absorbance to moles of product for GST and acetylcholinesterase, respectively.
Results and discussion
Status of phenotypic resistance
Insecticide susceptibility of An. gambiae (s.l.) from two study sites in Kome, southern Chad exposed to WHO diagnostic doses for nine insecticides (knockdown at 60 min and percent mortality at 24 h)
% knockdown at 60 min (95 % CI)
% 24 h mortality (95 % CI)
% knockdown at 60 min (95 % CI)
% 24 h mortality (95 % CI)
Permethrin, 0.75 %
Lamdacyhalothrin, 0.05 %
Deltamethrin, 0.05 %
DDT, 4.0 %
Fenithrothion, 1.0 %
Malathion, 5 %
Pirimiphos methyl, 0.9 %
Propoxur, 0.1 %
Bendiocarb, 0.1 %
Composition of the local population of An. gambiae (s.l.)
Molecular forms of Anopheles gambiae (s.s.)
Detection of knockdown resistance (kdr-w) gene mutation
The results add to the growing evidence that kdr mutations may not be homogeneously distributed in An. coluzzii and An. gambiae (s.s.) molecular forms of An. gambiae (s.s.) . We postulate that An. coluzzii probably face comparably higher selection pressure for kdr, from agriculture and related human activities, as a result of sustained presence throughout the year. The kdr-w resistance allele was also present in An. arabiensis (n = 206) with more homozygote susceptible (SS) alleles observed (Fig. 4).
As anticipated, there was a significantly (Pearson Chi-square test: χ 2 = 38, df = 1, P < 0.0001) higher frequency of kdr-w allele among the mosquitoes that survived the susceptibility tests, while those that were killed mostly had susceptible genes. The presence of about 36 % heterozygous kdr-w in the individuals that survived the WHO susceptibility tests, however indicates ongoing development of resistance and thus, a need to arrest a further spread of the resistance.
There is evidence suggesting that An. arabiensis maintains a permanent population spread over a large area in West Africa, with a potential high level of gene flow . This means that insecticide resistance genes could potentially spread rapidly within the country and sub-region. Therefore, although the current frequency of kdr-w in An. arabiensis in the Kome area is low, it is nonetheless present and could increase and spread rapidly, if effective management strategies are not put in place.
Acetylcholinesterase target site mutation
Of all the total 137 individuals tested, the homozygous susceptible Ace-1R allele was the most predominant. The same result was obtained for all categories investigated including An. arabiensis, both An. coluzzii and An. gambiae (s.s.), and status of the kdr-w allele (homozygous, heterozygous or resistant) and irrespective of whether or not they survived the WHO insecticide susceptibility test. Recalling that the vectors were fully susceptible to organophosphates while resistant to carbamates (negative correlation), the absence of cross resistance denotes a likelihood that the carbamate resistance was due to a mechanism other than Ace-1R target site mutation and that metabolic resistance may be the main mechanism involved in the development of resistance. Further evaluation is recommended.
Metabolic and biochemical resistance
Enzyme activity levels in An. gambiae (s.l.) samples from site A and B in Kome, southern Chad
Number of vectors tested
α esterase (μmol/min/mg protein)
Oxidase (μmol/min/mg protein)
GST (μmol/min/mg protein)
Mean ± SD
Mean ± SD
Mean ± SD
0.19 ± 0.08 (P = 0.496)
0.11 ± 0.05 (P = 0.211)
0.048 ± 0.027
0.21 ± 0.11 (P = 0.084)
0.12 ± 0.09 (P = 0.097)
0.06 ± 0.024
An. gambiae (s.s.) Kisumu reference
0.18 ± 0.085
0.1 ± 0.041
0.08 ± 0.037
The fact a few individual mosquitoes showed elevated levels of esterases may indicate that, biochemical mechanisms may be contributing, alongside the knockdown resistant gene in the phenotypic expression of resistance. Mixed function oxidases may be acting together with kdr to create pyrethroid resistance. The phenomenon has been linked to intervention failure involving An. coluzzii elsewhere in West Africa [26, 27]. The enzyme level activity of the vectors in Kome area must be carefully monitored because of this potential threat.
There is an urgent need for comprehensive entomological surveillance and monitoring program in south-western Chad, to inform the development and implementation of an effective insecticide resistance management strategy. The high level of resistance among the local malaria vectors in the area for three out of the four groups of WHOPES recommended insecticides, threatens the achievement of malaria control objectives. In particular, the significant resistance to LLIN insecticides in an area targeting universal LLIN coverage must elicit renewed efforts by national and developmental partners to address the problem. Immediate actions include active promotion of judicious use of insecticides, particularly in agriculture, to reduce the selective pressure; concerted evaluation of the utility of LLINs with insecticide-synergist combinations, which have been shown elsewhere to reduce pyrethroid resistance in An. gambiae  and is now recommended by WHO under certain conditions. The coinciding occurrence of a complete absence of Ace-1R gene mutation, in the presence of settled carbamate resistance, but full susceptibility to organophosphate, adds to the growing evidence of the complexity of the interplay of biochemical, biological and gene target site mutation in the phenotypic expression of resistance.
The authors would like to thank the National Malaria Control Program (NMCP) of the Ministry of Health, Republic of Chad, for facilitating access for the study; Stephane Nguembatoum and Klamadje Djimoko; the team at the entomology laboratory at Noguchi Memorial Institute of Medical Research, Ghana, for their outstanding support on the biomolecular assays; Kathryn Welter and the administrative team at RTI for project management support.
Availability of data and materials
Data supporting the conclusions of this article are included within the article.
JW conceived and coordinated the study. SD, MCA, DK, AF, CKH and JW collected the data. SD, MAA, MA conducted the biomolecular laboratory evaluations. JW, SD, MCA, CKH, MA analyzed the data, JW and SD drafted the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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