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Preliminary assessment of the potential role of urbanization in the distribution of carbamate and organophosphate resistant populations of Culex species in Ghana
© Kudom et al.; licensee BioMed Central. 2015
Received: 30 May 2014
Accepted: 22 December 2014
Published: 8 January 2015
Besides its role as a pathogen vector, Culex species also indirectly promotes the transmission of malaria if the use of bed nets or indoor residual spraying is discontinued due to a lack of insecticide efficacy against it. A recent survey revealed widespread occurrence of pyrethroid resistance among urban populations of this mosquito in Ghana. In order to plan and implement insecticide-based resistance management strategies, this study was carried out to assess resistance status of Culex species to organophosphate and carbamate in urban areas in Ghana and the possible mechanisms involved as well as environmental factors associated with its distribution.
Mosquito larvae were sampled from various land use and ecological settings and in different seasons. In adults, susceptibility to organophosphates (fenitrothion, malathion) and carbamates (propoxur, bendiocarb) were determined. Mixed function oxidase (MFO) and α- and β-esterase assays, as well as a PCR diagnostic assay to determine ace1 mutation were performed in individual mosquitoes.
Culex quinquefasciatus as well as C. decens and other unidentified Culex species were found breeding in polluted water bodies in the study sites. Across all sites and seasons, carbamate induced mortality was 94.1% ± 15.4 whereas mortality caused by organophosphate was 99.5% ± 2.2. In addition, ace1 mutation and high levels of esterases were detected in some of the mosquito populations. There was a strong correlation between susceptibility status of the mosquitoes and the level of absorbance of β-esterase (Pearson r = − 0.841, p = 0.004).
The study found low prevalence of resistance to carbamate and organophosphate insecticides among Culex species from Ghana. However, there were populations with ace1 mutations and high levels of esterases, which can confer high resistance to these classes of insecticides. Thus, it is important to monitor activities or behaviour that has the potential to select for carbamate and organophosphate resistance populations.
Recommended options for malaria control include the use of long-lasting insecticide-treated bed nets (LLIN) and indoor residual spraying (IRS) . Unfortunately, LLIN is highly dependent on a single class of insecticides, the pyrethroids, for which malaria vectors and other mosquitoes have developed resistance. In order to act before insecticide resistance compromises current vector control strategies, WHO has proposed various guidelines to encourage countries to plan and implement insecticide resistance management strategies .
Successful resistance management depends upon reducing the selection pressure exerted by a particular insecticide or a particular mode of action . Four classes of insecticides, namely pyrethroids, organochlorine (exclusively DDT), organophosphates and carbamates are used widely in the public health sector; however, pyrethroids and DDT share similar modes of action, thus making organophosphate and carbamate very important in resistance management strategies. Various resistance management strategies including rotations, mosaics and mixture of pyrethroid and/or organophosphate or carbamate for IRS and on nets have been demonstrated both in laboratory and field conditions [4,5]. As a result, knowledge on resistance status of vectors against organophosphate or carbamate and the mechanism involved as well as factors that influence the resistance have become important.
Until recently, most research efforts on insecticide resistance in mosquitoes have been focused on determining the mosquitoes’ resistance status and the mechanisms responsible. Less attention has been paid to the impact of environmental factors that can influence resistance in mosquitoes. Recent studies have found pollutants in urban water bodies, which are mostly generated by motor vehicles, industries and domestic waste, to affect mosquito detoxification enzymes leading to enhanced insecticide degradation . Several studies in Ghana have shown different levels of susceptibility to organophosphates and carbamates in Anopheles mosquitoes including the presence of ace1 mutation in some populations [7-9]. However, for Culex species, which also plays a critical role in malaria control, information on susceptibility to organophosphate and carbamate is limited. Besides its role as a pathogen vector itself, Culex species also indirectly promotes the transmission of malaria, if the use of bed nets or IRS is discontinued due to a lack of insecticide efficacy against it. A recent survey revealed widespread occurrence of pyrethroid resistance among urban populations of this mosquito in Ghana (Kudom et al. unpublished observations.). This study was therefore carried out to assess resistance status of Culex species to organophosphate and carbamate in urban areas in Ghana and the possible mechanisms involved as well as environmental factors associated with its distribution.
An adult susceptibility assay was carried out using WHO discriminating dosages of two insecticides each from organophosphate (Fenitrothion 1%, Malathion 5%) and carbamate (Bendiocarb 0.1%, Propoxur 0.1%). Mosquitoes were exposed to the insecticides according to WHO guidelines (WHO/VBC/81.806). Mortality resulting from tarsal contact with treated filter paper was measured using WHO test kits. Four batches of 20–25 unfed females, aged 2–4 days, were exposed to papers impregnated with malathion or bendiocarb for 1 h, fenitrothion or propoxur for 2 h.
Mixed function oxidase (MFO) and α- and β-esterase were assayed in individual 2–4 day old frozen (−80°C) adults that had been reared from larvae and not been previously exposed to insecticides in the laboratory. The procedures in preparing the solutions and conducting the experiments followed the method described by Hemingway . A total of about 450 mosquitoes, comprising fifty female individuals from each of the nine study sites were used for the biochemical assay. Mosquitoes were homogenized in potassium phosphate buffer (pH 7.2) and the homogenate was loaded into micro plate wells in duplicate on the same plate for each enzyme assay. A plate-reading spectrophotometer was used to collect data at the appropriate absorbing wavelength (nm).
For the esterase assay, 100 μl mosquito homogenate with 100 μl α- or β-naphthyl acetate was incubated at room temperature for 10 minutes. Then, 100 μl dianisidine solution was added to the mixture and incubated for 2 minutes. The plate was then read using a 620 nm filter for α-naphthyl and 540 nm filter for β-naphthyl.
For the oxidase assay, 100 μl mosquito homogenate with 200 μl of Tetramethyl-Benzidine and 25 μl of 3% hydrogen peroxide were incubated for 5 minutes. The plate was then read using a 620 nm filter.
Species identification and detection of ace1 mutations
Genomic DNA was extracted from 20 mosquitoes each from the nine urban sites. These mosquitoes had not been previously exposed to insecticide. The DNA was extracted with DNeasy extraction kit (QIAGEN) based on the protocol from the manufacturer.
Oligonucleotides (primers) used for identification of Culex species and detection of ace 1 mutation
Type of molecular assay
Detection of ace1 mutation
Also, the universal DNA primers, LCO1490 and HCO2198 (Table 1) of Folmer et al.  were used to amplify an 830 bp region of the mitochondrial cytochrome oxidase subunit I gene of four mosquitoes randomly selected from the mosquitoes that failed to amplify from the previous PCR assays.
Based on the results from the sequence, two primers (Cddir and Cdrev) (Table 1) were designed from C. decens cytochrome c oxidase subunit 1 (CO1), which was obtained from Genebank with accession number AY64524. The primer was designed with Primer 3® software. PCR was conducted with Cddir and Cdrev on the DNAs that failed to amplify in the previous PCR assay.
Each PCR contained 5 ml of 10 PCR buffer, 1.5 mM of MgCl2, 35 ml of distilled water, 200 mM of each dNTP, 1 unit of Taq polymerase, 0.3 mM of each primer and 3 μl of DNA template. The PCR thermal condition consisted of one cycle of 1 min at 94°C; five cycles of 1 min at 94°C, 1.5 min at 45°C and 1.5 min at 72°C; 35 cycles of 1 min at 94°C, 1.5 min at 50°C and 1 min at 72°C and a final cycle of 5 min at 72°C. To confirm a successful reaction, a 7 μl sample from each reaction was then run via electrophoresis through a 2% agarose gel with ethidium bromide and visualized using ultraviolet (UV) light.
The four PCR products amplified by the universal DNA primers were purified using a QIAGEN QIAquick® PCR purification kit according to the manufacturers protocol and sequenced in both forward and reverse directions on an ABI 377 automated sequencer (Applied Biosystems) using the Big Dye v. 3 sequencing kit.
PCR-RFLP diagnostic test to detect ace1 mutation were carried out on the extracted DNA from the nine study sites. In the PCR assay, 3 μl of genomic DNA was amplified with the primers “Moustidir1” and “Moustrev1” (Table 1) described by Weill et al. . PCR was conducted in 25 μl volumes containing 1× PCR buffer containing 1.5 mM MgCl2, 0.2 mM of each dNTP, 3.1 μl of each of the primers, one unit of Taq polymerase (HotstarTaq: Qiagen®). The PCR conditions included an initial denaturation step at 95°C for 15 min followed by thirty five cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min.
Fifteen μl of the PCR product were digested with 5 units of Alu1 restriction enzyme in the final volume of 20 μl and incubated at 37°C for 16 h. The restriction products were fractionated on a 2% agarose gel and visualised by ethidium bromide staining under UV light.
Percentage mortality was calculated from the results of the bioassay. Percentage mortalities from different land use and ecological zones were compared using Kruskal-Wallis test whereas difference in mortalities between the two classes of insecticides and seasons were compared using Mann Whitney test. Study sites were categorized into large urban area (metropolitan area with several sub-metro and human population more than one million) and small urban area (a metropolitan or municipal area with human population less than one million) as well as percentage mortality into resistant (mortality less than 98%) and susceptible (mortality more than 98%) based on WHO  criteria. Pearson’s Chi-square and correlation test was used to determine the association between percentage mortality and enzyme levels or resistance status and urban size. Results from the esterase assays were compared between study sites with ANOVA (Analysis of Variance) and post hoc test using Fisher’s Least Significant Difference. All the tests were done with SPSS® (version 20). Phylogenetic tree was constructed using Mega 6 software from aligned DNA sequences of mitochondrial cytochrome c oxidase (CO1) gene using 500 boot-straps.
Results and discussion
Distribution of Culex species
Distribution of ace 1 mutation (G119S) in Culex species from different urban areas in Ghana
ace 1 mutation
Besides C. quinquefasciatus and C. decens, existence of other Culex species in the study sites is possible but the result from this study was unable to identify or confirm the identity of such species. There is a need for further studies on the diversity and habitat ecology of Culex species in Ghana. Also the development of molecular tools to identify them is vital since existing molecular tools for the identification of Culex species mostly target members of the C. pipiens complex. With further evaluation, the primers ‘cddir and cdrev’ could be useful molecular tools in identifying C. decens.
Insecticide susceptibility assay
Percentage mortality (95% CI) of Culex species to organophosphate and carbamate insecticides and different environmental factors associated with it
Mean mortality (%)
Multiple insecticide resistance mechanisms
In mosquitoes, insensitive acetyl cholinesterase (AChE) is another common resistance mechanism to organophosphate and carbamate insecticides . A single mutation (G119S of the ace1 gene) is responsible for AChE insensitivity to the two classes of insecticides . The PCR diagnostic assay that was performed in the present study detected the ace1 mutation (G119S) in some of the mosquito populations (Table 2) especially in C. quinquefasciatus. Also, ace1 mutation was detected in C. decens; however, the frequency of the mutation should be interpreted with caution owing to the small sample size. Approximately equal numbers of mosquitoes (about 20 mosquitoes) from each site were used but most study sites failed to produce results (DNA failed to amplify) with the exception of mosquitoes from Accra and Kumasi. Since this method  was originally designed to detect ace1 mutation in Anopheles species and C. pipiens complex to which C. quinquefasciatus belongs, it is possible that the method is not sensitive enough for other Culex species particularly those found in this study. This may partly explain why most of the DNAs that were not from C. quinqefasciatus failed to produce results. Within the two C. quinquefasciatus populations, the distribution of the ace1 genotypes did not differ from the Hardy-Weinberg equilibrium (P > 0.05, Table 2).
Despite ace1 mutations being reported to provide cross resistance to organophosphate and carbamate , the resistance level greatly varied between the two classes of insecticides. However, some studies have suggested that ace1 mutations have a greater impact on carbamate than organophosphate resistance [20,26]. The reason for this is unclear at this time.
Impact of urbanization on insecticide resistance
Resistance to carbamate were observed in C. quinquefasciatus populations from Accra and Kumasi (Table 3). In addition, high levels of esterases and ace1 mutations (Table 2) were found in the same population. This indicates a higher insecticide selection pressure in Accra and Kumasi (large urban areas) than the rest of the study sites (small urban areas). There are strong arguments in favour of agricultural and domestic use of insecticides as the major cause of resistance in urban areas . Yet, despite the use of organophosphate in urban farms, mosquitoes collected from those areas were susceptible to the insecticides. Additionally, the use of carbamate and organophosphate for malaria control or domestic use of these insecticides for mosquito control is limited. Therefore, agricultural and domestic use of insecticides cannot fully explain the cause of resistance in the present study.
Another cause of resistance in mosquitoes which until recently has received little attention is the presence of pollutants in breeding habitats of mosquitoes. Exposure of mosquitoes to common urban pollutants has been shown to increase mosquito tolerance to insecticides in some studies [28,29]. We suspect pollutants found in breeding habitats to have played a role in resistance of C. quinquefasciatus to carbamate in this study. However, the result from the study cannot confirm this hypothesis. In a related study, Chandre and colleagues  were also not able to relate carbamate and organophosphate resistance in C. quinquefasciatus in Ivory Coast and Burkina Faso to the use of agricultural pesticides but rather implicated domestic use of insecticides to be associated with corresponding resistances. Similarly, Anopheles species that were sampled from an urban vegetable farm in Accra were susceptible to organophosphate, though several organophosphate insecticides were detected in the water body in which the mosquito was breeding . On the contrary, Essandoh and colleagues  implicated agricultural use of pesticides as a cause of carbamate and organophosphate in Anopheles species from peri-urban agricultural area near Accra.
The presence of C. decens in polluted breeding sites, which is not known to breed in such habitats, coupled with various reports that have observed A. gambiae, a major malaria vector, also breeding in polluted habitats [32,33] reinforces the need to take a critical look at the numerous polluted breeding sites scattered in the country. Both solid and liquid waste are poorly managed in urban areas in Ghana as well as many African countries and what enters into gutters or water bodies from commercial and domestic activities is not much regulated. This presents a situation where apart from pesticides officially sanctioned for public use, mosquitoes could also be exposed to unknown chemicals or insecticides in polluted breeding habitats, which can select for resistance mechanisms that can confer high level of resistance to new insecticides that officially have not been recognized to be used in the country.
The study found low prevalence of resistance to carbamate and organophosphate insecticides among Culex species from Ghana. Due to low levels of resistance to the insecticides, resistance management strategies comprising the use of organophosphate and carbamate may be successful against pyrethroid-resistant Culex species in Ghana. However, such strategies must be carried out with caution since populations with ace1 mutations and high levels of esterases, which can confer high resistance to these classes of insecticides, already exist.
This is the first study in Ghana showing evidence of the existence of multiple insecticide resistance mechanisms in C. quinquefasciatus to carbamates and organophosphates. With the existence of populations with multiple resistance mechanisms, it is important to monitor activities or behaviours that have the potential to select for resistance populations. Besides agriculture and domestic use of insecticides, urban pollutants found in mosquito breeding habitats have also been implicated as a cause of resistance. With the expansion of agricultural activities in urban areas, the amount of pesticides used is greatly increasing. In addition, inadequate urban infrastructure, partly due to rapid population growth, leads to creation of numerous polluted habitats that are suitable for mosquito breeding. For these reasons, proper management of waste, particularly in urban areas, and effective regulation of the use of pesticides appear to be critical for managing insecticide resistance.
We are grateful to Kwadwo Frimpong, Selasie P. Bansah and all the staff of Noguchi Memorial Institute for Medical Research (NMIMR) who helped during the molecular and biochemical assays. We also appreciate the contribution of Eddy Odari during the molecular work. We acknowledge the Federal Ministry for Economic Cooperation and Development (BMZ), the German Academic Exchange Services (DAAD) together with its Exceed-Programme for the offer of scholarship to AAK. We also appreciate the Parasitology Unit of NMIMR and Department of Entomology and Wildlife, University of Cape Coast for their contribution to the project. We are also grateful to the anonymous reviewers for their constructive criticism.
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