RESEARCH Open Access
Preliminary assessment of the potential role of
urbanization in the distribution of carbamate and
organophosphate resistant populations of Culex
Keywords: ace1, Carbamate, Culex decens, Culex quinquefasciatus, Ecology, Esterase, Organophosphate,
Kudom et al. Parasites & Vectors  (2015) 8:8 
DOI 10.1186/s13071-014-0621-4University of Cape Coast, Cape Coast, Ghana
Full list of author information is available at the end of the articleInsecticide-resistance, Urbanization
* Correspondence: akudom@ucc.edu.gh
1Center for International Health, Department of Infectious Diseases and
Tropical Medicine, Ludwig-Maximilians-Universität Munich, Munich, Germany
2Department of Entomology and Wildlife, School of Biological Sciences,has the potential to select for carbamate and organophospspecies in Ghana
Andreas A Kudom1,2*, Ben A Mensah2, Guenter Froeschl1, Daniel Boakye3 and Heinz Rinder1,4
Abstract
Background: 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.
Methods: 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.
Results: 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).
Conclusions: 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
hate resistance populations.© 2015 Kudom et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 2 of 10Background
Recommended options for malaria control include the
use of long-lasting insecticide-treated bed nets (LLIN)
and indoor residual spraying (IRS) [1]. Unfortunately,
LLIN is highly dependent on a single class of insecti-
cides, 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 [2].
Successful resistance management depends upon redu-
cing the selection pressure exerted by a particular insecti-
cide or a particular mode of action [3]. Four classes of
insecticides, namely pyrethroids, organochlorine (exclu-
sively DDT), organophosphates and carbamates are used
widely in the public health sector; however, pyrethroids
and DDT share similar modes of action, thus making or-
ganophosphate and carbamate very important in resistance
management strategies. Various resistance management
strategies including rotations, mosaics and mixture of pyr-
ethroid 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 influ-
ence the resistance have become important.
Until recently, most research efforts on insecticide re-
sistance in mosquitoes have been focused on determining
the mosquitoes’ resistance status and the mechanisms re-
sponsible. 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 vehi-
cles, industries and domestic waste, to affect mosquito
detoxification enzymes leading to enhanced insecticide
degradation [6]. Several studies in Ghana have shown dif-
ferent levels of susceptibility to organophosphates and car-
bamates 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 pyr-
ethroid resistance among urban populations of this mos-
quito 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 in-
volved as well as environmental factors associated with its
distribution.Methods
Study sites
Mosquito larvae were collected from ponds, polluted drains
and choked gutters in nine urban areas in Ghana (Figure 1).
Urban areas were selected according to ecological settings
and in each selected town, mosquitoes were sampled
from different land use settings; residential, urban agricul-
tural and swampy areas. Mosquito larvae were also sam-
pled in both rainy and dry seasons. Larvae were brought
to the laboratory for emergence and testing of adults.
Susceptibility test
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%). Mos-
quitoes were exposed to the insecticides according to
WHO guidelines (WHO/VBC/81.806). Mortality result-
ing 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, feni-
trothion or propoxur for 2 h.
Biochemical assay
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 [10]. A
total of about 450 mosquitoes, comprising fifty female in-
dividuals 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 spectropho-
tometer was used to collect data at the appropriate ab-
sorbing 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 dianisi-
dine 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
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 3 of 10extracted with DNeasy extraction kit (QIAGEN) based
on the protocol from the manufacturer.
Mosquitoes for this study were mostly collected from
polluted water bodies; hence C. quinquefasciatus was gen-
erally expected. PCR diagnostic assay was therefore car-
ried out to identify C. quinquefasciatus using the method
described by Smith and Fonseca [11]. Four primers
(“ACEquin”,“B1246s”, F1457” and “B1246) (Table 1) were
used to detect C. quinquefasciatus and any other species
that belongs to the C. pipiens complex.
Figure 1 Map of Ghana showing the three ecological zones and urbaAlso, the universal DNA primers, LCO1490 and
HCO2198 (Table 1) of Folmer et al. [12] were used to
amplify an 830 bp region of the mitochondrial cyto-
chrome oxidase subunit I gene of four mosquitoes ran-
domly 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
n towns where mosquitoes were sampled.
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 4 of 10AY64524. The primer was designed with Primer 3® soft-
ware. 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 con-
sisted 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 re-
action, 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 di-
Table 1 Oligonucleotides (primers) used for identification
of Culex species and detection of ace1 mutation
Type of molecular
assay
Primers
Species
identification
ACEquin 5’-CCTTCTTGAATGGCTGTGGCA-3’
B1246s 5’-TGGAGCCTCCTCTTCACGG-3’
F1457 5’-GAGGAGATGTGGAATCCCAA-3’
B1246 5’-TGGAGCCTCCTCTTCACGGC-3’
Cddir 5’-ACCTCGACGATACTCCGATTT-3’
Cdrev 5’-TGTGTTCTGCAGGAGGAAGA-3’
LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’
HCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’
Detection of ace1
mutation
Moustdir1 5’-CCGGGNGCSACYATGTGGAA-3’
Moustrev1 5’-ACGATMACGTTCTCYTCCGA-3’rections 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 ampli-
fied with the primers “Moustidir1” and “Moustrev1”
(Table 1) described by Weill et al. [13]. PCR was con-
ducted in 25 μl volumes containing 1× PCR buffer con-
taining 1.5 mM MgCl2, 0.2 mM of each dNTP, 3.1 μl of
each of the primers, one unit of Taq polymerase (Hotstar-
Taq: Qiagen®). The PCR conditions included an initial de-
naturation 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.Statistical analysis
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 mil-
lion) 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 [14] criteria. Pearson’s Chi-square and
correlation test was used to determine the association
between percentage mortality and enzyme levels or re-
sistance 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
Culex quinquefasciatus was generally expected due to
the breeding habitat in which the mosquitoes were col-
lected. Nevertheless, PCR diagnostic assay confirms C.
quinquefasciatus in only Accra and Kumasi. Besides C.
quinquefasciatus, no member of the C. pipiens complex
was found. Data from mitochondrial COI gene sequence
did not match 100% with any existing COI sequence in
GeneBank. The phylogenetic analysis (Figure 2) indi-
cated that the unidentified Culex species could be made
up of two or more different species. From the branches
of the phylogenetic tree, it could be inferred that Culex
species from Bolgatanga and Cape Coast were either C.
decens or closely related to it whereas Culex species
from Techiman and Sekondi were closely related to C.
fuscocephala or C. perexiguus. The genetic resemblance
of the Culex species from Techiman and Sekondi to C.
fuscocephala and C. perexiguus was unexpected since
such species have not been reported in Ghana before.
Further studies, combining morphology and molecular in-
formation are needed to identify these species. C. fuscoce-
phala and C. perexiguus are mostly distributed in South
East Asia and the Mediterranean countries respectively
(Walter Reed Biosystematics Unit, www.wrbu.org).
PCR diagnostic assay using primers designed from mito-
chondrial COI gene sequence of C. decens confirmed C.
decens in some of the study sites (Table 2; Figure 3). This
is not surprising since C. decens is a widely distributed
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 5 of 10species in Ghana [15,16]; however their presence in pol-
luted breeding habitats are not well known. The collection
of C. decens in polluted waters is in agreement with
Opoku and colleagues [16], who found C. decens occur-
ring sympatrically with C. quinquefasciatus in polluted
habitats in Accra.
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 mem-
bers of the C. pipiens complex. With further evaluation,
Figure 2 Phylogenetic tree constructed from aligned DNA sequences
boot-straps with Mega 6 software. Techiman, Sekondi, Cape Coast and Bthe primers ‘cddir and cdrev’ could be useful molecular
tools in identifying C. decens.
Insecticide susceptibility assay
Culex species from the study populations were suscep-
tible to organophosphate (malathion, fenitrothion). Also,
the level of carbamate (propoxur, bendiocarb) resistance
was generally very low (Table 3; Figure 4). In total the
carbamate-induced percentage mortality (± SD) was
94.1% ± 15.4 whereas mortality caused by organophos-
phate was 99.5% ± 2.2 and the difference between the two
mortalities was significant (Mann Whitney U: p < 0.05).
Resistance to carbamate was only found in C. quinquefas-
ciatus populations from Accra and Kumasi (large urban
areas). Complete susceptibility of Culex species in Ghana
of mitochondrial cytochrome c oxidase (CO1) gene using 500
olgatanga are the samples collected in this study.
Figure 3 Species diagnostic PCR for Culex decens using the
primers ‘cddir and cdrev’. Lane 1 – 1 kb ladder, lane 2 – Culex
mosquito from Cape Coast (sequence of mitochondrial cytochrome
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 6 of 10to organophosphate is good for vector control and resist-
ance management. Although, the level of resistance to car-
bamate observed in this study may not have any serious
implication on vector control, there is a need for constant
monitoring and resistance surveillance to prevent resist-
ance to these insecticides rising to a level that could affect
insecticide resistance management strategies in the coun-
try. Environmental variables such as ecology, seasons and
land use settings were marginally or non-significantly as-
sociated with carbamate and organophosphate resistance
(Table 3). Culex species in Ghana and most West African
countries are not epidemiologically important but many
households spent a considerable amount of money and re-
sources to prevent their nuisance [17,18]. Furthermore,
the influence of Culex species on the use of insecticide
treated net has been highlighted by several studies [19,20].
These reasons emphasise the importance of controlling
and monitoring insecticide resistance in Culex species.
Multiple insecticide resistance mechanisms
Overall mean absorbance of α-esterase was 0.448 ± 0.12,
β-esterase, 0.807 ± 0.22 and oxidase, 0.152 ± 0.08 (Figure 5).
Absorbance of all the three enzymes tested was higher in
C. quinquefasciatus populations from Accra and Kumasi
than the other Culex species. A strong negative correlation
was observed between percentage mortality from the bio-
Table 2 Distribution of ace1 mutation (G119S) in Culex
species from different urban areas in Ghana
Study sites Culex species ace1 mutation
C.q C.d SS RS RR F(R)
Accra 25 0 48% (12) 40% (10) 12%(3) 0.32
Cape Coast 0 10 100% (20) 0%(0) 0%(0) 0
Kumasi 16 0 81.3% (13) 12.5(2) 6.2%(1) 0.13
Sunyani 0 10 36.4% (4) 45.5% (5) 18.2% (2) 0.41
Techiman 0 5 80% (4) 20%(1) 0%(0) 0.1
Value in bracket represents sample size. C.q – Culex quinquefasciatus,
C.d – Culex decens, SS – homozygote susceptible, RS – heterozygote resistant,
RR – homozygote resistant, F(R) – frequency of resistant allele.assay and mean absorbance of β-esterase (Pearson
r = −0.841, p = 0.004). However, such significant association
was not observed between percentage mortality and α-
esterase (Pearson r = − 0.45, p = 0.22) or oxidase (Pearson
r = 0.131, p = 0.738). This may suggest the involvement of
enzyme activity particularly esterases in the resistance of
C. quinquefasciatus to carbamate insecticides. Enhanced
levels or modified activities of esterases and other detoxi-
fying enzymes have been reported to prevent some insecti-
cides from reaching their site of action [21]. Meanwhile,
several studies have shown close association between or-
ganophosphate and carbamate resistance and high levels
of esterases [22,23].
In mosquitoes, insensitive acetyl cholinesterase (AChE) is
another common resistance mechanism to organophosphateand carbamate insecticides [24]. A single mutation (G119S
of the ace1 gene) is responsible for AChE insensitivity to the
two classes of insecticides [13]. 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 fre-
quency of the mutation should be interpreted with cau-
tion owing to the small sample size. Approximately equal
numbers of mosquitoes (about 20 mosquitoes) from each
c oxidase (CO1) gene of this mosquito was close to existing COI of
Culex decens in Genebank), Lane 3 – Culex species from Cape Coast
(from the same breeding habitat with the one in lane 2), lane 4 –
Culex quinquefasciatus from Accra.
Table 3 Percentage mortality (95% CI) of Culex species to organophosphate and carbamate insecticides and different
environmental factors associated with it
Environmental factors Mean mortality (%)
Carbamate Organophosphate
Bendiocarb* Propoxur* Fenitrothion* Malathion*
Land use Residential 87a 93a 99a 99a
(78–95) (86–99) (98–100) (98–100)
Urban farm 98a 99a 99a 100b
(95–101) (99–101) (99–100) (100–100)
Swampy 98a 97a 99a 100b
(94–101) (94–100) (99–100) (100–100)
Season Rainy 89a 96a 99a 99a
(80–97) (93–99) (98–100) (98–100)
Dry 95.7a 96a 99a 100a
(92–99) (90–101) (99–100) (99–100)
Ecology Coastal savannah 94a,b 97a,b 99a 100a
(89–100) (95–100) (99–100) (99–100)
Forest 88b 93b 99a 99a
(80–97) (86–99) (98–100) (98–100)
Guinea savannah 100a 100a 100a 100a
(100–100) (100–100) (100–100) (100–100)
*In each insecticide, values in columns (environmental factors) sharing same letter are not significantly different.
Figure 4 Resistance status of Culex species to organophosphate and carbamate insecticides in urban areas in Ghana, a) bendiocarb
b) propoxur c) fenitrothion d) malathion.
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 7 of 10
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 8 of 10site were used but most study sites failed to produce re-
sults (DNA failed to amplify) with the exception of mos-
quitoes from Accra and Kumasi. Since this method [13]
was originally designed to detect ace1 mutation in Anoph-
eles species and C. pipiens complex to which C. quinque-
fasciatus 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. quinquefas-
ciatus 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 [25], the re-
sistance level greatly varied between the two classes of in-
secticides. 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
The distribution of Culex species in this study appears
to be influenced by the degree of urbanization. C. quin-
quefasciatus was found in the populous and most
Figure 5 Mean absorbance (optic density) of α-esterase (esterase A),
species from different urban areas in Ghana. (Error bar: 95% CI).urbanized areas in Ghana whereas C. decens and other
unidentified Culex species were found in relatively small
urban areas. Although the level of pollution was not
quantified in the breeding sites, it was nevertheless ob-
served that breeding sites where mosquitoes were col-
lected were more polluted in Accra and Kumasi than
those in other urban areas (Figure 6a-d). It was therefore
not surprising that C. quinquefasciatus was found in these
areas since C. quinquefasciatus are most common in pol-
luted heavily urbanized sites. The results also showed that
other Culex species such as C. decens are also breeding in
polluted water bodies in urban areas in Ghana.
Resistance to carbamate were observed in C. quinque-
fasciatus 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 indi-
cates 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 argu-
ments in favour of agricultural and domestic use of in-
secticides as the major cause of resistance in urban areas
[27]. 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
β-esterase (esterase B) and mixed function oxidase of Culex
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 9 of 10domestic 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 [30] were also
not able to relate carbamate and organophosphate resist-
ance in C. quinquefasciatus in Ivory Coast and Burkina
Figure 6 a-d Polluted breeding habitats where mosquito larvae
were collected - a) Tarkwa, b) Sunyani, c) Kumasi, d) Accra.Faso to the use of agricultural pesticides but rather im-
plicated domestic use of insecticides to be associated
with corresponding resistances. Similarly, Anopheles spe-
cies that were sampled from an urban vegetable farm in
Accra were susceptible to organophosphate, though sev-
eral organophosphate insecticides were detected in the
water body in which the mosquito was breeding [31].
On the contrary, Essandoh and colleagues [20] impli-
cated agricultural use of pesticides as a cause of carba-
mate 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 coun-
try. 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 fromcommercial and domestic activities is not much regu-
lated. This presents a situation where apart from pesti-
cides officially sanctioned for public use, mosquitoes
could also be exposed to unknown chemicals or insecti-
cides in polluted breeding habitats, which can select for
resistance mechanisms that can confer high level of re-
sistance to new insecticides that officially have not been
recognized to be used in the country.
Conclusions
The study found low prevalence of resistance to carba-
mate and organophosphate insecticides among Culex
species from Ghana. Due to low levels of resistance to
the insecticides, resistance management strategies com-
prising 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 behav-
iours that have the potential to select for resistance popula-
tions. 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 ex-
pansion of agricultural activities in urban areas, the
amount of pesticides used is greatly increasing. In addition,
inadequate urban infrastructure, partly due to rapid popu-
lation growth, leads to creation of numerous polluted habi-
tats 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AAK participated in the design and coordination of the study, carried out the
field work and data collection, performed statistical analysis of the study and
drafted the manuscript. BAM, GF, DB, and HR participated in the study
design and contributed to the writing of the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
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.
haplotypes in resistant Culex pipiens mosquitoes from Algeria. Insect
Biochem Mol Biol. 2011;41:29–35.
24. Fournier D, Mutero A. Modification of acetylcholinesterase as a mechanism
Kudom et al. Parasites & Vectors  (2015) 8:8 Page 10 of 10Author details
1Center for International Health, Department of Infectious Diseases and
Tropical Medicine, Ludwig-Maximilians-Universität Munich, Munich, Germany.
2Department of Entomology and Wildlife, School of Biological Sciences,
University of Cape Coast, Cape Coast, Ghana. 3Noguchi Memorial Institute for
Medical Research, University of Ghana, P.O. Box LG 581, Legon, Ghana.
4Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL),
Veterinärstraße 2, Oberschleißheim 85762, Germany.
Received: 30 May 2014 Accepted: 22 December 2014
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