PLOS ONE
RESEARCH ARTICLE
Influence of insecticide resistance on the
biting and resting preferences of malaria
vectors in the Gambia
Majidah Hamid-Adiamoh 1,2ID *, Davis Nwakanma2, Benoit Sessinou Assogba2, Mamadou
Ousmane Ndiath2, Umberto D’Alessandro2, Yaw A. Afrane1,3, Alfred Amambua-Ngwa2
1 West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) and Department of Biochemistry,
Cell and Molecular, University of Ghana, Legon, Accra, Ghana, 2 Medical Research Council Unit, The
Gambia at The London School of Hygiene & Tropical Medicine, Banjul, The Gambia, 3 Department of
a1111111111 Medical Microbiology, University of Ghana Medical School, University of Ghana, Accra, Ghana
a1111111111
a1111111111 * madiamoh@mrc.gm
a1111111111
a1111111111
Abstract
Background
OPEN ACCESS The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with
Citation: Hamid-Adiamoh M, Nwakanma D, other interventions have considerably reduced the malaria burden in The Gambia. This
Assogba BS, Ndiath MO, D’Alessandro U, Afrane study examined the biting and resting preferences of the local insecticide-resistant vector
YA, et al. (2021) Influence of insecticide resistance populations few years following scale-up of anti-vector interventions.
on the biting and resting preferences of malaria
vectors in the Gambia. PLoS ONE 16(6):
e0241023. https://doi.org/10.1371/journal. Method
pone.0241023
Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July
Editor: Berlin Londono, Kansas State University, and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray col-
UNITED STATES
lection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction
Received: October 3, 2020 assays were performed to identify molecular species, insecticide resistance mutations,
Accepted: June 11, 2021 Plasmodium infection rate and host blood meal.
Published: June 24, 2021
Results
Copyright: © 2021 Hamid-Adiamoh et al. This is an
open access article distributed under the terms of A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423,
the Creative Commons Attribution License, which 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%,
permits unrestricted use, distribution, and outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, out-
reproduction in any medium, provided the original
door: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae
author and source are credited.
s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed
Data Availability Statement: All relevant data are
in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii
within the paper and its S1 File.
(Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel
Funding: This work was supported by funds from a
(Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78–
Wellcome Trust DELTAS Africa grant (DEL-15-007:
Awandare). Majidah Hamid-Adiamoh was 1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76–0.87) An. arabiensis pop-
supported by a WACCBIP-Wellcome Trust DELTAS ulation. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor
PhD fellowship. The DELTAS Africa Initiative is an
(allele freq. = 0.92, 95%CI: 0.81–0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56–
independent funding scheme of the African
Academy of Sciences (AAS)’s Alliance for 0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-
Accelerating Excellence in Science in Africa (AESA) 1575Y and GSTe2-114T was high (allele freq. = 0.96–1), but did not vary by resting location.
PLOS ONE | https://doi.org/10.1371/journal.pone.0241023 June 24, 2021 1 / 13
PLOS ONE Biting and resting preferences of malaria vectors
and supported by the New Partnership for Africa’s The overall sporozoite positivity rate was 1.3% (95% CI: 0.5–2%) in mosquito populations.
Development Planning and Coordinating Agency Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabien-
(NEPAD Agency) with funding from the Wellcome
sis fed on animal blood.
Trust (107755/Z/15/Z: Awandare) and the UK
government. Additional support was also provided
by the H3Africa PAMGENe project, H3A/18/002, Conclusion
funded by the AAS; and the National Institutes of
In this study, high levels of resistance mutations were observed that could be influencing the
Health (D43 TW 011513).
mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour
Competing interests: The authors have declared
demonstrated in the mosquito populations suggest that larval source management could be
that no competing interests exist.
an intervention to complement vector control in this setting.
Introduction
Successful implementation of indoor residual spraying (IRS) and long-lasting insecticidal nets
(LLINs) has hugely contributed to the malaria decline observed in sub-Saharan Africa [1].
These interventions reduce transmission by primarily limiting human contact with human-
feeding (anthropophagic), indoor-feeding (endophagic) and indoor-resting (endophilic) vec-
tors [2]. Unfortunately, these measures also induce selection for physiological and behavioral
resistance in vector populations, resulting in reduced mosquito susceptibility to most of the
current insecticides used for LLINs and IRS [3], as well as increased exophilic behavioral phe-
notypes in primarily endophilic vectors [4]. Moreover, residual transmission partly driven by
high LLINs and IRS use, is maintained by vectors with physiological and behavioral resistance
[5]. Therefore, studying the behavioral dynamics of vector populations during the scale up of
vector control interventions will assist in determining the appropriate response to emerging
behavioral changes.
Malaria burden in the Gambia has declined significantly over the last decades with vector
control approaches being a major component of intervention, coordinated and implemented
by the Gambia National Malaria Control Program (GNMCP). Following the World Health
Organization (WHO) Global Plan for Insecticide Resistance Management (GPIRM), the
GNMCP has consistently implemented rotational use of different classes of insecticides for
IRS, to curtail dichlorodiphenyltrichloroethane (DDT) and deltamethrin resistance. For IRS,
DDT was replaced initially by deltamethrin and bendiocarb, and since 2017 by pirimiphos-
methyl (actellic 300CS) [6]. Similarly, LLINs intervention has been stable over the years and
Gambia has recorded successful LLINs coverage as high as 90% [7, 8].
Despite such successes, residual transmission has become increasingly spatially heteroge-
neous, with its intensity increasing from western to eastern Gambia, and could have been
driven by specific vector population dynamics [7]. The major vector species, namely Anopheles
arabiensis, An. coluzzii and An. gambiae sensu stricto (s.s.) are variably distributed throughout
the country. An. arabiensis is most prevalent in the eastern Gambia while An. coluzzii and An.
gambiae s.s. inhabit the western region [9, 10]. However, An. arabiensis has been recently
found throughout the country [11], indicating possible replacement due to successful control
of other sibling species [12, 13]. Moreover, the population prevalence of each vector species
varies by season, whereby An. arabiensis and An. coluzzii are dominant throughout the rainy
season, while An. gambiae s.s. become rarest early in the onset of dry season [9, 10]. DDT and
pyrethroid resistance has been reported at various degrees in all vectors, that continue to be
highly susceptible to carbamates and organophosphates [11, 14, 15].
Host seeking and resting behavior of vectors are important metrics to evaluate the impact
of control and resistance management strategies [16]. Vector behavioral adaptation, resistance
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PLOS ONE Biting and resting preferences of malaria vectors
selection and persistent transmission could increase during extensive scale-up of interven-
tions, and this information can only be captured by real-time surveillance [17, 18]. Hence,
national malaria control programs should actively monitor behavioral dynamics in the local
vector population, to inform decisions.
In the Gambia, DDT and pyrethroid resistance is widespread and associated with residual
transmission [11, 14]. However, the effect of control activities on vectors feeding and resting
behavior remains unclear. The biting and resting preferences of An. gambiae sensu lato (s.l.)
populations was investigated in the Gambia following few years of intensive vector control
interventions.
Materials and methods
Ethical clearance
Ethical approval was obtained from the Gambia Government/Medical Research Council
(MRC) Unit the Gambia at London School of Hygiene and Tropical Medicine (LSHTM) Joint
Ethics Committee. The permit Number was: SCC 1586.
Anopheles gambiae s.l. collection
Indoor and outdoor-resting adult mosquitoes were sampled from July to October 2019, during
the malaria transmission season across five administrative regions in the Gambia, namely Cen-
tral River Region (CRR), Lower River Region (LRR), North Bank Region (NBR), Upper River
Region (URR) and West Coast Region (WCR) (Fig 1). WCR is a coastal area characterized by
mangrove swamps. The remaining regions are mainly inland and have forest vegetation. Rice
is mainly cultivated in CRR while cereals farming is common in all regions. Two villages were
selected from each region and most of the villages are GNMCP surveillance sites with high
LLIN and IRS coverage. Malaria transmission is highest in URR compared to other regions in
the Gambia [7].
Indoor-resting mosquitoes were collected from sleeping rooms using pyrethrum spray col-
lection (PSC). Twenty houses per village, at least 50m apart from each other, were randomly
selected. In each village, collections were done for two consecutive days, with ten houses sam-
pled per day. Outdoor-resting mosquitoes were sampled from pit shelter traps using prokopak
aspirator. Three pit shelter traps that were 10m away from the selected compounds, were
placed at different parts in each village. Both indoor and outdoor collections were conducted
from 06.00 am to 09.00am in every collection day.
Mosquito identification
Morphological identification of female An. gambiae s.l. was done using identification keys as
described by Gillies & Coetzee [19]. Afterwards, mosquitoes were stored individually in 96%
ethanol in 1.5ml Eppendorf tube until DNA extraction. DNA was extracted separately from
Fig 1. Map of the Gambia showing the study sites, comprising two villages each from the 5 administrative regions
in the country.
https://doi.org/10.1371/journal.pone.0241023.g001
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abdomen and head/thoraces of individual mosquitoes using Qiagen QIAxtractor robot. Spe-
cies-specific genotyping PCR to identify An. arabiensis, An. melas and An. gambiae was per-
formed using specific primers to discriminate the species as previously done [20]. This was
followed by restriction enzyme digestion to specifically identify An. coluzzii, An. gambiae s.s.
and their hybrids (An. coluzzii-An. gambiae s.s.) [21].
Insecticide resistance markers identification
Screening for molecular markers of target-site resistance to carbamates, DDT, pyrethroids and
organophosphates was done on all samples using a probe-based assay (TaqMan SNP genotyp-
ing) [22]. The following markers were investigated: voltage-gated sodium (Vgsc)-1014F, Vgsc-
1014S and Vgsc-1575Y associated with target-site mutation to DDT and pyrethroids [23–25].
Acetylcholine esterase (Ace)-119S, marker for carbamate and organophosphate resistance [26]
and glutathione-S-transferase epsilon 2 (Gste2)-114T, involved in metabolic resistance to DDT
[27] were also assayed. The TaqMan allelic discrimination assay used is a multiplex real time
PCR, where primers and probes specific for each insecticide target gene were employed to dis-
criminate susceptible (wild type) and resistant (mutant) alleles based on probe fluorescence
signals [28].
Plasmodium sporozoite detection
DNA extracted from mosquito head and thoraces was used to detect sporozoites of Plasmo-
dium falciparum, P. ovale, P. malariae and P. vivax species, employing TaqMan SNP genotyp-
ing protocol [29] which enables discriminatory identification of circum-sporozoites (CSPs) of
P. falciparum from P. ovale, P. malariae and P. vivax CSPs. Genomic DNA specific to each of
these Plasmodium species were analyzed in each assay as positive controls.
Blood meal identification
Extracted DNA from engorged mosquito abdomens were amplified using modified multiplex
PCRs with specific primers that amplify cytochrome B genes of human and animal hosts
including chicken, cow, dog, donkey, goat, horse and pig [30, 31].
Statistical analyses
The proportion of each mosquito species in relation to the total number of mosquitoes cap-
tured from each region was calculated in percentage, as well as allele frequencies of indoor and
outdoor-resting mosquitoes. Sporozoite positivity rate was the proportion of PCR positive
mosquitoes among all mosquitoes tested. Human (HBI) and animal blood meal indices were
estimated as the proportion of mosquitoes positive for human or animal hosts among those
positive for all hosts. Mean differences between HBI and animal blood meal indices by vector
species and resting locations were analyzed by ANOVA. Statistical analyses were done using
Stata/IC 15.0 (2017 StataCorp LP).
Results
Anopheles species distribution and their resting behavior
A total of 844 An. gambiae s.l. mosquitoes were collected from the five regions. Four main vec-
tor species were identified, namely An. arabiensis (N = 350, 41%); An. coluzzii (N = 214, 25%),
An. gambiae s.s. (N = 224, 27%) and An. melas (N = 17, 2%). Hybrids of An. coluzzii-An. gam-
biae s.s. were also detected (N = 39, 5%). Most mosquitoes were collected from URR (642,
76%), followed by LRR (97, 11%) and then the other regions (Fig 2).
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Fig 2. Distribution of Anopheles gambiae s.l by region as collected indoors and outdoors. An. coluzzii-An. gambiae
s.s. are the hybrids of An. coluzzii and An. gambiae s.s. Mosquitoes were collected from 5 regions: CRR- central river
region, LRR-lower river region. NBR- north bank region. URR-upper river region and WCR- West coast region.
https://doi.org/10.1371/journal.pone.0241023.g002
Overall, the number of mosquitoes resting indoors (421, 49.9%) and outdoors (423, 50.1%)
were similar. Nevertheless, the resting preference varied by species. A significantly higher pro-
portion of An. arabiensis were found outdoor (26.1%) than indoor (15.4%) (Pearson X2 = 22.7,
df = 4, P<0.001) while both An. coluzzii (19.1% indoor and 6.3% outdoor, Pearson X2 = 55.0,
df = 4, P<0.001) and An. melas (1.9% indoor and 0.1% outdoor, Pearson X2 = 13.3, df = 4,
P<0.01) preferred resting indoor. For An. gambiae s.s. (10.9% indoor and 15.6% outdoor,
Pearson X2 = 7.0, df = 4, P = 0.14) and An. coluzzii-An. gambiae s.s. hybrids (2.6% indoor and
2% outdoor, Pearson X2 = 0.7, df = 4, P = 0.45), there was no significant difference between
resting indoor and outdoor. In URR, the region with the highest malaria transmission in the
Gambia, An. arabiensis was most abundant vector (45.8%, 294) (indoor: 14.5%, outdoor:
31.3%), followed by An. gambiae s.s. (28.4%, 182) (indoor: 12.8%, outdoor: 15.6%) and An.
coluzzii (21.5%, 138) (indoor: 13.6%, outdoor: 7.9%). No An. gambiae s.s. was collected in CRR
while An. melas was mainly found in LRR (N = 15). All mosquitoes collected from LRR and
NBR were resting indoors. The hybrids of An. coluzzii and An. gambiae s.s. were mainly found
in URR (indoor: 2.3%, outdoor: 1.9%) and WCR (indoor: 10%, outdoor: 8.3%).
Distribution of voltage-gated sodium channel (Vgsc) mutation markers in
the vectors
Vgsc point mutations associated with DDT and pyrethroid resistance were highly prevalent
and detected at varying frequencies in all vector species across all regions. Overall, An. arabien-
sis was found resting indoors when resistance allele frequency was higher in the indoor popu-
lation, whereas An. coluzzii were resting outdoors with higher outdoor resistance. No
consistent resting preference was observed in An. gambiae in the presence of mutations.
Vgsc-1014S mutation was found predominantly in indoor-resting vector populations
(Table 1). In An. arabiensis, the mutation was more frequent in the indoor-resting than out-
door-resting mosquitoes regardless of the region. Vgsc-1014S was also the only mutation iden-
tified in An. gambiae s.s. and An. melas when found resting indoors.
In An. arabensis resting indoors in URR, Vgsc-1014S frequency was significantly higher
(Z = 2.230, P = 0.03) in the indoor- (allele freq. = 0.91, 95%CI: 0.84–0.96) than outdoor-resting
(allele freq. = 0.82, 95%CI: 0.76–0.87) mosquitoes. Moreover, Vgsc-1014S was the only muta-
tion identified in this species when found resting indoors (allele freq. = 0.96, 95%CI: 0.78–1) in
CRR. Whereas in An. coluzzii in URR, Vgsc-1014S mutation was higher in the indoor (allele
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Table 1. Frequencies of insecticide resistance alleles on VGSC, GST and AChE loci in Anopheles gambiae s.l. populations from all study regions.
Region Anopheles species Vgsc-1014F Vgsc-1014S Vgsc-1575Y GSTe2-114T Ace1-119S
Indoor Outdoor Indoor Outdoor Indoor Outdoor Indoor Outdoor Indoor Outdoor
URR An. arabiensis 0.05 0.02 0.91 0.82 0 0.004 0 0.01 0 0
N = 5 N = 4 N = 85 N = 164 N = 1 N = 1
An. coluzzii 0.74 0.92 0.25 0.04 0.68 0.9 0.78 0.9 0 0
N = 64 N = 47 N = 22 N = 2 N = 59 N = 46 N = 68 N = 46
An. gambiae s.s. 1 0.99 0 0.01 0.96 0.98 0.98 0.99 0.05 0.04
N = 82 N = 99 N = 1 N = 79 N = 98 N = 80 N = 99 N = 4 N = 4
An. coluzzii-An. gam biae s.s 0.93 1 0 0 0.87 1 0.87 0 0.07 0
N = 14 N = 12 N = 13 N = 12 N = 13 N = 1
LRR An. arabiensis 0 - 0.9 - 0 - 0.1 - 0 -
N = 9 N = 4 N = 1
An. coluzzii 0.66 - 0.3 - 0 - 0 - 0 -
N = 47 N = 21
An. gambiae s.s 0 - 1 - 0 - 0 - 0 -
N = 1
An. melas 0 - 1 - 0 - 0 - 0 -
N = 15
WCR An. arabiensis - 0.13 - 0.88 - 0 - 0 - 0
N = 1 N = 7
An. coluzzii - 1 - 0 - 0 - 0 - 0
An. gambiae s.s. 0.25 0.13 0.75 0.84 0.13 0.06 0 0 0 0
N = 2 N = 4 N = 6 N = 27 N = 1 N = 2
An. coluzzii-An. gam biae s.s 0 0 1 1 0 0 0.17 0 0 -
N = 6 N = 5 N = 1
CRR An. arabiensis 0 1 0.96 0 0 0 0.1 0.27 0 0
N = 11 N = 22 N = 3 N = 3
An. coluzzii - 1 - 0 0 0 0 0 0 0
An. melas 0 - 1 - 0 - 0 - - 0
Vgsc- voltage-gated sodium channel. GSTe2-glutathione-s-transferase epsilon 2. Ace1-Acetylcholine esterase1. N = number of mosquitoes positive for respective
resistance marker.
https://doi.org/10.1371/journal.pone.0241023.t001
freq. = 0.25, 95%CI: 0.17–0.36) than outdoor-resting mosquitoes (allele freq. = 0.04, 95%CI:
0.005–1.3) but this was not statistically significant (Z = 0.965, P = 0.33). In LRR, the mutation
was found only in indoor-resting mosquitoes (allele freq. = 0.3, 95%CI: 0.19–0.42). In WCR,
the mutation was common in An. gambiae s.s. and higher among outdoor- (allele freq. = 0.84,
95%CI: 0.67–0.95) than indoor-resting (allele freq. = 0.75, 95%CI: 0.35–0.97) mosquitoes, with
no significant difference (Z = 0.510, P = 0.61).
Vgsc-1014F was almost fixed in most mosquitoes, except An. arabiensis. It was also more
common in the outdoor- than indoor-resting mosquitoes. Specifically in URR, the mutation
was found to be significantly higher (Z = 2.956, P = 0.003) in outdoor-resting (allele freq. =
0.92, 95%CI: 0.81–0.98) than the indoor-resting An. coluzzi population (allele freq. = 0.74, 95%
CI: 0.63–0.82). Likewise, in the hybrid population of An. coluzzi and An. gambiae s.s., the
mutation was fixed and higher in the outdoor-resting (allele freq. = 1, 95%CI: 0.74–1) than
indoor-resting (allele freq. = 0.93, 95%CI: 0.80–1) mosquitoes but not statistically significant
(Z = 1.027, P = 0.3). The mutation was similarly fixed in both the indoor (allele freq. = 1, 95%
CI: 0.96–1) and outdoor (allele freq. = 0.99, 95%CI: 0.95–1) An. gambiae s.s. populations.
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Although in WCR, Vgsc-1014F was more frequent in An. gambiae s.s. resting indoors (allele
freq. = 0.25, 95%CI: 0.03–0.65) than those outdoors (allele freq. = 0.13, 95%CI: 0.04–0.29), the
difference was not statistically significant (Z = 0.688, P = 0.49). Whereas in LRR, where only
mosquitoes resting indoors were caught, this mutation was most common in An. coluzzii
(allele freq. = 0.66, 95%CI: 0.81–0.98).
Vgsc-1575Y and GSTe2-114T were found mostly in URR and were more frequent in out-
door-resting mosquitoes. The mutations were almost fixed in An. gambiae s.s. regardless of
resting place (allele freq. = 0.96–1, 95% CI: 0.92–1.2). In An. coluzzii, these mutations were sig-
nificantly higher (Vgsc-1575Y: Z = 3.343, P = 0.001. GSTe2-114T: Z = 1.948, P = 0.05) in those
resting outdoors (allele freq: 0.9, 95% CI: 0.79–0.97) than in their indoor-resting counterpart
(allele freqs: Vgsc-1575Y = 0.68, 95% CI: 0.57–0.77. GSTe2-114T = 0.78, 95% CI: 0.68–0.86).
An. coluzzii -An. gambiae s.s. hybrids with higher and fixed Vgsc-1575Y mutation were equally
found resting outdoors (allele freq. = 1, 95% CI: 0.74–1) while those found resting indoors
were carrying only the GSTe2-114T mutation (allele freq. = 0.87, 95% CI: 0.60–0.98).
The carbamate and organophosphate resistance marker, acetylcholine esterase (Ace)-119S
was detected only in 8 (4 indoor and 4 outdoor) An. gambiae s.s. and in one hybrid specimen
in URR.
Sporozoite infection rate
Plasmodium falciparum sporozoites were detected in 11 out of 844 mosquitoes (Table 2), rep-
resenting a 1.3% (95% CI: 0.5–2%) infection rate. All the infected mosquitoes were caught in
URR, of which six were resting indoors and five resting outdoors. Outdoor-resting An. ara-
biensis were mostly infected (36%, 4/11), followed by indoor-resting An. gambiae s.s. (27%,
3/11) and indoor-resting An. arabiensis (18%, 2/11). One each of outdoor-resting An. coluzzii
and An. coluzzii-An. gambiae s.s. hybrid were also infected.
Host blood meal preference
Host blood meal origin was determined in 251 randomly selected engorged mosquito abdo-
mens. Overall, animal and human blood meal indices were higher for indoor- than outdoor-
resting mosquitoes (Table 3). In all vector species, most blood meal (91%) were from animal
origin. Indoor-resting An. coluzzii had the highest preference for human blood while indoor-
resting An. arabiensis had most preference for animal blood. Regardless of their resting loca-
tion, all vector species preferred cow and donkey blood meal compared to other animals. Most
vectors rarely fed on chicken and horse.
Discussion
Insecticide resistance is currently widespread among malaria vectors in the Gambia [11, 14],
resulting in insecticide rotation for IRS. Currently, actellic, an organophosphate insecticide is
being used, whilst LLINs impregnated with deltamethrin and permethrin as recommended by
WHO are distributed widely [32]. This study assessed how such vector interventions have
Table 2. Sporozoite positivity rate in the eleven vector species that were infected based on their resting locations.
An. arabiensis Proportion (n) An. coluzzii Proportion (n) An. gambiae s.s. Proportion (n) An. coluzzii-An. gambiae s.s. proportion (n)
Indoor 0.18 (2) 0.09 (1) 0.27 (3) 0
Outdoor 0.36 (4) 0 0 0.09 (1)
n = number of mosquitoes positive for sporozoite detection. Proportion = the number positive per species divided by overall positive (11).
https://doi.org/10.1371/journal.pone.0241023.t002
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Table 3. Human and animal blood meal preferences of the indoor and outdoor-resting vector species in combined study sites.
An. arabiensis An. coluzzii An. gambiae s.s. An. coluzzii-An. gambiae s.s.
Indoor(n) Outdoor(n) Indoor(n) Outdoor(n) Indoor(n) Outdoor(n) Indoor(n) Outdoor(n)
Human 0.01(3) 0.004(1) 0.03(7) 0 0.008(2) 0.008(2) 0 0.004(1)
Cow 0.09(22) 0.02(6) 0.06(14) 0.03(8) 0.09(22) 0.08(21) 0.03(7) 0.02(4)
Chicken 0 0 0 0 0 0.004(1) 0 0
Dog 0.008(2) 0.004(1) 0 0.004(1) 0 0.004(1) 0 0
Donkey 0.11(27) 0.12(31) 0.06(14) 0.04(11) 0.02(6) 0.03(8) 0.004(1) 0.008(2)
Goat 0.02(4) 0.008(2) 0.008(2) 0.008(2) 0.004(1) 0.004(1) 0.004(1) 0
Horse 0.004(1) 0 0 0 0 0 0 0
Human + animals 0.004(1) 0.004(1) 0.01(3) 0 0.008(2) 0 0 0
Mixed animals 0.008(2) 0 0 0.004(1) 0 0.004(1) 0 0
HBI 2 0.8 4 0 1 0.8 0 0.4
Animal blood indices 23 16 12 9 12 13 4 2
Proportion (number). HBI = Human blood index.
https://doi.org/10.1371/journal.pone.0241023.t003
influenced the feeding and resting behaviour, as well as malaria transmission dynamics of the
vector population. Anopheles arabiensis showed a marked preference for outdoor resting and
An. coluzzii and An. melas for indoor resting. An. gambiae s.s. and An. coluzzii-An. gambiae
s.s. hybrid populations were found mostly resting outdoors, but with no statistical difference
from those resting indoors. Moreover, local vectors had a marked preference for animal blood.
The overall sporozoite infection rate was low and infectious mosquitoes were mainly outdoor-
resting An. arabiensis.
In this study, high frequencies of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T were recorded
in the malaria vector populations resting indoors and outdoors. These mutations were particu-
larly at saturation in both indoor and outdoor-resting An. gambiae s.s. populations in URR, as
well as in most of the An. coluzzii-An. gambiae s.s. hybrid samples analyzed. This magnitude of
the DDT and pyrethroid-associated resistance mutations [23, 24, 27] recorded in the vector
populations is worrying. These might have been as a result of extensive coverage of IRS and
LLINs across the Gambia [7, 8]. These findings suggest that effectiveness of LLINs may be
compromised in the local vector populations as pyrethroids remain the only public health-
approved insecticide for LLINs [32]. Selection for resistance could have resulted from the
extensive use of pyrethroids for vector control [7, 8] and pest control in agriculture [33, 34] in
this region. Moreover, the level of resistance found in the An. coluzzii-An. gambiae s.s. hybrid
population suggests an extensive gene flow between An. gambiae and An. coluzzii [35–37] in
these settings. These observations are a concern for a possible setback to the ongoing anti-vec-
tor efforts; thus deserve close monitoring.
A possible influence of genotypic resistance on the resting behaviour of the vector popula-
tions [38], was observed from this study. A higher proportion of An. arabiensis that was found
resting indoors harboured the Vgsc-1014S mutation whereas An. coluzzii exhibited an out-
door-resting behavior when all mutation markers except Vgsc-1014S were higher in the out-
door than indoor population. Anopheles gambiae s.s. with extremely high mutations were also
found resting both indoors and outdoors. These indicate that resistance could be driving An.
coluzzii and An. gambiae s.s. from their usual indoor resting to outdoor resting behaviour [4,
39, 40], a trait that could promote residual transmission [5, 17, 40]. Indeed, indoor resting
behaviour demonstrated in the resistant vectors despite the presence of IRS and LLINs sug-
gests that resistance could be protecting these vectors against the effect of insecticides used
indoors [41, 42]. This has been previously reported from studies in Kenya [43, 44], where An.
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PLOS ONE Biting and resting preferences of malaria vectors
gambiae s.s and An. arabiensis that had higher frequencies of Vgsc-1014F and Vgsc-1014S
respectively, were predominantly found resting indoors. Similarly, association between geno-
typic resistance and outdoor-resting behavior was earlier found in An. coluzzii [45, 46]. Any
resistance-driven resting behavior in these vectors could further limit the success of anti-vector
interventions currently being scaled-up in these settings [47, 48].
The majority of the vector populations analyzed had a marked preference for animal than
human blood meal. Anopheles arabiensis demonstrated predominant preference for animal
blood meal than other vector species. This is not surprising as An. arabiensis is known to be
highly zoophilic [49, 50]. However, the proportion of vectors resting indoors and that have
taken a blood meal either from human and animal source, could be a concern for the effective-
ness of vector control measures. As animals were found only in outdoor locations in the study
sites, this finding shows that the vectors took blood meal from animals outdoors and later
went indoors to rest regardless of the presence of IRS and LLINs, indicating that these vectors
are resistant to the insecticides being used [51]. Furthermore, the observed choice of animal
blood by majority of the vectors could lead to increase in vector population that may eventu-
ally resort to biting humans in the long run and become difficult to control. Notably, alterna-
tive vector control methods such as treatment of animals with endectocides [52] and larval
source management [53] could be promising tools that could be adopted by the Gambia
National Malaria Control Program.
This study recorded an overall low sporozoite rate in the vector populations. Given the cur-
rent low malaria prevalence in the Gambia [7], a low sporozoite rate is expected. This may
reflect the impact of the scaled-up in IRS and LLINs program in the study sites which seems to
successfully limit mosquito access to human blood meal indoors and consequently reducing
transmission, as previously reported [54, 55]. Indeed, the study sites benefitted from improved
housing projects conducted in country which aimed at reducing mosquito survival as well as
malaria transmission [56].
The composition of the vector species was consistent with previous studies in the Gambia
where the most abundant vector was An. arabiensis, followed by An. gambiae s.s. and, An. coluzzii
along with their hybrids [11, 14, 15]. Low density of An. melas found was as a result of our choice
of villages in the West, which were not located in the coastal regions where this species breeds in
salty water [57]. Remarkably, predominance of An. arabiensis could be as a result of its outdoor-
resting preference to avoid insecticide used in IRS and LLINs [58, 59]. This leaves the highly
anthropophilic and endophilic species more exposed to vector interventions, possibly leading to
relative advantage that maintains the exophilic population and malaria transmission [59].
Conclusion
This study observed high levels of resistance mutations in the local vectors that could be influ-
encing their resting behaviour. As the Gambia is in earnest preparation for pre-elimination
phase of malaria, the magnitude of resistance mutations observed in the vectors in this study
suggests that vectors could pose great challenges for their control using the present control
paradigm. Therefore, use of larval source management as a complementary vector control
measure is highly recommended.
Supporting information
S1 File. Overall data including separate worksheets for genotypic resistance and species
genotypes; host blood meal and sporozoite positivity data.
(XLSX)
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PLOS ONE Biting and resting preferences of malaria vectors
Acknowledgments
We thank Messrs Musa Jawara, and Mamlie Touray for their assistance in the field work for
this study.
Author Contributions
Conceptualization: Yaw A. Afrane, Alfred Amambua-Ngwa.
Formal analysis: Majidah Hamid-Adiamoh.
Investigation: Majidah Hamid-Adiamoh.
Methodology: Majidah Hamid-Adiamoh.
Supervision: Davis Nwakanma, Yaw A. Afrane, Alfred Amambua-Ngwa.
Writing – original draft: Majidah Hamid-Adiamoh.
Writing – review & editing: Majidah Hamid-Adiamoh, Davis Nwakanma, Benoit Sessinou
Assogba, Mamadou Ousmane Ndiath, Umberto D’Alessandro, Yaw A. Afrane, Alfred
Amambua-Ngwa.
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