Malaria Journal

Stica et al. Malar J (2019) 18:244

https://doi.org/10.1186/s12936-019-2875-y

RESEARCH Open Access

Characterizing the molecular and metabolic

mechanisms of insecticide resistance
in Anopheles gambiae in Faranah, Guinea
Caleb Stica1, Claire L. Jeffries1, Seth R. Irish2,3, Yaya Barry4, Denka Camara4, Ismael Yansane5, Mojca Kristan1,
Thomas Walker1 and Louisa A. Messenger1,2,6*

Abstract
Background: In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS)
has greatly reduced malaria transmission. However, malaria remains a global public health concern with the major-
ity of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector
populations, with potential implications for the continued effectiveness of available control interventions. Improved
understanding of current resistance levels and underlying mechanisms is essential to design appropriate manage-
ment strategies and to mitigate future selection for resistance.
Methods: Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea
and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Syner-
gist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases
in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species,
presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative
expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3).
Results: In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to ben-
diocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin
and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F
kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y
mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant devia-
tions from Hardy–Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes
tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3
and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations,
compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors,
implicating its potential role in carbamate resistance in Faranah.
Conclusions: Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the
Guinea National Malaria Control Programme (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs
for vector control. Study findings will be used to guide current and future control strategies in the region.
Keywords: Insecticide resistance, Anopheles gambiae, Guinea, L1014F kdr, N1575Y, G119S Ace-1, Metabolic resistance,
CYP6M2, CYP6P3, GSTD3

*Correspondence: [email protected]
1
Department of Disease Control, Faculty of Infectious Tropical Diseases,
London School of Hygiene and Tropical Medicine, London, UK
Full list of author information is available at the end of the article

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/
publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Stica et al. Malar J (2019) 18:244 Page 2 of 15

Background Methods
Despite impressive progress made towards the control Study sites and mosquito collections
and elimination of malaria, this disease remains the lead- Human landing catches (HLCs) were conducted at
ing cause of morbidity and mortality in the tropics, where three sites in Faranah Prefecture (Fig. 1). The villages
it is estimated to have resulted in the deaths of approxi- of Balayani (10.1325, − 10.7443), Foulaya (10.144633,
mately 435,000 individuals in 2017 [1]. Between 2010 − 10.749717), and Tindo (9.9612230, − 10.7016560) were
and 2017, global malaria incidence has fallen by approxi- selected, due to their high malaria prevalence, and were
mately 18% globally (72 to 59 cases per 1000 at risk) and visited every other day until a sufficient sample size for
by 20% in the World Health Organization African region testing was acquired. Following consent from the house-
[1], which still bears the greatest disease burden [2]. hold owner, two to three fieldworkers, positioned outside
Malaria deaths have been decreasing annually, largely due of the house, collected mosquitoes landing and attempt-
to the scale-up of long-lasting insecticidal nets (LLINs) ing to feed on their exposed legs and feet from 18.00 to
[3] and implementation of indoor residual spraying (IRS) 07.00. Mosquitoes were transported back to the insectary
[2]. However, progress has stalled in some areas, with an in Faranah and provided with 10% sucrose solution prior
increase of 2 million cases from 2016 to 2017 [1]. to bioassay testing.
Long-term intensive insecticide use to control agri- Larval collections were also performed in Faranah
cultural pests and disease vectors has resulted in the (10.042423, − 10.740980), at sites selected through active
selection of resistance in many insect species [4]. searching and/or known to have been productive in pre-
The widespread use of dichlorodiphenyltrichloroeth- vious years. Larvae and pupae were collected using lar-
ane (DDT) in the 1950–1960s, followed by the recent val dippers, ladles, buckets, and pans. Collections were

they were fed on ground fish food (­TetraMin® Tropical

increase in distribution of LLINs impregnated with pyre- then transported back to the insectary in Faranah where
throids, and the broad use of the same insecticides in the
agricultural industry has led to the development of resist- Fish Food Flakes). Pupae were removed on a daily basis,
ance in mosquito populations worldwide [5]. This resist- placed in plastic cups, and transferred to mosquito cages.
ance poses a major threat to malaria control [4], where Emerging adult mosquitoes were provided 10% sucrose
vector control is reliant primarily on insecticide-based solution prior to bioassay testing. All mosquito samples
interventions [6]. Of the 80 malaria-endemic countries were collected between 25th June and 20th July 2018, at
for which data are available from 2010 onwards, 68 coun- the start of the long rainy season.
tries detected decreased susceptibility to at least one
insecticide among vector populations, with 57 countries CDC resistance intensity and synergist bioassays
reporting resistance to two or more chemical classes [1]. Centers for Disease Control and Prevention bottle bio-
In Guinea, malaria remains one of the most significant assays were performed using 250 mL Wheaton bottles
diseases of public health importance, with 92% of infec- with adult female mosquitoes of varying ages caught in
tions caused by Plasmodium falciparum [7]. The national HLCs from the three villages (each village was tested
malaria prevalence is approximately 15%, reaching up to separately), or 2–5-day old female mosquitoes raised
25% in Faranah Prefecture [8]. Guinea’s tropical climate from larvae in the insectary. Mosquitoes collected using
allows for year-round malaria transmission, with peak different methods were tested separately in bioassays.
transmission from July through to October in most areas. Bioassays were performed at the Centre de Santé Mar-
The Guinean national vector control strategy focuses ché in Faranah; mosquitoes were held in the insectary
almost exclusively on the distribution of LLINs, with IRS for no more than 48 h prior to testing. Following CDC
occurring in only 1.7% of households, primarily those of guidelines, bottles were coated with alpha-cypermethrin
workers engaged in mining operations [9]. The United (12.5 μg/bottle), deltamethrin (12.5 μg/bottle), perme-
States President’s Malaria Initiative (PMI) estimates that thrin (21.5 μg/bottle), and bendiocarb (12.5 μg/bottle) at
intervention coverage remains low, with only 48% of 1, 2, 5, and 10 times the diagnostic dose, and with pirimi-
households owning at least one insecticide-treated net phos-methyl (20 μg/bottle), clothianidin (90 μg/bottle),
(ITN) per every two members [9]. Given the reliance and chlorfenapyr (100 μg/bottle) at the diagnostic dose.
on LLINs for malaria control, the detection of nation- Stock solutions for all insecticides and synergists were
wide pyrethroid resistance is of concern [10]. In order to prepared using 95–98% ethanol as a solvent for all, clothi-
safeguard malaria control efforts in the country, current anidin also included 58.8 μg Mero (Sigma-Aldrich, USA),
insecticide resistance levels and underlying mechanisms to circumvent issues previously experienced with bottle
were characterized among vector populations, to design coating and insecticide crystallization. Approximately
appropriate management strategies and mitigate future 20–25 mosquitoes were introduced into each assay bot-
selection for resistance. tle and mortality was recorded in all bottles at the start
Stica et al. Malar J (2019) 18:244 Page 3 of 15

Esri, HERE, Garmin, © OpenStreetMap contributors, and the GIS user

0 75 150 300 Kilometers community, Source: Esri, DigitalGlobe, GeoEye, Earthstar Geographics,
CNES/Airbus DS, USDA, USGS, AeroGRID, IGN, and the GIS User
Community

Foulaya
Balayani

Faranah

Tindo
Esri, HERE, Garmin, © OpenStreetMap contributors, and the GIS user
0 2.5 5 10 Kilometers community, Source: Esri, DigitalGlobe, GeoEye, Earthstar Geographics,
CNES/Airbus DS, USDA, USGS, AeroGRID, IGN, and the GIS User
Community

Fig. 1 Map of study sites in Faranah Prefecture, Guinea. Human landing catches (HLCs) were performed in the villages of Balayani, Foulaya and
Tindo to collect adult An. gambiae s.l. Larval collections were undertaken at multiple sites in the city of Faranah
Stica et al. Malar J (2019) 18:244 Page 4 of 15

of the assay and at 15-min intervals for up to 30 min (for further identified using an end point PCR assay devel-
alpha-cypermethrin, deltamethrin, permethrin, bendio- oped by Santolamazza et al. [12]. This assay amplifies the
carb, and pirimiphos-methyl) or 60 min (for clothianidin SINE200 insertion, a highly repetitive ~ 200 bp element
and chlorfenapyr). Mosquitoes exposed to chlorfenapyr which is widespread in the An. gambiae sensu stricto
were held for an additional 24 h in paper cups, with (s.s.) genome [12]. Samples were prepared with forward
access to 10% sugar solution. In each bioassay, a control (5′-TCG​CCT​TAG​ACC​TTG​CGT​TA-3′) and reverse
bottle, coated with 98% ethanol, was run in parallel. Mor- (5′-CGC​TTC​AAG​AAT​TCG​AGA​TAC-3′) primers, and
tality was defined as the inability of a mosquito to stand amplifications performed in 20 µL reactions containing
or fly in a coordinated manner [11]. Synergist CDC bot- 2 µL cDNA, 2 µL of each primer (10 µM), 4 µL H ­ 2O, and

Biolabs). The cycling conditions in the Bio-Rad T100™

tle bioassays were performed using piperonyl butoxide 10 µL 2× Hot Start Taq PCR Master Mix (New England
(PBO) to investigate the potential role of detoxifying
enzyme families in resistance. Bottles were coated with Thermal Cycler were: 10 min at 94 °C; 35 cycles of 30 s
100 μg of PBO and mosquitoes were exposed for 60 min, at 94 °C, 30 s at 54 °C, and 60 s at 72 °C; then 10 min at

itrogen E-gel iBase™ Real-Time Transilluminator. Ampli-

followed by exposure to pyrethroid treated bottles. Adult 72 °C. Products were run on 2% agarose gels in an Inv-
mosquitoes reared from larvae were exposed for 30 min
to permethrin and deltamethrin for comparison to wild fication products of 479 bp or 249 bp were considered
caught adults. Multiple replicates were performed per indicative of Anopheles coluzzii or An. gambiae s.s.,
insecticide and study village. When limited by mosquito respectively; both bands indicate a hybrid individual. The
availability, for insecticides where there was a clear lack An. gambiae s.s. form has an identical banding pattern to
of resistance, i.e. 100% of exposed mosquitoes dying/ that of Anopheles melas and Anopheles quadriannula-
becoming knocked-down very quickly into the exposure tus (249 bp), however, due to the geographical location
period, priority was given to testing remaining mosqui- of sampling, it is highly unlikely that these species were
toes with doses of insecticides where initial resistance present.
to the diagnostic dose was observed. At the end of each
exposure period, separate individual surviving (resist- Plasmodium falciparum detection
ant), knocked-down or dead (susceptible), and control A total of 508 whole body mosquito samples (collected

in ­RNAlater® (Thermo Fisher Scientific, UK) at 4 °C in

mosquitoes were dipped in ethanol and then preserved in HLCs) were tested for the presence of P. falciparum
using a real-time assay targeting the cytochrome c oxi-
Faranah, for a maximum of 3 weeks, and subsequently at dase subunit 1 (cox1) mitochondrial gene of P. falciparum
− 80 °C for downstream molecular analyses at the Lon- according to Boissière et al. [13]. This sample includes all
don School of Hygiene and Tropical Medicine (LSHTM). specimens which underwent PCR for species identifica-
tion and were subsequently processed for metabolic gene
RNA extraction expression (see below). This assay is highly sensitive and
Single mosquitoes were homogenized using a Qiagen Tis- specific, capable of detecting the target gene at all stages
sue Lyser II (Qiagen, Hilden, Germany) with 3 mm stain- of the P. falciparum life cycle [13]. Samples were prepared
less steel beads and RNA was extracted using Qiagen 96 with forward (5′-TTA​CAT​CAG​GAA​TGT​TAT​TGC-3′)
RNeasy Kits according to the manufacturer’s instructions and reverse (5′-ATA​TTG​GAT​CTC​CTG​CAA​AT-3′) prim-
(Qiagen, Hilden, Germany). RNA was eluted in 45 μL of ers, and amplifications performed in 10 µL reactions con-
RNase-free water and stored at − 70 °C. Specimens were taining 2 µL cDNA, 1 µL of each primer (10 µM), 1 µL
selected from all three study villages, including repre- ­H2O, and 5 µL 2× Roche FastStart Essential DNA green

on a Roche ­Lightcycler® 96 Real-Time PCR system for

sentative susceptible and resistant individuals per insec- master mix containing SYBR Green. Samples were run
ticide per concentration. A High Capacity cDNA Reverse
Transcription kit (Applied Biosystems) was used to per- 15 min at 95 °C, followed by 35 cycles of 15 s at 95 °C and

were performed in a Bio-Rad T100™ Thermal Cycler Roche ­Lightcycler® 96 software. Positive controls were
form reverse transcription on eluted RNA. Reactions 30 s at 58 °C. Fluorescence results were analysed using

which cycled for 10 min at 25 °C, 120 min at 37 °C, and used from gDNA extracted from a cultured P. falciparum-
5 min at 85 °C. The resulting cDNA was then stored at infected blood sample (parasitaemia of ~ 10%) in addition
− 20 °C. to the inclusion of no template controls (NTCs).

Identification of Anopheles gambiae species complex Characterization of resistance mutations: target site
Mosquitoes were morphologically identified as Anoph- mutations
eles gambiae sensu lato (s.l.) in the field prior to CDC A sub-sample of 60 mosquitoes was selected to be tested
bottle bioassay testing. A sub-set of 480 samples were for the West African L1014F kdr mutation, given the high
Stica et al. Malar J (2019) 18:244 Page 5 of 15

frequency of this allele and its fixation in many parts of in 20 µL reactions containing 2 µL cDNA, 2 µL each
Guinea and West Africa [10, 14]. The PCR master mix primer (10 µM), 4 µL H ­ 2O, and 10 µL 2× Hot Start Taq
was prepared according to MR4 guidelines [15]. Prim-
loaded into a Biorad T100™ Thermal Cycler for 3 min at
PCR Master Mix (New England Biolabs). Samples were
ers IPCF (5′-GAT​AAT​GTG​GAT​AGA​TTC​CCC​GAC​
CATG-3′), AltRev (5′-TGC​CGT​TGG​TGC​AGA​CAA​ 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 52 °C,
GGATG-3′), WEST WT (5′-GGT​CCA​TGT​TAA​TTT​ and 1 min at 72 °C, and a final step of 10 min at 72 °C.
GCA​TTA​CTT​ACG​AAT​A-3′), and West:West (5′-CTT​ The resulting PCR fragments were then digested with
GGC​CAC​TGT​AGT​GAT​AGG​AAA​TGTT-3′) were used AluI restriction enzyme (Thermo Scientific) for 16 h,
to detect the L1014F allele. Amplifications were per-
on 2% agarose gels in an Invitrogen E-gel iBase™ Real-
according to the manufacturer’s instructions, and run
formed in 25 µL reactions containing 2 µL cDNA, 1 µL
IPCF (2.5 pmol/µL), 1 µL AltRev (2.5 pmol/µL), 1 µL Time Transilluminator. 194 bp undigested PCR products
West WT (25.0 pmol/µL), 3 µL West:West (8.0 pmol/µL), indicated the susceptible allele and 74 bp and 120 bp
4.5 µL ­H2O, and 12.5 µL 2× Hot Start Taq PCR Master digested fragments indicated the presence of the resist-

Rad T100™ Thermal Cycler and cycled for 5 min at 95 °C,

Mix (New England Biolabs). Samples were run on a Bio- ant allele. Presence of all three product sizes indicated
the sample was heterozygous. Positive controls from
followed by 35 cycles of 30 s at 95 °C, 30 s at 59 °C, and gDNA extracted from known An. gambiae s.s. that were
30 s at 72 °C, and a final extension step of 5 min at 72 °C. homozygous susceptible (SS), homozygous resistant (RR)

Invitrogen E-gel iBase™ Real-Time Transilluminator. A

PCR products were separated on 2% agarose gels in an and heterozygous individuals (RS) for G119S Ace-1 were
included in addition to no template controls (NTCs).
control band at 314 bp indicated a successful reaction, a
band at 214 bp indicated the susceptible wild type, and a Characterization of resistance mechanisms: metabolic
band at 156 bp indicated the resistance mutation. gene expression
Detection of the N1575Y mutation was carried out The relative gene expression of two cytochrome-
with 570 samples (including the 60 individuals who tested dependent monooxygenases: CYP6P3, CYP6M2, and
positive for L1014F kdr), using a TaqMan PCR assay glutathione-s-transferase, GSTD3, was analysed in 461
developed by Jones et al. [16]. Forward primer (5′-TGG​ individuals from Guinea and 41 susceptible G3 individu-
ATC​GCT​AGA​AAT​GTT​CAT​GAC​A-3′), reverse primer als from a colony at LSHTM, using quantitative reverse
(5′-CGA​GGA​ATT​GCC​TTT​AGA​GGT​TTC​T-3′), and transcriptase PCR (qRT-PCR) relative to the house-
two probes: Yprobe (5′- TTT​TTC​ATT​GCA​TAA​TAG​ keeping gene ribosomal protein S7 (RPS7), according
TAC-3′) and Nprobe (5′-ATT​TTT​TTC​ATT​GCA​TTA​ to Yahouédo et al. [18]. RPS7 was selected as an endog-
TAG​TAC​-3′) were used to detect the presence of the wild enous reference gene, commonly used to normalize RNA
type and the mutation allele. HEX and FAM fluorophores expression levels, between Anopheles populations. These
were used due to their different excitation wavelengths, genes were targeted based upon their significant overex-
ensuring no interference: excitation of HEX showed no pression in other neighbouring West African vector pop-
mutation, while excitation of HEX and FAM at similar Ct ulations [18, 19]. Each gene used the following primers:
values indicated the N1575Y mutation. 20 µL reactions RPS7 forward (5′-ATT​GCC​GAG​CGC​CGC​ATT​CT-3′)
containing 2 µL cDNA, 1 µL each primer (10 µM), 0.5 µL and reverse (5′-GAC​GCG​GAT​ACG​CTT​GCC​GA-3′)
each probe, 5 µL ­H2O, and 10 µL QuantiTect Probe Mas- primers, CYP6M2 forward (5′-TCG​GGA​TGT​GTG​CGT​
ter Mix were prepared in plates and run on an Agilent TCG​GC-3′) and reverse (5′-TCG​TGT​CTC​GCA​CCG​
cycled according to the Quantitect™ Probe PCR Hand-
Technologies Stratagene Mx3005P qPCR system and CGT​TC-3′) primers, CYP6P3 forward (5′-TGT​GAT​TGA​
CGA​AAC​CCT​TCG​GAA​G-3′) and reverse (5′-ATA​GTC​
book guidelines (15 min at 95 °C; 35 cycles of 15 s at 95 °C CAC​AGA​CGG​TAC​GCGGG-3′) primers, and GSTD3
and 60 s at 60 °C). Positive controls from gDNA extracted forward (5′-CTA​AGC​TTA​ATC​CGC​AAC​ATA​CCA​-3′)
from known An. gambiae s.s. with the N1575Y mutation and reverse (5′-GTG​TCA​TCC​TTG​CCG​TAC​AC-3′)
and without the mutation were included on each run in primers. For each gene, 10 µL reactions were prepared
addition to no template controls (NTCs). containing 2 µL cDNA, 1 µL each primer (10 µM), 1 µL
A subsample of 30 mosquitoes which were resistant or ­H2O, and 5 µL 2× Roche FastStart Essential DNA green
susceptible to bendiocarb were tested for the presence of master mix containing SYBR Green. Prepared reac-
the G119S Ace-1 mutation using a TaqMan PCR assay, tions were loaded into the Agilent Technologies Strata-
according to Weill et al. [17]. Samples were prepared with gene Mx3005P qPCR system which cycled for 10 min at
degenerate primers Moustdir1 (5′-CCGGGNGCSACY​ 95 °C; 35 cycles of 10 s at 95 °C, 22 s at 60 °C, and 10 s
ATG​TGGAA-3′) and Moustrev1 (5′-ACGATMACG​ at 72 °C; followed by a melt curve. Serial dilutions were
TTC​TCY​TCC​GA-3′), and amplifications performed performed on selected samples for each of the four genes
Stica et al. Malar J (2019) 18:244 Page 6 of 15

and relative standard curves produced using the Strata- Considering HLC-collected adult mosquitoes from
gene MxPro qPCR software (Agilent Technologies). Faranah Prefecture as a whole, resistance was consist-
Using the same software, sample Ct values could then be ently observed to permethrin and deltamethrin. Perme-
used to generate relative quantities, accounting for each thrin gave the lowest mortality of all insecticides tested
assays’ efficiency, and the expression level of each meta- with 4% [95% CI 1%, 11%] and 15% [95% CI 9%, 24%]
bolic gene tested could be normalized to the housekeep- mosquito mortality at 1× and 2× the diagnostic doses,
ing gene RPS7. respectively (Fig. 2 and Table 1). Resistance to deltame-
thrin was also evident, but to a lesser degree; average
mosquito mortality to the diagnostic dose was 86% [95%
Data analysis
CI 77%, 91%] (Fig. 2). Possible resistance was observed to
Data were recorded on pre-prepared data sheets and
bendiocarb, with mosquito mortality ranging between 94
entered into an Excel spreadsheet. Control mortality
and 97% at 1×, 2×, and 5× concentrations. Mosquitoes
in bioassays never exceeded 5%, thus correction using
were found to be susceptible to the diagnostic doses of all
Abbott’s formula was not necessary. Mosquito mortal-
other insecticides with mortalities > 98% for alpha-cyper-
ity was analysed according to WHO criteria: 98–100%
methrin, chlorfenapyr, clothianidin, and pirimiphos-
mortality at 30 min of exposure indicates ‘susceptibil-
methyl (Fig. 2 and Table 1).
ity’, 90–97% mortality suggests ‘possible resistance’ and
Minor variations in levels of resistance were apparent
< 90% indicates the presence of ‘resistance’ [4]. Graph-
between study villages. The most intense permethrin
Pad Prism 7 (GraphPad Software) was used for statisti-
resistance was measured in Foulaya, with average mos-
cal analysis (t-tests, Fisher’s exact tests and Chi-squared
quito mortality of 0% [95% CI 0%, 13%] following expo-
tests). Microsoft Excel was used to calculate proportions
sure to either 1× or 2× the diagnostic dose, relative to
and construct resistance graphs. Stratagene MxPro qPCR
6% [95% CI 2%, 19%] and 10% [95% CI 4%, 24%] in Bal-
software (Agilent Technologies) was used to produce rel-
ayani and 7% [95% CI 1%, 30%] and 43% [95% CI 24%,
ative standard curves for genotypic analysis.
63%] in Tindo, respectively (Table 1). Possible resistance
to permethrin 5× was identified in Foulaya and Balayani
Results (average mosquito mortality of 96% [95% CI 81%, 99%]
Mosquito collections and species identification and 97% [95% CI 87%, 100%], respectively). By compari-
A total of 2597 female An. gambiae s.l. mosquitoes were son, the lowest levels of resistance to the diagnostic dose
either wild-caught using HLCs from three villages in of deltamethrin were observed in Foulaya, with average
Faranah Prefecture (Balayani = 956, Foulaya = 914, and mosquito mortality of 95% [95% CI 84%, 99%], compared
Tindo = 589) or raised from larvae collected in Faranah to 79% [95% CI 62%, 89%] and 75% [95% CI 51%, 90%] in
(n = 138). Of those, 480 were selected for molecular spe- Balayani and Tindo, respectively.
cies identification, with 466 (97.1%) determined to be Two to five-day old mosquitoes raised in the insectary
An. gambiae s.s., 6 (1.3%) identified as An. coluzzii, and 6 from larvae demonstrated moderately similar resistance
(1.3%) identified as hybrids; results were inconclusive for profiles, when compared to locally-collected adult popu-
2 (0.4%) individuals. lations of varying physiological age (Table 1). Both per-
methrin and deltamethrin resistance was present, with
Insecticide resistance intensity
average mosquito mortality of 21% [95% CI 10%, 40%]
Levels of susceptibility to seven insecticides (perme- and 60% [95% CI 36%, 80%] for permethrin 1× and 2×
thrin, deltamethrin, alpha-cypermethrin, bendiocarb, doses, respectively, and 73% [95% CI 43%, 90%] mortal-
pirimiphos-methyl, clothianidin and chlorfenapyr) ity for deltamethrin 1×. Comparing resistance profiles
were assessed across three villages in Faranah (Balayani, in larvae to that observed in each village, a statistical
Foulaya, and Tindo). Overall, each study site displayed difference was observed for permethrin 1× in Foulaya
comparable resistance profiles for deltamethrin (pyre- (χ2 = 6.014, p = 0.0142) and in all villages combined
throid) and bendiocarb (carbamate); no significant asso- at 1× (χ2 = 7.407, p = 0.0065); permethrin 2× in Bal-
ciation between mosquito mortality and sample site was ayani (χ2 = 14.62, p = 0.0001) and Foulaya (χ2 = 20.753,
observed (χ2 = 6.495, p = 0.0899 for deltamethrin 1×; p < 0.0001), and all villages combined at 2× (χ2 = 15.2,
χ2 = 1.338, p = 0.5122, χ2 = 2.38, p = 0.304 and χ2 = 0.903, p = 0.0001); and deltamethrin 1× in Foulaya (χ2 = 4.72,
p = 0.637 for bendiocarb at 1×, 2× and 5×, respectively). p = 0.0298). Reared mosquitoes were found to be suscep-
However, mosquito mortality following permethrin tible to all other concentrations of permethrin and del-
(pyrethroid) exposure varied among villages in Faranah tamethrin with mortalities of 100%.
(χ2 = 8.573, p = 0.035 and χ2 = 29.58, p < 0.0001, for 1× Synergist bioassays were performed on a sub-sam-
and 2×, respectively). ple of mosquitoes from Balayani. Pre-exposure to PBO
Stica et al. Malar J (2019) 18:244 Page 7 of 15

Fig. 2 Pooled CDC resistance intensity assay data for all tested insecticides (permethrin, deltamethrin, alpha-cypermethrin, bendiocarb,
pirimiphos-methyl, clothianidin and chlorfenapyr) in Faranah Prefecture, Guinea for adult wild-caught mosquitoes. Mortality below 90% indicates
the presence of confirmed resistance. *Only tested at diagnostic dose

and subsequent permethrin or deltamethrin treatment Characterization of resistance mechanisms: target site
resulted in partial or complete abolishment of resistance. mutations
Mortality to permethrin increased from 6 to 87% at 1×, Of the sub-sample of 60 An. gambiae s.s. which were
and from 10 to 100% at 2×; mortality following deltame- tested for the L1014F kdr allele, this mutation was
thrin exposure increased from 79 to 100% at 1×. identified in all An. gambiae s.s. samples (100%). Of
the 570 individuals which were assayed for the pres-
Plasmodium falciparum detection ence of N1575Y, this mutation was detected in 20%
Of the 508 mosquitoes tested for the presence of P. fal- (113/561; 9 specimens failed to amplify) (Table 2). For
ciparum, 37 individuals were positive (indicating the PCR-confirmed An. gambiae s.s., N1575Y was observed
presence of any parasite lifecycle stage), giving an infec- in 19% (91/473) of specimens. Four (0.7%) individuals
tion rate of 7.3%. For PCR-confirmed An. gambiae s.s., P. were heterozygous for this mutation, while the remain-
falciparum infection prevalence was 6.1% (26/423). There der were homozygous. Across Faranah, the frequency
was no significant difference in P. falciparum infection of N1575Y did not vary significantly (χ2 = 4.4819,
among study villages (χ2 = 1.358, p = 0.507). Considering p = 0.214 for comparisons between Balayani, Fou-
pooled insecticide results (permethrin, deltamethrin, and laya, Tindo, and larvae from Faranah). In addition,
bendiocarb), susceptible mosquitoes were more likely to no significant association was observed between the
be infected with P. falciparum (7.6%; 33/432) than resist- presence of the N1575Y mutation and the ability to
ant mosquitoes (2.8%; 4/144) (χ2 = 4.236, p = 0.039). survive exposure to pyrethroid insecticides (perme-
However, no statistical association between survival to thrin 1× χ2 = 0.015, p = 0.902; 2× χ2 = 0.189, p = 0.664;
insecticide exposure and P. falciparum infection was 5× χ2 = 0.814, p = 0.359; deltamethrin 1× χ2 = 0.934,
observed per individual chemical (Fisher’s exact: perme- p = 0.334). Significant deviations from Hardy–Wein-
thrin 2× p = 0.37, 5× p = 0.54; deltamethrin 1× p = 0.43; berg equilibrium were observed in almost all phe-
bendiocarb 1× p = 0.58, 2× p = 0.73, 5× p = 0.6 and 10× notyped specimens, indicative of ongoing selection
p = 0.54). (Table 2).
Stica et al. Malar J (2019) 18:244 Page 8 of 15

Table 1 Percentage mortality (and numbers tested) of Anopheles gambiae s.l. in CDC resistance intensity assays
conducted at four sites in Faranah Prefecture, Guinea
Study site Insecticide % Mortality (numbers tested) after diagnostic time
1× 2× 5× 10×

Balayani Permethrin 6% (34) 10% (39) 97% (38) 100% (55)

Permethrin + PBO 87% (15) 100% (18) 100% (22) 100% (28)
Deltamethrin 79% (33) 100% (35) 100% (32) 100% (46)
Deltamethrin + PBO 100% (26) – – –
Alpha-cypermethrin 100% (20) – – –
Bendiocarb 97% (38) 98% (42) 96% (46) 100% (43)
Pirimiphos-methyl 100% (40) – – –
Clothianidin 100% (38) – – –
Chlorfenapyr 100% (21) – – –
Foulaya Permethrin 0% (26) 0% (28) 96% (25) 100% (24)
Deltamethrin 95% (41) 100% (39) 100% (41) 100% (24)
Alpha-cypermethrin 100% (42) – – –
Bendiocarb 92% (48) 93% (43) 96% (53) 100% (47)
Pirimiphos-methyl 100% (19) – – –
Clothianidin 100% (33) – – –
Chlorfenapyr 100% (40) – – –
Tindo Permethrin 7% (15) 43% (21) 100% (20) 100% (29)
Deltamethrin 75% (16) 100% (15) 100% (17) 100% (17)
Alpha-cypermethrin 100% (41) – – –
Bendiocarb 95% (21) 100% (23) 100% (21) 95% (21)
Pirimiphos-methyl 100% (38) – – –
Clothianidin 100% (53) – – –
Chlorfenapyr 100% (40) – – –
Faranah Permethrin 21% (28) 60% (15) 100% (12) 100% (17)
Larvae Deltamethrin 73% (11) 100% (11) 100% (13) 100% (14)

Of the subsample of 30 An. gambiae s.s. which sur- G3 colony. These three genes were significantly overex-
vived or died following bendiocarb exposure, the G119S pressed in the majority of wild-caught An. gambiae s.s.
Ace-1 mutation was present in 62% (18/29 individuals; 1 when compared to the susceptible G3 laboratory strain
specimen failed to amplify) (Table 3), with allele frequen- (Table 4 and Fig. 3).
cies ranging from 0.25 to 1.0. Seventeen specimens were The greatest changes in gene expression were
homozygous, the remaining individual was heterozygous. observed for GSTD3, with an average mean fold change
There was a statistically significant association between of 1.19 among wild An. gambiae s.s. compared to 0.44
the presence of the G119S Ace-1 mutation and mosquito in G3 colony individuals; average levels of CYP6M2
survival after bendiocarb exposure at 1× and 2× (Fisher’s and CYP6P3 were 0.84 and 0.79 compared to 0.09 and
exact: p = 0.00108 and p = 0.0238 respectively). Signifi- 0.12 between field and colony mosquitoes, respec-
cant deviations from Hardy–Weinberg equilibrium for tively. Among Guinean vectors, a significantly higher
G119S Ace-1, were limited to mosquitoes which died fol- expression of CYP6P3 was observed between individu-
lowing exposure to bendiocarb 2× and 5× (Table 3). als which survived bendiocarb exposure at 1× and 2×,
compared to those that died (1.44 vs. 0.62; p = 0.0524
Characterization of resistance mechanisms: metabolic and 1.68 vs. 0.55; p = 0.0366, respectively) (Table 4).
gene expression However, no significant changes in gene expression of
461 samples identified as An. gambiae s.s. were tested CYP6M2 and GSTD3 were apparent between wild An.
for the expression of CYP6M2, CYP6P3, and GSTD3 gambiae s.s. which survived or died after insecticide
genes relative to the housekeeping gene RPS7 and com- exposure at any dose.
pared to 41 An. gambiae s.s. samples from a susceptible
Stica et al. Malar J (2019) 18:244 Page 9 of 15

Table 2 N1575Y allele frequencies and p values for Chi-square tests for deviations from Hardy–Weinberg equilibrium
in An. gambiae s.s. from four sites in Faranah Prefecture, Guinea
Study village/sampling site

Balayani Foulaya Tindo Larvae

RS RR SS RS RR SS RS RR SS RS RR SS

Permethrin 1×
Alive 0 2 14 0 3 7 0 4 7 0 1 11
Dead 0 0 2 0 0 0 0 0 1 0 2 4
Permethrin 2×
Alive 0 2 22 0 2 12 0 2 10 0 0 6
Dead 0 0 3 0 0 0 0 3 6 0 0 9
Permethrin 5×
Alive 0 1 0 0 0 1 0 0 0 0 0 0
Dead 0 4 12 0 0 4 0 2 10 0 3 6
Permethrin 10×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 3 6 0 0 4 0 3 2 0 0 4
Deltamethrin 1×
Alive 0 2 4 0 0 2 0 2 2 0 1 2
Dead 0 2 13 0 2 9 0 4 8 0 1 4
Deltamethrin 2×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 1 7 0 2 6 0 1 2 0 1 4
Deltamethrin 5×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 1 10 0 1 5 0 1 2 0 2 2
Deltamethrin 10×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 2 6 0 2 3 0 1 5 0 1 4
Alpha-cypermethrin 1×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 2 3 0 0 9 0 4 6 0 0 0
Alpha-cypermethrin 2×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 1 0 4 0 4 7 0 1 7 0 0 0
Alpha-cypermethrin 5×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 1 5 0 3 14 0 0 0 0 0 0
Alpha-cypermethrin 10×
Alive 0 0 0 0 0 0 0 0 0 0 0 0
Dead 0 0 0 1 3 20 0 0 0 0 0 0
Bendiocarb 1×
Alive 0 4 1 0 0 0 0 0 1 0 0 0
Dead 0 3 17 0 2 8 0 1 2 0 0 0
Bendiocarb 2×
Alive 0 0 1 0 1 2 0 0 0 0 0 0
Dead 1 5 13 0 1 9 0 0 4 0 0 0
Bendiocarb 5×
Alive 0 0 2 0 0 2 0 0 0 0 0 0
Dead 0 4 17 1 0 7 0 0 3 0 0 0
Bendiocarb 10×
Alive 0 0 0 0 0 0 0 1 0 0 0 0
Dead 0 0 9 0 0 10 0 0 4 0 0 0
Stica et al. Malar J (2019) 18:244 Page 10 of 15

Table 2 (continued)
Study village/sampling site N1575Y allele frequency χ2 test p value

Total

RS RR SS R S

Permethrin 1×
Alive 0 10 39 0.2 0.8 49 < 0.00001
Dead 0 2 7 0.22 0.78 9 0.0027
Permethrin 2×
Alive 0 6 50 0.11 0.89 56 < 0.00001
Dead 0 3 18 0.14 0.86 21 < 0.00001
Permethrin 5×
Alive 0 1 1 0.5 0.5 2 0.157299
Dead 0 9 32 0.22 0.78 41 < 0.00001
Permethrin 10×
Alive 0 0 0 – – – –
Dead 0 6 16 0.27 0.73 22 < 0.00001
Deltamethrin 1×
Alive 0 5 10 0.33 0.67 15 0.00108
Dead 0 9 34 0.21 0.79 43 < 0.00001
Deltamethrin 2×
Alive 0 0 0 – – – –
Dead 0 5 19 0.21 0.79 24 < 0.00001
Deltamethrin 5×
Alive 0 0 0 – – – –
Dead 0 5 19 0.21 0.79 24 < 0.00001
Deltamethrin 10×
Alive 0 0 0 – – – –
Dead 0 6 18 0.25 0.75 23 < 0.00001
Alpha-cypermethrin 1×
Alive 0 0 0 – – – –
Dead 0 6 18 0.25 0.75 23 < 0.00001
Alpha-cypermethrin 2×
Alive 0 0 0 – – – –
Dead 1 5 18 0.23 0.77 18.6729 0.000016
Alpha-cypermethrin 5×
Alive 0 0 0 – – – –
Dead 0 4 19 0.17 0.83 23 < 0.00001
Alpha-cypermethrin 10×
Alive 0 0 0 – – – –
Dead 1 3 20 0.15 0.85 16.6434 0.000045
Bendiocarb 1×
Alive 0 4 2 0.67 0.33 6
Dead 0 6 27 0.18 0.82 33
Bendiocarb 2×
Alive 0 1 3 0.25 0.75 4
Dead 1 6 26 0.2 0.8 29.9806 < 0.00001
Bendiocarb 5×
Alive 0 0 4 0 1 – –
Dead 1 4 27 0.14 0.86 24.2602 < 0.00001
Bendiocarb 10×
Alive 0 1 0 1 0 – –
Dead 0 0 23 0 1 – –

Italic values indicate significance of p values (p < 0.05)

Stica et al. Malar J (2019) 18:244 Page 11 of 15

Table 3 G119S Ace-1 allele frequencies and p values for Chi-square tests for deviations from Hardy–Weinberg
equilibrium in An. gambiae s.s. from four sites in Faranah Prefecture, Guinea
Study village/sampling site G119S Ace-1 χ2 test p-value
allele frequency
Balayani Foulaya Tindo Larvae
RS RR SS RS RR SS RS RR SS RS RR SS R S

Bendiocarb 1×
Alive 0 1 0 0 4 0 0 1 0 0 6 0 1 0 – –
Dead 0 0 5 0 0 0 0 0 0 0 0 5 0 1 – –
Bendiocarb 2×
Alive 0 1 0 0 4 0 0 0 0 0 5 0 1 0 – –
Dead 0 1 1 0 0 1 0 0 1 0 1 3 0.25 0.75 4 0.0455
Bendiocarb 5×
Alive 0 2 0 1 0 0 0 0 0 1 2 0 0.83 0.17 0.12 0.729034
Dead 0 2 2 0 0 0 0 0 0 0 2 2 0.5 0.5 4 0.0455
Bendiocarb 10×
Alive 0 0 0 0 0 0 0 0 0 0 0 0 – – – –
Dead 0 0 0 0 0 1 0 1 0 0 1 1 0.5 0.5 2 0.157299
Italic values indicate significance of p values (p < 0.05)

Discussion carbamate, bendiocarb, which showed the development

The susceptibility of An. gambiae s.l. populations from of possible resistance with mortality ranging between
Faranah, Guinea to seven public health insecticides 94 and 97% in the region. This emerging resistance may
were assessed and underlying resistance mechanisms also be attributable to carbamate use in the agricultural
characterized. Intense resistance to permethrin and setting [10], as has been documented previously in other
deltamethrin was apparent, with survivors at 5× and parts of Guinea [20]. The organophosphate, pirimiphos-
2× the diagnostic doses, respectively; minor hetero- methyl, achieved complete mosquito mortality in this
geneity in mosquito mortality was also evident across study. Currently, large-scale government-funded, wide-
this restricted geographic area. By comparison, no evi- spread IRS activities are not underway in Guinea. How-
dence of alpha-cypermethrin resistance was observed ever, knowledge of susceptibility in relation to current
in Faranah, with 100% mosquito mortality follow- LLIN use will help to guide potential IRS implementa-
ing exposure. Increased tolerance to particular pyre- tion in the future [9]. Local mosquito populations were
throids in this region is not unexpected, given LLINs also susceptible to two new insecticides under considera-
are the sole insecticide-based malaria control strategy tion for public health use, clothianidin (neonicotinoid)
and chlorfenapyr (pyrrole), strengthening the evidence
tributed nationwide in 2013 ­(Netprotect®) and 2016
implemented in Guinea. Deltamethrin LLINs were dis-
that net impregnations and IRS formulations with these
­(PermaNet® 2.0 and ­Yorkool®) by the National Malaria insecticides may be successful in future vector control

­ 2® LLINs
Control Programme (NMCP) [10], thus a resulting efforts. Likewise, absence of alpha-cypermethrin resist-
increase in deltamethrin resistance would be antici- ance supports the deployment of Interceptor G
pated. However, the higher levels of permethrin resist- (containing a combination of alpha-cypermethrin and
ance demonstrated in this study could be the result chlorfenapyr) in prospective LLIN distributions.
of control efforts prior to 2013 or concurrent use of Regarding underlying resistance mechanisms,
this insecticide (and/or others capable of facilitating decreased susceptibility to pyrethroids in Faranah was
cross-resistance) in agriculture activities. Alternatively, mediated both by target site mutations and overexpres-
L1014F kdr, which our study indicated was fixed in Far- sion of metabolic enzymes. The L1014F kdr mutation
anah in 2018, as well as in other parts of Guinea [10, was detected in all tested samples, which is consistent
20], has been reported to contribute more to resistance with high frequencies of this mutation observed through-
to type I (permethrin) vs type II (alpha-cypermethrin out the country [10, 20]. This study did not screen for
and deltamethrin) pyrethroids [21]. the East African L1014S kdr mutation, which is known
In Faranah, susceptibility to all other classes of insec- to have variable frequencies in West Africa and may
ticides under evaluation was confirmed, excluding the warrant surveillance in Guinea in the future [22–24]. A
 Stica et al. Malar J

Table 4 Mean fold change in relative expression of CYP6M2, CYP6P3, and GSTD3, and associated p values, between An. gambiae s.s. populations in the Faranah
Prefecture and the An. gambiae s.s. susceptible G3 colony
Permethrin Permethrin Permethrin Permethrin Permethrin Permethrin Permethrin Deltamethrin Deltamethrin Deltamethrin Deltamethrin Deltamethrin Bendiocarb Bendiocarb Bendiocarb Bendiocarb Bendiocarb Bendiocarb Bendiocarb Bendiocarb G3
1× (R) 1× (S) 2× (R) 2× (S) 5× (R) 5× (S) 10× (S) 1× (R) 1× (S) 2× (S) 5× (S) 10× (S) 1× (R) 1× (S) 2× (R) 2× (S) 5× (R) 5× (S) 10× (R) 10× (S)

CYP6M2 Exposed 51 7 55 22 2 39 22 16 42 24 23 21 6 33 4 33 4 32 1 23 41
(2019) 18:244

(n)
Mean fold 1.88 (0.70, 0.74 (0.32, 1.32 (0.76, 0.57 (0.37, 0.44 (−0.74, 1.08 (0.73, 1.65 (0.95, 0.70 (0.49, 0.91) 0.92 (0.54, 1.30) 0.35 (0.26, 0.45) 2.50 (−1.86, 0.41 (0.20, 0.62) 0.48 (0.07, 0.41 (0.28, 0.22 (0.05, 0.54 (0.36, 0.41 (−0.15, 0.46 (0.24, 1.03 0.59 (0.40, 0.09 (0.07,
change 3.10) 1.08) 1.89) 0.78) 1.61) 1.43) 2.35) 6.86) 0.89) 0.54) 0.39) 0.73) 0.98) 0.68) 0.78) 0.10)
in gene
expres-
sion
Resistant v p = 0.4792 p = 0.1035 p = 0.4131 – p = 0.4804 – – – p = 0.6586 p = 0.2988 p = 0.8824 – –
Suscep-
tible
Wild v G3 p = 0.0077 p< 0 .0001 p = 0.0003 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.1274 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.0002 – p < 0.0001
CYP6P3 Exposed 51 7 55 22 2 39 22 16 42 24 23 22 6 33 4 33 4 32 1 23 41
(n)
Mean fold 0.87 (0.44, 0.72 (0.31, 1.17 (0.39, 0.91 (0.51, 0.12 (−0.90, 0.60 (0.38, 0.43 (0.14, 0.73 (0.15, 1.31) 0.45 (0.29, 0.61) 0.24 (0.17, 0.32) 1.05 (−0.77, 0.29 (0.09, 0.49) 1.44 (0.07, 0.62 (0.32, 1.68 (−0.77, 0.55 (0.26, 1.50 (1.05, 1.46 (−0.11, 0.6 0.30 (0.18, 0.12 (0.10,
change 1.29) 1.00) 1.95) 1.32) 1.14) 0.82) 0.72) 2.87) 2.80) 0.92) 4.13) 0.84) 1.95) 3.04) 0.41) 0.15)
in gene
expres-
sion
Resistant v p = 0.8045 p = 0.6836 p = 0.3372 – p = 0.1832 – – – p = 0.0524 p = 0.0366 p = 0.9872 – –
Suscep-
tible
Wild v G3 p = 0.0025 p < 0.0001 p = 0.022 p < 0.0001 p = 0.9417 p < 0.0001 p = 0.0047 p = 0.0007 p = 0.0001 p = 0.0003 p = 0.1605 p = 0.0261 p < 0.0001 p = 0.0004 p < 0.0001 p = 0.0013 p < 0.0001 p = 0.0534 – p = 0.0002
GSTD3 Exposed 50 7 55 22 2 39 22 16 41 24 23 22 6 33 4 32 4 32 1 23 41
(n)
Mean fold 2.25 (1.19, 1.14 (0.55, 1.40 (0.90, 0.87 (0.62, 0.63 (−2.06, 1.27 (0.68, 1.47 (0.35, 1.14 (0.84, 1.44) 1.29 (0.66, 1.93) 0.66 (0, 1.32) 2.01 (−1.57, 0.36 (0.19, 0.53) 1.61 (0.28, 1.07 (0.33, 2.76 (−2.88, 0.66 (0.41, 1.09 (0.06, 0.59(0.17, 1.16 0.42 (0.31, 0.44 (0.38,
change 3.32) 1.45) 1.90) 1.11) 3.30) 1.85) 2.59) 5.60) 2.94) 1.80) 8.40) 0.91) 2.11) 1.00) 0.52) 0.51)
in gene
expres-
sion
Resistant v p = 0.4106 p = 0.1916 p = 0.6208 – p = 0.7657 – – – p = 0.5427 p = 0.1609 p = 0.4062 – –
Suscep-
tible
Wild v G3 p = 0.0027 p < 0.0001 p = 0.0015 p < 0.0001 p = 0.2315 p = 0.0048 p = 0.0115 p < 0.0001 p = 0.0085 p = 0.3894 p = 0.2276 p = 0.2717 p < 0.0001 p = 0.0601 p < 0.0001 p = 0.0615 p < 0.0001 p = 0.4474 – p = 0.6432

Italic values indicate significance of p values (p < 0.05)

(R): resistant or alive; (S): susceptible
Page 12 of 15
Stica et al. Malar J (2019) 18:244 Page 13 of 15

second mutation in the voltage-gated sodium channel, observation strengthens the importance of utilizing the
N1575Y, was confirmed in 20% of vectors, with strong same methods of mosquito collection and assay prepa-
evidence for ongoing selection, as demonstrated by sig- ration when testing field-collected mosquitoes [31] and
nificant deviations from Hardy–Weinberg allele frequen- cautions against the use of such data interchangeably.
cies; however this mutation was not associated with Because mosquito availability was a limiting factor, test-
mosquito survival following insecticide exposure. A syn- ing priority for replicates was given to insecticides where
ergistic relationship between the L1014F kdr and N1575Y initial resistance was observed to the diagnostic dose,
mutations has been established in other parts of West to more accurately characterize levels of phenotypic
Africa, where it has been shown to enhance resistance resistance intensity. Additional bioassay testing of larger
to pyrethroids and DDT, and potentially compensate for samples sizes is required to corroborate these results in
fitness costs incurred by L1014F kdr [16]. By compari- Faranah.
son, in Maferinyah, Guinea, this mutation was present at Malaria prevalence in Faranah Prefecture is approxi-
similar frequencies to Faranah and directly implicated in mately 15%, reaching up to 25% [8]; mosquito infection
phenotypic resistance to permethrin [20]. rates in this study were 7.3%. Because whole mosquito
The ability of the synergist PBO to re-establish full or bodies were screened for P. falciparum by PCR, it is also
partial susceptibility to both permethrin and deltame- possible that, in addition to infection or infectivity, a
thrin indicates that mixed-function oxidases (MFOs) positive sample could be indicative of a mosquito which
play some role in the resistance reported. However, the recently fed upon an infected human blood meal. More
expression of metabolic enzymes examined in this study importantly, insecticide susceptible vectors were more
(CYP6M2, CYP6P3, and GSTD3), while significantly likely to be infected with P. falciparum, but this phe-
upregulated in wild An. gambiae s.s. compared to the sus- nomenon could not be ascribed to a single insecticide or
ceptible G3 colony, did not statistically differ between our resistance mechanism.
pyrethroid resistant and susceptible wild-caught mos- Additional considerations for the interpretation of
quitoes. Further exploratory analyses of our wild popula- study data include that in the tested population of An.
tions are warranted to characterize additional metabolic gambiae s.l., An. gambiae s.s. was the predominant spe-
resistance pathways. Given the influence of MFOs as a cies at 97.1%, with An. coluzzii representing 1.3%, and
predominant resistance mechanism to pyrethroids in the hybrid form 1.3%. Only two samples failed to pro-
the area and recent findings of improved protection with duce any result on the An. gambiae complex end-point
permethrin and PBO impregnated nets [25], the Guinea PCR assay. Anopheles gambiae s.s. breed in temporary,
NMCP should consider including these next-generation rain-dependent breeding sites [33], which could account
nets in future vector control activities. for its predominance during the rainy season. Future
The G119S Ace-1 mutation, which confers resistance collections at multiple timepoints, utilizing different
to carbamate and organophosphate insecticides [17] was collection methods would be necessary to determine
found in this study to be highly predictive of bioassay the principal species in the area. Regarding sampling
survival to bendiocarb. Furthermore, CYP6P3 was signif- methodology, in this study mosquitoes were primarily
icantly overexpressed in bendiocarb survivors, suggesting collected using HLCs as this is often the most efficient
it may also be responsible for decreased susceptibility to technique to obtain large catches for bioassay testing.
carbamates in Faranah. Similar results implicating a role Due to heavy rains in the areas, larval breeding sites can
for CYP6P3 in bendiocarb metabolism and resistance be completely washed away, rendering this sampling
have been reported from Côte d’Ivoire [26] and over- method unpredictable. HLCs were chosen in favour
expression of this CYP450 has been documented among of aspirating indoor resting, blood-fed adults and forc-
many multi-insecticide resistant field populations [5, ing oviposition, to generate an ­F1 population, because
27–30]. house collections frequently yield low numbers of mos-
In this study, mosquitoes that were reared from lar- quitoes, potentially introducing family effects as a bias.
vae collected from sites in the town of Faranah showed In addition, preference was given to testing mosquitoes
a higher mortality rate to permethrin at 1× and 2× the caught in HLCs, because the latter two techniques are
diagnostic dose, when compared to the wild-caught adult not appropriate for pathogen screening. Finally, sam-
population of varying physiological age. This finding pling took place in only three villages in one area of
contradicts previous studies which have suggested that Guinea and, therefore, these findings cannot be extrapo-
field-collected adults have higher mortality rates due to lated across the entire country. However, this informa-
the mixture of ages and blood-feeding statuses in these tion will contribute to an understanding of resistance in
populations [31], and the reported decline of pheno- this region, which will be valuable information for the
typic resistance with increasing mosquito age [32]. This NMCP [34].
Stica et al. Malar J (2019) 18:244 Page 14 of 15

Fig. 3 Relative expression of genes (CYP6M2, CYP6P3, and GSTD3) and associated 95% confidence intervals, for three insecticides that showed
resistance at varying concentrations, normalized to the housekeeping gene RPS7 in An. gambiae s.s. populations in Faranah Prefecture and in the An.
gambiae s.s. susceptible G3 colony. (R): resistant or alive; (S): susceptible or dead

Conclusions molecular assays. CS, LAM, TW and SRI drafted the manuscript, which was
revised by co-authors. All authors read and approved the final manuscript.
Resistance to the pyrethroids, permethrin and deltame-
thrin, as well as possible resistance to bendiocarb were Funding
observed in Faranah Prefecture in Guinea. Both target The authors would like to thank multiple partners for their financial support.
Study funding was provided by a Bayer Research and Travel Grant for Vector
site mutations (L1014F kdr, N1575Y and G119S Ace-1) Control and a London School of Hygiene and Tropical Medicine MSc Trust
and metabolic mechanisms of resistance are present Fund Grant, both awarded to CS, and a Sir Halley Stewart Trust grant, awarded
in this field population. The complete susceptibility to LAM. CLJ and TW were supported by a Wellcome Trust/Royal Society Sir
Henry Dale Fellowship awarded to TW (101285/Z/13/Z). LAM is supported
of vectors to alpha-cypermethrin, clothianidin, chlo- by an American Society for Microbiology/Centers for Disease Control and
rfenapyr, and pirimiphos-methyl is encouraging, and Prevention Fellowship. SRI is supported by the President’s Malaria Initiative
these insecticides should be considered by the NMCP (PMI)/CDC.
in future control efforts in the form of new LLIN and Availability of data and materials
IRS interventions as they become approved and readily Not applicable.
available.
Ethics approval and consent to participate
The study protocol was reviewed and approved by the Comité National
d’Ethique pour la Recherche en Santé (030/CNERS/17) and the institutional
Abbreviations
review boards (IRB) of the London School of Hygiene and Tropical Medicine
Ace-1: acetylcholinesterase; Cox-1: cytochrome c oxidase subunit 1; CDC:
(#14990). The protocol was reviewed at the Centers for Disease Control and
Centers for Disease Control and Prevention; CI: confidence interval; CNERS:
Prevention, USA and determined to be non-human subject research (refer-
Comité National d’Ethique pour la Recherche en Santé; CYP450: cytochrome-
ence number 2018-086); all study procedures were performed in accordance
dependent monooxygenase 450; DDT: dichlorodiphenyltrichloroethane; GST:
with relevant guidelines and regulations. Fieldworkers participating in human
glutathione-s-transferase; HLC: human landing catch; IRB: institutional review
landing catches were provided with malaria prophylaxis for the duration of
board; IRS: indoor residual spraying; ITN: insecticide-treated net; kdr: knock-
the study.
down resistance; LLIN: long-lasting insecticidal net; LSHTM: London School of
Hygiene and Tropical Medicine; MFO: mixed-function oxidase; NMCP: National
Consent for publication
Malaria Control Programme; PBO: piperonyl butoxide; PMI: President’s Malaria
Not applicable.
Initiative; qRT-PCR: quantitative reverse transcriptase PCR; RPS7: ribosomal
protein S7; WHO: World Health Organization.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
The authors would like to thank all mosquito collectors for their dedicated
Author details
work. 1
Department of Disease Control, Faculty of Infectious Tropical Diseases,
London School of Hygiene and Tropical Medicine, London, UK. 2 Entomology
Disclaimer
Branch, Division of Parasitic Diseases and Malaria, Center for Global Health,
The findings and conclusions in this report are those of the author(s) and
Centers for Disease Control and Prevention, Atlanta, GA, USA. 3 Division
do not necessarily represent the official position of the Centers for Disease
of Parasitic Diseases and Malaria, President’s Malaria Initiative, Center for Global
Control and Prevention.
Health, Centers for Disease Control and Prevention, Atlanta, GA, USA. 4 Pro-
gramme National de Lutte Contre le Paludisme, Ministère de la Santé, BP. 595,
Authors’ contributions
Conakry, Guinea. 5 RTI International, Conakry, Guinea. 6 American Society
LAM, CS, SRI, CLJ and TW designed the study and were responsible for data
for Microbiology, 1752 N Street NW, Washington, DC 20036, USA.
analysis and interpretation. CS, YB, DC, IY and MK led the entomology field
activities and participated in data collection. TW, CS and CLJ performed the
Stica et al. Malar J (2019) 18:244 Page 15 of 15

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