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 Author's personal copy
Opinion
Does kdr genotype predict insecticide-
resistance phenotype in mosquitoes?
Martin J. Donnelly1, Vincent Corbel2, David Weetman1, Craig S. Wilding1,
Martin S. Williamson3 and William C. Black IV4
1
Vector Group, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK
2
Institut de Recherche pour le Développement, Centre de Recherches Entomologiques de Cotonou, 01 BP 4414 RP, Cotonou, Benin
3
Department of Biological Chemistry, Rothamsted Research, Harpenden, AL5 2JQ, UK
4
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA
Several groups are developing and applying DNA-based
technologies to monitor insecticide-based disease con-
trol programmes. However, several recent papers have
concluded that the knockdown resistance (kdr) geno-
type–phenotype correlation that is observed in a wide
variety of taxa might not hold in all mosquitoes. In this
article, we review the evidence to support this putative
breakdown and argue that the conclusion follows from
unreliable data or the unparsimonious interpretation of
data. We assert that the link between kdr genotype and
DDT- and pyrethroid-susceptibility phenotype is clear.
However, we emphasize that kdr genotype might
explain only a portion of heritable variation in resistance
and that diagnostic assays to test the importance of
other resistance mechanisms in field populations are
required.
Resistance to DDT and pyrethroid insecticides
Knockdown resistance (kdr) is one of the two major forms of
resistance to dichlorodiphenyltrichloroethane (DDT) and
pyrethroid insecticides. The other major type of resistance,
termed ‘metabolic resistance’ (see Glossary), usually
results from enhanced expression of detoxification
enzymes (for a review, see Ref. [1]). Knockdown resistance
to DDT and pyrethroids has long been associated with
mutations in the sodium-channel gene, the target of these
insecticides. Two recent papers have reviewed the numer-
ous studies that show a clear association between a small
number of non-synonymous mutations in the voltage-gated
sodium channel and resistance phenotype in a diverse
array of taxa [2,3]. Furthermore, using in vitro expression
systems in Xenopus oocytes, it has been possible to demon-
strate that these mutations confer sodium-channel insen-
sitivity to DDT and pyrethroids [3]. From a public health
perspective, the presence of kdr in vector populations could
have severe consequences for the sustainable use of pyr-
ethroids and, also, DDT, given its recent re-emergence as
an insecticide for anti-malaria programmes [4].
In Anopheles gambiae s.s., the African malaria vector,
two point mutations in the voltage-gated sodium-channel
gene confer kdr to DDT and pyrethroid insecticides. Mar-
tinez-Torres et al. [5] identified a leucine–phenylalanine
substitution at position 1014 (L1014F) of the gene in
Corresponding author: Donnelly, M.J. (m.j.donnelly@liv.ac.uk).
1471-4922/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2009.02.007
strains from Burkina Faso and Côte d’Ivoire. A second
mutation, a leucine–serine substitution at the same codon
(L1014S), was identified in a colony from Kenya [6]. The
L1014F mutation has been observed in both M and S
molecular forms of An. gambiae s.s., whereas the
Glossary
Allele-specific PCR: a polymerase chain reaction in which amplification
depends upon the exact match of a primer to the DNA sequence of only one
of two or more alleles of interest, thus resulting in amplification of products
that are specific to particular alleles.
Heritable variation: proportion of the variability in a phenotype that is
transmitted across generations; primarily, this will be DNA sequence variation
but might sometimes represent non-genetic variation – for example, in
mechanisms that regulate gene expression.
LOD value: an acronym for logarithmic (base 10) odds. In QTL analysis, an
estimate of the strength of the association between a genotype (QTL) and a
trait of interest compared with null hypothesis of no QTLs by phenotype
association. A LOD score of 3 (1 in 1000) is often used as the significance level.
Metabolic resistance: partial or complete resistance to the harmful effects of
insecticides or other alien compounds provided by detoxifying enzymes, of
which cytochrome P450 monooxygenases are a well-known superfamily that is
important in both insects and humans.
Multigenic: involving the action, and potentially interaction, of multiple genes.
Non-synonymous mutation: a mutation in an exon of a protein-coding gene
coding that results in a change in the amino acid. Synonymous mutations do
not result in modification to the sequence of amino acids.
Odds ratio: a measure of effect size commonly used in association studies,
which can be calculated as p1q2  p2q1, where p and q are the frequencies of
two alleles at a locus (e.g. kdr1014F and kdr1014S) and 1 and 2 are the
comparison groups (e.g. insecticide resistant and susceptible).
Parsimonious interpretation: preference for the least complex explanation for
an observation (i.e. that requiring the fewest unproven caveats or assump-
tions).
Penetrance: the proportion of individuals carrying a particular variation of a
gene that also express an associated phenotype trait of interest; thus, genetic
variants carrying a high risk of susceptibility to a particular disease or
resistance to a particular insecticide have high penetrance for that trait.
Phylogenetically common: found across a wide range of taxa.
Post-transcriptional modification of RNA: changes to RNA transcripts before
formation of mature messenger RNA (mRNA), which is translated into amino
acids (e.g. the removal of non-coding introns, thus retaining only the coding
exonic regions in the mature mRNA for translation into amino acids).
Quantitative trait locus (QTL): a stretch of DNA that co-varies with genetic
variants that generate variation in the phenotype of interest. QTL studies
typically involve the crossing of strains that differ markedly in a particular
phenotype of interest to produce a hybrid generation, with subsequent
crossing back into one of the strains; the more generations of crossing are
made, the more that recombination will occur and the shorter the resulting QTL
region will be.
RNA editing: a special case of post-transcriptional modification of RNA
involving one or more changes in the base composition of an mRNA molecule,
such that the information in the mRNA differs from that of the DNA from which
it was transcribed, often resulting in translation into a different amino acid.
Reverse transcription PCR: a reaction in which single-stranded RNA is first
converted to its double-stranded DNA complement (cDNA), which is then PCR
amplified.
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Opinion Trends in Parasitology Vol.25 No.5

L1014S substitution has been reported only in the S mol-
ecular form [7]. More recently, both of these mutations
have been observed in the sister taxa Anopheles arabiensis
[8,9].
The association between these mutations and the pyre-
throid and/or DDT-resistance phenotype in An. gambiae s.s.
has been shown in several studies using quantitative trait
loci (QTL) [10] and genotype–phenotype association
approaches [11–15]. In the first article, which describes
kdr mutations in An. gambiae, Martinez-Torres et al. [5]
have shown that in seven samples from West Africa, the
frequency of the L1014F allele correlated strongly with
reduced mortality in a permethrin-World Health Organiz-
ation (WHO) tube test. Furthermore, by mixing different
proportions of wild-type homozygotes and wild-type/L1014F
heterozygotes, Chandre et al. [12] were able to show a strong
correlation between the frequency of heterozygotes and
knockdown time. These authors suggested that time to
knockdown might be a more sensitive discriminator of
kdr genotypes, in that it increased markedly before any
change in mortality rates was observed [12]. Ranson et al.
[6,10] also demonstrated clear linkage between inheritance
of the L1014S mutation and permethrin resistance.
Simulated field trials of insecticide-treated bed nets
(ITNs) in Côte d’Ivoire showed that kdr had a strong
impact on the efficacy of nets treated with either of the
pyrethroids a-cypermethrin and etofenprox; L1014F
homozygotes showed a survival advantage [16,17]. The
same trend was noted in Burkina Faso, where the protec-
tive effect of permethrin-treated plastic sheeting was
apparent against susceptible genotypes but not against
kdr homozygotes [18]. Similarly, in Benin, low mortality of
An. gambiae L1014F homozygotes was observed after
exposure to permethrin-treated nets [14].
There are two distinct sets of data that are used to
dispute the genotype–phenotype link [19]. The first chal-
lenge arises from a series of papers [20–22] that present
data interpreted as evidence for post-transcriptional modi-
fications of RNA. The second challenge to the link is posed
by a pair of studies on An. arabiensis that observed no
correlation between kdr genotype, as determined by allele-
specific PCR (AS-PCR), and resistance phenotype, as
determined by WHO tube bioassay [23,24]. Through a
careful examination of the two sets of data, we have shown
that there is, at present, insufficient evidence to draw into
question the causal association between kdr genotype and
pyrethroid and/or DDT-resistance phenotype, and we can
unequivocally state that the kdr genotype is an important –
although, certainly, not necessarily the only – predictor of
resistance phenotype.
Evidence for post-transcriptional modifications of RNA
in the Culicidae?
In a series of papers, Liu and colleagues present data that
is interpreted as evidence for post-transcriptional modifi-
cation of pre-mRNA (precursor mRNA) coding for the
voltage-gated sodium channel in Musca domestica, Blat-
tella germanica, Culex quinquefasciatus and Aedes albo-
pictus [20–22]. Pre-mRNA represents the first step in the
transcription process, through which the mRNA (mRNA) is
produced from the coding DNA sequence. Pre-mRNA is
produced in the cell nucleus and maintains the intron–exon
structure seen in the DNA copy (Figure 1). Introns are then
removed from the pre-mRNA to produce the mRNA, which
is transported out of the nucleus to the cytoplasm where
translation (the production of the protein) occurs. Post-
transcriptional modification of the pre-mRNA sequence
could mean that the translated protein sequence is not

Figure 1. The in vivo processes by which DNA is translated into protein through an RNA intermediate, and the in vitro techniques used to sequence mRNA and DNA. The
left-hand panel shows how single-stranded RNA is transcribed from double-stranded genomic DNA by RNA polymerase, after which the introns are spliced out to produce
the mRNA that is used as the template for protein synthesis. The start and stop sites for protein translation are marked. The right-hand panel demonstrates how an RNA-
editing event would be detected by in vitro PCR-based methods through a discordance between genomic DNA and mRNA sequences. There is a need for an intermediate
step in the sequencing of mRNA to produce a double-stranded template (cDNA). The lightening strike symbol indicates where the contamination of Aedes gDNA with Culex
cDNA is likely to have occurred (Figure 2).

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Opinion Trends in Parasitology Vol.25 No.5

that predicted from the coding DNA sequence. Liu and

colleagues observed that, although they were unable to
detect correlation between kdr genotype and levels of
susceptibility, a correlation was found when they
examined expression of kdr alleles (as determined by
reverse transcription PCR) and levels of susceptibility
[20–22]. Such a discrepancy could be explained by post-
transcriptional modification.
There is certainly evidence that post-transcriptional
modification of pre-mRNA can occur. One phylogenetically
common example is the chemical conversion of adenosine
to inosine (A to I) [25,26]. The translating ribosome then
recognizes the inosine as guanosine and, therefore, a
different amino acid might be incorporated into the protein
than that predicted from the genomic DNA (gDNA)
sequence [26]. Ten A-to-I editing sites have been observed
in the para voltage-gated sodium channel in Drosophila
melanogaster [25]. Although this A-to-I editing event
seems to be the most common in terms of the number of
taxa in which it is observed, other changes have been
observed. Uracil to cytidine (U-to-C) editing has been
observed in sodium channels of both D. melanogaster
and B. germanica and was associated with persistent
current in tetrodoxin-sensitive sodium channels [27,28],
although (at most) only 3.2% of the transcripts were edited
[28]. So, although the phenomenon of RNA editing is well
established, what is novel about the data from Liu and
colleagues is that the RNA edits they observe are both
unique (uracil to adenosine, cytidine to uracil and guani-
dine to cytidine) and extremely frequent, to the extent that
Figure 2. Knockdown, recovery and survival rates as a function of genotypes at the
in some instances, none of the transcripts they examined para 1106 locus in Aedes aegypti. Genotypes are from F3 offspring of Val1016 and
had the same sequence as the gDNA from which they were Iso1016 homozygous P1 parents. Val1016 is the wild-type allele inherited from the
transcribed [20–22]. The approach taken to investigate susceptible P1 parent, and the Iso1016 allele was inherited from the resistant P1
parent. Three-day-old F3 adults were exposed to 1.2 mg permethrin per bottle for
potential RNA-editing events in all three studies is essen- one hour, and adults still flying were classified as knockdown resistant (kdr).
tially the same [20–22] (Figure 1). DNA and total RNA was Knocked-down mosquitoes were removed from the bottle and transferred to the
extracted from pools of five insects, from colonies of known insectary, and 4 h later, flying and crawling mosquitoes were recorded as
recovered. The remaining mosquitoes were scored as dead. (a) Knockdown rate
phenotype. PCR amplification of the region of the sodium (1-(knocked down  all F3 offspring)). (b) Recovery rate (number recovered 
channel that contains the kdr 1014 codon was undertaken (number recovered or dead)). (c) Survival rate (number kdr or recovered  all F3
offspring). Solid lines, females; dashed lines, males. Data from Ref. [38].
on genomic DNA, whereas RNA was amplified after a
reverse-transcription step to form complementary DNA
(cDNA). The genotype at the 1014 locus was then screened. albopictus. However, as predicted by Davies et al. [29], the
To prevent amplification of gDNA in the cDNA reactions, codon usage at the 1014 location (CTA) means that, for a
Liu and colleagues designed primers that spanned intron– 1014F kdr allele to occur at this codon, two point mutations
exon boundaries, but then (curiously) reported that there would be required. As demonstrated by Saavedra-Rodri-
were no intronic regions in any of the four diverse taxa they guez et al. [30] in the closely related taxa Aedes aegypti, the
studied. As discussed by Davies et al. [29], the para-sodium mutations associated with a kdr phenotype in Aedes occur
channel is highly conserved between species, especially at the nearby codon 1016 (Figure 2). What is striking about
with respect to exon–intron structure (although not intron the Ae. albopictus data is that the putative RNA-editing
length); Liu and colleagues do acknowledge that their data events result in restoration of the CTA codon, the codon
are at odds with previously published information but that has been observed in the gDNA of several Aedes
provide no explanation why [21]. To explain their data, species [29]. The gDNA 1014 codon sequence for Ae. albo-
the authors of Refs [20–22] would have to propose that the pictus from the strains examined is either TTA or TTT,
four diverse species showed independent loss of the same sequences that have never been observed in Aedes to date,
introns. The probability of this occurrence is very small, despite considerable research effort [29–32]. However, this
and we would argue that the observations would be better codon sequence has been observed in Culex quinquefascia-
explained by either the co-extraction of gDNA and RNA or tus, and a consensus neighbour-joining tree (Figure 3)
one (or more) contamination events of gDNA samples with nests the two gDNA sequences for Ae. albopictus within
cDNA extracts. a Culex pipiens s.l. clade. Such evidence provides compel-
Indeed, there is overwhelming evidence of a contami- ling support for the contamination hypothesis in Ae. albo-
nation event in the data presented in Ref. [20]. This paper pictus (Figure 1). We are unable to determine if this was
examines potential RNA editing at the 1014 locus in Aedes also the case in the cDNA sequences used in the study of

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Figure 3. Bootstrap consensus neighbour-joining tree showing the position of the putative Aedes albopictus gDNA sequences (DQ538357; DQ538358) within the Culex
pipiens s.l. cluster. Sequence data were obtained from GenBank by performing a basic local alignment search tool (BLAST) search using sequence DQ538357. Sequences
were aligned in BioEdit [45] using the Clustal W routine and then manually truncated to 316 bp. The tree was constructed in Mega4 [46] using a Maximum Composite
Likelihood algorithm, gaps and missing data were handled by pairwise deletion, bootstrapping was performed 1000 times and nodes with <70% support were condensed.
GenBank accession numbers are given after the taxon names. Underlined taxon names are from Refs [20,22]. The sequence alignment file is available in Mega format from
http://pcwww.liv.ac.uk/mjames/kdralignment.meg.

RNA editing in Blatella and Musca [21] because the appro-
priate sequences were not available in the public data-
bases.
Thus contamination, rather than RNA editing, is the
cause of the discrepancy between gDNA and cDNA
sequences. This is a far more parsimonious explanation
than invoking multiple parallel intron loss in four diverse
taxa, three novel RNA-editing changes, multiple editing
events in the same codon in the same individual and the
high proportion of pre-mRNA transcripts that experienced
editing.
Dissociation of kdr genotype and insecticide-resistance
phenotype in Anopheles arabiensis?
Recently, the two kdr mutations known in An. gambiae s.s.
(L1014F and L1014S) have been observed in the sibling

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species An. arabiensis. The kdr mutations have been found
in An. arabiensis by independent groups in several widely
dispersed locations: Burkina Faso [8], Tanzania [33],
Sudan [9,23], Kenya [34] and Uganda [35]. It would be
reasonable to assume that the kdr alleles might show a
similarly high penetrance with respect to resistance phe-
notype, as has been observed previously [36,37]. However,
the authors of two studies of colonized and wild-caught
specimens of An. arabiensis from Sudan concluded that
there was no association between genotype and phenotype
[23,24]. In the study by Matambo et al. [24], a colony of An.
arabiensis from the Sennar region of Sudan was subjected
to 16 generations of selection by exposure to DDT. Over the
course of this selection, mortality, 24 h after a one-hour
exposure to 4% DDT, decreased from 90.6% to 12.1%. No
mortality was observed in the F16 generation after
exposure to 0.75% permethrin and only 24% mortality
after exposure to a 0.05% concentration of the class-II
pyrethroid deltamethrin. Because sequencing suggested
the L1014F mutation was at or near fixation in the F16

Figure 4. Plot of logarithmic odds (LOD) values associated with three different susceptibility-phenotype comparisons in Aedes aegypti. The three comparisons are ‘knock
down’, flies that were or were not knocked down after a one-hour exposure to permethrin; ‘survival’, those that were knocked down but did or did not recover four hours
post-exposure; and ‘3 phenotypes’, a comparison between knockdown-resistant, recovered and susceptible phenotypes. LOD scores (y axis) are given along all three
chromosomes, and the names of markers are listed along the x-axis to orient QTL positions. As an example of how a phenotype can be multigenic, the knockdown-
resistance phenotype (dotted line) is associated with two highly significant regions – one on Chromosome 3, around the sodium channel (marked ‘para’), and the other on
Chromosome 1, associated with sex locus and a cytochrome P450 (marked ‘Cyp9AE1’). Reproduced, with permission, from Ref. [38].

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Opinion
generation but at a low proportion in the unselected
parental colony [24], a reasonable conclusion would be that
a substantial proportion of the increase in resistance was
associated with the increase in kdr frequency. However,
the authors concluded that the lack of difference in the
frequency of the kdr mutation between individuals classi-
fied as susceptible or resistant to DDT in the F16 gener-
ation was evidence that kdr was not a predictor of
susceptibility phenotype. Tests of association are reliant
upon underlying variation in genotype, which was absent
for kdr in the F16 generation; therefore, a more appro-
priate conclusion would be that after 16 generations of
selection, some variation in resistance remained that was
independent of kdr genotype.
As discussed by Brooke [19], resistance could be multi-
genic, and the kdr genotype might not fully explain all the
variance in phenotype. For example, in QTL mapping the
genetic factors conditioning permethrin resistance in Ae.
aegypti, Saavedra-Rodriguez et al. [38] were able to esti-
mate the proportion of the phenotype variance in knock-
down that was attributable to broad genotypic effects
(Figure 4). The V1016I kdr allele accounted for 58.6% of
variance in the knockdown phenotype, and six other QTLs
accounted for 25.5% of the remaining variance. Only 23.1%
of the variance in eventual survival was accounted for by
the V1016I allele, and 13.7% was accounted for by three
additional QTLs.
In a second article, by Abdalla et al. [23], no significant
difference (x2 test p>0.05) was observed between the fre-
quency of the L1014F mutation in wild-caught Sudanese
specimens that were killed by, or survived, a standard
WHO exposure to DDT or permethrin (see Table 4 in
Ref. [23]). However, this analysis is confounded by the
AS-PCR method that was used to genotype the specimens.
Our own experience, and that of several other authors,
suggests that the AS-PCR method of genotyping can be
somewhat inaccurate compared to sequencing [39–41]; this
is borne out to an extreme extent in the study in question,
in which an error rate of 57% is reported (see Table 3 in Ref.
[23]), yielding a genotyping accuracy indistinguishable
from that expected by chance. Genotype–phenotype associ-
ation tests are obviously reliant upon accurate allele scor-
ing, and there are now several new assays available, based
upon a variety of platforms, that have been shown to
improve sensitivity and specificity [40]. Abdalla et al.
[23] performed tests of genotype–phenotype association
on the 14 sequenced specimens and concluded that there
was no relationship with the insecticide bioassay. A test
with such a small sample size would require a very strong
association (odds ratio  9) to detect significance at a level
of P<0.05. In fact, based on the sequencing data presented,
sample sizes of <50 resistant and susceptible individuals
(fewer than screened by AS-PCR by Abdalla et al.) would
have yielded a significant association between the
kdr1014F allele and insecticide resistance at P<0.05 (odds
ratio = 2.6) [42,43].
Concluding remarks
It is certainly not our intention here to contend that kdr
genotyping is the only predictor of susceptibility to DDT
and pyrethroids. Indeed, even in An. gambiae, in which the
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Trends in Parasitology Vol.25 No.5
association is best understood, equating kdr with resist-
ance is – at best – premature and probably misleading, and
we fully accept that the association in other mosquito
disease vector species requires further research. However,
almost all evidence to date has shown a strong causal
relationship between kdr genotype and susceptibility to
DDT and pyrethroids. As we have argued above, the papers
that purport to show a lack of such a relationship seem to
be based on flawed data and/or the unparsimonious
interpretation of results.
Attempts have been made to infer the impact of kdr on
the efficacy of ITNs and indoor residual spraying, but no
consensus has yet been reached [13,17,44]. Recently,
N’Guessan et al. [13] observed that pyrethroid control of
An. gambiae populations with low kdr frequencies (5%)
was possible in North Benin but not in the southern part of
the country, where An. gambiae populations showed high
kdr frequencies (>80%). What might partially confound
these studies is that it is not yet possible to determine the
role of additional resistance mechanisms, such as meta-
bolic resistance. When metabolic-resistance assays are
available, it will be possible to infer which resistance
mechanisms are having the greatest impact on vector
control programmes. Nevertheless, at present, as demon-
strated by Sharp et al. [15], kdr screening is our best
molecular diagnostic tool for predicting pyrethroid and
DDT efficacy.
Acknowledgements
We are grateful for the constructive comments of Amy Lynd, Raphael
N’Guessan and Hilary Ranson.
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