TITLE:
Use of Insecticide-Treated Bednets Drives Adaptive Introgression in African Malaria
Mosquito Species
AUTHORS:
Laura C. Norris*,1,2, Bradley J. Main1,3, Yoosook Lee1,3, Travis Collier1,3, Abdrahamane
Fofana4, Anthony J. Cornel1,2, Gregory C. Lanzaro1,3
*Corresponding author: Laura Norris, lcnorris@ucdavis.edu
1
Vector Genetics Laboratory, University of California, Davis CA 95616, USA
2
Department of Entomology and Nematology, University of California, Davis CA 95616, USA
3
Department of Pathology, Microbiology and Immunology, University of California, Davis CA
95616, USA
4
Malaria Research and Training Center, University of Bamako, Bamako, Mali
Classification: Biological Sciences: Genetics
Abstract: Malaria control across Africa is largely based on the wide-scale distribution of
insecticide treated mosquito nets. These campaigns have been successful; however widespread
insecticide resistance currently threatens this success (1). Animal species adapt to changes in
their environment, including man-made changes like the introduction of insecticides, via
selection for advantageous genes already present in populations or on those newly arisen through
mutation (2). A possible alternative mechanism is the acquisition of adaptive genes from related
species, known as adaptive introgression (3). While common in plant species, well-documented
examples of adaptive introgression are rare in animals (4). In the malaria vectors Anopheles
coluzzii and A. gambiae, divergence and adaptation to specific ecological niches is thought to be
mediated by three genomic islands of speciation located proximal to the X, 2, and 3 centromeres.
We demonstrate that during a brief breakdown in assortative mating, A. coluzzii inherited the
entire A. coluzzii 2L speciation island, including a suite of insecticide resistance alleles. This
introgression was coincident with the start of a major ITN distribution campaign, providing
selection pressure that swept the 2L island through the A. coluzzii population.
Significance statement: Our results demonstrate a rare example of adaptive introgression in an
animal species, and may help to develop effective methods to drive engineered anti-malaria
genes (5) into highly-structured malaria vector populations.
Keywords: Anopheles, vector biology, insecticide resistance, adaptive introgression
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INTRODUCTION
The major African malaria vector, A. gambiae, has long been known to exist in nature as
two distinct population groups, initially described as the M and S molecular forms (6), and
recently recognized as separate species (the M form now designated as A. coluzzii (7)). They
occur in sympatry over much of their range and exhibit varying degrees of reproductive isolation
(RI) across this range. Hybrids between the two species generated in the lab suffer no reduction
in overall fitness compared with the parental types; e.g. egg hatch rate, sex ratio, survival and
fertility are all normal in hybrids, including backcross hybrids (8). It has therefore been assumed
that the RI observed in nature is due in large part to behavioral differences that affect mate
choice (assortative mating), so that hybrid matings are relatively rare in most places.
Studies comparing the genomes of A. gambiae and A. coluzzii revealed that divergence is
restricted to three small pericentromeric regions on each of the three chromosomes, X, 2 and 3
(9, 10). Collectively these islands of divergence represent only ~3% of the genome (11). A panel
of species-specific single nucleotide polymorphisms (SNPs), with multiple SNPs distributed
within each island of divergence, was recently developed (12) and used to study patterns of
hybridization and introgression between the two species (13). This study included a longitudinal
survey of hybridization over a 21 year period (1991 – 2012, ~250 mosquito generations), in the
town of Selinkenyi in southern Mali. This study demonstrated that normally strong barriers to
gene flow periodically break down, presumably due to ecological changes. Hybridization
episodes are generally followed by the near complete loss of hybrid genotypes, presumably due
to reduced fitness. However, following one such episode in 2006, the chromosome 2L
divergence island introgressed from A. gambiae into A. coluzzii and is now maintained at
equilibrium frequency (13).
Here, we present data demonstrating that an insecticide resistance allele located within
the 2L island provided a selective advantage to A. coluzzii sufficient to drive introgression of the
entire island (~3Mbp). This event coincides with implementation of an organized insecticidetreated bed net (ITN) campaign across Mali, suggesting that increased insecticide exposure acted
as a selective force strong enough to overcome the previously observed reduced hybrid fitness,
and promote adaptive introgression in these species.
The L1014F knock down resistance (kdr) SNP (14) in the para voltage-gated sodium
channel gene is ~2.4 Mbp from the 2L centromere, and lies within the introgressed region found
by Lee et al. (13). The L1014F variant, termed kdr-west, first arose in West African A. gambiae
populations. The kdr-w allele confers target-site resistance to DDT and pyrethroid insecticides
used for malaria control (14), and has been highly selected for in A. gambiae (15, 16). Kdr-w has
been present in A. gambiae populations for at least 20 years (15), but was virtually absent in
most sympatric A. coluzzii populations as recently as 2004 (15). Subsequently, kdr-w has
emerged in A. coluzzii populations in several locations, including Mali, Burkina Faso, Cameroon
and Nigeria (17). Intron sequencing of insecticide-resistant A. coluzzii samples from Benin
showed that the kdr-w SNP in A. coluzzii emerged via introgression from A. gambiae (18).
However, several questions still remain unknown: the genomic extent of the introgression; how
introgression occurred in the face of reproductive isolation; and how selection for normally unfit
hybrids occurred. Here, we document the dynamics of introgression, using SNP genotyping and
whole-genome sequencing of a temporal series of field-collected mosquitoes from 2002-2012.
RESULTS
We genotyped 1076 specimens of A. gambiae and A. coluzzii for three SNPs that are
diagnostic for each divergence island, and for the kdr-w SNP (Table S1). Samples were collected
before, during, and after the 2006 hybridization event, from field sites in southwestern Mali
(Selenkenyi and Kela), southeastern Mali (Sidarebougou), and north central Mali (Tissana) (Fig.
S1).
The 2L island and kdr-w SNPs were found to be in very tight linkage disequilibrium (LD)
in introgressed A. coluzzii populations (LD; r2=0.81-0.96), with kdr-w nearly always cooccurring with the A. gambiae 2L SNP (Fig. 1, Fig. S2, Table S2, Table S3). LD was high in all
A. coluzzii populations sampled, even though the initial hybridization event was recorded only in
Selenkenyi. Interestingly, the introgressed region is present in allopatric A. coluzzii collected in
Tissana, nearly 400 km north of Selenkenyi. This is a dry region where A. gambiae is absent,
suggesting that once kdr-w emerged in A. coluzzii, it rapidly spread throughout Mali, via gene
flow between geographically distant populations. Between 2009 and 2012, LD decayed slightly,
indicating some recombination between the 2L SNP and kdr-w, but was still strong (r2=0.65)
(Fig. 1, Table S3).
Fifteen percent (n=19) of the 2006 Selenkenyi samples were F1 hybrids, indicating this
was a period of active hybridization (Fig. 1). The kdr-w frequency in A. gambiae at that time was
only 46.3%, and kdr-w was absent in A. coluzzii. As expected, the majority (59%), of F1 hybrids
were wildtype (Table S4). However, it appears that only hybrids possessing kdr-w survived and
backcrossed with parental populations. In Selenkenyi and Kela, only 2 out of 280 backcross
individuals were wild type at the kdr locus, a significant deficiency relative to expected numbers
(Table S5). Lee et al. showed that an earlier hybridization episode in Selenkenyi (in 2002) was
followed by strong selection against hybrids (13), presumably due to ecological maladaptation or
decreased mating success (19). It therefore appears that inheritance of kdr-w was the defining
factor that allowed hybrids to overcome their selective disadvantage and backcross with the
parental population, contributing to gene flow between the two species.
Selection for kdr-w was significantly stronger in A. coluzzii than for sympatric A.
gambiae (A. coluzzi: s = 0.13 and 0.14; A. gambiae: s = 0.065 and 0.014, for Selinkenyi and
Sidarebougou, respectively) (Fig. S3, Table S6), due to either a) necessity of having the kdr-w to
overcome the selective disadvantage inherent in being a hybrid, b) differences in selection
pressure in associated larval habitats, or c) phenotypic penetrance of kdr-w differing in the A.
gambiae genetic background.
To explore the chromosomal extent of introgression, pre- and post-introgression A.
coluzzii and A. gambiae were whole-genome sequenced. FST between pre- and post-2006 A.
coluzzii was near 0 throughout the genome, with the exception of a 3 Mbp region near the 2L
centromere, where FST peaked at 0.45, around the voltage-gated sodium channel gene containing
kdr-w (Fig. 2a). Proportion A. gambiae ancestry was calculated in this region, which showed
that homozygous backcrossed A. coluzzii were genetically more similar to A. gambiae from 0-3
Mbp, while heterozygous backcrossed A. coluzzii had intermediate ancestry (Fig. 2b), indicating
that the A. gambiae pericentromeric region was inherited as a whole segment and is
independently assorting. When considered with the genotyping data, this means that ~95% of A.
coluzzii individuals in Mali possess at least one copy of the A. gambiae 2L centromeric region,
resulting in the loss of approximately one third of the diverged genomic regions that define these
species. However, two recombinant individuals were identified by whole-genome sequencing,
both of which retained kdr-w while re-establishing a portion of the A. coluzzii divergence island.
Tajima’s D statistic shows evidence of a selective sweep on the voltage-gated sodium channel
gene in A. gambiae and post-2006 A. coluzzii (Fig. 2), implicating kdr, not neighboring genes, in
driving introgression and selection.
Adaptive introgression often involves transfer of complex adaptations via “cassettes” of
multiple, linked mutations (20), and previous work has shown that kdr-based resistance to
pyrethroids and DDT can be modulated by other mutations in the voltage-gated sodium channel
gene (21). Using the whole-genome sequencing data, we investigated two other SNPs in the
same voltage-gated sodium channel gene, which have been shown to affect pyrethroid resistance.
The N1575Y SNP is associated with increased pyrethroid resistance and is only found on an
L1014 (kdr-w) genetic background (22), and a second, synonymous SNP 4,974 bp upstream of
kdr-w was detected in association mapping for pyrethroid resistance (21). Both of these
mutations were absent in pre-2006 A. coluzzii, but present in 43% and 76% of post-2006 A.
coluzzii, respectively, and are linked to kdr-w (Fig. 3).
DISCUSSION
The hybridization event in 2006 was one of a series of recurring breakdowns of
assortative mating (13), however, this was the first time that significant gene flow was observed
(backcrossed individuals represented 55%-90% of the population in 2010-12 (13)). It is possible
that an unidentified ecological change affected mating cues, leading to higher hybridization rate.
However, the increased fitness of backcross hybrids is likely due to the widespread and
organized implementation of ITNs and indoor residual spraying (IRS), resulting in stronger
selection for insecticide-resistant individuals. In 2005, the Malian Ministry of Health began
providing free ITNs to children (23), while the President’s Malaria Initiative (PMI) began
operations in Mali shortly thereafter, providing 369,800 ITNs in 2007 (24). By 2007, 20-40% of
children <5 years slept under an ITN in the provinces where our study sites are located (25).
Although selection for kdr-w has been strong in Sidarebougou for decades, likely due to
widespread agricultural insecticide use on cotton in this region (26), kdr-w remained at
significantly lower levels in Selenkenyi prior to 2006 (15-45%).
The rapid scale-up of insecticide-based malaria control measures in Africa during the
mid-2000s has had measurable effects on malaria vectors, especially on A. gambiae and A.
coluzzi, which are highly dependent upon humans for blood meals and indoor resting spaces. In
some cases, selection pressure from these measures has been strong enough to drive A. gambiae
to local extinction (27), and has shifted the feeding behavior of A. arabiensis, a sister species of
A. gambiae and A. coluzzii, to biting outdoors and on non-human hosts (27). A. coluzzii seems to
have responded to insecticide selection pressure by co-opting pyrethroid and DDT resistance, in
the form of the kdr-w mutation, from A. gambiae, via adaptive introgression. Hybridization due
to anthropogenic habitat disturbance has been well-described in plant systems (28), but welldocumented examples are rare in animal taxa (4). The most well-characterized examples include
warfarin resistance in European house mice (29) and melanism in North American gray wolves
(30), but even the example of gray wolves lacks ancient DNA data to show that coat color is not
instead due to shared ancestral polymorphism. Conversely, the public health importance of
pyrethroid resistance in malaria vectors has made it a well-researched field over the past two
decades, and has provided an opportunity to study the process of adaptive introgression in great
detail.
Better understanding of factors underlying the breakdown of assortative mating and
adaptive introgression will not only contribute to a basic understanding of hybridization and
speciation in general, but will also help provide information on the spread of genes that affect
malaria control efficacy. The frequency of introgression events between A. coluzzii and A.
gambiae is still unknown, but independent examples of kdr introgression in Mali in 2006 and
Benin prior to 2000 (18) indicate that gene flow between these species in west Africa may be
more common than previously supposed. Elucidation of how genes move through structured
populations will become increasingly important in the development and implementation of
malaria control strategies based on the use of genetically modified, malaria-refractory
mosquitoes (5).
MATERIALS AND METHODS
Sample collection: A. coluzzii and A. gambiae were collected by aspiration inside houses, from
three sites in Mali: Selenkenyi/Kela (2002, 2004, 2006, 2009, 2010, 2011, 2012), Sidarebougou
(2002, 2009, 2011), and Tissana (2006, 2011), during the rainy season (August-October). DNA
was extracted using a Qiagen Biosprint (Valencia, CA) and A. gambiae sensu lato species were
distinguished by PCR (31, 32).
SNP genotyping: 1,076 samples were analyzed with an iPLEX Gold multiplexed SNP
genotyping array, at five loci: one in the X divergence island (28S rDNA intergenic sequence,
also used to differentiate A. coluzzii and A. gambiae), one in the 2L divergence island (10), one
in the 3L divergence island (10), and both the kdr-east (L1014S, TTA→TCA (33)) and kdr-west
(L1014F, TTA→TTT (14)) SNPs (Table1). Data were used to calculate standard molecular
indices, Hardy-Weinberg equilibrium, kdr-w frequency, linkage disequilibrium, and selection
coefficients.
Whole-genome sequencing: 33 samples from Selenkenyi and Kela were whole-genome
sequenced at 5-10x coverage per individual: 12 A. gambiae, 6 pre-hybridization (pre-2006) A.
coluzzii, and 15 introgressed (post-2006) A. coluzzii. Library preparations were made with 25-50
ng DNA input, using the Nextera DNA Sample Preparation Kit and TruSeq dual indexing
barcodes (Illumina). Samples were pooled in equimolar amounts and sequenced on the Illumina
HiSeq2500 platform with paired-end, 100 bp reads. Reads were mapped to the PEST AgamP3
reference genome, and data were used to call SNPs and calculate FST, Tajima’s D statistics, and
A. gambiae vs. A. coluzzii ancestry. Detailed methods are given in Supporting Information.
FIGURE LEGENDS
Figure 1: Genotypes for the three divergence island SNPs (DIS) and the kdr-w (L1014F) SNP in
Selenkenyi/Kela populations over 10 years. Columns represent SNPs (X divergence island, 3L
island, 2L island, L1014F/kdr-w), individual mosquitoes are represented by colored horizontal
lines, with individuals stacked vertically. Light blue = homozygous for A. coluzzii-associated
alleles, dark blue = homozygous for A. gambiae-associated alleles, green = heterozygous, white
= missing data. Kdr-w genotypes are shown as red for homozygous resistant (r/r), pink for
heterozygous (r/+), and gold for homozygous susceptible (+/+). Samples that are heterozygous
across the X, 2L, and 3L SNPs are assumed to be F1 hybrids. Population assignments (A.
coluzzii, hybrid, and A. gambiae) are indicated by brackets on the left of each heat map.
Figure 2: a) FST between non-introgressed A. coluzzii (pre-2006) and backcross A. coluzzii (post2006) across chromosome 2, * indicates position of kdr SNP, red shading indicates zoomed-in
region for Figures 2b and 2c. b) Proportion A. gambiae ancestry for A. gambiae (purple), pre-
introgression A. coluzzii (blue), homozygous introgressed A. coluzzii (red), heterozygous
introgressed A. coluzzii (yellow). Line width indicates standard error among samples. Shaded
area indicates the location of the voltage-gated sodium channel gene and kdr-w SNP. c) Tajima’s
D in non-introgressed A. coluzzii and A. gambiae, showing selection near kdr. Gray dashed lines
represent two standard deviations for Tajima’s D.
Figure 3: Genotype frequencies for the minor-effect SNPs that have co-introgressed with kdr,
identified in whole genome sequenced samples. The synonymous/intronic [C/T] SNP at chr
2L:2,417,678 was identified by Weetman et al. (21) and is significantly correlated with
permethrin resistance. The N1575Y [A/T] SNP at chr 2L: 2,429,745 was identified by Jones et
al. (34), and has been shown to increase phenotypic pyrethroid resistance in an L1014F
genotypic background. Susceptible alleles are shown in light blue, resistant alleles in dark blue,
and heterozygotes are shown in yellow.
Acknowledgments: The authors would like to acknowledge Allison Weakly and Catelyn
Nieman for assistance with DNA extraction and genotyping, Julia Malvick and the UC Davis
Veterinary Genetics Lab for assistance with iPLEX genotyping; the Malaria Research and
Training Center at the University of Bamako; the QB3 Vincent J Coates Genomics Sequencing
Laboratory at UC Berkeley; Adama Sacko, Rebecca Trout-Fryxell, Stephanie Seifert, and
Michelle Sanford for help with field collections; and Clare Marsden for valuable comments on
the manuscript. Funding sources: NIH grant T32AI074550, R01AI 078183, and D43TW007390.
Authors’ contributions: LCN carried out genotyping, whole-genome library preparation, and
data analysis, and drafted the manuscript. BJM carried out whole-genome library preparation and
data analysis. YL helped conceive the study, carried out 2009 collection and contributed to the
manuscript. AF participated in all field collections, and performed karyotyping on polytene
chromosomes. TC assisted with whole-genome sequencing data analysis and performed selection
coefficient estimation. GCL helped conceive the study and contributed to the manuscript. All
authors read and approved the final manuscript. AJC participated in all field collections,
participated in karyotyping and contributed to the manuscript.
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