Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.

Abstract

We surveyed an Anopheles gambiae population in a West African malaria transmission zone for naturally occurring genetic loci that control mosquito infection with the human malaria parasite, Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen resistance mechanisms in plants and mammals.

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DOI: 10.1126/science.1124153
, 577 (2006);
312
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et al.Michelle M. Riehle,
Regulated by a Single Genomic Control Region
IsAnopheles gambiaeNatural Malaria Infection in

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Natural Malaria Infection in
Anopheles gambiae Is Regulated
by a Single Genomic Control Region
Michelle M. Riehle,1* Kyriacos Markianos,2* Oumou Niare ´,3Jiannong Xu,1Jun Li,1
Abdoulaye M. Toure ´,3Belco Podiougou,3Frederick Oduol,1Sory Diawara,3
Mouctar Diallo,3Boubacar Coulibaly,3Ahmed Ouatara,3Leonid Kruglyak,4
Se ´kou F. Traore ´,3Kenneth D. Vernick1†
We surveyed an Anopheles gambiae population in a West African malaria transmission zone for
naturally occurring genetic loci that control mosquito infection with the human malaria parasite,
Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of
chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent
pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains
most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the
candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in
Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1),
encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen
resistance mechanisms in plants and mammals.
T
netically resistant and susceptible mosquitoes
exist in nature, and that resistance can seg-
regate as a simple Mendelian trait (1), but
until now the prevalence, strength, and genomic
location of such natural resistance loci in mos-
quitoes were not known. Genetic control of mos-
quito response to Plasmodium has been studied
in an inbred laboratory strain of A. gambiae se-
lected for the ability to melanize animal malaria
parasites (2), for which A. gambiae is not a
natural vector. However, associations uncovered
in laboratory genetic mapping experiments may
(3) or may not (4) hold when tested in nature.
We sampled genetic variation in a natural
mosquito population by initiating isofemale
pedigrees from wild mosquitoes captured in
village dwellings in Mali, and we used the ped-
igrees to map allelic variants that have major
effects on parasite development (5). Because
female A. gambiae mate only once (5, 6), each
mosquito pedigree was the progeny of a single-
pair crossthat occurredin nature.Mosquito ped-
igreeswerechallengedbyfeedingonbloodfrom
natural human malaria infections in the same
village.Thus,theseexperimentscapturedvector-
parasite interactions of the evolutionary pair re-
he mosquito Anopheles gambiae is the
major African vector of human malaria
caused by Plasmodium falciparum. Ge-
sponsible for malaria transmission in the local
population.
Each pedigree was fed malaria-infected
blood from a single P. falciparum gametocyte
carrier, and unfed mosquitoes were removed
from the analysis. Thus, absence of oocysts re-
sulted from failure of parasite development
rather than lack of feeding. At 7 to 8 days after
the infected blood meal, we dissected all mos-
quitoes and counted normal oocyst-stage
parasites on the midgut. The number of oocysts
constitutes the quantitative phenotype. If mela-
nization of parasites was observed, which is an
insect response to many pathogens (2), the frac-
tion of melanized oocysts (melanized oocysts
per total oocyst number) was also counted and
used as a second, distinct quantitative pheno-
type. DNA extracted from each mosquito was
genotyped with the use of 25 microsatellite
markers spaced throughout the genome (1, 7),
enabling us to perform a genome-wide scan
with È9–centimorgan (cM) resolution. Linkage
between DNA markers and either of the two
quantitative phenotypes was detected with the
use of nonparametric tests (1).
Of 101 pedigrees that were generated and
challenged, 27 met criteria for genotyping: (i)
920 mosquitoes, and (ii) at least 30% of mos-
quitoes with normal oocysts, or any mosquitoes
with melanized parasites (fig. S1). Notably, 22
pedigrees had no infected individuals despite
feeding on blood that contained gametocytes.
Such completely uninfected pedigrees are not
suitable for genetic analysis, but their large
number suggests that mosquito resistance to
P. falciparum is common.
Among 17 genotyped pedigrees Eincluding
two from our previous work (1)^, significant
linkage between quantitative infection pheno-
type and DNA marker(s) was detected in seven
pedigrees Egenome-wide P value G 0.05 by
permutation (5)^: five loci linked to P. falcipa-
rum infection intensity (Pfin) and two linked to
P. falciparum melanization (Pfmel) (Fig. 1 and
Table 1). Overall, 41% of pedigrees analyzed
revealed a significant locus, indicating that loci
withamajoreffectonparasitedevelopmentinthe
field population are widespread. All analyzed
pedigrees were independently generated from
wild isofemales. Each named locus was the only
significantlocusfoundinthepedigree.Secondary
loci with influence on Plasmodium may segre-
gate in some of these pedigrees, but if so, none
had a strong enough influence on parasite
development to be independently identified as
a distinct locus with genome-wide significance.
Although our experiments detect significant
linkage to loci on all three chromosomes, the
loci with the strongest effect colocalize in a
small region of chromosome arm 2L (Fig. 1 and
Table 1). Four of the seven independent loci
mapped to a 15–megabase (Mb) region of chro-
1Center for Microbial and Plant Genomics and Department of
Microbiology, University of Minnesota, St. Paul, MN 55108,
USA.2Program in Computational Biology, Fred Hutchinson
Cancer Research Center, 1100 Fairview Avenue North, M2-
B876, Seattle, WA 98109, USA.3De ´partement d’Epide ´miolo-
gie des Affectations Parasitaires, Universite ´ de Bamako, Boı ˆte
Postale 1805, Bamako, Mali.
Integrative Genomics and Department of Ecology and Evo-
lutionary Biology, Carl Icahn Laboratory, Princeton University,
Princeton, NJ 08544, USA.
4Lewis-Sigler Institute for
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
kvernick@umn.edu
Fig. 1. Genome-wide A. gambiae
linkage map. Genomic location of
loci linked with Pfin (number of
normal oocysts following infected
blood meal) or Pfmel (fraction of
melanized parasites) in wild iso-
female pedigrees from Mali, West
Africa. Each locus shows significant
linkage in a single F1- or F2-
generation pedigree to indicated
marker locus. Horizontal lines indi-
cate chromosomes (R, right arm; L,
left arm), and vertical lines indicate
microsatellite markers. Naturally
occurring P. falciparum resistance
loci are shown in black. Pen and
Pcen loci (gray) indicate the genomic
location of melanizationlocimapped
in a laboratory-selected Plasmodium-melanizing line of A. gambiae challenged with two different strains of the
simian model parasite, P. cynomolgi (Pen, P. cynomolgi B; Pcen, P. cynomolgi Ceylon) (15, 16). The box
highlights the genomic cluster of loci with linkage to P. falciparum infection outcome in four independent
mosquito pedigrees.
X
2R 2L
3R
H145
H36
H19
H711
H678
H71
Pfin3
Pfin2
Pen1
10 cM
H776
H525
H555
H158
H750
H544
H817
H341
H758
Pfmel1
Pcen3R
Pen2
Pcen3L
H417
H290
H197
H57
H786
H135
H603
H117
H325
H769
Pen3
Pcen2R
Pfin1
Pfin4
Pfin5
Pfmel2
3L
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mosome 2L, 5 Mb on either side of markers
H603 and H325 EP e 0.014 for clustering of
loci (5)^. For fine mapping of the clustered loci,
six to ten additional markers (5) were tested in
the four pedigrees (Fig. 2). Three of the four
clustered loci (Pfin1, Pfin4, and Pfin5) stand
out among all mapped loci, because they have
by far the lowest P values and the greatest in-
fluence on parasite numbers (Table 1). For all
three Pfin loci, the presence of a resistance
allele explains at least 89% of parasite-free
mosquitoes within a pedigree; the fourth locus
in the cluster, Pfmel2, explains 100% of mos-
quitoes with melanized parasites within the
pedigree. The four clustered loci display domi-
nant or semidominant effects on parasite infec-
tion.Incontrast,Pfin2andPfin3,locatedoutside
this cluster, display recessive inheritance of
resistance and explain little of the variation
ininfectionphenotype.Giventhedata,linkage
in this region could be due to four distinct loci
in the population, different allelic forms of the
same locus, or a single causative mutation.
Regardless,aspecificchromosomeregion,which
controls the majority of naturally segregating
variation for P. falciparum infection detected in
this study, is a major Plasmodium-resistance
island (PRI) of the A. gambiae genome. Finding
linkage in independent pedigrees over multiple
years, with each pedigree fed on infected blood
fromadifferentgametocytecarrier,furthersug-
gests that the resistance effect controlled by the
mosquito PRI is largely independent of parasite
genotype.
The small size of the PRI makes feasible a
search for candidate genes. Three filters were
applied to the 976 Ensembl-predicted tran-
scripts in this 15-Mb interval (Fig. 3 and fig.
S2) (5). First,theintervalcontained39predicted
members of mosquito immune gene families by
sequence annotation, including putative func-
tions in pathogen recognition, immune signal
modulation, immune effectors, and signal trans-
duction. Second, 13 genes in the interval were
transcriptionally regulated on whole-genome
microarrays after infection with P. berghei.
Third, 29 genes were represented in an enriched
A. gambiae immune expressed sequence tag
(EST) library containing a wide spectrum of
host defense genes, including transcriptional
response to Plasmodium. Taking into account
overlaps between categories, the filters high-
light 72 genes. Despite caveats of the filters
(e.g., causative genes might not belong to
known families of immune genes or be trans-
criptionally regulated by Plasmodium infec-
tion), these genes represent a plausible set of
candidate genes for initial screening.
Genesfortwonovelleucine-richrepeat(LRR)–
containingproteins(EnsemblA. gambiae genome
database accession codes ENSANGG00000012041
and ENSANGG00000019333) were the only
onestosatisfyallfiltercriteria(Fig.3). These genes
are named Anopheles Plasmodium-responsive
leucine-rich repeat 1 (APL1) and APL2 (respec-
tive to the accession codes above). Interestingly,
the 39 immune genes by annotation in the PRI
included 13 genes for LRR proteins, among
them APL1 and APL2, and the numerically
largest genomic cluster of LRR genes coincides
with the PRI (fig. S3). LRR proteins have known
roles in innate immunity in many phyla as direct
or indirect pathogen recognition factors such as
NACHT-LRR proteins, Toll-like receptors, and
plant R genes (8–10). Plant R genes form ge-
nomic clusters of rapidly evolving homologs (11).
Table 1. Pedigrees with significant linkage. We examined two P. falciparum
resistance phenotypes in A. gambiae 8 days after a malaria-infected blood
meal: (i) normal parasite count and (ii) rate of parasite melanization. Nominal
P values are derived from two-tailed Wilcoxon-Mann-Whitney tests, whereas
genome-wide P values result from 105to 108simulations per pedigree, as
appropriate. Neither of the pedigrees yielding Pfmel loci gave Pfin loci, nor
did the Pfin4 pedigree give a Pfmel locus. Gen, generation after wild mother.
N, number of females in the pedigree. Prevalence, percentage of mosquitoes
in pedigree with at least one normal oocyst per midgut. Melanization rate,
fraction of total malaria parasites melanized in individuals with at least one
parasite melanized. Allele effect, dominant or codominant inheritance;
genotype effect, recessive inheritance.
LocusLocationGenN Prevalence (%)
Oocyst range
Melanization rate (%)
P value
Effect
Normal MelanizedNominal Genome-wide
Pfin1
Pfin2
Pfin3
Pfin4
Pfin5
Pfmel1
Pfmel2
2L
2R
X
2L
2L
3R
2L
F2
F2
F2
F1
F1
F1
F1
83
82
152
45
62
40
21
67
96
72
80
53
100*
100†
0–116
0–66
0–31
0–69
0–37
4–151
1–87
0
0
0
0
0
0
1.47 ? 10–8
7.56 ? 10–4
1.12 ? 10–4
9.78 ? 10–7
1.37 ? 10–7
1.84 ? 10–4
2.30 ? 10–4
1.20 ? 10–7
2.66 ? 10–2
2.59 ? 10–3
3.02 ? 10–6
5.60 ? 10–7
4.98 ? 10–3
1.35 ? 10–3
‡Melanization prevalence is 22%.
Allele
Genotype
Genotype
Allele
Allele
Allele
Allele
0–51‡
0
0–117
0–43
46 (24–91)
0
39 (2–85)
60 (31–77)
*Melanization prevalence (fraction of individuals in pedigree with at least one melanized parasite) is 42.5%.
†Melanization prevalence is 29%.
0
1
2
3
4
5
6
7
8
9
1015 2025 3035
-1 x Log(p-value)
H325
Pfmel2
Pfin4
Pfin5
Pfin1
H603
Position on chromosome 2L (Mb)
SRPN10 CTL4
APL2SCRB3APL1
FBN31
Fig. 2. FinemappingofthePlasmodium resistance island. Nominal P values are shown as a function of
position for the four pedigrees defining the PRI. The map shows consistent evidence of significant
linkage over closely spaced microsatellite markers (points on graphed lines) in all four pedigrees with
significant linkage to two distinct infection phenotypes, oocyst intensity (Pfin) and parasite melanization
(Pfmel) (See (5) for markers).The locations of the markers yielding significant linkage signalinthe initial
9-cM scan, H325 and H603, are indicated by arrows. Only the most informative markers are shown (Q3
genotypes in a pedigree). For clarity, the labeled candidate genes above the x axis are those annotated
immune genes that were also either transcriptionally regulated after P. berghei infection or present in
an immune-enriched EST library. Predicted functions or functional domains: SRPN10, serpin; CTL4,
c-type lectin; SCRB3, scavenger receptor; and FBN31,fibrinogen. All candidate genes are shown in fig. S2.
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The roles of APL1 and APL2 were func-
tionally tested by RNA interference (RNAi)–
mediated reductions of gene expression in
laboratory colony mosquitoes infected with
P. berghei (5). APL1 knockdown mosquitoes
carriedsignificantlyhighernumbersofoocysts(by
a factor of 6 to 15, P G 0.007) than did controls
treated with green fluorescent protein (GFP)
double-stranded RNA (Fig. 4 and fig. S4). Few
or no melanized parasites were observed in the
APL1 and APL2 knockdown mosquitoes, similar
to controls. In contrast, reduction of APL2 did
not affect oocyst number (P 0 0.515). Thus,
APL1 mediates significant protection from Plas-
modium infection and could underlie, at least in
part, the phenotypic effect of the PRI. However,
APL1 is only a candidate that must await further
genetic and functional studies for confirmation of
a role in natural transmission. In this regard, the
phenotypes in the APL1 knockdowns and
controls (high versus moderate oocyst number)
do not precisely reproduce those observed in sus-
ceptible and resistant Pfin genotypes after natural
infection (many versus zero oocysts). Exact rep-
lication of the field phenotype may require
specific aspects of the genetic background found
in field mosquitoes or the specific APL1 allele
presentinthenaturalpedigrees.Also,responseto
P. berghei may be different from response to P.
falciparum. Resolving the exact role, if any, of
APL1 in the natural transmission system will
require additional fieldwork.
There is no previous information on genetic
control of malaria parasite melanization in na-
ture. Our study shows that control of melani-
zation maps to two different loci, Pfmel1 and
Pfmel2. Melanization was inefficient as a re-
sistance mechanism because parasite removal
by melanization was only partial, as compared
with complete elimination by the Pfin loci.
Moreover, pedigrees segregating Pfmel alleles
displayed 100% prevalence of normal parasites
despite the ability to melanize (Table 1). The
particularly high prevalence of normal parasites
could indicate that natural melanization alleles,
while killing the melanized parasites, may also
cause defects in other mechanisms that other-
wise limit parasite numbers. On balance, the
observed melanizing genotypes would likely
result in more, not less, efficient transmission
of Plasmodium in nature. Given the data from
these pedigrees, melanization of parasites in A.
gambiae is not a major component of natural
Plasmodium resistance.
We report a comprehensive population sur-
vey of A. gambiae in Mali using a study design
that allows identification and genetic mapping
of loci with major effect on P. falciparum
development in the field population. We de-
tected genetic loci responsible for two distinct
outcomes of Plasmodium infection, variation
in numbers of normal parasites and rate of
parasite killing by melanization. Mostlociwith
major effect were clustered, forming a significant
PRI in the A. gambiae genome. Annotation
information and laboratory-based experiments
were used to extract candidate genes for
functional testing.
Examination of malaria transmission in wild
populations is essential to understand and idea-
lly capitalize on the efficient malaria conrol
strategies already implemented by A. gambiae
in nature. For example, one possible malaria
control approach would increase the population
frequency of pre-existing natural resistance
alleles like those described here by elevating
the fitness cost of malaria parasite infection
(12). Recently reported entomopathogenic fungi
can disproportionately kill Plasmodium-infected
mosquitoes as compared with uninfected ones
(13, 14), and thus could have the properties
required of a selective agent to transform vector
populations to Plasmodium resistance. Such an
evolutionary engineering strategy would not
require introduction of new genetic information
into natural vector populations.
The most notable feature of the observed
mosquito resistance is that it segregates as a
simple Mendelian trait of major effect at rea-
sonably high frequency in randomly sampled
natural genotypes. Interestingly, many mosqui-
to pedigrees completely eliminated the parasite
despite feeding on infected blood. We speculate
that the Bwild-type[ mosquito phenotype is re-
sistance and that susceptibility should be at-
tributed to specific points of failure or loss of
function in the mosquito immune system.
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Supporting Online Material
www.sciencemag.org/cgi/content/full/312/5773/577/DC1
Materials and Methods
Figs. S1 to S4
References
21 December 2005; accepted 29 March 2006
10.1126/science.1124153
81 24
2
2
2
33
Transcriptional
regulation
Immune
ESTs
Annotated immune genes
Fig. 3. Candidate gene filtering. Venn diagram of
candidate genes in the 15-Mb interval surrounding
the PRI, 5-Mb outside markers H325 and H603
[nucleotide coordinates 2L:10398172 to 24623620
onthe2Lainvertedchromosome(5)].Venn segments
indicate numbers of genes with changes in transcript
abundance 24 hours after P. berghei infection by
whole-genome microarray (false discovery rate 0
0.05), predicted immune genes by annotation, and
genes represented by ESTs in a subtracted immune-
enriched library (5). Intersection of the three filtering
criteria within the PRI contains the two predicted
LRR-containing proteins APL1 and APL2.
0
50
100
150
200
250
APL1 kdAPL2 kd GFP kd
Oocysts per midgut
APL2kd
APL1kd
GFPkd
Fig. 4. Silencing of APL1 gene expression
increases Plasmodium infection. APL1 signifi-
cantly limits the efficiency of malaria parasite
infection in four independent knockdown experi-
ments. APL1 kd, oocyst numbers in mosquitoes 7
to 8 days after RNAi-mediated reduction of APL1
transcript abundance followed by infection with
P. berghei; APL2 kd, mosquitoes treated with
APL2 double-stranded RNA; GFP kd, control
mosquitoes treated with GFP double-stranded
RNA. The APL1 and GFP data are averages of the
mean oocyst number from four independent ex-
periments, and error bars indicate standard error.
APL2 data comes from three replicate experiments
that tested APL2 along with APL1 and GFP control.
The APL1 kd results in oocyst loads 6to 15 times as
highasthoseofGFPcontrols(P G 0.007).The APL1
kd effect was significant by Wilcoxon-Mann-
Whitney, Student’s t test, and Kolmogorov-Smirnov
when analyzed for pooled data across replicates or
in analysis of individual replicates. The APL2 kd
had no significant effect on parasite number (P 0
0.515). (Inset) Fluorescence micrographs of oocysts
[constitutively fluorescent line PbGFPCON (17)] in
midguts of APL1 kd, APL2 kd, and GFP kd
mosquitoes, respectively.
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