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Selection of Aedes aegypti (Diptera: Culicidae) strains that are

susceptible or refractory to Dengue-2 virus

Article  in  The Canadian Entomologist · April 2013

DOI: 10.4039/tce.2012.105

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 1

Selection of Aedes aegypti (Diptera: Culicidae)

strains that are susceptible or refractory to
Dengue-2 virus
Paola A. Caicedo, Olga L. Barón, Mauricio Pérez, Neal Alexander, Carl Lowenberger,
Clara B. Ocampo1

Abstract—The vector competence (VC) of Aedes aegypti (Linnaeus) (Diptera: Culicidae) varies
geographically and is affected by both genetic and environmental factors. Understanding the molecular
mechanisms that influence VC may help develop novel control strategies. The selection of susceptible and
refractory strains is the first step in this process. We collected immature A. aegypti in the field and
established strains that were susceptible and refractory to Dengue-2 virus by isofamily selection through
several generations. Infection was detected by immunofluorescence of head or midgut tissues to determine
infection barriers and the % of VC by tissue. We selected three strains: Susceptible (Cali-S) (96.4%
susceptible at F19), Refractory with a midgut escape barrier (Cali-MEB) (44.1% refractory at F15), and
Refractory with a midgut infection barrier (Cali-MIB) (40% refractory at F16). The effects of the infection
were measured using Kaplan–Meier survival rates over the first seven generations. All selected strains
showed a similar decrease in survival and in the number of eggs laid/female through the seven generations,
suggesting that changes were a result of the selection process rather than the virus infection. The results of
this study suggest that VC is associated with multiple genes, which have additive effects on susceptibility.

Résumé—La compétence vectorielle (CV) d’Aedes aegypti (Linnaeus) (Diptera: Culicidae) varie
géographiquement et est influencée par des facteurs génétiques et environnementaux. Comprendre les
mécanismes moléculaires qui affectent la CV pourrait aider à développer de nouvelles stratégies de
contrôle des moustiques. La sélection de souches susceptibles et réfractaires est la première étape dans
ce processus. Nous avons recueilli des A. aegypti immatures sur le terrain et des souches susceptibles
et réfractaires au virus dengue-2 on été établies par la sélection d’isofamilles sur plusieurs générations.
L’infection a été détectée par immunofluorescence sur des tissus de la tête ou de l’intestin moyen pour
déterminer les barrières d’infection et le % de CV par tissu. Nous avons sélectionné trois souches:
Susceptible (Cali-S) (96,4% sensibles à F19), Réfractaire avec une barrière contre la dissémination dans
l’intestin moyen (Cali-MEB) (44,1% réfractaire à F15) et Réfractaires avec une barrière contre l’infection
dans l’intestin moyen (Cali-MIB) (40% réfractaire à F16). Les effets de l’infection ont été mesurés en
utilisant les taux de survie de Kaplan–Meier au cours des sept premières générations. Toutes les souches
sélectionnées ont montré une diminution similaire du taux de survie et du nombre d’oeufs pondus/
femelle à travers les sept générations, ce qui suggère que les changements sont le résultat du processus
de sélection plutôt que l’infection par le virus. Les résultats de cette étude suggèrent que la CV est
associée à de multiples gènes qui ont des effets additifs sur la susceptibilité.

Introduction are endemic diseases throughout tropical and

Aedes aegypti (Linnaeus) (Diptera: Culicidae)
is the main vector of dengue viruses (serotypes
1–4), responsible for dengue fever, dengue shock
syndrome, and dengue haemorrhagic fever, which
subtropical countries. An estimated 2.5 billion
people live in areas where they are at risk of
becoming infected with dengue virus. Dengue
virus transmission accounts for , 100 million
new infections and 20 000 deaths per year

Received 17 May 2012. Accepted 27 August 2012.

P.A. Caicedo, O.L. Barón, M. Pérez, N. Alexander, C.B. Ocampo,1 Centro Internacional de Entrenamiento
e Investigaciones Médicas (CIDEIM), Carrera 125 No. 19–225, Santiago de Cali, Colombia
C. Lowenberger, Department of Biological Sciences, Simon Fraser University, 8888 University Drive,
Burnaby, British Columbia V5A 1S6, Canada
1
Corresponding author (e-mail: claraocampo@cideim.org.co).
Subject editor: Gilles Boiteau
doi:10.4039/tce.2012.105

Can. Entomol. 145: 1–10 (2013) 䉷 2013 Entomological Society of Canada

2 Can. Entomol. Vol. 145, 2013
(World Health Organization 2009). There are
no vaccines or antiviral treatments available for
dengue, and disease control is based on reducing
insect bites and vector populations (Mackenzie
et al. 2004). Despite extensive vector control
efforts, disease incidence continues to increase
due to the concurrent presence of multiple virus
serotypes circulating in many geographical regions
(World Health Organization 2009). It is imperative
to explore alternative strategies to decrease the
transmission of this disease. Advances in mole-
cular techniques such as genome sequencing and
genetic manipulation open new routes to improve
disease transmission control.
Vector competence (VC) is the intrinsic per-
missiveness of an arthropod vector to infection,
replication, and transmission of a virus (Hardy
1988; Woodring et al. 1996). VC is controlled
by genetic and environmental factors. Low VC,
or refractoriness, is associated with different
barriers that inhibit viral replication or dissemi-
nation (Black et al. 2002). An inability of the
virus to enter or replicate in the midgut is
referred to as a midgut infection barrier (MIB),
while an ability to infect the midgut but not
move subsequently into other regions of the
vector is known as a midgut escape barrier
(MEB). If the virus escapes the midgut it must
breach the salivary gland barrier and enter the
salivary glands for transmission (Woodring et al.
1996). Understanding the genetic factors asso-
ciated with VC and those factors that regulate
the barriers to viral development will allow us to
identify new strategies to decrease transmission
based on genetic manipulation, or may identify
DENv-specific molecules that may become
targets for new drugs or vaccines (Beerntsen
et al. 2000; Alphey et al. 2002). In A. aegypti,
the genetic components of VC have been studied
through selections of strains with different
susceptibilities to dengue virus (Gubler et al.
1979; Miller and Mitchell 1991; Bennett et al.
2002, 2005). Methodologies such as random
amplified polymorphic DNA-polymerase chain
reaction (RAPD-PCR) markers and quantitative
trait loci (QTL) have identified biological
markers associated with VC (Gubler et al. 1979;
Bosio et al. 2000; Black et al. 2002) but have not
identified specific genes that determine VC.
Once specific genes are identified these must
be evaluated in strains of A. aegypti that
demonstrate different levels of susceptibility and
well-known and characterised infection barriers.
It is unknown whether the same genetic regula-
tion of VC is conserved across geographic
strains of a vector species or sympatric vectors
of different species. Comparing the VC of strains
from different geographical origins might yield
different models to evaluate and validate general
antiviral control mechanisms.
Previous studies carried out in A. aegypti
collected from Cali, Colombia, demonstrated
a high variation in VC depending on the time and
location of collection (Ocampo and Wesson 2004),
and we have observed the presence of different
anatomical barriers. Based on these studies, we
began to select strains with different susceptibilities
to Dengue-2 virus (DENv-2) using the isofemale
selection methodology (families obtained from a
mother with a specific phenotype) (Tardieux et al.
1991) starting with field collected mosquitoes, to
allow us to study genetic components associated
with VC in these A. aegypti populations. The
objectives of this research were to select for higher
levels of specific phenotypes (resistance or sus-
ceptibility to DENv) and to measure the effects of
this selection on fitness parameters.
Materials and methods
Mosquitoes
Laboratory colonies were established from field
collected larvae and pupae from artificial larval
habitats in five locations at least 10 km apart in the
city of Cali, Colombia. The mosquitoes were
maintained under standard laboratory conditions:
26 7 2 8C, 70% relative humidity, and a 12:12
hour light:dark cycle. Larvae were maintained in
plastic containers at a density of 300 larvae/2 L of
water and were fed daily with 2 mL of a stock
solution (8 g/400 mL) of beef liver (DIFCO,
Becton, Dickinson and Company, Franklin Lakes,
New Jersey, United States of America). Adults
were fed with a 10% sugar solution. Blood feeding
was done through an artificial membrane feeder
using a pig intestine membrane and defibrinated
rabbit’s blood. The Filial 1 (F1) eggs were stored
and used to begin the selection process.
Virus strain
DENv-2 New Guinea C strain, freshly grown
in C6/36 HT (Aedes albopictus [Skuse] cells),
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Caicedo et al.
was used in oral challenges. Infected cells were
incubated for 14 days at 32 8C in L15 medium
supplemented with 2% heat-inactivated foetal
bovine serum, 1% penicillin/streptomycin, and
1% L-glutamine (Higgs et al. 1996). Virus and
cells were harvested and collected in a 15-mL
conical centrifuge tube. Aliquots of the infected
cell suspension and the mixture of blood and
virus before and after the infection process were
titered as described by Bennett et al. (2002).
Titres in the cell suspensions ranged from 108 to
108.5 TCID50/mL in all oral challenges. The viral
suspension was used in experimental infections
and also to microinject the virus intrathoracically
into mosquitoes, to serve as positive controls for
indirect immunofluorescence (IFI) studies.
Mosquito infection and virus titration
Five-day-old female A. aegypti (F1) that had
been starved for 12 hours were exposed for 2 hours
to an infectious bloodmeal consisting of a 1:1
mixture of defibrinated rabbit blood and the dengue
virus suspension via a membrane feeder described
above. Subsequently, engorged females were
maintained individually in plastic (2 cm 3 4 cm)
containers and given access to moist cotton and
a piece of moist filter paper for oviposition.
The presence and concentration of virus to
which the females were exposed was determined
in: (a) uninfected rabbit blood, (b) virus sus-
pension, and (c) the mixture of blood and virus
at the beginning and end of the exposure period
as described by Bennett et al. (2002).
Indirect IFI was used to determine the percent-
age of infections in the head and midgut of each
female as described by Wallis et al. (1985).
Fourteen days post exposure, the barriers to virus
dissemination were determined as described
previously (Bennett et al. 2002; Black et al.
2002). A murine IgG polyclonal antibody bound
to FITC (Dengue FITC conjugate 1 2% BSA;
obtained from the Center for Disease Control,
Puerto Rico, United States of America), in a
1:200 dilution, was used in the IFI.
Selection of Susceptible (S), refractory-
midgut infection barrier (R-MIB), or
refractory-midgut escape barrier
(R-MEB) strains
We determined the phenotype of the mothers
using IFI to begin the selection process. Heads of
3
females that were positive with IFI indicated
susceptible mothers with no midgut barriers
(Cali-S). We assumed that this population had no
barriers to virus dissemination. The midgut of
mothers whose heads were negative to IFI were
dissected to determine anatomical barriers
associated with virus infection. Positive IFI
analysis of the midgut indicated a MEB, and
these females and their descendents were classi-
fied as refractory MEB (Cali-MEB). Negative
midgut IFI analysis indicated a MIB and these
females and their offspring were classified as
refractory MIB (Cali-MIB).
The selection by phenotype after exposure to
DENv-2 was not done in every generation. The
strains were exposed to blood free of dengue
virus every second generation to allow them
to recover from any fitness limitations. This
protocol was initiated after the F1 generation
and continued throughout the selection process.
The offspring of females that demonstrated
the same phenotype in each selection process
were grouped with the aim of increasing
genetic variability and increasing the numbers of
each strain.
Survival analysis
The effects of the infection and initial selection
on the fitness of these selected strains were eval-
uated using Kaplan–Meier survival analyses for
the first seven generations. This technique esti-
mates the survival over the stages of egg hatching,
larvae, pupae, and adult emergence. Survival was
therefore measured in terms of the stage achieved,
rather than time in days. The Kaplan–Meier
survival probabilities are applicable because they
depend only on the ordering of the stages, not their
duration in days. The final point of the survival
curve is the proportion of eggs surviving to
emergent adults. Using Greenwood standard
errors, these proportions were analysed by
contrasts, in terms of trends over generations
(1–7), and differences between strains (Cali-S,
Cali-MIB, and Cali-MEB) (Armitage et al. 2001).
The average number of eggs laid per female in
generations exposed to DENv was evaluated using
the nonparametric Kruskal–Wallis test (K–W).
The proportion of eggs that hatched was evaluated
among strains and generations using a x2-test
for multiple comparisons. A negative binomial
analysis was used to compare differences in the
䉷 2013 Entomological Society of Canada
4 Can. Entomol. Vol. 145, 2013

Table 1. Effect of selection on the percentage of females from different selected strains that are susceptible to
infection with DENv-2.

% Infected females per phenotype

Strain Filial Total (n) S Refractory-MEB Refractory-MIB
Initial population F1 38 65.8 26.3 8
S F3 23 82.6 8.7 8.7
F4 36 80.5 19.4 0
F6 12 91.6 8.4 0
F12 27 89 7.4 3.7
F14 74 94.6 4.1 1.3
F16 48 96 4 0
F18 46 92 8 0
F19 56 96.4 0 3.5
Cali-MEB F4 20 75 25 0
F6 88 67 36.4 10.3
F9 22 28.6 71.4 0
F11 44 52.3 34.1 13.6
F12 55 40 33 23
F14 44 46.3 36.6 17.0
F15 59 5.1 44.1 50.8
Cali-MIB F3 5 40 40 20
F4 17 41 12 47
F6 48 54.5 10.1 35.4
F9 34 32.3 38.2 29.4
F11 77 41.5 28.6 29.9
F13 57 35.1 17.5 47.4
F15 53 32.1 25.9 42
F16 66 40.3 19.7 40
Notes: The selected phenotype is underlined.
DENv-2, dengue-2 virus; S, susceptible strain; MEB, midgut escape barrier; MIB, midgut infection barrier; Cali-MEB,
refractory with midgut escape barrier strain; Cali-MIB, refractory with midgut infection barrier strain.

percentage of mortality per developmental stage
between our strains and also to compare these
parameters between the parental generation and
subsequent virus-exposed generations. The repor-
ted effect measure is the incidence rate ratio (IRR)
with the 95% confidence intervals (95% CI).
Results
Strain selection
In offspring of Cali-S females, the percentage of
susceptibility (positive head infections) increased
from 65.8% (F1) to 96.4% (F19) through the
selection process. Any inherent midgut barriers
were reduced over the 19 generations (Table 1).
In offspring of mosquitoes with the Cali-MIB
phenotype, refractoriness (absence of infection)
increased from 8% (F1) to 40% (F16). During the
selection process we observed variability in the
frequency of individuals with MEB phenotype
(Table 1). The Cali-MEB strain first increased
from 26.3% (F1) to 71.4% in generation 9, then fell
to 34.1% (F11) and increased again to 44.1% in
generation 15 (Table 1). The titre of the DENv-2
preparation used was monitored throughout all
selections and ranged from 108 to 108.5 TCID50/mL
(50% tissue culture infective doses/mL) at the
beginning of virus exposure and from 107.2 to 107.4
TCID50/mL at the end of the exposure period. All
rabbit blood was free of infection.
Effects of DENv-2 infection on the
survival of eggs to emergent adults
of the selected strains
The average number of eggs laid per female
diminished during the selection process, from

䉷 2013 Entomological Society of Canada

Caicedo et al.
Table 2. Average number of eggs laid per female of Cali-S, Cali-MEB, and Cali-MIB Aedes aegypti strains
at different generations of selection against DENv-2.
2 4
X (7SD)
Strain [n]
S 76.05 (717.2)
[43]
Refractory-MEB 79 (712.5)
[13]
Refractory-MIB 67.5 (729.6)
[4]
Parental generation 110.5 (728.1)
[17]
Notes: The sample sizes are indicated in [ ].
Cali-S, susceptible strain; Cali-MEB, refractory through a midgut escape barrier strain; Cali-MIB, refractory through a
midgut infection barrier strain; MEB, midgut escape barrier; MIB, midgut infection barrier; DENv-2, dengue-2 virus.
110 eggs/female in the Parental (F1) generation
to ,40 in the F6 generation in all selected strains
(Table 2). There were significant differences in
the overall number of eggs laid between the
parental and the subsequent generations (K–W
x2 5 58.339, df 5 4, P , 0.001) and among the
generations (Cali-S: K–W x2 5 63.91, df 5 2,
P , 0.001; Cali-MEB: K–W x2 5 27.79, df 5 2,
P , 0.001; Cali-MIB: K–W x2 5 4.33, df 5 2,
P , 0.001).
The survival curves for the three strains are
shown in Figure 1. Of all the eggs laid, there was a
trend for the proportion of eggs hatching and
surviving to adults to be reduced over the genera-
tions (P , 0.001). On average over the seven first
generations, the proportion of eggs surviving to
adults fell to 29.4% for the S strain, 26.7% for the
Cali-MEB strain, and 21.5% for the Cali-MIB
strain (P , 0.001), relative to the F1 strain.
Most of these differences can be attributed to
the percentage of eggs that hatched. Mortality was
observed mainly in the first day during the egg
hatching period (Fig. 1). In the Cali-S strain the
percentage of eggs that hatched to produce viable
larvae was reduced from 78.9% to 40.5% between
generations 2 and 7 (Table 3). Similarly, between
generations 2 and 7, the hatching rates of eggs in
the Cali-MIB strain were reduced from 76.7% to
47.9%, and in the Cali-MEB strain from 90% to
54.3% (Table 3).
Of the first instars that did hatch, there were
no significant differences in the survival of
5
Generation
6
X (7SD) X (7SD)
[n] [n]
60.55 (724.3) 32.03 (715.0)
[64] [40]
83.94 (754.7) 29.32 (718.1)
[17] [22]
49.75 (731.7) 27.43 (77.07)
[4] [7]
L1–L4 larvae in any of the strains (negative
binomial regression, P . 0.05; Table 4). Simi-
larly, there were no differences in the survival of
larvae once they hatched between generations
within a strain (negative binomial regression,
P . 0.05). In the Cali-MEB strain there were
significantly more losses at the pupal stage over
generations of selection. Within the other strains
there were no significant differences in pupal
survival over the six generations.
Initially all selected strains spent more time as
4th instar and pupae than established laboratory
colonies, and emerging females were receptive
to a bloodmeal at later periods (5–8 days post
emergence) than established laboratory colonies
(3–4 days post emergence).
Discussion
Selection in nonconsecutive generations yielded
survival and allowed maintenance of significant
populations of these strains through .15 gene-
rations. Previously, when all generations were
exposed consecutively to selection pressure,
we were able to maintain the strains for only three
generations.
The selection-free generations allowed the
mosquitoes to recuperate from the stress and to
ensure maximum survival of all selected strains.
The decrease in survival, due to a decrease in
hatching, observed in the first three generations
of constant or alternate generation selection,
䉷 2013 Entomological Society of Canada
6 Can. Entomol. Vol. 145, 2013

Fig. 1. Kaplan–Meier analysis of the survival of three selected strains of Aedes aegypti in response to exposure
to Dengue-2 virus over seven generations. (A) Refractory through a midgut escape barrier (Cali-MEB);
(B) Refractory through a midgut infection barrier (Cali-MIB); (C) Susceptible strain (Cali-S).

may have resulted from recessive deleterious other horizontally transmitted arboviruses, has
alleles being expressed due to the high degree of been reported to have negative effects on
relatedness (Bennett et al. 2005). Dengue, and A. aegypti including a reduced fitness of the

䉷 2013 Entomological Society of Canada

Caicedo et al.
Table 3. Effect of infection with DENv-2 on the egg hatching rates over different generations in the selected
strains of Aedes aegypti.
Generation Cali-S (% of n)
1 (parental) 76.8 (250)
2 78.9 (3355)
3 32.2 (3265)
4 56.7 (1746)
5 58.2 (1746)
6 66.2 (4021)
7 40.5 (1281)
Notes: Generation 1 is the parental strain brought into the laboratory as field collected pupae.
Strains are Cali-S, Cali-MIB, and Cali-MEB.
Numbers represent % of eggs that hatched from (n) eggs.
DENv-2, dengue-2 virus; Cali-S, susceptible strain; Cali-MIB, refractory through a midgut infection barrier strain;
Cali-MEB, refractory through a midgut escape barrier strain.
Table 4. Parameters of the negative binomial regres-
sion analysis of larval mortality among the three strains
of Aedes aegypti: Cali-S, Cali-MIB, and Cali-MEB.
instar IRR P value 95% CI
L1 0.6942771 0.896 0.0028965–166.4137
L2 1.034371 0.967 0.2061835–5.189179
L3 9.202318 0.031 1.218485–69.4983
L4 0.861216 0.672 0.4310291–1.720749
Cali-S, susceptible strain; Cali-MIB, refractory through a
midgut infection barrier strain; Cali-MEB, refractory through
a midgut escape barrier strain; IRR, incidence rate ratio; CI,
confidence ratio.
arthropod hosts (Lambrechts and Scott 2009;
Maciel-de-Freitas et al. 2011). In addition, other
linked alleles that were deleterious or ‘‘side
effects’’ caused by the alleles that conferred
resistance, also may have contributed to the
reduced fitness we observed.
The selection of Cali-S, Cali-MEB, and Cali-
MIB strains, whose phenotypes are defined in
relation to infection with DENv-2, confirms the
heritable component of these characteristics
(Hardy 1988). Although we did not achieve 100%
susceptible or refractory phenotypes in these
selections, we did increase the values compared
with the original field collected mosquitoes. We do
not know, nor could we control for, the phenotype
of the male, and similar results were reported in
Culex tarsalis Coquillett (Hardy et al. 1978).
In this study, we experimentally infected
A. aegypti with titres of DENv-2 ranging from
107 to 108 TCID50/mL; similar levels to which
they would be exposed when biting a human
7
Cali-MIB (% of n) Cali-MEB (% of n)
76.7 (270) 90.0 (713)
46.1 (2588) 28.2 (2210)
33.9 (1303) 22.7 (643)
53.3 (199) 52.9 (1183)
32.0 (906) 55.1 (1953)
47.9 (1920) 54.3 (645)
suffering from dengue fever. Studies of the
variation in susceptibility to dengue virus infec-
tion suggest that a constant virus titre must be
used in each experiment because this quantitative
factor can affect the determination of the VC
(Gubler et al. 1979). Additionally, larval density,
food availability, and temperature conditions
were controlled for all the selected lines, which
produced homogenous populations. We did not
evaluate other variables such as viral dose,
mosquito size, the specific combination of vector
and virus genotypes, or changes in temperatures
found in the field that are known to affect virus
susceptibility (Gubler et al. 1979; Lambrechts
2011; Lambrechts et al. 2011).
The exact mode of inheritance of the suscept-
ible or refractory phenotypes was not determined
in this study. However, the susceptible strain was
dominant in all three selection schemes (Table 1).
In the S strain the proportion of individuals with
this phenotype increased progressively with gene-
rations of selection. In contrast, there was greater
variation in the selection results for refractory
strains (Table 1). Although there was an initial
increase towards refractory phenotypes (Cali-
MEB and Cali-MIB) under selection, this did not
continue consistently, suggesting that there are
adverse effects or fitness costs associated with the
selection. The relatively easy selection of the
susceptible phenotype differs from previous
reports that suggested that the DENv-2 resistant
phenotype in A. aegypti was dominant over the
susceptible phenotype (Gubler and Rosen 1976).
Instead, our results agree with Hardy et al. (1978)
who reported that in C. tarsalis there was a
䉷 2013 Entomological Society of Canada
8 Can. Entomol. Vol. 145, 2013
complete or partial dominance of the susceptible
over the resistant phenotype in response to infec-
tion with equine encephalitis virus. The results of
this study suggest that VC is associated with
multiple genes that have some additive effects
on susceptibility, as suggested previously (Bosio
et al. 1998).
Other studies on the genetics of VC for
flavivirus infections in A. aegypti also have
used strain selection by isolines. Wallis et al.
(1985) selected lines of A. aegypti susceptible and
refractory to infection with the yellow fever virus.
Similarly, Tardieux et al. (1991) selected lines of
A. aegypti from a French Polynesian colony using
isofemale lines, and concluded that the variation in
susceptibility to DENv-2 was partially controlled
by genetic factors. Bennett et al. (2005) selected
two strains of A. aegypti on the basis of isofamilies
that differed in their susceptibility to infection with
DENv-2 and Miller and Mitchell (1991) selected
A. aegypti strains that were completely refractory
or highly susceptible to the yellow fever virus.
Recent advances in molecular biology, genetics,
and statistics have allowed us to map QTL in
relation to parasite or pathogen infection (Severson
et al. 1994a, 1994b; Bosio et al. 1998, 2000;
Bennett et al. 2005). These studies have confirmed
that the genetic component of VC is a product of
multiple genes acting additively or in a dominant
manner. These results suggest that A. aegypti is
an excellent model for the study of selection
processes that will allow an evaluation of the
genetic complexities of specific aspects of VC.
The mortality levels in the selected strains may
be attributed to a differential tolerance of infection
or to an adaptation of strains to laboratory condi-
tions and/or the selection process used. It is
known that infection by pathogens may cause
pathological effects in mosquitoes, subsequently
reducing survival and fecundity of the vector
(Kershaw et al. 1954; Javadian and Macdonald
1974; Courtney et al. 1985; Lambrechts and Scott
2009; Lambrechts 2011; Lambrechts et al. 2011).
Whether these differences were caused directly by
the virus (Lambrechts and Scott 2009), a result of
the innate immune response towards the virus
(Maciel-de-Freitas et al. 2011), or a result of
the selection process itself was not addressed in
this study.
The present study supports the concept that we
can select for strains with different VC although
there is variability. These results could allow us to
use more quantitative techniques such as QTL, to
further identify the genetic components of VC.
The strains generated in this study were used in
suppressive subtraction hybridisation studies that
identified differentially expressed genes likely
involved in determining the susceptibility of
A. aegypti to infection with DENv-2 (Barón et al.
2010) and will be used for larger deep sequencing
and microarray analyses to delve further into the
mechanisms that determine phenotype. These
strains also will serve as a natural model of
A. aegypti: dengue virus to study specific innate
immune mechanisms related to DENv infection,
and to expand current in vivo studies on the effects
of knocking down specific genes using RNA
interference to evaluate their role in determining
the refractory phenotype.
Acknowledgements
The authors thank Luis Ernesto Ramirez for
technical support at the Centro Internacional
de Entrenamiento e Investigaciones Medicas
(CIDEIM). This research was funded in part by the
Departamento Administrativo de Ciencia, Tecno-
logı́a e Innovación (COLCIENCIAS) contract
349-2004 to C.B.O., by the Tropical Disease
Research contract TDA30848a to C.B.O., the
young investigator award by COLCIENCIAS
contract 159-2008 to O.L.B. and grant 261940 from
the Natural Sciences and Engineering Research
Council of Canada to C.L., and by the Canada
Research Chairs program to C.L.
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