# Challenges in estimating insecticide selection pressures from mosquito field data.

**ABSTRACT** Insecticide resistance has the potential to compromise the enormous effort put into the control of dengue and malaria vector populations. It is therefore important to quantify the amount of selection acting on resistance alleles, their contributions to fitness in heterozygotes (dominance) and their initial frequencies, as a means to predict the rate of spread of resistance in natural populations. We investigate practical problems of obtaining such estimates, with particular emphasis on Mexican populations of the dengue vector Aedes aegypti. Selection and dominance coefficients can be estimated by fitting genetic models to field data using maximum likelihood (ML) methodology. This methodology, although widely used, makes many assumptions so we investigated how well such models perform when data are sparse or when spatial and temporal heterogeneity occur. As expected, ML methodologies reliably estimated selection and dominance coefficients under idealised conditions but it was difficult to recover the true values when datasets were sparse during the time that resistance alleles increased in frequency, or when spatial and temporal heterogeneity occurred. We analysed published data on pyrethroid resistance in Mexico that consists of the frequency of a Ile1,016 mutation. The estimates for selection coefficient and initial allele frequency on the field dataset were in the expected range, dominance coefficient points to incomplete dominance as observed in the laboratory, although these estimates are accompanied by strong caveats about possible impact of spatial and temporal heterogeneity in selection.

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**ABSTRACT:**BACKGROUND: Insecticides are an effective and practical tool for reducing malaria transmission but the development of resistance to the insecticides can potentially compromise controls efforts. In this study a mathematical model was developed to explore the effects on mosquito populations of spatial heterogeneous deployment of insecticides. This model was used to identify important parameters in the evolution of insecticide resistance and to examine the contribution of new generation long-lasting insecticidal bed nets, that incorporate a chemical synergist on the roof panel, in delaying insecticide resistance. METHODS: A genetic model was developed to predict changes in mosquito fitness and resistance allele frequency. Parameters describing insecticide selection, fitness cost and the additional use of synergist were incorporated. Uncertainty and sensitivity analysis were performed followed by investigation of the evolution of resistance under scenarios of fully effective or ineffective synergists. RESULTS: The spread of resistance was most sensitive to selection coefficients, fitness cost and dominance coefficients while mean fitness was most affected by baseline fitness levels. Using a synergist delayed the spread of resistance but could, in specific circumstances that were thoroughly investigated, actually increase the rate of spread. Different spread dynamics were observed, with simulations leading to fixation, loss and most interestingly, equilibrium (without explicit overdominance) of the resistance allele. CONCLUSIONS: This strategy has the potential to delay the spread of resistance but note that in an heterogeneous environment it can also lead to the opposite effect, i.e., increasing the rate of spread. This clearly emphasizes that selection pressure acting inside the house cannot be treated in isolation but must be placed in context of overall insecticide use in an heterogeneous environment.Malaria Journal 08/2012; 11(1):258. · 3.49 Impact Factor - SourceAvailable from: Craig S Wilding[Show abstract] [Hide abstract]

**ABSTRACT:**Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide-based control efforts, necessitating regular monitoring. We assessed resistance in field-collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) biosassays. Only An. gambiae s.s. and An. arabiensis (≅70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT(50) > 2 h; deltamethrin LT(50) > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (≅95%), consistent with insecticide-mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately-powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s.×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid-impregnated bednets or indoor spraying of pyrethroids.Medical and Veterinary Entomology 10/2012; · 2.21 Impact Factor - SourceAvailable from: Adriana E. FloresAdriana E Flores, Gustavo Ponce, Brenda G Silva, Selene M Gutierrez, Cristina Bobadilla, Beatriz Lopez, Roberto Mercado, William C Black[Show abstract] [Hide abstract]

**ABSTRACT:**Seven F1 strains of Aedes aegypti (L.) were evaluated by bottle bioassay for resistance to the pyrethroids d-phenothrin, permethrin, deltamethrin, lambda-cyalothrin, bifenthrin, cypermethrin, alpha-cypermethrin, and z-cypermethrin. The New Orleans strain was used as a susceptible control. Mortality rates after a 1 h exposure and after a 24 h recovery period were determined. The resistance ratio between the 50% knockdown values (RR(KC50)) of the F1 and New Orleans strains indicated high levels of knockdown resistance. The RR(KC50) with alpha-cypermethrin varied from 10 to 100 among strains indicating high levels of knockdown resistance. Most of the strains had moderate resistance to d-phenothrin. Significant but much lower levels of resistance were detected for lambda-cyalothrin, permethrin, and cypermethrin. For zeta-cypermethrin and bifenthrin, only one strain exhibited resistance with RR(KC50) values of 10- and 21-fold, respectively. None of the strains showed RR(KC50) >10 with deltamethrin, and moderate resistance was seen in three strains, while the rest were susceptible. Mosquitoes from all strains exhibited some recovery from all pyrethroids except d-phenothrin. Regression analysis was used to analyze the relationship between RR(LC50) and RR(KC50). Both were highly correlated (R2 = 0.84-0.97) so that the slope could be used to determine how much additional pyrethroid was needed to ensure lethality. Slopes ranged from 0.875 for d-phenothrin (RR(LC50) approximately equal to RR(KC50)) to 8.67 for lambda-cyalothrin (-8.5-fold more insecticide needed to kill). Both RR(LC50) and RR(KC50) values were highly correlated for all pyrethroids except bifenthrin indicating strong cross-resistance. Bifenthrin appears to be an alternative pyrethroid without strong cross-resistance that could be used as an alternative to the current widespread use of permethrin in Mexico.Journal of Economic Entomology 04/2013; 106(2):959-69. · 1.60 Impact Factor

Page 1

Challenges in Estimating Insecticide Selection Pressures

from Mosquito Field Data

Susana Barbosa1*, William C. Black IV2, Ian Hastings1

1Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 2Department of Microbiology, Colorado State

University, Fort Collins, Colorado, United States of America

Abstract

Insecticide resistance has the potential to compromise the enormous effort put into the control of dengue and malaria

vector populations. It is therefore important to quantify the amount of selection acting on resistance alleles, their

contributions to fitness in heterozygotes (dominance) and their initial frequencies, as a means to predict the rate of spread

of resistance in natural populations. We investigate practical problems of obtaining such estimates, with particular emphasis

on Mexican populations of the dengue vector Aedes aegypti. Selection and dominance coefficients can be estimated by

fitting genetic models to field data using maximum likelihood (ML) methodology. This methodology, although widely used,

makes many assumptions so we investigated how well such models perform when data are sparse or when spatial and

temporal heterogeneity occur. As expected, ML methodologies reliably estimated selection and dominance coefficients

under idealised conditions but it was difficult to recover the true values when datasets were sparse during the time that

resistance alleles increased in frequency, or when spatial and temporal heterogeneity occurred. We analysed published data

on pyrethroid resistance in Mexico that consists of the frequency of a Ile1,016 mutation. The estimates for selection

coefficient and initial allele frequency on the field dataset were in the expected range, dominance coefficient points to

incomplete dominance as observed in the laboratory, although these estimates are accompanied by strong caveats about

possible impact of spatial and temporal heterogeneity in selection.

Citation: Barbosa S, Black WC IV, Hastings I (2011) Challenges in Estimating Insecticide Selection Pressures from Mosquito Field Data. PLoS Negl Trop Dis 5(11):

e1387. doi:10.1371/journal.pntd.0001387

Editor: Rhoel Ramos Dinglasan, Johns Hopkins Bloomberg School of Public Health, United States of America

Received June 15, 2011; Accepted September 19, 2011; Published November 1, 2011

Copyright: ? 2011 Barbosa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: S.B. was supported by research grant SFRH/BD/33534/2008 from Fundac ¸a ˜o para a Cie ˆncia e Tecnologia, Portugal and Siemens Portugal under a Ph.D.

Program in Computational Biology of the Instituto Gulbenkian de Cie ˆncia, Oeiras, Portugal. The funders had no role in study design, data collection and analysis,

decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: sbarbosa@liv.ac.uk

Introduction

Mosquito-borne diseases are prevalent in the tropics and

subtropics and constitute a large proportion of the health problems

in developing countries. The major mosquito vectors occur in the

genera Culex, Aedes, and Anopheles, which transmit Filaria spp,

Japanese encephalitis, dengue and yellow fever viruses and

malaria. The control of vector populations is often based on

insecticides, such as larviciding, indoor residual spraying (IRS) and

personal protection through insecticide treated materials (ITM)

and their use has been shown to have a powerful impact on

mosquito abundance and disease transmission [1]. The foraging

and resting behaviors of mosquitoes ensure a number of

potentially lethal interactions with insecticide-treated surfaces

during parts of the mosquito lifecycle [2], but prolonged exposure

to an insecticide over many generations runs the risk that

mosquitoes will develop resistance. In general only four different

chemical classes of synthetic insecticides are used in the field:

organochlorines, organophosphates, carbamates and pyrethroids.

Pyrethroids are particularly important because they are the only

class of insecticides recommended by The World Health

Organization to use on ITM. Since ITM are being widely

distributed across malaria and dengue affected countries and

pyrethroids are employed in some areas for agricultural pest

control, there is concern that the emergence of resistance will

compromise these efforts. The widespread use of a small portfolio

of compounds against large mosquitoes populations with many

generations per year (estimated at 12 per year for Anopheles gambiae

[3] and 20 for Aedes aegypti [4]) have raised fears that high levels of

resistance may arise very quickly.

We consider the problem of measuring the strength of selection

for insecticide resistance in mosquito field populations and show

how changes in the frequencies of the alleles at a single locus can

be used to estimate the selection acting on each genotype. This

type of data is collected for the identification of genetic

mechanisms of resistance and/or during monitoring programs of

vector control campaigns. The method we developed extends that

described earlier by DuMouchel and Anderson in 1968 [5] for

laboratory populations. Laboratory

significantly from the field. In the laboratory insecticide assays

are conducted over standardized range of doses and concentra-

tions that do not account for field situations such as decay rates

and exposure characteristics. Following insecticide deployment in

the field, concentration decreases and there is a selective window

of time at lower concentrations (see Figure 1), where resistant

heterozygotes do not die but susceptible homozygotes are still

killed, therefore acting as dominant when under more standard-

ized conditions it may appear to be recessive. This is relevant

because dominance relationships between susceptible and resis-

tance alleles affect the rate of spread of resistance.

basedconditions differ

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Page 2

Using a maximum likelihood (ML) procedure and a recursive

genetic model that tracks the changes in the resistance allele

frequencies at a single locus it is possible to estimate a selection

coefficient (s), a coefficient quantifying dominance (h) and the

initial frequency of the resistance allele (p0,), key parameters that

determine the dynamics of resistance. The model provides a

straightforward way to obtain these values with the least complex

dataset possible. However, field data on the spread of resistance is

often suboptimal: datasets may be small, may only track one

period of dynamics (typically early and late stage of spread) or may

be pooled from different locations. In this paper we discuss the

challenges associated with this approach. We used published data

on pyrethroid resistance from Aedes aegypti, throughout Mexico [6]

on the frequency of the Ile1,016 mutation, one of the mutations in

the voltage-gated sodium channel gene known to confer resistance

to pyrethroids (this is known as knockdown resistance, a term

applied to insects that fail to lose coordinated activity immediately

after exposure).

Model and Methods

The genetic model we employ assumes a single autosomal locus

conferring insecticide resistance in a diploid sexually reproducing

population, with non-overlapping generations and assuming

random mating; these are standard assumptions in population

genetics models. There are two possible alleles, resistant (R) or

susceptible (S), and three possible genotypes (SR, RR, SS). The

fitness coefficient, which is a measure of survival and reproduction

of the different genotypes, was defined as 1 for the susceptible

homozygotes SS, 1+s (s is the selection coefficient) for resistant

homozygotes RR and 1+hs (h is the dominance coefficient) for

heterozygotes SR. The level of dominance is a measure of the

relative position of the phenotype of the heterozygote relative to

the phenotype of the two corresponding homozygotes. Complete

dominance for susceptible allele is represented by h=0 and

complete dominance for resistance allele by h=1, alleles are

codominant or additive when h=0.5. The fitness coefficients are

composite measures of fitness in both the exposed and unexposed

mosquitoes groups and are assumed to be the same for males and

females.

We also assume a large population, so that genetic drift can be

ignored, which enabled us to predict the frequency of the resistant

allele at any time t according to the recursion expression:

ptz1~p2

t(1zs)zptqt(1zhs)

1zs(p2

tz2hptqt)

ð1Þ

Where:

pt: frequency of the resistant allele at time/generation t

qt=12pt: frequency of the susceptible allele at time/

generation t

This recursive equation is the basic formula of selection of a

favourable gene [7,8]. We defined the allele initial frequency p0, as

the frequency in the first sampling time point, set as generation 0.

Each subsequent generation can be converted onto a real

timescale of years by assuming a constant number of generations

per calendar year.

The ML approach to estimate the unknown parameters h and s

and p0, based on this genetic model, involved selecting initial

values of s, h, and p0, and then testing how well the predicted allele

frequencies matched those observed in the dataset.

Field datasets usually consist of the number of resistant alleles xt

and the total number of sampled alleles n at different time points t.

The probability of observing x resistant alleles among n alleles

follows a binomial distribution, with a probability of success (being

a resistance allele) p for each sampled time point t.

f(xtjnt,pt)~

nt

xt

??

pxt

t(1{pt)ntxt

ð2Þ

Where:

Figure 1. The typical change in insecticide concentration in the

field over time. As concentration decays with time after deployment

there is a differential survival of genotypes. In period A the RR genotype

will survive while the RS and SS dies: this makes the R allele recessive in

this period. In period B both RR and RS survive making the R allele

dominant in this period. In period C all genotypes can survive so no

selection occurs. These are windows of selection, adapted from [37].

doi:10.1371/journal.pntd.0001387.g001

Author Summary

The emergence and spread of insecticide resistance

compromise the control of mosquito borne diseases such

as dengue or malaria, which are responsible for millions of

deaths every year in tropical and subtropical areas. There

are currently no easily implemented methodologies to

quantify the strength of selection for resistance occurring

in nature. Using field data from Mexico on the frequency of

an allele mutation conferring resistance in the mosquito

Aedes aegypti we use maximum likelihood (ML) to estimate

the selection and dominance coefficients driving the

evolution of resistance. We explored the impact of poor

data collection, data that combine information from

different locations and the consequences of selection

and dominance coefficients varying over the sampling

time period. The ML method can accurately estimate these

parameters with simulated data in ideal sampling situa-

tions but it is difficult to recover true values when spatial

and temporal heterogeneity occurs. The analysis high-

lighted factors relevant to field work such as the need for

frequent surveillance in discrete sentinel sites.

Estimating Selection from Mosquito Field Data

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Page 3

pt: probability of sampling an R allele, i.e, probability of

a success

nt

xt

ways of sampling x resistant alleles among n total alleles

??

: combinatorial term to account for the number of

The corresponding binomial likelihood function is:

L(ptjxt,nt)~f(xtjnt,pt)

ð3Þ

The likelihood function returns the likelihood of the value ptgiven

the observed data of xtresistant alleles among the sample of ntat

each generation. Essentially, it tells us how consistent the data are

with predicted values of pt(Equation 1). The likelihood value for

the dataset is the product of the likelihoods across the entire

sample:

L pjx,n

ðÞ~ P

t

i~1

ni

xi

??

pxi

i

1{pi

ðÞni{xi

ð4Þ

We implemented this ML methodology in R [9] using constrOptim()

function (from stats package) for which it is not necessary to

provide analytic derivatives and that can minimize/maximize a

function subject to linear inequality constraints. Three constraints

on the parameter values were enforced: 0,p,1, 0,s,1, 0,h,1,

except when analysing the field data when a constraint on h (0–1.5)

was imposed. The Nelder-Mead optimization method algorithm

was used, that generates a new test position by extrapolating the

behavior of the objective function measured at each test point

arranged as a simplex. The algorithm then chooses to replace one

of these test points with the new test point and the algorithm

progresses. The simplest step is to replace the worst point with a

point reflected through the centroid of the remaining points. If this

point is better than the best current point, then it will expand

exponentially along this line. On the other hand, if this new point

is not much better than the previous value the simplex returns the

previous point. The standard error (s.e) of the estimates was

determined by inverting the Hessian matrix evaluated at the ML

estimate and the 95% confidence interval endpoints were

calculated as Parameter estimate61.96*s.e.

Maximum likelihood estimation is an optimization technique

and there is no guarantee that the set of parameters that uniquely

maximizes the likelihood will always be found because the

algorithm may converge onto local optima whose likelihood is

below the global maximum. To overcome this problem 1000 runs

of the ML iteration procedure were performed in every estimation,

with random starting values of the parameter estimates used to

initialise the optimization routine [10]. In the analyses described

here, the runs that converged to other estimates had ML values

sufficiently less than the global maximum that a likelihood ratio

test considered them different, so that the set of parameters could

be safely discarded. However a small percentage of the runs

converged to a set of different parameters with a similar likelihood

value that could not be considered different using a likelihood ratio

test. The criteria used to exclude these results as potential best

estimates was that the estimated value of h lay on the boundary of

the constrained parameter range and is expected to reflect erratic

behavior of the algorithm when using a small sample.

We tested the algorithm and program by analyzing 100 datasets

simulated under idealized conditions using Equations 1 and 2.

Initial frequency, dominance and selection coefficient were in the

ranges 0.01–0.04, 0.3–0.8 and 0.1 to 0.3 respectively, all

distributions were uniform. Three parameters values were selected

for each dataset and held constant during the simulation, i.e., there

was no temporal or spatial variation in parameter values and

populations sizes were sufficiently large that stochastic changes in

alleles frequencies could be ignored. Data were available for each

generation, 100 alleles (50 mosquitoes) were sampled each

generation (Equation 2) and the simulations were run until the

resistance allele frequency exceeded 0.99. Accuracy of analysis was

gauged by the correlation coefficient between true and estimated

parameter values, and by checking how frequently the true values

fell with the estimated 95% confidence intervals.

Next we examined the impact of suboptimal datasets. Equation

1 was used to predict allele frequency for 120 generations and we

assumed that 100 alleles were sampled in each generation. Two

optimal datasets with different dominance values were produced to

check if the ML method accurately recovered the parameters

when data from all generations was available (as above) and to

investigate the effect of different degrees of dominance on

estimations. Subsets of the data were used to examine the

influence of incomplete sampling when only a few generations of

data are available, or when only the initial stages of spread are

available for analysis.

Field data, collected and analyzed by Garcia et al. [6], was

available for analysis. There were a total of 78 field collections

containing 3,808 Aedes aegypti (some as much as 2000 km apart).

Each mosquito was genotyped at the Ile1,016 locus. We pooled

data from the different locations and analysed it assuming different

number of generations of mosquitoes per year (6,9,12,16 and 20),

to check the consistency of the estimations. Intuitively, we would

expect spatial and temporal variation in the selection parameter in

the Garcia et al. dataset and in many other datasets obtained under

field conditions. It was therefore vital to ascertain how heteroge-

neity would affect the algorithm’s ability to recover the underlying

parameters from pooled data. Spatial heterogeneity was investi-

gated by simulating allele frequencies for 80 different locations

over 50 generations using Equation 1; 100 alleles were sampled

from each generation (Equation 2) and data from each generation

in each location were used in the analyses. Parameters p0and h

were randomly selected from a uniform probability distribution

(p0,<(0,1), h,<(0,1)) while s was randomly drawn from a normal

distribution (s,N(0.15,0.025)), the constraints on s coefficient are

within a reasonable range for a field setting. Once selected for a

location, the values of h and s did not change, i.e., there was no

temporal heterogeneity. Two simulation strategies were used: (i) p0

and h were allowed to vary while s was held constant at 0.1, 0.3,

0.6, 0.8 or 1, (ii) p0, and s was allowed to vary while h was held

constant at 0, 0.25, 0.5. 0.75 or 1. The data across the simulated

locations were pooled for analyses. Each simulation strategy was

run 300 times giving a total of 30065=1500 per strategy. The

mean values of each parameter over all simulated locations was

assumed to be the true value and the accuracy of the program was,

as before, gauged by the correlation coefficient between the

estimated and true values, and by the proportion of the true values

falling within the 95% CI.

The effect of temporal heterogeneity in estimations was also

investigated by varying s and h over 50 generations in a single

location, i.e., different s and h values in different generations. The

distribution of values were the same as those used for spatial

heterogeneity. Three scenarios were considered: (i) s and h both

varied over generations, (ii) h could vary while s was held constant,

(iii) h could vary while s was held constant. In the simulations of

spatial heterogeneity the values of h and s had to be fixed across

locations (e.g. h=0, 0.25, 0.5. 0.75 or 1) but in the simulation of

temporal heterogeneity only one location was examined in each

simulation so the values could be drawn from the underlying

distributions. As before, 300 datasets were produced for each

Estimating Selection from Mosquito Field Data

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Page 4

scenario but because the fixed values of h and s could be drawn

from a distribution, the total number of runs was 30063=900. As

before, the performance of the algorithm under conditions of

temporal heterogeneity was assessed by defining the true value as

mean over the generations, and calculating the correlation

coefficient between the estimated and true values and how

frequently the true value was included in the 95% confidence

interval.

Finally, it is important to note two features of our analyses that

may not be obvious to non-specialists. Firstly, that the genetic

parameters h and s describe the overall, net rate of spread of

resistance alleles through natural populations and cannot formally

distinguish where selection is acting. For example, they cannot

determine whether selection was acting differentially on the adult

or larval stages, whether fitness costs were associated with

resistance, whether there was differential selection on the sexes,

nor whether killing was likely to be in early or later adult stages,

the latter being a topic of contemporary interest given recent

suggestion by Koella and colleagues [11] that killing older adults

will reduce the selective pressures for insecticide resistance.

Secondly, the analyses were designed to recover the genetic

parameters that resulted from past control program and, as such,

they cannot explore the issue of how differing patterns of

insecticide deployment drive resistance. This require a separate,

formal modelling approach explicitly designed to investigate the

differing impact of deployment strategies on driving resistance.

These analyses have been described elsewhere, particularly for the

agriculture pesticides [12–15].

Results

The analysis of idealized datasets (Table 1) suggest ML can

accurately recover the underlying parameter values from optimal

simulated data.

Figure 2 shows six example simulations of the increase in

resistance allele frequencies over 120 generations, under two

dominance conditions (semi-recessive, h=0.2 bottom panel, and

semi-dominant, h=0.8 top panel). Values of p0, s, and h appear in

Table 2. The program appears less accurate when analysing

subsets of the original data, particularly if the resistance allele is

semi-recessive. When the resistance allele was semi-dominant,

resistance increased rapidly and the true estimates were recovered

if the subset included points that captured the pattern of increase,

such as the subset 1. When the resistance allele was semi-recessive,

the frequency was maintained at low levels for a long period, the

true parameters values were either recovered (Subset 1) but within

confidence intervals that were so large as to be uninformative, or

were not even contained in the confidence intervals (Subset 2) even

with the inclusion of the last generation, the only sampling point in

the subset that captures the incipient frequency rise. The ML

parameter estimates in Table 2 were achieved in 34 to 83% of the

1000 ML runs indicating that a significant proportion of the

estimation routines converged onto local maxima.

Analysis of the Aedes aegypti dataset resulted in the parameter

estimates in Table 3. These ML estimations were obtained

assuming 6, 9, 12, 16 and 20 generations per year. The estimation

converged on the same ML value 76 to 96% of the runs. With a

small percentage of the runs (0.005 to 0.16%) converged to a set of

different parameters with a similar likelihood value but which were

excluded because the estimated value of h was on the boundary of

the constrained parameter range. The estimates of p0and h were

highly consistent irrespective of assumed number of generations

per year and ranged from 0.0032 to 0.0035 and from 0.77 to 0.78,

respectively. As expected the s was strongly dependent on the

assumed number of generations per year and ranged from 0.042 to

0.15.

Results from spatially heterogeneous datasets pooling data from

80 different locations are shown in Figures 3 and 4. The algorithm

appears unable to consistently obtain accurate estimations of the

parameters s and h under such heterogeneous settings, manifested

by low values of correlation coefficients and many true values

outside the 95% confidence interval of the estimate. For example,

with the dominance estimations in Figure 3 when selection was

constant at 0.6, only 12% of the true values fell within the

confidence interval. Initial frequency values were accurately

recovered in all simulated scenarios, possibly due to the the

recursion dependency on the initial frequency. However, the

estimation of selection and dominance coefficients was achieved

with very low values of correlation coefficients between the

estimates and the mean of the parameter over the 80 simulated

locations (not very precise), in all different hypothetical scenarios.

Additionally, if most of the values were in the confidence

interval, the mean range of the interval was as wide as the

parameter range. For example in Figure 4 note that when

dominance is constant at 0.75, 100% of the true values are in the

confidence interval, but the average mean range is 0.86 (the

parameter range is 0–1). The plotted simulated data and estimates

do not traverse the entire range of the parameters values because

they are the mean over the 80 locations, the central limit theorem

predicts that these estimates will converge to the center of the

distribution.

Simulations of a location with temporal heterogeneous selection

pressure (dominance and/or selection changing in every genera-

tion) are shown on Figure 5. Again, the model does not accurately

recover the true parameters under conditions of temporal

heterogeneity. The exception was the dominance parameter when

it was held constant in a particular location with selection varying

in each generation, the correlation coefficient between the

estimate and the mean dominance value over the 80 locations

was 0.86.

Discussion

Insecticide resistance research is largely focused on the

identification of the mechanisms responsible for resistance, and

whether the genetic mechanism is monogenic or polygenic,

general or population specific and if there are associated fitness

costs and developmental patterns [16]. The emergence and spread

of resistance is well documented, but there is still a worrying lack of

quantification of the evolution dynamics in populations under

control [17] and its persistence in populations following cessation

of control. The quantification of the strength of selection acting in

Table 1. Details of 100 idealized simulated datasets.

p0

hs

Parameter range0.01–0.04 0.2–0.80.1–0.3

r * 0.940.990.99

TV (%)* 9192 97

[ ]*0.021 0.0140.002

The simulated datasets were used to check the precision and accuracy of the

ML procedure.

*r correlation coefficient between original value and estimate, TV percentage of

true values in the estimates 95% confidence interval and [ ] mean range value of

the confidence interval.

doi:10.1371/journal.pntd.0001387.t001

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Page 5

the wild has previously been attempted using direct laboratory and

field trials, and indirect approaches using a variety of data,

including patterns of DNA variability and spatial and temporal

changes in allele frequencies [17,18]. Selection acting on

insecticide resistance genes in the field was first estimated using

genetic models for species in the genera Anopheles by Curtis et al.

[19] and Wood and Cook [20], both were based on the observed

changes in gene frequency over regular intervals and the latter also

discussed estimation by deviations from the expected Hardy-

Weinberg equilibrium frequencies. Both methods assumed a fixed

level of effective dominance under field conditions. A recent

example is the estimation of relative fitness by Livingston and

Fackler [21] for pyrethroid resistance in insects that infest crops. In

this case the magnitude of the estimates were similar to those

obtained using traditional laboratorial direct approaches using non

linear least squares estimation. The most refined work that we are

Figure 2. Simulated evolution of resistance allele frequency over 120 generations under two different scenarios of dominance

relationship and analysing the full dataset or subsets of data. Specifications in Table 2.

doi:10.1371/journal.pntd.0001387.g002

Table 2. Specifications of datasets of Figure 2.

True Estimates [95% CI]

Dataset Generationsp0

hsp0

hs

Complete1:120 0.001 0.20.20.0010 [0, 0.0018]0.17 [0.13, 0.38] 0.24 [0, 0.48]

0.001 0.80.20.0009 [0.0005, 0.0013]0.81 [0.77, 0.84]0.20 [0.19, 0.21]

Subset 1 1:5,15:18,116:120 0.0010.2 0.2 0.0025 [0, 0.0062]0.45 [0, 1.5]0.07 [0, 1]

0.0010.80.2 0.0016 [20.0017, 0.0049] 0.77 [0.36, 1.18]0.19 [0.08, 0.29]

Subset 2 1,13,25,37,73,85,97,1200.001 0.20.2 0.0014 [0, 0.0030]0.02 [0, 0.18]1.00 [0.96, 1]

0.0010.80.2 0.0009 [20.0007, 0.0025] 0.80 [0.64, 0.96]0.20 [0.16, 0.25]

Sampled generations and true parameter values and ML parameter estimates with respective 95% confidence intervals.

doi:10.1371/journal.pntd.0001387.t002

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aware of, quantifies selection coefficients and costs associated with

resistance for Culex pipiens in Southern France, using spatial

information from clines to estimate selective advantages and costs,

and temporal information from a long-term survey to estimate the

selection coefficients of alleles in each environment using a

standard ML estimation approach [22,23].

We have described a ML method for simultaneously estimating

the selection and dominance coefficients and an initial resistance

allele frequency similar to that of [5], but we also tackled the

effects of spatial and temporal differences in selection intensity that

can arise as a result of different strategies of deployment of the

insecticide, migration patterns and/or infrequent and sparse field

sampling of mosquitoes. The approach described in this paper was

accurate with simulated data but proved less robust when

analysing few intermediate allele frequencies, especially when the

resistance allele is recessive. The reason is that all resistance

dynamics start from the same point (very low frequency) and end

at the same point (very high frequencies) but in the absence of

intermediate time points it is impossible to reconstruct the

dynamics in between. If the sampling period covers only the

onset of resistance or the final stages, when resistance is close to

fixation, the accurate estimation of selection and dominance

coefficients can be difficult. The estimation is problematic because

in the early stages heterozygotes prevail in the population, with a

fitness Wrs=1+hs for which there are a range of values of h and s

that yield the same product hs. This is illustrated using subsets in

Figure 2. The true values of s and h were 0.2 and 0.2 but the

estimates were 0.45 and 0.07 (Table 2). The situation was even

worse for subset 2 of the data (Figure 2) where the analysis inferred

a completely different trajectory of resistance spread and the true

values of h=s=0.2 were estimated as h=0.02 and s=1.0

(Table 2). Once again, note that the fitness of the heterozygote

was estimated as 1+hs=1.02 which was relatively close to the true

value of 1.04 and that it is the predicted value of the homozygotes,

which were largely absent from this subset of the data, that were

badly estimated (as Wrr=2.0 rather than the true value of 1.2).

Nevertheless, the calculated fitness (1+hs) is very similar (1.04 and

1.03). On the other extreme, when resistance is almost fixed, there

will be mainly homozygotes in the dataset (with fitness Wrr=1+s),

so estimating a dominance value will also be problematic because

of the lack of heterozygotes (with fitness Wrs=1+hs). Unfortunate-

ly, this is a very common type of data where genetic surveys

initially indicate resistance was absent then, once its presence was

detected, a second survey was run and higher levels were detected.

Our analyses indicate that it is highly unlikely that any robust

genetic parameters can be obtained from these kind of fragmented

datasets. Future surveillance surveys should consequently be

optimized by choice of a proper sampling strategy and timeframe.

It is therefore of extreme importance to sample as many

generations as possible, even if it means collecting fewer

individuals. There is an important difference between standard

statistics and ML estimation. In standard statistics, the 95% CI

should capture the likely variation in magnitude of parameter

estimates. In ML it only captures the likely variation provided the

model has identified the correct trajectory of allele frequency

changes. This is problematic in incomplete datasets where many

trajectories may provide similar fits to the observed data. It is

absolutely essential to run numerous analyses from randomly

selected starting parameter values to check for the presence of

numerous trajectories of similar ML but with widely different

parameter estimates.

Pooling data from different locations can be seen as a

reasonable option to minimize the lack of sampled generations

and small sample size. The Ile1,016 mutation frequency dataset of

Garcia et al. [6] provided the opportunity to apply the model to

real field data. This data contains allele frequencies of mosquitoes

collected in 78 different locations around Mexico since 1999.

Insecticide use was not uniform across cities and towns in Mexico

and will probably differ between years and in addition migration

will probably lead to different initial resistance allele frequency.

The estimates obtained from simulated pooled data demonstrated

that this kind of data-pooling, which is probably inevitable in most

surveys, is not very robust. The coefficients reported in Table 3

should simply be recognized as a rough estimate between the years

1999 to 2008 and that they may vary, albeit by an unknown

amount, over time and space.

Equation 1 describes a highly idealized population, i.e., one

that is large, randomly mating, and homogenous in time and

space. It is therefore important to consider the extent to which

our population differs from this paradigm and what consequence

this may have for the results. A large population is required so

that we can ignore genetic drift, i.e., random fluctuations in allele

frequency around our predicted values. Drift is important in

laboratory studies (see [24] for discussion) but in natural

populations there is a consensus that genetic drift can be ignored

provided 4Ne~ s s.10 where Ne is effective population size [25] and

~ s s is the weighted mean fitness of the resistance heterozygotes and

homozygotes. Estimates of Ne provided by [26] for Aedes Aegypti

ranged from 10–22 in different regions of Mexico. These

estimates seem intuitively to be very small. The most likely

explanation is that they are measure of historical population size,

so may have been caused by founder effects and population

bottlenecks in the distant past. Estimates of contemporary

population sizes are more appropriate in the current context

and most estimates of contemporary effective population sizes of

vectors are much higher, for example, in the region of 1000+ for

Anopheles gambiae [27–29]. It would be possible to introduce the

effects of drift by simulating small populations sizes and sampling

(with replacement) the parents of the next generation. However

one of the key conclusions of this study is the difficulty of

Table 3. Estimated p0, h and s parameters from field data.

Parameter

Generations/

year

Best

value

95% Confidence

interval

p0

6 0.00320.00320.0032

9 0.0033 0.00330.0033

120.00340.0034 0.0034

16 0.00340.0034 0.0034

20 0.00350.00350.0035

h60.770.76 0.78

90.77 0.760.78

120.77 0.760.78

160.78 0.770.78

200.780.77 0.78

s60.15 0.140.16

90.096 0.0900.101

12 0.0710.0600.081

160.0530.048 0.057

200.0420.0380.046

The dataset corresponds to field collected data on Ile1,016 resistance allele

frequencies in Ae. Aegypti from Mexico. Assuming 6, 9, 12, 16 and 20

generations per year.

doi:10.1371/journal.pntd.0001387.t003

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Page 7

obtaining good quality estimates of genetic parameters from field

data, so we prefer to ignore the effects of drift, and simply point

out that the stochastic variation introduced by drift will likely

further decrease our ability to recover accurate genetic parameter

values from field data.

The second requirement, that mating occurs at random is

unlikely to be true given the geographical scale of our surveys. It

would be relatively straightforward to incorporate this effect by

including Wrights F statistics in Equation 1. However, there was

no evidence of significant departure from Hardy-Weinberg in our

Figure 3. Effect of spatial heterogeneity (pooled data from 80 simulated locations) on estimates of initial allele frequency,

dominance and selection parameters. The value of the selection coefficient was held constant at 0.1, 0.3, 0.6, 0.8 or 1 in all locations and in every

generation, hence there are five rows of results corresponding to each of the 5 values of the selection coefficient. Dominance (h,<(0,1)) varied

between simulated locations, but was constant over time within each location. The true value is the mean parameter value over all locations. The

Pearson correlation coefficient (r) is between estimated and true values. TV is the percentage of the true values that are included in the 95%

confidence interval of the estimate. [ ] is the mean width of the 95% confidence interval in all runs.

doi:10.1371/journal.pntd.0001387.g003

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Page 8

dataset (results not shown) so this strategy was not required. The

assumption that the population is homogenous in space is clearly

untrue. Pooling of data from different regions was required to

increase sample size and frequency because mosquito collections

were not uniform at the same location. The simulation results

demonstrate the dangers of this approach and work on malaria

vectors in Africa show unpredictably high levels of heterogeneities

in resistance even across relatively small distances [30]. As

mentioned by [5] simple models cannot account for the alteration

of selection pressure by long term changes in the environment.

More complex models that consider geographic clines and the

antagonist effect of selection-migration, should be more accurate,

Figure 4. Effect of spatial heterogeneity on estimates of initial allele frequency, dominance and selection parameters. The value of

the dominance coefficient was held constant at 0, 0.25, 0.5, 0.75 or 1 in all locations and in every generation, hence there are five rows of results

corresponding to each of the 5 values of the dominance coefficient. The value of the selection coefficient (s,N(0.15, 0.025)) varied between locations,

but was held constant over time in each location. The true value is the mean parameter value over all locations. The Pearson correlation coefficient (r)

is between estimated and true values. TV is the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is

the mean width of the 95% confidence interval in all runs.

doi:10.1371/journal.pntd.0001387.g004

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Page 9

but the amount of data necessary make the implementation

unlikely in most settings. This model in its simplicity presents a

straightforward way to obtain estimates of fitness parameters. The

fact that only information about resistant allele frequencies is

necessary should make it easier to apply, and yet even such a

simple data design is difficult to implement.

Nevertheless the estimated value for p0(0.0032–0.0035), was in

the higher range of 1022to 10213expected when a pesticide is

first introduced, based on mutation-selection equilibrium [31].

This initial p0value reflects the frequency prior to the sampling

period. Since 1950, vector control programs in Mexico have used

a series of insecticides. DDT was used extensively for indoor

Figure 5. Effect of temporal heterogeneity on estimates of initial allele frequency, dominance and selection coefficients

parameters. Three different scenarios were simulated: (A) dominance and selection are different in every generation, (B) selection coefficient was

held constant in all generations but dominance was allowed to vary, (C) dominance was held constant in all generations while the selection

coefficient was allowed to vary. The Pearson correlation coefficient (r) between estimate and true value is shown. TV refers to the percentage of the

true values that are included in the 95% confidence interval of the estimate. [ ] is the mean range of the 95% confidence interval in all runs.

doi:10.1371/journal.pntd.0001387.g005

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Page 10

house spraying from 1950–1960 and was still used in some

locations up until 1998. Malathion was later used for ultra-low

volume space spraying of wide areas from 1981 to 1999. In 2000,

programs switched to permethrin based insecticides [32]. The

spread of resistance genes in a treated region will depend on the

initial resistant allele frequency and it is known that resistance

development in pest organisms can occur within 5–100

generations [31]. The relatively high initial frequency estimated

explains the immediate, dramatic increase in frequencies of

Ile1,016 from the late 1990s to 2006–2008 [6] neglecting genetic

drift.

As expected, the strength of selection increased as the

number of generations per year decreased, whereas there was

less time to get to the same frequency of resistance allele.

Selection coefficients ranging from 0.042 to 0.053 (assuming 20

and 16 generations per year) are similar to the selection

coefficients of DDT and dieldrin resistant phenotypes in

Anopheles mosquitoes that have been previously estimated to

be on the order of 0.013–0.061 [16]. The values for 0.071 and

0.097 (12 and 9 generations per year) are in the range of what

was estimated for antimalarial drug resistance: 0.05–0.1 [33],

however the value of 0.147 for 6 generations was higher than

any previous estimates. This is the first time selection for

insecticide resistance has been quantified in this species and

should be seen as a preliminary estimate.

The estimated values of h, 0.77 to 0.78, point to partial

dominance of the resistance allele under field settings. Alleles

conferring knockdown resistance were found to be to be

recessive or semi-recessive in their influence in Anopheles gambiae

s.s. [34], but there is strong evidence for partial dominance or

additive effects of Ile1,016 from two laboratory studies of

knockdown and survival in strains or families of Aedes aegypti

segregating for the Ile1,016 allele. Saavedra-Rodriguez et al.

[35] found that 127 of 221 heterozygotes recovered from

permethrin knockdown and showed later [36] that when

considering overall survival the differences among the three

phenotypes appear additive. Dominance in the field is

dependent on the concentration and decay of the insecticide

(see Figure 1), under this situation the resistant allele will be

effectively dominant and we think that our results of

intermediate dominance of Ile1,016 reflect this effect [37].

This interpretation is supported by Roush and Tabashnik [31],

who reported the same situation of partial dominance for

cyclodienes and lindane, diazinon, malathion and also for

pyrethroids, where 20–60% of the heterozygotes survived

exposure in a field setting. There is ongoing debate about

differences between laboratory and field settings that extended

to the evolution of insecticide resistance itself, some suggesting

that resistance in the fields tends to be based on an allele of

major effect at a single locus whereas resistance obtained in the

laboratory is usually polygenically based [38]. Our results show

rapid selection of mutations at a single locus.

The number of generations under natural conditions for this

species was estimated at 20 or more among strains in field

conditions in Brazil, this leads us to consider the results with the

highest number of generations as the most likely, but because Aedes

aegypti eggs can survive desiccation for months and hatch once

submerged in water [39], the number of generations is variable.

Nevertheless, the predicted resistance frequency trajectory using

equation 1 and the different estimates obtained assuming different

generations per year will be approximately the same in a timescale

of 20 years.

Most mutations encoding insecticide resistance are expected to

incur a fitness penalty, compared to unmutated genes, in the

absence of insecticide. There is some field evidence of reduced

fitness of Ile1,016 mutations in Aedes aegypti in permethrin free

environments [6] which leads us to make two technical points.

Firstly, that the selection and dominance coefficients reported here

are overall values that combine the mutations benefit when

encountering insecticide and any fitness effect in insecticide-free

areas. Secondly, the method can equally be applied to measure

negative selection pressures, (i.e., when a mutation is being lost

from a population) from field data on the mutation after insecticide

is withdrawn.

Two factors are of particular relevance to field work. Firstly,

that surveillance needs to be continuous so that a full dataset

covering the whole period of resistance spread becomes available

upon which to base these estimates. This may mean monitoring

sentinel sites for long periods when resistance is rare or absent,

but a continuous dataset is a prerequisite for accurately

estimating the dynamics underlying the spread of resistance.

Note that a continuous dataset does not necessarily mean

collecting samples every generation. The reason the analysis

could fail to recover the true parameters (Table 2) was because of

large gaps in the survey: simulations of semi-dominant mutations

lacked samples from periods of intermediate frequency, while

simulations of semi-recessive mutations only contained data from

the early stages (Figure 2). Operationally, this suggests that

regular, rather than intensive but periodic, sampling is the best

strategy. As an example, we re-analysed the semi-dominant

dataset but just incorporated samples every 10 generation, i.e., at

generations 1, 10, 20, 30:120. This resulted in estimates of

p0=0.0006 (95% CI: 0.0001–0.0010 ), h=0.89 (95% CI: 0.84–

0.95), s=0.20 (95% CI: 0.18–0.21) which are similar to those

obtained using data from all generations (Table 2, the dominance

coefficient is higher but the confidence intervals overlap). The

second point is that dominance levels acting in the field may be

much higher than those observed in the laboratory. The most

plausible explanation is that mosquitoes in the wild are

encountering low levels of insecticide that are insufficient to kill

heterozygotes. Increasing dominance greatly increases the rate at

which resistance develops. This suggests that insecticide applica-

tions should be enforced in such a way that ensure high coverage

with high doses. Our results suggest that the doses being applied

may be inadequate and that pursuing the current deployment

settings will lead to the rapid increase of resistant mosquitoes and

eventually to the complete inefficiency of permethrin in the

combat of dengue in Mexico.

Acknowledgments

The authors would like to thank Hilary Ranson and Tiago Anta ˜o for

providing useful comments on earlier versions of the manuscript, and three

anonymous reviewers for helpful comments and discussion regarding the

manuscript.

Author Contributions

Conceived and designed the experiments: SB IH. Performed the

experiments: SB. Analyzed the data: SB IH. Contributed reagents/

materials/analysis tools: SB IH WCBIV. Wrote the paper: SB IH.

Estimating Selection from Mosquito Field Data

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Page 11

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