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Analytica Chimica Acta 706 (2011) 157163

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

journal homepage: www.elsevier .com/ locate /aca

Matrix-assisted laser desorption/ionization-mass spectrometry ofcuticular lipidprofiles can differentiate sex, age, and mating status ofAnophelesgambiaemosquitoes

Estrella Suarez a, Hien P. Nguyen a, Israel P. Ortiz a, KyuJong Lee c, Seoung Bum Kim c,,1,Jaroslaw Krzywinskib,,2, Kevin A. Schug a, ,3

a Department of Chemistry& Biochemistry, TheUniversity of Texas at Arlington, Arlington, TX,United Statesb VectorGroup, Liverpool School of Tropical Medicine, Liverpool, UKc Data Mining& Quality Management Lab, Schoolof Industrial Management Engineering, Korea University, Seoul,Republic of Korea

a r t i c l e i n f o

Article history:

Received 28 June 2011

Received in revised form 23 August 2011

Accepted 24 August 2011

Available online 31 August 2011

Keywords:

Malaria

Matrix-assisted laser desorption/ionization

Anopheles gambiae

Mosquito

Cuticular hydrocarbons

Cuticular lipids

Principal component analysisSupport vector machines

a b s t r a c t

Malaria is a devastating mosquito-borne disease, which affects hundreds ofmillions ofpeople each year.

It is transmitted predominantly by Anophelesgambiae, whose females must be >10 days old to become

infective. In this study, cuticular lipids from a laboratory strain ofthis mosquito species were analyzed

using a mass spectrometry method to evaluate their utility for age, sex and mating status differentia-

tion. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), in conjunction with an

acenaphthene/silver nitrate matrix preparation, was shown to be 100% effective in classifyingA.gambiae

females into 1, 710, and 14 days ofage. MALDI-MS analysis, supported by multivariate statistical meth-

ods, was also effective in detecting cuticular lipid differences between the sexes and between virgin and

mated females. The technique requires further testing, but the obtained results suggest that MALDI-MS

cuticular lipid spectra could be used for age grading ofA. gambiae females with precision greater than

with other available methods.

2011 Elsevier B.V. All rights reserved.

1. Introduction

Mosquitoes transmit devastating infectious diseases, such as

malaria, dengue, elephantiasis and yellow fever, which kill up to

three million people and debilitate hundreds of millions every

year [1]. Most of these clinical cases and deaths are attributable

to malaria in sub-Saharan Africa, where children under the age of

five and pregnant women are the primary victims; the mosquitoAnopheles gambiae is the main vector. Transmission of malaria

parasites occurs during repeated blood feeding by the Anopheles

Abbreviations: CL, cuticular lipids; PCA, principal component analysis; FDR-FS,

falsediscovery rate-based feature selection;DHB, 2,5-dihydroxy benzoic acid;SVM,

support vector machines. Corresponding author. Tel.: +82 02 32903397. Corresponding author. Tel.: +44 151 705 3155. Corresponding author. Tel.: +1 817 272 3541.

E-mail addresses: [email protected] (S.B. Kim),[email protected] (J. Krzywinski),

[email protected] (K.A. Schug).1 For Statistical Analysis.2 For Mosquito Biology.3 For Analytical Chemistry.

females, which require a protein-rich blood meal for egg devel-

opment. Males feed exclusively on sweet plant exudates and thus

have no role in spreading the disease.

Extensive control campaigns launched in an attempt to lessen

the enormous malaria burden have become increasingly inefficient

largely because of the emergence and spread of drug resistance

in pathogens and insecticide resistance in mosquito vectors. As a

result, the number of cases continues to be extremely high [1].

At present, no vaccine against malaria is available, and, thus far,

measures aiming to reduce humanvector contact, such as indoor

residual spraying with insecticides to kill mosquitoes entering

houses or use of insecticide impregnated bed nets, remain most

effective in decreasing transmission.

The insects body is covered by the cuticle, a hard exoskele-

ton providing support and protection from dehydration, ultraviolet

radiation, and bacterial and fungal pathogens [2]. The cuticle con-

sists of a number of layers, each with different physico-chemical

properties. The outermost waxy layer is composed of normal and

branchedsaturatedand unsaturated hydrocarbons, free fatty acids,

free alcohols, wax esters, glycerides, sterol esters, and aldehydes

[35]. Cuticular lipids (CL), in addition to protecting insects from

dessication [3], play a major role in species andmate recognition in

0003-2670/$ seefrontmatter 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.aca.2011.08.033
http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.aca.2011.08.033http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.aca.2011.08.033http://www.sciencedirect.com/science/journal/00032670http://www.elsevier.com/locate/acamailto:[email protected]:[email protected]:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.aca.2011.08.033http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.aca.2011.08.033mailto:[email protected]:[email protected]:[email protected]://www.elsevier.com/locate/acahttp://www.sciencedirect.com/science/journal/00032670http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.aca.2011.08.033


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158 E. Suarez et al./ AnalyticaChimica Acta 706 (2011) 157163

various insect groups [6]. Differences in CL composition, depending

on the femalemating status, suggest that these moleculesmay play

some role in chemical communication during mosquito courtship

[7]. Changes in the CL profiles also occur duringaging, and, in some

insect species, in response to seasonal alterations of the environ-

mental conditions[3,8]. Dependence of the lipidabundanceson age

has been the basis of one of the methods of female mosquito age

grading.

The age ofAnopheles females is of central importance to malaria

transmission. Because the malaria parasites must develop, multi-

ply, and invade salivary glands before transmission can occur, only

relatively old (>10 days) individuals become infectious [9]. Esti-

mation of mosquito age is therefore vital to the understanding of

malaria epidemiology. It allows estimation of mosquito survival

and response to control, and prediction of proportionof potentially

infectious females within population, which, combined, provides a

measure of local malaria risk.

The few available techniques of femaleAnopheles age-grading

include the analysis of dissected ovaries [10,11], analysis of CL pro-

files by GCMS [12,13], and near-infrared spectroscopy (NIRS) of a

whole individual [14]. They substantially differ in resolution, relia-

bility, ease of implementation, and amenability to high throughput

sample processing. Only the analysis of ovaries has been shown to

be 100% accurate. However, that technique, as well as the GCMSanalysis of lipids, is time consuming and allows only a coarse cat-

egorizing of females into


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E. Suarez et al./ AnalyticaChimica Acta 706 (2011) 157163 159

sonicated for 15min to ensure that the CL were mixed and that

residues were removed from the sides of the vial and concentrated

to the bottom of the vial. Again, the solution was dried under a

nitrogen stream. Lastly, another 15L of chloroform was added to

the glass vial containing the CL and labeled as the sample solution.

2.4. Sample preparation and replication

The samples for MALDI analysiswere prepared by premixingthe

CL extract solution (15L), squalene (15L) as internal standard,

acenaphthene (15L) or DHB (15L) as matrix, and silver nitrate

(7.5L) as cationization reagent prior to spotting. Following vor-

tex mixing,1-Laliquotsof themixture weredeposited three times

on selected target spots. The spots were allowed to air dry at ambi-

ent temperature, and the target was introduced into the MALDI

source for data collection. Two representative spectra per spot

were recorded. For each mosquito cohort, the procedure was repli-

cated three times using CL samples from different three-mosquito

pools. The replicate measurements were taken on different days

under the same conditions, totaling eighteen replicate spectra (3

spots/analysis2 spectra/spot3 replicate analyses on different

days) per cohort for statistical analysis.

2.5. Instrumentation

A Bruker-Daltonics Autoflex MALDI-TOF-MS instrument (Bil-

lerica, MA, USA) was used in reflectron mode to acquire the

spectra. Desorption/ionization was achieved using a nitrogen laser

(337nm). An extraction voltage of 20kV was used in the positive

ionization mode. Matrix ions were suppressed using a low mass

(m/z450480) cutoff in order to obtain better resolution for the

range of CL of interest. With acenaphthene as the matrix, the spec-

tra were obtained using 500 laser shots each, a laser energy of 57%,

and a pulsed ion extraction setting of 250 ns. 100% laser energy

corresponds to 87J pulse1. With DHB as the matrix, the spectra

were collected at optimal resolution with 300900 shots, a laser

energyof 48%, and a pulsed ion extraction of 150ns. Smoothed and

baseline-subtracted data were centroided and calibrated using iso-topic silver adducts of squalene. The calibration was carried out

based on the theoretical m/zvalues (517.2963, 519.296) for each

silver-adducted isotope. Data were collected and analyzed by Flex-

Analysis software running on a PC workstation.

2.6. Statistical analysis

PCA was used to facilitate the visualization of the high-

dimensional MS spectra and to identify the small subset of

important features (m/zvalues) to efficiently discriminate the dif-

ferent types of mosquitoes. The initial set of features was extracted

from mass list reported by the FlexAnalysis software across the

mass range collected (from400 to 1000m/z) during the analysis.

Signal intensities were binned (intensities of signals summed) tothe nearest 0.1m/zunit to obtain the initial set of features eval-

uated for statistical analysis. PCA is one of the most widely used

multivariate statistical methods for dimensionality reduction and

visualization [26]. PCA extracts a lower dimensional feature set,

which accounts for most of the variability of the original data set

throughthe linear transformation of theoriginal features.Extracted

features, called principal components (PCs), are uncorrelated with

each other andusually,the first fewPCs aresufficient to explain the

structure of the original data. Although PCA can significantly facili-

tate the visualization of high-dimensional spectral data by plotting

the samples with the reduced dimensions, interpretation of the

transformed features cannot be readily made because PCs are lin-

ear combinations of a larger number of the original features. To

overcome a limitation posed by the transformed features in PCA,

we used a feature selection approach based on weighted PCs. More

precisely, as shown Eq. (1), PCs are each a linear combination of

the original features with the corresponding coefficients, called

loadingsij (i,j=1, 2,. . ., p):

PC1 = 11X1 + 12X2 + + 1pXpPC2 = 21X1 + 22X2 + + 2pXp...

PCp = p1X1 + p2X2 + + ppXp

(1)

The loading values in each PC indicate the importance of the orig-

inal feature in the principal component domain. For instance, ijindicates the degree of significance of the jth feature in the ith PC.

Assuming that the first k PCs are sufficient to account for most of

the variability, a weighted PCA loading value for the jth original

feature can be computed by the following equation:

j =

z

i=1

|ij|i (j = 1,2, . . . , p) (2)

where z is the number of PCs of interest and i represents the

weight of the ith PC, which can be determined by the propor-

tion of total variance explained by the ith PC. Based on Eq. (2), afeature with a large value of indicates a significant feature. How-

ever, definition of how large is large is not obvious. To determine a

thresholdthatindicates thesignificanceof eachfeature, we usedEq.

(3), which is based on a moving range-based threshold technique

[27]:

t= +1(1 )

2, (3)

where is the average ofi and1 is the inverse cumulative stan-

dard normal distribution function. is the Type I error rate where

its range is between 0 and 1. can be estimated by the following

average of moving range between two successive observations.

MA =

i /=j

i j

p 1. (4)

Because there is no specific order of values, they are randomly

shuffled for H1000 times and the can be estimated by taking

the average ofHmoving average values.

=1

H

H

h=1

MAh. (5)

The estimated () is then fed into Eq. (6) and the final threshold

can be calculated as follows:

t

= +1

(1)

2 . (6)

Thus, we declare the feature xi significant if the corresponding

weighted PC (i) exceeds t*.

Once the set of important features was narrowed down, their

adequacy in terms of classification ability was evaluated. Thus,

feature selection results were validated using a classification algo-

rithm. In the present study, a support vector machines (SVM)

algorithm, one of the widely used classification methods that

can efficiently handle high-dimensional data, was used [28]. SVM

obtains a separating hyperplane by solving a convex optimiza-

tionproblem thatsimultaneously maximizes the geometricmargin

between the classes and minimizes the errors [29]. Nonlinear SVM

models can be established by incorporating the kernel functions

including polynomial, radial basis, and sigmoid functions.


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Table 1

Feature selection and classification results for MALDI-MS CL analysis methods.

MALDI-MS method Statistical method No. of features identified SVM classification accuracy (%)

Acenaphthene/AgNO3

Baseline 10,728 81.1 3.7

WPC + M R (= 0.01) 704 93.2 2.0

WPC + M R (= 0.10) 1084 93.0 2.0

DHB/AgNO3

Baseline 10,046 75.2 4.1

WPC + M R (= 0.01) 684 79.8 3.2

WPC + M R (= 0.10) 1208 80.1 2.9

Fig. 1. MALDI spectra of (A) oneday old virgin female with acenaphthenematrix/AgNO3 compared to (B)one day old virgin female with DHBmatrix/AgNO3.

3. Results and discussion

AseriesofCLspectrafromthe A. gambiaemosquitoesthat varied

with age, sex, and mating status, were measured by the optimizedmethods. The analysis incorporated variability in cohorts (3 sepa-

ratepools per cohort),time (measuredon 3 separate days), spotting

(3 spots plated per analysis), and ionization (2 spectra collected

per spot). The spectra were highly reproducible in each replicated

measurement of a given sample and highly consistent with regard

to signal presence and intensity among different samples from a

given mosquito cohort. Very similar results were obtained when

acenaphthene or DHB was used as the matrix, as exemplified by

spectra recorded from one-day-old virgin females, shown in Fig. 1.

Marked quantitativedifferenceswere observedin theCL profiles

originating from different mosquito cohorts. Because the whole

spectra consisted of over 10,000 features (m/zvalues of discernible

peaks), most of which were invariable in different cohorts, we

used statistical methods to identify a smaller subset of impor-tant discriminating features. The overall feature selection and

cross-validated classification results from SVM models are given

in Table 1.

The results showed thatthe number of features wassignificantly

reduced by the weighted PCA-based feature selection method pre-

sented in this paper. For example, in acenaphthene, the number of

features identified by the weighed PCA method (=0.01) is 704,

which amounts to only 7% of the total number of original features.

In order to evaluate the adequacy of the features selected, we used

an SVM algorithm with radial basis kernel functions. To compute

misclassification rates, we used a 10-fold cross-validation tech-

nique (a leave-one-out experiment) that splits 108 spectra into

10 groups. Each group contains 11 spectra, except for two groups

having 10 spectra. In each of 10 rounds, nine groups were used

for training a SVM model and the remaining group was used for

evaluating the constructed model based on its testing errors. The

overall cross-validated error rate of SVM was computed by taking

an average of testing errors obtained from these 10 rounds. Theclassification results showed that the SVM model constructed with

the features selected by the weighted PCA method yielded smaller

misclassification rates than those with all features (Table 1). This

demonstrated thatfeatureselection by a weightedPCA method was

adequate and that it successfully removed redundant features and

improved overall classification accuracy. While the classification

accuracy with each matrix method was high, a clearly higher per-

formance was obtained with the acenaphthene matrix preparation

and analysis.

Representative spectra recorded from different mosquito

cohorts using the acenaphthene matrix are displayed in

Figs. 2 and 3. The spectra in each figure have been normal-

ized to the same m/z and absolute intensity scales to facilitate

visual comparison of changes in various signals. Although, asdescribed above, a relatively large number of features significantly

vary between the cohorts, relative intensities of only a few signals

facilitated differentiation of each group presented in Figs. 2 and 3.

Fig. 2 shows representative spectra recorded from one-day-old

virgin females and seven to ten-day-old mated females. The spec-

tra were very similar for both groups, but for some compounds,

there were notable quantitative differences between them. In par-

ticular, in the latter group, an approximately 3-fold increase in the

signal intensity was found for signals at m/z 570 and 655660,

roughly corresponding to lipids C41 and C46C47. Based on the

GCMS analysis of CL fromA. gambiae, Caputo et al. [13] reported

an age-dependent increase in the amounts of four hydrocarbons

(n-C29, n-C31 and the centrally branched C31 and C33), but also a

trend of decrease in concentration of several other CL, in accord


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Fig. 2. MALDI spectra of (A) one day old virgin female compared to (B) seven-to-ten day old mated female both with acenaphthene as matrix and AgNO3 as cationization

reagent.

Fig. 3. MALDI spectra of (A) oneday old virgin females, (B)seven-to-ten day old mated females, and C) fourteen-day-old virgin females, all with acenaphthene/AgNO3.

with anearlier study byPolerstock et al.[7]. We alsoobservedsome

decrease in the levels of lower molecular weight CL in 710 days

old mated females, but the decrease became evident when the 14

days oldfemales were included in comparison (Fig.3; see signals at

m/z535545). Unexpectedly, in the oldest group we noticed a dra-matic increase in abundance of lipids atm/z670680 and 700710

(approximately, C48 and C51). Importantly, these dramatic changes

pertain to lipids with molecular weights that are beyond the limits

of range of the GCMS detection.

CL profiles inA. gambiae females have been reported to change

after mating, which led to a decrease in levels of low-abundance

n-henicosane (C21) and n-tricosane (C23) [7]. In our study we ana-

lyzed only mated 710 days oldindividuals. Thus, it is unclear if the

changes observed in the CL spectra from females of that cohort (as

compared to one day old virgin females) are related to aging or to

an altered mating status. However, further comparisons with the

14days oldvirgin females (Fig. 3) allowed us to untanglethe signals

with a certain amount of confidence. Of the two peaks, for which

signal increase in the 710 days old mated females was discussed

above, one (atm/z570) was almost absent in both the one day old

and the 14 days old virgin females, suggesting that change in the

intensity of the features at m/z570 is related to mating.

The comparison between the CL profile of mated males and

mated females is highlighted in Fig. 4. Consistent with the previ-ous studies based on GCMS [10], no gender-specific peaks were

found, butcertainquantitative differences wereobserved. The most

pronounced was a strong signal in males at m/z 770 (relative to

the neighboring peaks at m/z750760) and a very weak signal for

the corresponding hydrocarbon in females. This difference was not

reported in earlier studies, because the mass of the discriminating

CL is beyond the detection range of the GCMS.

Three-dimensional PCA score plots of all 108 spectra col-

lected using acenaphthene and DHB as matrix are displayed in

Figs. 5 and 6, respectively. In both data sets, all female samples

were well segregated into their respective age groups, but in the

spectra collected with acenaphthene matrix, the segregation was

more evident and each agegroupformed a relatively compact clus-

ter. Differences between male CL were less pronounced; however,


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Fig. 4. MALDI spectra of (A) seven-to-tenday old mated females and (B)seven-to-ten day old mated males with acenaphthene/AgNO3.

Fig. 5. 3D PCA score plot for the analysis ofA. gambiae cuticular lipids using MALDI-MS with a acenaphthene/AgNO3 matrix. Collections of points corresponding to female

cohorts areindicateswith thicker lines.

Fig. 6. 3DPCA score plotfor the analysisofA. gambiae cuticular lipidsusing MALDI-MS with a DHB/AgNO3 matrix. Collections of points corresponding to female cohorts are

indicates with thicker lines.


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in the majority of cases, young males could be distinguished from

the 7 days old or older individuals. Fig. 5 also shows a clear sepa-

ration of older males from females, while in 1 day old individuals,

the male and female spectra broadly overlapped.

Suitability of MALDI mass spectrometry for the analysis of com-

plex lipid mixtures from the insect cuticular waxy layer has been

demonstratedby Cvackaand coworkers [17,19,24]. We usedamod-

ified approach (silver ionization and acenaphthene as matrix) in

this study. The capability of this modified method for visualizing

lipids is evidenced by the intense signal for the squalene inter-

nal standard in all spectra (see e.g., Fig. 2; squalene signal marked

with an asterisk). Additional controls to ensure reproducible sig-

nal production for high molecular weight saturated hydrocarbons,

pentacontane (C50) and hexacontane (C60) standards (along with

appropriate blanks), were run extensively throughout the data col-

lection scheme (data not shown). In all the measured samples,

we detected high molecular weight lipids that were not reported

in mosquitoes before. The largest lipids identified corresponded

to those with approximately 7071 carbon atoms, while those

detected by GCMS had main chain lengths up to a maximum of

C47 [10].

While it is impossible for this method to discern the extent of

branching, saturation, or functionalization on the high molecular

weight lipid signals, broad envelopes of varying intensity for lipidsC47C55 in the m/z range 650760, and for lipids C59C70 in the

m/zrange 820970, were observed. In most cases, signals for dif-

ferent lipids incorporated a collection of features. This could be

attributed to the significant abundances of the two silver cationiza-

tion reagent isotopes, as well as differences in levels of branching,

saturation, or functional groups in each compound around a given

molecular weight. Some interferences from silver clusters were

apparent. These were clearly visualized in various blank analy-

sis experiments. The m/z ranges for the silver clusters can also

be easily calculated, and were observed (in the regions relevant

to the hydrocarbon signals of interest) at m/z534535, 747762,

861868, 962975, and 11771194.

4. Conclusions

Our study is the first to apply MALDI-TOF to the analysis

of cuticular lipids from mosquitoes. It allowed a much deeper

insight into the diversity of CL fromA. gambiae, than gained with

GCMS approaches used previously for this major malaria vec-

tor species. We detected a number of previously undiscovered

high molecular weight compounds in the mass range beyond the

capabilities of GCMS instruments. The incorporation of a silver

nitrate cationization/chemicalionization reagent facilitatedMALDI

analysis. Further studies to examine the use of atmospheric pres-

sure chemical ionization (APCI) techniques on instruments with a

mass range beyond that whichis available for GCMS analysis, and

potentially incorporating liquid phase separations, may be viable

for enhanced qualitative analysis in future studies.More importantly, we discovered dramatic age-related changes

that may allow unequivocal differentiating between mosquito

females old enough to potentially carry infective malaria para-

sites and younger females whose bites are non-infective. The CL

profiles may also allow further distinguishing within the latter

groupbetweenyoung and710daysold females. Here weanalyzed

spectra of CL sampled from pools of three mosquitoes, but the

intensity of the relevant signals indicates that single mosquitoes

should provide sufficient amounts of CL for age grading. Com-

pared to all the proposed age grading methods, MALDI-MS is

least demanding with regard to mosquito sample preservation

(mosquitoes can be dried) and potentially more accurate.However,

it shouldbe borne in mind that ourstudywas based on only a single

strain ofA. gambiae reared in a controlled laboratory environment.

Therefore, further research is necessary to establish whether the

age-related differences in CL profiles hold for samples from other

strains of different geographical origins and for individuals from

wild populations reared in semi-natural conditions. If validated,

these results would constitute a vast improvement in our ability to

estimate the age ofA. gambiae.

Acknowledgement

Support is acknowledged from the UT Arlington Research

Enhancement Program.

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