Analytica Chimica Acta 706 (2011) 157163

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Analytica Chimica Acta

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Matrix-assisted laser desorption/ionization-mass spectrometry of cuticular lipid proles can differentiate sex, age, and mating status of Anopheles gambiae mosquitoes
Estrella Suarez a , Hien P. Nguyen a , Israel P. Ortiz a , Kyu Jong Lee c , Seoung Bum Kim c,,1 , Jaroslaw Krzywinski b,,2 , Kevin A. Schug a, ,3
a

Department of Chemistry & Biochemistry, The University of Texas at Arlington, Arlington, TX, United States Vector Group, Liverpool School of Tropical Medicine, Liverpool, UK c Data Mining & Quality Management Lab, School of Industrial Management Engineering, Korea University, Seoul, Republic of Korea
b

a r t i c l e

i n f o

a b s t r a c t
Malaria is a devastating mosquito-borne disease, which affects hundreds of millions of people each year. It is transmitted predominantly by Anopheles gambiae, whose females must be >10 days old to become infective. In this study, cuticular lipids from a laboratory strain of this mosquito species were analyzed using a mass spectrometry method to evaluate their utility for age, sex and mating status differentiation. 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 classifying A. gambiae females into 1, 710, and 14 days of age. MALDI-MS analysis, supported by multivariate statistical methods, 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 of A. gambiae females with precision greater than with other available methods. 2011 Elsevier B.V. All rights reserved.

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 analysis Support vector machines

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 ve and pregnant women are the primary victims; the mosquito Anopheles 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, false discovery rate-based feature selection; DHB, 2,5-dihydroxy benzoic acid; SVM, support vector machines. Corresponding author. Tel.: +82 02 3290 3397. Corresponding author. Tel.: +44 151 705 3155. Corresponding author. Tel.: +1 817 272 3541. E-mail addresses: sbkim1@korea.ac.kr (S.B. Kim), jarek@liv.ac.uk (J. Krzywinski), kschug@uta.edu (K.A. Schug). 1 For Statistical Analysis. 2 For Mosquito Biology. 3 For Analytical Chemistry. 0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.08.033

females, which require a protein-rich blood meal for egg development. 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 inefcient 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 exoskeleton providing support and protection from dehydration, ultraviolet radiation, and bacterial and fungal pathogens [2]. The cuticle consists of a number of layers, each with different physico-chemical properties. The outermost waxy layer is composed of normal and branched saturated and 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 and mate recognition in

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various insect groups [6]. Differences in CL composition, depending on the female mating status, suggest that these molecules may play some role in chemical communication during mosquito courtship [7]. Changes in the CL proles also occur during aging, and, in some insect species, in response to seasonal alterations of the environmental conditions [3,8]. Dependence of the lipid abundances on age has been the basis of one of the methods of female mosquito age grading. The age of Anopheles females is of central importance to malaria transmission. Because the malaria parasites must develop, multiply, and invade salivary glands before transmission can occur, only relatively old (>10 days) individuals become infectious [9]. Estimation 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 proportion of potentially infectious females within population, which, combined, provides a measure of local malaria risk. The few available techniques of female Anopheles age-grading include the analysis of dissected ovaries [10,11], analysis of CL proles by GCMS [12,13], and near-infrared spectroscopy (NIRS) of a whole individual [14]. They substantially differ in resolution, reliability, 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 GCMS analysis of lipids, is time consuming and allows only a coarse categorizing of females into <4 days of age and older, or <2 days of age and >4 days of age, which is not optimal, when attempting to evaluate a proportion of infective mosquitoes in a population. NIRS performs better; it is fast and enables differentiation between <7 days old and >7 days old females, although the spectra from individuals of different ages are broadly overlapping, leaving substantial room for improvement in accuracy. Two recent studies proposed a quantitative analysis of transcript levels of marker genes responsive to aging [15,16]. Performance of that approach is yet to be seen, but its efcacy may be compromised by a rigorous requirement for a non-degraded RNA, which is rarely achievable during mosquito eld collections. A fundamental limitation of GCMS is the molecular weight range of lipids that can be separated and detected. The analysis of compounds with higher molecular weight requires special instrumentation, but the high temperature necessary to vaporize long-chain molecules may itself destabilize the samples [17]. In contrast, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) offers an extended range of mass analysis when coupled to time-of-ight detection (TOF) and may be a suitable alternative technique to study the mosquito CL [18,19]. However, as extracted mixtures of lipids are expected to contain a signicant amount of unfunctionalized hydrocarbons, the absence of heteroatom-based functional groups can lead to poor protonation, cationization, and/or anionization prior to mass spectrometry detection. To address this problem, researchers have shown that the incorporation of transition metals during sample preparation can lead to effective cationization of hydrocarbons with laser desorption/ionization (LDI) [20,21]. Additionally, with silver nitrate as a cationization reagent, LDI-MS has been successfully applied to ngerprint hydrocarbons in a set of petroleum (crude oil) samples [22]. In that work, multivariate statistical methods, including principal component analysis (PCA) [23] and false discovery ratebased feature selection (FDR-FS), were used to extract information from the mass spectra to discriminate between the sample classes. Cva cka et al. have also used MALDI-MS and preparations of lithium dihydroxybenzoate matrices in order to ngerprint lipids extracted from the cuticle of a variety of insects [17,24]. In this work, MALDI-MS was used for ngerprinting the cuticular lipids of A. gambiae mosquitoes. The objective of the study was to test whether lipid proles generated by MALDI-MS could

distinguish different age groups and mating status of males and females of this species. Silver nitrate was used as a cationization reagent, in combinations with 2,5-dihydroxybenzoic acid (DHB) or acenaphthene as traditional MALDI matrices. The data were rigorously evaluated using a weighted PCA algorithm for dimensionality reduction and a support vector machines algorithm for classication. The method effectively discriminated between different cohorts of mosquito adults with high precision, accuracy, and repeatability. As such, our data demonstrate that MALDI-MS-based cuticular lipid analysis provides a very promising way of evaluating age structure of A. gambiae populations, which is necessary for effective monitoring of malaria control efforts. 2. Experimental 2.1. Mosquitoes A. gambiae G3 laboratory strain was used in the experiments. Mosquitoes were reared according to established procedures [25]. For the virgin cohort, they were sorted according to sex at the pupal stage; males and females were kept in separate cages. All mosquitoes were provided with 10% sucrose solution ad libitum. At day 1, days 710, and day 14 from eclosion, they were collected with an aspirator, quickly killed by ash freezing at 80 C, put in tubes containing silica gel, and kept in a desiccator until CL analysis. 2.2. Chemicals and materials Two different matrices, acenaphthene (C12 H10 : FW = 154.21 g mol1 ) and 2,5-dihydroxybenzoic acid (DHB: FW = 154.12 g mol1 ) (SigmaAldrich, St. Louis, MO, USA), were prepared at concentrations of 0.1 M and 0.01 M in tetrahydrofuran (THF), respectively. Solvents used for overall sample preparation, including chloroform (EMD Chemicals, Inc., Gibbstown, NJ, USA), methanol (Mallinckrodt-Baker, Inc., Phillipsburg, NJ, USA), and THF (Merck, Darmstadt, Germany), were analytical grade. Pentacontane (C50 ) and hexacontane (C60 ), used as high molecular weight control standards to guide method development, were from SigmaAldrich. Squalene (C30 H50 : MW = 410.7 g mol1 ), minimum 98% purity (SigmaAldrich), was added to each sample as the internal standard for calibration purposes at a concentration of 0.256 mM and 1.023 mM in chloroform for use with the DHB and acenaphthene matrices, respectively. Silver nitrate puriss (SigmaAldrich Laborchemikalien, Riedel-de-Haen, Germany) at a concentration of 3 mg mL1 and 4 mg mL1 in MeOH for DHB and acenaphthene preparations, respectively, was used as the cationization reagent. An uncoated polished stainless steel MALDI sample target plate (100 positions; Bruker Daltonics, Billerica, MA) was washed stepwise with methanol, chloroform, and 2% nitric acid (VWR International, West Chester, PA, USA) to remove contaminations, and air-dried, prior to sample spotting. 2.3. Hydrocarbon extraction Since hexane as a solvent has proven to have a limited ability to extract long-chain hydrocarbons and lipids from complex matrices, CHCl3 , which can effectively dissolve the entire wax layer on the insect cuticle, was selected for lipid extractions [17]. Three mosquitoes from the same cohort (of the same age, sex and mating status) were carefully placed in a 5-mL glass vial with 100 L of chloroform and gently swirled for 5 min to extract the CL. Care was taken not to break any appendages. Subsequently, the chloroform was removed (without touching the mosquitoes; via a thin syringe needle maneuvered between the mosquitoes), transferred into a 1-mL glass vial, and evaporated under a gentle nitrogen gas stream. Chloroform (15 L) was added to the glass vial and then

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sonicated for 15 min 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 15 L 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 analysis were prepared by premixing the CL extract solution (15 L), squalene (15 L) as internal standard, acenaphthene (15 L) or DHB (15 L) as matrix, and silver nitrate (7.5 L) as cationization reagent prior to spotting. Following vortex mixing, 1-L aliquots of the mixture were deposited three times on selected target spots. The spots were allowed to air dry at ambient 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 replicated 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/analysis 2 spectra/spot 3 replicate analyses on different days) per cohort for statistical analysis. 2.5. Instrumentation A Bruker-Daltonics Autoex MALDI-TOF-MS instrument (Billerica, MA, USA) was used in reectron mode to acquire the spectra. Desorption/ionization was achieved using a nitrogen laser (337 nm). An extraction voltage of 20 kV was used in the positive ionization mode. Matrix ions were suppressed using a low mass (m/z 450480) cutoff in order to obtain better resolution for the range of CL of interest. With acenaphthene as the matrix, the spectra 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 87 J pulse1 . With DHB as the matrix, the spectra were collected at optimal resolution with 300900 shots, a laser energy of 48%, and a pulsed ion extraction of 150 ns. Smoothed and baseline-subtracted data were centroided and calibrated using isotopic silver adducts of squalene. The calibration was carried out based on the theoretical m/z values (517.2963, 519.296) for each silver-adducted isotope. Data were collected and analyzed by FlexAnalysis software running on a PC workstation. 2.6. Statistical analysis PCA was used to facilitate the visualization of the highdimensional MS spectra and to identify the small subset of important features (m/z values) to efciently discriminate the different types of mosquitoes. The initial set of features was extracted from mass list reported by the FlexAnalysis software across the mass range collected (from 400 to 1000 m/z) during the analysis. Signal intensities were binned (intensities of signals summed) to the nearest 0.1 m/z unit to obtain the initial set of features evaluated 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 through the linear transformation of the original features. Extracted features, called principal components (PCs), are uncorrelated with each other and usually, the rst few PCs are sufcient to explain the structure of the original data. Although PCA can signicantly facilitate 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 linear 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 coefcients, called loadings ij (i,j = 1, 2,. . ., p): PC1 = 11 X1 + 12 X2 + + 1p Xp PC2 = 21 X1 + 22 X2 + + 2p Xp . . . PCp = p1 X1 + p2 X2 + + pp Xp

(1)

The loading values in each PC indicate the importance of the original feature in the principal component domain. For instance, ij indicates the degree of signicance of the jth feature in the ith PC. Assuming that the rst k PCs are sufcient to account for most of the variability, a weighted PCA loading value for the jth original feature can be computed by the following equation:
z

j =
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 proportion of total variance explained by the ith PC. Based on Eq. (2), a feature with a large value of indicates a signicant feature. However, denition of how large is large is not obvious. To determine a threshold that indicates the signicance of each feature, we used Eq. (3), which is based on a moving range-based threshold technique [27]: + 1 (1 ) t= 2 , (3)

is the average of i and 1 is the inverse cumulative stanwhere 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. i j MA =
i= / j

p1

(4)

Because there is no specic order of values, they are randomly shufed for H 1000 times and the can be estimated by taking the average of H moving average values. = 1 H
H

MAh .
h=1

(5)

The estimated ( ) is then fed into Eq. (6) and the nal threshold can be calculated as follows: + 1 (1 ) t = 2 . (6)

Thus, we declare the feature xi signicant if the corresponding weighted PC (i ) exceeds t*. Once the set of important features was narrowed down, their adequacy in terms of classication ability was evaluated. Thus, feature selection results were validated using a classication algorithm. In the present study, a support vector machines (SVM) algorithm, one of the widely used classication methods that can efciently handle high-dimensional data, was used [28]. SVM obtains a separating hyperplane by solving a convex optimization problem that simultaneously maximizes the geometric margin 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 classication results for MALDI-MS CL analysis methods. MALDI-MS method Acenaphthene/AgNO3 Statistical method Baseline WPC + MR ( = 0.01) WPC + MR ( = 0.10) Baseline WPC + MR ( = 0.01) WPC + MR ( = 0.10) No. of features identied 10,728 704 1084 10,046 684 1208 SVM classication accuracy (%) 81.1 93.2 93.0 75.2 79.8 80.1 3.7 2.0 2.0 4.1 3.2 2.9

DHB/AgNO3

Fig. 1. MALDI spectra of (A) one day old virgin female with acenaphthene matrix/AgNO3 compared to (B) one day old virgin female with DHB matrix/AgNO3 .

3. Results and discussion A series of CL spectra from the A. gambiae mosquitoes that varied with age, sex, and mating status, were measured by the optimized methods. The analysis incorporated variability in cohorts (3 separate pools per cohort), time (measured on 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 exemplied by spectra recorded from one-day-old virgin females, shown in Fig. 1. Marked quantitative differences were observed in the CL proles originating from different mosquito cohorts. Because the whole spectra consisted of over 10,000 features (m/z values of discernible peaks), most of which were invariable in different cohorts, we used statistical methods to identify a smaller subset of important discriminating features. The overall feature selection and cross-validated classication results from SVM models are given in Table 1. The results showed that the number of features was signicantly reduced by the weighted PCA-based feature selection method presented in this paper. For example, in acenaphthene, the number of features identied 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 misclassication rates, we used a 10-fold cross-validation technique (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. The classication results showed that the SVM model constructed with the features selected by the weighted PCA method yielded smaller misclassication rates than those with all features (Table 1). This demonstrated that feature selection by a weighted PCA method was adequate and that it successfully removed redundant features and improved overall classication accuracy. While the classication accuracy with each matrix method was high, a clearly higher performance 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 gure have been normalized to the same m/z and absolute intensity scales to facilitate visual comparison of changes in various signals. Although, as described above, a relatively large number of features signicantly 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 spectra were very similar for both groups, but for some compounds, there were notable quantitative differences between them. In particular, 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 C46 C47 . Based on the GCMS analysis of CL from A. 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) one day old virgin females, (B) seven-to-ten day old mated females, and C) fourteen-day-old virgin females, all with acenaphthene/AgNO3 .

with an earlier study by Polerstock et al. [7]. We also observed some decrease in the levels of lower molecular weight CL in 710 days old mated females, but the decrease became evident when the 14 days old females were included in comparison (Fig. 3; see signals at m/z 535545). Unexpectedly, in the oldest group we noticed a dramatic increase in abundance of lipids at m/z 670680 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 proles in A. 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 analyzed only mated 710 days old individuals. 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 14 days old virgin females (Fig. 3) allowed us to untangle the signals with a certain amount of condence. Of the two peaks, for which signal increase in the 710 days old mated females was discussed

above, one (at m/z 570) 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/z 570 is related to mating. The comparison between the CL prole of mated males and mated females is highlighted in Fig. 4. Consistent with the previous studies based on GCMS [10], no gender-specic peaks were found, but certain quantitative differences were observed. The most pronounced was a strong signal in males at m/z 770 (relative to the neighboring peaks at m/z 750760) 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 collected 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 age group formed a relatively compact cluster. Differences between male CL were less pronounced; however,

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

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

Fig. 6. 3D PCA score plot for the analysis of A. gambiae cuticular lipids using 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 separation 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 complex lipid mixtures from the insect cuticular waxy layer has been cka and coworkers [17,19,24]. We used a moddemonstrated by Cva ied approach (silver ionization and acenaphthene as matrix) in this study. The capability of this modied method for visualizing lipids is evidenced by the intense signal for the squalene internal standard in all spectra (see e.g., Fig. 2; squalene signal marked with an asterisk). Additional controls to ensure reproducible signal production for high molecular weight saturated hydrocarbons, pentacontane (C50 ) and hexacontane (C60 ) standards (along with appropriate blanks), were run extensively throughout the data collection 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 identied 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 lipids C47 C55 in the m/z range 650760, and for lipids C59 C70 in the m/z range 820970, were observed. In most cases, signals for different lipids incorporated a collection of features. This could be attributed to the signicant abundances of the two silver cationization 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 analysis 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/z 534535, 747762, 861868, 962975, and 11771194. 4. Conclusions Our study is the rst to apply MALDI-TOF to the analysis of cuticular lipids from mosquitoes. It allowed a much deeper insight into the diversity of CL from A. gambiae, than gained with GCMS approaches used previously for this major malaria vector 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/chemical ionization reagent facilitated MALDI analysis. Further studies to examine the use of atmospheric pressure chemical ionization (APCI) techniques on instruments with a mass range beyond that which is 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 parasites and younger females whose bites are non-infective. The CL proles may also allow further distinguishing within the latter group between young and 710 days old females. Here we analyzed

spectra of CL sampled from pools of three mosquitoes, but the intensity of the relevant signals indicates that single mosquitoes should provide sufcient amounts of CL for age grading. Compared 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 should be borne in mind that our study was based on only a single strain of A. gambiae reared in a controlled laboratory environment. Therefore, further research is necessary to establish whether the age-related differences in CL proles 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 of A. gambiae. Acknowledgement Support is acknowledged from the UT Arlington Research Enhancement Program. References
[1] J.G. Breman, M.S. Alilio, A. Mills, Am. J. Trop. Med. Hyg. 71 (2004) 115. [2] R.W. Howard, G.J. Blomquist, Annu. Rev. Entomol. 50 (2005) 371393. [3] R.H. Hackman, in: J. Bereiter-Hahn, A.G. Matoltsy, K.S. Richards (Eds.), Biology of the Integument Volume 1: Invertebrates, Springer-Verlag, 1984, pp. 583610. [4] K.H. Lockey, Comp. Biochem. Physiol. 89 (1988) 595645. [5] D.R. Nelson, G.J. Blomquist, in: R.J. Hamilton (Ed.), Waxes: Chemistry, Molecular Biology and Functions, The Oily Press, 1995, pp. 190. [6] T.L. Singer, Am. Zool. 38 (1998) 394405. [7] A.R. Polerstock, S.D. Eigenbrode, M.J. Klowden, J. Med. Entomol. 39 (2002) 545552. [8] D.R. Nelson, R.E. Lee Jr., Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 138 (2004) 313320. [9] J.C. Beier, Ann. Rev. Entomol. 43 (1998) 519543. [10] T. Detinova, Monogr. Ser. World Health Organ. 47 (1962) 13191. [11] M.A. Gillies, Ann. Trop. Med. Parasitol. 52 (1958) 261273. [12] B. Caputo, F.R. Dani, G.L. Horne, S. NFale, A. Diabate, S. Turillazi, M. Coluzzi, C. Costantini, A.A. Priestman, V. Petrarca, A. della Torre, Insect Biochem. Mol. Biol. 37 (2007) 389398. [13] B. Caputo, F.R. Dani, G.L. Horne, V. Petrarca, S. Turillazzi, M. Coluzzi, A.A. Priestman, A. della Torre, J. Mass Spectrom. 40 (2005) 15951604. [14] V.S. Mayagaya, K. Michel, M.Q. Benedict, G.F. Killeen, R.A. Wirtz, H.M. Ferguson, F.E. Dowell, Am. J. Trop. Med. Hyg. 81 (2009) 622630. [15] P.E. Cook, S.P. Sinkins, Insect Mol. Biol. 19 (2010) 745751. [16] M.H. Wang, O. Marinotti, A.A. James, E. Walker, J. Githure, G. Yan, PLoS One 5 (2010) e13359. cka, P. Jiro s, J. Sobotnk, R. Hanus, A. Svato s, J. Chem. Ecol. 32 (2006) [17] J. Cva 409434. [18] M. Karas, F. Hillenkamp, Anal. Chem. 60 (1988) 22992301. cka, A. Svato s, J. Am. Soc. Mass Spectrom. 21 (2001) [19] V. Vrkoslav, A. Muck, J. Cva 220231. [20] R. Chen, L. Li, J. Am. Soc. Mass Spectrom. 12 (2001) 367375. [21] M.S. Kahr, C.L. Wilkins, J. Am. Soc. Mass Spectrom. 4 (1993) 453460. [22] H.P. Nguyen, I.P. Ortiz, C. Temiyasathit, S.B. Kim, K.A. Schug, Rapid Commun. Mass Spectrom. 22 (2008) 22202226. [23] R. Goodacre, E.V. York, J.K. Heald, I.M. Scott, Phytochemistry 62 (2003) 859863. cka, A. Svato s, Rapid Commun. Mass Spectrom. 17 (2003) 22032207. [24] J. Cva [25] M.Q. Benedict, in: J.M. Crampton, C.B. Beard, C. Louis (Eds.), The Molecular Biology of Insect Disease Vectors, Chapman & Hall, London, 1997, pp. 312. [26] I.T. Jollife, Principal Component Analysis, Springer, New York, 2002. [27] M.B. Vermaat, R.A. Ion, R.J.M.M. Does, C.A.J. Klaassen, Qual. Reliab. Eng. Int. 19 (2003) 337353. [28] J. Shawe-Taylor, N. Cristianini, An Introduction to Support Vector Machines and Other Kernel-Based Learning Methods, Cambridge University Press, New York, 2000. [29] V.N. Vapnik, Statistical Learning Theory, Wiley, New York, 1998.