SHORT COMMUNICATION

Identification of European mosquito species by MALDI-TOF MS

Amina Yssouf & Philippe Parola & Anders Lindström &

Tobias Lilja & Grégorie L’Ambert & Ulf Bondesson &

Jean-Michel Berenger & Didier Raoult & Lionel Almeras

Received: 3 January 2014 /Accepted: 24 March 2014 /Published online: 16 April 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract MALDI-TOF MS profiling has proved to be effi-cient for arthropod identification at the species level. However,prior to entomological monitoring, the reference spectra data-base should cover relevant species. Here, 74 specimens werefield-collected from 11 mosquito species captured in twodistinct European areas and used either to increment ourdatabase or for blind tests. Misidentification was not noted,underlining the power of this approach. Nevertheless, threeout of the 26 specimens used for the blind test did not reachthe significant identification threshold value set, attributed tolower spectral quality. In the future, the quality control spectraparameters need to be defined to avoid not achieving signifi-cant threshold identification.

Keywords MALDI-TOFmass spectrometry .Mosquito .

Identification

Short communication

The recent epidemics of mosquito-borne diseases, includingChikungunya, West Nile Fever, and Dengue in Europe, and

the high capacity of some mosquito species to colonize newareas (Weaver and Reisen 2010) demonstrate the need tomonitor mosquito distribution and accurately identify mos-quitoes at the species level. Although mosquito species areprimarily identified using morphological characteristics anddetermination keys, damaged specimens or morphologicalinterspecies similarities limit accurate species determination(Matson et al. 2008). PCR-basedmethods have been effectivelyused to discriminate morphologically undistinguishable mos-quito species for closely related species groups (e.g., molecularforms of Anopheles gambiae M and S) (Zapata et al. 2007).Nevertheless, molecular approaches are technically expensiveand time consuming (Freiwald and Sauer 2009); thus, thedevelopment of an alternative cheap and rapid tool that allowsthe monitoring of Culicidae fauna populations is required.Therefore, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology wassuccessfully evaluated for its ability to identify different arthro-pod groups, such as Drosophila (Feltens et al. 2010; Campbell2005), Culicoides (Kaufmann et al. 2012), Ixodidae (Yssoufet al. 2013a), and Culicidae species (Yssouf et al. 2013b), usingwhole specimens or different body parts. Recent studies per-formed in our laboratory based on legs dissected fromIxodidae (Yssouf et al. 2013a) or Culicidae (Yssouf et al.2013b) and submitted to MALDI-TOF MS profiling analysisdemonstrated that this compartment is sufficient to accuratelyidentify hematophagous arthropod species. A reference spec-tra database including 20 mosquito species from tropical areas(i.e., 7 and 13 species collected in the field of La ReunionIsland and Senegal, respectively) was established in our lab-oratory. Prior to performing longitudinal studies to followmosquito fauna changes or monitor the introduction of newspecies in an area, the Culicidae reference spectra databasemust be improved. Therefore, 74 specimens collected in thefield from either the southern (i.e., Camargue, France, n=35)or northern (i.e., Luleå, Uppsala, and Gotland, Sweden, n=39)

A. Yssouf : P. Parola : J.<M. Berenger :D. Raoult : L. Almeras (*)Unité de Recherche en Maladies Infectieuses et TropicalesEmergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar,Sénégal), Inserm 1095, WHO Collaborative Center for Rickettsiosesand Other Arthropod-Borne Bacterial Diseases, Faculté deMédecine, Aix Marseille Université, 27 bd Jean Moulin,13385 Marseille CEDEX 5, Francee-mail: almeras.lionel@gmail.com

A. Lindström : T. Lilja :U. BondessonDepartment of Chemistry, Environment and Feed Hygiene,National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden

G. L’AmbertEntente Interdépartementale de Démoustication Méditerranée,165, avenue Paul-Rimbaud, 34184 Montpellier CEDEX 4, France

Parasitol Res (2014) 113:2375–2378DOI 10.1007/s00436-014-3876-y

parts of Europe were submitted to MALDI-TOFMS analysis.Two Culiseta longiareolata specimens collected in Aix-en-Provence (France) that were not included in the database weretested to control for misidentification.

Field-collected female adult mosquitoes from Camargue(Entente Interdépartementale pour la Démoustication, EID,Montpellier, France) or Uppsala (National Veterinary Institute,Sweden), captured using Mosquito Magnet traps (WoodstreamCorp. USA), were identified morphologically (Becker et al.2010). The morphological identification of the Swedishmosquitoes was confirmed by molecular methods (PCRamplification of COI and then sequencing). Six distinctmosquito species were collected per site (Table 1), andone was found in both areas (i.e., Aedes vexans). Eachspecimen was stored directly in tubes containing silica geluntil MS analysis.

All legs from each specimen were submitted toMALDI-TOFMS (Microflex LT, Bruker Daltonic, Germany), as describedpreviously (Yssouf et al. 2013b). Flex control, Flex analysissoftware 3.3, and MALDI-Biotyper v3.0 software (BrukerDaltonic) were used for acquisition, assessment of reproduc-ibility, and analysis of protein mass spectra profiles. To controlfor the misidentification of mosquito species not yet includedin the reference spectra database, one specimen from eachmosquito species was first subjected to analysis using theMALDI-Biotyper software. A threshold log score value(LSV) of 1.8 was a relevant identification (Yssouf et al.2013b). Among the 11 mosquito species tested, a preciseidentification was obtained uniquely for Culex pipiens, whichwas already included in the database. The unambiguous iden-tification of Cx. pipiens despite the distinct geographical origin

of the tested specimen compared to theCx. pipiens used for thereference spectra database supported the idea that MALDI-TOF MS profiling is a robust method (Yssouf et al. 2013b).The first query that was performed on each specimen againstthe initial database revealed misidentifications with LSVsbetween 0.598 and 1.404 for the other 10 species tested.

Based on these results, 46 adult specimens from these 10species and four to seven specimens per species were submit-ted to MALDI-TOF MS. These last spectral profiles wereanalyzed with Flex analysis software, as described previously(Yssouf et al. 2013b). The analysis revealed that mosquito legprotein extracts still provided consistent and reproduciblespectra profiles with high intensity peaks in the range of2–20 kDa (Fig. 1). These findings agree with those ofprevious MALDI-TOF MS evaluations of different mos-quito species. The resulting spectra have been added toour database, which initially contained 20 species. Finally,incrementing these spectra yielded a database with 30distinct mosquito species.

To determine the identification accuracy, the remainingspecimens from each species (n=26) newly included in thereference spectra database were analyzed using MALDI-Biotyper software for automated comparison against all of theCulicidae spectra sets available (Table 1). All of the specimenswere correctly identified, with an identification LSV between1.628 and 2.378 (Table 1), among which 88.5 % (23/26)possessed an identification LSV that was higher than thethreshold (i.e., LSV>1.8). Conversely, for Cs. longiareolataspecimens tested blindly, the corresponding LSVs were ex-tremely low, confirming that spectra should be included in thedatabase for correct identification. In a previous evaluation of

Table 1 Mosquito species usedeither to increment the MALDI-TOF MS reference spectra data-base or to perform blind tests

a Range of log score values.b Species not included in thedatabase

Species Geographical origin/source Number ofspecimensadded to thedatabase

Number ofspecimens usedfor the blindtest procedure

ID log score-valuesa

[low-high]

Ae. cinereus Luleå, Sweden 4 2 [1.978–1.999]

Ae. vexans Camargue, France 3 2 [2.106–2.357]

Gotland, Sweden 4 1 [2.015]

Marseille, France 0 1 [1.855]

Ae. caspius Camargue, France 5 3 [1.762–2.239]

An. claviger Uppsala, Sweden 4 2 [1.908–2.252]

An. hyrcanus Camargue, France 5 2 [2.111–2.272]

An. maculipennis Camargue, France 5 2 [2.042–2.090]

Cq. richiardii Uppsala, Sweden 4 2 [1.628–2.096]

Cx. pipiens Camargue, France 0 3 [1.837–1.936]

Cx. modestus Camargue, France 4 2 [1.795–2.016]

Ae. rusticus Gotland, Sweden 4 2 [2.218–2.378]

Ae. excrucians Luleå, Sweden 4 2 [2.124–2.212]

Cs. longiareolatab Aix en Provence/France 0 2 [1.315–1.351]

Total 46 28

2376 Parasitol Res (2014) 113:2375–2378

mosquito identification using the MALDI-TOF MS approach(Yssouf et al. 2013b), a threshold LSV for unambiguous iden-tification was set at 1.8. Although one specimen from Aedescaspius, Culex modestus, and Coquillettidia richiardii did not

reach the significant identification threshold value set in thepresent study, all of these specimens were correctly identified.The lower identification LSVobtained for these last specimenscould be attributed to lower spectral quality (i.e., ≤1,500). In the

D10 0:D10 MS, BaselineSubtracted

0

500

1000

1500

Inte

ns. [

a.u.

].]

0

F3 0:F3 MS, BaselineSubtracted

0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

A7 0:A7 MS Raw

0

2000

4000

6000

8000

Inte

ns. [

a.u.

]]

C6 0:C6 MS, BaselineSubtracted

0

250

500

750

1000

1250

1500

Intens.

[a.u.]

.]

0G7 0:G7 MS, BaselineSubtracted

0

500

1000

1500

2000

2500

3000

Inten

s. [a.

u.].] 0

B12 0:B12 MS, BaselineSubtracted

0

1000

2000

3000

4000

Inten

s. [a.

u.]

0A7 0:A7 MS, BaselineSubtracted

0

2000

4000

6000

Inten

s. [a.u

.].]

0A12 0:A12 MS, BaselineSubtracted

0

500

1000

1500

2000

Intens.

[a.u.]

.] 0

B7 0:B7 MS, BaselineSubtracted

0

1000

2000

3000

4000

Inten

s. [a.

u.]

410.]

Ae. rusticus

Cq. richiardii

An. claviger

An. maculipennis

An. hyrcanus

Cx. modestus

Cx. pipiens

Ae. caspius

Ae. vexans

m/z

Intensity

[a.u.]

0G5 0:G5 MS Raw

0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

0

0

Ae. excrucians

0

Fig. 1 MALDI-TOFMS spectra profiles obtained from the leg protein extracts of 10 distinct mosquito species ranging from 2 to 20 kDa. The mosquitospecies are indicated at the right corner of each protein profile spectrum. a.u. arbitrary units, m/z mass-to-charge ratio

Parasitol Res (2014) 113:2375–2378 2377

future, the spectra should be quality controlled prior to interro-gating the reference spectra database.

The present work confirmed that specimen leg proteinextracts are sufficient for the identification of mosquito spe-cies using the MALDI-TOFMS approach when a counterpartspecies spectrum is already uploaded in the reference data-base. In addition, the accurate identification of specimensfrom one species collected from distinct regions highlightsthat this alternative method is robust and reliable in addition tobeing quick and cheap, and it may be able to revolutionizearthropod identification in the future. To standardize the inter-pretation of the spectral analysis, spectral quality controlparameters need to be developed to avoid misidentificationor the failure to achieve a significant identification threshold.The mosquito database was upgraded by adding 10 speciespresent in Europe. The enrichment of this database with themost abundant Palearctic mosquito species is indispensablefor longitudinal and/or transversal monitoring of Culicidaefauna from Europe. The rapid identification of mosquitopopulations is an essential starting point to establish a mos-quito distribution map and to evaluate the risk of mosquito-borne diseases.

References

Becker N, Petrić D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A(2010) Mosquitoes and their Control. Springer, Heidelberg, p 577

Campbell PM (2005) Species differentiation of insects and other multi-cellular organisms using matrix-assisted laser desorption/ionizationtime of flight mass spectrometry protein profiling. Syst Entomol 30:186–190

Feltens R, Gorner R, Kalkhof S, Groger-Arndt H, von Bergen M (2010)Discrimination of different species from the genus Drosophila byintact protein profiling using matrix-assisted laser desorption ioni-zation mass spectrometry. BMC Evol Biol 10:95

Freiwald A, Sauer S (2009) Phylogenetic classification and identificationof bacteria by mass spectrometry. Nat Protoc 4(5):732–742. doi:10.1038/nprot.2009.37

Kaufmann C, Schaffner F, Ziegler D, Pfluger V, Mathis A (2012)Identification of field-caught Culicoides biting midges using matrix-assisted laser desorption/ionization time of flight mass spectrometry.Parasitology 139(2):248–258. doi:10.1017/S0031182011001764

Matson R, Rios CT, Chavez CB, Gilman RH, Florin D, Sifuentes VL,Greffa RC, Yori PP, Fernandez R, Portocarrero DV, Vinetz JM, KosekM (2008) Improved molecular technique for the differentiation ofneotropical anopheline species. Am J Trop Med Hyg 78(3):492–498

Weaver SC, Reisen WK (2010) Present and future arboviral threats.Antiviral Res 85(2):328–345. doi:10.1016/j.antiviral.2009.10.008

Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger JM,Raoult D, Parola P (2013a) Matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid identification of tick vectors.J Clin Microbiol 51(2):522–528. doi:10.1128/JCM.02665-12

Yssouf A, Socolovschi C, Flaudrops C, Ndiath MO, Sougoufara S,Dehecq JS, Lacour G, Berenger JM, Sokhna CS, Raoult D, ParolaP (2013b) Matrix-assisted laser desorption ionization–time of flightmass spectrometry: an emerging tool for the rapid identification ofmosquito vectors. PLoS One 8(8):e72380. doi:10.1371/journal.pone.0072380

Zapata MA, Cienfuegos AV, Quiros OI, Quinones ML, Luckhart S,CorreaMM (2007) Discrimination of seven Anopheles species fromSan Pedro de Uraba, Antioquia, Colombia, by polymerase chainreaction-restriction fragment length polymorphism analysis of itssequences. Am J Trop Med Hyg 77(1):67–72

2378 Parasitol Res (2014) 113:2375–2378