BACKGROUND: Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches. METHODS: Six laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes. RESULTS: Specimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens. CONCLUSIONS: Species identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.

Department of Parasitology Faculty of Science Charles University Prague Prague Czech Republic

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Maroli M, Feliciangeli MD, Bichaud L, Charrel R, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27(2):123–147. doi: 10.1111/j.1365-2915.2012.01034.x. PubMed DOI

Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M. WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012;7(5):e35671. doi: 10.1371/journal.pone.0035671. PubMed DOI PMC

World Health Organization. Control of the Leishmaniasis. WHO Technical Report Series 949. Geneva: WHO; 2010. PubMed

Ready P. Biology of Phlebotomine sand flies as vectors of disease agents. Annu Rev Entomol. 2013;58:227–250. doi: 10.1146/annurev-ento-120811-153557. PubMed DOI

Mukhopadhyay J, Ghosh K, Braig H. Identification of cutaneous leishmaniasis vectors, Phlebotomus papatasi and P. duboscqi using random amplified polymorphic DNA. Acta Trop. 2000;76:277–283. doi: 10.1016/S0001-706X(00)00130-3. PubMed DOI

Boudabous R, Bounamous A, Jouet D, Depaquit J, Augot D, Ferte H, Berchi S, Couloux A, Veuille M, Babba H. Mitochondrial DNA differentiation between two closely related species, Phlebotomus (Paraphlebotomus) chabaudi and Phlebotomus (Paraphlebotomus) riouxi (Diptera: Psychodidae), based on direct sequencing and polymerase chain reaction-restriction fragment length polymorphism. Ann Entomol Soc Am. 2009;102(3):347–353. doi: 10.1603/008.102.0301. DOI

Khalid N, ElNaiem D, Aboud M, Al Rabba F, Tripet F. Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies. Med Vet Entomol. 2010;24(4):352–360. doi: 10.1111/j.1365-2915.2010.00893.x. PubMed DOI

Manonmani A, Mathivanan A, Srinivasan R, Janbulingam P. Species-diagnostic polymerase chain reaction assays for Phlebotomus argentipes and Phlebotomus papatasi, Vectors of Leishmania. J Med Entomol. 2010;47(5):743–747. doi: 10.1603/ME09195. PubMed DOI

Sauer S, Kliem M. Mass spectrometry tools for the classification and identification of bacteria. Nat Rev Microbiol. 2010;8(1):74–82. doi: 10.1038/nrmicro2243. PubMed DOI

Cassagne C, Pratlong F, Jeddi F, Benikhlef R, Aoun K, Normand A-C, Faraut F, Bastien P, Piarroux R. Identification of Leishmania at the species level with matrix-assisted laser desorption/ ionization time of flight mass spectrometry. Clin Microbiol Infect. in press. PubMed

Campbell PM. Species differentiation of insects and other multicellular organisms using matrix-assisted laser desorption/ionization time of flight mass spectrometry protein profiling. Syst Entomol. 2005;30:186–190. doi: 10.1111/j.1365-3113.2004.00279.x. DOI

Kaufmann C, Ziegler D, Schaffner F, Carpenter S, Pfluger V, Mathis A. Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for characterization of Culicoides nubeculosus biting midges. Med Vet Entomol. 2011;25(1):32–38. doi: 10.1111/j.1365-2915.2010.00927.x. PubMed DOI

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

Kaufmann C, Steinmann IC, Hegglin D, Schaffner F, Mathis A. Spatio-temporal occurrence of Culicoides biting midges in the climatic regions of Switzerland, along with large scale species identification by MALDI-TOF mass spectrometry. Parasit Vectors. 2012;5:246. doi: 10.1186/1756-3305-5-246. PubMed DOI PMC

Hoppenheit A, Murugaiyan J, Bauer B, Steuber S, Clausen P-H, Roesler U. Identification of Tsetse (Glossina spp.) using matrix-assisted laser desorption/Ionisation time of flight mass spectrometry. PLoS Negl Trop Dis. 2013;7(7):e2305. doi: 10.1371/journal.pntd.0002305. PubMed DOI PMC

Muller P, Pfluger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C. Identification of cryptic Anopheles mosquito species by molecular protein profiling. PLoS One. 2013;8(2):e57486. doi: 10.1371/journal.pone.0057486. PubMed DOI PMC

Yssouf A, Socolovschi C, Flaudrops C, Ndiath M-O, Sougoufara S, Dehecq J-S, Lacour G, Berenger J-M, Sokhna S, Raoult D, Parola P. Matrix-assisted laser desorption ionization - time of flight mass spectrometry: an emerging tool for the rapid identification of mosquito vectors. PLoS One. 2013;8(8):e72380. doi: 10.1371/journal.pone.0072380. PubMed DOI PMC

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36(Suppl 1):S1–S9. PubMed

van Veen SQ, Claas EC, Kuijper EJ. High throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol. 2010;48(3):900–907. doi: 10.1128/JCM.02071-09. PubMed DOI PMC

Seng P, Rolain J-M, Fournier PE, La Scola B, Drancourt M, Raoult D. MALDI-TOF-mass spectrometry applications in clinical microbiology. Future Microbiol. 2010;5(11):1733–1754. doi: 10.2217/fmb.10.127. PubMed DOI

Pesson B, Ready J, Benabdennbi I, Martin-Sánchez J, Esseghir S, Cadi-Soussi M, Morillas-Marques F, Ready P. Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif. Med Vet Entomol. 2004;18(1):25–37. doi: 10.1111/j.0269-283x.2004.0471.x. PubMed DOI

Ilango K. A taxonomic reassessment of the Phlebotomus argentipes species complex (Diptera: Psychodidae: Phlebotominae) J Med Entomol. 2010;47(1):1–15. doi: 10.1603/033.047.0101. PubMed DOI

Kasap OE, Votypka J, Alten B. The distribution of the Phlebotomus major complex (Diptera:Psychodidae) in Turkey. Acta Trop. 2013;127(3):204–211. doi: 10.1016/j.actatropica.2013.05.001. PubMed DOI

Feltens R, Gorner R, Kalkhof S, Groger-Arndt H, von Bergen M. Discrimination of different species from the genus Drosophila by intact protein profiling using matrix-assisted laser desorption ionization mass spectrometry. BMC Evol Biol. 2010;10:95. doi: 10.1186/1471-2148-10-95. PubMed DOI PMC

Depaquit J, Ferté H, Léger N, Lefranc F, Alves-Pires C, Hanafi H, Maroli M, Morillas-Marques F, Rioux JA, Svobodova M, Volf P. ITS2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): Possible consequences in their ability to transmit Leishmania tropica. Int J Parasitol. 2002;32(9):1123–1131. doi: 10.1016/S0020-7519(02)00088-7. PubMed DOI

Dvorak V, Aytekin AM, Alten B, Skarupova S, Votypka J, Volf P. A comparison of the intraspecific variability of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae) J Vector Ecol. 2006;31(2):229–238. doi: 10.3376/1081-1710(2006)31[229:ACOTIV]2.0.CO;2. PubMed DOI

Dvorak V, Votypka J, Aytekin AM, Alten B, Volf P. Intraspecific variability of natural populations of Phlebotomus sergenti, the main vector of Leishmania tropica. J Vector Ecol. 2011;36(Suppl 1):S49–S57. PubMed

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