Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Abstract

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.

Figure 1

MALDI-TOF MS protein profiles of five different species of the genus Phlebotomus showing species-unique peaks for conclusive species identification. The peaks in the mass spectra represent peptides or small proteins obtained from the sand fly bodies using acidic extraction.

Figure 2

Dendrogram obtained by cluster analysis of MALDI-TOF MS spectra of five Phlebotomus representatives. Five female individuals of each species were analyzed. Distance is displayed in relative units.

Figure 3

Dendrogram obtained by cluster analysis of MALDI-TOF MS spectra of specimens of Phlebotomus sergenti from laboratory colonies originating from Turkey and Israel. Distance is displayed in relative units.

Figure 4

Comparison of MALDI-TOF MS spectra of two males and two females of Phlebotomus tobbi. Nearly identical spectra were observed for both sexes with all species-specific peaks present.

Figure 5

Effect of the storage conditions on the spectra quality. Comparison of MALDI-TOF MS spectra of Phlebotomus tobbi females stored in 70% ethanol for 3, 39 and 75 days.

Figure 6

Species clustering observed for five female individuals of three Phlebotomus species after storage in 70% ethanol for 75 days. Distance is displayed in relative units.