Abstract
BackgroundPhlebotomine 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.MethodsSix 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.ResultsSpecimens 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.ConclusionsSpecies 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.
Highlights
Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens
Accurate species identification is profound mainly in epidemiological studies conducted in endemic areas of leishmaniases, where the presence of morphologically similar species with different vectorial capacities can obscure the vector – parasite relationships with consequences in adequate control measures
Preparation of samples for MALDI-TOF MS Insect bodies, stored at various conditions, were dried at room temperature and dissected, cutting off the head and abdomen so that body parts bearing decisive characters could be mounted on slides for morphological analysis, the rest of the abdomen was spared for DNA isolation
Summary
Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. Sand flies transmit other human pathogens such as bacteria (Bartonella) and viruses of families Bunyaviridae, Reoviridae and Rhabdoviridae [1] Despite their undisputable importance in human and veterinary medicine, inadequate and inconsistent attention has been paid to sand fly species identification. The conventional approach to sand fly species identification, based on morphological features, requires the mounting of each specimen’s head and abdomen, which bear the decisive characteristics (genitalia, cibarium and pharyngeal armature). Both slide preparation and species identification are laborious and time-consuming, demanding a certain degree of proficiency and expertise. Accurate species identification is profound mainly in epidemiological studies conducted in endemic areas of leishmaniases, where the presence of morphologically similar species with different vectorial capacities can obscure the vector – parasite relationships with consequences in adequate control measures
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