engleski

Proceedings of the IX International Symposium on Phlebotomine Sandflies (ISOPS IX), Reims, France, June 28th–July 1st, 2016

Conclusive species identification of sand flies by morphological analysis remains a challenging task due to limited availability of robust characters, their intraspecific variability among different populations and a need of expertise to assess them. According to proposed scenarios of future climatic trends, sand flies may emerge in regions with a lack of expertise for their species identification by classical morphological approach. Therefore, especially for high-throughput assessment of field surveys in endemic areas, there is a need for complementary methods of species identification relying on molecular techniques. Protein profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry emerged as a molecular method recently used for species identification of various medically important arthropod vectors including phlebotomine sand flies. With a standardized protocol of specimen capture, storage and sample preparation and a robust reference database, it provides characteristic and unique protein fingerprints that allow conclusive species identification. It requires minimal sample preparation that enables utilization of a single sand fly specimen for several purposes. Balkan countries are in a transition area in the European part of the Mediterranean basin where sand fly fauna of the western part meets with eastern elements and where several species have the limit of their geographical distribution. At the same time, data on sand fly presence and species composition are scarce due to recent upheavals. Due to two field surveys in the Balkan area and adjacent countries (Bosnia and Hercegovina, Bulgaria, Croatia, Hungary, Macedonia, Montenegro, Serbia and Slovenia) in summer of 2015, approx. 700 sand fly specimens of a total of 8000 captured were analyzed by protein profiling. The sand flies were trapped by CDC traps placed in domestic, peridomestic and sylvatic sites and processed according to standardized protocol. An in-house reference spectrum database was used as reference comprising 16 sand fly species based on specimens from both laboratory colonies as well as field-collected specimens whose species identity was conclusively confirmed by morphological analysis and DNA sequencing. Analyzed specimens belonged to species of four subgenera of the genus Phlebotomus : Adlerius ( Ph. balcanicus and Ph. simici ), Larroussius ( Ph. neglectus , Ph. perfiliewi , Ph. tobbi ), Paraphlebotomus ( Ph. sergenti )and Phlebotomus ( Ph. papatasi ). Moreover, two species of the genus Sergentomyia were also recorded ( Se. minuta and Se. dentata ). The method proved very suitable for identification of female specimens of the subgenus Larroussius (492 analyzed specimens) where species-specific diagnostic markers were characterized beside the overall protein spectra. All dubious specimens where morphological and protein-based identification did not agree were further characterized by a sequencing analysis (cytochrome oxidase 1 gene) which confirmed the identification by MALDI-TOF MS in all cases. MALDI-TOF protein profiling proved to be a time- and cost-effective method of choice for species identification of large sets of field-caught sand flies if these are collected, stored and analyzed using a standard, optimized protocol. Financial support: This project is supported by the Czech Science Foundation (GA15-04329S) and by VectorNet.

sandfly, MALDI-TOF

nije evidentirano