Leishmaniasis, caused by the protozoan Leishmania parasites, by the bite of infected female sand flies (Phlebotomus spp.), is one of the most important vector-borne diseases in the Eastern Europe. Zoonotic visceral... [ view full abstract ]
Leishmaniasis, caused by the protozoan Leishmania parasites, by the bite of infected female sand flies (Phlebotomus spp.), is one of the most important vector-borne diseases in the Eastern Europe. Zoonotic visceral leishmaniasis (VL) is widely distributed in the countries of the former Soviet Union with human cases in Central Asia and the Caucasus, including Georgia. Field collections of sand flies were conducted in Georgia and the DNA barcoding method was used to identify Phlebotomus fauna present in Georgia. Identifications were confirmed using morphological characters of vouchered specimens preserved during the study. This study established a DNA barcode library and a reference collection for the Phlebotomus sand fly species found in Georgia, as well as provided distribution data that was previously unknown for VL vectors.
Sand fly specimens were collected in CDC light traps then pooled by collection site and separated into males and females. Specimens were dissected and the thorax, wings, and legs were stored in 95% ethanol for DNA extraction. The head and abdomens of males were stored in 70% ethanol and those of females stored in lactophenol in preparation of permanent slide mounting. DNA extractions were carried out using the Autogen® DNA extraction platform, and, PCR reactions were completed on 605 samples with similar morphological features. Mitochondrial DNA (MtDNA) cytochrome oxidase I (COI) DNA barcode sequencing was generated from 656 specimens; in total, we were able to generate 513 (78%) sequences.
Six different species of Phlebotomus (Larrousius) were recovered, with P. kandelaki being the most common at 55% (282/513), followed by P. sergenti 17% (n=87), P. tobbi 0.01% (n=4), and P. galileaus 0.004% (n=2). The Leishmaniasis vector P. galileaus was reported for the first time in Georgia. In addition, our data confirmed the presence of three species of Phlebotomus (Adlerius) – P. halepensis, P. balcanicus and P. sp. nr. balcanicus. The identity of the latter is unclear as no males were included in our samples, thus we are unable to confirm whether this is Phlebotomus (Adlerius) brevis, previously reported from Georgia, or in fact a new taxon for the country. Two specimens were collected in the eastern part of Georgia and seemed most likely to have originated from Iran. Our results also identified two species of Phlebotomus (Larroussius) that require additional morphological verification.
This research established a DNA barcode library and a reference collection for the Phlebotomus sand fly species found in Georgia. We were able to provide distribution data that was previously unknown for VL vectors. Further research efforts will be required to collect more samples and apply an integrated morphological and molecular approach to verify the identity of these taxa species investigated in the frame of the project are submitted to the worldwide database at http://boldsystems.org/.
