Molecular identification of Phlebotomus longiductus collected from an endemic focus of cutaneous leishmaniasis in Himachal Pradesh (India)

The state of Himachal Pradesh is the most endemic region for cutaneous leishmaniasis transmission in northern India. Phlebotomus adlerius longiductus and Phlebotomus Larrousisus major have a wide distribution in Himachal Pradesh (India) and mostly found in mountainous areas. There is very little data available on the identification and distribution of sand flies in Himachal Pradesh. Therefore, the present study was carried out with the aim of identifying and distinguishing the sand flies species found in endemic focus of CL in Himachal Pradesh (HP), India, using PCR and sequencing methods. Collections of sand flies were made from endemic villages of cutaneous leishmaniasis in Shimla, Kullu and Kinnaur districts of HP during 2017-2019. A total of 291 sand flies were collected and all the collected sandflies were identified morphologically. In addition, genus specific identification of 52 sand flies was carried by Polymerase Chain Reaction (PCR). The PCR amplified products were sequenced for confirmation of sand-fly species. The sequences obtained were submitted to NCBI GenBank under accession no. MT126505 to MT126510 and compared for similarities to the NCBI database. The morphological identification revealed Phlebotomus adlerius longiductus and Phlebotomus Larrousisus major. Of 291 sand flies,>99% were Phlebotomus adlerius longiductus with Phlebotomus Larrousisus major in low numbers. The sequences obtained were compared for similarities to NCBI database and showed 99-100% similarity with Phlebotomus longiductus (Parrot). The phylogenetic tree analysis showed that the isolates were geographically closest to the sand flies from Bhutan and China. The study revealed that the P. longuductus (Parrot) is the predominant species found in the CL endemic area in HP. The study confirms the identity of P. longiductus (Parrot) through molecular tools as well as morphologically. As, P. longiductus (Parrot) has been reported to transmit L.donovani, it is important to distinguish the sand-fly species using molecular tool.