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Background: In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands. Methodology/Principal Findings: Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase. Conclusions: This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition. Author Summary: Phlebotomus orientalis is the vector of visceral leishmaniasis (VL) caused by Leishmania donovani in Northeast Africa. Immunization with sand fly saliva or with individual salivary proteins has been shown to protect against leishmaniasis in different hosts, warranting the intensive study of salivary proteins of sand fly vectors. In our study, we characterize the salivary compounds of P. orientalis, thereby broadening the repertoire of salivary proteins of sand fly species belonging to the subgenus Larroussius. In order to find out whether there is any connection between the composition of P. orientalis saliva and the epidemiology of VL in two distinct Ethiopian foci, Addis Zemen and Melka Werer, we studied the transcriptomes, proteomes, enzymatic activities, and the main salivary antigens in two P. orientalis colonies originating from these areas. We did not detect any significant difference between the saliva of female sand flies originating in Addis Zemen (endemic area) and Melka Werer (non-endemic area). Therefore, the different epidemiology of VL in these Ethiopian foci cannot be related to the distant salivary gland protein composition. Identifying the sand fly salivary gland compounds will be useful for future research focused on characterizing suitable salivary proteins as potential anti-Leishmania vaccine candidates. [ABSTRACT FROM AUTHOR]