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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.

Comparative Analysis of Salivary Gland Transcriptomes of Phlebotomus orientalis Sand Flies from Endemic and Non-endemic Foci of Visceral Leishmaniasis