Salivary transcriptome and proteome analysis of P. duboscqi
has resulted in a better understanding at the molecular level of the repertoire of proteins present in the saliva of this sandfly (Tables and ). Most salivary transcripts identified from the P. duboscqi
cDNA libraries are very similar to those of the salivary proteins previously identified in P. papatasi
. This is not surprising, because both P. papatasi
and P. duboscqi
belong to the same subgenus (Phlebotomus) and are proven natural vectors of L. major
. A clear difference between P. duboscqi
and P. papatasi
cDNA libraries was the presence in P. duboscqi
of an adenosine deaminase (the transcript and the protein). Adenosine deaminase has been reported in Aedes and Culex mosquitoes [31
] and in the sandfly L. longipalpis
; however, not in sandflies from the genus Phlebotomus [15
This salivary transcriptome analysis allowed us to compare the salivary proteins of a sandfly from two different geographical locations. We investigated whether the salivary proteins from two different sites (Mali and Kenya) would be divergent, as previously reported with the salivary protein maxadilan when comparing L. longipalpis
sandflies from Costa Rica, Colombia, and Brazil [12
]. In the present work, we performed a global comparative analysis of the most abundant salivary proteins of sandflies from two locations, and searched for orthologues using phylogenetic analysis. We found the majority of the proteins to be highly conserved at both the aa and the nucleotide levels. We found that at least five families of proteins (SP15-like, SP12-like, D7-like, antigen 5-like, and yellow-related protein) were 100% identical in sandflies from Mali and Kenya. The other families were also highly conserved (94.6% to 99.8%) with the exception of three proteins that had moderate homology (of 18 orthologous sequences): two SP12-like members that were 84.4% and 71.6% identical, respectively, and a SP32-like member that was 84% identical.
Because cellular immune responses to sandfly saliva – particularly a DTH response – was previously associated with protection against Leishmania infection [5
], we wanted first to identify potential MHC class II T-cell epitopes, which are required for DTH T cell-dependent responses and then determine whether these putative epitopes were also conserved among salivary proteins from these sandflies. The majority of potential T-cell epitopes were highly conserved among the different sandfly proteins; in fact, the majority of potential T cell epitopes were 100% identical, with the exception of only five epitopes that were 75% to 90% identical. These data suggest that even if the overall level of identity of some salivary proteins (Mali vs Kenya) is not 100%, the proteins have the potential to cross-react, at least at the level of cellular immune response (DTH) because of the high conservation of their T-cell epitopes that can be presented in the proper MHC class II context. This assumption needs to be tested experimentally.
A possible explanation for the conservation of salivary proteins include recent establishment of these sandflies in these regions with little or no evolutionary pressure from host immune response on these salivary proteins; or evolutionary pressure to keep these sequences constant (negative selection). Additionally, the location of these sand flies is more than 2000 thousand of kilometers apart. Then, it is difficult to suggest that there is a continuous exchange of sand flies in the whole sub-Saharan Africa moving from Kenya all the way to Mali or is also possible that the gene flow may be very low. In history, this area was affected by dramatic aridization (~5 millions years ago) [32
] and consequent creation of Sahel as a unique transient formation (~3 milions years ago) [33
], events that might led to separation and later rejoining of Eastern and Western populations of P. duboscqi
. Further studies are needed to determine if these two populations are genetically isolated.
A DTH response to P. papatasi
bites in mice was experimentally demonstrated to help these sandflies to probe and feed faster [34
]. It was shown that this type of response considerably increased blood flow at the site of the bite (after subsequent sandfly challenge), creating a favorable environment for feeding. It is thus possible that this type of immune response may favor sandfly survival in nature and therefore will also favor the presence of highly conserved sequences in their salivary proteins.
The data presented in this work are in contrast to previous studies performed with the salivary protein maxadilan from the sandfly L. longipalpis
, which was shown to be highly divergent between sandflies of distinct locations [12
]. In contrast, PpSP15 from P. papatasi
was shown to be highly conserved when comparing sandflies from different locations and isolates from field and laboratory colonies [35
]. Therefore, it is possible that Phlebotomus salivary proteins are more conserved in general than proteins present in the saliva of Lutzomyia sandflies, perhaps due to the benefit accrued in increased feeding due to the host DTH response. It is also important to take into account that L. longipalpis
is allegedly a complex of cryptic species [36
], hence the larger variability observed in their salivary protein. Additionally, if P. duboscqi
is a much older sand fly than L. longipalpis
, it may be possible that Phlebotomus sand flies are more stable species which could explain the high conservancy of salivary proteins in the two different Phlebotomus species (P. papatasi
and P. duboscqi
Sandfly salivary components are potential vaccine candidates to control Leishmania infection. Our results suggest that P. duboscqi salivary protein that may be able to produce a protective cellular immune response should be able to induce the same immune response in hosts from distant geographical locations in the Sub-Saharan Africa where P. duboscqi is present.