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Background  Sand flies (Diptera, Psychodidae, Phlebotominae) in the genus  Lutzomyia  are the predominant vectors of the protozoan disease leishmaniasis in the New World. Within the watershed of the Panama Canal, the cutaneous form of leishmaniasis is a continuous health threat for residents, tourists and members of an international research community. Here we report the results of screening a tropical forest assemblage of sand fly species for infection by both  Leishmania  and a microbe that can potentially serve in vector population control, the cytoplasmically transmitted rickettsia,  Wolbachia pipientis . Knowing accurately which  Lutzomyia  species are present, what their evolutionary relationships are, and how they are infected by strains of both  Leishmania  and  Wolbachia  is of critical value for building strategies to mitigate the impact of this disease in humans.    Methodology and Findings  We collected, sorted and then used DNA sequences to determine the diversity and probable phylogenetic relationships of the Phlebotominae occurring in the understory of Barro Colorado Island in the Republic of Panama. Sequence from CO1, the DNA barcoding gene, supported 18 morphology-based species determinations while revealing the presence of two possible “cryptic” species, one ( Lu.  sp. nr  vespertilionis ) within the  Vespertilionis  group, the other ( Lu. gomezi ) within the Lutzomyia-cruciata series. Using ITS-1 and “minicircle” primers we detected  Leishmania  DNA in 43.3% of  Lu. trapidoi , 26.3% of  Lu. gomezi  individuals and in 0% of the other 18 sand fly species. Identical ITS-1 sequence was obtained from the  Leishmania  infecting  Lu. trapidoi  and  Lu. gomezi , sequence which was 93% similar to  Leishmania (viannia) naiffi  in GenBank, a species previously unknown in Panama, but recognized as a type of cutaneous leishmaniasis vectored broadly across northern and central South America. Distinct strains of the intracellular bacterium  Wolbachia  were detected in three of 20 sand fly species, including  Lu. trapidoi , in which it frequently co-occurred with  Leishmania .    Conclusions  Both morphological and molecular methods were used to examine an assemblage of 20 sand fly species occurring in the forests of the Panama Canal area. Two of these species, members of separate clades, were found to carry  Leishmania  at high frequency and hence are likely vectors of leishmaniasis to humans or other mammal species. A single  Leishmania  species, identified with high confidence as  Le. naiffi , was carried by both species. That  Le. naiffi  is known to cause cutaneous lesions in South America but has hitherto not been reported or implicated in Panama opens the possibility that its range has recently expanded to include the Isthmus or that it occurs as a recent introduction. The occurrence of  Leishmania  and  Wolbachia  in  Lu. trapidoi  identifies one important vector of the disease as a potential target for gene introductions using  Wolbachia  population sweeps.

Lutzomyia Sand Fly Diversity and Rates of Infection by Wolbachia and an Exotic Leishmania Species on Barro Colorado Island, Panama