A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi.

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.