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Phlebotomus duboscqi is the principle vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL), in West Africa and is the suspected vector in Mali. Although found throughout the country the seasonality and infection prevalence of P. duboscqi has not been established in Mali. We conducted a three year study in two neighboring villages, Kemena and Sougoula, in Central Mali, an area with a leishmanin skin test positivity of up to 45%. During the first year, we evaluated the overall diversity of sand flies. Of 18,595 flies collected, 12,952 (69%) belonged to 12 species of Sergentomyia and 5,643 (31%) to two species of the genus Phlebotomus, P. duboscqi and P. rodhaini. Of those, P. duboscqi was the most abundant, representing 99% of the collected Phlebotomus species. P. duboscqi was the primary sand fly collected inside dwellings, mostly by resting site collection. The seasonality and infection prevalence of P. duboscqi was monitored over two consecutive years. P. dubsocqi were collected throughout the year. Using a quasi-Poisson model we observed a significant annual (year 1 to year 2), seasonal (monthly) and village effect (Kemena versus Sougoula) on the number of collected P. duboscqi. The significant seasonal effect of the quasi-Poisson model reflects two seasonal collection peaks in May-July and October-November. The infection status of pooled P. duboscqi females was determined by PCR. The infection prevalence of pooled females, estimated using the maximum likelihood estimate of prevalence, was 2.7% in Kemena and Sougoula. Based on the PCR product size, L. major was identified as the only species found in flies from the two villages. This was confirmed by sequence alignment of a subset of PCR products from infected flies to known Leishmania species, incriminating P. duboscqi as the vector of CL in Mali. Author Summary: Female sand flies transmit a parasite called Leishmania that causes a disease called cutaneous leishmaniasis (CL). Several species of sand flies are found in West Africa, but only one species, Phlebotomus duboscqi, has been proven to transmit the parasite. Cutaneous Leishmaniasis has also been reported from Mali, Central West Africa, but the sand fly transmitting the parasite and its annual abundance has not been established, until now. Sand flies were collected during three consecutive years from two neighboring villages in Central Mali, Kemena and Sougoula, where CL is present. P. duboscqi was collected year-round and was the dominant sand fly inside of and surrounding human dwellings. Other sand fly species, known not to be vectors of CL, were primarily found outside the village. Additionally, P. duboscqi females were found infected with L. major, the same Leishmania species identified from human CL cases in Mali. The estimated infection prevalence of P. duboscqi females was 2.7%. Interestingly, the sand fly abundance and infection prevalence was similar in the two villages despite a previous report indicating a disparate L. major exposure rate in humans. This study greatly enhances our knowledge of CL transmission in Mali, poorly studied in this country to date. [ABSTRACT FROM AUTHOR]