Leishmaniasis is a vector-borne parasitic disease caused by Leishmania spp., transmitted to humans by phlebotomine sand flies. The development of Leishmania into infective metacyclic promastigotes occurs within the sand fly gut, where the bacterial microbiota plays a pivotal role in parasite development and transmission dynamics. This study aimed to characterize the gut bacterial composition of phlebotomine sand flies collected from both endemic (Lalla Aaziza) and non-endemic (Marrakech) regions of leishmaniasis in Morocco. We investigated the microbiota of Phlebotomus papatasi, P. sergenti, P. perniciosus, and P. longicuspis, all proven vectors of cutaneous and visceral leishmaniasis in the Old World, including Morocco, as well as Sergentomyia minuta, a potential vector in the Mediterranean basin. Gut bacteria were isolated using conventional microbiological techniques and identified by MALDI-TOF mass spectrometry. Fifteen bacterial strains from three phyla were identified, with Bacillus pumilus being the most frequently detected species. Significant differences in colony-forming unit (CFU) counts and bacterial richness were observed between sand fly species and collection sites. Notably, Bacillus simplex (in P. papatasi), Nocardia ignorata (in P. sergenti), and Serratia spp. (in P. longicuspis) were identified for the first time in these vectors. This study is the first to investigate the gut bacterial composition of sand flies in Morocco, revealing species and locality-dependent differences in microbial communities. The predominance of Bacillus spp., particularly B. pumilus, suggests a potentially influential role in sand fly physiology and vector competence. Furthermore, the novel detection of B. simplex, N. ignorata, and Serratia spp. underscores previously unrecognized microbial associations that warrant further investigation. These findings provide a critical baseline for future studies exploring the microbiota-mediated modulation of sand fly-Leishmania interactions.
Keywords: Leishmania; MALDI-TOF; Morocco; gut bacteria; sand fly.