Leishmania (Sauroleishmania) tarentolae and Trypanosoma platydactyli are reptile-associated trypanosomatids transmitted by Sergentomyia minuta sand flies, posing challenges for accurate diagnosis due to the fact that they often occur in sympatry. This study aimed to isolate and characterize new strains of these parasites from reptiles and sand flies using morphological, molecular and genomic approaches. Fifty-five reptiles were captured in Apulia, Italy, and sand flies were collected and dissected under a surveillance framework. Blood samples and gut contents were cultured in Schneider's Drosophila (SC) and Tobie-Evans (TEv) media. Two positive cultures underwent whole-genome sequencing, and a new conventional PCR (cPCR) protocol targeting the β-tubulin gene was developed. T. platydactyli was isolated from 27% of Tarentola mauritanica geckos using SC medium and 12.5% with TE, while L. (S.) tarentolae was isolated from 4.15% of geckos exclusively with SC. Cytology confirmed T. platydactyli in 25% of gecko blood smears. cPCR revealed T. platydactyli in 18.75%, L. (S.) tarentolae in 12.5% and co-infections in 14.6%. No infections were found in Podarcis siculus or Hemidactylus turcicus. Out of 208 S. minuta sand flies tested, 19 (9.1%) were positive for T. platydactyli, 30 (14.4%) for L. (S.) tarentolae, and 15 (7.2%) were co-infected with both. The newly developed cPCR assay robustly differentiated these parasites in both reptile and sand fly samples. Monitoring of natural infections in geckos revealed persistent, low-level L. (S.) tarentolae infections, detectable only by molecular methods, in contrast to the intermittent parasitaemia of T. platydactyli, which was detectable by cytology and culture. Kimura 2-parameter (K2P) divergence profiles indicate no evidence of a recent mobilome in T. platydactyli, whereas L. (S.) tarentolae retains a small but detectable fraction of low-divergence transposable-element copies (≤5-10% K2P; <0.05% of the genome). These findings confirm the sympatric circulation of L. (S.) tarentolae and T. platydactyli in geckos and sand flies in southern Italy, highlighting T. mauritanica as the most common reptilian host. The successful isolation and genome assembly of these trypanosomatids, along with the newly developed molecular tool, lay a solid foundation for future epidemiological and comparative genomic investigations, emphasizing the role of reptilian hosts in maintaining trypanosomatid diversity.

Department of Biosciences Pediatric CRC Romeo ed Enrica Invernizzi University of Milan 20133 Milan Italy

Department of Chemical Biological Pharmaceutical and Environmental Sciences University of Messina 98166 Messina Italy

Department of Parasitology Faculty of Science Charles University 12800 Prague Czechia

Department of Veterinary Clinical Sciences City University of Hong Kong 518057 Hong Kong PR China

Department of Veterinary Medicine Università degli Studi di Bari Aldo Moro 70124 Bari Italy

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