To elucidate portions of the transmission cycles of American tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) occurring in the region surrounding the Lençóis Maranhenses National Park, an important tourist center in Brazil, the present study objectives were to determine the rate of natural infection by Leishmania spp. and the blood meal in caught sand flies species in the region. Sand flies were captured over 36 mo in 62 locations of the municipality of Barreirinhas, Maranhão with notifications of disease incidence. Species identification of parasites was performed with internal transcribed spacer 1 (ITS1) PCR-RFLP using HaeIII enzyme. Blood meal identification was performed with cytochrome b (cytb) gene PCR-RFLP using HaeIII and MboI enzyme. The species Lutzomyia longipalpis (Lutz and Neiva 1912) presented a positivity rate of 3.7% for Leishmania infantum. Species not considered vectors of this parasite such as Lu. lenti (Mangabeira 1938) and Lu. whitmani (Antunes & Coutinho 1939) showed infection rates of 0.6% and 0.9%, respectively. Among the vectors of Leishmania spp. was Lu. whitmani with detection rate of 0.3% for Le. braziliensis and Lu. flaviscutellata (Mangabeira 1942) with a detection rate of 8% for Le. amazonensis. After restriction of amplification product encoding a 359bp sequence of the cytb recognized in as follows: pigs (37.9%); dogs (27.4%); chickens (20.9%); horses (9%), rodents (3.3%), and humans (1.4%). The presence of Leishmania DNA in sand flies fed with human blood and domestic animals in villages with transmission of VL and TL suggests that transmission could be occurring in the locations of the infected patients.

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed. Graphical Abstract

Sand fly (Diptera: Psychodidae) taxonomy is complex and time-consuming, which hampers epidemiological efforts directed toward controlling leishmaniasis in endemic regions such as northeastern Brazil. Here, we used a fragment of the mitochondrial cytochrome c oxidase I (COI) gene to identify sand fly species in Maranhão State (northeastern Brazil) and to assess cryptic diversity occurring at different spatial scales. For this, we obtained 148 COI sequences of 15 sand fly species (10 genera) from Maranhão (fine spatial scale), and joined them to COI sequences from other Brazilian localities (distant about 2,000 km from Maranhão, broad spatial scale) available in GenBank. We revealed cases of cryptic diversity in sand flies both at fine (Lutzomyia longipalpis (Lutz and Neiva) and Evandromyia termitophila (Martins, Falcão and Silva)) and broad spatial scales (Migonemyia migonei (França), Pressatia choti (Floch and Abonnenc), Psychodopygus davisi (Root), Sciopemyia sordellii (Shannon and Del Ponte), and Bichromomyia flaviscutellata (Mangabeira)). We argue that in the case of Bi. flaviscutellata, the cryptic diversity is associated with a putative new species. Cases in which DNA taxonomy was not as effective as morphological identification possibly involved recent speciation and/or introgressive hybridization, highlighting the need for integrative approaches to identify some sand fly species. Finally, we provide the first barcode sequences for four species (Brumptomyia avellari (Costa Lima), Evandromyia infraspinosa (Mangabeira), Evandromyia evandroi (Costa Lima and Antunes), and Psychodopygus complexus (Mangabeira)), which will be useful for further molecular identification of neotropical species.