{'fraction_non_alphanumeric': 0.036353580048270315, 'fraction_numerical': 0.02659895414320193, 'mean_word_length': 4.979855682501503, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador

Abstract The detection and identification of natural infections in sand flies by Leishmania protozoan species in endemic areas is a key factor in assessing the risk of leishmaniasis and in designing prevention and control measures for this infectious disease. In this study, we analyzed the Leishmania DNA using nuclear ribosomal internal transcript spacer (ITS) sequences. Parasite DNA was extracted from naturally infected, blood-fed sand flies collected in nine localities considered leishmaniasis-endemic foci in Ecuador. The species of parasites identified in sand flies were Leishmania major-like, Leishmania naiffi, Leishmania mexicana, Leishmania lainsoni, and “Leishmania sp. siamensis”. Sand fly specimens of Brumptomyia leopoldoi, Mycropigomyia cayennensis, Nyssomyia yuilli yuilli, Nyssomyia trapidoi, Pressatia triacantha, Pressatia dysponeta, Psychodopygus carrerai carrerai, Psychodopygus panamensis, and Trichophoromyia ubiquitalis were found positive for Leishmania parasite. These findings contribute to a better understanding of the epidemiology and transmission dynamics of the disease in high-risk areas of Ecuador.

The first documented human case of leishmaniasis in Ecuador was reported in 1920 (Rodriguez 1974). Since then, 25 species of sand flies have been identified from four endemic localities (Hashiguchi et al. 1991). According to Kato et al. (2016b) and Hashiguchi et al. (2016), Leishmania guyanensis, L. braziliensis, L. naiffi, L. lainsoni, L. panamensis, L. amazonensis, L. mexicana, and Leishmania major-like have been isolated from human samples from tropical and subtropical areas of Ecuador. Lutzomyia ayacuchensis, Lutzomyia trapidoi, Lu. gomezi, Lutzomyia tortura, and P. maranonensis have been incriminated as the vectors for Leishmania spp. (Kato et al. 2005, Hashiguchi et al. 2016. Lainson and Shaw (1987) classified Leishmania species based on their geographical distribution, vector origin, tropism, and clinical manifestations. Owing to the difficulty of identifying the species of Leishmania and associating them with specific clinical manifestations, modern approaches to taxonomic identification use molecular markers (Fraga et al. 2009, Schö nian et al. 2010, Bates et al. 2015. Several techniques of genotyping and molecular characterization have been used to identify Leishmania species (Van der Auwera and Dujardin 2015). These techniques include multilocus-sequence typing (MLST; Van der Auwera and Dujardin 2015), multilocusmicrosatellite typing (MLMT), restriction-fragment length polymorphism PCR-RFLP (Schö nian et al. 2003, Terayama et al. 2008, and sequencing of specific genetic markers such as Hsp70 (Fraga et al. 2009), ribosomal DNA (rDNA), and kinetoplast DNA (kDNA; Orlando et al. 2002), internal transcript spacer ITS, and cytochrome B (Asato et al. 2008, Yang et al. 2013. Sequence analysis of ITS1 and ITS2 have been shown to be suitable for species discrimination of Leishmania parasites (Orlando et al. 2002, Mahdy et al. 2016, Nazma et al. 2016). There are between 150 and 200 copies of these rRNA genes in Leishmania genome (Berzunza-Cruz et al. 2002). Several authors have used the ITS region to determine the polymorphism of clinical samples and the genetic variability of strains of the Leishmania species complex with different PCR methods (Schö nian et al. 2001, Toz et al. 2013, Maia et al. 2015.
The detection and identification of Leishmania parasites in sand flies may help in understanding the transmission dynamics of the parasite species in high transmission areas among vectors and human and other animal reservoirs. In the present study, we use ITS1 sequences to identify the Leishmania species from engorged sand flies collected in nine localities of Ecuador.

Sand Fly Sample Collection
Phlebotomine sand flies were collected between 2014 and 2016 in nine tropical and subtropical localities, five in the Pacific lowlands and three in the Amazon basin of Ecuador (Fig. 1). Collections were made during both the rainy and dry seasons using CDC light traps (John W. Hock USA). Traps were placed in the forest at three different distances from inhabited houses: at 150 m (peridomiciliary), 300 m (forest), and 600 m (forest). All light traps operated from 1800 to 0600 hours for three consecutive nights, during each of the dry and rainy seasons. Samples were sorted using a Discovery V12 stereo microscope (Zeiss, Germany). Females were separated as to whether blood-fed or unfed. Blood-fed females were identified using the taxonomic keys of Young andDucan 1994 andGalati 2016. The abdomens of fed females were dissected and individually stored at �20 � C for DNA extraction. The head, wings, and thorax of each specimen were cleared in 10% potassium hydroxide and mounted in a temporary fructose-gum Arabic medium for taxonomic identification. The natural infection rates (NIRs) for the identified species of sand flies were calculated including only the total number of engorged females (Pinheiro et al. 2010).

Positive Control Cultures and Genomic DNA Extraction
Three reference strains of Leishmania were used as positive controls in the molecular studies. The species used were Leishmania tarentolae Wenyon, 1921 (LT2I UCLA) and clinical isolates of L. mexicana LM379 and L. braziliensis LB2903 (Instituto de Medicina Tropical "Alexander von Humboldt" Universidad Peruana Cayetano Heredia, Lima, Peru). Leishmania promastigotes were grown at 24 � C in Schneider's Drosophila medium (Gibco USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U/ml) solution (Gibco USA). Promastigotes were harvested on the third day of growth for genomic DNA extraction and posterior-sequence comparison with the DNA from the parasites extracted from the sand flies.
Genomic DNA extraction was conducted using abdomens of engorged field-collected sand flies and promastigotes from Leishmania cultures (positive controls). All the samples were then processed using DNeasy Blood & Tissue Kit (Qiagen, Germany)

ITS1 Amplification and Sequencing
Polymerase chain reaction was performed to generate a 330-bp ITS1 amplicon using the forward primer LITSR (5 0 -CTGGATCATTTT CCGATG-3 0 ) and the reverse primer L5.8S (5 0 -TGATACCACTTAT CGCACTT-3 0 (Schö nian et al. 2003). The PCR protocols for amplification were: 94 � C for 3 min followed by 40 cycles of 94 � C for 40 s, 53 � C for 45 s, and 72 � C for 60 s, followed by a final elongation step at 72 � C for 10 min. The amplified products were purified on a 2.0% agarose gel stained with SYBR safe (Invitrogen USA). T he purified PCR product was then sequenced (Macrogen, Seoul).
Polymerase chain reactions included DNA from the reference strains as positive controls (L. tarentolae LT2I, L. mexicana M379, and L. braziliensis LB2903) and a negative control (water).
Phylogenetic analysis was conducted in Geneious v 9.0 software using the neighbor-joining (NJ) method, 100-replicates and 70 bootstrap (BP) test. For species identification of Leishmania, ITS1 sequences from studied isolates were multiple-aligned using ClustalW-Geneious v 9.0 software (Biomatters Ltd, New Zealand). In the analysis, a sequence of Leptomonas seymouri was included as an outgroup (GenBank ATCC 30220).

Results
In total, 11,307 sand flies were captured with CDC light traps in nine locations of Ecuador. There were 6,764 males, 4,200 unfed females, and 343 blood-fed females. The total capture effort per location was six traps per night (total ¼ 18 traps in three nights) in each season (dry and rainy). The capture effort at each collecting ecotope was two traps per night (total ¼ six traps in three nights).
The peridomiciliary ecotope showed higher diversity of sand flies, higher number of infected sand flies, and higher NIRs than the forest ecotope. Natural infection rate showed values that ranged from 2.9% to 50% in the dry season, and from 6.3% to 12.5% during the rainy season (Table 1). "Leishmania sp. siamensis" was detected in five different species of sand flies from five localities. Leishmania major-like was only detected in Psychodopygus panamensis from Puerto Quito-Pichincha. The sequence that clustered with L. tropica was detected in Nyssomyia trapidoi collected in Joya de los Sachas-Orellana. Leishmania mexicana was detected in Micropygomyia cayennensis from El Progreso in the province of Guayas, in the coastal region (Table 1).

Discussion
One step in vector incrimination for leishmaniasis is to determine the occurrence of natural infection in sand fly populations. The dissection of the digestive tract of sand flies for observing Leishmania promastigotes by light microscopy has been considered the gold standard test for detecting natural infections in these vectors (Kato et al. 2005). However, identification of Leishmania parasites within sand flies requires examination of a large number of sand flies and isolation and culture of Leishmania parasites from each dissected fly. In some cases, it is necessary to inoculate parasites into laboratory animals to find true positives. Owing to the laboriousness of this procedure, molecular techniques have been standardized to easily detect Leishmania DNA in sand flies (Bates et al. 2015).

Leishmania tropica
One ITS1 sequence from a sand fly collected in Joya de los Sachas-Orellana corresponded to 98% homology with the Leishmania tropica cluster (BP ¼ 98). Although there are cases of visceralizing L. tropica, this parasite is more commonly the causative agent of cutaneous leishmaniasis, such as Leishmaniasis recidivans (Akhoundi et al. 2016, Sarkari et al. 2016. In Ecuador and South America, there are no epidemiological records of visceral leishmaniasis caused by L. tropica. It is known that L. tropica is genetically very heterogeneous (Schö nian et al. 2001, Schnur et al. 2004) and for this reason further studies should include a larger number of samples and genetic markers, along with studies of clinical samples in the province of Orellana. On the other hand, our analysis includes L. tropica and L. naiffi in the same cluster, implying a sequence similarity that requires further analysis of different alleles to differentiate between these two species.

Leishmania major-Like
The analysis of ITS1 sequences clustered two samples with sequences of L. major reported from the Old World. Leishmania major is one of the causative agents of cutaneous leishmaniasis in the Old World, which is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi (Ayari et al. 2015, Wu et al. 2015. Thus, the strong relationship of our sequence with L. major is remarkable. However, isoenzyme electrophoretic mobility patterns using RAPD, SSR-PCR, and enzyme electrophoresis (zymodeme), and digestion profiles of kinetoplast DNA with restriction enzymes (schizodeme) showed that parasites from the Americas, including those from Paraguay, Brazil, and Ecuador, are different and have been denominated as L. major-like (Yamasaki et al. 1994, De Oliveira Silva et al. 2009, Wu et al. 2015. Berzunza-Cruz et al. (2002) reported L. major in clinical isolates using RFLP studies in Mexico, and it has been suggested that this species may have been introduced to the New World. In Ecuador, several studies also reported L. major-like in clinical isolates from patients with cutaneous leishmaniasis from Andean highland areas (Hashiguchi et al. 1991, Kato et al. 2016a). This is the first recorded natural infection of L. major-like parasites in sand flies of the genus Psychodopygus (Ps. panamensis) in Ecuador (from Puerto Quito-Pichincha). The comparison of ITS1 sequences obtained of L. major-like was not possible because there are not registered sequences for this strain in Table. 1. Leishmania species detected in sand flies collected in two ecotopes in eight localities of Ecuador during the rainy and dry seasons. the GenBank. Further sampling and analysis from the same area are needed to determine the identity and relationships of this group of Leishmania parasites and their vectors.

Leishmania lainsoni
The ITS1 sequences from two sand fly specimens collected in Macas, Morona Santiago province, were grouped with Leishmania lainsoni (BP ¼ 74; 97). This species has already been reported from a human lesion collected in the province of Sucumb� ıos, Ecuador (Kato et al. 2016a). Leishmania lainsoni has been detected in Lutzomyia nuneztovari (Young & Duncan 1994) in Bolivia (Bastrenta et al. 2002), in Lutzomyia auraensis in Per� u (Valdivia et al. 2012), and in Trichophoromyia ubiquitalis in Brazil (Silveira et al. 1991, Lainson et al. 1992. The latter report is consistent with the identification of L. lainsoni in the sand fly Th. ubiquitalis in our study.

Leishmania naiffi
Psychodopygus carrerai carrerai collected in Macas-Morona Santiago province and Presatia dysponeta from Cumand� a-Chimborazo were infected with parasites placed in the Leishmania naiffi group (BP ¼ 79). This is the first record for Ecuador of these sand fly species naturally infected with L. naiffi. This species of Leishmania is known to occur widely in South America and the Caribbean, where it causes cutaneous leishmaniasis (CL) in humans (Pratlong et al. 2002). Previous studies in the province of Orellana, located in the Amazon region of Ecuador, detected Leishmania naiffi in clinical samples from patients with cutaneous leishmaniasis and Lutzomyia yuilli and Lu. tortura as the vectors (Kato et al. 2005, Kato et al. 2013. Azpurua et al. (2010) found Lu. trapidoi and Lu. gomezi infected with L. naiffi in Barro Colorado Island in Panam� a. The analysis of the gene sequences placed our sample in the cluster that is related to the subgenus Sauroleishmania and Leishmania braziliensis complex with the same BP value (79; Fig. 2). A further analysis with other genes may allow species differentiation in the cluster.

Leishmania mexicana
The sand flies collected in Progreso, province of Guayas, were identified as Micropygomyia cayennensis and the parasites extracted from them grouped in the cluster with Leishmania mexicana. Sand flies of the genus Micropygomyia are known to feed on coldblooded animals, which does not correspond to the habitat requirements of the Leishmania parasite (Rêgo et al. 2015). However, this result is consistent with findings reported in Colombia and Venezuela, where Mi. cayennensis was reported infected with Leishmania (Feliciangeli 1987, Alveiro et al. 2015. In Colombia, this sand fly attacks humans and is very common inside homes (Cochero et al. 2007, Cortés andFern� andez 2008). Detection of L. mexicana in Mi. cayennensis expands the distribution of the parasite in the lowlands along with a new vector. Hashiguchi et al. (1991) reported the possible vector incrimination of Lutzomyia ayacuchensis with Leishmania mexicana. Kato et al. (2005) registered the presence of Lutzomyia ayacuchensis in the provinces of Manab� ı, Pichincha, Chimborazo, and Azuay, and Gomez et al. (2014) report this species at altitudes >650 m. The analysis of the geographic distribution of cases of cutaneous leishmaniasis by phylogenetic relationships based on cytochrome b revealed the presence of Leishmania mexicana in the Andes of Ecuador (Kato et al. 2016a). However, Calvopiña et al. (2006) reported an isolate of L. mexicana from a human clinical case in the Pacific lowlands. Owing to the wide distribution of the parasite that includes the highlands and the lowlands, it is necessary to identify the parasite using other molecular markers such as cytochrome b and ITS2, and also determine the polymorphisms.
"Leishmania sp. siamensis" ITS1 sequences isolated from sand flies corresponded to 100% homology with "Leishmania sp. siamensis" and Leishmania sp. According to Akhoundi et al. (2016), this species has been reported in both Old and New World. However, the name "Leishmania siamensis" is not a valid taxon and is used with quotation marks (Akhoundi et al. 2016). This species has been reported in Central Europe, Thailand, and United States, where it causes visceral and cutaneous leishmaniasis (Sukmee et al. 2008, Bualert et al. 2012, Reuss et al. 2012, Kanjanopas et al. 2013, Akhoundi et al. 2016.
The isolates are grouped in the cluster Leishmania sp. and "Leishmania siamensis" (BP ¼ 70), but they did not group with the subgenus Sauroleishmania as was expected according to the classification proposed by Fraga et al. (2009). A detailed phylogeny of the genus Leishmania is required with a greater number of genes from parasites in different geographic ranges to determine the degree of polymorphisms to clarify the evolution and epidemiology of the genus (Berzunza-Cruz et al. 2002, Yang et al. 2013.
The sequences from samples corresponded to 98% homology with "Leishmania sp. siamensis." The sand flies hosting these parasites were identified as Br. leopoldoi (F005), Ps. panamensis (F006), Ps. carrerai carrerai (F008), Ny. yuilli yuilli (F010), Ny. trapidoi (F016), and Pr. triacantha (F017). Valdivia et al. (2012) reported Pressatia sp. sand flies infected with Leishmania sp. for the first time in Per� u. Araujo-Pereira et al. (2017) also reported Pressatia sp. infected with Leishmania brazilienzis. These findings, along with ours, underscore the importance of further investigations to determine the species involved as the potential vectors of leishmaniasis. Leishmania naiffi has been found infecting Lutzomyia trapidoi and Lu. gomezi (Azpurua et al. 2010), whereas the transmission of Leishmania lainsoni is associated with the sand fly Trichophoromyia ubiquitalis. Our results indicate that under these conditions, humans potentially can become infected with more than one Leishmania species. This is the first record of natural infection of T. ubiquitalis with L. lainsoni in Ecuador, which may help to establish the distribution of the parasite and its vector. According to (Forattini 1973), Brumptomyia species do not have epidemiological importance in the transmission of Leishmania spp. to humans, and some species are known for feeding on other mammals like armadillos.
The high number of sand fly species and engorged females found in the peridomiciliary ecotope may be related to the availability of blood sources. It has already been reported that the feeding behavior of sand flies in this ecotope is highly diversified and includes blood from humans, chickens, cows, and dogs (Anaguano et al. 2015). However, it is essential to determine if the domesticated animals are reservoirs of the disease, as the risk for humans would be higher in these areas than in the forest.
Crithidia fasciculata (Kinetoplastida: Trypanosomatidae) found in some of the sand flies is a known exclusive parasite of several orders of insects, including Diptera (Wallace 1966). Trypanosoma sp. detected in some sand flies may be parasites of anurans and reptiles reported in other studies in the neotropics (Viola et al. 2008).
Determining the distribution and identify of Leishmania species requires standardization of methodologies to identify DNA isolated from clinical samples and sand fly vectors. Therefore, only whole-genome sequencing of the isolated parasite would generate consensus regions and establish target nucleotide sequences of mitochondrial or nuclear genes for interspecies difference. Similarly, the correct vector incrimination requires the right identification of the collected sand fly specimens.
The high diversity of sand flies and high rates of Leishmaniainfected insects, especially in the peridomiciliary ecotope, suggests a very complex epidemiology of the disease. Consequently, determination of vector-parasite-reservoir relationships is needed to quantify the risk in these areas and implement preventive and control strategies.