Natural infections of Pintomyia verrucarum and Pintomyia maranonensis by Leishmania (Viannia) peruviana in the Eastern Andes of northern Peru

The natural infection of sand flies by Leishmania was investigated in Andean areas located between the Central and Eastern Cordilleras of northern Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured at five locations along the Utcubamba River in the Department of Amazonas, and morphologically identified under a microscope. Among 422 female sand flies dissected, the most dominant species was Pintomyia verrucarum (320 flies), followed by Pi. maranonensis (83 flies), Pi. robusta (13 flies), and Lutzomyia castanea (6 flies). Genetic analysis of sand flies from these areas together with those from other areas revealed that individuals of Pi. verrucarum were closely related regardless of morphological variation of their spermathecae. On the other hand, individuals of Pi. maranonensis collected in the study area were distant from those of other areas with genetic distances over the intraspecific level but mostly below the interspecific level, suggesting the unique characteristics of sand flies in this area. The natural infection of sand flies by flagellate parasites was detected mainly in the hindgut of each one of Pi. verrucarum and Pi. maranonensis. Both parasite species were identified as L. (V.) peruviana based on cytochrome b and mannose phosphate isomerase gene analyses. In addition, parasite species obtained from the lesion of a patient with cutaneous leishmaniasis in the study area in this period was identified as L. (V.) peruviana. These results strongly suggest that Pi. verrucarum and Pi. maranonensis are responsible for the transmission of L. (V.) peruviana in these areas. This is the first report of the natural infection of Pi. maranonensis by L. (V.) peruviana.

Introduction Phlebotomine sand flies are blood-sucking insects belonging to the family Psychodidae in the order Diptera [1,2]. To date, 1,020 sand fly species have been recorded in the world, of which about 550 species are in the New World [3]. The identification of sand fly species is medically important since approximately 10 percent of them are responsible for the transmission of human pathogens such as Leishmania protozoa [2,4,5]. In addition, each vector species transmits specific Leishmania species, and the infecting species is the major determinant of the clinical outcomes, such as cutaneous, mucocutaneous, and visceral disorders [4][5][6]. Therefore, studies on sand fly fauna and the identification of vector species of leishmaniasis in endemic and surrounding areas are important for predictions of the risk of transmission and expansion of the disease.
Peru is one of the most highly endemic countries for cutaneous leishmaniasis (CL), distributed through the country from highlands to lowlands, whereas mucocutaneous leishmaniasis (MCL) in this country is endemic mostly in Amazonian areas [7,8]. Six Leishmania species and several hybrids have been recorded as responsible for leishmaniasis in this country [7][8][9]. Of these, predominant causative agents are L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis, mainly circulating in the Andean highlands, tropical rainforest, and northern to central rainforest areas, respectively [7,8,10,11]. Approximately 190 sand fly species have been recorded in Peru, and information on prevalent sand fly species is accumulating, especially in Andean areas; however, the vector species responsible for the transmission of Leishmania remains to be identified in most endemic areas [12]. The vector species of L. (V.) peruviana was identified as Lutzomyia ayacuchensis in the western valley of central Andes [13] and Lu. peruensis in northern and central Andes [14][15][16][17]. Pintomyia verrucarum, a widely distributing species in Andean highlands, was reported to have the capacity to transmit L. (V.) peruviana under experimental conditions [18], and the natural infection of Pi. verrucarum by Leishmania species was detected by PCR using pooled sand fly samples from the west Andean slope in central Peru, although the parasites were not identified at the species level [19]. In addition, Lu.
tejadai was reported as a vector of a hybrid of L. (V.) braziliensis and L. (V.) peruviana in the central Andes of Peru [20].
In the present study, to further disclose circulating sand fly species and identify the vectors of Leishmania protozoa, sand flies were investigated in endemic areas along the Utcubamba River in Provinces of Chachapoyas, Luya, and Bongara, Department of Amazonas located in the Eastern Andes of northern Peru where CL caused by L. (V.) peruviana is endemic [7,8].

Ethics statement
Verbal informed consent was obtained prior to the sample collection, providing information on the process of diagnosis and Leishmania species analysis, following the guidelines of the Ethics Committee of the Ministry of Health, Peru. The study was approved by the ethics committee of Jichi Medical University (approval number: 17-080) [8,9].

Sand fly collection
Sand flies were captured with a mouth aspirator on protected human bait between 18:30 and 21:00 and CDC light traps operated throughout the night from 18:00-06:00 for 11 nights in July 2019 around patients' houses in the rural area at five localities along the Utcubamba River in the Provinces of Chachapoyas, Luya, and Bongara, Department of Amazonas (Fig 1). Female sand flies were dissected and identified at the species level mainly based on the morphology of their spermathecae [6,21]. They were also examined under light microscopy for natural flagellate infections, and samples were fixed individually in absolute ethanol. Ethanolfixed specimens were dried up and individually lysed in 50 μL of DNA extraction buffer [150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA and 0.1% sodium dodecyl sulfate (SDS)] containing proteinase K (100 μg/mL). The samples were incubated at 37˚C overnight, heated at 95˚C for 5 min, and then 0.5 μL of each sample was directly used as a template for PCR amplification [16,17,20,22,23].

Clinical samples
A clinical sample was collected from a patient suspected of having CL. A tissue sample was taken by scraping the margins of an active cutaneous lesion of the patient, spotting the scrapings onto an FTA Classic Card (Whatman, Newton Center, MA) and storing it at room temperature. Two-mm-diameter disks containing the sample spot were punched out from the card and washed three times with FTA Purification Reagent (Whatman) and once with Tris-EDTA buffer. The disks were air-dried and directly subjected to PCR amplification [7,8,20].

Phylogenetic analysis
The obtained sequences were aligned with CLUSTAL W software [27] and examined using the program MEGA (Molecular Evolutionary Genetics Analysis) version 6 [28]. Phylogenetic trees were constructed by the maximum likelihood (ML) method with the best ML model selected based on the lowest BIC score (Bayesian Information Criterion) in MEGA 6 [28]. Branch support for the ML tree was calculated using the bootstrapping method with 1,000 replicates [28].

Detection and identification of flagellates in sand flies
In the present study, 422 female sand flies were dissected for identification at the species level, and four species were recognized. Among them, the most dominant species, Pi. verrucarum (320 flies), was captured in all localities, and the remaining three species, Pi. maranonensis (83 flies), Pi. robusta (13 flies), and Lu. castanea (6 flies), were captured only in Nuevo Churuja (Table 1). Interestingly, Pi. verrucarum showed variations in the morphology of spermathecae, regardless of the collection sites; Some were shrinking, some were round, and the others were elongated (S1 Fig). Natural infection of sand flies by flagellates was detected mainly in the hindgut of each one of Pi. verrucarum from Tingo Bajo and Pi. maranonensis from Nuevo Churuja under the microscope (Fig 2). The infection rates of Pi. maranonensis and Pi. verrucarum with Leishmania in the present study were 0.3% and 1.2%, respectively. The cyt b gene fragments were amplified from the parasites and subjected to sequence analyses.

Identification of Leishmania species from a patient's specimen
During this field research, a clinical sample was obtained from the cutaneous lesion of a patient with CL in Tingo Bajo where a flagellate-infected sand fly was detected. The cyt b gene fragment was analyzed, and the sequence (19-7TI-1) (GenBank accession number: LC593673) had a greater degree of homology with those of L. (V.) peruviana and L. (V.) braziliensis (99.5-99.7%) than others. The result was supported by a phylogenetic analysis (Fig 3A). PCR-RFLP analysis of the mpi gene showed that the parasite species was L. (V.) peruviana (Fig 3B).

COI gene analyses of Pintomyia verrucarum and Pintomyia maranonensis
Since Pi. verrucarum showed morphological variations of spermathecae regardless of their habitat, genetic analysis based on the COI gene was performed on Pi. verrucarum and Pi. maranonensis, in which Leishmania infection was detected in this study, to identify their genetic diversities in comparison with those from other areas. Sequences of 630-bp COI gene fragments were determined in 7 Pi. verrucarum including the Leishmania-positive sample (Ting-p) collected in this study (GenBank accession numbers: LC593641-LC593647), and a phylogenetic analysis was performed together with those from other areas registered in GenBank (Fig 1). Pintomyia verrucarum captured in Tingo Bajo (Ting1-1, Ting1-2, Ting2-1, Ting3-1, Ting3-2, and Ting-p) and Nuevo Churuja (Chur-v2) of the Department of Amazonas were closely related to each other with genetic distances of 0.0-0.2% independent of the morphological variation of spermathecae ( Fig 4A). COI gene sequences of Pi. verrucarum from the Department of Amazonas registered in GenBank (Ama10, Ama11, and Ama14) were located in the same clade as our samples. On the other hand, COI gene sequences of Pi. verrucarum from Departments of Piura (Piu01 and Piu17), Cajamarca (Caj09, Yuram02, and Yumpe15), and Lima (Lim01 and Lim07) composed different clades from those of Amazonas samples (Fig 4A). Genetic distances based on Kimura 2-parameter values were 2.4-2.8% between Amazonas and Piura samples, 2.1-3.1% between Amazonas and Cajamarca samples, and 2.3-2.8% between Amazonas and Lima samples, all of which were intraspecific diversity levels (<6.0%) [26].
The study area is located between the Central and Eastern Cordilleras of the Andes in northern Peru [29]. Epidemiological studies on leishmaniasis in the Peruvian Andes, including sand fly research, have been carried out mainly in endemic areas located in the North, Center, and South of the Western Andes, and occasionally in the North and Central inter-Andean valleys of the Central Andes and in the southern zone of the Eastern Andes. Lu. ayacuchensis and Lu. peruensis, which distribute in western valleys of the Central Andes and Northern and Central Andes, respectively, were identified as vectors of L. (V.) peruviana, the primary etiological agent of CL in the Peruvian Andes [13][14][15][16]. On the other hand, the vector responsible for the transmission of CL remains to be elucidated in endemic areas of the Eastern Andes. Interestingly, the distribution of both Lu. ayacuchensis and Lu. peruensis has not been reported in these areas, suggesting that some factors that may affect distributing sand fly species, such as the ecosystem, fauna, and flora, are unique in these areas.
In the study areas, L. (V.) peruviana was identified as the causative agent of CL in previous studies [7,8], and this was confirmed in the present study. The vector species responsible for the transmission of L. (V.) peruviana has not been identified. The present study detected flagellate parasites mainly in the hindguts of Pi. verrucarum and Pi. maranonensis, and both parasites were identified as L. (V.) peruviana, strongly suggesting that these sand fly species are responsible for the transmission of CL in these areas. Pi. verrucarum is widely distributed in Andean mountainous areas of the Eastern and Central Cordillera, and well-known as the primary vector of Bartonella bacilliformis, the etiologic agent of Carrion's disease, also known as Oroya fever [12]. Pintomyia verrucarum was reported to have the capacity to transmit L. (V.) peruviana under experimental conditions [18], and the natural infection of the sand fly by unidentified Leishmania species was detected by PCR in an endemic area [19]. These findings strongly suggest that Since morphological variation of spermathecae was noted in Pi. verrucarum during the microscopic examination, genetic analysis of these flies was performed to identify the difference depending on the morphological characteristics. Interestingly, individuals of Pi. verrucarum in the study areas were closely related to each other regardless of the morphological variation, and their genetic diversities were within the intraspecific level when compared with those from other areas, suggesting that spermathecae of Pi. verrucarum are more flexible than those of other species. This finding should be taken into account on the morphological identification of sand flies, especially when unfixed specimens are used. Another Leishmania-positive sand fly species, Pi. maranonensis, was also subjected to genetic analysis, although morphological variation was not observed. Unexpectedly, Pi. maranonensis in the study area was genetically distant from flies of other areas with genetic distances between intraspecific and interspecific levels. This is the first report on the transmission of Leishmania species by Pi. maranonensis. Since Pi. maranonensis in the study area may have unique characteristics of the intestinal environment that result in acquiring the vectorial capacity, further studies associated with vector competence, such as mid-and hindgut molecules and the microbiome, would be interesting [30][31][32].
In the present study, the natural infection of sand flies by Leishmania was microscopically examined in the northern Peruvian Andes on the Cordillera Central where CL caused by L.
(V.) peruviana is endemic. Both flagellates detected within Pi. verrucarum and Pi. maranonensis were identified as L. (V.) peruviana, strongly suggesting that Pi. verrucarum and Pi. maranonensis are responsible for the transmission of leishmaniasis in these areas. Genetic divergence of Pi. verrucarum in these areas was confirmed to be at the intraspecific level regardless of their morphological variations, whereas Pi. maranonensis was genetically characteristic regarding the genetic distance between intra-and interspecific levels when compared with flies from other areas. Since Pi. maranonensis has never reported to transmit Leishmania species, this species in the study area is expected to have unique characteristics associated with the vectorial capacity. Further studies on the microenvironment of the mid-and hindgut, in which particular parasite species can develop, will be expected to provide further insight into the vector competence of sand flies. On the other hand, more detailed morphological and molecular analyses of Pi. maranonensis in the study area will be needed since the possibility of a new species cannot be ruled out, based on the finding that genetic distances of the COI gene with those from other areas were over the intraspecific level.