Diversity, distribution and natural Leishmania infection of sand flies from communities along the Interoceanic Highway in the Southeastern Peruvian Amazon

The Peruvian-Brazilian border is a highly endemic tegumentary leishmaniasis region in South America. The interoceanic highway is a commercial route that connects Peru and Brazil through Madre de Dios and has raised concerns about its impact on previously undisturbed areas. In order to assess leishmaniasis transmission risk along this highway, we conducted a surveillance study of the sand fly populations in this area. Sand flies were collected between 2009 and 2010 along transects at 200 m, 600 m and 1000 m from six study sites located along the highway (Iberia, La Novia, Alto Libertad, El Carmen, Florida Baja, Mazuko and Mavila) and an undisturbed area (Malinowski). Collected specimens were identified based on morphology and non-engorged females of each species were pooled and screened by kinetoplast PCR to detect natural Leishmania infections. A total of 9,023 specimens were collected belonging to 54 different Lutzomyia species including the first report of Lu. gantieri in Peru. Four species accounted for 50% of all specimens (Lutzomyia carrerai carrerai, Lu. davisi, Lu. shawi and Lu. richardwardi). El Carmen, Alto Libertad, Florida Baja and Malinowski presented higher Shannon diversity indexes (H = 2.36, 2.30, 2.17 and 2.13, respectively) than the most human disturbed sites of Mazuko and La Novia (H = 1.53 and 1.06, respectively). PCR detected 10 positive pools belonging to Lu. carrerai carrerai, Lu. yuilli yuilli, Lu. hirsuta hirsuta, Lu. (Trichophoromyia) spp., and Lu. (Lutzomyia) spp. Positive pools from 1,000 m transects had higher infectivity rates than those from 600 m and 200 m transects (9/169 = 5.3% vs 0/79 = 0% and 1/127 = 0.8%, p = 0.018). El Carmen, accounted for eight out of ten positives whereas one positive was collected in Florida Baja and Mazuko each. Our study has shown differences in sand fly diversity, abundance and species composition across and within sites. Multiple clustered Lutzomyia pools with natural Leishmania infection suggest a complex, diverse and spotty role in leishmaniasis transmission in Madre de Dios, with increased risk farther from the highway.


Introduction
Leishmaniasis is a complex parasitic disease caused by digenetic protozoans belonging to the genus Leishmania that is transmitted by the bite of infected phlebotomine sand flies. This disease is endemic in 98 countries with up to 350 million people at risk; causing more than 1.5 million new cases per year with an estimated 12 million people living with the infection [1].
Leishmaniasis is caused by at least 20 different Leishmania species and presents a wide spectrum of clinical manifestations that are divided into tegumentary (TL) and visceral leishmaniasis (VL) [2]. In the New World, TL is the most common clinical form of leishmaniasis and causes skin sores that can lead to mutilation and disfigurement. Most of these cases are caused by species of the Viannia subgenus, including L. (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) panamensis [3,4].
Leishmania transmission relies on the interaction between an infected phlebotomine sand fly of the genus Phlebotomus in the Old World or Lutzomyia in the New World, with a mammal host [5]. Therefore, the identification of putative Leishmania vectors provides key information for epidemiological control and risk assessment in endemic areas.
Peru is one of the ten countries where more than 75% of TL cases worldwide occur, with more than 6,563 cases reported in 2017 [1,6]. The Peruvian Amazon is an endemic leishmaniasis region due to the broad diversity of sand fly vectors, reservoirs and human activities [7]. However, information about the geographic distribution of Leishmania vectors, their role in disease transmission and the variables affecting their distribution is still scarce.
The Interoceanic Highway opened in 2005 and is an international route that interconnects Peru and Brazil through Madre de Dios (Peru) and Acre (Brazil) States. Currently, there is major concern about its potential impact on previously undisturbed areas due to human activities and their effects on the diversity, abundance and distribution of leishmaniasis vectors and disease [8,9].
In this study, we conducted sand fly surveillance in communities in Madre de Dios and Puno states along the Interoceanic Highway in Peru and employed molecular tools to identify natural Leishmania infections in sand flies collected in this region of the Peruvian-Brazilian Amazon Basin. Our results will serve as a basis for the identification of putative Leishmania vectors in this area to better assess the long term impact of human activities conducted along the highway on the transmission of leishmaniasis in the region.

Ethics statement
This study was exempt from NAMRU-6 IRB review as this project did not involve humans as the subject of the study evaluation. Therefore, this study did not meet the definition of research involving human subjects.
Sand fly collections were performed under approval from the General Directorate of Forestry and Wild Fauna from the Ministry of Agriculture and Irrigation of Peru.

Study area
The study was conducted in the communities of Iberia, La Novia, Mavila, Alto Libertad, Florida Baja, Mazuko (Madre de Dios, Peru), and El Carmen (Puno, Peru). All communities are located along the Interoceanic Highway route. An additional site (Malinowski), which is located within the limits of Tambopata Natural Reserve and Bahuaja-Sonene National Park (Madre de Dios) was selected as an undisturbed habitat control site (Fig 1).
These sites have a humid sub-tropical climate with annual temperatures between 22˚C to 34˚C and annual precipitation of more than 3,000 mm. The presence of Leishmania has been extensively documented in

Sand fly collection and taxonomy
Fieldwork took place between October 2009 and October 2010. In each study site, three 350 m transects were set-up at 200 m, 600 m and 1,000 m from the left and right margins of the highway. All transects were located in settings nearby urban and peri urban areas. Human activities were observed more frequently in the 200 m transects were the secondary forest is predominant; transects at 600 m present intermediate, primary and secondary forest whereas transects at 1,000 m were characterized by the presence of primary forest, wild animals and limited human activity. Sand flies were collected at each site for five consecutive nights using six Mini CDC light traps Model 512 (John W. Hock) placed every 50 m along each transect (Fig 2). Protected human bait was performed at one end of each transect and a Shannon trap was placed at the midpoint between the 600 m and 1,000 m transects.
Collected sand flies were stored in 70% ethanol and transported to the NAMRU-6 Entomology Department laboratory in Lima where they were identified based on keys developed by Young and Duncan [11] and Galati [12]. Non-engorged females were pooled in groups of 2-10 specimens or stored individually depending on species, study site, collection date and trap type.
Female specimens were processed using a protocol that allows molecular analysis. Briefly, the head and the two last abdominal segments were separated and placed in lactophenol (Acid Lactic:Phenol 3:2) for two hours at room temperature. These body parts contain key taxonomic structures (cibarium, palpal segments, flagellomeres and spermathecae) which are used for species identification. The remaining parts of the sand fly were preserved in ethanol 70% at -20˚C for molecular testing. Male specimens were prepared for mounting and identification with slight modifications from those described by Young & Duncan [11]. Male specimens were placed in 20% KOH for 12 to 24 hours at room temperature. Then, specimens were clarified with lactophenol for two hours at room temperature. Male and female specimens were mounted permanently on Euparal as previously described [13].

Data analysis
Shannon-Wiener (H) diversity indexes were calculated for each study site including both male and female specimens collected by all trapping methods using the equation H ¼ À P n i¼1 p ln p where "p" represents the proportion in which each species "n" was collected (∑p = 1). Statistical significant differences among Shannon-Wiener (H) diversity indexes were assessed by the Hutcheson t-test adjusting the false discovery rate using the Benjamini Hochberg method [14,15]. The Index of Species Abundance (ISA) [16] was calculated using the formula ISA = (a + Rj)/ k. This method implies establishing a rank for species abundance per site and then calculating "a" as the number of zero observations for each species in all sites multiplied by "c" which is the single largest rank in all the data set plus 1. The Rj value corresponds to the sum of ranks for a given species in all the sites whereas "k" corresponds to the number of sites. The resulting ISA values were converted into the Standardized Index of Species Abundance (SISA) using the formula SISA = (c − ISA)/(c − 1).
Transect based analysis was performed only on data obtained from collections performed at the left side of the highway because not all transects were sampled on the right margin. Comparisons of Shannon Diversity indexes between transects of the same location were conducted using the Hutcheson T-test. Mavila was excluded from the analysis since collections were only performed at the 1,000 m transect.

DNA extraction and polymerase chain reaction
Pools of less abundant species were not analyzed. In addition, only 50 specimens were analyzed out of pools with more than 50 specimens in a given species, site and transect.
DNA was isolated using the Gentra Puregene kit (QIAGEN, Germantown, US) following the standard manufacturer's protocol for isolation of genomic DNA.
Detection of Lutzomyia DNA was done by targeting the 12S ribosomal DNA using primers T1B 5 0 -AAA CTA GGA TTA GAT ACC CT-3 0 and T2A 5 0 -AAT GAG AGC GAC GGG CGA TGT-3 0 as previously described [17]. Reactions were prepared in 25 μL that contained 1X Taq polymerase buffer (Invitrogen, Carlsbad, US), 1.5 mM MgCl 2 , 125 μM dNTPs (Invitrogen, Carlsbad, US), 0.5 μM of each primer, 1 unit of Taq DNA polymerase (Invitrogen, Carlsbad, US), and 5 μL of DNA sample. The PCR was run under the following cycling conditions: initial denaturation at 94˚C for 5 minutes followed by 35 cycles of denaturation 94˚C for 20 sec, annealing at 56˚C for 30 sec, and extension at 72˚C for 25 sec; and a final extension step at 72˚C for 5 min. The reaction generates a product of approximately 400 base pairs (bp).
The PCR conditions consisted of an initial denaturation for 5 minutes at 94˚C; followed by 35 cycles for 45 seconds at 94˚C, 45 seconds at 58˚C, and 1 minute at 72˚C; and a final extension step for 5 minutes at 72˚C. This reaction generated a 70 bp product specific for the Viannia subgenus.
Minimum infection rates were calculated for all species with positive Leishmania PCR results (number of positive pools/total number of specimens tested x 1,000). This estimate is based on the assumption that positive pools contains one infected sand fly.

Sand fly species diversity
During the study period, we collected 9,045 sand flies belonging to the genera Lutzomyia Species identification using taxonomical keys developed by Young & Duncan (1994) and Galati (2003) resulted in 54 Lutzomyia species and one Brumptomyia species (S1 Table). Among the Lutzomyia species, we report for the first time the presence of Lutzomyia gantieri in Peru, collected in the Mazuko site (S1 Fig).
Some subgenus Trichophoromyia specimens were not identified at the species level due to the high similarity of the female morphology within the subgenus, the lack of male specimens or the detection of multiple circulating species based on male morphology (for instance Lu. auraensis, Lu. clitella, Lu. nemorosa, Lu. sinuosa and Lu. ubiquitalis).
The  Table).
The lower sand fly diversity in the highly disturbed, illegal gold mining sites of La Novia and Mazuko coincided with single species predominance. Lutzomyia shawi accounted for 59% of all collected specimens in La Novia, and Lu. carrerai carrerai for 42% in Mazuko.
Collection methods resulted in significant differences between Shannon diversity indexes estimated with CDC light traps and Shannon traps in El Carmen, Florida Baja and Malinowski (FDR t-test adjusted p < 0.05, S2 Table).

Transect analysis
Transect based analysis was conducted on collections performed at the left margin of all study sites except for Mavila which was excluded because collections were only performed at the 1,000 m transect, and Iberia where sand fly collections were performed only in the 200 m and 600 m transects. Higher abundance was observed in the 600 m transect compared to each the 200 m and 1000 m transects across all sites (p < 0.05) (Fig 4).
The analysis per location and transect showed differences in species abudance in each site (S3 Table and

Natural Leishmania infections
Non-engorged female sand fly specimens were grouped into 567 pools (56% of the total sand fly females collected) based on species, trap type, transect and site. PCR analysis of the 12S ribosomal DNA confirmed the presence of Lutzomyia DNA and ruled out the presence of PCR inhibitors on our samples.
El Carmen accounted for eight out of ten positive pools (7 at 1000m, and 1 at 200m transects) whereas the other two positive pools were from Mazuko and Florida Baja ( Table 1). Shannon traps accounted for most of the positive pools (eight out of ten positive pools) whereas CDC accounted for the remaining two positive pools.

Discussion
The South Eastern Peruvian Amazon is an endemic foci of TL [7,24] that is experiencing dramatic land use changes due to different activities such as illegal mining, logging, agriculture and others that are related to the Transoceanic Hhighway that connects Peru, Brazil and Bolivia.
Given the locations of our study sites along the highway, our results were compared to those obtained in the other side of the border [25,26]. In our study, we found a high density of Lutzomyia species of the subgenera Psychodopygus (55.4%), Nyssomyia (26.0%) and Trichophoromyia (13.9%) on the Peruvian side of the border; whereas Teles et al. [25] found a higher  https://doi.org/10.1371/journal.pntd.0009000.g005 Table 1. Positive pools according to Lutzomyia species, study site and transect. The table shows that the majority of positive pools were collected in El Carmen, at the 1,000 m transect. The SISA index for each species per locality is also shown and the trap types as CDC Light traps (CDC) or Shannon trap (SH). The last column shows reports of Leishmania species naturally infecting these sand fly species [19][20][21][22][23]. density of the subgenera Trichophoromyia (39.51%), Psychodopygus (27.34%), Pressatia (9.89%) and Nyssomyia (8.58%) in the municipality of Assis, located in Acre (Brazil). A similar study conducted in Rio Branco, Brazil [26] reported a lower number of sand flies and diversity (23 sand fly species) with the subgenera Trichophoromyia (53.3%) and Nyssomyia (23.5%) as the most prevalent [26].

Species
These comparisons indicate that sand fly distribution and species composition is substantially different between Peruvian and Brazilian border regions. It is possible that environmental and anthropogenic variables including increased urbanization or lower precipitation rates in the Brazilian sites are responsible for these differences [27,28]. Additionally, differences in the study designs, time and year of collection could also explain the differences found.
Our collections showed not only high heterogeneity of sand fly abundance and diversity between regions but also within the same region as shown by the significant differences found at the transect level. These micro-environments with specific sand fly species can be susceptible to anthropogenic change. However, it is important to consider that our study was limited to a single collection at each site and therefore could not assess changes in sand fly diversity and abundance through time.
It has been suggested that residential development in previously forested regions may prompt the spread of TL in these areas [27]. These environmental modifications probably result in shifts in the sand fly population, vector replacement or the introduction of other sand fly species that could in turn transmit the disease [8]. Moreover, the presence of domestic and wild animals in the peridomicile may attract large number of sand flies, thus increasing the risk of transmission [29].
The highest impact of anthropogenic activities was observed in Mazuko and La Novia, where the lowest sand fly species diversity was found. Reduction of species diversity on these sites seemed to happen together the rise of single species predominance (>40% for each Lu. carrerai carrerai and Lu. shawi, respectively). Such high abundance suggests that these two sand fly species, which were found to be infected with Leishmania, may have adapted well to these areas of transitional ecology. It has been reported that multiple Lutzomyia species are involved in leishmaniasis transmission in the Peruvian-Brazilian Amazon Basin, forming hotspots of multiple infected sand fly species cohabitating in the same region [7,26]. Our study clearly reflects that pattern, with 80% of all positive pools found in the same transect (1,000m) of a single site (El Carmen), and across three distinct Lutzomyia species (Lu. yuilli yuilli, Lu. (Helcocyrtomyia) spp. and Lu. hirsuta hirsuta).
We were unable to identify the infecting Leishmania species on the positive pools but all the Lutzomya species found positive in our study have previous reports of natural Leishmania infection.
Previous studies have shown that species of the Helcocyrtomyia subgenus such as Lu. peruensis, Lu. ayacuchensis and Lu. tejadai are highly anthropophilic and have major a role in the transmission of Leishmania (Viannia) spp. in Peru [22].
In summary, we found that the sand fly fauna in the Peruvian-Brazilian Amazon Basin is differentially distributed between and within study sites and transects with substantial heterogeneity. Local hot spots of TL transmission appear to occur due to the presence of different species of infected sand flies cohabitating in the same area. Therefore, prevention and control strategies should consider focusing in such areas first to reduce the risk of leishmaniasis transmission. Finally, this study may serve as a basis to better assess the effects that human activities and land use change have in the sand fly fauna and leishmaniasis in the Peruvian southeastern Amazon.  Table. Species abundance per location and transect. The table shows the number of collected specimens per species at each transect in each study site. Differences were found between sites and within transects at the same site. (XLSX) Some authors of this manuscript are military service members and employees of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that "Copyright protection under this Title is not available for any work of the United States Government". Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.