Figures
Abstract
Leishmaniasis is a zoonotic disease where parasites of the genus Leishmania are transmitted among hosts by sandfly vectors of the subfamily Phlebotominae. In Paraguay, studies on sandfly biodiversity are limited, even though the disease is present in its tegumentary and visceral forms. This pioneering ecoepidemiological study analyzes the diversity, abundance, and detection of Leishmania spp. DNA in sandflies from primary, secondary, and degraded forest habitats within the Mbaracayú Forest Biosphere Reserve. Over a year of sampling, 954 sandflies were captured, comprising 599 females and 355 males, belonging to 12 species, 10 of which are reported for the first time in Canindeyú. The species with the highest positivity for Leishmania spp. DNA were the Evandromyia cortelezzii complex (20.3%), Pintomyia monticola (18.8%), Brumptomyia brumpti (17.4%), Brumptomyia avellari (15.9%), and Psathyromyia lanei (11.6%). In contrast, Evandromyia evandroi, Micropygomyia quinquefer, Nyssomyia neivai, and Migonemyia migonei displayed rates of 4.3% or lower, while Psathyromyia shannoni complex, Brumptomyia guimaraesi, and Evandromyia termitophila were negative. The identified Leishmania species were L. amazonensis (65.2%) and L. infantum (34.8%). Additionally, dogs and their ectoparasites from an indigenous community were analyzed, detecting L. amazonensis DNA in 18 flea groups (54.5%) and one tick (16.7%). However, all dogs tested negative for the Rk39 test. This study highlights the risk of leishmaniasis transmission in forested and degraded ecosystems, which could have significant public health consequences, calling for deeper analyses to assess associated risks.
Author’s summary
The interaction between natural ecosystems and human activities in the Mbaracayú Forest Biosphere Reserve reveals a complex leishmaniasis transmission network that challenges traditional control strategies. This study uncovers a remarkable diversity of sandflies, coupled with the detection of Leishmania spp. DNA in vectors and dog ectoparasites, highlighting unexplored ecoepidemiological dynamics in the region. The findings emphasize the potential role of previously underestimated species in the transmission of L. amazonensis and L. infantum, even in the absence of traditionally incriminated vectors. The detection of L. amazonensis DNA in dog ectoparasites underscores a complex and multifaceted transmission process, where domestic and wild reservoirs interact within a delicate ecological balance. Furthermore, ecotourism activities in the region, particularly during peak vector activity seasons, could inadvertently increase human exposure to pathogens. Therefore, a one health approach is vital to integrate surveillance, environmental management, and community education measures to mitigate disease expansion and protect both people and the ecosystems they inhabit.
Citation: Rolón MS, Rojas de Arias A, Velázquez M, Britos M, Arze Selich P, Alfonso Ruiz Díaz J, et al. (2025) The diversity of sandflies (Psychodidae: Phlebotominae) and the presence of Leishmania spp. DNA in potential vectors of the Mbaracayú Forest Biosphere Reserve, Canindeyú, Paraguay: New records and findings. PLoS Negl Trop Dis 19(12): e0013806. https://doi.org/10.1371/journal.pntd.0013806
Editor: Clarence Mang'era, Egerton University, KENYA
Received: May 27, 2025; Accepted: November 30, 2025; Published: December 10, 2025
Copyright: © 2025 Rolón et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was funded by National Council of Science and Technology CONACYT-Paraguay, grant numbers: PINV18-178 (MSR) and 14-INV-052 (MV) and the Organization for the Structural Convergence in the Mercosur Region FOCEM, grant number COF N°03/11 (ARA). The funders did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1 Introduction
Leishmaniasis is a public health issue in 98 countries distributed across four continents (the Americas, Europe, Africa, and Asia), with mandatory reporting in 32 countries, including Paraguay [1]. It is estimated that 350 million people are at risk of contracting the infection, with an annual occurrence of 700,000–1 million new cases of its various clinical forms over the last 20 years [1]. In the Americas, leishmaniasis manifests as two main clinical forms: American visceral leishmaniasis (AVL) and American tegumentary leishmaniasis (ATL), which can present in cutaneous, mucocutaneous, and diffuse forms [2,3].
Leishmaniasis is a zoonotic, vector-borne disease with a complex transmission cycle involving a diverse array of parasites, reservoirs, and vectors. The disease is caused by protozoan parasites of over 20 Leishmania species, transmitted primarily through the bites of infected female phlebotomine sandflies (Psychodidae: Phlebotominae), hematophagous insects with nocturnal or crepuscular habits [1]. Sandflies exhibit great diversity and a wide geographic distribution, with over 1,000 species described worldwide. Of these, 555 (538 extant and 17 fossil) species are found in the Americas [4] and Leishmania spp. has been detected in 60 species [5].
Following Theodor’s 1948 classification of phlebotomine sand flies, in which American species were divided into two genera, Brumptomyia and Lutzomyia, several authors have included the species considered vectors of Leishmania in the latter genus; however, Lutzomyia species were divided into several genera, and many of them into subgenera, in the classification proposed in 1995 by Galati [4]. Adopting Galati’s classification, Paraguay’s most recent official data report 29 species from 10 different genera, three of them (Pintomyia, Evandromyia and Psathyromyia) with different subgenera [6,7]. This anthropophilic sandfly species is widely distributed across the eastern region of Paraguay and in the western region’s Boquerón department [6]. Species reported locally as responsible for ATL transmission include Nyssomyia neivai, Nyssomyia whitmani, Migonemyia migonei, and Pintomyia pessoai, all of which are widely distributed, as well as Psathyromyia shannoni, reported only in the eastern region [6].
In Paraguay, several Leishmania species have been identified from patient samples [8]. Leishmania infantum has been isolated in cases of visceral leishmaniasis while various species [9–11], including Leishmania braziliensis [12,11], Leishmania lainsoni [13,14], Leishmania amazonensis [15], and Leishmania guyanensis have been identified in tegumentary forms [15,16]. Additionally, two cases of Leishmania major-like parasites were reported in 1994 [17].
In the zoonotic transmission cycle, dogs are key reservoir hosts for L. infantum, highlighting their public health importance [18,19]. However, L. infantum is not the only species involved in zoonotic transmission [20], as other Leishmania species,including L. braziliensis, L. amazonensis, L. guyanensis, Leishmania mexicana, Leishmania panamensis, Leishmania peruviana, Leishmania naiffi, Leishmania major, Leishmania tropica and Leishmania tarentolae, have been detected in dogs in various regions worldwide, particularly in the Americas [21–25].
Although sandflies are the only recognized biological vectors of Leishmania parasites, alternative transmission modes have been discussed and speculated. In areas where cases of leishmaniasis have been reported without the presence of primary vectors [26,27] or even without any described Phlebotominae species, alternative infection routes have been suggested [28]. Various non-vector transmission pathways have been proposed for humans [29–32] and dogs, including sexual transmission [33,34] and transmission through the bites of infected fleas or ticks or their accidental ingestion [35,36].
Scientific studies on the biodiversity of phlebotomine sandflies in Paraguay remain limited, with the first nationwide mapping reported in 2021 [6]. Despite this advancement, significant gaps persist regarding the natural infection status and the identification of Leishmania species present in sandflies classified as confirmed, suspected, or potential vectors. To date, only one study in Paraguay has employed PCR for sandfly analysis, detecting L. infantum DNA for the first time in Evandromyia cortelezzii [37].
To contribute to the understanding of the transmission cycle of leishmaniasis in the northeast of the country, we have conducted this study in the Mbaracayú Forest Natural Reserve (RNBM by its Spanish abbreviation), a central area of the Mbaracayú Forest Biosphere Reserve (RBBM by its Spanish abbreviation), located in the Department of Canindeyú, which borders Brazil to the north and east. This department, along with San Pedro, reports the highest number of notified cases of ATL in the country, with Canindeyú also being the only department with a high number of cases across all age categories [38]. The RNBM presents ideal conditions for conducting this type of study, as it is not only a protected wilderness area but also hosts research, education, and ecotourism activities. Surrounding communities of indigenous peoples enter the reserve for hunting [39]. These characteristics foster the interaction between humans and wildlife, including vectors and reservoirs, thereby increasing the risk of transmission of various diseases.
Detailed information on the presence and distribution of parasites in vectors and reservoirs is critical to understanding the transmission cycles and dynamics of leishmaniasis in endemic areas. This knowledge is essential for developing effective control strategies.
Therefore, our primary objective was to study eco-epidemiological aspects, such as the diversity, abundance, and presence of Leishmania spp. DNA in sandflies across different habitats in the RNBM. Additionally, we focused on studying the presence of Leishmania spp. DNA in dogs and their associated ectoparasites within an indigenous community settled in the RBBM.
2 Materials and methods
2.1 Ethics statement
The collection of fleas, ticks, and blood from dogs was carried out with the authorization of the Ministry of the Environment and Sustainable Development (MADES - Paraguay) under scientific collection permits N° 036/2020. Canine samples were collected with informed consent from dog owners. Sampling was performed in accordance with the ethical standards of the Declaration of Helsinki.
2.2 Study area
The study was conducted in the Mbaracayú Forest Biosphere Reserve (RBBM), located in the Department of Canindeyú, northeastern Paraguay (Fig 1). This reserve includes a core area known as the Mbaracayú Forest Nature Reserve (RNBM), which covers 64,405 hectares of protected private wilderness area, managed by Fundación Moisés Bertoni (FMB). Only research, ecotourism, and subsistence hunting by the Aché indigenous people using traditional methods are permitted within this reserve [39]. The climate in the region is typically continental, classified as temperate rainy according to Köppen. The average temperature ranges between 21 and 22°C, with frosts occurring between June and October. There are two distinct seasons: a dry and cold season, and a wet and hot season, with large daily temperature fluctuations. The average annual precipitation is 1,800 mm, with the highest rainfall recorded between October and March [39].
This map was created using ArcGIS software by Esri and data from the Cartographic Atlas of Paraguay (GIS data set). ArcGIS and ArcMap are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. World Imagery: https://services.arcgisonline.com/ArcGIS/rest/services/World_Imagery/MapServer. Basemaps: World Topographic Map https://cdn.arcgis.com/sharing/rest/content/items/7dc6cea0b1764a1f9af2e679f642f0f5/resources/styles/root.json.
2.3 Collection sites
The entomological sampling design was carried out by stratifying the collection areas into three distinct environments based on the conditions of their natural surroundings (primary forest, secondary forest, and modified areas with significant human intervention). Ten sites were selected within each of the defined strata, totaling 30 sampling units in the study area. All geographic coordinates of the sampled sites were recorded using a global positioning system (GPS) (Garmin eTrex10 and Trimble Juno D).
For sandfly capture, 20 sentinel sites were selected in the RNBM, distributed across two different ecotopes. Ten CDC-type traps were placed in a secondary forest area (Zone A: traps A1-A10), and ten traps were placed in a primary forest area (Zone B: traps B1-B10) (Fig 1). Additionally, 10 sentinel sites were selected in the RBBM, where 10 traps were placed in a degraded area (Zone C: traps C1-C10), where anthropogenic activities occur (Fig 1).
Vegetation classification was based on studies by Naidoo and Hill [40] where, the primary forest consists of high forest, bamboo forest, and liana forest, while the secondary forest is composed of riparian vegetation, bamboo understory, high forest, and bamboo forest. The degraded zone is a 20-hectare area altered by human activity, primarily dedicated to small-scale agricultural production, and is composed of meadow, pasture, degraded land, and grassland vegetation.
For the study of dogs and their ectoparasites, the Arroyo Bandera indigenous community was selected. This community is located in the RBBM, 2 km away from the RNBM (Fig 1), and forms part of their ancestral territory, where they continue to engage in hunting and gathering activities. The Aché community consists of 31 households, 169 inhabitants, and 50 dogs that roam freely and do not receive veterinary care [41].
2.4 Sandfly collection and taxonomical identification
Sandfly capture was carried out using CDC mini light traps (BioQuip, CA 90220, USA) [42]. Ten traps were placed in a rotating manner in Zones A, B, and C over two consecutive nights, from 6 pm to 6 am, once a month from October 2020 to October 2021 (except April 2021). The traps were positioned at distances of approximately 100–150 meters from each other, 1.5 meters above the ground. The captured sandflies were transported under cold conditions to the RNBM laboratory, where they were frozen at -20°C for two hours for subsequent identification.
Taxonomic identification of the sandflies was performed using the taxonomic keys of Galati [43]. with the generic abbreviations proposed by Marcondes [44]. Sandflies were separated by sex using a stereomicroscope (SMZ-171, Motic), and the female specimens were mounted on glass slides for microscopic observation of the genital tract and head structures (palps and cibarium). Each dissected specimen was stored individually in tubes with 70% ethanol, transported under cold conditions, and frozen at -20°C until DNA extraction.
2.5 Collection and taxonomic identification of fleas and ticks from domestic dogs
Fleas and ticks were collected from 40 domestic dogs belonging to the inhabitants of the Arroyo Bandera indigenous community. The dogs were sampled in May 2021, after obtaining informed consent from the community. All specimens were collected using entomological forceps and placed individually in vials without preservatives. They were then classified morphologically under a magnifying lens using taxonomic keys for fleas [45–47] and ticks [48]. Once classified, they were stored in tubes with 95% ethanol and frozen at -20°C until DNA extraction.
2.6 DNA extraction from sandflies, ticks, and fleas for Leishmania detection
The processing of female sandflies was performed individually using the thorax and abdomen of each specimen [49]. Ticks were processed individually [50], while fleas were processed individually or grouped into pools of 2–4 specimens [36], depending on the infestation level of each animal.DNA extraction was carried out using the GeneJET Genomic DNA Purification Kit (K0722, Thermo Scientific), following the manufacturer’s instructions. The purity of the extracted genetic material was evaluated using a spectrophotometer (DeNovix DS-11FX+).
2.7 Molecular detection of Leishmania DNA
To detect Leishmania spp., DNA extracted from sandflies, ticks, and fleas was analyzed. The primers LSGITS1-F193 (CATTTTCCGATGATTACAC) and LSGITS1-R1 (CGTTATGTGAGCCGTTATC) were used, designed to amplify a fragment of the internal transcribed spacer 1 (ITS-1) of ribosomal RNA. These primers generate fragments ranging from 220–275 bp, depending on the species of Leishmania [51]. For amplification, the Mix GoTaq Green Master Mix (Promega) was used in a final volume of 50 µL, containing 4 µL of DNA (25 ng/µL), 200 µM of each dNTP, 1.5 mM of MgCl2, 2 units of Taq polymerase, and 25 pmol of each primer. The samples were amplified in a thermal cycler (MultiGene OptiMax, Labnet) with the following conditions: initial denaturation at 95°C for 2 minutes, followed by 34 cycles of denaturation at 95°C for 20 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 1 minute. This was followed by a final extension at 72°C for 6 minutes [52]. Positive controls for the PCR reaction included DNA extracted from L. infantum (MHOM/BR/1972/LD), L. braziliensis (MHOM/CO/88/UA301), and L. amazonensis (IFLA/BR/67PH8). Negative controls were included in all reactions. Amplicons were visualized on a 1.5% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen), with a 100 bp DNA ladder (Gene Ruler, Thermo Scientific) as a molecular weight size standard.
2.8 Identification of Leishmania species by sequencing
For the molecular determination of Leishmania species, PCR products were purified using the commercial GeneJET PCR Purification Kit (Thermo Scientific), following the manufacturer’s instructions. A volume of 20 µL of the purified product was subjected to sequencing using the Sanger method, performed by Macrogen (Geumcheon-gu, Seoul, Korea). The sequences were analyzed using MEGA7 software by comparisons with sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST), considering the most similar organism according to an identity of at least 95% for species level.
2.9 rK39 rapid diagnostic test for canine visceral leishmaniasis
A total of 40 domestic dogs of different breeds, sex, and ages belonging to the inhabitants of the Arroyo Bandera indigenous community were analyzed. After obtaining authorization from the owners, data pertaining to the dogs were recorded in a database, and serum samples were collected. Veterinarians extracted 2 mL of blood from the central vein of each dog, and characteristics and clinical signs compatible with leishmaniasis were recorded. For the rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios), 20 µL of serum were placed on the test strips and incubated with the running buffer, following the manufacturer’s instructions. The results were read after 10 minutes and were interpreted by the same person. The rK39 RDT strips were provided by the National Zoonosis Control Program and the National Rabies Center (Ministry of Public Health and Social Welfare). The serological survey results were provided to the program for monitoring and control of the canine population in the community.
2.10 Environmental variables around sentinel sites
To characterize the environmental conditions surrounding the sentinel sites, we used Sentinel-2A MSI satellite imagery with a spatial resolution of 10 m. Image selection followed two criteria: (i) cloud cover ≤ 10% and (ii) correspondence with the study period. For each trap, buffer zones with radii of 25 m, 50 m, and 100 m were established. Within these buffers, two vegetation and water-related indices were calculated:
NDVI (Normalized Difference Vegetation Index):
where B8 (NIR) corresponds to the near-infrared band and B4 (RED) to the red band of the visible spectrum.
This index is based on the strong contrast between the high reflectance of vegetation in the near-infrared and its strong absorption in the red band, making it a reliable indicator of vegetation vigor.
NDWI (Normalized Difference Water Index):
where B3 (GREEN) represents the green visible band and B8 (NIR) the near-infrared band.
This index enhances water features by exploiting the difference between the higher reflectance of water in the green band and its strong absorption in the near-infrared region.
For both indices, the value assigned to each buffer corresponded to the zonal mean of all pixel values within the defined area.
Two sets of variables were generated according to their temporal criteria:
NDVI 1 and NDWI 1: obtained from the Sentinel-2A image closest in date to each sandfly and reservoir sampling campaign, reflecting point-in-time environmental conditions around the traps.
NDVI 2 and NDWI 2: obtained from the monthly averages of NDVI and NDWI, integrating all eligible images per month under the cloud cover criteria, representing the average monthly environmental conditions in the surroundings of each sentinel site.
In order to study how environmental variables affect the abundance of sandflies in the sampled traps, variance analysis of the following variables: total phlebotominae abundance, minimum temperature, maximum temperature, minimum relative humidity, maximum relative humidity and win speed was performed. Normality and homoscedasticity assumptions were applied. The analysis considered only traps in which sandflies were found, taking those with p values under 0.05 as significant results.
Data collected during trapping months were analyzed using the Kruskal-Wallis test (5% error) and a post-hoc test. Multivariate analysis was also applied to the data, to obtain the Principal Component Analysis (PCA), which seeks to summarize the variation pattern of the original variables. The software Past 4. 3.00 and Infostat version 2020 were used [53,54]. Additionally, the PCA indicated the percentage of variation explained by each axis [55,56]. Subsequently, differences between sites were evaluated using discriminant analysis (DA).
3 Results
3.1 Sandfly collection by ecotypes and taxonomic identification
During a 12-month period, a total of 228 traps were placed, with 76 traps in each ecotope (Table 1). Of the traps placed, 64% of the sandfly captures were recorded in the secondary forest, 33% in the primary forest, and 3% in the degraded zone, the latter being the least effective (Table 1). Out of the 30 sites sampled, 24 (80%) recorded sandflies at least once, while 6 sites (20%) had no captures (Table 1). The traps with no captures were in zones B (B1 and B6) and C (C2, C7, C8, and C9) (Table 1).
In total, 954 sandflies were collected from the three ecotopes, consisting of 599 females and 355 males, representing 12 different species: Ev. cortelezzii complex (n = 340), Brumptomyia brumpti (n = 312), Pintomyia monticola (n = 161), Psathyromyia lanei (n = 53), Nyssomyia neivai (n = 31), Evandromyia evandroi (n = 19), Brumptomyia avellari (n = 11), Micropygomyia quinquefer (n = 8), Brumptomyia guimaraesi (n = 7), Migonemyia migonei (n = 5), Evandromyia termitophila (n = 4), and Psathyromyia shannoni complex (n = 3) (Table 2). The species collected in each of the 30 traps are detailed in Table S1.
Regarding the sex distribution of the collected specimens, a predominance of females was observed in all the species collected, except in Br. guimaraesi and Br. brumpti, where a higher number of males were recorded (Table 2).
When analyzing each studied ecotope, it was observed that in the secondary forest, 230 males and 381 females were collected, totaling 611 specimens (64%) from 10 different species (Tables 1 and 2). The most abundant species in this zone were Ev. cortelezzii complex, followed by Br. brumpti, Pi. monticola, Pa. lanei, and Ny. neivai. Meanwhile, species such as Ev. evandroi, M. quinquefer, Br. guimaraesi, Br. avellari, and Mg. migonei were captured in smaller quantities (Table 2). All traps in this zone collected specimens at least once, with traps in sites A5, A6, and A7 being the most effective, capturing 111, 113, and 117 sandflies, respectively, while trap A1 was the least effective, with only 5 specimens captured (Table 1). Notably, Ny. neivai was captured exclusively in this zone, and only male specimens of Br. guimaraesi and female specimens of Br. avellari and M. quinquefer were collected (Table 2). There were no captures of Ev. termitophila and Pa. shannoni complex in this zone.
In the primary forest ecotope, 113 males and 206 females were collected, totaling 319 specimens (33%) from 11 different species (Tables 1 and 2). Br. brumpti was the most abundant species, followed by Ev. cortelezzii complex and Pi. monticola, while species such as Pa. lanei, Ev. evandroi, Br. avellari, M. quinquefer, Br. guimaraesi, Mg. migonei, Ev. termitophila, and Pa. shannoni complex were captured in smaller quantities (Table 2). Traps B8 and B10, which captured 33 and 99 sandflies, respectively, were the most effective compared to the rest, in contrast to traps B1 and B6, where no captures were recorded (Table 1). Notably, Ev. termitophila and Pa. shannoni complex were captured exclusively in this zone (Table 2). Regarding the species captured in low quantities, almost all were females, except for Br. guimaraesi and Ev. evandroi, where one male of each species was also collected (Table 2).
In the degraded zone ecotope, 12 males and 12 females were collected, totaling 24 specimens (3%) from 3 different species (Tables 1 and 2). The predominant species was Br. brumpti, followed by Ev. cortelezzii complex, and a single female specimen of Pi. monticola (Table 2), which were also present in zones A and B. Captures in this zone were limited to only 6 traps, with trap C10 being the most productive, capturing 12 specimens, while traps C2, C7, C8, and C9 recorded no captures (Table 1).
3.2 Seasonality
The highest number of sandflies captures occurred in the spring, with 709 specimens collected, of which 70% were females. In contrast, during the summer, 194 specimens were captured, with males being more abundant, representing 59%. Sandfly captures decreased considerably in autumn and winter. In autumn, 37 specimens were recorded, with similar proportions of males and females, while in winter, with only 14 specimens, males represented 86% of the captures (Fig 2).
The diversity of sandfly species showed variation throughout the year, with 11 species collected in spring, 8 in summer, 6 in autumn, and 5 in winter (Table 3). The species Br. brumpti, Ev. cortelezzii complex, Ev. evandroi, and Br. avellari were present during all seasons, while Br. guimaraesi, Ev. termitophila, and Pa. shannoni complex were only detected in spring, and Ny. neivai was only present in summer (Table 3).
3.3 Molecular detection of Leishmania DNA in Phlebotominae
To determine the presence of Leishmania spp. DNA in sandflies, 599 females were processed individually. Of these, 69 specimens from 9 different species tested positive (11.5%) (Table 4). The species with the highest positivity rates were Ev. cortelezzii complex (20.3%), Pi. monticola (18.8%), and Br. brumpti (17.4%), which were also the most captured, followed by Br. avellari (15.9%) and Pa. lanei (11.6%). Species like Ev. evandroi, M. quinquefer, Ny. neivai, and Mg. migonei showed positivity rates of 4.3% or lower (Table 4). A significant finding was that all females of Br. avellari were positive. Species that tested negative for Leishmania spp. DNA were Pa. shannoni complex, Br. guimaraesi, and Ev. termitophila.
The Leishmania spp. species circulating in the study areas were L. amazonensis (65.2%) and L. infantum (34.8%) (Table 4). Species from the Ev. cortelezzii complex, Pi. monticola, Br. brumpti, Pa lanei, Br. avellari, Mg. quinquefer, and Mg. migonei carried either L. amazonensis or L. infantum DNA (Table 3). In Ev. evandroi, only L. infantum DNA was detected, while in Ny. neivai, only L. amazonensis DNA was identified (Table 4). No sandfly tested positive for both species simultaneously.
3.4 Leishmania spp. DNA presence in collection sites and seasonality
Regarding the collection sites of the 69 specimens positive for Leishmania spp., 48 were collected from the secondary forest (69.6%), 20 from the primary forest (29.0%), and 1 from the degraded area (1.4%) (Table 4). In the secondary forest, 9 positive species were detected, 6 in the primary forest, and 1 in the degraded area (Table 5). It should be highlighted that in the secondary forest, L. amazonensis predominated, while in the primary forest, the circulation of L. amazonensis and L. infantum was similar. In the only positive specimen captured in the degraded area, L. amazonensis DNA was detected.
Sequencing analysis of the amplified ITS1 fragments revealed that 45 (65.2%) of the L. amazonensis-positive specimens had an identity percentage between 98.84% and 99.22%, while the 24 (34.8%) L. infantum-positive specimens had an identity percentage between 99.17% and 100% (Tables 4 and 6).
Out of the 30 sites where light traps were placed, 17 of them captured sandflies positive for Leishmania spp. (Fig 3). In all the traps placed in the secondary forest, positive specimens were captured, occasionally with higher abundance in traps A7, A3, A6, and A5 (Fig 4). In the primary forest, 6 out of 10 traps captured positive specimens, with traps B8, B7, and B10 showing the highest abundance (Fig 3). In the degraded area, only trap C5 captured a sandfly positive for L. amazonensis (Fig 3).
The greatest diversity of species positive for Leishmania spp. was observed in spring and summer, with 8 and 7 species registered, respectively (Fig 4). In contrast, autumn and winter showed lower diversity, with only 4 and 2 positive species recorded in each season, respectively (Fig 5). Notably, Br. avellari was the only species detected as positive for Leishmania spp. in all seasons of the year, followed by Pi. monticola, which was detected in spring, summer, and autumn (Fig 4).
A. Skyn ulcer B. Alopecia C. Nail overgrowth D. Hair loss and skin ulcers.
3.5 rK39 rapid diagnostic test for canine visceral leishmaniasis and molecular detection of Leishmania DNA in ectoparasites
A parallel study was conducted in 40 mixed-breed dogs (15 females and 25 males) belonging to 26 households in the Arroyo Bandera indigenous community (Table 7).
The dogs underwent an immunological analysis using the rapid immunochromatographic rK39 test, which detects antibodies against the Leishmania donovani species complex. All dogs tested negative for the rK39 test (Table 7), however, all displayed clinical signs associated with leishmaniasis (Fig 5), such as overgrowth of nails (78%), skin ulcers on ears, face, or body (75%), cachexia (58%), hair loss (55%), seborrhea (8%), eye discharge (5%), alopecia (5%), lymph node hypertrophy (5%), and dry hair (5%) (Fig 6).
Additionally, ectoparasites were collected from 37 of the 40 dogs (92.5%), which were analyzed for the presence of Leishmania spp. using molecular techniques. In total, 81 fleas of the Ctenocephalides genus were collected from 33 dogs (82.5%), and 6 ticks of the Rhipicephalus genus were collected from 4 dogs (12.5%) (Table 7).
Fleas collected from each dog were processed individually or in groups, totaling 33 samples, of which 18 (54.5%) were positive for Leishmania spp. Ticks were processed individually, with DNA from Leishmania spp. detected in only one specimen. All positive ectoparasites were identified by sequencing as L. amazonensis (Table 7).
3.6 Meso-scale environmental variables and sandfly abundance
The subsequent correlation analysis between the aforementioned mesoscale environmental variables and the abundance of sandflies revealed significant patterns, thereby facilitating a more profound comprehension of the environmental factors that influence the distribution of these vectors. Among the climatic variables, mean temperature exhibited a modest yet statistically significant positive correlation (r = 0.235; p < 0.001), suggesting that moderately high temperatures may favor the activity and abundance of sandflies. Conversely, relative humidity, maximum humidity, and wind speed exhibited no statistically significant correlations. However, minimum humidity exhibited a modest positive correlation (r = 0.179; p = 0.0032), suggesting that low levels of nocturnal humidity might still permit the activity of these insects under specific conditions (S2 Table).
Regarding the relationship between vegetation and humidity indices and the abundance of sandflies, the NDVI exhibited a significant positive correlation at the three evaluated scales (25, 50, and 100 m), with consistent values around r ≈ 0.29 and very high levels of significance (p < 0.001) (S2 Table). These findings corroborate the hypothesis that areas with higher vegetation density are associated with a higher presence of sandflies, likely due to the greater availability of shelter, moisture, and favorable microclimatic conditions. Conversely, NDWI exhibited a negative correlation across all scales (r ≈ -0.20; p < 0.01), suggesting that the lower moisture content in surface vegetation might be associated with higher sandfly populations (S2 Table).
In terms of land cover, a negative correlation was observed with agricultural and bare soil areas, particularly at distances of 50 and 100 meters from the sentinel site (r = -0.29 to -0.18; p < 0.01) (S2 Table). These negative associations suggest that more disturbed landscapes reduce the presence of sandflies, possibly due to loss of suitable habitat and microenvironmental alterations. In contrast, forest cover exhibited a significant positive correlation (r = 0.26 to 0.259; p < 0.001) at 50 and 100 meters (S2 Table), thereby reinforcing the hypothesis that forested environments, both primary and secondary, provide optimal conditions for the survival and reproduction of sandflies.
Finally, no statistically significant relationships were identified between proximity to water and abundance of sandflies in any of the evaluated scales. These findings suggest that, at least within the context of this particular ecological study, the presence of water does not significantly influence the population density of these insects (S2 Table).
Principal component analysis (PCA) revealed clear differentiations between the evaluated types of environments in terms of sandfly species composition. The first principal component (PC1) explained 49% of the observed variability, while the second component (PC2) accounted for 29.9%, indicating a high discrimination capacity between sites (Fig 7A).
Within this distribution, the secondary forest exhibited the most extensive dispersion of points in the multivariate space, suggesting a greater degree of diversity and heterogeneity in species composition. In contrast, the primary forest exhibited intermediate variability, while degraded areas demonstrated more pronounced clustering, indicating lower variability and more homogeneous communities (Fig 8A).
The secondary forest exhibited the highest species richness and abundance, including Ev. cortelezzii complex, Pi. monticola, Br. brumpti, M. quinquefer, Mg. migonei, Ny. neivai, Br. guimaraesi, and Pa. lanei. Conversely, the primary forest exhibited a distinct biodiversity, featuring species such as Pa. shannoni complex, Pa. lanei, Ev. termitophila and Mg. migonei. In contrast, degraded areas showed lower specific richness, with a predominance of Mg. migonei and M. quinquefer (Fig 7B).
Discriminant analysis (DA) confirmed significant differences in the different species composition and abundance of sandflies between habitat types (Fig 8A). While primary and secondary forests exhibited some species overlap, the secondary forest demonstrated a higher density of sandflies. A statistically significant increase in total abundance was observed in the secondary forest compared to the primary forest (p < 0.05) and degraded areas (p < 0.001), which recorded the lowest number of sandflies (Fig 8B). The findings indicate that secondary forests offer particularly favorable ecological conditions for the proliferation of sandflies, both in terms of diversity and density.
4 Discussion
This is the first study exploring the abundance and presence of Leishmania DNA in sandflies in the RBBM, a protected area in the Atlantic Forest (Bosque Atlántico) of Alto Paraná, Paraguay, near the border with Brazil. We selected the RBBM as our study area because it offers a unique and representative environment of the Atlantic Forest (Bosque Atlántico) biodiversity and the transitional ecosystem with the Cerrado. As the largest and most protected remnant of this biome, the RBBM provides a relatively intact habitat where wildlife can be studied as natural reservoirs of pathogens, which is vital for understanding the dynamics of zoonotic disease transmission [57]. The reserve is a critical area for monitoring and researching vector-borne pathogens due to its high biodiversity, conservation of natural habitats, and the presence of numerous vertebrate species acting as reservoirs. These characteristics make the RBBM a strategic site to advance the understanding of the ecology and transmission dynamics of zoonotic diseases in Paraguay and the region.
The capture of sandflies using CDC mini light traps allowed us to sample various ecotopes and proved highly efficient, providing accurate data on the species richness and diversity of sandflies present, as observed in other studies [58].
The highest abundance and diversity of sandflies were recorded in the primary and secondary forests of RBBM, forest areas with less anthropogenic impact compared to the degraded area (farm). These less disturbed areas act as ecological refuges, maintaining a higher species distribution richness [59]. This trend aligns with studies in southern Brazil, where a clear correlation was found between insect diversity and the presence of remnant forests [60]. These findings emphasize the importance of conserving these ecosystems, as their protection is a pillar in maintaining the biological diversity of sandflies and other organisms coexisting in these habitats.
Studies aimed at identifying molecular targets for the accurate diagnosis of leishmaniasis have shown that the ITS-1 region is highly effective in detecting all Leishmania species, supporting its choice as a marker in this study [61]. Studies on natural infection rates have revealed that PCR is more sensitive and specific than dissection for demonstrating the presence of Leishmania in sandflies [62–64]. In our study, amplification of the ITS1 fragment was successful in 69 females, distributed across nine species captured in the three ecotopes studied. The largest number of sandflies was captured in spring and summer, periods known for intense sandfly activity, as temperature and humidity conditions during these months favor their proliferation and the transmission of Leishmania spp. [65,66]. The study of potential vectors is relevant to leishmaniasis epidemiology, especially in regions where specific vectors are absent. These vectors may occasionally intervene in parasite transmission, allowing their persistence and favoring the expansion of their geographic distribution to non-endemic areas [67–72].
This study observed a high diversity of sandflies, with a total of 12 different species identified, 10 of which are reported for the first time in the Department of Canindeyú (Paraguay), highlighting the importance of this forest area and the impressive biodiversity of the region, which had not been previously sampled.
The Ev. cortelezzii complex is distributed across Argentina, Bolivia, Brazil, Paraguay, Peru, and Uruguay [73]. In Paraguay, it has been recorded in the departments of Alto Paraná, Itapúa, Cordillera, Central, and Boquerón [6]. This study marks the first record of the species in Canindeyú, with specimens captured in all three ecotopes studied. In Argentina, it inhabits a variety of habitats, including tree trunks and hollows, roots, marginal areas, peridomestic constructions with animals, and both external and internal walls of human dwellings [68]. In this study, we found specimens with L. infantum DNA in secondary forests and L. amazonensis in primary and secondary forests. Natural infections of this species with L. braziliensis have been reported in Argentina’s Chaco province, reinforcing the hypothesis of its potential role in transmitting ATL [74]. In Brazil, cases of specimens infected with L. infantum [75–77] and L. braziliensis [78] have been documented, suggesting its potential role in the transmission cycles of tegumentary and visceral leishmaniasis in wild or rural environments [79]. This study documents for the first time the presence of L. amazonensis DNA in Ev. cortelezzii complex, observed in specimens collected from primary and secondary forests. The detection of L. amazonensis DNA suggests its potential involvement in the transmission cycles of cutaneous and mucocutaneous leishmaniasis and highlights the need for further studies to confirm its vector competence and ecological role in the region.
Pi. monticola is distributed in Argentina, Brazil, and Peru [73]. In Paraguay, it has been recorded in the departments of San Pedro and Caaguazú [68], and this study represents the first report of its presence in Canindeyú. In Brazil, it has been captured in areas of fragmented and closed native forests, showing a notable preference for CDC light traps [80,81]. Regarding the presence of Leishmania spp., in Minas Gerais, Brazil, specimens with L. braziliensis have been reported in rural and forested areas [82], while protected areas have recorded specimens with L. infantum DNA [83]. In Argentina, this sandfly species has been recorded in an endemic area of ATL in the Corrientes province, though no infections by Leishmania spp. have been reported [84]. Our study is pioneering in documenting Pi. monticola with L. amazonensis and L. infantum in primary and secondary forests, marking the first global report of the presence of L. amazonensis DNA in this species. Additionally, P. monticola was the species with the highest number of specimens positive for L. amazonensis, most of which were captured in the secondary forest area, where there is high human circulation due to the ecotourism and education zone of the RBBM. This finding is particularly relevant, as previous studies have indicated that Pi. monticola is an essentially forest-dwelling, highly anthropophilic species susceptible to infections by Leishmania spp. [85–87].
Brumptomya species occur in the Atlantic Forest (Bosque Atlántico) area and primarily inhabit wild environments. Their presence is considered an indicator of environmental health [88], although recent habitats also include domestic animal annexes and marginal areas [89]. Unfortunately, perhaps due to its lack of epidemiological importance and low or absent anthropophily [90], this genus has received limited attention. Nevertheless, recent reports of molecular detection of Leishmania DNA in species of this genus have been recorded in Ecuador [91], Mexico [92] and Brazil [60,58,93]. These detections raise questions about the feeding habits of this genus, which is thought to feed exclusively on the blood of armadillos (Dasypodidae), or about the possible role of armadillos as reservoirs of Leishmania spp. in wild transmission cycles. Information on infections in Brumptomyia species with Leishmania spp. is still limited. Documented cases so far come from Acre, Brazil, where specimens infected with L. braziliensis [59] and Leishmania spp. [58] have been found; from Br. leopoldoi in Ecuador [91]; from Br. mesai in Mexico [92]; and from Br. nitzulescui in Minas Gerais, Brazil [93]. In this study, we collected several species of this genus, whose findings are described below.
Br. brumpti is found in Argentina, Bolivia, Brazil, and Paraguay, where it has been captured in the departments of Amambay, San Pedro, Canindeyú, Alto Paraná, Cordillera, Paraguarí, Caazapá, and Itapúa in intra, peri, and extra-domiciliary environments [6]. This species occupies a wide range of habitats, from armadillo shelters and tree trunks to caves, cracks, rural areas, and recently deforested zones [68]. In our study, we detected Leishmania spp. DNA in all three ecotopes studied. Notably, Br. brumpti was the only species positive for L. amazonensis collected in the farm zone. In specimens captured in the secondary forest, we detected L. infantum and L. amazonensis, while in the primary forest, only L. amazonensis was circulating.
Br. avellari, distributed in Argentina, Bolivia, Brazil, Colombia, Panama, Peru, and Venezuela [73], has been recorded in Paraguay in the departments of Concepción, San Pedro, Cordillera, Caaguazú, Ñeembucú, Itapúa, and Misiones, inhabiting intra- and peridomiciliary areas [6]. In this study, we report for the first time its presence in Canindeyú in primary and secondary forest and with the presence of DNA of L. amazonensis and L. infantum.
Br. guimaraesi, distributed in Argentina, Brazil, and Paraguay, has been previously recorded in the departments of San Pedro, Misiones, and Itapúa [6]. This study represents the first finding of the species in the department of Canindeyú. In Brazil, it has been found in fragmented native forest areas, while in Argentina, it was found in the urban environment of Clorinda, a border town with Paraguay [81]. During our research, we captured specimens in primary and secondary forests; however, none tested positive for Leishmania spp.
Pa. lanei is found in Brazil, Argentina, and Paraguay [73], with previous records in San Pedro and Caaguazú [68]. Our study marks the first finding of this species in Canindeyú. This sandfly is found in various environments, from domiciles and peridomiciles to chicken coops, pigsties, and in tree hollows and canopies. In Argentina, it has been reported in Misiones [94], while in Brazil, it has been found in a protected park dedicated to ecotourism in the state of Alagoas [95], as in the Parque Estadual do Alto Ribeira (PETAR), located within an Atlantic Forest reserve, an important ecotourism attraction in the Ribeira Valley and an endemic area for ATL [85]. In our study, we captured specimens in both primary and secondary forests, where several specimens tested positive for L. infantum or L. amazonensis, all linked to the secondary forest. This study represents the first report of Pa. lanei with DNA of L. infantum and L. amazonensis.
Ev. evandroi has been observed in both semi-arid regions [96] and conservation areas with high anthropogenic activity [97]. In northeastern Argentina, it has been captured in peridomiciliary peri-urban areas [68]. A study conducted in northeastern Brazil identified specimens with L. amazonensis DNA [98]. In this study, we identified several specimens with DNA of L. infantum exclusively, from both the primary and secondary forests. This discovery is especially relevant given the anthropophilic behavior of Ev. evandroi and its association with peridomiciliary environments, where it feeds on a wide variety of animal species [99].
Ny. neivai is found in Argentina, Bolivia, Brazil, and is widely known in Paraguay, where it occurs throughout the eastern region, except for Ñeembucú, and in the department of Presidente Hayes in the western region [6]. This species adapts to various environments, including urban, rural, periurban, wild, and secondary vegetation areas [100]. In Argentina and Brazil, it is the main vector of ATL caused by L. braziliensis [100,101] and the presence of L. infantum DNA has been documented in different states of Brazil [76,102,103]. In Paraguay, this species is associated with the transmission of L. braziliensis in the Central department, according to epidemiological evidence [10]. In our study, we report for the first time Ny. neivai with the presence of DNA of L. amazonensis captured in a secondary forest. It is worth noting that Ny. neivai can disperse distances between 250–520 meters across pastures or peridomiciliary areas, showing its ability to spread in open areas [104], facilitated by the degradation of primary environments.
Mi. quinquefer has been recorded in Argentina, Bolivia, and Brazil [73] and was recently identified in Paraguay, with captures in intradomiciliary environments in San Pedro, Misiones, and Itapúa [6]. This study marks the first record of its presence in Canindeyú. In this study, several specimens were captured in secondary forests, positive for L. infantum DNA, and in primary forests, positive for L. amazonensis DNA. These findings are consistent with observations in other regions, such as in Puerto Iguazú, Argentina, where L. infantum DNA was detected [98], and in northeastern Brazil, where L. amazonensis was found [105].
Mg. migonei is a sandfly species with a distribution across much of South America, including Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, Trinidad and Tobago, and Venezuela [73]. In Paraguay, it has been documented in urban areas such as Asunción and in border regions with Brazil, like Amambay and Canindeyú, highlighting its adaptability to various environments [6]. Historically, Mg. migonei has been considered a species typically found in dense primary forests, although it can also be found in less dense vegetation areas. While primarily associated with forested environments, it is not uncommon to find it in indoor areas and animal shelters, reflecting its anthropophilic behavior [106–109]. This adaptability may explain its role as a vector of zoonotic diseases in urban and peri-urban areas. In our study, we captured specimens of Mg. migonei in primary and secondary forests. Specimens positive for L. infantum and L. amazonensis DNA were found in secondary forests, suggesting that these areas, despite being less dense and more disturbed by human activity, may serve as key habitats for the transmission of Leishmania spp. This finding is particularly relevant in the epidemiological context, as Mg. migonei has been implicated as a vector of L. braziliensis in Paraguay [10] and Brazil [110]. Additionally, previous studies in Santiago del Estero, Argentina, and the states of Rio de Janeiro and Pernambuco, Brazil, have suggested that Mg. migonei could be a potential vector of L. infantum, given that natural infections have been identified in these regions [111–113]. An experimental study has also shown that L. amazonensis can develop in this species [114], further reinforcing its significance in transmitting different species of Leishmania. Due to the epidemiological importance of this species, it is worth noting that this study is the first report of Mg. migonei with L. amazonensis DNA and the first record for Paraguay of its positivity to both L. infantum and L. amazonensis.
In the Pa. shannoni complex, the species Pa. shannoni is widely distributed across the Americas, with documented presence in Belize, Bolivia, Colombia, Costa Rica, French Guiana, Guatemala, Honduras, Mexico, Nicaragua, Panama, Peru, Suriname, Trinidad and Tobago, the United States, and Venezuela [73]. This species shows a notable preference for anthropogenic environments, frequently captured in intra- and peridomiciliary areas [115]. In Paraguay, it is common in the eastern region of the country and has been documented in nearly all departments except Canindeyú, Ñeembucú, and Misiones [6]. Therefore, with the findings of this study, the presence of Pa. shannoni complex in Canindeyú is documented for the first time. Regional studies have revealed the high susceptibility of this species to infection with L. chagasi/infantum in Peru, where its behavior is comparable to that of Lu. longipalpis [116]. Additionally, it has been documented that this species can present natural infections with Trypanosoma rangeli and has been identified as a vector of L. chagasi in Venezuela [117]. Laboratory studies have shown it is susceptible to L. chagasi and L. panamensis, and it is considered a competent vector for L. mexicana. This transmission capability, particularly in the United States, reinforces its status as a potential vector of leishmaniasis, especially L. braziliensis [118–120]. However, during our investigation, none of the three female specimens collected in primary forest tested positive for Leishmania spp. DNA. The taxonomic revision by Sábio et al. clarified that several taxa previously grouped under Pa. shannoni actually represent distinct species (Pa. limai, Pa. bigeniculata, Pa. ribeirensis and Pa. baratai sp. n.) each with specific geographic ranges in Brazil and other Neotropical regions [121,122]. Consequently, historical records attributed to Pa. shannoni in southern and central Brazil, such as those reported by Dorval et al., may include misidentified members of this complex [123]. Extending the confirmed distribution of Pa. shannoni sensu stricto beyond its core range (from the southern United States to northern South America) will require the integration of morphological, molecular, and ecological data to minimize taxonomic uncertainty. Although Pa. shannoni has occasionally been found infected with Leishmania (Viannia) spp. in various localities, its role as a vector remains secondary or incidental compared to species such as Ny. whitmani or Mg. migonei. The recognition of cryptic diversity within the Shannoni complex further emphasizes the need for updated assessments of both distribution and vector competence, particularly in areas where sympatric Viannia species circulate. In such regions, genetic exchange—such as that demonstrated by Stocco Lima et al. for L. guyanensis/ L. shawi hybrids—may additionally complicate eco-epidemiological interpretations [124].
Ev. thermitophila is distributed across Brazil, Bolivia, and Argentina [125] and has been recently recorded in Paraguay, with a male specimen captured in the San Pedro department [6]. This species is typically associated with the burrows of wild animals and is found in rock crevices or in proximity to farm animals in rural areas. Our study represents the first finding of Ev. thermitophila in Canindeyú, where it was collected in the primary forest, extending its known geographic range but without evidence of positivity to Leishmania spp. DNA. However, previous research in Brazil has documented the presence of L. infantum DNA [78] and L. guyanensis DNA [126], reflecting the potential of this species to serve as a host for these parasites.
The RNBM harbors the largest and most protected remnant of the Atlantic Forest, composed of late-stage secondary forest with minimal human impact over the past 35 years [57]. The activities allowed in the RNBM include traditional hunting by the Ache indigenous people, limited biological research, and ecotourism. During our study, we collected sandflies in five vegetation classes present in the RNBM: high forest, bamboo and liana forest, bamboo understory, and riparian vegetation [40]. However, the traps were placed in two well-differentiated forest zones: the primary forest, which includes high forest, bamboo, riparian vegetation, and bamboo understory, and the secondary forest, composed of high forest, bamboo, and lianas. The bamboo forest is characterized by having few trees and a predominance of bamboo, which can reach heights of 10–15 meters. In contrast, the liana forest features abundant lianas that dominate the understory, while in the high forest, tree heights reach around 20 meters, with an open understory [57].
The results of this study confirm the association of a higher abundance of sandflies in areas with greater vegetation cover and their decrease as soil cover or agricultural areas increase. Therefore, the largest number of specimens was collected in the secondary forest, specifically in areas of liana and bamboo forests, followed by the primary forest where the vegetation consists of tall forests and bamboo. The main difference between primary and secondary forests lies in the greater human presence in the latter, including ecotourism areas. These ecotourism practices can facilitate the interaction between domestic animals, humans, and wildlife, increasing the risk of disease transmission [127].
The collection area in degraded soil was conducted on a farm near the entrance of the RNBM, characterized by meadows, grasslands, and degraded areas. In this area, we obtained a low quantity and diversity of sandflies, with the highest number captured in a trap located on a forest island within a pasture frequented by cows, near the primary forest of the RNBM. This behavior is not surprising, considering that sandflies are originally wild. The distribution and abundance of species are directly influenced by the presence of forested areas, as observed in a study on the faunal diversity of sandflies in southern Brazil, which established a correlation between insect richness and the existence of forested areas [59].
Although the anthropized species Ev. evandroi and Ny. neivai showed low capture rates, they showed DNA rates of 4.3% for L. infantum and L. amazonensis, respectively, suggesting a significant risk of transmission for nearby peri-domestic and domestic populations. On the other hand, the wild species Pi. monticola and Br. brumpti, attracted to light and typical of edge areas, also tested positive for L. infantum and L. amazonensis DNA, with DNA rates of 18.8% and 17.4%, respectively. These species could play a role in the transmission of leishmaniasis, especially in areas where human intervention has altered the forest, potentially facilitating their movement towards more populated areas.
However, certain methodological factors may have influenced the detection of the main vectors of L. amazonensis and L. infantum. The sampling strategy relied primarily on CDC light traps placed in sylvatic environments, with limited peridomestic coverage. This approach may have underestimated Lutzomyia species, which are often concentrated in peridomiciliary and animal shelter areas, particularly in anthropized environments [128]. Similarly, Bichromomyia flaviscutellata–the established vector of L. amazonensis in Brazil, though not yet reported in Paraguay–shows low attraction to light traps and exhibits cryptic behavior, typically inhabiting rodent burrows and humid forest soil microhabitats [129]. Its apparent absence in our captures may therefore reflect sampling bias rather than true ecological scarcity. Future studies should adopt a broader trapping strategy, including emergence traps, aspiration near rodent nests, and more intensive peridomestic sampling, to improve vector detection and provide a more accurate representation of transmission foci diversity.
Although several of the dogs evaluated showed evident clinical signs of leishmaniasis, all tested negative when analyzed using the immunochromatographic rk39 test. This result, though initially puzzling, aligns with previously documented limitations of this test in studies conducted in Brazil [130,131] and Argentina [132]. These studies have shown that the low sensitivity of the rk39 test makes it difficult to accurately detect L. infantum infections in canines, particularly those with mild clinical manifestations or low parasitic loads. This limitation could be even more significant if the presence of other Leishmania species that can infect canines [20] is considered, as the test is specifically designed to detect species of the L. donovani complex. Therefore, exclusive dependence on this diagnostic tool in endemic areas could lead to an underestimation of the real prevalence of canine leishmaniasis, highlighting the need to use complementary diagnostic approaches for a more thorough evaluation. In this study, molecular assays were not performed because we only obtained authorization from the dog owners for the extraction of peripheral blood, which does not represent the most suitable tissue for detecting Leishmania spp. However, the ectoparasites collected from these dogs revealed a notably high positivity for L. amazonensis DNA when analyzed using molecular techniques. This finding is particularly significant, as although sandflies are recognized as the exclusive biological vectors of Leishmania spp., speculation has begun regarding potential secondary modes of transmission, particularly in areas where cases of leishmaniasis have been reported without confirmed presence of known vectors [26,27,111,133], or even in the absence of sandflies [28]. In this context, ticks and fleas, highly specialized hematophages, have been identified as potential transmitters [36,130,134], as they feed on a variety of mammals, including humans, livestock, dogs, cats, rabbits, squirrels, rats, and mice [135]. The transmission of Leishmania spp. by infected fleas or ticks, accidentally ingested by dogs or other mammals, has also been suggested in previous studies [35,136], as occurs with the protozoan parasite Hepatozoon canis, which is effectively transmitted through the ingestion of infected ticks [137].
Analysis of environmental variables revealed that the abundance and species composition of phlebotomine sandflies at the sentinel sites were strongly influenced by both land cover characteristics and local climatic conditions, displaying spatial patterns consistent with those reported for other Neotropical regions. The negative association between sandfly abundance and agricultural land cover within 25–100 m suggests that areas subject to greater anthropogenic disturbance provide less suitable conditions for sustaining sandfly populations. This pattern is consistent with the findings of Rêgo et al. in the Xakriabá Indigenous Reserve [138], where vegetation loss and soil compaction reduced local diversity, and with those of Pereira et al. who reported lower species richness and abundance in urbanized areas of Itaúna, Minas Gerais [139].
Conversely, the positive correlations observed between sand fly abundance and forest cover at 50 and 100 m underscore the importance of vegetation in maintaining suitable microhabitats by moderating temperature and increasing humidity. Silva et al. demonstrated that both primary and secondary forest fragments harbor significantly greater sand fly richness and density compared with built-up zones, emphasizing their role as ecological refuges in urban and periurban contexts [140]. In our study, secondary forest showed the highest total abundance, reinforcing the hypothesis that regenerating environments provide an optimal balance of organic matter, shading, and blood sources conditions particularly favorable for species such as the Ev. cortelezzii complex and Ny. neivai.
Discriminant analysis further revealed marked differences among habitat types, with some species overlap between primary and secondary forests. This pattern reflects the ecological plasticity of certain taxa. Fernandes et al. reported similar results in Mato Grosso do Sul, where generalist species such as Lu. longipalpis and Mg. migonei successfully colonized both natural and periurban environments [141]. Such coexistence may be linked to the simultaneous availability of shelter, organic matter, and hosts, as well as tolerance to variable microclimatic conditions.
Regarding climatic variables, the positive correlation between mean temperature and sand fly abundance supports previous studies associating higher temperatures with increased development rates, survival, and dispersal capacity [141–143]. Oliveira et al. documented a direct relationship between temperature and Lu. longipalpis density, indicating that warmer periods promote reproductive and feeding activity [142]. Similarly, Carvalho et al. demonstrated that rising temperatures expand the ecological niche of Bi. flaviscutellata, enabling its projected southward range expansion in South America under climate change scenarios [143].
The weak but negative correlation with minimum humidity suggests that environmental desiccation may limit sand fly abundance, particularly during immature stages that depend on moist, organic-rich substrates. This finding agrees with Silva et al., who reported abundance declines during drier months [140], and Fernandes et al., who identified relative humidity as a key determinant of population success [141]. The absence of a significant relationship with wind speed is consistent with Oliveira et al., who observed a limited effect of wind in vegetated areas where canopy cover reduces its impact on sand fly flight activity [142].
The species–habitat associations observed here—Ps. shannoni and Ps. lanei in primary forest, Mg. migonei and Ev. evandroi in farm areas—mirror the ecological partitioning described by Rêgo et al. [138] and Pereira et al. [139]. In these studies, specialist forest species were distinguished from generalist taxa adapted to modified landscapes. This differentiation suggests a successional gradient in community structure, in which secondary forests act as transitional zones facilitating coexistence and dispersal of medically important species.
Taken together, our findings, supported by recent literature, reinforce the role of environmental and climatic factors in shaping sand fly population dynamics in tropical settings. Forest fragments—particularly secondary ones—represent key habitats for the persistence and expansion of vector species and may play a central role in Leishmania transmission across fragmented landscapes. Therefore, entomological surveillance in transitional and periurban zones, combined with fine-scale monitoring of vegetation cover and local climate, should be prioritized as part of integrated leishmaniasis control strategies in South America.
5 Conclusions
The results presented in this study reveal a remarkable diversity of sandfly species that could be involved in the transmission of Leishmania spp. in this remnant of the Atlantic Interior Forest, some of which are reported for the first time in the region. The detection of L. amazonensis and L. infantum in the absence of their traditional vectors raises questions about new transmission dynamics, especially considering that several of the species captured and positive for Leishmania spp. DNA have previously been identified as potential vectors of LTA and LV. The high number of positive specimens captured in the secondary forest highlights its relevance in terms of risk, as it could facilitate the transmission of Leishmania spp. to tourists and people involved in educational and scientific activities in the area. Furthermore, the high detection of L. amazonensis DNA in ectoparasites collected from dogs in the indigenous community represents a relevant finding, as these arthropods could facilitate parasite transmission, with key implications regarding the role of dogs as reservoirs of the pathogen. It is crucial to incorporate molecular methods in future studies to detect and identify the pathogen in both domestic and wild reservoirs, which will facilitate a more detailed understanding of the complex transmission dynamics of the disease. In this sense, addressing the transmission of leishmaniasis in wild environments requires adopting strategies beyond traditional approaches, establishing entomo-epidemiological surveillance as a strategic tool within the One Health paradigm.
Supporting information
S1 Table. Sandfly species recorded per trap in different ecotopes of the RBBM, Canindeyú Department, Paraguay, between October 2020 and October 2021.
https://doi.org/10.1371/journal.pntd.0013806.s001
(DOCX)
S2 Table. Correlation analysis between environmental variables and the abundance of sandflies captured in the RBBM, Canindeyú, Paraguay.
https://doi.org/10.1371/journal.pntd.0013806.s002
(DOCX)
Acknowledgments
The authors would like to thank Laura Rodríguez of the Moisés Bertoni Foundation for the design and preparation of the map. We would also like to thank Fredy Ramírez for his assistance in the field during the collection of phlebotomines and Luciano Franco for his assistance in the sandflies’ classification. All authors are grateful to the National System of Researchers of the National Council of Science and Technology (in its Spanish acronym SISNI-CONACYT).
References
- 1.
World Health Organization. Leishmaniasis. 2023. https://www.who.int/news-room/fact-sheets/detail/leishmaniasis
- 2. Lainson R. The Neotropical Leishmania species: A brief historical review of their discovery, ecology and taxonomy. Rev Pan-Amazônica Saúde. 2010;1(2):13–32.
- 3.
World Health Organization. Neglected tropical diseases. World Health Organization. 2021. http://www.who.int/gho/neglected_diseases/leishmaniasis/en/
- 4.
Galati EAB, Rodrigues BL. A review of historical Phlebotominae taxonomy (Diptera: Psychodidae). Neotrop Entomol. 2023;52(4):539–59.
- 5. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27(2):123–47. pmid:22924419
- 6. Martínez N, Benítez IR, Cacciali P, Muñoz M, Aragón MA. Contributions to the knowledge of the distribution of sand flies (Diptera: Psychodidae: Phlebotominae) of Paraguay. Bol Mus Nac Hist Nat Parag. 2021;25(2):0–100.
- 7. Salomón OD, Rossi GC, Cousiño B, Spinelli GR, Rojas de Arias A, López del Puerto DG, et al. Phlebotominae sand flies in Paraguay: abundance distribution in the Southeastern region. Mem Inst Oswaldo Cruz. 2003;98(2):185–90. pmid:12764432
- 8.
Organización Panamericana de la Salud. Leishmaniasis: informe epidemiológico de las Américas. Organización Panamericana de la Salud: OPS. 2021. https://iris.paho.org/handle/10665.2/51742
- 9. Migone LE. Un caso de kalazar en Assunción (Paraguay). Bull Soc Pathol Exot. 1913;6:118–20.
- 10. Torales MR, Martínez NJ, Franco L. Phlebotominae (Diptera: Psychodidae) y especies consideradas como vectores de leishmaniosis en Paraguay. Rev Parag Epidemiol. 2010;1(1):33–5.
- 11. Chena L, Nara E, Canese A, Oddone R, Morán M, Russomando G. Caracterización de cepas de Leishmania, por medio de la técnica de PCR-RFLP de la región del Spliced Leader Miniexon (SLME), aisladas de humanos y caninos en Paraguay. Mem lnst lnvestig Cienc Salud. 2012;10(1):14–23.
- 12.
Torales M, Segovia L, Martínez N, Benítez I, Viveros C, Ruiz Díaz P, et al. Manual de diagnóstico y tratamiento de las leishmaniasis. 1st ed. Asunción: Ministerio de Salud Pública y Bienestar Social, Servicio Nacional de Erradicación del Paludismo. 2018.
- 13. Benítez IR, Andrade-Zampieri R, Silveira-Elkhoury AN, Floeter-Winter LM, Lauletta-Lindoso JA, Pereira J. Detección molecular de Leishmania (Viannia) lainsoni Silveira, Shaw, Braga & Ishikawa, 1987 (Kinetoplastida: Trypanosomatidae) en humanos de Paraguay. Rev del Inst Med Trop. 2019;14(1):21–8.
- 14. Aldama A, Wattiez V, Aldama F, Pereira J, Zampieri R, Lindoso J. Cutaneous leishmaniasis caused by Leishmania (Viannia) lainsoni in Paraguay. Apropos of 2 cases. Med Cutan Iber Lat Am. 2019;47(2):119–22.
- 15. Salvioni Recalde OD, Pereira Brunelli J, Rolon MS, Rojas de Arias A, Aldama O, Gómez CV. First Molecular Report of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis in Paraguayan Inhabitants Using High-Resolution Melt-PCR. Am J Trop Med Hyg. 2019;101(4):780–8. pmid:31407656
- 16. Canese A. Leishmaniosis tegumentaria en el Paraguay. Evolución de 22 años (1975-1996). Rev Parag Microb. 1998;18(1):25–9.
- 17. Yamasaki H, Agatsuma T, Pavon B, Moran M, Furuya M, Aoki T. Leishmania major-like parasite, a pathogenic agent of cutaneous leishmaniasis in Paraguay. Am J Trop Med Hyg. 1994;51(6):749–57. pmid:7810807
- 18. Dantas-Torres F. The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Vet Parasitol. 2007;149(3–4):139–46. pmid:17703890
- 19. Quinnell RJ, Courtenay O. Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology. 2009;136(14):1915–34. pmid:19835643
- 20. Dantas-Torres F. Canine leishmaniasis in the Americas: etiology, distribution, and clinical and zoonotic importance. Parasit Vectors. 2024;17(1):198. pmid:38689318
- 21. Dantas-Torres F. Canine leishmaniosis in South America. Parasit Vectors. 2009;2 Suppl 1(Suppl 1):S1. pmid:19426440
- 22. Marcondes M, Day MJ. Current status and management of canine leishmaniasis in Latin America. Res Vet Sci. 2019;123:261–72. pmid:30708238
- 23. Baneth G, Yasur-Landau D, Gilad M, Nachum-Biala Y. Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology. Parasit Vectors. 2017;10(1):113. pmid:28285601
- 24. Mendoza-Roldan JA, Latrofa MS, Iatta R, R S Manoj R, Panarese R, Annoscia G, et al. Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities. Parasit Vectors. 2021;14(1):461. pmid:34493323
- 25. Baneth G, Nachum-Biala Y, Adamsky O, Gunther I. Leishmania tropica and Leishmania infantum infection in dogs and cats in central Israel. Parasit Vectors. 2022;15(1):147. pmid:35534906
- 26. Rêgo FD, Souza GD, Miranda JB, Peixoto LV, Andrade-Filho JD. Potential Vectors of Leishmania Parasites in a Recent Focus of Visceral Leishmaniasis in Neighborhoods of Porto Alegre, State of Rio Grande do Sul, Brazil. J Med Entomol. 2020;57(4):1286–92. pmid:32112089
- 27. Galvis-Ovallos F, Ueta AE, Marques G de O, Sarmento AMC, Araujo G, Sandoval C, et al. Detection of Pintomyia fischeri (Diptera: Psychodidae) With Leishmania infantum (Trypanosomatida: Trypanosomatidae) Promastigotes in a Focus of Visceral Leishmaniasis in Brazil. J Med Entomol. 2021;58(2):830–6. pmid:33047129
- 28. Gaskin AA, Schantz P, Jackson J, Birkenheuer A, Tomlinson L, Gramiccia M, et al. Visceral leishmaniasis in a New York foxhound kennel. J Vet Intern Med. 2002;16(1):34–44. pmid:11822802
- 29. Rosypal AC, Troy GC, Zajac AM, Frank G, Lindsay DS. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J Parasitol. 2005;91(4):970–2. pmid:17089780
- 30. Dey A, Singh S. Transfusion transmitted leishmaniasis: a case report and review of literature. Indian J Med Microbiol. 2006;24(3):165–70. pmid:16912434
- 31. Monge-Maillo B, Norman FF, Cruz I, Alvar J, López-Vélez R. Visceral leishmaniasis and HIV coinfection in the Mediterranean region. PLoS Negl Trop Dis. 2014;8(8):e3021. pmid:25144380
- 32. Carrasco-Antón N, López-Medrano F, Fernández-Ruiz M, Carrillo E, Moreno J, García-Reyne A, et al. Environmental Factors as Key Determinants for Visceral Leishmaniasis in Solid Organ Transplant Recipients, Madrid, Spain. Emerg Infect Dis. 2017;23(7):1155–9. pmid:28628447
- 33. Diniz SA, Melo MS, Borges AM, Bueno R, Reis BP, Tafuri WL, et al. Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs. Vet Pathol. 2005;42(5):650–8. pmid:16145211
- 34.
Quintal APN. Leishmaniose visceral em hamsters experimentalmente infectados: doença venérea em machos com transmissão sexual. Uberlândia (UB): Universidade Federal de Uberlândia. 2015.
- 35. Coutinho MTZ, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, et al. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol. 2005;128(1–2):149–55. pmid:15725545
- 36. Colombo FA, Odorizzi RMFN, Laurenti MD, Galati EAB, Canavez F, Pereira-Chioccola VL. Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs. Parasitol Res. 2011;109(2):267–74. pmid:21221638
- 37. Recalde SO, González N, Giménez-Ayala A, Vega Gomez MC, González Sander M, Ferreira Coronel M, et al. First DNA report of Leishmania infantum in Evandromyia (complex) cortelezzii and Lutzomyia longipalpis in Alto Paraná, Paraguay. Int J Curr Res. 2017;9(08):55931–4.
- 38. Benítez I, Cacciali P, Elkhoury A, Muñoz M, Aragón M. Análisis de datos basados en evidencia para la caracterización epidemiológica de leishmaniasis en Paraguay: I - Leishmaniasis tegumentaria. Rev Inst Med Trop. 2020;15(2):29–44.
- 39.
Salas D, Bartrina L, Rodríguez L, Ferreira I, Rolon C, Velazquez M. Plan de manejo reserva natural del bosque Mbaracayu 2020-2030. 1st ed. Asunción: Fundación Moises Bertoni. 2019.
- 40. Naidoo R, Hill K. Emergence of indigenous vegetation classifications through integration of traditional ecological knowledge and remote sensing analyses. Environ Manage. 2006;38(3):377–87. pmid:16832592
- 41. Kowalewski M, Pontón F, Pinto F, Velázquez M. Estado sanitario de perros en áreas de interfase entre animales domésticos y silvestres de la reserva natural del bosque Mbaracayú, Paraguay. Rev Soc Cient Py. 2019;24(1):114–25.
- 42. Sudia WD, Chamberlain RW. Battery-operated light trap, an improved model. Mosquito News. 1962;22:126–9.
- 43.
Rangel EF, Shaw JJ. Morfologia e taxonomia: Classificação de Phlebotominae. Flebotomíneos do Brasil. Rio de Janeiro: Fiocruz. 2003. 23–52.
- 44. Marcondes CB. A proposal of generic and subgeneric abbreviations for phlebotomine sandflies (diptera: psychodidae: phlebotominae) of the world. Entomological News. 2007;118(4):351–6.
- 45.
Guimarães JH, Tucci EC, Barros-Battesti DM. Ectoparasitos de importância médico-veterinária. FAPESP. São Paulo: Plêiade. 2001. 155–73.
- 46. Linardi PM, Guimarães LR. Sifonápteros do Brasil. Mem Inst Oswaldo Cruz. 2003;98(3):433.
- 47. Linardi PM, Santos JLC. Ctenocephalides felis felis vs. Ctenocephalides canis (Siphonaptera: Pulicidae): some issues in correctly identify these species. Rev Bras Parasitol Vet. 2012;21(4):345–54. pmid:23295817
- 48. Nava S, Lareschi M, Rebollo C, Benítez Usher C, Beati L, Robbins RG, et al. The ticks (Acari: Ixodida: Argasidae, Ixodidae) of Paraguay. Ann Trop Med Parasitol. 2007;101(3):255–70. pmid:17362600
- 49.
Molina R, Jiménez M, Alvar J. Methods in sand fly research. 1st ed. Alcalá: Editorial Universidad de Alcalá. 2017.
- 50. Ammazzalorso AD, Zolnik CP, Daniels TJ, Kolokotronis S-O. To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis). PeerJ. 2015;3:e1147. pmid:26290800
- 51. De Almeida ME, Koru O, Steurer F, Herwaldt BL, Da Silva AJ. Detection and differentiation of Leishmania spp. in clinical specimens by use of a SYBR green-based realtime PCR assay. J Clin Microbiol. 2017;55:281–90.
- 52. El Tai NO, Osman OF, El Fari M, Presber W, Schönian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg. 2000;94(5):575–9. pmid:11132393
- 53. Hammer Ø, Harper DAT, Ryan PD. Past: Paleontological statistics software package for education and data analysis. Palaeontol Electron. 2001;4(1):1–9.
- 54.
Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW. InfoStat versión 2011.
- 55.
Zelditch ML, Swiderski DL, Sheets HD, Fink WL. Geometric morphometrics for biologists: a primer. 1st ed. San Diego: Elsevier Acad Press. 2004.
- 56.
Strauss RE. Discriminating groups of organisms. In: Elewa AMT. Morphometrics for nonmorphometricians. Berlin: Springer Berlin. 2010. 73–91.
- 57. Eastwood G, Camp JV, Chu YK, Sawyer AM, Owen RD, Cao X, et al. Habitat, species richness and hantaviruses of sigmodontine rodents within the Interior Atlantic Forest, Paraguay. PLoS One. 2018;13(8):e0201307. pmid:30067840
- 58. Brilhante AF, Lima L, de Ávila MM, Medeiros-Sousa AR, de Souza JF, Dos Santos NP, et al. Remarkable diversity, new records and Leishmania detection in the sand fly fauna of an area of high endemicity for cutaneous leishmaniasis in Acre state, Brazilian Amazonian Forest. Acta Trop. 2021;223:106103. pmid:34416187
- 59. De Araujo-Pereira T, de Pita-Pereira D, Baia-Gomes SM, Boité M, Silva F, Pinto I, et al. An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil. Mem Inst Oswaldo Cruz. 2020;115(10):1–17.
- 60. Da Silva AM, de Camargo NJ, dos Santos DR, Massafera R, Ferreira AC, Postai C, et al. Diversidade, distribuição e abundância de flebotomíneos (Diptera: Psychodidae) no Paraná. Neotrop Entomol. 2008;37(2):209–25.
- 61. Tian Y, Chen J, Hu X. Cloning and sequence analysis of the ribosomal DNA ITS gene of Leishmania donovani isolates from hill foci of China. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2004;22(5):294–6. pmid:15830885
- 62. Dinesh DS, Kar SK, Kishore K, Palit A, Verma N, Gupta AK, et al. Screening sandflies for natural infection with Leishmania donovani, using a non-radioactive probe based on the total DNA of the parasite. Ann Trop Med Parasitol. 2000;94(5):447–51. pmid:10983557
- 63. Medeiros ACR, Rodrigues SS, Roselino AMF. Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis. Braz J Med Biol Res. 2002;35(4):421–4. pmid:11960189
- 64. Michalsky EM, Fortes-Dias CL, Pimenta PFP, Secundino NFC, Dias ES. Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae). Rev Inst Med Trop Sao Paulo. 2002;44(5):255–9. pmid:12436164
- 65. Alexander B, Maroli M. Control of phlebotomine sandflies. Med Vet Entomol. 2003;17(1):1–18. pmid:12680919
- 66. Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17(3):279–89. pmid:10384867
- 67. Dantas-Torres F, de Paiva-Cavalcanti M, Figueredo LA, Melo MF, da Silva FJ, da Silva AL, et al. Cutaneous and visceral leishmaniasis in dogs from a rural community in northeastern Brazil. Vet Parasitol. 2010;170(3–4):313–7. pmid:20227186
- 68. Salomón OD, Quintana MG, Bezzi G, Morán ML, Betbeder E, Valdéz DV. Lutzomyia migonei as putative vector of visceral leishmaniasis in La Banda, Argentina. Acta Trop. 2010;113(1):84–7. pmid:19716797
- 69. De Carvalho MR, Valença HF, da Silva FJ, de Pita-Pereira D, de Araújo Pereira T, Britto C, et al. Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera: Psychodidae: Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil. Acta Trop. 2010;116(1):108–10.
- 70. Laurenti MD, Silveira VM dos S, Secundino NFC, Corbett CEP, Pimenta PPF. Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis. Parasitol Int. 2009;58(3):220–6. pmid:19454323
- 71. Tolezano JE, Uliana SRB, Taniguchi HH, Araújo MFL, Barbosa JAR, Barbosa JER, et al. The first records of Leishmania (Leishmania) amazonensis in dogs (Canis familiaris) diagnosed clinically as having canine visceral leishmaniasis from Araçatuba County, São Paulo State, Brazil. Vet Parasitol. 2007;149(3–4):280–4. pmid:17720321
- 72. Dantas-Torres F, Faustino MA da G, Lima OC da C, Acioli RV. Epidemiologic surveillance of canine visceral leishmaniasis in the municipality of Recife, Pernambuco. Rev Soc Bras Med Trop. 2005;38(5):444–5. pmid:16172765
- 73. Shimabukuro PHF, de Andrade AJ, Galati EAB. Checklist of American sand flies (Diptera, Psychodidae, Phlebotominae): genera, species, and their distribution. Zookeys. 2017;(660):67–106. pmid:28794674
- 74. Santini MS, Salomón OD. Eco-epidemiología de las leishmaniosis, Argentina. Rev Arg Parasitol. 2012;1:16–24.
- 75. Carvalho GML, Andrade Filho JD, Falcao AL, Rocha Lima ACVM, Gontijo CMF. Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil. Vector Borne Zoonotic Dis. 2008;8(3):407–14. pmid:18429695
- 76. Saraiva L, Carvalho GML, Gontijo CMF, Quaresma PF, Lima ACVMR, Falcão AL, et al. Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil. J Med Entomol. 2009;46(5):1159–63. pmid:19769049
- 77. de Andrade ARO, Dorval MEMC, de Andrade SMO, Marques A, da Costa Lima Júnior MS, da Silva BAK, et al. First report of natural infection of phlebotomines for Leishmania (Leishmania) chagasi captured in Ponta Porã, on the border between Brazil and Paraguay. Asian Pacific Journal of Tropical Disease. 2011;1(4):253–8.
- 78. Saraiva L, Andrade Filho JD, Silva S de O, de Andrade ASR, Melo MN. The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil. Mem Inst Oswaldo Cruz. 2010;105(8):1033–9. pmid:21225201
- 79. Nascimento BWL, Saraiva L, Neto RGT, Meira PCLS, Sanguinette C de C, Tonelli GB, et al. Study of sand flies (Diptera: Psychodidae) in visceral and cutaneous leishmaniasis areas in the central-western state of Minas Gerais, Brazil. Acta Trop. 2013;125(3):262–8. pmid:23178219
- 80. Cutolo AA, Von Zuben CJ. Flebotomíneos (Diptera, Psychodidae) de área de cerrado no município de Corumbataí, Centro-Leste do estado de São Paulo, Brasil. Rev Bras Parasitol Veterinária. 2008;17(1):45–9.
- 81. Cutolo AA, Galati EAB, Von Zuben CJ. Sandflies (Diptera, Psychodidae) from forest areas in Botucatu municipality, central western São Paulo State, Brazil. J Venom Anim Toxins Incl Trop Dis. 2013;19(1):15. pmid:23849624
- 82. Margonari C, Soares RP, Andrade-Filho JD, Xavier DC, Saraiva L, Fonseca AL, et al. Phlebotomine sand flies (Diptera: Psychodidae) and Leishmania infection in Gafanhoto Park, Divinópolis, Brazil. J Med Entomol. 2010;47(6):1212–9. pmid:21175074
- 83. Donalisio MR, Paiz LM, da Silva VG, Richini-Pereira VB, von Zuben APB, Castagna CL, et al. Visceral leishmaniasis in an environmentally protected area in southeastern Brazil: Epidemiological and laboratory cross-sectional investigation of phlebotomine fauna, wild hosts and canine cases. PLoS Negl Trop Dis. 2017;11(7):e0005666. pmid:28704391
- 84. Villarquide ML, Andreo V, Salomon OD, Santini MS. Distribution of Phlebotominae (Diptera: Psychodidade) species and human cases of leishmaniasis in the Province of Corrientes, Argentina. Rev Soc Entomol Arg. 2022;81(04).
- 85. Galati EAB, Marassá AM, Gonçalves-Andrade RM, Consales CA, Bueno EFM. Phlebotomines (Diptera, Psychodidae) in the speleological province of the Ribeira Valley: 2. Parque Estadual do Alto Ribeira (PETAR), São Paulo State, Brazil. Rev Bras Entomol. 2010;54(3):665–76.
- 86. de Souza CM, Pessanha JE, Barata RA, Monteiro EM, Costa DC, Dias ES. Study on phlebotomine sand fly (Diptera: Psychodidae) fauna in Belo Horizonte, state of Minas Gerais, Brazil. Mem Inst Oswaldo Cruz. 2004;99(8):795–803. pmid:15761593
- 87. Souza NA, Andrade-Coêlho CA, Vilela ML, Rangel EF. The Phlebotominae sand fly (Diptera: Psychodidae) fauna of two Atlantic Rain Forest Reserves in the State of Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz. 2001;96(3):319–24. pmid:11313637
- 88. Quintana MG, Santini MS, Cavia R, Martínez MF, Liotta DJ, Fernández MS, et al. Multiscale environmental determinants of Leishmania vectors in the urban-rural context. Parasit Vectors. 2020;13(1):502. pmid:33008441
- 89. Moya SL, Szelag EA, Manteca-Acosta M, Quintana MG, Salomón OD. Update of the Phlebotominae Fauna with New Records for Argentina and Observations on Leishmaniasis Transmission Scenarios at a Regional Scale. Neotrop Entomol. 2022;51(2):311–23. pmid:34936066
- 90. de Aguiar GM, Vieira VR. Regional Distribution and Habitats of Brazilian Phlebotomine Species. Brazilian Sand Flies. Springer International Publishing. 2018. 251–98.
- 91. Quiroga C, Cevallos V, Morales D, Baldeón ME, Cárdenas P, Rojas-Silva P, et al. Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador. J Med Entomol. 2017;54(6):1704–11. pmid:28981860
- 92. Lozano-Sardaneta YN, Sánchez-Montes S, Sánchez-Cordero V, Becker I, Paternina LE. Molecular detection of Leishmania infantum in sand flies (Diptera: Psychodidae: Phlebotominae) from Veracruz, Mexico. Acta Trop. 2020;207:105492. pmid:32298655
- 93. Serra E Meira PCL, Abreu BL, de Almeida Zenóbio APL, de Castilho Sanguinette C, Rêgo FD, de Lima Carvalho GM, et al. Phlebotominae Fauna (Diptera: Psychodidae) and Molecular Detection of Leishmania (Kinetoplastida: Trypanosomatidae) in Urban Caves of Belo Horizonte, Minas Gerais, Brazil. J Med Entomol. 2022;59(1):257–66. pmid:34532734
- 94. Fernández MS, Lestani EA, Cavia R, Salomón OD. Phlebotominae fauna in a recent deforested area with American tegumentary leishmaniasis transmission (Puerto Iguazú, Misiones, Argentina): seasonal distribution in domestic and peridomestic environments. Acta Trop. 2012;122(1):16–23. pmid:22155061
- 95. Afonso MMS, Costa WA, Azevedo ACR, da Costa SM, Vilela ML, Rangel EF. Data on sand fly fauna (Diptera, Psychodidae, Phlebotominae) in Itatiaia National Park, Rio de Janeiro State, Brazil. Cad Saude Publica. 2007;23(3):725–30.
- 96. Gómez-Bravo A, German A, Abril M, Scavuzzo M, Salomón OD. Spatial population dynamics and temporal analysis of the distribution of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in the city of Clorinda, Formosa, Argentina. Parasites and Vectors. 2017;10(1):1–9.
- 97. Pinheiro MPG, Silva MMDM, Silva Júnior JB, da Silva JHT, Alves MDL, Ximenes MDFFDM. Sand flies (Diptera, Psychodidae, Phlebotominae), vectors of Leishmania protozoa, at an Atlantic Forest Conservation Unit in the municipality of Nísia Floresta, Rio Grande do Norte state, Brazil. Parasit Vectors. 2016;9:83. pmid:26864023
- 98. Carvalho-Silva R, Ribeiro-da-Silva RC, Cruz LNPD, Oliveira M da S de, Amoedo PM, Rebêlo JMM, et al. Predominance of Leishmania (Leishmania) amazonensis DNA in Lutzomyia longipalpis sand flies (Diptera: Psychodidae) from an endemic area for leishmaniasis in Northeastern Brazil. Rev Inst Med Trop Sao Paulo. 2022;64:e32. pmid:35544910
- 99. Sherlock IA. Ecological interactions of visceral leishmaniasis in the state of Bahia, Brazil. Mem Inst Oswaldo Cruz. 1996;91(6):671–83. pmid:9283643
- 100. Aramayo LV, Copa GN, Hoyos CL, Almazán MC, Juarez M, Cajal SP, et al. Leishmaniasis tegumentaria y flebótomos en la localidad de Colonia Santa Rosa del norte de Argentina. Rev Argent Microbiol. 2022;54(2):143–51.
- 101. Pita-Pereira D, Souza GD, Zwetsch A, Alves CR, Britto C, Rangel EF. First report of Lutzomyia (Nyssomyia) neivai (Diptera: Psychodidae: Phlebotominae) naturally infected by Leishmania (Viannia) braziliensis in a periurban area of south Brazil using a multiplex polymerase chain reaction assay. Am J Trop Med Hyg. 2009;80(4):593–5. pmid:19346382
- 102. Dias ES, Michalsky ÉM, do Nascimento JC, Ferreira E de C, Lopes JV, Fortes-Dias CL. Detection of Leishmania infantum, the etiological agent of visceral leishmaniasis, in Lutzomyia neivai, a putative vector of cutaneous leishmaniasis. J Vector Ecol. 2013;38(1):193–6. pmid:23701627
- 103. Thomaz-Soccol V, Gonçalves AL, Baggio RA, Bisetto A Jr, Celestino A, Hospinal-Santiani M, et al. One piece of the puzzle: Modeling vector presence and environment reveals seasonality, distribution, and prevalence of sandflies and Leishmania in an expansion area. One Health. 2023;17:100581. pmid:37332885
- 104. Galati EAB, Fonseca MB, Marassá AM, Bueno EFM. Dispersal and survival of Nyssomyia intermedia and Nyssomyia neivai (Diptera: Psychodidae: Phlebotominae) in a cutaneous leishmaniasis endemic area of the speleological province of the Ribeira Valley, state of São Paulo, Brazil. Mem Inst Oswaldo Cruz. 2009;104(8):1148–58. pmid:20140376
- 105. Moya SL, Giuliani MG, Santini MS, Quintana MG, Salomón OD, Liotta DJ. Leishmania infantum DNA detected in phlebotomine species from Puerto Iguazú City, Misiones province, Argentina. Acta Trop. 2017;172:122–4. pmid:28476601
- 106.
Barretto MP. Observações sobre a biologia em condições naturais dos flebótomos do estado de São Paulo (Diptera: Psychodidae). São Paulo: Faculdade de Medicina da USP. 1943.
- 107.
Forattini OP. Entomologia Médica. 1st ed. São Paulo: Edgard Blücher & Editora da Universidade de São Paulo. 1973.
- 108.
Araújo Filho NA. Epidemiologia da leishmaniose tegumentar na Ilha Grande. Rio de Janeiro: Universidade Federal do Rio de Janeiro. 1979.
- 109. Rangel EF, Souza NA, Wermelinger ED, Barbosa AF, Andrade CA. Biology of Lutzomyia intermedia Lutz & Neiva, 1912 and Lutzomyia longipalpis Lutz & Neiva, 1912 (Diptera, Psychodidae), under experimental conditions. I. Feeding aspects of larvae and adults. Mem Inst Oswaldo Cruz. 1986;81(4):431–8. pmid:3613978
- 110. Guimarães VCFV, Pruzinova K, Sadlova J, Volfova V, Myskova J, Filho SPB, et al. Lutzomyia migonei is a permissive vector competent for Leishmania infantum. Parasit Vectors. 2016;9:159. pmid:26988559
- 111. de Souza MB, Marzochi MC de A, de Carvalho RW, Ribeiro PC, Pontes C dos S, Caetano JM, et al. Absence of Lutzomyia longipalpis in some endemic visceral leishmaniasis areas in Rio de Janeiro municipality. Cad Saude Publica. 2003;19(6):1881–5. pmid:14999355
- 112. de Carvalho MR, Lima BS, Marinho-Júnior JF, da Silva FJ, Valença HF, Almeida F de A, et al. Phlebotomine sandfly species from an American visceral leishmaniasis area in the Northern Rainforest region of Pernambuco State, Brazil. Cad Saude Publica. 2007;23(5):1227–32. pmid:17486244
- 113. Salomón OD, Filho JDA, Fernández MS, Rosa JR, Szelag EA, Santini MS. Nuevos registros de Phlebotominae (Diptera: Psychodidae) para la Argentina. Rev la Soc Entomológica Argentina. 2010;69(3–4):261–5.
- 114. Nieves E, Pimenta PF. Development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae). J Med Entomol. 2000;37(1):134–40. pmid:15218917
- 115. Rojas-Jaimes J, Requena EZ-, Correa-Nuñez G. Lutzomyia shannoni a potential vector of Leishmania chagasi in Madre de Dios, Peru. Rev Peru Med Exp Salud Publica. 2018;35(3):534–6. pmid:30517491
- 116. Travi BL, Ferro C, Cadena H, Montoya-Lerma J, Adler GH. Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas. Res Vet Sci. 2002;72(1):83–6. pmid:12002643
- 117. Cazorla-Perfetti D. Lista comentada de los flebotominos (Diptera: Psychodidae, Phlebotominae) citados para Venezuela. Rev Multidiscip del Cons Investig la Univ Oriente. 2015;27:178–231.
- 118. Killick-Kendrick R. Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Trans R Soc Trop Med Hyg. 1995;89(1):125.
- 119. Ferro C, Cárdenas E, Corredor D, Morales A, Munstermann LE. Life cycle and fecundity analysis of Lutzomyia shannoni (Dyar) (Diptera: Psychodidae). Mem Inst Oswaldo Cruz. 1998;93(2):195–9. pmid:9698892
- 120. Pech-May A, Escobedo-Ortegón FJ, Berzunza-Cruz M, Rebollar-Téllez EA. Incrimination of four sandfly species previously unrecognized as vectors of Leishmania parasites in Mexico. Med Vet Entomol. 2010;24(2):150–61. pmid:20604861
- 121. Sábio PB, Andrade AJ, Galatit EAB. Assessment of the taxonomic status of some species included in the shannoni complex, with the description of a new species of Psathyromyia (Diptera: Psychodidae: Phlebotominae). J Med Entomol. 2014;51(2):331–41. pmid:24724281
- 122. PB Sábio, AJ Andrade, EAB Galati. Description of Psathyromyia (Psathyromyia) barataisp. n. (Diptera: Psychodidae: Phlebotominae) from Cantareira State Park, São Paulo, Brazil. J Med Entomol. 2016;53(1):83–90.
- 123. Dorval MEC, Cristaldo G, da Rocha HC, Alves TP, Alves MA, Oshiro ET, et al. Phlebotomine fauna (Diptera: Psychodidae) of an American cutaneous leishmaniasis endemic area in the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz. 2009;104(5):695–702. pmid:19820827
- 124. Lima ACS, Gomes CMC, Tomokane TY, Campos MB, Zampieri RA, Jorge CL, et al. Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil. Genet Mol Biol. 2021;44(2):e20200123. pmid:33949621
- 125. Szelag EA, Andrade Filho JD, Rosa JR, Parras MA, Stein M, Quintana MG. Argentinian phlebotomine fauna, new records of Phlebotominae (Diptera: Psychodidae) for the country and the province of Chaco. Zootaxa. 2016;3(7):427–43.
- 126. Guimarães-E-Silva AS, Silva S de O, Ribeiro da Silva RC, Pinheiro VCS, Rebêlo JMM, Melo MN. Leishmania infection and blood food sources of phlebotomines in an area of Brazil endemic for visceral and tegumentary leishmaniasis. PLoS One. 2017;12(8):e0179052. pmid:28837565
- 127. Esposito MM, Turku S, Lehrfield L, Shoman A. The Impact of Human Activities on Zoonotic Infection Transmissions. Animals (Basel). 2023;13(10):1646. pmid:37238075
- 128. Dorval MEC, Cristaldo G, da Rocha HC, Alves TP, Alves MA, Oshiro ET, et al. Phlebotomine fauna (Diptera: Psychodidae) of an American cutaneous leishmaniasis endemic area in the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz. 2009;104(5):695–702. pmid:19820827
- 129. Dorval MEC, Alves TP, Cristaldo G, da Rocha HC, Alves MA, Oshiro ET, et al. Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil. Rev Soc Bras Med Trop. 2010;43(5):491–5. pmid:21085855
- 130. Ferreira MGPA, Fattori KR, Souza F, Lima VMF. Potential role for dog fleas in the cycle of Leishmania spp. Vet Parasitol. 2009;165(1–2):150–4. pmid:19595512
- 131. Quinnell RJ, Carson C, Reithinger R, Garcez LM, Courtenay O. Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis: longitudinal study and meta-analysis. PLoS Negl Trop Dis. 2013;7(1):e1992. pmid:23326615
- 132. Cruz I, Acosta L, Gutiérrez MN, Nieto J, Cañavate C, Deschutter J, et al. A canine leishmaniasis pilot survey in an emerging focus of visceral leishmaniasis: Posadas (Misiones, Argentina). BMC Infect Dis. 2010;10:342. pmid:21122107
- 133. Pinto IS, dos Santos CB, Grimaldi G, Ferreira AL, Falqueto A. American visceral leishmaniasis dissociated from Lutzomyia longipalpis (Diptera, Psychodidae) in the State of Espírito Santo, Brazil. Cad Saude Publica. 2010;26(2):365–72.
- 134. Azarm A, Dalimi A, Mohebali M, Mohammadiha A, Pirestani M, Zarei Z, et al. Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran. J Arthropod Borne Dis. 2022;16(3):243–50. pmid:37056639
- 135. Darvishi MM, Youssefi MR, Changizi E, Lima RR, Rahimi MT. A new flea from Iran. Asian Pacific Journal of Tropical Disease. 2014;4(2):85–7.
- 136. Reimann MM, Torres-Santos EC, de Souza CSF, Andrade-Neto VV, Jansen AM, Brazil RP, et al. Oral and Intragastric: New Routes of Infection by Leishmania braziliensis and Leishmania infantum?. Pathogens. 2022;11(6):688. pmid:35745542
- 137. Baneth G, Samish M, Shkap V. Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris). J Parasitol. 2007;93(2):283–99. pmid:17539411
- 138. Rêgo FD, Shimabukuro PHF, Quaresma PF, Coelho IR, Tonelli GB, Silva KMS, et al. Ecological aspects of the Phlebotominae fauna (Diptera: Psychodidae) in the Xakriabá Indigenous Reserve, Brazil. Parasit Vectors. 2014;7:220. pmid:24886717
- 139. Pereira RA, Pereira AM, Pinto IS, Falqueto A. Ecology of phlebotomine sand flies (Diptera: Psychodidae) in Itaúna, Minas Gerais, Brazil. Rev Soc Bras Med Trop. 2020;53:e20190538.
- 140. da Silva BQ, Afonso MMDS, Freire LJM, da Santana ALF, Pereira-Colavite A, Rangel EF. Ecological Aspects of the Phlebotominae Fauna (Diptera: Psychodidae) among Forest Fragments and Built Areas in an Endemic Area of American Visceral Leishmaniasis in João Pessoa, Paraíba, Brazil. Insects. 2022;13(12):1156. pmid:36555066
- 141. Fernandes WS, Oliveira EF, Sampaio LFC, Sanguinette CC, Ribeiro JR, Casaril AE. Phlebotomine sandfly (Diptera: Psychodidae) fauna and the association between climatic variables and the abundance of Lutzomyia longipalpis in central western Brazil. J Med Entomol. 2022;59(3):954–63.
- 142. de Oliveira EF, dos Santos Fernandes CE, Araújo e Silva E, Brazil RP, de Oliveira AG. Climatic factors and population density of Lutzomyia longipalpis (Lutz & Neiva, 1912) in an urban endemic area of visceral leishmaniasis in midwest Brazil. J Vector Ecol. 2013;38(2):224–8. pmid:24581349
- 143. Carvalho BM, Rangel EF, Ready PD, Vale MM. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change. PLoS One. 2015;10(11):e0143282. pmid:26619186