Predominance of Leishmania (Leishmania) amazonensis DNA in Lutzomyia longipalpis sand flies (Diptera: Psychodidae) from an endemic area for leishmaniasis in Northeastern Brazil.

Leishmaniasis is a serious public health concern in the Northeastern region of Brazil, where the sand fly fauna is well studied, although few species have been identified as competent vectors. The detection of Leishmania spp. parasites in wild-caught sand flies could help sanitary authorities draw strategies to avoid the transmission of the parasites and, therefore, the incidence of leishmaniases. We detected Leishmania DNA in wild-caught sand flies and correlated that data with aspects of sand fly ecology in the Caxias municipality, Maranhao State, Brazil. The sand flies were sampled in the peridomicile (open areas in the vicinity of human residences) and intradomicile (inside the residences) from July/2019 to March/2020. Leishmania DNA was detected in females, targeting a fragment of the Internal Transcribed Spacer (ITS1) from ribosomal DNA. Among the fourteen species of sand flies identified, five (Lutzomyia longipalpis, Nyssomyia whitmani, Evandromyia evandroi, Micropygomyia trinidadensis, and Micropygomyia quinquefer) harbored DNA of Leishmania (Leishmania) amazonensis. The most abundant species in rural (Ny. whitmani: 35.2% and Ev. evandroi: 32.4%) and urban areas (Lu. longipalpis: 89.8%) are the permissive vectors of L. (L.) amazonensis, especially Ny. whitmani, a known vector of causative agents of cutaneous leishmaniasis. Although Lu. longipalpis is the vector of L. (L.) infantum, which was not detected in this study, its permissiveness for the transmission of L. (L.) amazonensis has been reported. We suspect that visceral leishmaniasis and cutaneous leishmaniasis are caused by L. (L.) amazonensis, and the transmission may be occurring through Lu. longipalpis, at least in the urban area.

INTRODUCTION

Leishmaniases are neglected tropical diseases that affect numerous people annually; caused by protozoans from the genus Leishmania (Kinetoplastida, Trypanosomatidae), transmitted among vertebrate hosts by sand flies (Diptera: Psychodidae). Traditionally, leishmaniases are divided according to the causative agent, disease progression, and symptoms into visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), depending on the species of the pathogen .

Leishmaniases are found in the Americas from South-Central Texas to Central and South America, except for Chile . The North and Northeast regions of Brazil have the most cases of visceral and cutaneous leishmaniasis . In Maranhao State, Northeastern Brazil, where both diseases are endemic, more than 90 sand fly species are distributed throughout several ecosystems . Molecular surveys have shown the occurrence of DNA from at least seven Leishmania species in sand flies sampled in Maranhao . Such diversity of sand flies and parasites is determined by the intersection of three biomes in Maranhao State: the Amazon Rainforest, the Cerrado (savannah-like vegetation), and the Caatinga (seasonal dry forest), which promotes the formation of several ecotones .

In Caxias, a municipality located in the Cerrado biome of Maranhao, 27 species of sand flies have already been recorded . The most medically relevant species are: i) Lutzomyia longipalpis (Lutz and Neiva, 1912), the main vector of Leishmania (Leishmania) infantum, which has large populations in urban areas; and ii) Nyssomyia whitmani (Antunes and Coutinho, 1939), the competent vector of Leishmania (Viannia) braziliensis and a putative vector of L. (V.) shawi, and usually found in rural zones of this region .

The vegetation cover of the municipality of Caxias has been severely deforested by the real estate business, with the construction of large popular housing estates, malls, roads, and other ventures . This process occurs in peri-urban areas (suburbs), thus contributing to a higher density and adaptation of vector species in these environments, primarily Lu. longipalpis and Ny. whitmani, thereby promoting and increasing the number of leishmaniases cases . In the last four years (2017 to 2020), forty-four confirmed cases of VL, twenty-nine cases of CL in humans, and 1,001 cases of seropositive dogs with VL were reported.

Recently, Guimarães-e-Silva et al. detected the DNA of Leishmania spp. belonging to seven distinct sand fly species from urban and rural areas of the municipality of Caxias. This surprising result motivated us to obtain more consistent information about these parasites circulating in the city. Therefore, we monitored, through molecular biology approaches, the circulation of different Leishmania species in sand flies from urban and rural areas of Caxias, where cases of both forms of leishmaniasis occur.

MATERIAL AND METHODS

Characterization of the sample collection sites and ethical considerations

Sand flies were collected in two localities of the Caxias municipality, Maranhao State, Brazil: Povoado Mulata/rural area (04°54’55” S and 43°23’15” W) and Volta Redonda/urban area (04°52’57” S and 43°21’00” W). These localities were chosen based on the presence of domestic animals within the residences and the record of human leishmaniases cases during 2018 and 2019.

The Povoado Mulata includes approximately ninety-seven residences; it is characterized by unpaved streets, mud-built houses (the majority), large green and shaded areas, yards with fruit trees, and secondary vegetation located close to the houses. The Volta Redonda neighborhood contains 2,002 residences, high human density, paved streets, an intense flow of people, commercial areas, and several environmental alterations caused by the man, surrounded by the presence of secondary vegetation (Figure 1).

Figure 1

– Location of the capture sites of phlebotomine sand flies in Povoado Mulata and Volta Redonda, municipality of Caxias, Maranhao State, Brazil.

The Research Ethics Committee from the Universidade Estadual do Maranhao - CEP/UEMA approved this study under protocol N° 3.893.983. The field studies were conducted on private properties. The landowners authorized the installation of traps in their peridomicile areas (yards and open areas next to the residence, but still within the property) and intradomicile areas (living rooms or kitchens), which did not involve endangered or protected species or contact with people.

Sampling and identification of the sand flies

Sand flies were sampled from 6:00 p.m. to 6:00 a.m., on two consecutive nights, once a month, from July 2019 to March 2020. Two light traps of the CDC (Center of Disease Control) type were set 1.5 meters above ground level in five residences in each area: one in the peridomicile (near animal shelters such as pigsties, hen houses, and stables) and another in the intradomicile (installed in the living room or the kitchen), for a total of ten traps per night in each location. The capture effort was 2 traps x 12h x 2 nights x 5 residences x 2 locations x 9 months = 4,320h. The locations of the residences were georeferenced using the Global Positioning System (GPS) with the consent of the residents.

After transportation to the laboratory, the insects were stored at -20 ºC in 1.5 mL plastic microtubes. The specimens were clarified and prepared for identification following the technique described by Vilela et al. , and identified morphologically according to the taxonomy and classification of Galati . The female specimens’ thorax and abdomen were properly conditioned in a -20 ºC freezer for molecular assays.

Molecular assays

Total DNA from each specimen was extracted by Phenol:Chloroform:Isoamyl Alcohol (25:24:1), according to Michalsky et al. . The DNA of Leishmania spp. was detected by Polymerase Chain Reaction (PCR), with amplification of a fragment of approximately 300 to 350 base pairs (bp) of the Internal Transcribed Spacer (ITS1) ribosomal DNA, using the primers LITSR (5’-CTGGATCATTTTCCGATG-3’) and L5.8S (5’-TGATACCACTTATCGCACTT-3’), according to Schonian et al. . The Personal Cycler Thermal Cycler (BiometraTM, Jena, Germany) was used for sample amplification, and the PCR reaction mixture (26µL) was prepared with a final concentration of 1µM of each primer; 0.2mM dNTP (Promega®, Madison, WI, USA), 1U of Taq DNA Polymerase (Promega®), 3mM of MgCl2, enzyme buffer and 3µL of total DNA (about 30 ng/µL for each sample). The amplified products were stored at -20 °C until analysis.

For each PCR assay, a negative control (reaction mixture + 3µL sterile water) and a positive control (reaction mixture + 90 ng of DNA from L. (L.) amazonensis (IFLA/BR/1968/PH8), extracted from cultures) were used. Amplicons were stained with GelRed™ (Nucleic Acid Gel Stain - Biotium, Hayward, CA, USA) and visualized under UV light after electrophoresis through a 1% agarose gel. All PCR products that showed the same band patterns as the positive control were purified with the commercial ExoSAP-IT PCR Product Cleanup Reagent kit (Applied Biosystems™, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, 1µL of a purified sample (30-40 ng/µL), 1µL (4.5 µM) of the LITSR primer, and 4µL of nuclease-free water were added to sterile microtubes of 0.2 mL, dried and shipped to the sequencing service by the ACTGene (Analises Moleculares Ltda, Brazil). After our analysis, the DNA sequences were deposited on the GenBank platform of the National Center for Biotechnological Information (NCBI) website, under the access numbers available in Supplementary Table S1.

Sequence identification

The identification of Leishmania species was based on the identity of the ITS1 sequences obtained by PCR from the total DNA of sand flies. The sixty-two sequences obtained were preliminarily aligned by the ClustalW algorithm in Bioedit v7.2.5 software . Sixty of those were checked preliminary for identity using the NCBI’s online BLAST (Basic Local Alignment Search Tool) tool . Only fifty sequences presented an acceptable size (at least 230 base pairs) for further phylogenetic analysis, which resembles sequences from other studies deposited in the NCBI GenBank (final alignment of 260 base pairs), representing different species and complexes within the genus Leishmania, and sequences from Trypanosoma cruzi as an outgroup (Supplementary Table S2). For haplotype identification, we used the software DnaSP v6 (Barcelona, Spain).

Statistical analyses

An F-test (Quasi-Poisson model with log-link function) was used to verify whether the frequencies of the sand fly species found in the areas (rural and urban) were equal or different from each other. The Chi-square (χ2) test was used to verify this difference by type of environment (intradomicile and peridomicile). The tests were performed with the R software package version 2020 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Sand fly fauna

A total of 2,244 sand flies belonging to fourteen species and seven genera were collected: Lutzomyia (França, 1924), Nyssomyia (Barreto, 1962), Evandromyia (Mangabeira, 1941), Micropygomyia (Barreto, 1962), Sciopemyia (Barreto,1962), Psathyromyia (Barreto,1962) and Brumptomyia (França & Parrot, 1921). The most abundant species were Ny. whitmani (31.4%), Lu. longipalpis (23.4%), Ev. evandroi (Costa Lima & Antunes, 1936) (28.1%), Ev. lenti (Mangabeira, 1938) (9.63%) and Mi. trinidadensis (Newstead, 1922) (5.84%) (Table 1).

Table 1

Distribution of phlebotomine sand fly species collected from July 2019 to March 2020 according to the area, rural (Povoado Mulata) and urban (Volta Redonda), and gender (male and female) of the municipality of Caxias, Maranhao State, Brazil.

Species	Rural area			Urban area			Overall Total
	♂	♀	♂/♀	♂	♀	♂/♀	N	%
Lu. longipalpis Ny. whitmani Ev. evandroi Ev. lenti Mi. trinidadensis Mi. oswaldoi Mi. quinquefer Ev. termitophila Br. avellari Sc. sordellii Ev. cortelezzii Ny. intermedia Ps. hermanlenti Ev. sallesi	168 453 326 134 39 9 4 0 3 1 0 1 0 0	83 230 303 82 90 3 5 3 0 1 0 0 0 0	251 683 629 216 129 12 9 3 3 2 0 1 0 0	212 16 1 0 0 0 0 1 0 0 2 0 1 1	63 6 1 0 2 0 0 0 0 0 0 0 0 0	275 22 2 0 2 0 0 1 0 0 2 0 1 1	526 705 631 216 131 12 9 4 3 2 2 1 1 1	23.4 31.4 28.1 9.63 5.84 0.53 0.40 0.18 0.13 0.09 0.09 0.045 0.045 0.045
TOTAL	1.138	800	1.936	234	72	306	2.244	100
%	58.7	41.3	100	76.5	23.5	100	100

Lu. = Lutzomyia; Ny. = Nyssomyia; Ev. = Evandromyia; Mi. = Micropygomyia; Sc. = Sciopemyia; Ps. = Psathyromyia; Br. = Brumptomyia; ♂ = male; ♀ = female; N = absolute number; % = percentage.

Association with rural and urban environments

The abundance of sand flies was higher in rural areas (86.4%) than in urban areas (13.6%) (F = 8,8494, p < 0.0001). In addition, the rural area also showed higher species diversity (n = 11) than the urban area (n = 8). Seven species were only found in the rural area whereas three exclusively in the urban area. Four were sampled in both areas (Table 1). In the rural area, the dominant species were Ny. whitmani (35.2%), Ev. evandroi (32.4%), Lu. longipalpis (12.9%) and Ev. lenti (11.1%). In the urban area, Lu. longipalpis was predominant (89.9%) (Table 1).

Association with intra- and peridomicile environments

In both rural (53.7%; X2 = 147.8571 p < 0.05) and urban (59.1%; X2 = 25.28571 p < 0.05) areas, abundance was higher intradomicile (Table 2). In the rural area, the prevalent intradomicile species were Ev. evandroi (44.6%) and Ny. whitmani (20.9%), and the prevalent peridomicile species were Ny. whitmani (51.8%) and Lu. longipalpis (19.1%). In the urban area, Lu. longipalpis was the prevalent species in both environments, intradomicile (95.6%) and peridomicile (81.6%) (Table 2).

Table 2

Distribution of phlebotomine sand fly species collected from July 2019 to March 2020 according to intra- and peridomicile environments in rural and urban areas, in the Caxias municipality, Maranhao State, Brazil.

Species	Rural area				Urban area
	Intradomicile		Peridomicile		Intradomicile		Peridomicile
	N	%	N	%	N	%	N	%
Lu. longipalpis	80	7.68	171	19.1	173	95.6	102	81.6
Ny. whitmani	218	20.9	465	51.8	4	2.21	18	14.4
Ev. evandroi	464	44.6	165	18.4	0	0	2	1.60
Ev. lenti	152	14.6	64	7.13	0	0	0	0
Mi. trinidadensis	104	9.99	25	2.79	0	0	2	1.60
Mi. oswaldoi	10	0.96	2	0.22	0	0	0	0
Mi. quinquefer	7	0.67	2	0.22	0	0	0	0
Ev. termitophila	3	0.29	0	0	1	0.55	0	0
Br. avellari	0	0	3	0.33	0	0	0	0
Sc. sordellii	2	0.19	0	0	0	0	0	0
Ev. cortelezzii	0	0	0	0	2	1.10	0	0
Ny. intermedia	1	0.10	0	0	0	0	0	0
Ps. hermanlenti	0	0	0	0	0	0	1	0.80
Ev. sallesi	0	0	0	0	1	0.55	0	0
Total	1,041	100	897	100	181	100	125	100

Lu. = Lutzomyia; Ny. = Nyssomyia; Ev. = Evandromyia; Mi. = Micropygomyia; Sc. = Sciopemyia; Ps. = Psathyromyia; Br. = Brumptomyia; N= absolute number; % = percentage.

Identification of Leishmania spp. DNA sequences in phlebotomine sand flies

The presence of Leishmania spp. DNA was investigated in a total of 123 individually prepared samples of female sand flies (68 rural and 55 urban) belonging to eight species (Table 3). The ITS1 fragment was successfully amplified from sixty-two females belonging to five species, found in both, intradomicile and peridomicile environments, in rural and urban areas. The species Lu. longipalpis showed the highest positivity (44%), followed by Ny. whitmani (27%), Ev. evandroi (13%), Mi. trinidadensis (13%) and Mi. quinquefer (3%) (Table 3 and Figure 2A).

Table 3

Species of phlebotomine sand flies analyzed, and the number of females positive for Leishmania DNA captured in the intra- and peridomicile of rural and urban areas of Caxias municipality, Maranhao State, Brazil.

Species	Rural area			Urban area			Total samples analyzed	Total positive samples
	Intra.	Peri.	Positive samples	Intra.	Peri.	Positive samples		N	%
Lu. longipalpis	8	7	9	28	17	18	60	27	44
Ny. whitmani	4	14	14	0	6	3	24	17	27
Ev. evandroi	10	4	8	0	1	0	15	8	13
Mi. trinidadensis	11	3	7	2	1	1	17	8	13
Mi. quinquefer	1	2	2	0	0	0	3	2	3
Ev. lenti	2	0	0	0	0	0	2	0	0
Ev. termitophila	1	0	0	0	0	0	1	0	0
Sc. sordellii	1	0	0	0	0	0	1	0	0
Total	38	30	40	30	25	22	123	62	100

Lu. = Lutzomyia; Ny. = Nyssomyia; Ev. = Evandromyia; Mi. = Micropygomyia; Sc. = Sciopemyia; N = absolute number; % = percentage; Intra.= Intradomicile; Peri.= Peridomicile.

Figure 2

– Identification of Leishmania (Leishmania) amazonensis DNA sequences in phlebotomine sand flies (A): Agarose gel (1%) showing amplified products of 300-350 bp with the Leishmania ITS1 primers. PC: positive control; NC: negative control; 1-7: positive phlebotomine sand fly samples. (B) Phylogenetic tree of ITS1 DNA sequences of Leishmania spp. amplified from phlebotomine sand flies captured in two areas, rural and urban, in the Caxias municipality, Maranhao State, reconstructed by the Neighbor-Joining method. The percentage of replicate trees in which the associated taxons clustered by a bootstrap test (1000 replicates) is shown next to the nodes. Branch lengths are in the same units as the evolutionary distances used to infer the phylogenetic tree. Haplotype 1, highlighted in bold, represents the only haplotype identified in all ITS-1 sequences (all positive samples). The brackets indicate sequences from the Mexican complex (including Leishmania (L.) amazonensis and Leishmania (L.) mexicana). Trypanosoma cruzi is the external group.

Only sixty ITS1 sequences presented more than 90% similarity with sequences from Leishmania spp. and more than 97% similarity with sequences of Leishmania (L.) amazonensis stored in the GenBank database (Supplementary Table S1). Among them, the fifty sequences of an acceptable size for phylogenetic analysis belonged to a single haplotype (Figure 2B). The sequences of two PCR-positive samples were noticeably short, preventing us from checking their identity, and 10 sequences were just about long enough just for preliminary species checking by BLAST, although not for a phylogeny confirmation.

Monthly distribution of sand flies

In the rural area, Ny. whitmani and Lu. longipalpis was predominant and showed opposite trends regarding abundance throughout the sampling period. During the rainy season, Ev. evandroi and Ny. whitmani predominated, also with opposite trends (Figure 3A). Lu. longipalpis was abundant throughout the period in urban areas (Figure 3B).

Figure 3

– Monthly distribution of phlebotomine sand fly species according to the collection area, rural (A), and urban (B).

DISCUSSION

Sand flies are widely distributed in urban and rural areas in the Caxias municipality , maybe as a result of human population growth and subsequent deforestation of areas previously inhabited by sand fly fauna. Meanwhile, a sizable proportion of the population in these areas keeps domestic animals (such as dogs, hens and pigs) in their backyards . Domestic animals and their shelters play a significant role in attracting sand flies, especially when backyards are not clean .

Based on the distribution pattern of the sand flies, it is possible that some species such as Lu. longipalpis, Ny. whitmani, Ev. evandroi, and Ev. lenti are well integrated into the Povoado Mulata (rural area), abundantly invading the peridomiciles and easily entering human dwellings, where large numbers accumulate. These species have anthropophilic habits and adapt to different environments . The adjoining area has suffered from the impact of human activity and, consequently, has experienced outbreaks of leishmaniasis. This study aimed to evaluate the composition, abundance, species richness and seasonal distribution of sand flies in the region and to determine the constancy of the insect population. Methods: The survey was conducted at three sites located in the municipalities of Barreirinhas and Santo Amaro between September 2012 and August 2013. Sampling was performed monthly using automatic light traps installed 1.5 m above the soil adjacent to 13 randomly selected rural dwellings. At each site, one trap was placed in the peridomicile near to animal enclosures and another (extradomicile. Therefore, the infestation in the rural locality of Povoado Mulata by sand fly species such as Ny. whitmani and Lu. longipalpis, on its own, justifies the occurrence of CL and VL, respectively, since those sand flies are competent vectors for the parasites that cause these diseases . Despite occurring in very low density, Ny. intermedia deserves attention since it has considerable epidemiological importance, being identified as a vector of parasites that cause CL , as well as Mi. quinquefer, which has been found harboring L. (L.) infantum DNA in Puerto Iguazu, Argentina .

In this study, we found that, unlike other sand flies, Lu. longipalpis appeared abundantly in the urban environment. This vector is highly adapted to different degrees of urbanization and shows opportunistic feeding habits compared to other sand flies, feeding on the blood of humans and of a wide range of domestic animals . The high abundance of Lu. longipalpis in the Volta Redonda urban neighborhood warrants the occurrence of VL in this environment, corroborating previous observations and presenting a risk for the maintenance and expansion of L. (L.) infantum or, maybe, L. (L.) amazonensis . However, the species related to the transmission of causative agents of CL, like Ny. whitmani, had a low abundance. In this case, it is interesting to note the predominance of Lu. longipalpis in the sampling. According to previous reports, this vector is permissive for the infection by Leishmania species that cause CL, such as L. (L.) amazonensis .

Among the sand fly species found in the municipality of Caxias, several are identified as vectors of CL, while Lu. longipalpis is the only vector of L. (L.) infantum, which causes VL. Nevertheless, we detected Leishmania DNA in only five species, and they all only had L. (L.) amazonensis DNA fragments. Interestingly, in previous studies conducted in the same city, but in the urban neighborhood of Salobro and the Bom Jardim rural village, DNA fragments of at least seven species of these parasites were detected: L. (L.) infantum, L. (V.) shawi, L. (L.) mexicana, L. (V.) braziliensis, L. (V.) guyanensis, L. (L.) amazonensis, L. (V.) lainsoni, and L. (V.) naiffi, including putative mixed infections in sand flies .

The possible reduction in the diversity of circulating Leishmania spp. may be explained by the fact that our study was conducted in different areas (Povoado Mulata and Volta Redonda) from those in the previous survey. It is unlikely that several Leishmania species disappeared by competitive exclusion in such a brief period (three years), leaving only L. (L.) amazonensis to be transmitted by the sand fly populations. It is more feasible to assume that Leishmania species may segregate along the municipality area due to differences in local environmental characteristics: hosts, vectors, circulating parasites, backyard sanitary conditions, presence or absence of natural and cultivated vegetation, among others.

The presence of L. (L.) amazonensis could be related to the occurrence of twelve cases of VL during the study period, considering the absence of L. (L.) infantum in the results of this research, albeit in rural areas, where Ny. whitmani was abundant and could be an associated vector. However, it could not explain the urban cases or the occurrence of ten cases of VL. As mentioned, L. (L.) infantum DNA was not found in any sand fly in the area and period of this study. Although the clinical manifestation of VL is traditionally associated only with L. (L.) infantum, studies point to the participation of L. (L.) amazonensis . This parasite has been often recovered from sources in urban environments infecting dogs , synanthropic rodents , and humans ; however, its known vector, Bichromomyia flaviscutellata, is rarely found in this environment.

It has also been shown, by an experimental study, that a healthy hamster (Mesocricetus auratus) showed clinical manifestation of VL three months after being bitten by Lu. longipalpis infected with L. (L.) amazonensis , and that this sand fly is capable of sustaining infection of metacyclic forms of L. (L.) amazonensis inside its gut . Furthermore, L. (L.) amazonensis is already known to be genetically diverse in different areas of Northeastern Brazil and is associated with VL cases in humans . Moreover, recently, Ribeiro-da-Silva et al. demonstrated that Lu. longipalpis was able to transmit L. (L.) amazonensis to an immunosuppressed Balb/c mouse (Mus musculus), which developed clinical symptoms of diffuse cutaneous leishmaniasis. In this context, the data suggest that L. (L.) amazonensis cannot be disregarded as a causative agent of CL and VL in the urban area of our study.

Hence, we suspect the participation of Lu. longipalpis as a vector of L. (L.) amazonensis and the possible involvement of this parasite in the production of CL and VL for the following reasons:

Lu. longipalpis was found with L. (L.) amazonensis DNA;

it constitutes the only sand fly highly abundant in the urban area during nine months of entomological survey;

occurrence of urban cases of CL in the absence of other sand fly species;

the notification of VL cases during the study period in the absence of circulating L. (L.) infantum in the vector;

Lu. longipalpis is known to be a permissive vector for L. (L.) amazonensis transmission. Similarly, we suspect the participation of L. (L.) amazonensis as the etiologic agent of the reported cases of CL and VL due to: 1) the absence of evidence of other Leishmania spp., including L. (L.) infantum, in the sand flies we studied.

CONCLUSION

To better elucidate our findings and demonstrate whether L. (L.) amazonensis is predominating in previous endemic regions of L. (L.) infantum, we suggest that surveys (through integrative approaches) of the causative agents of leishmaniasis infecting dogs, humans and synanthropic reservoirs be carried out. Considering that in the Caxias municipality the diagnosis of dogs positive for leishmaniasis is performed only by serology – a non-specific method for identifying Leishmania species – and by microscopy of smears in human patients, molecular surveys, along with a genomic characterization of the parasites from those sources, must be performed. In addition to deeper ecological studies about the sand fly fauna, different sampling methods must be used in an integrative manner to verify whether some species, like Bi. flaviscutellata, are rare or just undersampled, as in surveys based on CDC-type traps, Lu. longipalpis is often the predominant sand fly in the sampling.