Contribution to the Knowledgment of Feeding Habbits of Lutzomyia (Lutzomyia) Longipalpis (Lutz & Neiva, 1912) in Association to Natural Infection by Leishmania (Leishmania) Infantum Chagasi (Cunha & Chagas, 1937)

Background: Brazil faces the expansion and urbanization of American Visceral Leishmaniasis. The presence of the vector in the urban area is one of the major challenges of the Brazilian Program for Control of Visceral Leishmaniasis, which refers the need for a better understanding the behavior of Lutzomyia longipalpis, as well as the factors of its adaptation to new habitats. The aim of the study was to combine diagnostic molecular tools capable to identifying natural infection by Leishmania (L.) and the origin of sources in L. longipalpis. Methods: The specimens were captured in the municipalities of Araguaína/Tocantins, Fortaleza/Ceará, Sobral/Ceará, and Rio de Janeiro/Rio de Janeiro. Molecular diagnoses were performed through Polymerase Chain Reaction using primers that amplify kinetoplast deoxyribonucleic acid in Leishmania sp. and the cacophony gene in phlebotomines for the diagnosis of natural infection, primers that amplify the cytochrome b gene, in addition to the sequencing technique, for the study of alimentary habit. Results: Among the analyzed females, 28.6% were diagnosed in the food evaluation. Among the total of 141 samples that were analyzed, showed a higher positivity for the human food source (62.5%), followed by dogs (27.5%) and birds (10%). In Araguaína, the samples showed positivity for human blood (61.5%) and dogs (38.5%). In Fortaleza and Sobral, specimens showed a positive percentage for human blood (54.5% and 66.7%), followed by dogs (27.3% and 20.0%) and birds (18.2% and 13.3%). In the females of Rio de Janeiro was detected exclusive feeding of human blood. In the natural infection, a general index of positivity was obtained equal to 4.3%. When associated, detection of the power source and natural infection, two infected specimens were found in Araguaína, with dog and human feeding; and in Sobral, 3 specimens infected and positive for human blood. Conclusions: The results corroborate


Background
In a global setting, leishmaniasis is among the most neglected diseases [1]. Leishmaniases are emerging endemic in Brazil, being the country with the highest prevalence of those diseases in the Americas [2,3].
In the context of the expansion and the urbanization of American Visceral Leishmaniasis (AVL), with a high case fatality rate, Brazil has not been successful in actions to combat the vector, although it has a well elaborated planning with well de ned policies of surveillance and control. It is hypothesized that the presence of the vector in the urban area is one of the major challenges for the Brazilian Visceral Leishmaniasis Control Program, which clearly points to the need to better understand the behavior of Lutzomyia (Lutzomyia) longipalpis, in the urban area and the factors of its adaptation to the new habitats, which is unequivocal proof of its ecological valence.
The present study aims to associate molecular diagnostic tools capable of identifying natural infection by Leishmania (Leishmania) infantum chagasi and the origin of the food source in L. (L.) longipalpis specimens from municipalities with different AVL transmission pro les: Araguaína/Tocantins (intense transmission area, urban pro le), Fortaleza/Ceará (intense transmission area, urban pro le), Sobral/Ceará (intense transmission area, rural pro le), and Rio de Janeiro/Rio de Janeiro sporadic transmission, urban pro le). It is worth mentioning that the focus on the locality of Caju, a port area, is the rst urban focus of the city of Rio de Janeiro. Finally, we intend to de ne and better elucidate aspects concerning the epidemiology of AVL in different transmission areas with different epidemiological pro les in Brazil.

Methodology
Municipalities of the origin of the specimens. The municipalities were chosen according to the categorization of AVL transmission and epidemiological pro le [3] (Figures 1).
Catch stations. The catch sites were de ned considering Probable Infection Sites and sites whose entomological survey had previously identi ed the presence of L. (L.) longipalpis. Phlebotomines were captured using modi ed CDC (Centers for Disease Control) light traps [18] (HP model, Hoover Pugedo) [19]. The traps were randomly installed from 6:00 p.m. to 6:00 a.m., in the peridomiciliary environment during the years of 2013 and 2016, in periods which were favorable to productive catches. The specimens were identi ed according to the Galati criteria [20]. Females from all municipalities, containing blood residues and males (used as negative control), were individualized in eppendorf tubes and stored in freezer -200C, with the exception of one sample from the city of Rio de Janeiro, which was analyzed as a pool (10 copies).

Molecular analyses
Obtaining total extract. The samples were analyzed in 100 μl of lysis buffer (10 mM Tris-HCl pH 9.2 containing 10 mM EDTA and 100 μg/ml proteinase K) and stored (-20 ° C) until extraction of the total deoxyribonucleic acid (DNA). DNA Extraction DNA extraction was performed from the lysates using the Wizard SV Genomic DNA Puri cation System (PROMEGA) commercial kit, according to the manufacturer's speci cations. All the steps for DNA extraction were monitored by the inclusion of samples for negative control (male insects captured in the eld). Such samples determine the control to possible contaminations, since they do not present DNA of Leishmania spp. The laminar uxes proper for DNA extraction, as well as all the material used during DNA extraction, were properly decontaminated with the use of chlorine and exposure to ultraviolet rays [21,22].
Diagnosis of Natural Infection. For the analysis of natural infection, the Polymerase Chain Reaction (PCR) technique was utilized, where a multiplex assay was used [21] via two pairs of primers that ampli ed simultaneously. A rst 120 bp product for Leishmania kDNA (samples of positive females) and another of 220 bp corresponding to phlebotomine DNA (samples of males and females). The rst pair ampli es the constant region of the mini-circle of kDNA of the genus Leishmania; primer A [5 'GGC CCA  TAC TAC ACC AAC CCC 3'] and primer B [5 'GGG GTA GGG GCG TTC TGC GAA 3 because it is known that coops near houses act as an attractive for phlebotomines, as well as functioning as breeding grounds, which facilitates the domiciliation of the vector [27]; dogs, which are considered important domestic reservoirs [17] and the opossums suggested as possible reservoirs [2], and in addition to these, humans. Primers that amplify a region of the cytochrome b (cyt b) gene were also used for all vertebrates: 3'CCC CTC AGA ATG ATA TTT GTC CTC 5 'and 3' CCA TCC AAC ATC TCA GCA TGA TGA AA 5 ' (Table 1).
Sequencing -Given the importance of the rst urban focus in the city of Rio de Janeiro (Porto area), a new methodology was applied to part of its samples (total of seven individualized samples and one pooled sample). The products obtained for the cyt b gene, obtained by the above methodology, were puri ed using the Wizard SV PCR Clean-up System kit (Promega), then sequenced with the same primers used for PCR. Sequencing was performed in an automatic sequencer (ABI PRISM ® BigDye ™ Terminator Cycle Sequencing) at Oswaldo Cruz Foundation (IOC) (Genomic Platform -DNA sequencing, PDTIS-FIOCRUZ). The sequences obtained were aligned and compared with those already deposited in the NCBI nucleotide database (http://blast.ncbi.nlm.nih.gov/Blast).

Results
Identi cation of eating habits From the total of L. (L.) longipalpis females tested (141), a general positivity index of 29.3% was obtained, with the municipality of Sobral having the highest index, 36.6%. In none of the localities multiple feeds were observed ( Table 2).
The different populations of L. (L.) longipalpis presented a constant pattern of blood tests in the digestive tract, with a higher positivity for human blood (63.4%), followed by dogs (26.8%) and birds (9,8%); no females fed on opossum were found (Table 3, Fig. 2).
From the positive samples for the city of Rio de Janeiro, only positive results were obtained for human blood (100%). These samples had their results sequenced, one sample analyzed individually (identity index equal to 96%, Query Cover value of 94% and Accession number: KX697544.1), and one sample in the form of a pool equal to 98%, Query Cover value of 97% and Accession number: KX697544.1), although in the pool there were 10 females for evaluations and in order to avoid any false positive result in the pool, only one female was considered positive (Table 3).

Identi cation of the natural infection by Leishmania (Leishmania) infantum chagasi
One of the analyzed specimens was obtained with a general index of positivity of 4.3%. A total of 6 positive specimens were found, with a positive index of 4.9% in Araguaína and 9.8% in Sobral (Table 4

Association between identi cation of eating habits and natural infection
In 141 female L. (L.) longipalpis, in 5 (3.6%) specimens, natural infection and the food source were identi ed, four of which were fed in human blood and one in dog blood (Table 5).
In the municipality of Araguaína, a L. (L.) longipalpis female was found infected with L. (L.) infantum chagasi fed on dog blood and a female fed on human blood. In the municipality of Sobral, 3 females were naturally infected and fed on human blood, and one female with unidenti ed food source (Table 5).
The literature has recorded, on the basis of eld work, the eclecticism of L. (L.) longipalpis in feeding on a wide range of mammals, including dogs, pigs, horses, cattle, and birds [17,43]. In addition to the favorable environmental conditions, the abundance of food sources is a determinant factor in the population growth of the vector, especially in periurban areas, which brings it closer to humans.
The data from the present study revealed that the specimens of the populations of Araguaína, Fortaleza and Sobral, areas that were classi ed as intensive transmission for AVL, fed mainly on humans. The anthropophilia of L. (L.) longipalpis has already been described in several studies [12,17,43], being one of the essential criteria for the species to be incriminated as a vector [44]. In Ceará we have already intense transmission to AVL, which is attributed to these municipalities. On the other hand, the data so far do not suggest that the populations of the different biomes (Cerrado and Caatinga) have different habits regarding the attraction to humans. The fact is that the species is highly anthropophilic, but due to its opportunistic character, characteristic of some species of phlebotomines, one can not expect the same pattern of feeding.
In Araguaina, the feeding of the analyzed specimens showed the attraction to dogs, as observed in the populations of, Fortaleza and Sobral, which are areas that present a high incidence of canine visceral leishmaniasis. In the 1950s, in Sobral [43], dogs had already been suggested as important domestic reservoirs of L. (L.) infantum chagasi and as sources of infection for L. (L.) longipalpis, in the home environment; in Araçatuba (São Paulo), the highest percentage of canine blood female feeding was observed, correlating its epidemiological role in the AVL transmission chain, with the hypothesis that canine enzootia precedes human transmission [30]. In other countries of South America, such as Colombia, in an area endemic to AVL, the population of L. (L.) longipalpis showed a strong attraction to dogs [28]. Studys in Montes Claros (Minas Gerais) shows that even with the adoption of euthanasia for seropositive AVL dogs, the canine population will be continue serving as a source of Leishmania sp. infection for sand ies [45]. The data obtained in this study con rm the importance of dogs in the maintenance of the AVL transmission.
However, it is possible to observe a weak attraction of L. (L.) longipalpis to birds, different from the analyzes that demonstrate opposite behavior, in Northeast areas [15] and in Raposo (Maranhão) [14]. It is well known that coops near houses act as attractive for phlebotomines [43], which serve as breeding grounds, since immature forms develop in soil that is rich in organic matter. In this context, the contact between vectors and humans increases, besides allowing the domiciliation of the vector, and consequently the transmission of AVL within the home, a situation that is observed both in the rural area and in the periphery of the cities [12,17,27]. In terms of environmental determinants of occurrence of AVL transmission in the home environment, the maintenance of coops near homes increases the risk of human exposure to the phlebotomine vector [40].
The analyses of the present study did not reveal positivity of L. (L.) longipalpis to opossum blood, a synanthropic animal, which was suggested as a reservoir of L. (L.) infantum chagasi, as already observed in populations of the vector from Sobral (Ceará ), Massapê (Ceará) and Jequié (Bahia), Brazil [15] and Colombia [28]. Some studies have reported the natural infection of Didelphis marsupialis, by Leishmania spp. possibly L. (L.) infantum chagasi, thus discussing the role of these mammals as potential reservoirs for AVL [2,46,47].
Literature has demonstrated that L. (L.) longipalpis is an eclectic, and opportunistic, species in the search for food sources [7,12,14,15,[28][29][30]. Thus, the non-positivity in the general context of this study, about 71% of the analyzed specimens, may have a direct relation with the chosen targets. In the catch areas, besides the sources tested (humans, dogs, birds and opossums), it was possible to observe other potential sources of food, such as pigs, cats, oxen, horses, rodents, goats, sheep and foxes. Another possibility is the viability of DNA. Studies indicate that molecular assays are able to detect host DNA only within 5 days after the blood supply [33, 34,40].
In relation to the other females that were fed, where it was not possible to detect the food source, probably other animals may be involved in the maintenance of the local population of L. (L.) longipalpis. Rodents must be taken into account, which are frequently observed during catches in areas of work and in the home environment. Rattus norvegivus and Rattus Rattus were found to be infected with L. infantum (= L. (infantum chagasi)), with analysis using Leishmania Nested PCR and hsp70 PCR-RFLP, suggesting the possibility of participation of this rodent in the zoonotic cycle of the LVA [48,49].
From the analyses performed with the few (13) copies from Rio de Janeiro (RJ), one specimen was detected with human blood DNA. This fact corroborates with the ndings that already indicated the local transmission, demonstrating the rst urban focus of the city in the Bairro do Caju; the autochthonous human case, canine cases due to visceral leishmaniasis and vector ndings (L. (L.) longipalpis) [50][51][52][53].
New studies should be done, seeking to better understand the ecoepidemiological dynamics of LVA in the city of Rio de Janeiro. The literature suggests that this recent (and rst) urban focus has been installed from passive vector dispersion, due to the transfer of used soil to the ower pots of the Caju Cemetery, from presence of L. (L.) longipalpis vector [54]. On the other hand, it can be assumed that in the area there was already the presence of the vector, a population that was in equilibrium and well adapted to the dens of rodents, which are very common in the cemetery. The canine transmission occurred by the introduction of an infected animal, because in the area adjacent to the cemetery there was a kennel, which triggered the local transmission to a considerable number of dogs.
The rate of naturally infected phlebotomines in endemic areas as well as the correct identi cation of the Leishmania species are of great importance for a better understanding of the epidemiology of leishmaniasis. The classic methodology, used until then, consists of the direct search of the parasite by microscopy, through dissection of the digestive tract, followed by microscopic analysis, isolation and culture of parasites, with subsequent identi cation/diagnosis of the species [55][56][57][58]. Although it is considered a "gold standard" technique, in some cases it is very laborious and unfeasible, given the amount of material to be analyzed, time and speci c training for this process, sometimes rendering the technique imprecise and not always allowing the isolation and the correct identi cation of the parasite.
Diagnostic assays using PCR have been developed based on different molecular targets such as ribosome minor subunit genes, mini-exon gene spacer sequences, single-copy nuclear sequences such as the DNA polymerase alpha gene, but the minicircle kDNA are the ones that present the highest sensitivity for detection of Leishmania [22, [58][59][60][61][62][63].
In the present study, the molecular analyses were performed individually, since the real scenario of infection rate and of identi cation of the food source was searched. When working with a female pool, some information may be lost as it would not be possible to detect the exact amount of positive females (for infection or food source analysis). In addition, we sought to associate the two approaches, food source and natural infection with Leishmania sp, in view of this association, individual analysis became mandatory. When parasite load analyses are performed on phlebotomines, one should consider the possibility that parasitic load may be different in each infected female, and the level of individual infection may interfere with the total pool load. Thus, if a sample showed a high parasitic load, this may be due to one or more infected phlebotomines [64]. In Camaçari (Salvador) was performed a compared study in diferents periods of captures from L. longipalpis, the results of parasite load was low and did not vary regardless of the season, despite the number of collected sand ies [65].
In this study, it was possible to detect the natural infection of L. (L.) longipalpis by L. (L.) infantum chagasi in Araguaína and Sobral, which are areas of intense transmission of AVL, with rates of 4.9% and 9, 8% respectively. In Fortaleza, a minimum natural infection rate of 3.7% was detected [61]. These results reinforce the practicality of the use of molecular techniques in the identi cation of natural infection, since it is known that infection rates are low in nature, even in areas endemic for AVL.
It was also possible to carry out the association between food identi cation and the detection of natural infection by L. (L.) infantum chagasi in 5 specimens; four specimens of L. (L.) longipalpis performed blood meal in human, and one in dog, the latter with its role already widely discussed in the transmission of AVL [2,12,43]. The non-identi cation of the source of the food source in a female L. (L.) longipalpis, from Sobral, corroborates the idea that, possibly, other mammals (in this untested study) can serve as food sources for well-known eclectic phlebotomines.

Conclusions
The presence of L. (L.) longipalpis in the urban area is one of the major challenges for the Brazilian Program for Control of Visceral Leishmaniasis. Studies that contribute to the knowledge of the rate of naturally infected phlebotomines, the correct identi cation of phlebotomines infected by L. (L.) infantum chagasi, and the analysis of the vector food identi cation, can help to understand the behavior of L. (L.) longipalpis in urban areas, as well as their adaptation factors to the new habitats, besides helping to elucidate the expansion of AVL in the Brazilian territory, which is clearly evidenced in the South region. The results generated in this study may contribute to the strategies of more effective controls, besides reinforcing the pepticity of the vector as an important aspect associated with its domiciliation.

Abbreviations
Deoxyribonucleic Acid DNA Centers for Disease Control CDC Declarations Ethics approval and consent to participate Not applicable for that section.

Consent for publication
Not applicable for that section.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.