Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia

In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

The Amazon basin probably has the highest diversity of phlebotomine sandflies worldwide (Alves et al. 2012). Besides that, the Amazon basin has a high species endemicity (Ramos et al. 2014, Pereira Jr et al. 2015. In spite of this diversity, information about sandfly fauna in the state of Acre in the Brazilian Amazon, is still scarce (Teles et al. 2013b, Araujo-Pereira et al. 2014, Oliveira et al. 2015. These insects are relatively well studied in several places worldwide, due to their importance as vectors of protozoans, bacteria and viruses (WHO 2015).
In In northern Brazil there is a wide range of geographically different places where sandflies can be found and also a variety of reservoirs and vector species from the subgenera Nyssomyia; Psychodopygus; Lutzomyia and Trichophoromyia (Castellón 2009, Feitosa et al. 2012). In the state of Pará, the species that stand out are Lutzomyia complexa, Lu. wellcomei and Lu. longipalpis (Aguiar & Medeiros 2003). In Amazonas the main vectors are Lu. umbratilis, Lu. anduzei, Lu. flaviscutellata and Lu. wellcomei (Aguiar & Medeiros 2003, Guerra et al. 2006.
The findings of different epidemiological profiles of leishmaniasis, together with records of outbreaks both in the wild as well as in peri-urban environments (WHO 2015), reinforces the hypothesis of the possible adaptation of vectors to changing environments and also the incorporation of domestic animals as reservoirs (Teles et al. 2013a, Araujo-Pereira et al. 2014. The municipality of Assis Brasil, in the state of Acre, an area that borders Peru and Bolivia, located in the microregion of Brasiléia, is one of the most important ACL endemic areas of Brazil with high rates of the disease (43.6% of the ACL cases in Acre between 2011-2012 were concentrated in this municipality). In Assis Brasil, during the period from 2007-2012, the average detection rate was 98.2 cases/10,000 inhabitants, with 21.4% being mucosal form cases (SINAN 2014). In addition to the high rate of detection, this municipality has some important epidemiological characteristics like the permanence of a significant percentage (40%) of the population living in rural areas (IBGE 2014). Furthermore, after the opening of the Pacific Highway, migration across the triple border and the growth of tourism in the region increased (Cesário et al. 2011). This scenario may play an important role in the spread of ACL.
According to Teles et al. (2015), the most prevalent Leishmania species in humans from this municipality are L. braziliensis and L. shawi. Little is known about the vectors of this region, but some species suspected of or implicated in transmitting Leishmania spp. were reported in the state such as Lutzomyia davisi, Lu. whitmani, Lu. antunesi, Lu. ubiquitalis andLu. umbratilis (Azevedo et al. 2008, Silva-Nunes et al. 2008). Sandfly studies in Acre are still scarce. Some entomological studies have been published to date, describing the sandfly fauna involving the cities of Cruzeiro do Sul, Feijó, Bujari, Xapuri, Rio Branco, Acre and Assis Brasil (Martins & Silva 1964, Arias & Freitas 1982, Azevedo et al. 2008, Silva-Nunes et al. 2008, Teles et al. 2013b, Araujo-Pereira et al. 2014, Oliveira et al. 2015.
Assis Brasil is one of the main epicenters of Amazonian ACL; however, there are information gaps on the epidemiological, clinical, vectorial and special aspects of Leishmania spp. circulating in this and various microregions of the state. The study reports for the first time, vectors and etiological agents from this border region in Acre.

MATERIALS AND METHODS
Study area -The municipality of Assis Brasil ( Fig. 1) occupies an area of 4.974 km 2 in the state of Acre, Brazil, in the mesoregion of the Acre valley, Brasiléia microregion (10º56'29"S and 69º34'01"W). This area of Brazil borders Bolivia and Peru (Acre-Brazil, Madre de Dios-Peru and Pando-Bolivia). The Acre River marks the border between Assis Brasil and Bolivia. A population of 6,610 inhabitants occupies the area. The study area has a landscape formed by a mosaic of indigenous lands, ex-tractive reserves, riverine communities, and small and large settlements. The local economy is focused on rubber, Brazil nuts, wood, vegetable oils and wild fruit. There are some small crop and livestock farms (Acre 2008).
The equatorial weather is hot and humid and the average annual temperature is 26.5ºC; the relative humidity is 80-90% all year round. There are two very distinct seasons: a wet season between November-April, and a dry season between May-October (IBGE 2014).
Phlebotomine sandfly survey -The phlebotomine sandfly surveys were done between August 2009-June 2010 with three or five CDC light traps/localities, placed approximately 150 cm above the ground with a distance of approximately 200 metres between them. Collections were carried out on five to eight consecutive nights from 18:00-06:00; a total of 780 captures were performed. During the dry season 26 visits were carried out totaling 448 traps installed in peridomiciliary environment (rural areas -Irecê road -capture August/2009; São Francisco roadcapture September/2009; and Museu road -capture April and June/2010) according to the accessibility of roads and reports of recent cases of ACL (Figs 1-2). During the rainy season, the collections were held at four fixed points in riparian environments along the left bank of the river where 23 visits were carried out totaling 332 traps (capture November andDecember/2009 andFebruary/2010).
Points 1-3 of the riparian environment of the Acre River had native vegetation consisting of continuous rainforest and point 4 was in a peridomicile environment located in a fragment of forest near a riverside residence.
The sandflies collected were stored in 70% ethanol and then identified by the taxonomic key according to Young and Duncan (1994). The captured females were treated as follows: the head and the last three segments of the abdomen were clarified and mounted in the dorsal-ventral position in Berlese fluid; and the remainder of the body was stored in a microtube containing 70% ethanol for later molecular analysis. The males were slide mounted normally and then identified. Recently, Lu. (Thrychophoromyia) ruifreitasi was described after the identification from individuals collected for this study (Oliveira et al. 2015), and due to the fact that the females of Lu. auraensis and Lu. ruifreitasi are probably indistinguishable, we decided to temporarily name them Lu. auraensis/Lu. ruifreitasi.
Detection of natural infection of sandflies by Leishmania sp. -The collected females were assembled in groups (2-20 specimens per group) in micro test tubes. The single pool is composed with belonging the same sandfly species, date and place of capture. Total DNA was extracted from each pool based on the method adapted by Mukhopadhyay et al. (2000) and Paiva et al. (2007). After the extraction they were stored at 20ºC for polymerised chain reaction (PCR) in order to identify the genus Leishmania, complexes and Leishmania species.
The size of the expected SL RNA Multiplex PCR product was 146 to 149 bp for the L. braziliensis complex, 218 to 240 bp for the L. mexicana complex, and 351 to 397 bp for the L. donovani complex.
The final products amplified using mkDNA gene PCR, SL RNA Multiplex PCR and hsp70 PCR were analysed on a 2% agarose gel stained with GelRed and examined under UV light.
For the RFLP analysis from the hsp70 amplicons of 240 bp, the restriction enzymes HaeIII (Invitrogen®, USA) and BstUI (Bio Labs®, New England) were used in independent reactions according to the manufacturer's instructions. The resulting products of the restriction digestion were analysed on 12% silver-stained polyacrylamide gels.
Negative controls such DNA from male sandflies was used for each PCR. The following DNA reference strains of the Leishmania collection from the Instituto Oswaldo Cruz ( Sequencing of Leishmania DNA copies was carried out with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Brazil, São Paulo, Brazil) in an Applied Biosystems3130xl DNA sequencer from Genomic Company (São Paulo, Brazil). The hsp70 genes of the Leishmania species were compared with the sequences obtained from analysis with reference sequences deposited in GenBank. Comparisons were done using BLAST program searches (Basic Local Alignment Search Tool, NCBI) (http://blast.ncbi.nlm.nih.gov).
Pool screening -Prevalence studies of infection in the sandflies by Leishmania spp. was assessed by estimating the prevalence of infection in positive pools using the Pool Screening Program (http://www.soph.uab. edu/bst/poolscreen) Version 2.0 (Katholi et al. 1995).
Most of the positive pools were found in riparian environments of the Acre River (28 pools -87.0% covering all points), of which 14 pools (44.0%) were collected near riverside houses (point 4 of the riparian environment). Another four positive pools (13.0%) were collected at points 1 and 2 along Museu road (Table II).
In this study, the hsp70 PCR-RFLP analysis confirmed the Leishmania species of 15 phlebotomine pools (Table II)  Five samples with higher amplification yield by hsp70 PCR were confirmed by genetic sequencing. Of these, three pools corresponding to Lu. davisi and two Lu. auraensis/Lu. ruifreitasi confirmed the presence and similarity (95-100%) of L. (V.) guyanensis deposited in GenBank under code number GU368213.1.

DISCUSSION
Of the 67 captured species, three were recorded for the first time in Acre: Lu. georgii, Lu. complexa and Lu. evangelistai. The first species mentioned is considered endemic in northern Brazil. According to past records and this study, there are 92 known species for the state of Acre which corresponds to 34.6% of the registered species in Brazil (Aguiar & Medeiros 2003, Teles et al. 2013b, Galati 2014, Oliveira et al. 2015. The number of species found in Assis Brasil was higher than that reported by other authors in Acre: Martins and Silva (1964) recorded 30 species of sandflies with a predominance of Lu. nevesi in Rio Branco and Arias and Freitas (1982), in a study carried out in the cities of Cruzeiro do Sul, Feijó and Rio Branco, identified 50 species, the most abundant being Lu. auraensis. Azevedo et al. (2008) identified 52 species in the municipalities of Bujari, Xapuri and Rio Branco, 67% of them being Lu. auraensis, Lu. antunesi, Lu. whitmani and Lu. davisi. Silva-Nunes et al. (2008) in the municipality of Acrelândia observed that Lu. antunesi (45%) and Lu. whitmani (15%) were the most abundant of the 40 collected specimens. Araujo- Pereira et al. (2014) in Rio Branco captured 23 species of sandflies with 53.3% Lu. auraensis and 18.8% Lu. whitmani. These studies indicated that knowledge of sandfly fauna is still scarce compared to other Brazilian states of the Amazon Basin.
The abundance the subgenera of Trichophoromyia, Psychodopygus, Pressatia and Nyssomyia found in this study is consistent with these studies performed in Acre (Arias & Freitas 1982, Azevedo et al. 2008, Silva-Nunes et al. 2008  The record of Lu. shawi in this study (frequency of 2.7%) deserves attention when considering the epidemiological surveillance of leishmaniasis around the border. This species has recently been implicated in the dissemination of Leishmania in Bolivia supported by evidence of its anthropophilic character, its occurrence and abundance in endemic areas of ACL and in peridomiciliary environments, as well as its proven infection by L. (V.) braziliensis and L. (V.) guyanensis (Garcia et al. 2007, Bustamante et al. 2012.
Thus, the data from this study demonstrate the great diversity of sandflies species with potential involvement in the leishmaniasis transmission cycle in Assis Brasil. In addition, the abundance of Lu. davisi and Lu. auraensis/ Lu. ruifreitasi with several positive pools for the L. braziliensis complex increases the data about vector suspects in the north Brazil and suggests the need for new studies proving such species as vectors in the ACL cycle.