Phlebotomine sandflies (Diptera: Psychodidae) from Lábrea, state of Amazonas, Brazil, with a description of Evandromyia (Aldamyia) apurinan Shimabukuro, Figueira & Silva, sp. nov.

An entomological survey was conducted from July-December 2009 and September-December 2010, as part of the epidemiological monitoring of American cutaneous leishmaniasis (ACL) in the municipality of Lábrea, state of Amazonas (AM), Brazil. Sandflies were collected using CDC light traps installed in intra and peridomiciliary locations, as well as the border of forested areas around houses where autochthonous cases of ACL were recorded. A total of 510 sandflies belonging to 26 species were collected. The most abundant species was Nyssomyia antunesi (44.5%) followed by Evandromyia walkeri (10.6%) and Micropygomyia rorotaensis (9.8%). Here we also describe Evandromyia (Aldamyia) apurinan sp. nov. and report new records for Trichophoromyia flochi and Evandromyia sipani in AM and Brazil, respectively. Our results describe the composition of the sandfly fauna in the south of AM and suggest Ny. antunesi as the putative vector in the transmission of Leishmania in this area of the Amazon Region.

online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro,Vol. 108(2): 280-287, April 2013 The Amazon Region accounts for the largest diversity of Leishmania Ross, 1903 parasites and sandfly vector species in the Neotropical Region (Rangel & Lainson 2003b, Rotureau 2006. Of the 30 described species of Leishmania in the world, at least seven have been found infecting humans in the Amazon Region, while at least 15 sandfly species are known or putative vectors (Rotureau 2006). In the Amazon, the transmission cycle is maintained between wild animal reservoirs -which typically present non-pathogenic Leishmania infection -and sandflies.
In Brazil, the region to the north of the rivers Negro and Amazonas, including the states of Amazonas (AM), Roraima, Pará (PA) and Amapá, Leishmania (Viannia) guyanensis Floch, 1954 is the main etiological agent of American cutaneous leishmaniasis (ACL) and it is associated with most cases of human leishmaniasis (> 50%) recorded for the whole Amazon (Arias & Freitas 1978, Romero et al. 2002, Rotureau 2006. In these states, human infections typically occur as a result of human activity (hunting, fishing, farming, mining, civil construction etc.), resulting from the exploitation and/or destruction of primary tropical forest.
In contrast, little is known about the epidemiology of ACL in the Amazonian Region, south of the rivers Negro and Amazonas (Arias & Freitas 1978, Justiniano et al. 2004. Recently, Guerra et al. (2011) described the epidemiology of mucosal leishmaniasis (ML) in the Ama-zon Region and not only found a higher prevalence of this form of the disease south of the river, but also reported similar distribution of Leishmania species among patients from north and south of the Amazonas River. However, the species of Leishmania parasites infecting humans, the non-human animal reservoir hosts and the sandfly vectors involved in transmission of cutaneous leishmaniasis in this region have not yet been identified.
The municipality of Lábrea is located in the south region of AM and has been recording an increasing number of cases of ACL (MS/SINAN 2012). In Lábrea, ACL is associated with sustainable subsistence and economic activities such as Brazil-nut collection, latex extraction, agriculture, fishing and hunting, performed mostly by indigenous and traditional riverine communities (Guerra et al. 2011). The objective of this work was to identify the sandfly fauna associated with autochthonous cases of ACL in Lábrea as part of the entomological monitoring program for leishmaniasis performed by the Amazonas Health Surveillance Foundation (FVS/AM).

MATERIALS AND METHODS
Study area -The municipality of Lábrea is located in the southern of AM, close to the border with state of Rondônia (RO) and between two main rivers (Purús and Madeira). The population is 37,574 inhabitants and the territorial area is 68,233.96 km² (IBGE 2012). Lábrea, along with the municipalities of Canutama, Tapauá and Pauini, comprises the area known as the Middle Purús. The main economic activity is Brazil nut collection, performed by indigenous and non-indigenous local populations, making Lábrea the second biggest producer in AM (IBGE 2012).
Sandfly collection -Sandflies were collected from July-December 2009 and September-December 2010, using modified CDC light traps placed in intra and peri-domiciliary locations, as well as along the border of forested areas around the houses where autochthonous cases of human ACL were recorded. The four sites studied were: Vila Falcão, a neighbourhood in Lábrea, Apairal, a rural settlement along the Trans-Amazonian Road -BR-230 and Aldeia Castanheira and Aldeia Irmã Cleusa, settlements of indigenous people of the Apurinã ethinc group, located in the Caititu Indian Reserve.
Specimens were placed in containers with ethanol 70%. At the FVS/AM laboratory, the insects were separated and sandflies were prepared according to the method described by Forattini (1973) and mounted on microscope slides in Canada Balsam medium.
The specimens were identified and measured using an AxioScope A1 Zeiss ® microscope with AxioVision ® software. All measurements are given in micrometers. The average measurements and standard deviation for paratypes are given in brackets. For definitions of the terminology used to describe sandfly morphology and a key for identification of all Evandromyia species see Bermúdez et al. (1991) and Galati (2003). Shimabukuro,Figueira and Silva, Description -Holotype (male): total body length: 2,309 (2,346 ± 156; n = 12). Predominantly light brown, with light brown pronotum, paratergite and mesonotum and thoracic pleura, except for brown katepimeron, katepisternum and coxae, but without clear contrast.

Type-locality
Taxonomic discussion -Among the Evandromyia (Aldamyia) species in which the male possesses a bifurcated aedeagus, the ventral margin of the paramere is without pre-apical lobe and the apex of the ejaculatory ducts are round-shaped with one distinct tooth, such as Sandflies collected in Lábrea -Overall, a total of 510 specimens (319 females and 191 males) were collected and 476 were identified to species level. They belonged to 26 species and represented 12 genera and three subtribes (Sergentomyiina, Lutzomyiina and Psychodopygina). Due to loss of structures of taxonomic importance, 15 specimens could only be identified to genus level and 19 specimens were too damaged to allow any identification (Table).
Most of the sandflies were collected in the peridomicile (232; 45.5%) and in the forest border (226; 44.3%), followed by intradomicile (52; 10.2%). The largest diversity was found in the forest border (25 species) and in the peridomicile (11 species); the intradomicile accounted for the smallest number of species collected (6).
Here we also report a new record in AM, Th. flochi collected at Apairal, and a new record for Brazil, Ev. sipani collected at Vila Falcão. Both species were collected in the peridomicile.

DISCUSSION
The sandflies collected from July-December 2009 and September-December 2010 in the municipality of Lábrea represent over 10% of the total of sandflies recorded in Brazil (Shimabukuro & Galati 2010). Our results describe the composition of the sandfly fauna from the south of AM, which is becoming part of the "arc of deforestation", an area under strong anthropic pressure due to deforestation for logging and cattle ranching (Fearnside 2005). The majority of the sandflies were collected in Vila Falcão, which is expected as most of the collection effort was concentrated in this location, which is a nearby rural neighbourhood of Lábrea easily accessed by car. In contrast, the other three localities studied have limited access and can only be reached during dry months of the year.
Nyssomyia was the most abundant genus collected in our study mainly because of the dominance of Ny. antunesi. This anthropophilic genus has been previously reported as highly abundant in other areas of AM (Barrett et al. 1996, Castellón et al. 2000, Dias-Lima et al. 2002, Feitosa & Castellón 2006, Guerra et al. 2006, Silva et al. 2007. Varying degrees of environmental change caused by human activities might be responsible for the differences in the sandfly fauna composition found throughout the Amazon Region (Ready et al. 1986). For example, Psychodopygus is more abundant in non-disturbed primary forest, while Nyssomyia is more abundant in areas under human impact found in AM, state of Acre (AC) and state of Mato Grosso (Azevedo et al. 2002, Alves et al. 2012. The most abundant sandfly species, Ny. antunesi (44.51%), is a suspected vector of Leishmania (V.) lindenbergi Silveira, Ishikawa, Souza & Lainson (Silveira et al. 2002) in PA. This sandfly has also been found infected with promastigotes, which did not produce skin lesions in hamsters and could possibly be Leishmania (Le.) infantum [syn. Le. (Le.) chagasi] (Ryan et al. 1984). In some areas of the Colombian Amazon Forest this species has developed intradomiciliary habits and is a suspected vector of Leishmania spp (Vásquez-Trujillo et al. 2008, Cabrera et al. 2012. Only five females from Nyssomyia umbratilis (Ward & Fraiha, 1977) and Nyssomyia yuilli yuilli (Young & Porter, 1972) were collected at the border of the forest at Apairal and one female of Ny. umbratilis at Vila Falcão. Ny. umbratilis is the main vector of L. (V.) guyanensis north of the Amazonas River and is the most abundant species sampled from the base of tree trunks during the day (Dias-Lima et al. 2002, Guerra et al. 2006. Ny. yuilli yuilli was found infected with Leishmania mexicana-like organisms (Arias et al. 1985) and this species has also been found in forest habitats in the states of Bahia (BA) and Espírito Santo where it has been implicated as a vector of Leishmania spp (Vexenat et al. 1986, Dias-Lima et al. 2003, Pinto et al. 2010. The low numbers of these two species collected in our study in the forest border, as opposed to the high number of Ny. antunesi collected in intra and peridomiciliary locations implicates the latter in the transmission of ACL in Lábrea.
The other species we collected of medical importance include Th. ubiquitalis, incriminated as a vector of Leishmania (Viannia) lainsoni Silveira, Shaw, Braga & Ishikawa, 1987(Silveira et al. 1991, Lainson et al. 1992, Bichromomyia flaviscutellata (Mangabeira, 1942), the main vector of Leishmania (Leishmania) amazonensis Lainson & Shaw, 1972, and species from the genus Psychodopygus: Psychodopygus ayrozai (Barretto & Coutinho, 1940), Psychodopygus davisi (Root, 1934) and Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979), which are associated with ACL transmission in both enzootic and zoonotic cycles in the Amazon Region (Rangel & Lainson 2003a, Rotureau 2006. Despite the fact that most collection efforts were spent in Vila Falcão, all the four areas surveyed in this study have reported autochthonous cases of ACL and ML. The inhabitants of three localities (Apairal, Aldeia Irmã Cleusa and Aldeia Castanheira) are involved in forest activities such as açaí and Brazil nut collection, hunting and fishing. Most of these inhabitants also live in wooden traditional houses built very close to the forest. Three cases of ML have been reported from Aldeia Castanheira, an indigenous community with approximately 60 people (FUNASA 2012, SEMSA 2012. Guerra et al. (2011) reported Lábrea as the municipality with the highest number of ML cases in AM, which are mainly associated with activities related to exploitation of forest products.
Despite the low number of specimens collected, we found a high diversity of species, with 26 species collected, most of them collected at the border of the forest. These collections also included one new species, as well as two species which had not been previously recorded, either in AM or the whole of Brazil. The new record for AM, Th. flochi, was collected at Apairal and has been previously recorded in a discontiguous region spanning, in the north, the Amazon Forest of French Guyana and, in the south, in BA, AC and RO (Galati 2003, Castellón 2009). Our record for AM fills in this gap, in the previously discontinuous description of the distribution of this species, between the south of the Brazilian Amazon and French Guyana. In this context, it is worth mentioning that a specimen of Th. flochi was found by one of us (PHFS) at the MZUSP. The specimen was collected by Dr RG Damasceno in the municipality of Coari, Fonte Boa, AM, in April 1943, but was misidentified by him as Trichophoromyia octavioi (Vargas, 1949). Even though Th. flochi and Th. octavioi are similar, they can be distinguished by the apical lobe of the paramere, which is glabrous in Th. flochi and is covered by setae in Th. octavioi. The new record for Brazil, Ev. sipani, was collected at Vila Falcão and has only been previously recorded in the Amazonian lowlands of Peru and Colombia (Fernandez et al. 1994, Barreto et al. 2000. With these two new records, the total of recorded sandfly species in AM increases from 131 to 133.
Entomological surveillance of sandflies is very important in providing information on the fauna composition, bio-ecology and behaviour of vectors of Leishmania. In our study, the observed presence and high abundance of Ny. antunesi in intra and peridomiciliary locations suggests that this species may be a vector of ACL in Lábrea.