Phlebotominae sand fl ies ( Diptera : Psychodidae ) : potential vectors of American cutaneous leishmaniasis agents in the area associated with the Santo Antônio H ydroelectric System in Western Amazonian , Brazil

Introduction: An entomological study was conducted as part of a vector-monitoring program in the area associated with the Santo Antônio hydroelectric system in State of Rondônia, Western Amazonian Brazil. Methods: Fourteen sampling sites were surveyed to obtain data on the potential vectors of Leishmania spp. in the area. Sand fl ies were collected from 2011 to 2014 during the months of January/February (rainy season), May/June (dry season), and September/October (intermediary season) using light traps arranged in three vertical strata (0.5, 1, and 20m). Results: A total of 7,575 individuals belonging to 62 species/subspecies were collected. The fi ve most frequently collected sand fl ies were Psychodopygus davisi (Root) (36.67%), Trichophoromyia ubiquitalis (Mangabeira) (8.51%), Nyssomyia umbratilis (Ward & Fraiha) (6.14%), Bichromomyia fl aviscutellata (Mangabeira) (5.74%), and Psychodopygus complexus (Mangabeira) (5.25%). These species have been implicated in the transmission of American cutaneous leishmaniasis agents in the Brazilian Amazon region and described as potential vectors of this disease in the study area. Conclusions: Additional surveillance is needed, especially in areas where these fi ve species of sand fl y are found.

Phlebotominae sand fl ies (Diptera: Psychodidae: Phlebotominae) belong to a group of insects with public health relevance that are vectors of certain pathogens, including agents of leishmaniasis (1) (2) .Currently, there are 274 recognized species of sand fl y in Brazil, and approximately 10% of these species have been implicated in the transmission of American cutaneous leishmaniasis (ACL) (3) (4) .Most of these species are endemic to the Brazilian Amazon region.The high biodiversity of the region may contribute to the variety of ecological systems that Leishmania Ross parasites and their respective mammalian reservoir hosts inhabit (5) .
As in other states of the Brazilian Amazon region, the territory of Rondônia contains infrastructure such as hydroelectric systems that impact the environment by suppressing the growth of vegetation.Because this human activity might infl uence the emergence of unknown epidemiological profi les of ACL, it is necessary to improve current knowledge of Leishmania species diversity and the temporal/spatial distribution of vector fauna in these areas using surveillance programs aimed at monitoring the transmission of disease.Thus, the aim of the present study was to provide data on the sand fl y fauna of an area associated with a hydroelectric system in western Amazonian Brazil and identify potential vectors of ACL agents in this area.

Study area
Porto Velho (8° 45′ 43″ S, 63° 54′ 14″ W) is the capital of and largest city in the State of Rondônia.The average altitude ranges from 70 to 600m above sea level.The climate has been classifi ed as tropical super humid (medium annual temperature 25.5°C), and the predominant vegetation is Amazonian tropical rain forest.This area is directly impacted by the construction of the Santo Antônio Hydroelectric System in the Madeira River.Porto Velho, which has an estimated population of 495,000 inhabitants, experienced 314 reported autochthonous cases of ACL in 2011, 2012, and 2013, accounting for more than 10% of the cases of ACL in the State of Rondônia (6) .Fourteen collection sites were selected as priority areas for a survey based on the recommendations of previous studies conducted as part of the Basic Environmental Plan (BEP) of the Santo Antônio Hydroelectric System (Table 1).These sites are located in the terra fi rme forest, which is characterized by distantly spaced apart trees (open canopy) with an average of 40m of canopy.Larger trees with a canopy of up to 55m such as Castanha-do-Pará (Lecythidaceae), Muiaracatiara (Anacardiaceae), Tauari (Lecythidaceae) and Angelim (Fabaceae) were also observed.The composition of palms (Arecaceae) is variegated in these dense forest environments.

Sampling
Before and after the dam was fi lled, eleven samples were collected between January 2011 and June 2014 during the rainy (January/February), dry (May/June), and intermediary (September/October) Amazonian seasons using light traps from the Center of Diseases Control (CDC) that were run from 06:00 p.m. to 06:00 a.m. for two consecutive nights.To ensure that the sampling effort was suffi cient, a cumulative curve was constructed to calculate the Chao 1, Chao 2, Jackknife 1, and Jackknife 2 diversity indices using the data from all 121 sampled points.The sampling points comprised three traps positioned in different vertical strata, i.e., 0.5, 1, and 20m into the tree canopy, providing 72h of sampling time per collection site.Individual data on the vertical stratifi cation were not available, and each collection site provided a single trapping sample.All collections were approved by the Brazilian Institute of Environment and Renewable Natural Resources (Authorization IBAMA n. 219/2013).

Sand fl y processing
The collected individuals were identified according to their external morphological characteristics.Specimens that could not be identifi ed in the fi eld were stored in 70% alcohol, transported to the laboratory and mounted on glass slides with Berlese liquid (G.B.I.Laboratories, Manchester, England) using a modifi ed version of the Young & Duncan (1) temporary mounting technique.The phylogenetic taxonomic criteria of Galati (19) were used to identify species, and generic abbreviations proposed by Marcondes (20) were used.

Rev
The sampling sufficiency curve showed stabilization, reaching an asymptote for the four analyzed diversity indices (Figure 1 As shown in Figure 2, an increasing number of sand fl ies was collected between the rainy and dry seasons (i.e., January/ June), and the number of sand fl ies collected tended to decrease during the intermediary season (July/October).In 2012-2013, the fi rst and second years after the dam was fi lled, the number of individuals collected increased during all of the collection seasons; thereafter, the number plateaued until June 2014.
The fi ve most frequently collected species of sand fl ies have been implicated in the epidemiology of ACL in the Brazilian Amazonian region.These species included Psychodopygus davisi (Root) (36.7%),Trichophoromyia ubiquitalis (Mangabeira) (8.51%), Nyssomyia umbratilis (Ward & Fraiha) (6.14%), Bicrhomomyia flaviscutellata (Mangabeira) (5.74%), and Psychodopygus complexus (Mangabeira) (5.25%).The frequency with which each species was encountered in 2012 and 2013 was compared with the rainfall; the population size of these insects was found to increase at the beginning of the dry season (May/June) when pluviometric precipitation began to decrease (Figure 3).

DISCUSSION
A total of 118 species were identifi ed in State of Rondônia, including the present six new records.Two previous studies conducted in this state had identifi ed 22 new records (14) (16) .Therefore, it appears that new records of phlebotomine sand fl ies are continuously being discovered in Rondônia.The Chao/ Jackknife indices calculated in the present study indicated that the sampling effort was suffi cient to completely estimate the species richness in the studied region.
Several recent studies have described a high level of biodiversity of sand fl ies (>50 species registered in some of environments) in areas associated with hydroelectric systems in Brazil (21) (22) (23) .The present study is the fi rst to provide a longitudinal evaluation of sand fl y populations before and after the fi lling of a dam.The results of this study indicate that the fi lling of the dam may be associated with an immediate increase in the density of the sand fl y population.
The proportion of sand fl y populations that had been disturbed by environmental changes tended do decline over time, as evidenced by comparison with subsequent collections, suggesting an apparent reestablishment of the ecological system.The factors associated with these fl uctuations have not been well defi ned.Environmental changes infl uence the biodiversity, population size, and proportion of dominant species of sand fl y (24) .Long-term surveys are needed to evaluate whether sand fl y populations are able to be replenished after a long period.A preliminary analysis found no correlation between the number of cases of ACL and variations in the number of fl ies (data not shown).
Psychodopygus davisi is a medically important species of sand fly.The anthropophilic behavior and widespread distribution of these insects in the Amazon basin (25) and previous records of natural Leishmania infections with this species suggest that this species is a potential vector of ACL agents in the studied region.This species has been shown to carry L. (V.) braziliensis and L. (V.) naiffi Lainson and Shaw in Serra dos Carajás (26) (Southern State of Pará, Brazil) and L. (V.) braziliensis in State of Rondônia (14) (27) .Recently, two females of this species from the area associated with the Belo Monte Hydroelectric System in State of Pará were found to carry peripylarian Leishmania -like fl agellates (unpublished observations).These fi ndings and data from the present study further suggest that Ps. davisi is involved in the transmission of Leishmania (Viannia) parasites (mainly L. (V.) naiffi ) in the Amazon region (14) .Trichophoromyia ubiquitalis, Nyssomyia umbratilis, Bichromomyia fl aviscutellata, and Ps.complexus are also likely involved in the transmission of agents of ACL in Brazilian Amazonia.
Trichophoromyia ubiquitalis is the only sand fl y vector associated with the transmission of L. (V.) lainsoni (28) , a parasite recently identified in State of Rondônia (8) .However, Trichophoromyia species are also thought to be carriers of L. (V.) lainsoni, as deoxyribonucleic acid (DNA) of this parasite was found in Trichophoromyia auraensis specimens in the Peruvian Amazon (29) .Furthermore, two pooled samples of Th. ubiquitalis were recently observed to carry the DNA of an ambiguously identifi ed Leishmania (Viannia) parasite [most likely L. (V.) braziliensis] in the rural area of Cerrado in the State of Mato Grosso (30) .
In the present study, Th. ubiquitalis, a known vector of L. (V.) lainsoni, was the second most frequently identifi ed species of sand fly in the study area.These observations are in confl ict with the hypothesis that other species more anthropophilic than Th.ubiquitalis transmit L. (V.) lainsonilike parasites in State of Rondônia (8) which was suggested by the fact that Th. ubiquitalis was infrequently encountered in entomological studies conducted in the municipality of Monte Negro (14) .Although it has previously been suggested that Th. ubiquitalis does not exhibit high levels of anthropophilic behavior under field conditions (28) , previous studies have described the intradomiciliar transmission of L. (V.) lainsoni via indoor-collected Th. ubiquitalis in an island of the metropolitan area of Belém, State of Pará (unpublished observations).These fi ndings suggest that Th. ubiquitalis is a putative vector of L. (V.) lainsoni in State of Rondônia.Nyssomyia umbratilis has been identifi ed as the main vector of L. (V.) guyanensis in northern Brazil, particularly in the Guianan ecoregion complex (31) .The fi ndings of the present study suggest that Ny. umbratilis is a frequently encountered species in the Porto Velho municipality, which is consistent with fi ndings of Azevedo et al (13) .These authors found that Ny. umbratilis was the second most (19.9%)prevalent species in the Samuel Ecological Station, accounting for 48.5% of the specimens collected in the canopy at 20m.Although the distribution of L. (V.) guyanensis near the southern Amazon River remains unknown, recent findings related to the circulation of a L. (V.) guyanensis-like parasite in State of Rondônia (8) suggest that there is a transmission cycle for this parasite/vector in the studied region.However, additional observations are needed to confi rm this hypothesis.
Bichromomyia flaviscutellata (2) (7) , the major vector of L. (L.) amazonensis, was frequently encountered in the present survey.This was surprising because light traps were used for collection, and these species typically cannot be collected in large numbers without the use of rodent-baited traps (32) .This unexpected result could be explained by the fact that the traps were placed 50cm above the ground at each CDC collection station where low-fl ying specimens may have been attracted to the light and intercepted.
Bichromomyia olmeca nociva (Young & Arias), a suspected vector (33) closely related to Bichromomyia species which are associated with the epidemiology of L. (L.) amazonensis, was detected in this study.This species is similar to Bi. reducta (Feliciangeli, Ramirez Pérez & Ramirez), a species that was not observed in the present survey but has previously been described as a natural vector of L. (L.) amazonensis in studies conducted in Cachoeira Samuel, State of Rondônia (12) .Because Bi. fl aviscutellata was detected most frequently in the present study, this species is suspected to be the main species involved in the transmission of ACL attributed to L. (V.) amazonensis in Rondônia (7) .
Psychodopygus complexus is currently recognized to be associated with L. (V.) braziliensis-associated ACL in areas where the closely related species Psychodopygus wellcomei is absent (34) .In the present study, Ps. complexus was unambiguously identifi ed in the collection because of the absence of Ps. wellcomei males.However, the transmission of L. (V.) braziliensis by Ps. complexus cannot be assumed because no reports of natural infection with this species or evidence of ecological association between the presence of this species and ACL have been reported in the study region.L. (V.) braziliensis is widely distributed throughout Brazil, and several species of sand fl y that were not encountered in this study, including Ps. wellcomei Fraiha, Shaw & Lainson, Migonemyia migonei (França), Nyssomyia whitmani (Antunes & Coutinho), and Ny.intermedia (Lutz & Neiva) (2) (7) , have been implicated in the transmission of these agents.
The epidemiological background of the highly anthropophilic species Ps. davisi should be considered in the context of the transmission of various L. (Viannia) species in areas of the Amazon region such as Rondônia (14) .The evidence encountered in the present study cannot exclude the possibility that this particular species of sand fl y was involved in human infections recently attributed to L. (V.) braziliensis (8) in Rondônia.
The area associated with the Santo Antônio hydroelectric system currently has the highest recorded diversity of Phlebotominae in State of Rondônia.The seasonal fl uctuations in sand fl y populations from the studied region have been shown to be associated with the end of the rainy period, which is consistent with fi ndings from other entomological studies.However, changes in the populations of these insects are also infl uenced by the fi lling of the dam.The increased number of sand fl ies was not found to be associated with an increase in the incidence of ACL.
Psychodopygus davisi, Trichophoromyia ubiquitalis, Nyssomyia umbratilis, Bichromomyia flaviscutellata, and Ps.complexus were the fi ve most frequently collected species in this study, and they are all considered to be potential vectors of ACL agents in the Amazon region.The large number of Ps. davisi specimens collected in the present study suggests a need for the entomological surveillance of this sand fl y species, as these insects are potential vectors of zoonotic cutaneous leishmaniasis in Rondônia.

FIGURE 1 -
FIGURE 1 -Accumulation curve for sand fl y species richness in 121 sites sampled from 2011 to 2014.