Sandfly frequency in a clean and well-organized rural environment in the State of Paraná , Brazil

Introduction: Sandflies caught in Santa Juliana Farm in Sarandi, State of Paraná, Brazil, were assessed in terms of their fauna, seasonality, and frequency in the homes and in shelters of domestic animals around the homes, as well as in the nearby forest. Methods: In Santa Juliana Farm, there are no records of cases of ACL, differing from other relatively clean and organized areas where surveys of sandflies have been conducted in Paraná. Samples were collected with Falcão light traps, fortnightly from 22:00 to 02:00 hours, from November 2007 to November 2008. Results: A total of 4,506 sandflies were captured, representing 13 species, predominantly Nyssomyia whitmani (71.8%). More sandflies were collected in the forest (52.6%) than outside the forest (residences and pigsty) (47.4%). However, Ny. whitmani was collected in greater numbers outside (38.3%) than inside the forest (33.5%). Most sandflies were collected in the warmer months and during periods with regular rainfall. Conclusions: The results suggest that cleaning and organization around the houses could reduce sandfly population in peridomicile. Constructing shelters for animal at a distance of approximately 100m from domiciles is recommended to prevent the invasion of sandflies, as this farm has an area of preserved forest, with wild animals and sandflies present to maintain the enzootic cycle of Leishmania.

Leishmaniases are among the six tropical diseases of major public-health significance.They are caused by protozoans of the genus Leishmania, which exploit wild mammals (reservoirs) and sandflies (vectors) in their natural life cycles 1,2 .In Latin America, where the number of cases of American cutaneous leishmaniasis (ACL) has remained relatively high, Brazil accounts for most cases, with 527,976 persons affected from 1990 to 2008 3 .Of these, 12,115 were recorded in Southern Brazil -95.4% of them in the State of Paraná 3 .
The large number and wide distribution of ACL cases in Brazil underscore the importance of studies on the control of sandflies.Traditionally, sandfly control has been accomplished with the use of chemical insecticides [4][5][6][7] .However, these measures have been proven ineffective for use during interrupted and/or inappropriate periods [8][9][10] .
In Paraná [11][12][13][14] and in other states of Brazil 7,10,15-17 , research on the ecology and control of sandflies in areas where ACL is endemic shows that human dwellings are usually located close to areas of vegetation.In endemic areas, lack of sanitation and peridomestic occupation can attract sandflies and create breeding sites for these insects 12,15,[18][19][20][21] , hence, the importance of studying the ecology, behavior, and development of alternative ways to control sandflies.The objectives of this study were to investigate the fauna; seasonality; and frequency of sandflies in houses, peridomestic animal shelters, and in the forest.No ACL has been recorded in the area of study, and the area is clean, well-kept, and maintained.

METHODS
The study was carried out in Santa Juliana Farm (Figure 1), located at 23° 30'21" S and 51º 54'14" W, at an altitude of 535m, and with an area of 169.4 hectares in the municipality of Sarandi in North-Central Paraná.
According to the 2000 Census, Sarandi has a population of 71,422 inhabitants -1,929 of these are residing in rural areas and 69,493 in the urban area 22 .
The predominant climate in the region, according to the Köppen classification, is Cfa (humid subtropical) in which the mean temperature of the coldest month is below 18ºC and the mean annual temperatures are above 20ºC.
The mean temperature in the collection period from November 2007 to November 2008 was 23.5ºC.The lowest temperature recorded in the period was in 2008 (13.1ºC), and the highest was in 2007 (30.9ºC).The mean monthly precipitation during this same period was 130mm; the lowest rainfall for the period was 8mm in July 2008, and the highest was 235mm in December 2007.The rainfall data were provided by the Climatological Station of the Universidade Estadual de Maringá (UEM).
The region has a basaltic lithology, with a high proportion of clay in its constitution, and the soils are red oxisols (ferric) and alfisols (ferric) 23,24 .The vegetation includes remnants of native semideciduous submontane forest 23 .Santa Juliana Farm contains 50.8ha of forest, of which 34.3ha is in legal reserves, and a permanent preserve of 16.5ha, where there is still a mammal fauna remaining (rodents, capybaras, wild cats, etc.)The farm has a small population of domestic animals (dogs, pigs, chickens) around the homes and cattle in the vicinity.

Soybeans
The study was conducted in this farm because there is no record of cases of ACL in this area.The organization of peridomicile distinguishes it from other locations in the state of Paraná where surveys of phlebotomines have been carried out 12,25,26 and the cleanliness and good organization of the farm are evident.
Sandflies were collected with Falcão light traps, twice a month from 22:00 to 02:00 hours, from November 2007 to November 2008.This resulted in a total of 104h per trap collection and 728h in total.
The traps were installed in the following ecotypes: E1 -a pigsty (Pi) with a roof and beaten earth floor, cleaned regularly, and housing two pigs; E2 -on the porch of the farm keeper's residence (R1), with a beaten earth floor, where several bags containing food were stored and 2-5 dogs rested at night, as well as several nearby trees where chickens roosted at night; E3 -on the porch of the residence (R2)  of the owner of the farm, which was frequently cleaned, the grass trimmed, and the peridomestic area always dry; E4 -on the edge of the forest beside a country road; E5 -in dense forest, 25m from the edge; E6 -on the shore of a lake, with grass and fruit trees, where the residents sometimes fish; and E7 -a grassy space between trees on the shore of another lake (Figure 1).
In the extra-forest environment, traps were installed in domiciliary areas (E1, E2, and E3).The forest environment refers to the traps installed in the forest and the lakeside (E4, E5, E6, and E7).
The sandflies collected were killed with chloroform and placed in small cardboard boxes impregnated with naphthalene.The specimens were prepared and subsequently identified in the Laboratory of Parasitology, Department of Basic Health Sciences of UEM.The nomenclature used here follows Galati 27 .
Statistical analysis was performed with data for Nyssomyia whitmani, which was collected in the largest numbers and because it is the most important species in the epidemiology of leishmaniasis in Paraná 11,28 1).Altogether, these species represented 97.7% of the sandflies collected.
We collected 2,765 females and 1,741 males.The number of female Ny.whitmani was significantly higher than the number of males of the same species (p<0.0001).
We collected a larger number of sandflies in the forest (52.6%) than in the extra-forest environments (47.4%).However, the number of specimens of Ny. whitmani was higher (p<0.0001) in the extraforest (38.3%) compared with the forest (33.5%).
The largest numbers of sandflies in E1 (79.9%) were collected in January, March, September, and November.In E2, 74.1% of the sandflies were collected in February, May, and September.In E3, 73% of the sandflies were collected in February, March, May, and September.In E4, 77.8% of the sandflies were collected in November 2007 and January, May, July, September, and November 2008.In E5, 71.9% of the sandflies were collected in January, May, and September (Table 2).Note that in all the ecotopes together, in January, February, March, May, September, and November 2008, 82.1% of the sandflies were collected (Table 2 and Figure 2).The 13 species collected have been recorded in several locations in the State of Paraná 11,29.30 .We note particularly the species Ny. whitmani, Ny. neivai, Pi. pessoai, and Mi.migonei because of their high frequency in domiciliar and peridomiciliar situations and because they have been found naturally infected with Leishmania in this state 28,31 and in other Brazilian states 17,30,32,33 .

DISCUSSION
The slightly higher number of sandflies collected in the forest was probably due to environmental conditions -i.e., humidity and organic matter necessary for the development of the immature stages of sandflies -and an available blood supply for the adult females -i.e., birds and mammals.The regular distribution of rainfall throughout the year helped to maintain soil moisture.The small number of sandflies collected in the lakeshores, despite the proximity of the forest, may have been due to the installation of traps in areas with low vegetation (grass).Ny. whitmani was collected in greater numbers outside the forest.In this environment, the high proportion of sandflies collected in ecotope E2 can be explained by the presence of several dogs that rested there and some chickens that roosted in the nearby trees at night.This confirms the attractiveness of domestic animals to phlebotomines 18,19,21 .Ny. whitmani is anthropophilic 34 and is important in the epidemiology of ACL.
The forest preserve, the presence of wild mammals, as well as the cleanliness and organization and the small supply of blood available in the peridomicile (domestic animals) are factors that may explain the collection of similar numbers of sandflies in the forest and extra-forest areas in Santa Juliana Farm.These insects have often been collected in larger numbers in peridomiciliar and extra-forest environments 12,35,36 .In Palmital Farm in the municipality of Terra Boa, Paraná, after deactivation of a colony of houses and the absence of residents and domestic animals (dogs, chickens, pigs, and others), which had served as sources of blood for the sandflies, the sandfly population decreased sevenfold 37 .In Santa Juliana Farm, we also observed the absence of garbage and other waste in the vicinity of the houses and in the edge of the forest 37 .
During the months of collection, the lowest mean temperatures occurred in May and June, and the lowest rainfall rates were observed in June and July.However, rainfall was well distributed throughout the collection period, and the mean, minimum, and maximum temperatures remained roughly constant.Most (65.1%) sandflies were collected in January, February, March, September, and November 2008, months that were warmer and with regular rainfall.In May, 17% of the specimens were collected, at a time when the temperature is usually milder.The finding of the highest densities of sandflies in the warmer months and with regular rainfall concords with observations in several previous surveys of these insects 36,38-42 .

FIGURE 1 -
FIGURE 1 -Distribution of Falcão traps in the different ecotypes in Santa Juliana Farm, municipality of Sarandi, State of Paraná, Brazil, from November 2007 to November 2008.

FIGURE 2 -
FIGURE 2 -Seasonality of sandflies in all the traps in different ecotypes in Santa Juliana Farm, mean temperatures, and monthly rainfall in the municipality of Sarandi, Paraná, Brazil, from November 2007 to November 2008.