Sandfl ies ( Diptera : Psychodidae ) in an urban area of Northeastern Brazil

Introduction: The sandfl y fauna is well studied globally. In Brazil, sandfl y fauna is very diverse in the Northeast region, especially in states such as Maranhão, Ceará, and Bahia. However, in the State of Pernambuco, the distribution of these insects is still not well known. Therefore, the objective of this study was to identify the different species that constitute the sandfl y fauna in an urban area in the Northeast region of Brazil, where an outbreak of visceral leishmaniasis (VL) was recently reported. Methods: The sandfl ies were collected from an urban area endemic for VL, at fi ve collection points. The collection of samples was carried out from November 2014 to December 2015, using CDC light traps installed in intradomiciliary and peridomiciliary fashion. Results: The collected sandfl ies (n = 297) belonged to eight species: Lutzomyia lenti, Lutzomyia longipalpis, Lutzomyia sallesi, Lutzomyia migonei, Lutzomyia walkeri, Lutzomyia capixaba, Lutzomyia carmelinoi, and Lutzomyia whitmani. Most of the specimens collected were peridomiciliary (247/297, 83%). L. lenti (154/297, 52%) was the most frequently sampled species, followed by L. longipalpis (88/297, 29.6%), and L. sallesi (42/297, 14.1%), which together accounted for over 90% of the collected sandfl y specimens. Conclusions: The continued presence of L. longipalpis in urban areas, including that in intradomiciliary areas, with a predominance of females, is crucial because of the high possibility of them causing VL outbreaks, since this species is the main vector of Leishmania infantum in Brazil.


INTRODUCTION
Sandfl ies (Diptera: Psychodidae) are important insects that are widespread globally, with approximately 800 species; there are approximately 243 known sandfl y species in Brazil (1) (2) .It is known that several species of sandfl ies are vectors of pathogens, including the Leishmania protozoan (Kinetoplastida: Trypanosomatidae), which is an etiologic agent of leishmaniasis (3) .In Brazil, two species are involved in transmission of visceral leishmaniasis (VL), namely Lutzomyia longipalpis and Lutzomyia cruzi (4) .Lutzomyia migonei specimens were found to be naturally infected with Leishmania infantum, in São Vicente Ferrer, Pernambuco (5) and Rio de Janeiro (6) (7) .An experimental infection with L. infantum was reported in a previous study (8) .However, further studies are needed to elucidate the role of this species in the epidemiology of VL in Brazil.
The occurrence of VL and American cutaneous leishmaniasis (ACL) in the Americas involves complex ecology and epidemiology, because it is directly related to the distribution of the species of sandfl ies (9) (10) .Because of environmental changes resulting from the deforestation and urbanization processes observed in various regions of Brazil, some species of wild sandfl ies settled around human dwellings and animal shelters, demonstrating their adaptation to the environments modifi ed by humans.This phenomenon contributed to the gradual spread of VL in all the regions of the State of Pernambuco over the last two decades (5) .For example, from 2005 to 2010 16.9% of canine visceral leishmaniasis (CVL) was observed in the municipality of Caruaru (11) .More recently, in 2015, an outbreak of VL was reported in the same study area, with six human cases (12) .
Despite recent progress in mapping the geographical distribution of vector-borne diseases, the occurrence and expansion of VL vectors have not been systematically monitored or documented.This is because of the epidemiological characteristics   and insuffi cient knowledge about the various elements involved in the transmission chain of VL in some areas where control strategies are still ineffective.Therefore, for performing surveys on sandfl ies, an increased familiarity with the areas of occurrence and an understanding of the population dynamics of VL vectors are important factors that can help expand the epidemiological control policies.The aim of the present study was to identify different species that constitute the sandfl y fauna in a specifi c urban area in northeastern Brazil where VL cases have been reported.

Study area
The study was conducted in the municipality of Caruaru (08°16'58" South and 35°58'33" West), which has an estimated population of 351,686 people and is considered an urban VL endemic area in the State of Pernambuco, Northeastern Brazil.
Over the recent years, the municipality has grown inordinately, presenting a close interface between the urban and rural areas.The main economic activity of the area is textile production, followed by subsistence agriculture (13) .It has a semi-arid climate with winter rainfall.Its pluviometric index is approximately 662mm, unevenly distributed throughout the year.The average annual temperature is 22.5°C, and the annual average of relative humidity (RH) is 75%.The predominant vegetation is Caatinga, with the presence of some remnant Atlantic Rainforest.The climatic data on temperature, RH, and rainfall were recorded by the Agronomic Institute of Pernambuco [Instituto Agronômico de Pernambuco (IPA)] and the Pernambuco Agency for Water and Climate [Agência Pernambucana de Águas e Clima (APAC)].

Collection and identifi cation of sandfl ies
From November 2014 to December 2015, samples were collected from fi ve districts in the municipality of Caruaru: Morada Nova, Rendeiras, Campo Novo, Nova Caruaru, and Alto do Moura.The criteria for selection of the capture sites were based on the occurrence of human and/or canine VL cases (12) , as well as on the presence of dogs and/or other pets in homes.
The capture of insects was performed monthly for three consecutive nights.Twelve Centers for Disease Control (CDC) light traps (14) were placed in each of the districts, 1.5m above the ground level, in the evening (at 5:00pm) and gathered at dawn (at 6:00am).The traps were placed in different intradomicile and peridomicile ecotypes: sheepfold, pen, kennel, barn, chicken coop, and pigsty.Households with traps were georeferenced; each trap was numbered and mapped using satellite remote sensing global positioning system (GPS).
The collected insects were fixed in 70% alcohol and identifi ed using taxonomic keys (15) .

Data analysis
The data were submitted for descriptive statistical analysis.The Lilliefors test was used to verify the normality of the data.The relationship between the number of collected sandfl ies and climatic variables was measured using cross correlation coeffi cient and t-test (16) .The Friedman test was used to compare the monthly variation in species among the collected sandfl ies.The Chi-square test (χ 2 ) with Yates correction was used to compare the presence of males and females in the environment (intradomiciliary or peridomiciliary), as well as the occurrence of various sandfl y species in the different ecotypes.A 5% signifi cance level was considered.The BioEstat software version 5.3 was used for statistical calculations (17) .

RESULTS
A total of 297 sandfl ies were collected during the study, of which 122 (41%) were males and 175 (59%) were females; i.e., a sex ratio (M:F) of 1:1.43.No monthly statistical difference between the number of collected females and males was observed (Fr = 0.0714; p = 0.7893).
Eight species were identifi ed (Table 1), and Lutzomyia lenti was the species that was collected most often (154/297, 52%), followed by L. longipalpis (88/297, 29.6%) and Lutzomyia sallesi (42/297, 14.1%), which together accounted for more than 90% of the collected sandfl ies.L. lenti, L. longipalpis, and L. sallesi were the most commonly occurring species throughout the study, with monthly variations (Friedman test, p = 0.0429).It is important to note that L. longipalpis was more predominant in June (n = 16) and L. lenti was more frequently found in July 2015 (n = 36).These three species were found in all the surveyed neighborhoods.
The species were more abundant in May and July 2015, at average temperatures 25.1 and 21.9ºC and RH 66 and 81%, respectively.However, in the present study any infl uence of temperature on the number of sandfl ies was observed through the analysis of cross correlation coeffi cient (cross correlation coeffi cient = -0.21; Figure 1).Moreover, RH directly infl uenced the number of specimens collected (cross correlation coeffi cient = -0.53).The rainfall ranged from 0.0 (December 2014) to 118.7mm (June 2015).No signifi cant (p > 0.05) correlation between the number of samples collected per month and precipitation was observed (cross correlation coeffi cient = 0.21).

DISCUSSION
The present study identifi ed the sandfl y fauna in an urban area, endemic for VL, located in the Northeast of Brazil.The presence of some species of sandfl ies in urban areas confi rms the ability of these insects to adapt to environments with profound difference from their natural habitats (18) .Three sandfl y species, L. lenti, L. longipalpis, and L. sallesi were found in all the surveyed neighborhoods.From an epidemiological perspective, this fi nding is very important because of the spread of insects in the city, especially L. longipalpis, which is considered the main vector of L. infantum in Brazil.
Most (247; 83%) sandfl ies collected during the study period were peridomiciliary.An increase in the abundance and diversity of peridomiciliary sandfl ies has been observed in several areas of Brazil in recent years (5) .It is important to correlate the vector density with favorable environmental aspects for peridomiciliary sandfl ies, such as the presence of vegetation, roots, tree trunks, and organic matter, which are the possible shelters and breeding grounds for the vector (19) .In locations, from where the samples were collected, there were mostly poor households, with defi cient basic sanitation and high pet population, resulting in the accumulation of organic matter.The families maintained diverse types of animal breeding outside their houses as means of subsistence.These conditions were more pronounced in places like Alto do Moura and Nova Caruaru, where a higher frequency of sandfl ies was found.These environmental factors associated with low economic status of residents have been reported to contribute considerably to the transmission of VL (20) .
Lutzomyia lenti represented 51.8% of the captured sandfl ies.This fi nding is notable, given the fact that this species feeds on the blood of humans, horses, and dogs.It has wide distribution in the country and although it is not presumably a vector of Leishmania, it is found in ACL and VL transmission areas (21) .L. lenti was found naturally infected with promastigotes in the City of Jacobina, Bahia (22) .Therefore, the possible epidemiological role of L. lenti needs to be further clarifi ed.
Lutzomyia longipalpis is the main vector of L. infantum in the New World.This species represented 29.6% of the specimens captured in this study.It is possible that this species is more adaptive to the environmental changes than the other Lutzomyia spp., as it is perpetuated in different ecotypes and is most synanthropic (23) (24) .This species was collected in large numbers from chicken coops, pigsties, and corrals.In fact, it is known that chickens are among the preferred hosts for the blood meal of L. longipalpis (20) (26) .
Lutzomyia sallesi represented 14.1% of the sandfl ies collected during the study period.This species commonly spread in the State of Minas Gerais and has been found naturally infected with L. infantum (27) .There are no records to date suggesting that this species uses humans as hosts.However, it may be involved in a wild or rural cycle of Leishmania transmission (28) .
The species L. migonei, L. walkeri, L. capixaba, L. whitmani, and L. carmelinoi together amounted to 4.37% of the collected sandflies.L. migonei, although not as anthropophilic as L. intermedia, can reach man and domestic animals by adapting to their home environment.Its presence inside the homes and outdoors indicates its adaptation to human environments (5) (29) .L. migonei was found naturally infected in the municipality of São Vicente Ferrer, Pernambuco (5) and in the State of Rio de Janeiro (6) (7) .However, its role in the epidemiology of VL in Brazil needs to be elucidated.
Lutzomyia walkeri, Lutzomyia capixaba, and Lutzomyia carmelinoi are usually found in forest environments, probably because of more abundant resources.However, in this study they were also observed in locations showing effects of human intervention, suggesting some adaptation to the changing environments (5) .
Lutzomyia whitmani is well known for its anthropophilic action and involvement in the transmission of leishmaniasis in Brazil and Paraguay (25) (30) (31) .The data on the ecology of this species is still incipient, especially in Northeastern Brazil (32) .However, even at low density, it may be contributing to the transmission of ACL in the urban area of Caruaru.
The seasonal dynamics of sandfl ies can vary greatly from region to region, and the varying climatic factors, over time, might cause species to have different patterns in the same geographical area (5) (26) .The specimens collected in this study showed abundance in the months of May and July 2015, corresponding to the months before and after the wettest period of the year.The greatest abundance of sandfl ies occurred in June 2015, the month with the highest rainfall of the year; this pattern was also observed in several other regions of Brazil (18) .The present study showed a moderate positive correlation between the number of sandfl ies and rainfall, but no signifi cant correlation could be deduced.On the other hand, a negative correlation was observed, with direct infl uence between the number of insects and humidity.Finally, a negative, albeit non-signifi cant, correlation between the number of sandfl ies and temperature was observed.
This study provides additional data on the ecology of the sandfl y, L. longipalpis, which is the main vector of L. infantum in South America.Moreover, the study demonstrated the ability of this species to adapt to anthropic environments.This species was consistently present in the studied locations and showed general intradomicile adaptation in the urban areas, which suggest a high possibility of VL outbreaks in these environments.The rainy season presented the highest risk, since it was the period showing the highest occurrence of the vector.The role of other sandfl y species in the epidemiology of VL in Brazil needs to be elucidated in future studies.

FIGURE 1 .
FIGURE 1. Number of sandfl ies collected and climatic variations in Caruaru, State of Pernambuco, from November 2014 to December 2015.RH: relative humidity.

TABLE 1
Sandfl y species collected in an urban area of Caruaru, State of Pernambuco, from November 2014 to December 2015.