Sandflies (Diptera: Psychodidae) are important insects that are widespread globally, with approximately 800 species; there are approximately 243 known sandfly species in Brazil1) (2. It is known that several species of sandflies are vectors of pathogens, including the Leishmania protozoan (Kinetoplastida: Trypanosomatidae), which is an etiologic agent of leishmaniasis3. In Brazil, two species are involved in transmission of visceral leishmaniasis (VL), namely Lutzomyia longipalpis and Lutzomyia cruzi4. Lutzomyia migonei specimens were found to be naturally infected with Leishmania infantum, in São Vicente Ferrer, Pernambuco5 and Rio de Janeiro6) (7. An experimental infection with L. infantum was reported in a previous study8. 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 sandflies9) (10. Because of environmental changes resulting from the deforestation and urbanization processes observed in various regions of Brazil, some species of wild sandflies settled around human dwellings and animal shelters, demonstrating their adaptation to the environments modified by humans. This phenomenon contributed to the gradual spread of VL in all the regions of the State of Pernambuco over the last two decades5. For example, from 2005 to 2010 16.9% of canine visceral leishmaniasis (CVL) was observed in the municipality of Caruaru11. More recently, in 2015, an outbreak of VL was reported in the same study area, with six human cases12.
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 insufficient 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 sandflies, 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 sandfly fauna in a specific urban area in northeastern Brazil where VL cases have been reported.
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 agriculture13. 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 identification of sandflies
From November 2014 to December 2015, samples were collected from five 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 cases12, 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 traps14 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 identified using taxonomic keys15.
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 sandflies and climatic variables was measured using cross correlation coefficient and t-test16. The Friedman test was used to compare the monthly variation in species among the collected sandflies. The Chi-square test (χ2) 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 sandfly species in the different ecotypes. A 5% significance level was considered. The BioEstat software version 5.3 was used for statistical calculations17.
A total of 297 sandflies 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 identified (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 sandflies. 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 sandflies showed preference for peridomicile environments (247/297, 83%), followed by dwellings (50/297, 17%). Both males and females were abundant in animal sheds (105/247, 42.5%; 142/247, 57.5%, respectively), and no statistical difference between them was observed (χ2 = 0.917; p = 0.3381).
Lutzomyia longipalpis (73/247, 29.6%) and L. lenti (133/247, 54%) were captured mainly in animal sheds and no statistical difference was observed between them (χ2 = 0.514; p = 0.5967). It is important to note that L. longipalpis females (9/48, 18.7%) were collected from bedrooms of the houses.
When observing the preference of sandflies to the peridomicile ecotypes, the study found a high prevalence in chicken coops, followed by kennels, sheepfolds, pigsties, barns, and stalls. L. longipalpis was mainly detected in chicken breeding sites (34/73, 46.6%) and L. lenti was detected in sheepfold (51/132, 38.6%).
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 influence of temperature on the number of sandflies was observed through the analysis of cross correlation coefficient (cross correlation coefficient = - 0.21; Figure 1). Moreover, RH directly influenced the number of specimens collected (cross correlation coefficient = - 0.53). The rainfall ranged from 0.0 (December 2014) to 118.7mm (June 2015). No significant (p > 0.05) correlation between the number of samples collected per month and precipitation was observed (cross correlation coefficient = 0.21).
The present study identified the sandfly fauna in an urban area, endemic for VL, located in the Northeast of Brazil. The presence of some species of sandflies in urban areas confirms the ability of these insects to adapt to environments with profound difference from their natural habitats18. Three sandfly species, L. lenti, L. longipalpis, and L. sallesi were found in all the surveyed neighborhoods. From an epidemiological perspective, this finding 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%) sandflies collected during the study period were peridomiciliary. An increase in the abundance and diversity of peridomiciliary sandflies has been observed in several areas of Brazil in recent years5. It is important to correlate the vector density with favorable environmental aspects for peridomiciliary sandflies, such as the presence of vegetation, roots, tree trunks, and organic matter, which are the possible shelters and breeding grounds for the vector19. In locations, from where the samples were collected, there were mostly poor households, with deficient 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 sandflies was found. These environmental factors associated with low economic status of residents have been reported to contribute considerably to the transmission of VL20.
Lutzomyia lenti represented 51.8% of the captured sandflies. This finding 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 areas21. L. lenti was found naturally infected with promastigotes in the City of Jacobina, Bahia22. Therefore, the possible epidemiological role of L. lenti needs to be further clarified.
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 synanthropic23) (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. longipalpis20) (26.
Lutzomyia sallesi represented 14.1% of the sandflies collected during the study period. This species commonly spread in the State of Minas Gerais and has been found naturally infected with L. infantum27. 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 transmission28.
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 environments5) (29. L. migonei was found naturally infected in the municipality of São Vicente Ferrer, Pernambuco5 and in the State of Rio de Janeiro6) (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 environments5.
Lutzomyia whitmani is well known for its anthropophilic action and involvement in the transmission of leishmaniasis in Brazil and Paraguay25) (30) (31. The data on the ecology of this species is still incipient, especially in Northeastern Brazil32. However, even at low density, it may be contributing to the transmission of ACL in the urban area of Caruaru.
The seasonal dynamics of sandflies 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 area5) (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 sandflies occurred in June 2015, the month with the highest rainfall of the year; this pattern was also observed in several other regions of Brazil18. The present study showed a moderate positive correlation between the number of sandflies and rainfall, but no significant correlation could be deduced. On the other hand, a negative correlation was observed, with direct influence between the number of insects and humidity. Finally, a negative, albeit non-significant, correlation between the number of sandflies and temperature was observed.
This study provides additional data on the ecology of the sandfly, 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 sandfly species in the epidemiology of VL in Brazil needs to be elucidated in future studies.