Acta Tropica 136 (2014) 104–107
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Natural breeding sites of phlebotomine sand flies (Diptera: Psychodidae) on Marambaia Island, Rio de Janeiro State, Brazil T.L. Souza a,∗ , F.B. Figueiredo b , A.B. Almeida b , C.V. Benigno a , C.S. Pontes a , M.B. Souza a a b
Departamento de Ciências Biológicas – Escola Nacional de Saúde Pública – Fiocruz, Brazil Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos – Instituto de Pesquisa Evandro Chagas – Fiocruz, Brazil
a r t i c l e
i n f o
Article history: Received 24 January 2014 Accepted 5 April 2014 Available online 15 April 2014 Keywords: Breeding sites Phlebotomine sand flies Immature forms Emergence traps Leishmaniasis
a b s t r a c t Immature phlebotomine sand flies develop in soils with essential and ideal characteristics for their life cycle, such as organic matter, humidity, temperature and low levels of light. Information regarding the potential breeding places of these dipterans is fundamental to understand the epidemiology and ecology of leishmaniasis, in addition to its importance to control them. In the present study, we aimed to find natural breeding sites of sand flies on Marambaia Island with the aid of emergence traps and direct search of immature forms using the flotation technique with saturated sugar solution in organic substrates of the region. Both methods were effective, with a total of 42 specimens of six different species – including some species that participate in the transmission cycle of American Tegumentary Leishmaniasis – collected by the emergence traps, and five immature forms obtained by floatation technique. However, further studies are still necessary, mainly with respect to the ecology and biology of immature sandfly stages, so that control measures focused on breeding sites can produce positive sustainable results in natural environments. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phlebotomine sand flies are dipterans belonging to the subfamily Phlebotominae. They are of great importance to public health because they are vectors of several Leishmania species (Forattini, 1973). There are over 800 known phlebotomine sandfly species (Young and Duncan, 1994), but only a few of them are capable of transmitting those parasites (Killick-Kendrick, 1990). These protozoa have wild or peridomestic cycles, in which man is infected by the bite of an infected phlebotomine sand fly (Rangel and Lainson, 2003). Information on the ecology and biology of phlebotomine sand flies allows us to point out relevant characteristics for planning control measures of leishmaniasis. The knowledge about natural breeding places for sand flies is still not well defined (Alencar, 2007), but its importance for the understanding of the population dynamics of adult insects of a given region is recognized (Warburg and Faiman, 2011). Characterizing the local development of immature forms of these insects is epidemiologically important because of the possible relation between breeding sites and anthropic environments, representing useful information for directing efforts to
∗ Corresponding author. Tel.: +55 02186995020. E-mail addresses: [email protected], tls2 [email protected] (T.L. Souza). http://dx.doi.org/10.1016/j.actatropica.2014.04.008 0001-706X/© 2014 Elsevier B.V. All rights reserved.
the biological control of these zoonoses through less environmentally invasive methods, that is, with environmental management. Reorganizing and cleaning the peridomestic environment, including soil drainage to prevent moisture and removal of its organic matter – decomposing leaves and fruits, may reduce the population density of adult insects, consequently diminishing the risk to humans and domestic animals (Teodoro et al., 2003; Marzochi et al., 2009). Immature stages of sand flies develop at ground level and the soil should provide essential characteristics for their development, such as organic matter, humidity, temperature, and low levels of light (Deane and Deane, 1957). Thus, female sand flies do not lay eggs indiscriminately as other dipterans; they conduct such life cycle in specific places that foster larval development. Both adult and immature forms present behaviors associated with the environmental conditions (Killick-Kendrick, 1999). The direct search for immature stages of sand flies is mostly performed using emergence traps seated on the soil of interest (Feliciangeli, 2004). Another technique employed was described by Hanson (1961), who used saturated sugar solution to search for immature forms on the soil. The main objective of this study was to characterize microhabitats able to provide favorable conditions for the successful development of breeding sites of phlebotomine sand flies on Marambaia Island. This region reports a varied fauna of sand flies, including important vector species of American
T.L. Souza et al. / Acta Tropica 136 (2014) 104–107
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Tegumentary Leishmaniasis and Visceral Leishmaniasis. In 2009, 13 different species of these flies were identified, four of them of epidemiological importance in the transmission of leishmaniases: Lutzomyia longipalpis, Lutzomyia migonei, Lutzomyia intermedia and Lutzomyia fischeri (Novo et al., 2013). 2. Materials and methods This is a cross-sectional study in which an entomological survey was conducted to identify breeding sites of phlebotomine sandflies on Marambaia Island. To this end, three different methods were employed: emergence traps, throughout the 12 months of the study (August 2012 to July 2013); analysis of soil with saturated sugar solution, in the first six months; and CDC light traps, in the last six months. 2.1. Study area Marambaia Island is located at Sepetiba Bay, in the southern coast of Rio de Janeiro State, and is part of the municipality of Mangaratiba, Rio de Janeiro State, Brazil. The area is administered by the Armed Forces of Brazil and is used to perform training activities, receiving military personnel from several regions of the country. Two sampling points were selected in a region known as Vacaria (23◦ 03 S; 43◦ 59 W), according to the density of adult insects (Novo et al., 2013). Sampling occurred in an anthropic environment with no domiciles. Both sampling points were characterized as forest edge: Point 1 comprised a region with fewer trees and the presence of a path, while Point 2 was located further into the forest presenting greater vegetation cover. 2.2. Search for breeding sites The phlebotomine sand flies used in this study were captured both in their winged forms, using emergence traps, adapted from the trap used by Casanova (2001), and in their immature stages, by the flotation technique in saturated sugar solution, proposed by Hanson (1961). The emergence trap was composed of a black rectangular (34 cm × 56 cm) plastic bowl, 14 cm tall. They were fixed to the ground, allowing the opening to stay in contact with the substrate. The base of the bowl had two 5.5 cm diameter holes, to which plastic tubes were connected in order to fix transparent pots where the collected insects remained trapped. The transparent pots (collection pots) were 12 cm tall and, besides allowing the passage of light so that the insects were attracted, they contained two 2.5 cm diameter holes covered by a fine nylon mesh for air circulation, unlike the trap proposed by Casanova (2001). Each trap contained two collection pots (Fig. 1). A total of 10 emergence traps were used: five in Point 1 and five in Point 2. The monitoring of the emergence traps was carried out every 15 days, in addition to a rotation every 2 months, according to what was proposed by Casanova (2001) and Casanova et al. (2013). After two months, the traps were transferred to other places, and they were considered as new samples, because they represented different areas, adjacent to those that had already been considered. The specimens collected were placed in plastic tubes containing 70% alcohol, cleared in potassium hydroxide solution (KOH) at 10% for 2 h, and then classified according to the nomenclature proposed by Young and Duncan (1994). The choice of time for this rotation was based on the total time that the immature forms of the species found in the region spend to go from egg stage until hatching, as performed by Alencar (2007). Upon rotation of the emergence traps, an amount of soil equivalent to 1 m2 area by 1.5 cm height was collected during the first six months of the study. Prior to this procedure, all leaf litter was
Fig. 1. Emergence trap adapted for the study.
also collected for later analysis with the rest of the material. The samples, a total of 10 per collection, were placed in resistant and identified plastic bags for subsequent washing and filtering. Sieves of different mesh were used in the washing process aiming to concentrate the smaller particles and discard the larger ones. At the end of this process, the material was retained in a cloth capable of preventing its total passage. A similar process was carried out by Deane and Deane (1957) and Mccombie-Young et al. (1926). Leaves, twigs and other components were washed and sieved. Samples of material from substrates were processed by the flotation technique with saturated sugar solution proposed by Hanson (1961); since collection, throughout the study, the material remained packed under refrigeration and 70% alcohol.
2.3. Light traps The captures with CDC light traps were carried out monthly during the last 6 months of the study. The two light traps – one in Point 1 and one in Point 2 – were operated from 6 pm to 7 am, totaling 13 h of collection per day. The specimens collected were placed in plastic tubes containing 70% alcohol, cleared in potassium hydroxide solution (KOH) at 10% for 2 h, and then classified according to the nomenclature proposed by Young and Duncan (1994).
3. Results 3.1. Search for breeding sites Throughout the 12 months of the study, the emergence traps captured a total of 42 specimens from six distinct species. Lutzomyia schreiberi species was found most frequently, with a predominance of 78.57% when compared with the total of sand flies captured (Table 1). In addition, L. ferreirana was registered for the first time on Marambaia Island. The month of May presented the greatest density, totaling 47.6% of the phlebotomines collected. No adult specimen was captured during the months of September, January and March. A total of five immature stages of phlebotomine sand flies (two 1st instar larvae; two 4th instar larvae; one pupae) were found in a total amount of 30 m2 of soil collected.
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T.L. Souza et al. / Acta Tropica 136 (2014) 104–107
Table 1 Frequency of species and sexes of phlebotomine sand flies captured by emergence traps in each collection point (P1 and P2). Species
Point 1
Total (P1)
♂
♀
n◦
L. intermedia L. migonei L. schreiberi L. capixaba L. ferreirana L. edwardsi
1 – 1 – – –
– – – – – –
1 – 1 – – –
Total
2
0
2
Point 2
3.2. Light traps The light traps were operated for 65 h and captured 556 phlebotomine sand flies distributed in ten different species (Table 2). 4. Discussion The study of natural breeding sites of phlebotomine sand flies contributes to elucidate characteristics with respect to the population dynamics of these insects and its implication in the transmission of leishmaniasis (Warburg and Faiman, 2011). Nevertheless, studies detailing the biology of the life cycle of sand flies are scarce, especially concerning the number of immature stages captured in natural environments (Alencar, 2007). The results obtained in this research confirmed that the region studied provides favorable conditions for the successful development of breeding sites of phlebotomine sand flies. Of the six species caught by the emergence traps, L. intermedia and L. migonei are vectors of American Tegumentary Leishmaniasis and L. ferreirana was registered for the first time on Marambaia Island. L. ferreirana has already been described in other areas in southern Brazil (Shimabukuro et al., 2011; Forattini, 1973; Young and Duncan, 1994), but had not yet been reported in the municipalities of Mangaratiba (Novo et al., 2013) and Rio de Janeiro (Souza et al., 2003). With the exception of this species, L. intermedia, L. migonei, L. schreiberi, L. capixaba and L. edwardsi represent approximately 38.5% of the total 13 species encountered so far on Marambaia Island (Novo et al., 2013). Also using an adapted trap, but with changes in method, Alencar (2007) recorded the presence of 9 of the 55 species (16%) present in the study area. Other studies have also started from the high concentration of phlebotomines to perform the search for breeding sites (Hanson, 1961; Feitosa and Castellon, 2009; Müller et al., 2011; Sangiorgi et al., 2012; Casanova et al., 2013), as well as from the presence of essential conditions for the development of immature forms.
Total (P2)
♂
♀
n◦
50 – 50 – – –
– 1 14 – 1 –
3 1 18 1 – 1
3 2 32 1 1 1
100
16
24
40
%
% 7.5 5 80 2.5 2.5 2.5 100
The relatively low emergence of phlebotomine sand flies encountered in this study compared with those found by Casanova (2001), Casanova et al. (2013), Sangiorgi et al. (2012) and Rutledge and Ellenwood (1975), for instance, may not be directly reflecting the efficiency of the traps employed, but the method and other conditions inherent to the location of the study, such as the climate, time of year, and type of biome. Casanova (2001) captured 73 specimens in less than 12 months in a greater number of analyzed environments. In the same study, only three different species were caught, fewer than in the present study. Alencar (2007), analyzed ground microhabitats of the Amazon Rainforest and captured 27 specimens using 30 traps in only 6 months – a high number compared with what was accomplished in this study. Therefore, the number of traps used, the larger size of the analyzed area, the residence time of traps in the sites, the total time of study, and the adverse conditions of the different environments in different studies (Deane and Deane, 1957; Casanova, 2001; Alencar, 2007; Müller et al., 2011; Sangiorgi et al., 2012; Casanova et al., 2013) are examples of factors which can influence the capture of adult insects by emergence traps. Moreover, the spatial distribution pattern of breeding sites, influenced by oviposition, hinders the finding of breeding sites in natural environments (Deane and Deane, 1957; Feliciangeli, 2004; Sangiorgi et al., 2012; Casanova et al., 2013). The emergence of sand flies in areas with predominance of trees and leaf litter seems to be related to the protection afforded to immature forms in these environments (Ferreira et al., 1938; Deane and Deane, 1957; Hanson, 1961; Rutledge and Mosser, 1972). In Point 2, which presented the highest frequency of phlebotomines, leaf litter was present throughout the study. In addition, this point was more protected by trees. In contrast, leaf litter in Point 1 was present inconsistently, making it more vulnerable to the action of the rain, which can easily wash the soil. Casanova et al. (2013), reported that leaf litter can actually impart conditions for the development of breeding sites and that these environments are important because they are many times located near domiciles.
Table 2 Frequency of species and sexes of phlebotomine sand flies captured by CDC light traps in each collection point (P1 and P2) over a period of six months. Species
Pont 1
♂
Total (P1)
♀
◦
n
Pont 2 %
Total (P2)
♂
♀
n◦
%
L. intermedia L. migonei L. fischeri L. cortelezzii L. schreiberi L. ferreirana L. edwardsi L. bianchigalatiae L. pelloni L. pessoai
232 57 – – – – 1 – 1 –
66 23 22 – 5 – – 2 – –
298 80 22 – 5 – 1 2 1 –
72.86 19.56 5.38 – 1.22 – 0.24 0.49 0.24 –
46 7 – 1 10 1 – – – –
59 9 4 – 9 – – – – 1
105 16 4 1 19 1 – – – 1
71.43 10.88 2.72 0.68 12.93 0.68 – – – 0.68
Total
291
118
409
100.00
65
82
147
100.00
T.L. Souza et al. / Acta Tropica 136 (2014) 104–107
It is known through laboratory analysis that certain species may show differences in the biology of immature stages. Some studies with colonies of phlebotomine sand flies show important parameters. It has been suggested that certain species prefer to lay their eggs on less flat surfaces or in cracks, as it has already been observed in laboratory with females of L. longipalpis and L. migonei (Elnaiem and Ward, 1992; Nieves et al., 1997). Pupae, for example, generally prefer to rest on vertical surfaces; therefore, the methodology employed in this study was not able to capture any pupae or adults that hatched from them. Such fact was registered by Ivovic´ et al. (2010) in colonies of Phlebotomus neglectus raised in laboratory. Some larvae, in turn, may be distributed differently in relation to soil depth and also in the leaf litter, as already observed by Hanson (1961). The emergence traps used in this research may have been ineffective in achieving microhabitats with these characteristics present in Points 1 and 2, such as bases of trees and rocks. The immature forms found in this study confirm the existence of natural breeding sites of sand flies and the effectiveness of emergence traps, even if they have only been found in Point 2. Just as in emergence traps, the low number of specimens collected does not seem to be associated with the effectiveness of the technique, but with the method instead. Comparison between emergence traps and CDC light traps was carried out only during the months of the study in which both traps were used concomitantly. Differences found in the patterns of species distribution and density of phlebotomine sandflies captured by distinct traps within the same area seem to corroborate previously published studies. Deane and Deane (1957) reported the rarity of immature stages of L. longipalpis in an area where its winged form is fairly abundant, and suggested that this species presents different breeding sites or that it can develop in a more scattered manner on the environment, dispersing its oviposition broadly, therefore hindering its capture. Hanson (1961) also registered different pattern of catches, in which not all species captured as adults were found in soil samples and vice versa, as in the case of P. skannom and P. hamatus, respectively. In contrast, Casanova et al. (2013) confirms the predominance of L. longipalpis in both types of trap: emergence traps and CDC light traps; as well as Alencar (2007), who reported that the same species were found in similar densities by both methodologies. This lack of correlation between emergence traps and CDC light traps is not rare, as reported by (Deane and Deane, 1957; Hanson, 1961; Rutledge and Ellenwood, 1975). The level of effectiveness of different chemical compounds and biological agents for the control of immature forms is already known in laboratory (Alexander and Maroli, 2003), but studies on the biology and ecology of natural breeding sites of sand flies are still scanty (Alencar, 2007) so that control measures directly focused on immature stages could obtain positive and mainly viable results under natural conditions. Environments containing live vegetation cover, in the form of trees, or in decomposition, in the form of leaf litter, enable the development of breeding sites even though these environments are affected and modified by anthropogenic actions, which are quite often present in peridomiciles. Acknowledgements This study was supported by Fundac¸ão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) – Estudo de doenc¸as Negligenciadas e Reemergentes/Jovem Cientista do Nosso Estado and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Universal 14/2013. Fabiano Borges Figueiredo holds a grant from CNPq for productivity in research.
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Am. 54, 317–322. ´ V., Chaniotis, V., Vujanic, ´ M., Bobic, ´ B., Nikili, A., Klun, I., Zˇ ivkovic, ´ T., Djurkovic´ Ivovic, ´ O., 2010. Life tables and reproductive parameters of Phlebotomus Djakovic, neglectus Tonnoir 1921 (Diptera, Psychodidae) under laboratory conditions. Arch. Biol. Sci. Belgrade 3, 420–424. Killick-Kendrick, R., 1990. Phlebotomine vectors of the leishmaniases: a review. Med. Vet. Entomol. 4, 1–24. Killick-Kendrick, R., 1999. The biology and control of phlebotomine sand flies. Clin. Dermatol. 17, 279–289. Marzochi, M.C., Fagunder, A., Andrade, M.V., Souza, M.B., Madeira, M.F., MoutaConfort, E., Schubach, A.O., Marzochu, B.F., 2009. Visceral leishmaniasis in Rio de Janeiro, Brazil: eco-epidemiological aspects and control. Revista Soc. Bras. Med. Trop. 42, 570–580. Mccombie-Young, T.C., Richmond, A.E., Brendish, G.R., 1926. Sandflies and sandfly fever in the Peshawar District. Indian J. M. Res. 13, 961–1021. 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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Natural breeding sites of phlebotomine sand flies (Diptera: Psychodidae) on Marambaia Island, Rio de Janeiro State, Brazil T.L. Souza a,∗ , F.B. Figueiredo b , A.B. Almeida b , C.V. Benigno a , C.S. Pontes a , M.B. Souza a a b
Departamento de Ciências Biológicas – Escola Nacional de Saúde Pública – Fiocruz, Brazil Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos – Instituto de Pesquisa Evandro Chagas – Fiocruz, Brazil
a r t i c l e
i n f o
Article history: Received 24 January 2014 Accepted 5 April 2014 Available online 15 April 2014 Keywords: Breeding sites Phlebotomine sand flies Immature forms Emergence traps Leishmaniasis
a b s t r a c t Immature phlebotomine sand flies develop in soils with essential and ideal characteristics for their life cycle, such as organic matter, humidity, temperature and low levels of light. Information regarding the potential breeding places of these dipterans is fundamental to understand the epidemiology and ecology of leishmaniasis, in addition to its importance to control them. In the present study, we aimed to find natural breeding sites of sand flies on Marambaia Island with the aid of emergence traps and direct search of immature forms using the flotation technique with saturated sugar solution in organic substrates of the region. Both methods were effective, with a total of 42 specimens of six different species – including some species that participate in the transmission cycle of American Tegumentary Leishmaniasis – collected by the emergence traps, and five immature forms obtained by floatation technique. However, further studies are still necessary, mainly with respect to the ecology and biology of immature sandfly stages, so that control measures focused on breeding sites can produce positive sustainable results in natural environments. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phlebotomine sand flies are dipterans belonging to the subfamily Phlebotominae. They are of great importance to public health because they are vectors of several Leishmania species (Forattini, 1973). There are over 800 known phlebotomine sandfly species (Young and Duncan, 1994), but only a few of them are capable of transmitting those parasites (Killick-Kendrick, 1990). These protozoa have wild or peridomestic cycles, in which man is infected by the bite of an infected phlebotomine sand fly (Rangel and Lainson, 2003). Information on the ecology and biology of phlebotomine sand flies allows us to point out relevant characteristics for planning control measures of leishmaniasis. The knowledge about natural breeding places for sand flies is still not well defined (Alencar, 2007), but its importance for the understanding of the population dynamics of adult insects of a given region is recognized (Warburg and Faiman, 2011). Characterizing the local development of immature forms of these insects is epidemiologically important because of the possible relation between breeding sites and anthropic environments, representing useful information for directing efforts to
∗ Corresponding author. Tel.: +55 02186995020. E-mail addresses: [email protected], tls2 [email protected] (T.L. Souza). http://dx.doi.org/10.1016/j.actatropica.2014.04.008 0001-706X/© 2014 Elsevier B.V. All rights reserved.
the biological control of these zoonoses through less environmentally invasive methods, that is, with environmental management. Reorganizing and cleaning the peridomestic environment, including soil drainage to prevent moisture and removal of its organic matter – decomposing leaves and fruits, may reduce the population density of adult insects, consequently diminishing the risk to humans and domestic animals (Teodoro et al., 2003; Marzochi et al., 2009). Immature stages of sand flies develop at ground level and the soil should provide essential characteristics for their development, such as organic matter, humidity, temperature, and low levels of light (Deane and Deane, 1957). Thus, female sand flies do not lay eggs indiscriminately as other dipterans; they conduct such life cycle in specific places that foster larval development. Both adult and immature forms present behaviors associated with the environmental conditions (Killick-Kendrick, 1999). The direct search for immature stages of sand flies is mostly performed using emergence traps seated on the soil of interest (Feliciangeli, 2004). Another technique employed was described by Hanson (1961), who used saturated sugar solution to search for immature forms on the soil. The main objective of this study was to characterize microhabitats able to provide favorable conditions for the successful development of breeding sites of phlebotomine sand flies on Marambaia Island. This region reports a varied fauna of sand flies, including important vector species of American
T.L. Souza et al. / Acta Tropica 136 (2014) 104–107
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Tegumentary Leishmaniasis and Visceral Leishmaniasis. In 2009, 13 different species of these flies were identified, four of them of epidemiological importance in the transmission of leishmaniases: Lutzomyia longipalpis, Lutzomyia migonei, Lutzomyia intermedia and Lutzomyia fischeri (Novo et al., 2013). 2. Materials and methods This is a cross-sectional study in which an entomological survey was conducted to identify breeding sites of phlebotomine sandflies on Marambaia Island. To this end, three different methods were employed: emergence traps, throughout the 12 months of the study (August 2012 to July 2013); analysis of soil with saturated sugar solution, in the first six months; and CDC light traps, in the last six months. 2.1. Study area Marambaia Island is located at Sepetiba Bay, in the southern coast of Rio de Janeiro State, and is part of the municipality of Mangaratiba, Rio de Janeiro State, Brazil. The area is administered by the Armed Forces of Brazil and is used to perform training activities, receiving military personnel from several regions of the country. Two sampling points were selected in a region known as Vacaria (23◦ 03 S; 43◦ 59 W), according to the density of adult insects (Novo et al., 2013). Sampling occurred in an anthropic environment with no domiciles. Both sampling points were characterized as forest edge: Point 1 comprised a region with fewer trees and the presence of a path, while Point 2 was located further into the forest presenting greater vegetation cover. 2.2. Search for breeding sites The phlebotomine sand flies used in this study were captured both in their winged forms, using emergence traps, adapted from the trap used by Casanova (2001), and in their immature stages, by the flotation technique in saturated sugar solution, proposed by Hanson (1961). The emergence trap was composed of a black rectangular (34 cm × 56 cm) plastic bowl, 14 cm tall. They were fixed to the ground, allowing the opening to stay in contact with the substrate. The base of the bowl had two 5.5 cm diameter holes, to which plastic tubes were connected in order to fix transparent pots where the collected insects remained trapped. The transparent pots (collection pots) were 12 cm tall and, besides allowing the passage of light so that the insects were attracted, they contained two 2.5 cm diameter holes covered by a fine nylon mesh for air circulation, unlike the trap proposed by Casanova (2001). Each trap contained two collection pots (Fig. 1). A total of 10 emergence traps were used: five in Point 1 and five in Point 2. The monitoring of the emergence traps was carried out every 15 days, in addition to a rotation every 2 months, according to what was proposed by Casanova (2001) and Casanova et al. (2013). After two months, the traps were transferred to other places, and they were considered as new samples, because they represented different areas, adjacent to those that had already been considered. The specimens collected were placed in plastic tubes containing 70% alcohol, cleared in potassium hydroxide solution (KOH) at 10% for 2 h, and then classified according to the nomenclature proposed by Young and Duncan (1994). The choice of time for this rotation was based on the total time that the immature forms of the species found in the region spend to go from egg stage until hatching, as performed by Alencar (2007). Upon rotation of the emergence traps, an amount of soil equivalent to 1 m2 area by 1.5 cm height was collected during the first six months of the study. Prior to this procedure, all leaf litter was
Fig. 1. Emergence trap adapted for the study.
also collected for later analysis with the rest of the material. The samples, a total of 10 per collection, were placed in resistant and identified plastic bags for subsequent washing and filtering. Sieves of different mesh were used in the washing process aiming to concentrate the smaller particles and discard the larger ones. At the end of this process, the material was retained in a cloth capable of preventing its total passage. A similar process was carried out by Deane and Deane (1957) and Mccombie-Young et al. (1926). Leaves, twigs and other components were washed and sieved. Samples of material from substrates were processed by the flotation technique with saturated sugar solution proposed by Hanson (1961); since collection, throughout the study, the material remained packed under refrigeration and 70% alcohol.
2.3. Light traps The captures with CDC light traps were carried out monthly during the last 6 months of the study. The two light traps – one in Point 1 and one in Point 2 – were operated from 6 pm to 7 am, totaling 13 h of collection per day. The specimens collected were placed in plastic tubes containing 70% alcohol, cleared in potassium hydroxide solution (KOH) at 10% for 2 h, and then classified according to the nomenclature proposed by Young and Duncan (1994).
3. Results 3.1. Search for breeding sites Throughout the 12 months of the study, the emergence traps captured a total of 42 specimens from six distinct species. Lutzomyia schreiberi species was found most frequently, with a predominance of 78.57% when compared with the total of sand flies captured (Table 1). In addition, L. ferreirana was registered for the first time on Marambaia Island. The month of May presented the greatest density, totaling 47.6% of the phlebotomines collected. No adult specimen was captured during the months of September, January and March. A total of five immature stages of phlebotomine sand flies (two 1st instar larvae; two 4th instar larvae; one pupae) were found in a total amount of 30 m2 of soil collected.
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Table 1 Frequency of species and sexes of phlebotomine sand flies captured by emergence traps in each collection point (P1 and P2). Species
Point 1
Total (P1)
♂
♀
n◦
L. intermedia L. migonei L. schreiberi L. capixaba L. ferreirana L. edwardsi
1 – 1 – – –
– – – – – –
1 – 1 – – –
Total
2
0
2
Point 2
3.2. Light traps The light traps were operated for 65 h and captured 556 phlebotomine sand flies distributed in ten different species (Table 2). 4. Discussion The study of natural breeding sites of phlebotomine sand flies contributes to elucidate characteristics with respect to the population dynamics of these insects and its implication in the transmission of leishmaniasis (Warburg and Faiman, 2011). Nevertheless, studies detailing the biology of the life cycle of sand flies are scarce, especially concerning the number of immature stages captured in natural environments (Alencar, 2007). The results obtained in this research confirmed that the region studied provides favorable conditions for the successful development of breeding sites of phlebotomine sand flies. Of the six species caught by the emergence traps, L. intermedia and L. migonei are vectors of American Tegumentary Leishmaniasis and L. ferreirana was registered for the first time on Marambaia Island. L. ferreirana has already been described in other areas in southern Brazil (Shimabukuro et al., 2011; Forattini, 1973; Young and Duncan, 1994), but had not yet been reported in the municipalities of Mangaratiba (Novo et al., 2013) and Rio de Janeiro (Souza et al., 2003). With the exception of this species, L. intermedia, L. migonei, L. schreiberi, L. capixaba and L. edwardsi represent approximately 38.5% of the total 13 species encountered so far on Marambaia Island (Novo et al., 2013). Also using an adapted trap, but with changes in method, Alencar (2007) recorded the presence of 9 of the 55 species (16%) present in the study area. Other studies have also started from the high concentration of phlebotomines to perform the search for breeding sites (Hanson, 1961; Feitosa and Castellon, 2009; Müller et al., 2011; Sangiorgi et al., 2012; Casanova et al., 2013), as well as from the presence of essential conditions for the development of immature forms.
Total (P2)
♂
♀
n◦
50 – 50 – – –
– 1 14 – 1 –
3 1 18 1 – 1
3 2 32 1 1 1
100
16
24
40
%
% 7.5 5 80 2.5 2.5 2.5 100
The relatively low emergence of phlebotomine sand flies encountered in this study compared with those found by Casanova (2001), Casanova et al. (2013), Sangiorgi et al. (2012) and Rutledge and Ellenwood (1975), for instance, may not be directly reflecting the efficiency of the traps employed, but the method and other conditions inherent to the location of the study, such as the climate, time of year, and type of biome. Casanova (2001) captured 73 specimens in less than 12 months in a greater number of analyzed environments. In the same study, only three different species were caught, fewer than in the present study. Alencar (2007), analyzed ground microhabitats of the Amazon Rainforest and captured 27 specimens using 30 traps in only 6 months – a high number compared with what was accomplished in this study. Therefore, the number of traps used, the larger size of the analyzed area, the residence time of traps in the sites, the total time of study, and the adverse conditions of the different environments in different studies (Deane and Deane, 1957; Casanova, 2001; Alencar, 2007; Müller et al., 2011; Sangiorgi et al., 2012; Casanova et al., 2013) are examples of factors which can influence the capture of adult insects by emergence traps. Moreover, the spatial distribution pattern of breeding sites, influenced by oviposition, hinders the finding of breeding sites in natural environments (Deane and Deane, 1957; Feliciangeli, 2004; Sangiorgi et al., 2012; Casanova et al., 2013). The emergence of sand flies in areas with predominance of trees and leaf litter seems to be related to the protection afforded to immature forms in these environments (Ferreira et al., 1938; Deane and Deane, 1957; Hanson, 1961; Rutledge and Mosser, 1972). In Point 2, which presented the highest frequency of phlebotomines, leaf litter was present throughout the study. In addition, this point was more protected by trees. In contrast, leaf litter in Point 1 was present inconsistently, making it more vulnerable to the action of the rain, which can easily wash the soil. Casanova et al. (2013), reported that leaf litter can actually impart conditions for the development of breeding sites and that these environments are important because they are many times located near domiciles.
Table 2 Frequency of species and sexes of phlebotomine sand flies captured by CDC light traps in each collection point (P1 and P2) over a period of six months. Species
Pont 1
♂
Total (P1)
♀
◦
n
Pont 2 %
Total (P2)
♂
♀
n◦
%
L. intermedia L. migonei L. fischeri L. cortelezzii L. schreiberi L. ferreirana L. edwardsi L. bianchigalatiae L. pelloni L. pessoai
232 57 – – – – 1 – 1 –
66 23 22 – 5 – – 2 – –
298 80 22 – 5 – 1 2 1 –
72.86 19.56 5.38 – 1.22 – 0.24 0.49 0.24 –
46 7 – 1 10 1 – – – –
59 9 4 – 9 – – – – 1
105 16 4 1 19 1 – – – 1
71.43 10.88 2.72 0.68 12.93 0.68 – – – 0.68
Total
291
118
409
100.00
65
82
147
100.00
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It is known through laboratory analysis that certain species may show differences in the biology of immature stages. Some studies with colonies of phlebotomine sand flies show important parameters. It has been suggested that certain species prefer to lay their eggs on less flat surfaces or in cracks, as it has already been observed in laboratory with females of L. longipalpis and L. migonei (Elnaiem and Ward, 1992; Nieves et al., 1997). Pupae, for example, generally prefer to rest on vertical surfaces; therefore, the methodology employed in this study was not able to capture any pupae or adults that hatched from them. Such fact was registered by Ivovic´ et al. (2010) in colonies of Phlebotomus neglectus raised in laboratory. Some larvae, in turn, may be distributed differently in relation to soil depth and also in the leaf litter, as already observed by Hanson (1961). The emergence traps used in this research may have been ineffective in achieving microhabitats with these characteristics present in Points 1 and 2, such as bases of trees and rocks. The immature forms found in this study confirm the existence of natural breeding sites of sand flies and the effectiveness of emergence traps, even if they have only been found in Point 2. Just as in emergence traps, the low number of specimens collected does not seem to be associated with the effectiveness of the technique, but with the method instead. Comparison between emergence traps and CDC light traps was carried out only during the months of the study in which both traps were used concomitantly. Differences found in the patterns of species distribution and density of phlebotomine sandflies captured by distinct traps within the same area seem to corroborate previously published studies. Deane and Deane (1957) reported the rarity of immature stages of L. longipalpis in an area where its winged form is fairly abundant, and suggested that this species presents different breeding sites or that it can develop in a more scattered manner on the environment, dispersing its oviposition broadly, therefore hindering its capture. Hanson (1961) also registered different pattern of catches, in which not all species captured as adults were found in soil samples and vice versa, as in the case of P. skannom and P. hamatus, respectively. In contrast, Casanova et al. (2013) confirms the predominance of L. longipalpis in both types of trap: emergence traps and CDC light traps; as well as Alencar (2007), who reported that the same species were found in similar densities by both methodologies. This lack of correlation between emergence traps and CDC light traps is not rare, as reported by (Deane and Deane, 1957; Hanson, 1961; Rutledge and Ellenwood, 1975). The level of effectiveness of different chemical compounds and biological agents for the control of immature forms is already known in laboratory (Alexander and Maroli, 2003), but studies on the biology and ecology of natural breeding sites of sand flies are still scanty (Alencar, 2007) so that control measures directly focused on immature stages could obtain positive and mainly viable results under natural conditions. Environments containing live vegetation cover, in the form of trees, or in decomposition, in the form of leaf litter, enable the development of breeding sites even though these environments are affected and modified by anthropogenic actions, which are quite often present in peridomiciles. Acknowledgements This study was supported by Fundac¸ão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) – Estudo de doenc¸as Negligenciadas e Reemergentes/Jovem Cientista do Nosso Estado and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Universal 14/2013. Fabiano Borges Figueiredo holds a grant from CNPq for productivity in research.
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