Phlebotomine (Diptera: Psychodidae) fauna in the Fiocruz Atlantic Forest Biological Station, Rio de Janeiro, Brazil

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1 ISSN: ORIGINAL / ORIGINAL ARTICLE / ORIGINALE Phlebotomine (Diptera: Psychodidae) fauna in the Fiocruz Atlantic Forest Biological Station, Rio de Janeiro, Brazil Fauna flebotomínica (Diptera: Psycodidae) na Estação Biológica Fiocruz Mata Atlântica, Rio de Janeiro, Brasil Fauna de flebotomos (Diptera: Psychodidae) en la Estación Biológica Fiocruz Bosque Atlántico, Río de Janeiro, Brasil Rodrigo Espíndola Godoy¹ Elizabeth Ferreira Rangel² Fernanda Moreira Alves³ Wagner Alexandre Costa 4 Descriptors Psychodidae, Biodiversity, American Cutaneous Leishmaniasis. Descritores Psychodidae. Biodiversidade. Leishmaniose Cutânea Americana. Descriptores Psychodidae. Biodiversidad. Leishmaniasis Cutánea Americana. Sources of funding: No Conflict of interest: No Date of first submission: Accepted: Publishing: Corresponding Address Rodrigo Espíndola Godoy Departamento de Epidemiologia Faculdade de Saúde Pública, Universidade de São Paulo Avenida Doutor Arnaldo, 715. Bairro: Pinheiros CEP: São Paulo (SP), Brazil Phone: ReonFacema (Esp): ABSTRACT Objective: Phlebotominae fauna studies are important to inform about diversity and leishmaniasisvectors. Fiocruz Atlantic Forest Biological Station, has an Atlantic Forest remnant area in Rio de Janeiro. Our study aim was to record sand fly fauna in this area. Methodology: Three Biodiversity monitoring sites were assessed through their anthropogenic level disturbance. Captures were performed using light-traps during three months. Results: Sixteen species were captured, Psychodopygus hirsutus hirsutus the most abundant, four being new records and two species leishmaniasis-vectors. Diversity and abundance were higher in more preserved areas. Conclusion. Monitoring stations enable the surveillance of changes and its impacts on Phlebotominae diversity. RESUMO Objetivo: Estudos da fauna de Phlebotominae são importantes para informar sobre diversidade e possíveis vetores de leishmaniose. A Estação Biológica da Fiocruz Mata Atlântica se localiza em um remanescente da Mata Atlântica no Rio de Janeiro. O objetivo do estudo foi registrar a fauna de flebotomíneos nesta área. Metodologia: Três estações de monitoramento da biodiversidade foram avaliados através de seu nível de distúrbio antropogênico. As capturas foram realizadas usando armadilhas luminosas durante três meses. Resultados: Dezesseis espécies foram capturadas, sendo Psychodopygus hirsutus hirsutus a mais abundante, quatro sendo novos registros e duas espécies vetores de leishmaniose. A diversidade e a abundância foram maiores em áreas mais preservadas. Conclusão: As estações de monitoramento permitem a vigilância de mudanças e seus impactos na diversidade de Phlebotominae. RESUMEN Objetivo. Los estudios de la fauna de Phlebotominae son importantes para informar sobre diversidad y vectores de leishmaniasis. La Estación Biológica Fiocruz Bosque Atlántico tiene una zona remanente de la Mata Atlántica en Río de Janeiro. El objetivo de nuestro estudio fue registrar la fauna de flebotomos en esta área. Metodología: Se evaluaron tres sitios de monitoreo de biodiversidad a través de su alteración de nivel antropogénico. Las capturas se realizaron con trampas de luz durante tres meses. Resultados: Dieciséis especies fueron capturadas, Psychodopygus hirsutus hirsutus fue la más abundante, cuatro son nuevos registros y dos especies vectores de leishmaniasis. La diversidad y la abundancia fueron mayores en las áreas más preservadas. Conclusione: Las estaciones de monitoreo permiten la vigilancia de los cambios y su impacto en la diversidad de Phlebotominae. ¹Biólogo. Doutorando do Programa de Saúde Pública da Faculdade de Saúde Pública - USP. Mestre em Ciências IOC. Departamento de Epidemiologia, Faculdade de Saúde Pública, USP, São Paulo, São Paulo, Brasil. rodrigoeg@usp.br ²Biológa. Pesquisadora em Saúde Pública IOC. Doutora em Zoologia - USP. Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera - IOC, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brasil. efrangel@ioc.fiocruz.br ³Médica Veterinária. Tecnologista em Saúde Pública - FIOCRUZ. Mestre em Medicina Tropical IOC. Campus Fiocruz da Mata Atlântica, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brasil. fernandmor@gmail.com 4 Biólogo. Pós-doutorando IOC. Doutor em Ciências.. Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera - IOC, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brasil. wagnerac_1@hotmail.com 911

2 INTRODUCTION Sand flies (Diptera: Psychodidae: Phlebotominae) are represented by more than 1,000 known species. Currently, over 530 sand flies species are registered in the Americas, more than half being recorded in Brazil (1), where several species are involved in the transmission cycles of American cutaneous leishmaniasis (ACL) and American visceral leishmaniasis (AVL) (2). Sand flies essentially occur in the wild. However, some Leishmania vector species are adapting to deforested areas, housing sites, as well as to urban environments and outskirts of large cities. The sand fly adaptation to new habitats, especially those with environmental impacts, indicates a new leishmaniasis transmission scenario (3). Considering the environmental changes occurring in Brazil, it is important to register the biological diversity of green areas protected and regarded as environmental preservation sites, and to examine the sylvatic sand fly fauna, not only to contribute to the knowledge of Phlebotominae biodiversity, but to provide important information about the potential sand fly vector species occurrence. Environmental effects of anthropogenic activities or natural disasters can result in biodiversity loss. Therefore, biodiversity conservation studies should focus on the historic occurrence of deforestation and inadequate occupations in forested areas in Rio de Janeiro. The city of Rio de Janeiro registers transmission of both ACL and AVL, and sand fly fauna studies are related mainly to vector species. Nevertheless, some studies have focused on the state parks of Rio de Janeiro, considered as environmental preservation areas, in order to contribute to the knowledge on Phlebotominae fauna of the Atlantic Forest biome (4). In this context, a project of inventorying fauna is being carried out in permanent monitoring sites (Permanent Plots, PPs), as part of the Biodiversity Monitoring Program of the Fiocruz Atlantic Forest Biological Station (Estação Biológica Fiocruz Mata Atlântica, EFMA). These PPs were outlined in nonbuildable areas according to the campus master plan, and were projected in accordance with different levels of human disturbance (LAD). METHODOLOGY The EFMA is situated Jacarepaguá, Western region of Rio de Janeiro municipality, comprising an area of 500 ha. Eighty percent of this area is covered by Atlantic Forest remnant, and part of its territory overlaps with the Pedra Branca State Park. The western region of the campus belongs to a preservation area characterized by Atlantic Forest vegetation, Dense Ombrophilous Forest, predominantly secondary. As an area with old colonization, there are several houses around the EFMA settled near forested areas. Wild and sinanthropic mammals are found in the preserved forest, and can be observed near dwellings. Previous study in old rural communities around the EFMA has indicated part of this area as endemic for ACL (5). The present study was conducted at three sites for biodiversity monitoring: (a) PP1, a highly affected area within a forest under edge effect surrounded by human dwellings, characterized by dispersed trees, few shrubs, some open areas (22 25 m above sea level), constant anthropogenic influence, and the presence of domestic animals; (b) PP2, situated in a forest continuous to the Pedra Branca State Park, 800 m away from PP 1, 270 m far from human settlements, and with controlled people access (42 55 m above sea level). Although this area was strongly altered by the agriculture in the past, it now presents innumerous fructiferous exotic trees, many shrubs, and represents an intermediate stage of the forest restoration; (c) PP 3 is the most preserved, in a more remote area, 1772 m away from PP1, with difficult access ( m above sea level), without direct anthropogenic influence presenting a scenario closely related to the PP2, but without fructiferous exotic trees. The sand fly captures were performed for four days per month, between March and May In each ReonFacema (Esp):

3 month, samples were obtained from a different PP, using five HP model light traps, from 18h-06h. These traps were installed at equidistant points, being arranged every 50 m. Traps were assigned codes (TR1 to TR5) according to the plot point. The geographic coordinates related to the capture areas were obtained by GPS (Table 1). Temperature and humidity data were recorded at the time of placement and withdrawal of each trap. Rainfall data were obtained from "Sistema Alerta Rio" of the Rio de Janeiro City Hall, using the information from Jacarepaguá/Cidade de Deus Station. Table 1 Traps location in the Permanent Parcels. Trap Geographic coordinate TR1.1 S 22º56.314' W 043º24.193' TR2.1 S 22º56.295' W 043º24.165' PP1 TR3.1 S 22º56.310' W 043º24.133' TR4.1 S 22º56.294' W 043º24.099' TR5.1 S 22º56.318' W 043º24.075' TR1.2 S ' W ' TR2.2 S ' W ' PP2 TR3.2 S ' W ' TR4.2 S ' W ' TR5.2 S ' W ' TR1.3 S 22º56.825' W 043º25.171' TR2.3 S 22º56.823' W 043º25.140' PP3 TR3.3 S 22º56.808' W 043º25.115' TR4.3 S 22º56.803' W 043º25.111' TR5.3 S 22º56.789' W 043º25.089' The captured sand flies were fixed in 70% ethanol, and identified for each capture and location data point. Species identification was performed according to Galati s proposal. Dives software version (6) was used to access species diversity, equitability, and dominance. Moreover, the index of species abundance (ISA) and the standardized index of species abundance (SISA) analysis were performed according to Roberts & His formula (7) to evaluate species abundance comparing all the PPs. Species abundance was considered higher when the SISA value was close to 1. The results provide information on the relative abundance of species. RESULTS In 180 hours, 343 sand flies were captured, belonging to 16 species: Brumptomyia cardosoi (Barretto & Coutinho, 1941); Brumptomyia cunhai (Mangabeira, 1942); Brumptomyia nitzulescui (Costa Lima, 1932); Evandromyia edwardsi (Mangabeira, 1941); Expapilata firmatoi Barretto, (Martins & Pellegrino, 1956); Micropygomyia schreiberi Martins, (Falcão & Silva, 1975); Migonemyia migonei (França, 1920); Pintomyia bianchigalatiae (Andrade Filho, Aguiar, Dias & Falcão, 1999); Pintomyia fischeri (Pinto, 1926); Pintomyia misionensis (Castro, 1959); Psathyromyia lanei (Barreto & Coutinho, 1941); Psathyromyia lutziana (Costa Lima, 1932); Psathyromyia pascalei (Coutinho & Barretto, 1940); Psathyromyia pelloni (Sherlock & Alencar, 1959); Psychodopygus hirsutus hirsutus (Mangabeira, 1942); and Sciopemyia microps (Mangabeira, 1942) (Table 2). Psathyromyia pascalei, Sc. microps, Pi. bianchigalatiae, and Pi. misionensis were recorded for the first time in the Rio de Janeiro municipality. The most frequent species were Br. cunhai, Br. nitzulescui, and Ps. hirsutus hirsutus. Abundance analysis (SISA) demonstrated that the most abundant species were Ps. hirsutus hirsutus (0.89), Mi. schreiberi (0.80), Ev. edwardsi (0.72), Br. cunhai (0.64), Br. nitzulescui (0.47), and Mg. migonei (0.47). Of the 16 captured species, only Ev. edwardsi, Mi. schreiberi, Mg. migonei, and Ps. hirsutus hirsutus were collected from all PPs (Table 2). Expapilata firmatoi was not captured in PP3. The number of sand fly species was higher in PP3 (14 species) than in PP2 (12) and PP1 (4). However, the Shannon Wiener diversity test revealed that PP 2 (H = ) was the most diverse, followed by PP3 (H = ), and PP1 (H = ). This is explained by the Simpson Equitability Index, which shows whether the captured specimens were evenly distributed among the species. As the equitability was in PP2, in ReonFacema (Esp):

4 PP1, and in PP3, the diversity score in PP2, even with fewer captured species than in PP3, was higher, according to the diversity test. The number of specimens of Br. cunhai and Br. nitzulescui in PP3 led to a higher Simpson Dominance Index (SDI) in this area (D = ). Permanent Plot 1 had an SDI (D = 0.2) higher than PP2 (D = ), as half of the captured specimens belonged to one single species (Mi. schreiberi) in the first capture site. Both sand fly diversity and number of captured specimens were higher in less disturbed and hence more preserved areas. Table 2 Sand flies captured in the Permanent Parcels Species Permanent Parcel 1 Permanent Parcel 2 Permanent Parcel 3 Total SISA Brumptomyia cunhai Brumptomyia nitzulescui Brumptomyia cardosoi Brumptomyia sp Evandromyia edwardsi Expapilata firmatoi Micropygomyia schreiberi Migonemyia migonei Pintomyia bianchigalatiae Pintomyia fischeri Pintomyia misionensis Psathyromyia lanei Psathyromyia lutziana Psathyromyia pascalei Psathyromyia pelloni Psychodopygus hirsutus hirsutus Sciopemyia microps Total DISCUSSION Regardless of the higher frequency of sand fly species in wild environments, previous studies have shown that the presence of human dwellings near PP1 is an important attractive factor for sand flies, as observed by the presence of animals such as dogs, chickens, and pigs in the domestic and peridomestic environments (5). Therefore, the low abundance of captured specimens might be partially explained by the environmental conditions. At the first two days of capture at PP1, the mean precipitation was 10.4 mm, enhancing the mean humidity to 77.8% and reducing mean temperature to 25.3 C. Indeed, we could capture sand flies only on the last two days of the expedition at PP1. This is first record of Psathyromyia pascalei, Sc. microps, Pi. bianchigalatiae, and Pi. misionensis based on the literature and studies conducted in the Rio de Janeiro municipality (3) (5) (8) (9). These species were detected only at the most preserved areas (PP2 and PP3), without direct anthropogenic influence. ReonFacema (Esp):

5 The large presence of the genus Brumptomyia species in PP2 and PP3, and their absence in PP1, was probably due to its essentially wild behavior. Studies in the communities around the EFMA have been carried out in the past few years; however, the majority of them have focused on peridomestic sand fly captures, since the studied areas are endemic for ACL (5) (9). Of the species already registered in this area, only Mi. quinquefer, Lu. longipalpis, and Ny. intermedia were not captured in the present study. Nyssomyia intermedia was the most abundant species in previous studies in communities from anthropogenic impact around the EFMA, occurring throughout the year (5) (9). The absence of Ny. intermedia in the present study suggests that this species occurs in sylvatic environments at a very low frequency and in specific months in PP, and could be undergoing adaptation to peridomestic environments. This sand fly vector has been recorded in many other studied regions of Rio de Janeiro State (4) (8). Interestingly, Rodrigues et al. (10) also reported the absence of Ny. intermedia in sylvatic environments of Serra da Tiririca State Park in Niterói, Rio de Janeiro state. Among the identified species Mg. migonei and Pi. fischeri are considered Leishmania vectors. Migonemyia migonei, the vector of L. (V.) braziliensis in some endemic areas of ACL in Brazil (2), has been found naturally infected with L. (V.) braziliensis in EFMA (11). This sand fly species was registered in all PPs, showing a wide distribution and ability to adapt to different environments. Since it was indicated as an ACL secondary vector at EFMA, we hypothesize that it would also be responsible for the enzootic transmission cycle in PP2 and PP3 and their surroundings. Moreover, the presence of Mg. migonei in domestic environments potentially suggests its adaptation as a risk factor for the occurrence of ACL (5) (9). Regarding Pi. fischeri, some studies have evaluated its occurrence in transmission areas of L. (V.) braziliensis in the Southeastern and Southern Brazil. In peridomestic areas around the EFMA this sand fly species was registered at a very low frequency (9). Although sylvatic species have been observed to adapt to rural peridomestic habitats, by inhabiting the proximities of domestic animal shelters (2) (12), the occurrence of this species at PP3 suggests a predominantly wild behavior of this population at the EFMA. Strong indications of the incrimination of this sand fly as a vector were observed for the first time at the Espírito Santo state, where Pi. fischeri was found naturally infected with L. (V.) braziliensis in rural endemic areas (13). In an ACL transmission area, in the forests of Porto Alegre (Rio Grande do Sul state) periphery, it was found naturally infected with L. (V.) braziliensis, leading the authors to hypothesize that this species is a local secondary vector (14). Rangel & Lainson (2) discussed the potential vectors of ACL in Brazil, and indicated this sand fly species can maintain the infection by L. (V.) braziliensis in sylvatic habitats. This could possibly be occurring in the studied areas of the Atlantic Forest, especially in PP3, in a primary zoonotic cycle of ACL. In southeastern Brazil, Ps. hirsutus hirsutus was found naturally infected with Leishmania (Viannia) sp. (15), and according to Rangel & Lainson (2), three specimens infected with flagellates have been recorded in Pará state, but there is no evidence of this sand fly participation in the transmission cycle of ACL. Psychodopygus hirsutus hirsutus was previously recorded in forested areas of the EFMA (9). In the present study, this species was more frequently found in PP2 and PP3, probably reflecting better adaptation to a more preserved forest. CONCLUSION In conclusion, the results of the present study contribute to knowledge on the interface of health environment biodiversity, obtaining data, and enabling the monitoring of changes in ecological processes in Atlantic Forest, and their influence on the sand fly fauna in Rio de Janeiro municipality. REFERENCES 1. Shimabukuro PHF, Andrade AJ, Galati EAB. Checklist of American sand flies (Diptera, Psychodidae, Phlebotominae): ReonFacema (Esp):

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