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Entomological survey of phlebotominae sand flies (diptera: psychodidae) and vector species in the tegumentary leishmaniasis endemic area in eastern brazilian Amazon, Amapá state

Levantamento entomológico de flebotomíneos (Diptera: Psychodidae) e espécies vetoras na área endêmica de leishmaniose tegumentar na Amazônia oriental brasileira, Estado do Amapá

Abstract:

American tegumentary leishmaniasis is an endemic that has increased considerably in recent decades in the Amazon region, sand flies are the vectors of the transmission of the protozoan that causes leishmaniasis, so the objective of this study was to carry out a survey of the diversity of species and the presence of Leishmania DNA in vectors circulating in three endemic counties for tegumentary leishmaniasis in the eastern Brazilian Amazon (Amapá state, Brazil). Using CDC light traps, a total of 10,773 specimens were collected between February 2019 and February 2020, representing 64 species in 15 genera. The vector specie Nyssomyia umbratilis Ward and Frahia, 1977 was the predominant species (13.20% of the total), being collected in all three counties, followed by Trichopygomyia trichopyga Floch & Abonnenc, 1945 (11.41%), Trichophoromyia ubiquitalis Mangabeira,1942 (9.47%) and Nyssomyia anduzei Rozeboom, 1942 (7.61%). For the identification of Leishmania DNA, 775 pools of unengorged females were used, of which 5 tested positive, 2 of Nyssomya umbratilis Ward & Fraiha,1977, 1 of Nyssomyia anduzei and 2 of Psychodopygus davisi Root,1934, demonstrating a natural total infection rate of 0.64%. This study increases the knowledge of vector diversity, as well as identifying Leishmania spp. in circulation in the eastern region of the Amazon.

Keywords:
Entomological surveillance; PCR; Molecular detection; Sand flies Diversity

Resumo:

A leishmaniose tegumentar americana é uma endemia que aumentou consideravelmente nas últimas décadas na região amazônica, os flebotomíneos são os vetores da transmissão do protozoário causador da leishmaniose, portanto o objetivo deste estudo foi realizar um levantamento da diversidade de espécies e a presença de DNA de Leishmania em vetores que circulam em três municípios endêmicos de leishmaniose tegumentar na Amazônia oriental brasileira (Amapá, Brasil). Usando armadilhas luminosas do tipo CDC, um total de 10.773 espécimes foram coletados entre fevereiro de 2019 e fevereiro de 2020, representando 64 espécies em 15 gêneros. As espécie vetoras - singular Nyssomyia umbratilis Ward e Frahia 1977 foram as espécies predominantes (13,20% do total), sendo coletadas nos três municípios, seguido por Trichopygomyia trichopyga Floch & Abonnenc, 1945 (11,41%), Trichophoromyia ubiquitalis Mangabeira, 1942 (9,47%) e Nyssomyia anduzei Rozeboom, 1942 (7,61%). Para a identificação do DNA de Leishmania, foram utilizados 775 pools de fêmeas não ingurgitadas, dos quais 5 foram positivos, 2 de Nyssomya umbratilis Ward & Fraiha, 1977, 1 de Nyssomyia anduzei e 2 de Psychodopygus davisi Root, 1934, demonstrando uma taxa de infecção total de 0,64%. Este estudo aumenta o conhecimento da diversidade de vetores, bem como a identificação das espécies de Leishmania spp. em circulação na região oriental da Amazônia.

Palavras-chave:
Vigilância Entomológica; PCR; Detecção Molecular; Diversidade de Flebotomíneos

Introduction

Sand flies (Diptera: Psychodidae) are small insects that play a fundamental role in the transmission of protozoa of the genus Leishmania Ross (Kinetoplastida: Trypanosomatidae), which are the etiological agents of American Tegumentary Leishmaniasis (ATL) and Visceral Leishmaniasis (VL) in humans (Ready 2013READY, P.D. 2013. Biology of phlebotomine sand flies as vectors of disease agents. Annu. Rev. Entomol. 58:227-250. https://doi.org/10.1146/annurev-ento-120811-153557
https://doi.org/10.1146/annurev-ento-120...
). Leishmaniasis infects approximately 12 million people around the world, with approximately 600,000 new cases being reported each year. It is estimated that 90% of ATL cases occur in Latin America (Bolivia, Peru, and Brazil) and Middle East (Iran, Saudi Arabia, Syria, Afghanistan) (WHO 2021WHO, 2021. Leishmaniasis factsheets. https://www.who.int/news-room/fact-sheets/detail/leishmaniasis (last acces 28/04/2021).
https://www.who.int/news-room/fact-sheet...
).

In Brazil, ATL is a disease that requires attention due to the growing number of cases and the characteristics of the country that are conducive to the development of the disease, such as the great diversity of phlebotomine vectors of several Leishmania species that occur in the country (Brazil et al. 2014BRAZIL, R.P., RODRIGUES, A.A.F. & ANDRADE-FILHO, J.D. 2014. Sand Fly Vectors of Leishmania in the Americas - A Mini Review. Entomol. Ornithol. Herpetol. Curr. Res. 4: 144. https://doi.org/10.4172/2161-0983.1000144
https://doi.org/10.4172/2161-0983.100014...
). Currently there are approximately 1,000 species of sand flies described in the world, 530 in the Americas and approximately 280 species have already been found in Brazil (Shimabukuro et al. 2017SHIMABUKURO, P.H.F., DE ANDRADE, A.J. & GALATI, E.A.B. 2017. Checklist of American sand flies (Diptera, psychodidae, phlebotominae): Genera, species, and their distribution. Zookeys 660:67-106., Galati 2018GALATI, E.A.B. 2018. Phlebotominae (Diptera, Psychodidae): Classification, Morphology and Terminology of Adults and Identification of American Taxa. In Brazilian Sand Flies Springer International Publishing, Cham, p.9-212.), of these, at least 19 species are identified as proven or probable vectors of medical-veterinary importance (Aguiar & Medeiros 2003AGUIAR, G. & MEDEIROS, W. 2003. Distribuição regional e habitats das espécies de flebotomíneos do Brasil. In Flebotomíneos do Brasil (E. F. Rangel & R. Lainson, eds) Fiocruz, Rio de Janeiro, p.207-255.). According to the Notifiable Diseases Information System (SINAN), from the Ministry of Health, between 2003 and 2018, more than 300,000 cases of ATL were reported, with an average of 21,158 cases per year. ATL is largely neglected, and Brazil is responsible for the majority of human cases in the West (Alvar et al. 2012ALVAR, J., VÉLEZ, I.D., BERN, C., HERRERO, M., DESJEUX, P., CANO, J., JANNIN, J., DEN BOER, M. & WHO LEISHMANIASIS CONTROL TEAM. 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7(5):e35671. https://doi.org/10.1371/journal.pone.0035671
https://doi.org/10.1371/journal.pone.003...
, Da Silva et al. 2020DA SILVA, Y.Y., SALES, K.G.D.S., MIRANDA, D.E.D.O., FIGUEREDO, L.A., BRANDÃO-FILHO, S.P. & DANTAS-TORRES, F. 2020. Detection of Leishmania DNA in Sand Flies (Diptera: Psychodidae) from a Cutaneous Leishmaniasis Outbreak Area in Northeastern Brazil. J. Med. Entomol. 57(2):529-533.). The northern region of the country, where the state of Amapá is located, it is responsible for the largest number of cases in the period (Brasil 2019BRASIL. 2019. Boletim Epidemiológico: Vigilância em Saúde no Brasil 2003-2019. Ministério da Saúde. https://portalarquivos2.saude.gov.br/images/pdf/2019/setembro/25/boletim-especial-21ago19-web.pdf (last acces 25/04/2021)
https://portalarquivos2.saude.gov.br/ima...
).

The state of Amapá, located in the Eastern Amazon, has environmental conditions that support one of the greatest biodiversity in the world, which provide opportunities for the development of vector-host relations and, consequently, of the leishmaniasis cycle (Ellwanger et al. 2020ELLWANGER, J.H., KULMANN-LEAL KULMANN-LEAL, BRUNA KAMINSKI, V.L., VALVERDE-VILLEGAS, J.M., VEIGA, A.B.G. DA, SPILKI, F.R., FEARNSIDE, P.M., CAESAR, L., GIATTI, L.L., WALLAU, G.L., ALMEIDA, S.E.M., BORBA, M.R., HORA, V.P. DA & CHIES, J.A.B. 2020. Beyond diversity loss and climate change: Impacts of Amazon deforestation on infectious diseases and public health. An. Acad. Bras. Cienc. 92(1):1-33.https://doi.org/10.1590/0001-3765202020191375
https://doi.org/10.1590/0001-37652020201...
). To date there has been a record of five species of Leishmania coexisting in the region: Leishmania (Viannia) guyanensis Floch, 1954, L. (V.) braziliensis Vianna, 1911, L. (V.) lainsoni Silveira, Shaw, Braga & Ishikawa, 1987, L. (V.) naiffi Laison and Shaw, 1989, L. (Leishmania) amazonensis Lainson and Shaw, 1972. L. (V.) guyanensis being responsible for 80% of ATL cases in this area (Brasil 2017BRASIL.2017. Manual De Vigilância Da Leishmaniose Tegumentar. Secretaria de Vigilância das Doenças Transmissíveis. Manual de vigilância da leishmaniose tegumentar.Brasília. http://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf (last acces 15/05/2021)
http://bvsms.saude.gov.br/bvs/publicacoe...
, de Souza et al. 2017DE SOUZA, A.A.A., DA ROCHA BARATA, I., DAS GRAÇAS SOARES SILVA, M., LIMA, J.A.N., JENNINGS, Y.L.L., ISHIKAWA, E.A.Y., PRÉVOT, G., GINOUVES, M., SILVEIRA, F.T., SHAW, J. & DOS SANTOS, T.V. 2017. Natural Leishmania (Viannia )infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil. Parasite 24:13. https://doi.org/10.1051/parasite/2017016
https://doi.org/10.1051/parasite/2017016...
). Despite the high incidence of ATL in the region, there are few studies that study the diversity of the sandfly and Leishmania fauna (Costa et al. 2021COSTA, T.S. da, FERREIRA, R.M. dos A., SANTOS, G.S., GARCIA JÚNIOR, M.D., PINTO, C.B. & SOUTO, R.N.P. 2021. Ecological aspects and molecular detection of Leishmania DNA (Kinetoplastida: Trypanosomatidae) in phlebotomine sand flies (Diptera: Psychodidae) from a rural settlement in the Eastern Amazon, Brazil. Rev. Bras. Entomol. 65(3). https://doi.org/10.1590/1806-9665-rbent-2021-0065
https://doi.org/10.1590/1806-9665-rbent-...
). The aim of this study is to characterize the sandfly fauna, as well as their natural infection by Leishmania spp. in an endemic region for ATL in the state of Amapá (AP), eastern Amazon.

Material and Methods

1 Study area

This study was conducted in the rural area of three counties in the state of Amapá (AP): Mazagão - P1 (0° 6’ 54” S, 51° 17’ 20” W), Porto Grande - P2 (0° 53’ 45” N, 52° 0’ 7” W) and Serra do Navio - P3 (0° 53’ 45” N, 52° 0’ 7” W) (Figure 1), located in the eastern Amazon, northern region of Brazil. According to Köeppen’s classification criteria, the region’s climate is in the humid tropical category, predominantly in the Am category, with the mildest month temperature above 18°C and monthly average rainfall below 60mm (Tavares 2014TAVARES, J.P.N. 2014. Características Da Climatologia De Macapá-Ap. CAMINHOS Geogr. - Rev. online 15(50):138-151., Garcia et al. 2020GARCIA, M.D.N., DAMASCENO, M.T.D.S., MARTINS, M.J.L., DA COSTA, T.S., FERREIRA, R.M.D.A. & SOUTO, R.N.P. 2020. New records of dragonflies and damselflies (Insecta: Odonata) from Amapá State, Brazil. Biota Neotrop. 21(1):1-7. https://doi.org/10.1590/1676-0611-BN-2020-1074 .
https://doi.org/10.1590/1676-0611-BN-202...
). The collections were carried out in areas characterized as dry land with predominant vegetation of dense rainforest. The research was carried out in areas of proven ATL transmission in the three counties, in which an increase in cases was detected in recent years by the state agency for Health Surveillance (SVS 2019SVS (SISTEMA DE VIGILÂNCIA EM SAÚDE). 2019. Boletim Epidemiológico - Situação Epidemiológica da Leishmaniose Tegumentar no Estado do Amapá: Período de 2017 a junho de 2019.https://editor.amapa.gov.br/arquivos_portais/publicacoes/SVS_4d77b443923909a984f01e74bf38240f.pdf (last acces 14/01/2021)
https://editor.amapa.gov.br/arquivos_por...
). Records have shown that human ATL cases have increased in rural areas in villages that are close to forested areas, and where villagers engage in agriculture and extractive activities of forest products.

Figure 1
Map of the study area, location counties of Mazagão (P1), Porto Grande (P2) and Serra do Navio (P3), Amapá State, Brazil, where the Sand flies collections with the CDC light traps were carried out.

2 Sandfly collections and morphological identification

The sand flies were captured in villages in rural areas of the three counties with Center for Disease Control (CDC) light traps placed in the peridomicile of homes that were selected for their proximity to the forest and the presence of chicken coops and pig breeding, characteristics that favor the presence of sand flies. The traps worked for three consecutive nights from 6:00 pm to 7:00 am, monthly for one year (February 2019 to February 2020), totalizing 468 hours of capture at each collection point. The 30 traps were distributed in locations with ATL human case records, in each of the three selected counties.

The collected insects were taken to the Arthropoda Laboratory (ArtroLab) at the Federal University of Amapá (UNIFAP) for screening by sex and dissection process. The final three segments of the abdomen and the head were removed for mounting on Berlese liquid glass slides. The rest of the phlebotomine bodies were conditioned in 94% ethanol at-20ºC for the subsequent extraction of genomic DNA. Species identification was based on the morphology of male genitalia and the spermatheca and by the characters present in female’s head,, using the updated classification key developed by Galati in 2003 (Galati 2019GALATI, E.A.B. 2019. Phlebotominae (Diptera, Psychodidae). Classificação, morfologia, terminologia e identificação de Adultos. Apostila da Disciplina HEP 5752—Bioecologia e Identificação de Phlebotominae 2019. I. https://edisciplinas.usp.br/mod/resource/view.php?id=2796254 (last acces 18/02/2021)
https://edisciplinas.usp.br/mod/resource...
), and following the abbreviation of the genera proposed by Marcondes (2007)MARCONDES, C. B. 2007. A Proposal of Generic and Subgeneric Abbreviations for Phlebotomine Sandflies (Diptera: Psychodidae: Phlebotominae) of the World. Entomol. News 118:351-356..

3 Molecular detection of Leishmania

The thorax of unengorged female sand flies were grouped according to date, species, and county of collection to form pools with 2 to 10 specimens of the 65 species collected. DNA extraction and Polymerase Chain Reaction (PCR) were performed to amplify the molecular targets of the region of kinetoplast DNA minicircles (kDNA) and hsp70, as described elsewhere (Pereira Júnior et al. 2015PEREIRA JÚNIOR, A.M., TELES, C.B.G., DE AZEVEDO DOS SANTOS, A.P., DE SOUZA RODRIGUES, M., MARIALVA, E.F., PESSOA, F.A.C. & MEDEIROS, J.F. 2015. Ecological aspects and molecular detection of Leishmania DNA Ross (Kinetoplastida: Trypanosomatidae) in phlebotomine sandflies (Diptera: Psychodidae) in terra firme and várzea environments in the Middle Solimões Region, Amazonas State, Brazil. Parasites Vectors 8(1):1-11., Resadore et al. 2017RESADORE, F., PEREIRA, A.M., CARVALHO, L.P.C., DE AZEVEDO DOS SANTOS, A.P., TELES, C.B.G. & MEDEIROS, J.F. 2017. Phlebotomine sand fly composition (Diptera: Psychodidae) and putative vectors of American cutaneous leishmaniasis in Porto Velho Municipality, Western Amazon, Brazil. J. Med. Entomol. 54(3):798-803.). For the negative control, purified water was used and for the positive controls, DNA from Le. (L.) amazonensis Lainson & Shawn, 1972 and Le. (V.) braziliensis Vianna, 1911 strains.

4 Data analysis

The collection effort and the number of species in the study were measured using the non-parametric Jackknife1 estimator, generating a rarefaction curve for each of the collection points using the R software (R Core Team 2021R CORE TEAM (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/
https://www.R-project.org/...
) using the vegan package (Oksanen et al. 2019OKSANEN, J., BLANCHET, F.G., FRIENDLY, M., KINDT, R., LEGENDRE, P., MCGLINN, D., MINCHIN, P.R., O’HARA, R.B., SIMPSON, B.L., SOLYMOS, P., STEVENS, M.H.H., SZOECS, E. & WAGNER, H. 2019. vegan: Community Ecology Package. R package version 2.5-5. https://CRAN.R-project.org/package=vegan
https://CRAN.R-project.org/package=vegan...
).

The sequences of the hsp70 molecular target were analyzed using Phred, Phrap and Consend software (Ewing & Green 1998EWING, B. & GREEN, P. 1998. Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities. Genome Res. 8(3): 175-185. http://dx.doi.org/10.1101/gr.8.3.175
http://dx.doi.org/10.1101/gr.8.3.175...
), with the minimum value defined as Q=30. The identification of Leishmania species was performed by comparing the consensus sequences obtained in the study with reference sequences deposited in the GenBank database (http: www.ncbi.nlm.nih.gov/genbank) using the BLAST tool (Basic Local Alignment Search Tool) (Altschul et al. 1990ALTSCHUL, S.F., GISH, W., MILLER, W., MYERS, E.W. & LIPMAN, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215(3):403-410.).

Results

The total number of sand flies collected in the three locations (P1, P2, P3) was 10,773 specimens, being: 4,512 males (41.89%) and 6,261 females (58.11%), totalizing 15 genera and 64 species (Table 1). The most abundant genera were Nyssomyia Barretto (3,559 individuals, 33.04%), Trichophoromyia Barreto (1,619, 15.02%), Trichopygomyia Barretto (1,278, 11.87%) and Psychodopygus Mangabeira (1,176, 10.92%). The genera collected in lesser abundance were Pintomyia Costa Lima (72 individuals, 0.67%), Vianamyia Mangabeira (65, 0.60%) and Pressatia Mangabeira (35, 0.32%). The most abundant species were Ny. umbratilis (n=1.422, 13.20%), Ty. trichopyga (n=1.229, 11.41%), Th. ubiquitalis (n=1020, 9.47%) and Ny. anduzei (n=820, 7.61%); these species accounted for 40.69% of the sand flies collections. The least abundant species were Pa. bigeniculata Floch & Abonnenc, 1941 (n=11,0.10%), Br. beaupertuiy Ortiz, 1954 (n=11,0.10%), Pa. lutziana Costa Lima, 1932 (n=8, 0.07%), Lu. spatotrichia Martins, Falcão & Silva, 1963 (n=6, 0.06%); among the less collected species is the vector Ps. ayrozai Barretto & Coutinho, 1940 (n=45, 0.42%).

Table 1
Species of Sand flies from Amapá State, Eastern Amazon, Brazil, collected with CDC light traps from February 2019 to February 2020.

Based on the data from the study, the rarefaction curves demonstrated a good result of the sampling efforts, with collection efficiency approaching 100% at the three collection points. The Jackknife 1 estimator showed that species richness corresponded to 96% in Mazagão (P1) and Porto Grande (P2), and 100% in Serra do Navio (P3) (Figure 2), demonstrating a tendency to stabilization in the three sampling points of the curve in an asymptote.

Figure 2
The rarefaction curves (observed and estimates by jackknife 1) of sand flies species collected in the Mazagão (P1), Porto Grande (P2) and Serra do Navio (P3), Amapá State, Brazil.

A total of 775 pools of unengorged females were formed for the detection of Leishmania DNA, of which PCR for molecular targets kDNA and hsp70 identified that a total of five pools were positive: two pools of Ny. umbratilis infected with L. (V.) guyanensis (query cover=100%, identity=100%, Genbank accession MW094227.1) collected in Porto Grande (P1) and Serra do Navio (P3), a pool of Ny. anduzei infected with L. (V.) naiffi (query cover=100%, identity=100%, Genbank accession MT469994.1) in Serra do Navio (P3), and two pools of Ps. davisi infected with L. (V.) braziliensis (query cover = 98%, identity = 98%, Genbank accession MT543301.1) collected in Mazagão (P2) and Serra do Navio (P3). The minimum infection rate (number of positive samples / total samples tested x 100) was calculated for each of the species that tested positive for Leishmania DNA: Ny. umbratilis (1.0%), Ny. anduzei (1,5%) and Ps. davisi (5.7%).

Discussion

Our sampling included 64 species of sand flies among the 77 recorded in the state of Amapá (Galati 2019GALATI, E.A.B. 2019. Phlebotominae (Diptera, Psychodidae). Classificação, morfologia, terminologia e identificação de Adultos. Apostila da Disciplina HEP 5752—Bioecologia e Identificação de Phlebotominae 2019. I. https://edisciplinas.usp.br/mod/resource/view.php?id=2796254 (last acces 18/02/2021)
https://edisciplinas.usp.br/mod/resource...
), 50 in Mazagão (P1),49 in Porto Grande (P2) and 56 in Serra do Navio (P3). The highest number of species was found in Serra do Navio, which corroborates the 55 species already found in the county in a previous study (de Souza et al. 2017DE SOUZA, A.A.A., DA ROCHA BARATA, I., DAS GRAÇAS SOARES SILVA, M., LIMA, J.A.N., JENNINGS, Y.L.L., ISHIKAWA, E.A.Y., PRÉVOT, G., GINOUVES, M., SILVEIRA, F.T., SHAW, J. & DOS SANTOS, T.V. 2017. Natural Leishmania (Viannia )infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil. Parasite 24:13. https://doi.org/10.1051/parasite/2017016
https://doi.org/10.1051/parasite/2017016...
), the present study found the highest number (83.1%) of species among all species with occurrence for the state. Our survey demonstrated a great range of sandfly species in the three collection sites, demonstrating the great diversity of the sandfly fauna in the Eastern Amazon. The diversity found in the study generally corresponded to the pattern of studies carried out in forest areas that demonstrate the phlebotomine fauna usually composed of a few dominant species and many species with specimens (Rosário et al. 2016ROSÁRIO, I.N.G., ANDRADE, A.J., LIGEIRO, R., ISHAK, R. & SILVA, I.M. 2016. Evaluating the Adaptation Process of Sandfly Fauna to Anthropized Environments in a Leishmaniasis Transmission Area in the Brazilian Amazon. J. Med. Entomol. 54(2): 450-452. doi: 10.1093/jme/tjw182
https://doi.org/10.1093/jme/tjw182...
)

The genus Nyssomia is of great importance for surveillance studies and understanding of the ecopidemiology of ATL, occurring from North America (Mexico) to South America (Argentina), with a total of seven species proven or suspected of transmission of ATL (Marcondes et al. 1998MARCONDES, C.B., LOZOVEI, A.L. & VILELA, J.H. 1998. Distribuição geográfica de flebotomíneos do complexo Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera, Psychodidae). Rev. Soc. Bras. Med. Trop. 31(1):51-58.). Three species of the genus showed great abundance: Nyssomyia umbratilis (13.2%), Nyssomyia anduzei (7.61%) and Nyssomyia whitmani Antunes & Coutinho, 1939 (5.71%), these species identified as vectors in Brazil (Brasil 2017BRASIL.2017. Manual De Vigilância Da Leishmaniose Tegumentar. Secretaria de Vigilância das Doenças Transmissíveis. Manual de vigilância da leishmaniose tegumentar.Brasília. http://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf (last acces 15/05/2021)
http://bvsms.saude.gov.br/bvs/publicacoe...
) and already associated with ATL transmission in the state of Amapá in previous studies (De Souza et al. 2017DE SOUZA, A.A.A., DA ROCHA BARATA, I., DAS GRAÇAS SOARES SILVA, M., LIMA, J.A.N., JENNINGS, Y.L.L., ISHIKAWA, E.A.Y., PRÉVOT, G., GINOUVES, M., SILVEIRA, F.T., SHAW, J. & DOS SANTOS, T.V. 2017. Natural Leishmania (Viannia )infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil. Parasite 24:13. https://doi.org/10.1051/parasite/2017016
https://doi.org/10.1051/parasite/2017016...
). Ny. umbratilis is a constant presence in endemic areas for ATL (Pinheiro et al. 2008PINHEIRO, F.G., LUZ, S.L.B. & FRANCO, A.M.R. 2008. Infecção natural por tripanosomatídeos (Kinetoplastida: Trypanosomatidae) em Lutzomyia umbratilis (Diptera: Psychodidae) em áreas de leishmaniose tegumentar americana no Amazonas, Brasil. Acta Amaz 38(1):165-172.), and is considered one of the main vectors for L. (V.) guyanensis in most of Latin America, in countries such as Brazil, Bolivia, Colombia, Peru, Venezuela, Suriname and French Guiana (Brazil et al. 2014BRAZIL, R.P., RODRIGUES, A.A.F. & ANDRADE-FILHO, J.D. 2014. Sand Fly Vectors of Leishmania in the Americas - A Mini Review. Entomol. Ornithol. Herpetol. Curr. Res. 4: 144. https://doi.org/10.4172/2161-0983.1000144
https://doi.org/10.4172/2161-0983.100014...
), as well as in the Amazon region (Brasil 2019BRASIL. 2019. Boletim Epidemiológico: Vigilância em Saúde no Brasil 2003-2019. Ministério da Saúde. https://portalarquivos2.saude.gov.br/images/pdf/2019/setembro/25/boletim-especial-21ago19-web.pdf (last acces 25/04/2021)
https://portalarquivos2.saude.gov.br/ima...
). Nyssomyia anduzei is considered a secondary vector of L. (V.) guyanensis and appears as the fourth species with the largest sampling in the present study, which corroborates studies carried out in the Western Amazon region (Barbosa et al. 2008BARBOSA, M.D.G.V., FÉ, N.F., MARCIÃO, A.H.R., SILVA, A.P.T., MONTEIRO, W.M. & GUERRA, J.A.D.O. 2008. Fauna de flebotomíneos (Diptera: Psychodidae) em um foco de leishmaniose tegumentar americana na área periurbana de Manaus, Estado do Amazonas. Rev. Soc. Bras. Med. Trop. 41(5):485-491. https://doi.org/10.1590/S0037-86822008000500010
https://doi.org/10.1590/S0037-8682200800...
, Grimaldi et al. 1991aGRIMALDI, G., MOMEN, H., NAIFF, R.D., MCMAHON-PRATT, D. & BARRETT, T. V. 1991a. Characterization and Classification of Leishmanial Parasites from Humans, Wild Mammals, and Sand Flies in the Amazon Region of Brazil. Am. J. Trop. Med. Hyg. 44(6):645-661. https://doi.org/10.4269/ajtmh.1991.44.645
https://doi.org/10.4269/ajtmh.1991.44.64...
), Ny. whitmani is considered one of the vectors of great medical importance in the Amazon region as it can transmit L (V.) braziliensis, L. (V.) shawi and L. (V.) guyanensis (Lainson et al. 1994LAINSON, R., SHAW, J.J., SILVEIRA, F.T., SOUZA, A.A.A. de, BRAGA, R.R. & ISHIKAWA, E.A.Y. 1994. The dermal leishmaniases of Brazil, with special reference to the eco-epidemiology of the disease in Amazonia. Mem. Inst. Oswaldo Cruz 89(3):435-443.), this species is considered a wild species and in the northern region of Brazil it has a lower anthropophilic habit (Silveira et al. 1991SILVEIRA, F.T., SOUZA, A.A.A., LAINSON, R., SHAW, J.J., BRAGA, R.R. & ISHIKAWA, E.E.A. 1991. Cutaneous leishmaniasis in the Amazon region: natural infection of the sandfly Lutzomyia ubiquitalis (Psychodidae: Phlebotominae) by Leishmania (Viannia) lainsoni in Pará state, Brazil. Mem. Inst. Oswaldo Cruz 86(1):127-130.), having been identified previously infected with Leishmania in the region (Rangel & Lainson 2009RANGEL, E.F. & LAINSON, R. 2009. Proven and putative vectors of American cutaneous leishmaniasis in Brazil: Aspects of their biology and vectorial competence. Mem. Inst. Oswaldo Cruz 104(7):937-954.).

Other species with medical importance such as Bichromomyia flaviscutellata Mangabeira 1941, Tricophoromyia ubiquitalis. Psychodopygus squamiventris maripaensis Floch & Abonnenc, 1946 and Migonemia migonei França,1920 were found at the three collection points in peridomicile areas. This is a factor that should be carefully observed as finding females of these species in these environments may indicate that the species are looking for blood meal sources in the domestic environment or being attracted because of the lights in the houses; the presence of these vectors near the dwellings greatly increases the risk of contracting ATL in the home environment (Tanure et al. 2015TANURE, A., PEIXOTO, J.C., AFONSO, M.M. dos S., DUARTE, R., PINHEIRO, A. da C., COELHO, S.V.B. & BARATA, R.A. 2015. Identification of Sandflies (Diptera: Psychodidae: Phlebotominae) Blood Meals in an Endemic Leishmaniasis Area in Brazil.. Rev. Inst. Med. Trop. 57(4):321-324.).

For the incrimination of Leishmania vectors, one of the crucial points is to determine the occurrence of natural infection in sandfly populations, identifying possible vector species. The test considered that the gold standard for the natural detection of infection is the dissection of the digestive tract to indicate the presence of Leishmania promastigotes by light microscopy (Kato et al. 2005KATO, H., UEZATO, H., KATAKURA, K., CALVOPIÑA, M., MARCO, J.D., BARROSO, P.A., GOMEZ, E.A., MIMORI, T., KORENAGA, M., IWATA, H., NONAKA, S. & HASHIGUCHI, Y. 2005. Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction. Am. J. Trop. Med. Hyg. 72(1):87-93.), but this method is extremely laborious and requires the dissection of a large number of specimens, isolation and culture of protozoa from dissected sand flies. Thus, molecular techniques such as PCR for the detection of Leishmania DNA have been increasingly used in studies with sand flies (Teles et al. 2016TELES, C.B.G., SANTOS, A.P. de A. dos, FREITAS, R.A., OLIVEIRA, A.F.J. de, OGAWA, G.M., RODRIGUES, M.S., PESSOA, F.A.C., MEDEIROS, J.F. & CAMARGO, L.M.A. 2016. Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia. Mem. Inst. Oswaldo Cruz 111(7):423-432., Da Silva et al. 2020DA SILVA, Y.Y., SALES, K.G.D.S., MIRANDA, D.E.D.O., FIGUEREDO, L.A., BRANDÃO-FILHO, S.P. & DANTAS-TORRES, F. 2020. Detection of Leishmania DNA in Sand Flies (Diptera: Psychodidae) from a Cutaneous Leishmaniasis Outbreak Area in Northeastern Brazil. J. Med. Entomol. 57(2):529-533.). The minimum infection rate of Leishmania DNA detected with molecular methods was 0.64%, a result compatible with a study conducted in the state of Amapá (0.78%) (Vasconcelos Dos Santos et al. 2019VASCONCELOS DOS SANTOS, T., DE PITA-PEREIRA, D., ARAÚJO-PEREIRA, T., BRITTO, C., SILVEIRA, F.T., PÓVOA, M.M. & RANGEL, E.F. 2019. Leishmania DNA detection and species characterization within phlebotomines (Diptera: Psychodidae) from a peridomicile-forest gradient in an Amazonian/Guianan bordering area.. PLoS One 14(7):e0219626.), as well as in other states in the Amazon region, such as Rondônia (0.28%) (Resadore et al. 2019RESADORE, F., JÚNIOR, A.M.P., DE PAULO, P.F.M., GIL, L.H.S., RODRIGUES, M.M.D.S., ARAÚJO, M.D.S., JULIÃO, G.R. & MEDEIROS, J.F. 2019. Composition and Vertical Stratification of Phlebotomine Sand Fly Fauna and the Molecular Detection of Leishmania in Forested Areas in Rondônia State Municipalities, Western Amazon, Brazil. Vector-Borne Zoonotic Dis. 19(5):347-357.), Amazonas (0.83%) and Acre (0.99%).

The DNA of the species L. (V.) guyanensis was detected in two samples of Ny. umbratilis in the collection locations (P1 and P3). This sandfly species is pointed out as the main vector of L. (V.) guyanenis in northern Brazil (Gil et al. 2009GIL, L.H.S., ARAÚJO, M. da S., VILLALOBOS, J.M., CAMARGO, L.M.A., OZAKI, L.S., FONTES, C.J.F., RIBOLLA, P.E.M., KATSURAGAWA, T.H., CRUZ, R.M., SILVA, A. de A. e & SILVA, L.H.P. da. 2009. Species structure of sand fly (Diptera: Psychodidae) fauna in the Brazilian western Amazon. Mem. Inst. Oswaldo Cruz 104(7):955-959. https://doi.org/10.1590/S0074-02762009000700002
https://doi.org/10.1590/S0074-0276200900...
). Several other studies conducted in areas endemic to ATL found this sandfly species infected with L. (V.) guyanensis (Lainson et al. 1981LAINSON, R., SHAW, J.J., READY, P.D., MILES, M.A. & POVOA, M. 1981. Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sandflies, wild mammals and man in north Pará state, with particular reference to L. braziliensis guyanensis causative agent of “pian-bois. Trans. R. Soc. Trop. Med. Hyg. 75(4): 530-536. https://doi.org/10.1016/0035-9203(81)90192-9
https://doi.org/10.1016/0035-9203(81)901...
, Pinheiro et al. 2008PINHEIRO, F.G., LUZ, S.L.B. & FRANCO, A.M.R. 2008. Infecção natural por tripanosomatídeos (Kinetoplastida: Trypanosomatidae) em Lutzomyia umbratilis (Diptera: Psychodidae) em áreas de leishmaniose tegumentar americana no Amazonas, Brasil. Acta Amaz 38(1):165-172.), including in studies previously conducted in the state of Serra do Navio (P3) (de Souza et al. 2017DE SOUZA, A.A.A., DA ROCHA BARATA, I., DAS GRAÇAS SOARES SILVA, M., LIMA, J.A.N., JENNINGS, Y.L.L., ISHIKAWA, E.A.Y., PRÉVOT, G., GINOUVES, M., SILVEIRA, F.T., SHAW, J. & DOS SANTOS, T.V. 2017. Natural Leishmania (Viannia )infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil. Parasite 24:13. https://doi.org/10.1051/parasite/2017016
https://doi.org/10.1051/parasite/2017016...
) and in Oiapoque, a region on the state border with French Guiana (Vasconcelos Dos Santos et al. 2019VASCONCELOS DOS SANTOS, T., DE PITA-PEREIRA, D., ARAÚJO-PEREIRA, T., BRITTO, C., SILVEIRA, F.T., PÓVOA, M.M. & RANGEL, E.F. 2019. Leishmania DNA detection and species characterization within phlebotomines (Diptera: Psychodidae) from a peridomicile-forest gradient in an Amazonian/Guianan bordering area.. PLoS One 14(7):e0219626.), where it is considered the main vector (Vasconcelos dos Santos et al. 2018VASCONCELOS DOS SANTOS, T., PRÉVOT, G., GINOUVÈS, M., DUARTE, R., SILVEIRA, F.T., PÓVOA, M.M. & RANGEL, E.F. 2018. Ecological aspects of Phlebotomines (Diptera: Psychodidae) and the transmission of American cutaneous leishmaniasis agents in an Amazonian/ Guianan bordering area. Parasit. Vectors 11(1):612.). Infection by L. (V.) guyanensis represents a high risk of complications for human health, as the protozoan is resistant to the drug most used in clinical practice in Brazil, Glucantime (Brasil 2017BRASIL.2017. Manual De Vigilância Da Leishmaniose Tegumentar. Secretaria de Vigilância das Doenças Transmissíveis. Manual de vigilância da leishmaniose tegumentar.Brasília. http://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf (last acces 15/05/2021)
http://bvsms.saude.gov.br/bvs/publicacoe...
).

Nyssomyia anduzei was found infected with the DNA of L. (V.) naiffi in P3 (Serra do Navio). This species is considered a secondary vector of L. (V.) guyanensis in the region (Rangel & Lainson 2009RANGEL, E.F. & LAINSON, R. 2009. Proven and putative vectors of American cutaneous leishmaniasis in Brazil: Aspects of their biology and vectorial competence. Mem. Inst. Oswaldo Cruz 104(7):937-954., Chagas et al. 2018CHAGAS, E.C.D.S., SILVA, A.S., FÉ, N.F., FERREIRA, L.S., SAMPAIO, V.D.S., TERRAZAS, W.C.M., GUERRA, J.A.O., SOUZA, R.A.F. De, SILVEIRA, H. & GUERRA, M.D.G.V.B. 2018. Composition of sand fly fauna (Diptera: Psychodidae) and detection of Leishmania DNA (Kinetoplastida: Trypanosomatidae) in different ecotopes from a rural settlement in the central Amazon, Brazil. Parasites Vectors 11:180. https://doi.org/10.1186/s13071-018-2743-6
https://doi.org/10.1186/s13071-018-2743-...
), but has already been found infected with L. (V.) naiffi DNA in the state of Amapá (de Souza et al. 2017DE SOUZA, A.A.A., DA ROCHA BARATA, I., DAS GRAÇAS SOARES SILVA, M., LIMA, J.A.N., JENNINGS, Y.L.L., ISHIKAWA, E.A.Y., PRÉVOT, G., GINOUVES, M., SILVEIRA, F.T., SHAW, J. & DOS SANTOS, T.V. 2017. Natural Leishmania (Viannia )infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil. Parasite 24:13. https://doi.org/10.1051/parasite/2017016
https://doi.org/10.1051/parasite/2017016...
), demonstrating its likely participation in the cycle of leishmaniasis in the region. This species of Leishmania has been commonly associated with the Ps. squamiventris maripaensis vector in northern Brazil (Naiff et al. 1991NAIFF, R.D., FREITAS, R.A., NAIFF, M.F., ARIAS, J.R., BARRET, T. V., MOMEN, H. & GRIMALDI JÚNIOR, G. 1991. Epidemiological and nosological aspects of Leishmania naiffi Lainson & Shaw, 1989. Mem. Inst. Oswaldo Cruz 86(3):317-321.), French Guiana (Fouque et al. 2007FOUQUE, F., GABORIT, P., ISSALY, J., CARINCI, R., GANTIER, J.-C., RAVEL, C. & DEDET, J.-P. 2007. Phlebotomine sand flies (Diptera: Psychodidae) associated with changing patterns in the transmission of the human cutaneous leishmaniasis in French Guiana. Mem. Inst. Oswaldo Cruz 102(1):35-40. https://doi.org/10.1590/S0074-02762007000100005
https://doi.org/10.1590/S0074-0276200700...
) and Suriname (Kent et al. 2013KENT, A.D., DOS SANTOS, T. V., GANGADIN, A., SAMJHAWAN, A., MANS, D.R.A. & SCHALLIG, H.D.F.H. 2013. Studies on the sand fly fauna (Diptera: Psychodidae) in high-transmission areas of cutaneous leishmaniasis in the Republic of Suriname. Parasit. Vectors 6(1):318.). In Brazil, L.(V.) naiffi has already been reported in other states of the Amazon region such as Pará (Lainson et al. 1981LAINSON, R., SHAW, J.J., READY, P.D., MILES, M.A. & POVOA, M. 1981. Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sandflies, wild mammals and man in north Pará state, with particular reference to L. braziliensis guyanensis causative agent of “pian-bois. Trans. R. Soc. Trop. Med. Hyg. 75(4): 530-536. https://doi.org/10.1016/0035-9203(81)90192-9
https://doi.org/10.1016/0035-9203(81)901...
), Amazonas (Grimaldi et al. 1991aGRIMALDI, G., MOMEN, H., NAIFF, R.D., MCMAHON-PRATT, D. & BARRETT, T. V. 1991a. Characterization and Classification of Leishmanial Parasites from Humans, Wild Mammals, and Sand Flies in the Amazon Region of Brazil. Am. J. Trop. Med. Hyg. 44(6):645-661. https://doi.org/10.4269/ajtmh.1991.44.645
https://doi.org/10.4269/ajtmh.1991.44.64...
) and Acre (Tojal da Silva et al. 2006TOJAL DA SILVA, A.C., CUPOLILLO, E., VOLPINI, A.C., ALMEIDA, R. & SIERRA ROMERO, G.A. 2006. Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil. Trop. Med. Int. Heal. 11(9):1388-1398.).

The Psychodopygus davisi species was found infected with L. (V.) braziliensis. The species is considered a potential vector for this species of Leishmania, having already been found in previous studies in forest environments infected by both L. (V.) braziliensis and L. (V.) naiffi (Grimaldi et al. 1991bGRIMALDI, G., MOMEN, H., NAIFF, R.D., MCMAHON-PRATT, D. & BARRETT, T. V. 1991b. Characterization and Classification of Leishmanial Parasites from Humans, Wild Mammals, and Sand Flies in the Amazon Region of Brazil. Am. J. Trop. Med. Hyg. 44(6):645-661., Gil et al. 2003GIL,L.H.S., BASANO, S.A., SOUZA, A.A., SILVA, M.G.S., BARATA, I., ISHIKAWA, E.A., CAMARGO, L.M.A. & SHAW, J.J. 2003. Recent observations on the sand fly (Diptera: Psychodidae) fauna of the State of Rondônia, Western Amazônia, Brazil: the importance of Psychdopygus davisi as a vector of zoonotic cutaneous leishmaniasis. Mem. Inst. Oswaldo Cruz 98(6):751-755. https://doi.org/10.1590/S0074-02762003000600007
https://doi.org/10.1590/S0074-0276200300...
). This species of sandfly is considered one of the main vectors of the etiological agent of Leishmaniasis, so it should be considered carefully in entomological surveys as it has relevant characteristics such as a high level of anthropophily. In Brazil, the transmission of L. (V.) braziliensis has as potential vectors 17 species of sand flies. In Amapá the DNA of L. (V.) braziliensis was found in other species of sand flies such as Th. ininni Floch & Abonnenc 1943, Ny. umbratilis and Ev. infraspinosa Mangabeira, 1941 (Vasconcelos Dos Santos et al. 2019VASCONCELOS DOS SANTOS, T., DE PITA-PEREIRA, D., ARAÚJO-PEREIRA, T., BRITTO, C., SILVEIRA, F.T., PÓVOA, M.M. & RANGEL, E.F. 2019. Leishmania DNA detection and species characterization within phlebotomines (Diptera: Psychodidae) from a peridomicile-forest gradient in an Amazonian/Guianan bordering area.. PLoS One 14(7):e0219626.).

The sandfly fauna of the eastern Amazon region is still poorly known. In this study we demonstrate the high level of species diversity of sand flies in the state of Amapá, the three collection counties demonstrate a diversity of proven or putative vectors of Leishmania in the region, as well as the detection of three different species of the Leishmania Viannia complex in sandfly species that already have a history of vectors in the literature. Thus, our studies suggest that the sand flies found are acting as vectors in the ATL transmission cycle, as well as indicating a high risk of transmission in the three collection counties of the three main Leishmania species. Thus, it is necessary that measures against ATL transmission be planned for the state of Amapá, based on entomological inventories to monitor the vector arthropod species as well as the species infected by Leishmania spp. that cause ATL in Eastern Brazilian Amazon.

Acknowledgements

We would like to thank the residents of the Mazagão, Porto grande and Serra do Navio counties, who allowed sample collection on their property. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) [Finance Code 001]

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Edited by

Associate Editor Gustavo Graciolli

Publication Dates

  • Publication in this collection
    04 Apr 2022
  • Date of issue
    2022

History

  • Received
    05 Aug 2021
  • Accepted
    28 Feb 2022
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