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Sand flies on Paraná River Islands and natural infection of Nyssomyia neivai by Leishmania in southern Brazil

2016; Wiley; Volume: 41; Issue: 1 Linguagem: Inglês

10.1111/jvec.12211

1948-7134

Barbara Andreo dos Santos, Kárin Rosi Reinhold-Castro, Edilson Colhera Cristóvão, Thaís Gomes Verzignassi Silveira, Ueslei Teodoro,

Trypanosoma species research and implications

Leishmaniasis occurs worldwide and has a great impact on global public health. The geographical distribution and importance of sand flies in the epidemiology of leishmaniasis have guided studies of these insects in order to search for ways to control the sand fly population and prevent Leishmania infection in humans. Changes that occur naturally and anthropogenically cause adaptations of sand fly species in new environments. Mutum Island has had three reported human cases of leishmaniasis (two permanent residents and one temporary resident) and eight reported canine cases (E.C. Cristóvão and N.A. Membrive, personal communication). However, no studies have investigated the fauna, sand fly behavior, and rate of natural infection specifically on islands on the Paraná River. Knowledge of the behavior of sand flies may reveal ways to control these insects, which the human population living on the islands can incorporate into their daily routines. The objectives of this study were to identify the fauna on four islands of the Paraná River and determine the frequency and natural rate of Leishmania infection in sand flies in the woods, peridomiciles, and domiciles. Sand fly collection was performed on Mutum, Bandeira, and Carioca Islands on the Paraná River. These islands are close together and located in the municipality of Porto Rico (53°16’ W and 22°46’ S). Collection was also performed on Fina Island in the municipality of Querência do Norte, approximately 60 km from the other islands. Mutum and Bandeira Islands have ten and five inhabitants, respectively. The four islands are situated in the middle part of the Upper Paraná River in the Northwest Paraná Mesoregion (Figure 1) within the Environmental Protection Area of Islands and Lowlands of the Paraná River. The climate is subtropical humid mesothermal, with hot summers and a tendency toward concentrated rainfall and occasional frosts. The average temperature is above 22°C in the summer and below 18°C in the winter, without a dry season (Aguiar et al. 2007). The Upper Paraná River has a system of rivers with archipelago islands that are largely formed by attaching sidebars (Souza-Filho and Stevaux 1997). Sand fly collection was performed with Falcão traps in June, August, and October, 2012, in several ecotopes of the following islands: Mutum, Bandeira, Carioca, and Fina. In June, two collections were performed on Mutum and Bandeira Islands from 18:00 to 06:00, for a total of 24 h. In August, one collection was performed on Mutum, Bandeira, and Carioca Islands from 18:00 to 23:00 for a total of 5 h. On Fina Island one collection was performed in October, from 18:00 to 06:00, for a total of 12 h. We calculated the hourly mean (HM) number of sand flies captured in each of the ecotypes on all of the islands. Two traps were installed in domiciles on Bandeira Island, three traps in peridomiciles (animal shelters) and three traps in the forest. On Carioca Island, one trap was in an uninhabited domicile, one trap in a peridomicile (abandoned pigsty), and three in the forest. On Mutum Island, one trap was in a domicile, three in a peridomicile (animal shelters), and two in the forest. On Fina Island, one trap was placed in a domicile, two traps in peridomiciles (animal shelters), and five in the forest. The domiciles were about 20 m distant from animal shelters and were about 150 m from the forest. The sand flies were killed with chloroform and placed in tubes containing 80% ethanol for preservation and later identification. The insects were processed (Oliveira et al. 2011) and identified in the Laboratory of Entomology, Universidade Estadual de Maringá. The nomenclature followed Galati (2003) and abbreviations followed Marcondes (2007). The sand flies were conserved in tubes containing isopropanol, with nine to ten females of the same species in each tube, for subsequent DNA extraction. For the detection of Leishmania DNA in sand flies, we performed multiplex PCR, which is independent of the stage, location, and number of parasites in the digestive tract in the sand fly (Perez et al. 1994). DNA extraction was performed according to Oliveira et al. (2011). For every 22 samples extracted, we used a positive control (male sand flies plus 105 promastigotes of L. [V.] braziliensis) and a negative control (male sand flies).Two pairs of primers were used for DNA amplification. The first pair, MP3H (5’-GAA CGG GGT TTC TGT ATG C-3’) and MP1L (5’-TAC TCC CCG ACA TGC CTC TG-3’), amplifies a 70 bp fragment of the conserved region of DNA from the minicircle of the kinetoplast (kDNA) of the Leishmania (Viannia) subgenus (Lopez et al. 1993). The second pair, 5Llcac (5’-GTG GCC GAA CAT AAT GTT AG-3’) and 3Llcac (5’-CCA CGA ACA AGT TCA ACA TC-3’), amplifies a 220 bp fragment from the IVS6 gene region of the cacophony in insects of the genus Lutzomyia (Lins et al. 2002). The second pair of primers were used as an internal control in the reaction, detecting the possibility of interference from the arthropod exoskeleton (Higgins and Azard 1995, Siridewa et al. 1996). The reaction mixture (final volume 25 μl) contained 0.5 μM of each primer (Invitrogen Life Technologies, São Paulo, Brazil), 0.2 mM dNTP (Invitrogen, Carlsbad, CA, U.S.A.), 1 U Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, U.S.A.), 1.5 mM MgCl2, 1× enzyme buffer, and 2 μl DNA. The amplification was performed in a PC thermocycler (Biometra, Germany). The tubes were maintained at 4°C until the amplification analysis. The amplified products were subjected to electrophoresis in agarose gel (Invitrogen, Paisley, Scotland, UK) and stained with 0.1 μg/ml ethidium bromide (10-15 V/cm). For every six samples, we used a positive control (1 pg DNA from L. [V.] braziliensis) and a negative control (water). The presence of bands was observed in a transilluminator (L-PIX-HE, Loccus Biotecnologia, São Paulo, Brazil). We collected a total of 19,818 specimens of Nyssomyia neivai (=Lutzomyia neivai) (of which 16,746 were females and 3,072 were males), two female specimens of Nyssomyia whitmani, and one female specimen of Psathyromyia shannoni. The largest number of specimens of Ny. neivai (HM 489.00) was captured on Mutum Island, especially on a chicken roost tree (peridomicile) (HM 408.14). Of the total Ny. neivai that were captured on Bandeira Island (HM 134.59), the majority (HM 71.83) were also captured in a chicken roost tree (peridomicile). On Carioca Island, the specimens of Ny. neivai were captured mainly at the forest edges (HM 134.59) and in an abandoned pigsty (HM 67.00). On Fina Island, the specimens of Ny. neivai were captured mainly in a domicile (HM 16.66), a henhouse (HM 23.66), and at the forest edge (in only one of the five traps in the forest; HM 23.50). The largest number of specimens (HM 529.91) was collected in chicken roost trees and henhouses, especially in the chicken roost tree on Mutum Island. Ny. neivai predominates in several municipalities in Paraná, mainly in domestic animal shelters (Teodoro et al. 2006, Reinhold-Castro et al. 2008). The predominance of Ny. neivai is also observed in municipalities where the soil is well-drained and sandy as soils derived from the Caiuá sandstone (Teodoro et al. 2006, Reinhold-Castro et al. 2008). Only three species, Ny. neivai, Ny. whitmani, and Ps. shannoni, were collected and identified on the islands in the present study. Specimens of Ny. neivai and a single Br. cunhai female were previously reported on Mutum Island (J.C. Gasparotto et.al., unpublished data). The predominance of Ny. neivai (99.9%) suggests that this species is very well adapted to the islands on the Paraná River, where it finds a favorable breeding environment. This species may have migrated from the riparian margins of this river in the last phase of island formation (Aguiar et al. 2007, Leli et al. 2013), which for Mutum Island occurred about 8,200 years ago (Leli et al. 2013). A small number of species was collected in relation to the 49 described species in the state of Paraná (Aguiar and Medeiros 2003, Galati et al. 2007, Silva et al. 2008, Cruz et al. 2012, 2013, Santos et al. 2012), possibly because of the isolation of the islands and inability of other sand fly species to adapt to the insular environment. Nyssomyia neivai is a species well adapted to the forest edges and modified environments and may develop around domiciles and invade them (Teodoro et al. 2006). This species is present in the southern region, southern coast, western São Paulo, southern and western Minas Gerais, southern Goiás, and southern Pará in Brazil, as well as in Argentina, Bolivia, and Paraguay (Andrade-Filho et al. 2007). A total of 378 females (38 pools) of Ny. neivai was submitted to multiplex PCR. The minimum infection rates in Ny. neivai was 0.26% or 1/378 analyzed females, considering that one sand fly was infected in each positive pool (Figure 2). Autochthonous cases of CL have been reported in the Doutor Camargo municipality, and the detection of Ny. neivai with natural Leishmania (Viannia) sp. infection demonstrate the importance of this species in the epidemiology of CL (Oliveira et al. 2011, Neitzke-Abreu et al. 2013). Studies of the natural infection of sand flies show that the number of infected insects may be low because of the influence of biotic factors such as the population size of mammal reservoirs, rate of infection, and barriers to reproduction of the parasite in the digestive tract in the insect vector (Pimenta et al. 2003). Córdoba-Lanús et al. (2006), Marcondes et al. (2009), Pita-Pereira et al. (2009), and Oliveira et al. (2011) have found Ny. neivai naturally infected with Leishmania using PCR, in the Argentina and Brazilian states of Santa Catarina, Rio Grande do Sul, and Paraná. They reported different infection rates of 9.1% in 440 females, 13.1% in 562 females, 1.11% in 270 females, and 0.23% in 1,755 females, respectively. Despite the low diversity of sand fly species on Paraná River Islands, the frequency of these insects in woods, peridomiciles, and domiciles and detection of Leishmania in Ny. neivai show the existence of the enzootic cycle of this parasite in island environments. The behavior of sand flies needs to be investigated on Paraná River islands, given that these islands are frequented by permanent residents and tourists and such results may guide public health services to implement entomological surveillance. The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their financial support. To José Luiz Filho, Valmir Ortiz da Silva and José do Porto dos Santos from the Núcleo de Entomologia de Porto Rico for their assistance in collecting sand flies.To Herintha Coeto Neitzke-Abreu for aid in the drawing of Figure 1, performance of multiplex PCR, and critical revision of the article. To Thais Regina Ranucci for aid in identifying sand flies.