Bat trypanosomes from Tapajós-Arapiuns Extractive Reserve in Brazilian Amazon Tripanossomas de morcegos da Reserva Extrativista Tapajós-Arapiuns na Amazônia Brasileira

Trypanosoma comprises flagellates able to infect several mammalian species and is transmitted by several groups of invertebrates. The order Chiroptera can be infected by the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon. In this study, we described the diversity of bat trypanosomes and inferred phylogenetic relationships among the trypanosomes from bats caught in Tapajós-Arapiuns Extractive Reserve (Resex) in Pará state. Trypanosomes from bats were isolated by means of hemoculture, and the molecular phylogeny was based on the trypanosome barcode (SSUrDNA V7V8 variable region). A total of 111 bats were caught in the area, belonging to three families (Emballonuridae, Molossidae and Phyllostomidae) and 12 species. The bat trypanosome prevalence, as evaluated through hemoculture, was 9% all positive cultures were cryopreserve (100% of isolation success). Phylogenetic trees grouped nine isolates in T. cruzi marinkellei branch and only one in T. dionisii branch. Studies on bat trypanosome diversity are important for identifying pathogenic species and for generating support for control measures, especially in such areas where humans inhabit the forest with close contact with bat species. In addition, this is the first study in Resex Tapajós-Arapiuns extractive reserve and further studies should be conducted to elucidate the role of these parasites as environmental degradation biomarkers.


Introduction
The genus Trypanosoma comprises protozoan flagellates that are able to infect several vertebrate species and are transmitted by several groups of invertebrates.More than 30 trypanosome species belonging to the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon have been described in bats (order Chiroptera) (HOARE, 1972;MARINKELLE, 1976).
The Most descriptions of the species of trypanosomes of bats are based on host of origin and morphological characters.Phylogenetic studies on trypanosomes of bats are scarce and only T. dionisii, T. vespertilionis, Trypanosoma sp.bat (isolated in Africa) and T. cruzi marinkellei were included in phylogenetic trees (STEVENS & GIBSON, 1999;STEVENS & RAMBAUT, 2001;HAMILTON et al., 2007).Recently, T. erneyi and T. livingstonei from African bats and T. teixeirae from flying fox in Australia were described and positioned in phylogenetic trees (LIMA et al., 2012(LIMA et al., , 2013;;BARBOSA et al., 2016).
The subgenus Schizotrypanum comprises the species Trypanosoma erneyi and T. dionisii, which only infect bats, and T. cruzi marinkellei, which has the ability to infect many orders of mammals (MARINKELLE, 1976;LIMA et al., 2012).
T. dionisii is transmitted by bugs of the family Cimicidae, which are widely distributed, while T. cruzi marinkellei is transmitted by bugs of the family Reduviidae (Cavernicola pilosa) and only occurs in the Americas.In Brazil, the studies conducted have found T. cruzi (TCI and TcBat group), T. cruzi marinkellei and T. dionisii through hemoculture evaluations (MARCILI et al., 2009a;CAVAZZANA et al., 2010).
Trypanosoma dionisii has been detected in the bat families Phyllostomidae, Molossidae, Vespertilionidae and Noctilionidae in all biomes analyzed, from northern to southern Brazil, while T. cruzi marinkellei has been found in phyllostomid and vespertilionid bats in northern, central and southeastern areas and T. cruzi in the bat families Phyllostomidae, Vespertilionidae, Noctilionidae and Thyropteridae in the Amazon, Pantanal and Atlantic rainforest biomes (COSTA et al., 2016(COSTA et al., , 2015;;ACOSTA et al., 2014;MARCILI et al., 2009a;CAVAZZANA et al., 2010).Additionally, Trypanosoma erneyi has been described in bats in Africa (LIMA et al., 2012).
There are few studies on Trypanosoma in wild mammals or vectors in the state of Pará.Trypanosoma cruzi lineages have been described in marsupials, rodents, carnivores, armadillos and triatomines (MARCILI et al., 2009a, b).New species of Trypanosoma were described recently in the bat genus Pteronotus in the state of Rondônia and in blood from bats in Pará (LIMA et al., 2015).In Altamira, T. cruzi, T. cruzi marinkellei and T. wauwau were isolated (COSTA et al., 2016).The Tapajós-Arapiuns Resex is a protected area designated for sustainable use of natural resources with difficult access with humans inhabit small villages inside the Amazon rainforest, with close contact with bats and limited information regarding trypanosome infection.
In the present study, we inferred phylogenetic relationships among the trypanosomes from bats caught in the Tapajós-Arapiuns Resex area, based on SSU rDNA sequences.Two species were described: T. cruzi marinkellei and T. dionisii from phyllostomid bats.

Study areas and bats caught
The bats were caught in the Tapajós-Arapiuns extractive reserve (Resex), mostly located in municipality of Santarem, in the Pará state (02º20'-03º40' S; 55º00'-56º00' W) on the left bank of the Tapajós river (Figure 1).The entire area of the Tapajós-Arapiuns Resex (677,513.24ha) is included in the Amazon biome (annual average temperature 27 °C; annual precipitation greater than 2400 mm).More than 3000 families inhabit the area of the Tapajós-Arapiuns Resex and conduct extractive activities consisting of fishing and hunting for their own consumption (ICMbio, 2014).
Bat-catching was undertaken in each area for five nights in April 2015.Six mist nets (7 × 3 m) were set up from 18:00 to 23:00 h and were checked every 30 min for the presence of bats.Artificial shelters on the roofs of schools were also set up and checked.The bats caught were anesthetized and blood samples were collected by means of heart puncture.
The animals were caught and manipulated in accordance with the recommendations of the Brazilian Institute for the Environment and Renewable Natural Resources and Chico Mendes Institute for Biodiversity Conservation (IBAMA-ICMBio) and the procedures used were approved by the Animal Research Committee of the University of Santo Amaro.

Isolation of bat trypanosomes
The blood samples were collected by means of heart puncture and were inoculated into vacutainer tubes containing a biphasic medium (15% sheep red blood cells with 4% blood agar base) and liquid LIT medium supplemented with 20% FBS (COSTA et al., 2016).The culture was incubated at 28 °C, and the isolates were cryopreserved in liquid nitrogen in the Brazilian Trypanosomatid Collection (Coleção Brasileira de Tripanossomatídeos, CBT), in the Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, Brazil.No primary samples (blood samples) were obtained to molecular diagnosis.The most caught bats were released and the amount of blood collected was prioritized for isolation.

Molecular and phylogenetic analysis
Samples of DNA from the trypanosome cultures were extracted using the phenol-chloroform method.The V7V8 barcode region of the SSU rDNA gene was amplified by means of a conventional polymerase chain reaction (PCR), as previously described (FERREIRA et al., 2008;VIOLA et al., 2008).PCR products of the expected size were purified by means of Exosap-IT (Affymetrix) and were sequenced in an automated sequencer (Applied Biosystems/PerkinElmer, model ABI Prism 3500 Genetic, Foster City, California, USA), in accordance with the manufacturer's recommendations.
The sequences obtained were aligned with sequences that had previously been determined for other Schizotrypanum species available in GenBank, using ClustalX (THOMPSON et al., 1997), and were adjusted manually using GeneDoc (NICHOLAS et al., 1997).
The phylogenetic tree was constructed using maximum parsimony (MP) and Bayesian analysis (B).MP was implemented in PAUP version 4.0b10 (SWOFFORD, 2002) with 500 bootstrap replicates, random stepwise addition starting trees (with random addition sequences) and TBR branch swapping.Bayesian analysis was performed through MrBayes v3.1.2 (HUELSENBECK & RONQUIST, 2001) with 1,000,000 MCMC generations.The first 25% of the trees represented burn-in, and the remaining trees were used to calculate Bayesian posterior probability.

Results
A total of 111 bats were caught in the area, belonging to three families (Emballonuridae, Molossidae and Phyllostomidae) and 12 species (Table 1).The bat trypanosome prevalence, as evaluated through hemoculture, was 9%.Ten positive hemocultures were  obtained, only from phyllostomid bats, and all of the cultures were established and cryopreserved in CBT (Table 2).The morphology of the cultured forms and the biological behavior in culture media were similar to those of the subgenus Schizotrypanum in all isolates from phyllostomid bats.
Phylogenetic relationships based on the trypanosome barcode were inferred by means of maximum parsimony and Bayesian analysis, and congruent topologies were generated for Schizotrypanum trypanosomes (Figure 2).The subgenus Schizotrypanum is a monophyletic group (100% of similarity of sequences, 100%  ) and grouped with a reference strain (B7) of T. cruzi marinkellei (99% of similarity of sequences, 100% bootstrap and 1.0% Bayesian posteriori probability) (Figure 2).One isolate, CBT214 from Glossophaga soricina, was identified as on the T. dionisii branch with other sequences from Brazilian T. dionisii isolates (98% of similarity of sequences, 100% bootstrap and 1.0% Bayesian posterior probability) (Figure 2).
The biomes with the highest prevalences of bat trypanosomes in Brazil are the Amazon biome with 45.2% according to microhematocrit and hemoculture (CAVAZZANA et al., 2010) and the Amazon/Cerrado biome with 15.5% according to hemoculture (MARCILI et al., 2013).The prevalence found in the Tapajós-Arapiuns Resex in the present study (9%) was low in comparison with other areas and studies.
The most isolates from Resex were positioned with T. cruzi marinkellei and only one isolate with T. dionisii isolates.In different studies of bat trypanosomes conducted in Brazil, T. cruzi marinkellei predominated, followed by T. dionisii and T. cruzi.Furthermore, T. dionisii and T. cruzi were isolated in all areas studied; meanwhile, T. cruzi marinkellei was not detected in southern or southeastern Brazil, except in Espirito Santo state (CAVAZZANA et al., 2010;ACOSTA et al., 2014).
The absence of T. cruzi isolates from the bats trypanosomes isolated from this study does not exclude their occurrence in the region.The diversity of bats species and trypanosomes associated of bats can be increase in new studies with more days, different area in Resex and sazonal variation.
Studies on diversity and associated parasites may provide information about the biology of hosts and host-parasite-environment relationships (GALLI et al., 2001;MADI & UETA, 2012).Neotropical bats are excellent quality indicators for different ecosystems (MEDELLÍN et al., 2000;WILLIG et al., 2007).
Parasites generally respond to environmental changes faster than their hosts.This provides a means of assessing environmental stress levels and creating a density regulator for the populations of their hosts.This has a great impact on the environment and on animal communities (SILVA-SOUZA et al., 2006).
Upward or downward variations in the amount of parasitism may be an indicator of human actions within the environment that can positively or negatively influence parasite development (LAFFERTY & KURIS, 1999).In Panamá, habitat fragmentation has increased the prevalence of Trypanosoma in the bat Artibeus jamaicensis caught in forest fragments, compared with those caught in areas of continuous forest (COTTONTAIL et al., 2009).
Studies aiming towards isolation and molecular characterization of bat trypanosomes in areas that have never previously been studied are extremely important for understanding the diversity of parasites in bats, as well as for identifying pathogenic species and generating support for control measures.In addition, studies should be conducted to confirm the role of these parasites as environmental degradation biomarkers.

Table 1 .
Hosts species and haemoculture positivity of bats examined in this study.

Table 2 .
Trypanosome isolates, host and geographic origin and sequences of SSU rDNA used for phylogenetic analysis.