Phylogenetic Analysis of Bolivian Bat Trypanosomes of the Subgenus Schizotrypanum Based on Cytochrome b Sequence and Minicircle Analyses

The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed.


Introduction
Short-tailed bats of the genus Carollia are widely distributed in the New World tropics. Also, there are detailed altitudinal records from the Peruvian Andes and samples of the three South American species found on both sides of the Andes are available, allowing testing of models of diversification across the Andes. Given the ability to fly, it would be expected dispersal might be expected to play a stronger role than vicariance in shaping bat phylogeographical patterns of variation [1]. Although Artibeus, Carollia, and Glossophaga generally feed on plant sources, it is clear that they also frequently consume significant quantities of insects [2]. Bats play a crucial role in tropical ecosystems by dispersing seeds, pollinating flowers, and controlling insect populations. C. perspicillata may be considered as understorey specialists (from 0-2.5 m high). The short-tailed fruit-eating bats, C. perspicillata and C. brevicauda, feed primarily on understorey plants such as Piper, Solanum and Vismia [3]. Roosting habits of these bats are caves, abandoned mine and rail tunnels, active road tunnel, hollow trees, drain pipes and culverts, unused/abandoned buildings or rooms, attics, basements, under bridges, unused cisterns, darkened recesses in rock formations or stream banks. Although there is limited field and experimental evidence, haematophagous arthropods can act as vectors of trypanosomes among bats [2]. Trypanosomes (genus Trypanosoma) are widespread blood parasites of vertebrates, usually transmitted by arthropod or leech vectors. Most trypanosome-infected bats are insectivorous and infection could also occur through the ingestion of infected arthropods. Bats are long-lived species and infections persist for years, with trypanosomes localising in skeletal, cardiac and stomach muscle cells [4,5]. Variable prevalence of trypanosomes in bats has been reported in surveys conducted throughout the world. In South American bats, prevalence varied widely. Colombian bats had a prevalence of approximately 9.0% infected with Schizotrypanum spp. [6,7]. Surveys performed in the Amazonia of Brazil; detected trypanosomes prevalence of 2.4-4.6%, by means of blood smears [8,9]. The strong association between Chiroptera order and all Schizotrypanum spp. suggests a long shared evolutionary history. Trypanosomatids parasitize many vertebrate and invertebrate phyla. Several trypanosome species are agents of disease in humans and/or livestock particularly in the tropics. For example, Trypanosoma brucei causes human African trypanosomiasis or sleeping sickness, while Trypanosoma cruzi causes Chagas disease in South and Central America. There is also strengthened support for two deep clades, one comprising a wide selection of mammalian trypanosomes and a tsetse fly-transmitted reptilian trypanosome, and the other combining two bird trypanosome subclades. Most clades are associated with a type of vertebrate or invertebrate host, or both, indicating that 'host fitting' has been the principal mechanism for evolution of trypanosomes [10]. The type species of the subgenus Schizotrypanum is T. cruzi, which infects man and a wide variety of mammalian hosts. Six different T. cruzi lineages have been described, named TcI-TcVI [11] . In the southern  cone of South America, isolates from humans and vectors of  domestic and peridomestic transmission cycles are predominantly  of lineages TcII,Tc V and Tc VI. Tc I and Tc bat have been  reported in the sylvatic cycle throughout Latin America (Tc I  present in bat genus such as Thyroptera, Carollia and Tc bat in  Myotis, Noctilio). Tc I predominantly infects humans in endemic areas northwest of the Amazon basin [12]. In contrast, all other species traditionally classified as Schizotrypanum are restricted to bats. Trypanosoma cruzi marinkellei is indigenous to South and Central America, and restricted to bats [13]. T. c. marinkellei is, apparently, only transmitted by triatomines of the genus Cavernicola, which is found associated with bat colonies in caverns, hollow trees and palms [6,5]. Also strains of Trypanosoma vespertilionis and Trypanosoma dionisii from European bats have been distinguished from other Schizotrypanum species [13,14]. T. dionisii, T.c. marinkellei and T. cruzi, belonging to the subgenus Schizotrypanum, can invade mammalian cells. These Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry [15]. Anti -T. dionisii monoclonal antibodies were tested against various strains of T. dionisii, T. vespertilionis, T. cruzi and T. c. marinkellei. The cross reactions between T. dionisii and T. cruzi demonstrate a strong correlation between T. dionisii and TcII-TcVI. Similarly TcI and T. c. marinkellei show very similar antigenic pattern [16]. The subgenus Schizotrypanum includes several trypanosome species that are difficult to discriminate by morphological examination [17]. Molecular phylogenetic data based on the SSU rRNA indicated that the broad host-range trypanosome Trypanosoma rangeli and the rat trypanosome Trypanosoma cornohini should also be reclassified in the subgenus Schizotrypanum [18]. T. rangeli are kinetoplastid protozoa which have been largely recognized and defined in several Latin American countries in relation to T. cruzi, because the two trypanosome species are frequently found in mixed infections in triatominae vectors, humans and a variety of wild and domestic mammals [19].
Trypanosomes are protozoa belonging to the Kinetoplastida order. The characteristic of this order is a highly unusual, concatenated mitochondrial DNA structure, the kinetoplast DNA (kDNA). Two types of DNA molecules are present, the maxicircles and minicircles. The maxicircles are 22,000 to 33,000 bp in size; they encode mitochondrial proteins. Along with other mitochondrial genes cytochrome b (cytB) are present in 10 to 20 identical copies. The cytB genes are transcribed but they suffer a posttranscriptional modification at the 59end called editing, in which the mature messenger RNA changes its sequence by multiple insertions and deletions of uridines [20]. In contrast, minicircles are highly heterogeneous in nucleotide sequence; however, the size of minicircles is virtually conserved in T. cruzi populations [21]. Restriction endonuclease and sequence analyses showed that a T. cruzi minicircle is composed of 4,3,2 or 1 conserved regions of approximately 100 to 150 bp that contain 3 hyper conserved sequence blocks used as universal probes, which are flanked by variable regions with sequences that diverge almost completely as determined in T. cruzi and T. rangeli [22]. No information is available about trypanosomes minicircles size circulating in bats.
With the goal to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats, we determined the nucleotide sequence of a portion of the cytB gene and characterized the size of the minicircle variable region in trypanosome stocks isolated from Amazonian bats of Bolivia. We include in this work T. cruzi and Schizotrypanum stocks available information of the cytB in GenBank from Brazilian bats for comparative purposes.

Origin of the Stocks and Ethics Statement
Bats were captured and manipulated using nets and procedures permitted by the Viceministerio de Medio Ambiente, Biodiversidad, Cambios Climáticos y Gestión y Desarrollo Forestal of Bolivia.
Peripheral blood samples were taken from all bats through xenodiagnosis to further culture in NNN agar medium for T.c. marenkellei isolation and haemoculture for T.dionisii. All bats were analyzed by xenodiagnosis using five nymphs each of the species R. robustus and T. infestans. All positive isolates were finally cloned [23] and harvested by centrifugation in the log phase. DNA purification was performed using the High Pure DNA preparation kit from ROCHE according to the manufacturer instructions.

PCR Amplification and Analysis of Cytochrome b Sequences
PCR amplification and sequencing of the partial sequences (< 516 bp) of cytB from eighteen bat isolates was performed as described previously. The primers used for amplification of the 59 half of cytB were: p18 (59-GACAGGATTGAGAAGCGAGA-GAG-39) and p20 (59-CAAACCTATCACAAAAAGCATCTG-39). Reaction conditions were the same as described before. [24]. Thirty-five cycles (94uC, 1 min; 50uC, 30 s; 72uC, 90 s) followed by a final elongation step (5 min, 72uC) were performed. Sequence determination of PCR products was carried out with the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) on an ABI-373 Automated DNA Sequencer. Sequences of bat trypanosomes derived from this study were aligned with sequences determined in previous studies of bat T. cruzi stocks, other trypanosomes isolated from bats and T. rangeli available in GenBank [24,25]. Sequences obtained from this work have accession numbers JN651278 to JN651295. Reference sequences used for tree construction are the following: FJ900248, FJ002262, FJ900247, AJ130927, AJ130932, AJ130933, EU856368, AJ439725, AJ439721, FJ555642, FJ555651, FJ002261, FJ002258, AJ130938, FJ549392, FJ555639, FJ900255, FJ002263 and FJ900249. Sequences Tcm B3 and Tcm B34 were provided directly by Dr. S. Brisse [24]. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories). All positions containing gaps and missing data were eliminated. There were a total of 382 positions in the final dataset. Alignments were made using ClustalW and manually refined. Phylogenetic analysis was performed using maximum likelihood (ML) method (using the Kimura two-parameter model) listed in the MEGA 5.05 analytical package.

Minicircle PCR Assay
The amplification reactions were performed in triplicate with oligonucleotides 121 (59-AAATAATGTACGGGT/GGAGATG-CATGA-39) and 122 (59-GGTTCGATTGGGGTTGGTGTAA-TATA-39), which anneal to the four conserved regions present in trypanosomes minicircles [26]. The DNA samples for PCR were boiled for 15 minutes and 5 ml of supernatant was used as DNA template in 50 ml final volumen [22]. Each experiment included a negative control that contained water instead of DNA and a positive control that contained purified DNA of T. cruzi. The PCR products were analyzed by electrophoresis in 2% agarose gels and visualized by staining with ethidium bromide.

Results
A total of 115 bats were caught in three Amazonian areas of Chapare Province, Bolivia [ Fig. 1]. All 22 bats from San Cristobal, belongs to Carollia perspicillata species. From 24 bats found at Ivirgarzama, 2 were Desmodus rotundus, 2 Glossophaga soricina and 20 C. perspicillata. In Guacharos, 68 bats were found; 6 Platyrrhinus helleri, 14 Desmodus rotundus and 48 C. perspicillata. All 18 trypanosomes isolated in this study were recovered from bats belonging to C. perspicillata the most abundant species in the area (78%). Tc. marinkellei was present in bats from San Cristóbal (5), Ivirgarzama (4) and Guacharos (1). T. dionisii was isolated only from bats caught in Ivirgarzama (1) and Guacharos (7) [ Table 1]. Isolates of T. dionisii were obtained only by haemoculture, while T.c. marinkellei isolates were obtained by haemoculture, and xenodiagnosis, showing that these species were able to establish infection in triatomines of the genus Rhodnius, the endemic triatomine species in the studied Amazonian biodeme. There was no positive xenodiag- nosis when T. infestans was used. The comparison of sequences from Bolivian bat isolates with those previously studied from Brazil allowed the classification of the isolates in two species, T. c. marinkellei and T. dionisii; both equally frequent in infected bats. Ten trypanosome isolates (24, 26, 27, Fig. 2]. This figure also shows phylogenetic relationships between T. cruzi clones and T. cruzi from bats (Tc bat), including the T. rangeli San Agustín stock (FJ 900255). The total DNA of each bat trypanosome isolates and T. cruzi clone was used as a template for minicircle PCR amplification with the universal primers 121 and 122 which align in the hyper conserved sequences present in all trypanosomes. The T. cruzi clones belonging to all DTUs (Tc I-TcVI) displayed a unique band close to 330 bp, while the PCR of a T. rangeli isolate generated bands close to 330 and 380 bp [ Fig. 3]. However, isolates 80, 82, 84 belonging to T. c. marinkellei and 24, 26, 27, 79, Tcm 1909, Tcm B3 (not shown) displayed a different pattern of amplicons close to 280 and 350 bp. Other bat isolates belonging to T. dionisii, on the contrary, displayed a unique band around 600 bp. Results not shown indicate the smaller size of the cultured forms of the Bolivian T. dionisii isolates compared to those of T. c. marinkellei.

Discussion
We show in this work that T. cruzi is different from T.c. marinkellei and T. dionisii in the minicircle variable region size. We used the faster-evolving gene cytB to investigate further the genetic distinctness and phylogenetic relationships among Schizotrypanum taxa. Phylogenetic relationships among cytB sequences have recently been shown to be congruent with rRNA promoter sequence data [27]. CytB phylogenetic analysis fully supported the high distinctness among T. rangeli, T. dionisii, T. cruzi and T. c. marinkellei. Bolivian trypanosomes from bats, studied here, were grouped as T. c. marinkellei or T. dionisii, in contrast to Brazil, which also includes the T. cruzi lineages TcI, TcII and TcIII (present in bat genus such as Carollia, Myotis, Noctilio and Thyroptera) [25], and T. rangeli as described [28,29]. All taxa appeared to be roughly equidistant in our analysis, with the exception of T. cruzi and T. c. marinkellei, which appeared to be more closely related, as described for Brazilian bat trypanosomes. However the Bolivian T. c. marinkellei are genetically distant from the Brazilian ones, indicating that trypanosomes of this species are genetically heterogeneous as described [24]. Most of bats studied here belong to the family Phyllostomidae, which exhibit varied alimentary habits, including insectivorous, therefore this represents the probable infection route by feeding of Cavernicola triatomines. A common ancestry of T. cruzi and T.c.marinkellei was suggested by 18S rRNA data [18] and phylogenetic analyses demonstrated that Tc bat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances Tcbat is closest to lineage Tc I [12].
In the present study, comprising a survey for bat trypanosomes in an Amazonian biome of Bolivia, the majority of cultures were identified as T.c. marinkellei and T. dionisii based on cytB gene. The prevalence of T.c. marinkellei was 9.0% and 7% of T. dionisii. The results strongly supported the suitability of this sequence for analysis of phylogenetic relationships among Schizotrypanum, as previously demonstrated for other clades of trypanosomes from mammals [30].
Phylogenetic relationships inferred using ssrRNA, gGAPDH and cytB generated trees with similar topologies and were also congruent with results based on cytB sequences. Three major clades of bat trypanosomes within the subgenus Schizotrypanum were strongly supported in all phylogenies regardless of data sets and analytical methods in which the clade containing T. cruzi was closer to that containing T.c. marinkellei than to T. dionisii. No other species of Schizotrypanum besides these species before mentioned were isolated from bats in this study, suggesting that other species of this subgenus are rare in this area of Bolivia and/or difficult to cultivate. Closest to the T. cruzi clade is T. rangeli, another American trypanosome of wild mammals also transmitted by triatomine bugs but rarely found in bats, except in Brazil. Only two cultures of T. rangeli from bats have been confirmed using morphological, biological and molecular parameters [29]. Phylogeographical, ecological and biological analyses of isolates classified as Schizotrypanum disclosed some patterns of association with bat species, biomes and geographic origin, as well as with their behavior in culture, triatomine bugs and mice. Our results show overlapping geographic areas of the two Schizotrypanum species in the Amazonia of Bolivia. T. c. marinkellei was found in bats from phyllostomid species (insectivorous, frugivorous) corroborating a strong association with this bat family, as suggested previously [6]. However, bats of this family were also infected by T. dionisii as shown in this and other studies [31]. The prevalence of T. c. marinkellei may be explained by the abundance of phyllostomid bat, whereas its distribution may be determined by that or its triatomine vector, Cavernicola pillosa, which shares caves, holes in trees, and palm leafs with bats.
We have demonstrated the existence in Bolivia of T. dionsii, another trypanosome found in neotropical bats. However the scarcity of T. dionisii in two areas studied here can be explained by the abundance and distribution of its vectors. The genetic distances of bat trypanosomes provided by this study are better explained by the ability of bats to disperse over large areas, crossing oceans and continents rather than by vicariance events. The reconstruction of the evolutionary histories of parasites has been linked to the comparable histories of their host. Phylogenetic and biogeographic analyses have suggested that Africa is the centre of origin of modern-day bat families, with a Southern Hemisphere origin in the Cretaceous. Two scenarios could account for the dispersal of bats from Africa in the Eocene: northwards dispersal to Eurasia and via Beringia into America or transatlantic dispersal from Africa to America through island hopping or direct flight [32,33].
The estimates of divergence time based on nuclear and mitochondrial genes suggested that T. cruzi may have evolved from bat-restricted trypanosomes 10-20 mya [34]. Limited divergence among Schizotrypanum spp. is compatible with recent diversification, and their present day distribution is equally consistent with hypotheses that T. cruzi evolved from a batrestricted trypanosome or vice versa [18,24].
Comparative analyses performed in this study showed that the morphology of blood and axenic culture forms (data not shown) and minicircle variable region size should be considered as the preliminary parameters to assign trypanosomes to the subgenus Schizotrypanum. A broad phylogeographical analysis including to determine the abiotic factors affecting the distribution patterns of flora and fauna, to compare the adaptations of organisms to different environmental conditions, to explain the historical and geographical reasons that determine the distribution of an organism in space and time, to evaluate the biological interactions that affect the distribution pattern of organisms, to recognize pattern of distribution of bat trypanosomes at the regional and global from Africa, Europe and America, is still required to understand the evolutionary history of Schizotrypanum and bat trypanosomes in general.