C. Miguel Pinto

TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification.

We report TcBat, a recently described genetic lineage of Trypanosoma cruzi, in fruit-eating bats Artibeus from Panama. Infections were common (11.6% prevalence), but no other T. cruzi cruzi genotypes were detected. Phylogenetic analyses show an unambiguous association with Brazilian TcBat, but raise questions about the phylogenetic placement of this genotype using the 18S rRNA gene alone. However, analyses with three concatenated genes (18S rRNA, cytb, and H2B) moderately support TcBat as sister to the discrete typing unit (DTU) TcI. We demonstrate that short fragments (>500 bp) of the 18S rRNA gene are useful for identification of DTUs of T. cruzi, and provide reliable phylogenetic signal as long as they are analyzed within a matrix with reference taxa containing additional informative genes. TcBat forms a very distinctive monophyletic group that may be recognized as an additional DTU within T. cruzi cruzi.

Evolution of the Sweet Taste Receptor Gene Tas1r2 in Bats

Taste perception is an important component of an animals fitness. The identification of vertebrate taste receptor genes in the last decade has enabled molecular genetic studies of the evolution of taste perception in the context of the ecology and dietary preferences of organisms. Although such analyses have been conducted in a number of species for bitter taste receptors, a similar analysis of sweet taste receptors is lacking. Here, we survey the sole sweet taste-specific receptor gene Tas1r2 in 42 bat species that represent all major lineages of the order Chiroptera, one of the most diverse groups of mammals in terms of diet. We found that Tas1r2 is under strong purifying selection in the majority of the bats studied, with no significant difference in the strength of the selection between insect eaters and fruit eaters. However, Tas1r2 is a pseudogene in all three vampire bat species and the functional relaxation likely started in their common ancestor, probably due to the exclusive feeding of vampire bats on blood and their reliance on infrared sensors rather than taste perception to locate blood sources. Our survey of available genome sequences, together with previous reports, revealed additional losses of Tas1r2 in horse, cat, chicken, zebra finch, and western clawed frog, indicating that sweet perception is not as conserved as previously thought. Nonetheless, we found no common dietary pattern among the Tas1r2-lacking vertebrates, suggesting different causes for the losses of Tas1r2 in different species. The complexity of the ecological factors that impact the evolution of Tas1r2 calls for a better understanding of the physiological roles of sweet perception in different species.

Taxonomic revision of the olingos (Bassaricyon), with description of a new species, the Olinguito

Abstract We present the first comprehensive taxonomic revision and review the biology of the olingos, the endemic Neotropical procyonid genus Bassaricyon, based on most specimens available in museums, and with data derived from anatomy, morphometrics, mitochondrial and nuclear DNA, field observations, and geographic range modeling. Species of Bassaricyon are primarily forest-living, arboreal, nocturnal, frugivorous, and solitary, and have one young at a time. We demonstrate that four olingo species can be recognized, including a Central American species (Bassaricyon gabbii), lowland species with eastern, cis-Andean (Bassaricyon alleni) and western, trans-Andean (Bassaricyon medius) distributions, and a species endemic to cloud forests in the Andes. The oldest evolutionary divergence in the genus is between this last species, endemic to the Andes of Colombia and Ecuador, and all other species, which occur in lower elevation habitats. Surprisingly, this Andean endemic species, which we call the Olinguito, has never been previously described; it represents a new species in the order Carnivora and is the smallest living member of the family Procyonidae. We report on the biology of this new species based on information from museum specimens, niche modeling, and fieldwork in western Ecuador, and describe four Olinguito subspecies based on morphological distinctions across different regions of the Northern Andes.

INFECTION BY TRYPANOSOMES IN MARSUPIALS AND RODENTS ASSOCIATED WITH HUMAN DWELLINGS IN ECUADOR

Small mammals trapped in domestic and peridomestic environments of rural Ecuador were screened for trypanosome infection by direct microscopy and hemoculture. Identification of species of trypanosomes was then performed by morphological characteristics and by polymerase chain reaction (PCR) assays. Of 194 animals collected, 15 were positive for infection (7.73%). Eight (4.12%) were infected with Trypanosoma cruzi (1 of 33 Didelphis marsupialis; 7 of 61 Rattus rattus). Eleven R. rattus (18.03%) harbored T. lewisi, 5 of which presented mixed infections with T. cruzi. Additionally, 1 of 3 Oryzomys xanthaeolus was infected with T. rangeli. No trypanosome infection was detected in Philander opossum (n = 1), Mus musculus (n = 79), Rattus norvegicus (n = 8), Akodon orophilus (n = 4), Sigmodon peruanus (n = 3), or Proechimys decumanus (n = 2). Many of the isolates belong to T. cruzi, the causative agent of Chagas disease, and R. rattus had the highest prevalence. Because of its abundance in the study areas, this species is considered an important reservoir for Chagas disease. This is the first report of T. lewisi and T. rangeli in Ecuador. This study is also the first to describe natural mixed infections of T. cruzi–T. lewisi.

Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit).

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI-TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI-Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU.

Bats, Trypanosomes, and Triatomines in Ecuador: New Insights into the Diversity, Transmission, and Origins of Trypanosoma cruzi and Chagas Disease

The generalist parasite Trypanosoma cruzi has two phylogenetic lineages associated almost exclusively with bats—Trypanosoma cruzi Tcbat and the subspecies T. c. marinkellei. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time T. cruzi Tcbat and T. c. marinkellei in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines Cavernicola pilosa and Triatoma dispar sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for T. c. marinkellei than any of the T. c. cruzi genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of T. c. marinkellei supports a long evolutionary relationship between T. cruzi and bats, implying that bats are the original hosts of this important parasite.

High Local Diversity of Trypanosoma in a Common Bat Species, and Implications for the Biogeography and Taxonomy of the T. cruzi Clade

The Trypanosoma cruzi clade is a group of parasites that comprises T. cruzi sensu lato and its closest relatives. Although several species have been confirmed phylogenetically to belong to this clade, it is uncertain how many more species can be expected to belong into this group. Here, we present the results of a survey of trypanosome parasites of the bat Artibeus jamaicensis from the Panamá Canal Zone, an important seed disperser. Using a genealogical species delimitation approach, the Poisson tree processes (PTP), we tentatively identified five species of trypanosomes – all belonging to the T. cruzi clade. A small monophyletic group of three putative Trypanosoma species places at the base of the clade phylogeny, providing evidence for at least five independent colonization events of these parasites into the New World. Artibeus jamaicensis presents a high diversity of these blood parasites and is the vertebrate with the highest number of putative trypanosome species reported from a single locality. Our results emphasize the need for continued efforts to survey mammalian trypanosomes.

Using museum collections to detect pathogens.

To the Editor: Natural history museum collections have evolved in recent years to meet the challenges of current and future interdisciplinary scientific studies. Many natural history museums have built tissue collections and made digital information (e.g., photographs, publications, geographic coordinates) freely available on the Internet. These collections provide endless opportunities to conduct studies, including temporal and spatial surveys of emerging and reemerging pathogens (1). We report an example of a museum collection being useful in detecting Trypanosoma cruzi, the etiologic agent of Chagas disease, in the southern plains woodrat (Neotoma micropus) in southern Texas. This finding is of interest in the epidemiology of Chagas disease because the climatic characteristics and demographics of the region are similar to areas in Latin America where Chagas disease is an important zoonotic agent that infects ≈20 million persons (2). Tissue samples from N. micropus woodrats archived in the Natural Science Research Laboratory at the Museum of Texas Tech University were evaluated for T. cruzi DNA by PCR methods. All samples were originally collected during March 2001–June 2003 from the Chaparral Wildlife Management Area in southern Texas (28o18′N, 99o24′W), 86 km west of the Mexico–US border; some samples had been used previously in other research projects (3). Individual rodents were captured with live traps (n = 13) or by excavating middens in which all the nest occupants were collected by hand (n = 146). Animals were later euthanized and tissue samples (heart, kidney, liver, lung, muscle, spleen) were obtained. Tissues were immediately frozen in liquid nitrogen and permanently stored in ultralow-temperature freezers. We extracted 1 DNA sample from each animal’s liver for use in this survey. DNA amplification was performed by using primers specific to T. cruzi (TCZ1 and TCZ2) (4) under previously standardized conditions and positive controls (5). T. cruzi DNA was detected in 42 (26.4%) of 159 woodrat samples tested. Males were infected significantly more often (31/82) than females (11/73); sex was not determined for 4 individuals (Score test for a binomial proportion, z = –4.0, p<0.01). Adults had a nonsignificant higher prevalence (24/92) than all other individuals in the remaining age categories combined (14/54) (age was not determined for 13 individuals) (Score test for a binomial proportion, z = –0.02, p = 0.98). Middens that harbored infected individuals (n = 28, mean = 1.8) were not significantly (t = 0.79, df = 84, p = 0.43) more populated than middens that harbored uninfected individuals (n = 58, mean = 1.6). Woodrats had been shown by using microscopy to be infected by T. cruzi and T. cruzi–like organisms (6); however, no definitive DNA-based confirmation had been performed (6,7). The results of this research confirm the infection of N. micropus woodrats with T. cruzi and show a higher prevalence than that reported in previous studies that used other diagnostic methods. These results also point to woodrats as a potentially important reservoir of T. cruzi in North America. We hypothesize that the high prevalence is a consequence of the nest-building habits of these rodents. These nests are complexes of dry branches, grasses, and leaves, with a mean diameter of 84 cm, and offer easy access and permanent refuge to triatomine bugs. Woodrats have been found in association with at least 5 triatomine species: Triatoma gerstaeckeri, T. lecticularia, T. neotomae, T. protracta, and T. sanguisuga (8). Another factor for consideration is woodrats’ multigenerational midden use, which may enable the permanent occurrence of triatomine colonies and therefore maintain long-term circulation of T. cruzi. Whereas recent characterizations of North American strains have included isolates from other mammalian reservoir hosts (9), the genotyping of parasites from N. micropus woodrats and other woodrats is still to be done. Despite successful results from tracking pathogens by using material deposited in natural history museum collections (10), this practice is not common. We suggest that natural history museum collections be used more frequently, especially for surveying and genotyping T. cruzi in mammals, because of the importance of such information in clarifying the epidemiology and the evolutionary history of this pathogen.

Hepatozoon Parasites (Apicomplexa: Adeleorina) in Bats

Abstract: We provide the first evidence of Hepatozoon parasites infecting bats. We sequenced a short fragment of the 18S rRNA gene (∼600 base pairs) of Hepatozoon parasites from 3 Hipposideros cervinus bats from Borneo. Phylogenies inferred by model-based methods place these Hepatozoon within a clade formed by parasites of reptiles, rodents, and marsupials. We discuss the scenario that bats might be common hosts of Hepatozoon.

Distribution, abundance and roosts of the fruit bat Artibeus fraterculus (Chiroptera: Phyllostomidae)

Where does a species live? How common is it? Where does it spend its inactive periods? These are basic questions about the biology of a species, which bring key information for application in conservation and management. Unfortunately, this information is available for only a minimum fraction of all animal species. Using 1) ecological niche modeling with maximum entropy (Maxent), 2) relative abundance estimates using museum records, and 3) field surveys of roosting sites, we report the fraternal fruit-eating bat, Artibeus fraterculus, as having a distribution limited to the Tumbesian ecoregion in Ecuador and west central Peru, being the relatively most abundant bat species throughout its range, with healthy populations which are primarily sustained by cultivated and introduced plants, and using human-made constructions as roost sites. Additionally, we described a large congregation of individuals of this species in a single roost, representing the largest colony reported for the genus Artibeus. These results may indicate resilience of A. fraterculus to human disturbance.