Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosomo cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TO presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TO and TCIIa from wild primates (16 TO and five TCIIa), Rhodnius spp, (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TO and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region. (C) 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Natural Plasmodium infections in Brazilian wild monkeys: Reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N = 140), semideciduous Atlantic forest (N = 257) and Cerrado (a savannah-like habitat) (N = 51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the Sao Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N = 30) and Cebus apella (N = 39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C apella specimens were negative. Parasitological examination of I he samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P.falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions. (C) 2008 Elsevier B.V. All rights reserved.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3 mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 +/- 1.8 mu m in total length, 12.9 +/- 0.8 mu m in body length, 3.4 +/- 0.3 mu m in width, 3.1 +/- 0.1 mu m in thickness and 24.6 +/- 2.2 mu m in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 +/- 0.5 mu m in length and 0.7 +/- 0.1 mu m in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size. (C) 2011 Elsevier B.V. All rights reserved.

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wildtype (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Effects of kinetin and nitrogen on growth rates, pigment and protein contents in wild and phycoerythrin-deficient strains of Hypnea musciformis (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 mu M were tested in ASP 12-NTA synthetic medium with 10 mu M nitrate (N-limited) and 100 mu M nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.

Importance of estimating matrix quality for modeling species distribution in complex tropical landscapes: a test with Atlantic forest small mammals

Information to guide decision making is especially urgent in human dominated landscapes in the tropics, where urban and agricultural frontiers are still expanding in an unplanned manner. Nevertheless, most studies that have investigated the influence of landscape structure on species distribution have not considered the heterogeneity of altered habitats of the matrix, which is usually high in human dominated landscapes. Using the distribution of small mammals in forest remnants and in the four main altered habitats in an Atlantic forest landscape, we investigated 1) how explanatory power of models describing species distribution in forest remnants varies between landscape structure variables that do or do not incorporate matrix quality and 2) the importance of spatial scale for analyzing the influence of landscape structure. We used standardized sampling in remnants and altered habitats to generate two indices of habitat quality, corresponding to the abundance and to the occurrence of small mammals. For each remnant, we calculated habitat quantity and connectivity in different spatial scales, considering or not the quality of surrounding habitats. The incorporation of matrix quality increased model explanatory power across all spatial scales for half the species that occurred in the matrix, but only when taking into account the distance between habitat patches (connectivity). These connectivity models were also less affected by spatial scale than habitat quantity models. The few consistent responses to the variation in spatial scales indicate that despite their small size, small mammals perceive landscape features at large spatial scales. Matrix quality index corresponding to species occurrence presented a better or similar performance compared to that of species abundance. Results indicate the importance of the matrix for the dynamics of fragmented landscapes and suggest that relatively simple indices can improve our understanding of species distribution, and could be applied in modeling, monitoring and managing complex tropical landscapes.

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy-Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species` genetic diversity.

Diet and food selection by small mammals in an old-growth Atlantic forest of south-eastern Brazil

Here we describe the stomach contents of nine small mammal species (seven rodents and two didelphid marsupials) co-occurring in an old-growth Atlantic forest area. For four terrestrial rodents, we also compared the importance of arthropods in the diet and the selection of arthropod groups by comparing consumption with availability. Small mammals and arthropods were sampled in a 36-ha grid containing 25 sampling stations spaced every 150 m, and 47 stomach contents were analysed. While plant matter was the predominant item in the stomach contents of two rodents (Oligoryzomys nigripes and Rhipidomys mastacalis), four species presented arthropods as the main food item (the rodents Brucepattersonius soricinus and Oxymycterus dasytrichus, and the marsupials Monodelphis n. sp. and Marmosops incanus) and three consumed more plant matter than arthropods, but had significant amounts of both items (the rodents Delomys sublineatus, Euryoryzomys russatus and Thaptomys nigrita). Our results suggest that differences in diet, coupled with differences in habit and microhabitat preferences, are important factors allowing resource partition among species of the diverse group of co-occurring terrestrial small mammals in Atlantic forest areas. Moreover, arthropods were not preyed opportunistically by any of the four terrestrial rodents, since consumption was not proportional to availability. Rather, selection or rejection of arthropod groups seems to be determined by aspects other than availability, such as nutritional value, easiness of capture and handling or palatability.

Taxon surrogates among Amazonian mammals: Can total species richness be predicted by single orders?

Limited financial sources and the difficulty in performing complete surveys, allied to the speed of habitat fragmentation and the urgent necessity in select conservation areas, create the necessity of using some methodologies which bypass these problems. One possibility is the use of surrogate taxa that might be used as indicator of others groups richness and even total richness of an area. We investigated if the use of surrogate taxon is useful among seven mammal orders in Amazon. We tested through Pearson`s correlation (Bonferroni`s adjusted) if (1) there was a correlation between richness of total species and some order; (2) there was a significant pair wise correlation between species richness of each order; and (3) the combination of two orders would give better results as a surrogate for the total richness. The correlations found, in general, were positive. It means that the increase in the richness of an order was followed by its increase in another order, as well as in the total species richness. Only Didelphimorphia was significantly correlated with the total species richness. In the pair wise analyses only one assembly, Primates and Artiodactyla, was significantly correlated with total richness. Since indicator species are more effective within taxonomic groups (life-history characteristics are likely to be more different among than within major taxonomic groups), we suggest that an indicator group might be chosen for each one. In this case, for mammals from Amazon, it would be Didelphimorphia. (C) 2008 Elsevier Ltd. All rights reserved.

Comparing methods for sampling large- and medium-sized mammals: camera traps and track plots

Activities involving fauna monitoring are usually limited by the lack of resources; therefore, the choice of a proper and efficient methodology is fundamental to maximize the cost-benefit ratio. Both direct and indirect methods can be used to survey mammals, but the latter are preferred due to the difficulty to come in sight of and/or to capture the individuals, besides being cheaper. We compared the performance of two methods to survey medium and large-sized mammal: track plot recording and camera trapping, and their costs were assessed. At Jatai Ecological Station (S21 degrees 31`15 ``- W47 degrees 34`42 ``-Brazil) we installed ten camera traps along a dirt road directly in front of ten track plots, and monitored them for 10 days. We cleaned the plots, adjusted the cameras, and noted down the recorded species daily. Records taken by both methods showed they sample the local richness in different ways (Wilcoxon, T=231; p;;0.01). The track plot method performed better on registering individuals whereas camera trapping provided records which permitted more accurate species identification. The type of infra-red sensor camera used showed a strong bias towards individual body mass (R(2)=0.70; p=0.017), and the variable expenses of this method in a 10-day survey were estimated about 2.04 times higher compared to track plot method; however, in a long run camera trapping becomes cheaper than track plot recording. Concluding, track plot recording is good enough for quick surveys under a limited budget, and camera trapping is best for precise species identification and the investigation of species details, performing better for large animals. When used together, these methods can be complementary.

Carnivore mammals in a fragmented landscape in northeast of Sao Paulo State, Brazil

Sao Paulo is the most developed state in Brazil and little of its native vegetation remains. In Luiz Antonio and Santa Rita do Passa Quatro municipalities, only small fragments of cerrado (Brazilian savanna) physiognomies (cerrado, cerrado sensu stricto) and of semideciduous forest have been left, surrounded by eucalyptus silviculture and sugar-cane agriculture. However, that vegetation mosaic still shelters large mammals, including several carnivore species. To detect the carnivores present in such a mosaic area (50,000 ha), and to find out how they use the landscape, we recorded them through 21 camera traps and 21 track plots, during 18 months. Species richness, diversity and relative frequency were evaluated according to the habitat. Ten species were recorded, some of them locally threatened to extinction (Puma concolor, Leopardus pardalis, Chrysocyon brachyurus). Species diversity did not significantly differ among fragments, and although most species preferred one or another habitat, the carnivore community as a whole explored all the study area regardless of the vegetation cover;eucalyptus plantations were as used by the carnivores as the native fragments. Therefore, it seems possible to maintain such animals in agricultural landscapes, where some large native fragments are left and the matrix is permeable to native fauna.

Small mammals of the mid-Araguaia River in central Brazil, with the description of a new species of climbing rat

The mid-Araguaia River basin in central Brazil is considered a priority area for biodiversity conservation, and Parque Estadual do Cantao (PEC) is one of the most important protected areas in this ecotone between Cerrado and Amazonia. This area suffers an intensive human pressure with high rates of deforestation, and still remains poorly studied in terms of biodiversity. From June 2007 to November 2008 we sampled small mammals from both banks of the mid-Araguaia River, in the states of Tocantins and Para. Data are given about morphological traits, geographic distribution and natural history of 22 species of small non-volant mammals (eight marsupials and 14 rodents) surveyed at PEC and its surroundings. We also present mitochondrial phylogenetic analyses that allow species identification within the genera: Oecomys, Oligoryzomys and Rhipidomys, and delineate an undescribed species of Thrichomys. Based on morphologic and molecular data, we describe a new species of Rhipidomys previously assigned to R. nitela, which is apparently endemic to the Araguaia-Tocantins basin in the Cerrado. Additionally, our phylogenetic analyses provide support for the role played by the Araguaia River as an important geographic barrier for two sister species of Rhipidomys.

Influence of multi-scale landscape structure on the occurrence of carnivorous mammals in a human-modified savanna, Brazil

So Paulo is the most developed state in Brazil and contains few fragments of native ecosystems, generally surrounded by intensive agriculture lands. Despite this, some areas still shelter large native animals. We aimed at understanding how medium and large carnivores use a mosaic landscape of forest/savanna and agroecosystems, and how the species respond to different landscape parameters (percentage of landcover and edge density), in a multi-scale perspective. The response variables were: species richness, carnivore frequency and frequency for the three most recorded species (Puma concolor, Chrysocyon brachyurus and Leopardus pardalis). We compared 11 competing models using Akaike`s information criterion (AIC) and assessed model support using weight of AIC. Concurrent models were combinations of landcover types (native vegetation, ""cerrado"" formations, ""cerrado"" and eucalypt plantation), landscape feature (percentage of landcover and edge density) and spatial scale. Herein, spatial scale refers to the radius around a sampling point defining a circular landscape. The scales analyzed were 250 (fine), 1,000 (medium) and 2,000 m (coarse). The shape of curves for response variables (linear, exponential and power) was also assessed. Our results indicate that species with high mobility, P. concolor and C. brachyurus, were best explained by edge density of the native vegetation at a coarse scale (2,000 m). The relationship between P. concolor and C. brachyurus frequency had a negative power-shaped response to explanatory variables. This general trend was also observed for species richness and carnivore frequency. Species richness and P. concolor frequency were also well explained by a second concurrent model: edge density of cerrado at the fine (250 m) scale. A different response was recorded for L. pardalis, as the frequency was best explained for the amount of cerrado at the fine (250 m) scale. The curve of response was linearly positive. The contrasting results (P. concolor and C. brachyurus vs L. pardalis) may be due to the much higher mobility of the two first species, in comparison with the third. Still, L. pardalis requires habitat with higher quality when compared with other two species. This study highlights the importance of considering multiple spatial scales when evaluating species responses to different habitats. An important and new finding was the prevalence of edge density over the habitat extension to explain overall carnivore distribution, a key information for planning and management of protected areas.