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.

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (similar to 99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 degrees C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01. (C) 2009 Elsevier Inc. All rights reserved.

Characterization of a novel freshwater gigartinalean red alga from Belize, with a description of Sterrocladia belizeana sp nov (Rhodophyta)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

Abstract Background Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli. Methods Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy. Results New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. Conclusion Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene

Abstract Background Little is known about the diversity, phylogenetic relationships, and biogeography of trypanosomes infecting non-mammalian hosts. In this study, we investigated the influence of host species and biogeography on shaping the genetic diversity, phylogenetic relationship, and distribution of trypanosomes from South American alligatorids and African crocodilids. Methods Small Subunit rRNA (SSU rRNA) and glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) genes were employed for phylogenetic inferences. Trypanosomes from crocodilians were obtained by haemoculturing. Growth behaviour, morphology, and ultrastructural features complement the molecular description of two new species strongly supported by phylogenetic analyses. Results The inferred phylogenies disclosed a strongly supported crocodilian-restricted clade comprising three subclades. The subclade T. grayi comprised the African Trypanosoma grayi from Crocodylus niloticus and tsetse flies. The subclade T. ralphi comprised alligatorid trypanosomes represented by Trypanosoma ralphi n. sp. from Melanosuchus niger, Caiman crocodilus and Caiman yacare from Brazilian river basins. T. grayi and T. ralphi were sister subclades. The basal subclade T. terena comprised alligatorid trypanosomes represented by Trypanosoma terena n. sp. from Ca. yacare sharing hosts and basins with the distantly genetic related T. ralphi. This subclade also included the trypanosome from Ca. crocodilus from the Orinoco basin in Venezuela and, unexpectedly, a trypanosome from the African crocodilian Osteolaemus tetraspis. Conclusion The close relationship between South American and African trypanosomes is consistent with paleontological evidence of recent transoceanic dispersal of Crocodylus at the Miocene/Pliocene boundaries (4–5 mya), and host-switching of trypanosomes throughout the geological configuration of South American hydrographical basins shaping the evolutionary histories of the crocodilians and their trypanosomes.

The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene

Background: Little is known about the diversity, phylogenetic relationships, and biogeography of trypanosomes infecting non-mammalian hosts. In this study, we investigated the influence of host species and biogeography on shaping the genetic diversity, phylogenetic relationship, and distribution of trypanosomes from South American alligatorids and African crocodilids. Methods: Small Subunit rRNA (SSU rRNA) and glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) genes were employed for phylogenetic inferences. Trypanosomes from crocodilians were obtained by haemoculturing. Growth behaviour, morphology, and ultrastructural features complement the molecular description of two new species strongly supported by phylogenetic analyses. Results: The inferred phylogenies disclosed a strongly supported crocodilian-restricted clade comprising three subclades. The subclade T. grayi comprised the African Trypanosoma grayi from Crocodylus niloticus and tsetse flies. The subclade T. ralphi comprised alligatorid trypanosomes represented by Trypanosoma ralphi n. sp. From Melanosuchus niger, Caiman crocodilus and Caiman yacare from Brazilian river basins. T. grayi and T. ralphi were sister subclades. The basal subclade T. terena comprised alligatorid trypanosomes represented by Trypanosoma terena n. sp. from Ca. yacare sharing hosts and basins with the distantly genetic related T. ralphi. This subclade also included the trypanosome from Ca. crocodilus from the Orinoco basin in Venezuela and, unexpectedly, a trypanosome from the African crocodilian Osteolaemus tetraspis. Conclusion: The close relationship between South American and African trypanosomes is consistent with paleontological evidence of recent transoceanic dispersal of Crocodylus at the Miocene/Pliocene boundaries (4–5 mya), and host-switching of trypanosomes throughout the geological configuration of South American hydrographical basins shaping the evolutionary histories of the crocodilians and their trypanosomes.

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

BACKGROUND: Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli. METHODS: Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy. RESULTS: New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. CONCLUSION: Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

Relationships among the three major lineages of the Acari (Arthropoda : Arachnida) inferred from small subunit rRNA: Paraphyly of the parasitiformes with respect to the opilioacariformes and relative rates of nucleotide substitution

We inferred phylogeny among the three major lineages of the Acari ( mites) from the small subunit rRNA gene. Our phylogeny indicates that the Opilioacariformes is the sister-group to the Ixodida+Holothyrida, not the Ixodida+Mesostigmata+Holothyrida, as previously thought. Support for this relationship increased when sites with the highest rates of nucleotide substitution, and thus the greatest potential for saturation with nucleotide substitutions, were removed. Indeed, the increase in support ( and resolution) was despite a 70% reduction in the number of parsimony-informative sites from 408 to 115. This shows that rather than 'noisy' sites having no impact on resolution of deep branches, 'noisy' sites have the potential to obscure phylogenetic relationships. The arrangement, Ixodida+Holothyrida+Opilioacariformes, however, may be an artefact of long-branch attraction since relative-rate tests showed that the Mesostigmata have significantly faster rates of nucleotide substitution than other parasitiform mites. Thus, the fast rates of nucleotide substitution of the Mesostigmata might have caused the Mesostigmata to be attracted to the outgroup in our trees. We tested the hypothesis that the high rate of nucleotide substitution in some mites was related to their short generation times. The Acari species that have high nucleotide substitution rates usually have short generation times; these mites also tend to be more active and thus have higher metabolic rates than other mites. Therefore, more than one factor may affect the rate of nucleotide substitution in these mites.

Evolution of the diatoms: major steps in their evolution and a review of the supporting molecular and morphological evidence.

Over the years, many reviews of different aspects of diatom biology, ecology and evolution have appeared. Since 1993 many molecular trees have been produced to infer diatom phylogeny. In 2004, Medlin & Kaczmarska revised the systematics of the diatoms based on more than 20 years of consistent recovery of two major clades of diatoms that did not correspond to a traditional concept of centrics and pennates and established three classes of diatoms: Clade 1 = Coscinodiscophyceae (radial centrics) and Clade 2 = Mediophyceae (polar centrics + radial Thalassiosirales) and Bacillariophyceae (pennates). However, under certain analytical conditions, an alternative view of diatom evolution, a grades of clades, has been recovered that suggests a gradual evolution from centric to pennate symmetry. These two schemes of diatom evolution are evaluated in terms of whether or not the criteria advocated by Medlin & Kaczmarska that should be met to recover monophyletic classes have been used. The monophyly of the three diatom classes can only be achieved if (1) a secondary structure of the small subunit (SSU) rRNA gene was used to construct the alignment and not an alignment based on primary structure and (2) multiple outgroups were used. These requirements have not been met in each study of diatom evolution; hence, the grade of clades, which is useful in reconstructing the sequence of evolution, is not useful for accepting the new classification of the diatoms. Evidence for how these two factors affect the recovery of the three monophyletic classes is reviewed here. The three classes have been defined by clear morphological differences primarily based on gametangia and auxospore ontogeny and envelope structure, the presence or absence of a structure (tube process or sternum) associated with the annulus and the location of the cribrum in those genera with loculate areolae. New evidence supporting the three clades is reviewed. Other features of the cell are examined to determine whether they can also be used to support the monophyly of the three classes.

Evolution of the diatoms: major steps in their evolution and a review of the supporting molecular and morphological evidence.

Over the years, many reviews of different aspects of diatom biology, ecology and evolution have appeared. Since 1993 many molecular trees have been produced to infer diatom phylogeny. In 2004, Medlin & Kaczmarska revised the systematics of the diatoms based on more than 20 years of consistent recovery of two major clades of diatoms that did not correspond to a traditional concept of centrics and pennates and established three classes of diatoms: Clade 1 = Coscinodiscophyceae (radial centrics) and Clade 2 = Mediophyceae (polar centrics + radial Thalassiosirales) and Bacillariophyceae (pennates). However, under certain analytical conditions, an alternative view of diatom evolution, a grades of clades, has been recovered that suggests a gradual evolution from centric to pennate symmetry. These two schemes of diatom evolution are evaluated in terms of whether or not the criteria advocated by Medlin & Kaczmarska that should be met to recover monophyletic classes have been used. The monophyly of the three diatom classes can only be achieved if (1) a secondary structure of the small subunit (SSU) rRNA gene was used to construct the alignment and not an alignment based on primary structure and (2) multiple outgroups were used. These requirements have not been met in each study of diatom evolution; hence, the grade of clades, which is useful in reconstructing the sequence of evolution, is not useful for accepting the new classification of the diatoms. Evidence for how these two factors affect the recovery of the three monophyletic classes is reviewed here. The three classes have been defined by clear morphological differences primarily based on gametangia and auxospore ontogeny and envelope structure, the presence or absence of a structure (tube process or sternum) associated with the annulus and the location of the cribrum in those genera with loculate areolae. New evidence supporting the three clades is reviewed. Other features of the cell are examined to determine whether they can also be used to support the monophyly of the three classes.

Structure et biogéographie des communautés de pico- et de nanoeurcaryotes pélagiques autour de l'archipel du Svalbard

L’Arctique s’est réchauffé rapidement et il y a urgence d’anticiper les effets que cela pourrait avoir sur les protistes à la base de la chaîne alimentaire. Le phytoplancton de l’Océan Arctique inclut les pico- et nano-eucaryotes (0.45-10 μm diamètre de la cellule) et plusieurs de ceux-ci sont des écotypes retrouvés seulement dans l’Arctique alors que d’autres sont introduits des océans plus méridionaux. Alors que les océans tempérés pénètrent dans l’Arctique, il devient pertinent de savoir si ces communautés microbiennes pourraient être modifiées. L’archipel du Svalbard est une région idéale pour observer la biogéographie des communautés microbiennes sous l’influence de processus polaires et tempérés. Bien qu’ils soient géographiquement proches, les régions côtières entourant le Svalbard sont sujettes à des intrusions alternantes de masses d’eau de l’Arctique et de l’Atlantique en plus des conditions locales. Huit sites ont été échantillonnés en juillet 2013 pour identifier les protistes selon un gradient de profondeur et de masses d’eau autour de l’archipel. En plus des variables océanographiques standards, l’eau a été échantillonnée pour synthétiser des banques d’amplicons ciblant le 18S SSU ARNr et son gène pour ensuite être séquencées à haut débit. Cinq des sites d’étude avaient de faibles concentrations de chlorophylle avec des compositions de communauté post-efflorescence dominée par les dinoflagellés, ciliés, des alvéolés parasites putatifs, chlorophycées et prymnesiophytées. L’intrusion des masses d’eau et les conditions environnementales locales étaient corrélées avec la structure des communautés ; l’origine de la masse d’eau contribuant le plus à la distance phylogénétique des communautés microbiennes. Au sein de trois fjords, de fortes concentrations de chlorophylle sous-entendaient des activités d’efflorescence. Un fjord était dominé par Phaeocystis, un deuxième par un clade arctique identifié comme un Pelagophyceae et un troisième par un assemblage mixte. En général, un signal fort d’écotypes liés à l’Arctique prédominait autour du Svalbard.

Iron biogeochemistry and associated greenhouse gas evolution in a forested subtropical Australian coastal catchment : Poona Creek, Southeast Queensland

Iron (Fe) is the fourth most abundant element in the Earth’s crust. Excess Fe mobilization from terrestrial into aquatic systems is of concern for deterioration of water quality via biofouling and nuisance algal blooms in coastal and marine systems. Substantial Fe dissolution and transport involve alternate Fe(II) oxidation followed by Fe(III) reduction, with a diversity of Bacteria and Archaea acting as the key catalyst. Microbially-mediated Fe cycling is of global significance with regard to cycles of carbon (C), sulfur (S) and manganese (Mn). However, knowledge regarding microbial Fe cycling in circumneutral-pH habitats that prevail on Earth has been lacking until recently. In particular, little is known regarding microbial function in Fe cycling and associated Fe mobilization and greenhouse (CO2 and CH4, GHG) evolution in subtropical Australian coastal systems where microbial response to ambient variations such as seasonal flooding and land use changes is of concern. Using the plantation-forested Poona Creek catchment on the Fraser Coast of Southeast Queensland (SEQ), this research aimed to 1) study Fe cycling-associated bacterial populations in diverse terrestrial and aquatic habitats of a representative subtropical coastal circumneutral-pH (4–7) ecosystem; and 2) assess potential impacts of Pinus plantation forestry practices on microbially-mediated Fe mobilization, organic C mineralization and associated GHG evolution in coastal SEQ. A combination of wet-chemical extraction, undisturbed core microcosm, laboratory bacterial cultivation, microscopy and 16S rRNA-based molecular phylogenetic techniques were employed. The study area consisted primarily of loamy sands, with low organic C and dissolved nutrients. Total reactive Fe was abundant and evenly distributed within soil 0–30 cm profiles. Organic complexation primarily controlled Fe bioavailability and forms in well-drained plantation soils and water-logged, native riparian soils, whereas tidal flushing exerted a strong “seawater effect” in estuarine locations and formed a large proportion of inorganic Fe(III) complexes. There was a lack of Fe(II) sources across the catchment terrestrial system. Mature, first-rotation plantation clear-felling and second-rotation replanting significantly decreased organic matter and poorly crystalline Fe in well-drained soils, although variations in labile soil organic C fractions (dissolved organic C, DOC; and microbial biomass C, MBC) were minor. Both well-drained plantation soils and water-logged, native-vegetation soils were inhabited by a variety of cultivable, chemotrophic bacterial populations capable of C, Fe, S and Mn metabolism via lithotrophic or heterotrophic, (micro)aerobic or anaerobic pathways. Neutrophilic Fe(III)-reducing bacteria (FeRB) were most abundant, followed by aerobic, heterotrophic bacteria (heterotrophic plate count, HPC). Despite an abundance of FeRB, cultivable Fe(II)-oxidizing bacteria (FeOB) were absent in associated soils. A lack of links between cultivable Fe, S or Mn bacterial densities and relevant chemical measurements (except for HPC correlated with DOC) was likely due to complex biogeochemical interactions. Neither did variations in cultivable bacterial densities correlate with plantation forestry practices, despite total cultivable bacterial densities being significantly lower in estuarine soils when compared with well-drained plantation soils and water-logged, riparian native-vegetation soils. Given that bacterial Fe(III) reduction is the primary mechanism of Fe oxide dissolution in soils upon saturation, associated Fe mobilization involved several abiotic and biological processes. Abiotic oxidation of dissolved Fe(II) by Mn appeared to control Fe transport and inhibit Fe dissolution from mature, first-rotation plantation soils post-saturation. Such an effect was not observed in clear-felled and replanted soils associated with low SOM and potentially low Mn reactivity. Associated GHG evolution post-saturation mainly involved variable CO2 emissions, with low, but consistently increasing CH4 effluxes in mature, first-rotation plantation soil only. In comparison, water-logged soils in the riparian native-vegetation buffer zone functioned as an important GHG source, with high potentials for Fe mobilization and GHG, particularly CH4 emissions in riparian loam soils associated with high clay and crystalline Fe fractions. Active Fe–C cycling was unlikely to occur in lower-catchment estuarine soils associated with low cultivable bacterial densities and GHG effluxes. As a key component of bacterial Fe cycling, neutrophilic FeOB widely occurred in diverse aquatic, but not terrestrial, habitats of the catchment study area. Stalked and sheathed FeOB resembling Gallionella and Leptothrix were limited to microbial mat material deposited in surface fresh waters associated with a circumneutral-pH seep, and clay-rich soil within riparian buffer zones. Unicellular, Sideroxydans-related FeOB (96% sequence identity) were ubiquitous in surface and subsurface freshwater environments, with highest abundance in estuary-adjacent shallow coastal groundwater water associated with redox transition. The abundance of dissolved C and Fe in the groundwater-dependent system was associated with high numbers of cultivable anaerobic, heterotrophic FeRB, microaerophilic, putatively lithotrophic FeOB and aerobic, heterotrophic bacteria. This research represents the first study of microbial Fe cycling in diverse circumneutral-pH environments (terrestrial–aquatic, freshwater–estuarine, surface–subsurface) of a subtropical coastal ecosystem. It also represents the first study of its kind in the southern hemisphere. This work highlights the significance of bacterial Fe(III) reduction in terrestrial, and bacterial Fe(II) oxidation in aquatic catchment Fe cycling. Results indicate the risk of promotion of Fe mobilization due to plantation clear-felling and replanting, and GHG emissions associated with seasonal water-logging. Additional significant outcomes were also achieved. The first direct evidence for multiple biomineralization patterns of neutrophilic, microaerophilic, unicellular FeOB was presented. A putatively pure culture, which represents the first cultivable neutrophilic FeOB from the southern hemisphere, was obtained as representative FeOB ubiquitous in diverse catchment aquatic habitats.

Cross-species chromosome painting in the golden mole and elephant-shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora

Cross-species painting (fluorescence in situ hybridization) with 23 human (Homo sapiens (HSA)) chromosome-specific painting probes (HSA 1-22 and the X) was used to delimit regions of homology on the chromosomes of the golden mole (Ghrysochloris asiaticus) and elephant-shrew (Elephantulus rupestris). A cladistic interpretation of our data provides evidence of two unique associations, HSA 1/19p and 5/21/3, that support Afrotheria. The recognition of HSA 5/3/21 expands on the 3/21 synteny originally designated as an ancestral state for all eutherians. We have identified one adjacent segment combination (HSA2/8p/4) that is supportive of Afroinsectiphillia (aardvark, golden mole, elephant-shrew). Two segmental combinations (HSA 10q/17 and HSA 3/20) unite the aardvark and elephant-shrews as sister taxa. The finding that segmental syntenies in evolutionarily distant taxa can improve phylogenetic resolution suggests that they may be useful for testing sequence-based phylogenies of the early eutherian mammals. They may even suggest clades that sequence trees are not recovering with any consistency and thus encourage the search for additional rare genomic changes among afrotheres.

Comportamento biológico "in vitro" de "novas espécies" de Leishmania no Velho Mundo

As leishmanioses são um grupo de doenças causadas pelo parasita protozoário Leishmania sp. Na Bacia mediterrânica, Leishmania infantum, é a principal espécie causadora de leishmaniose visceral, a forma mais severa da doença, sendo L. major um dos agentes etiológicos da leishmaniose cutânea. Apesar de se considerar que estes parasitas têm uma reprodução essencialmente clonal, nos últimos 20 anos tem vindo a ser descrita a recombinação genética entre diferentes estirpes e espécies, com ocorrência de híbridos naturais, quer no Velho quer no Novo Mundo. Recentemente, em Portugal, foram isoladas e identificadas pela primeira vez, estirpes híbridas de L. infantum/L. major. O presente estudo teve como principais objetivos, a pesquisa de “novas espécies” de Leishmania e a análise do comportamento “in vitro” de estirpes parentais e híbridas de L. infantum e L. major. Numa primeira parte do trabalho efetuou-se a cultura e pesquisa de DNA de Leishmania sp., em amostras de sangue medular de 229 cães provenientes de uma região endémica de Portugal, utilizando diferentes marcadores moleculares (kDNA, ITS1 e SSU rRNA) e protocolos de PCR. Não foi encontrado DNA de espécies híbridas, tendo-se no entanto, identificado DNA de Leishmania sp. em 45,85% (105/229) das amostras, incluindo cães sem sinais clínicos. Na segunda parte do trabalho, realizaram-se diversos ensaios “in vitro” com estirpes híbridas naturais L. infantum/L. major e parentais L. infantum e L. major. Em condições normais de crescimento, observou-se um padrão de crescimento distinto para cada estirpe estudada. Em condições de “stress” oxidativo, destacou-se uma diferença significativa entre as duas estirpes híbridas estudadas. Em condições de “stress” nutricional, as estirpes não apresentaram diferenças entre si. Após avaliação da suscetibilidade das estirpes na presença de Anfotericina B, todas se mostraram suscetíveis, com concentrações inibitórias (CI50) entre 0.21 e 1.15 μg/mL. Após infeção em linhas celulares monocíticas, não se verificaram diferenças estatisticamente significativas na taxa e intensidade de infeção das estirpes híbridas em comparação às putativas parentais. Os resultados obtidos, contribuíram para um melhor conhecimento sobre o comportamento biológico destas estirpes híbridas naturais L. infantum/L. major. Estas demonstraram um comportamento “in vitro” intermédio, relativamente às estirpes parentais. Estes resultados poderão servir de base para o desenvolvimento de outros estudos com estas “novas espécies”, nomeadamente estudos de patogenicidade “in vivo” e o papel de biomarcadores de virulência, que permitam um potencial prognóstico da infeção e avaliação do seu risco epidemiológico.

Floral morphology and development in quillajaceae and surianaceae (Fabales), the species-poor relatives of leguminosae and polygalaceae

Background and Aims: Molecular phylogenies have suggested a new circumscription for Fabales to include Leguminosae, Quillajaceae, Surianaceae and Polygalaceae. However, recent attempts to reconstruct the interfamilial relationships of the order have resulted in several alternative hypotheses, including a sister relationship between Quillajaceae and Surianaceae, the two species-poor families of Fabales. Here, floral morphology and ontogeny of these two families are investigated to explore evidence of a potential relationship between them. Floral traits are discussed with respect to early radiation in the order. Methods: Floral buds of representatives of Quillajaceae and Surianaceae were dissected and observed using light microscopy and scanning electron microscopy. Key Results Quillajaceae and Surianaceae possess some common traits, such as inflorescence morphology and perianth initiation, but development and organization of their reproductive whorls differ. In Quillaja, initiation of the diplostemonous androecium is unidirectional, overlapping with the petal primordia. In contrast, Suriana is obdiplostemonous, and floral organ initiation is simultaneous. Independent initiation of five carpels is common to both Quillaja and Suriana, but subsequent development differs; the antesepalous carpels of Quillaja become fused proximally and exhibit two rows of ovules, and in Suriana the gynoecium is apocarpous, gynobasic, with antepetalous biovulate carpels. Conclusions: Differences in the reproductive development and organization of Quillajaceae and Surianaceae cast doubt on their potential sister relationship. Instead, Quillaja resembles Leguminosae in some floral traits, a hypothesis not suggested by molecular-based phylogenies. Despite implicit associations of zygomorphy with species-rich clades and actinomorphy with species-poor families in Fabales, this correlation sometimes fails due to high variation in floral symmetry. Studies considering specific derived clades and reproductive biology could address more precise hypotheses of key innovation and differential diversification in the order.