Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

HYDRODYNAMICS AND SUSPENDED SEDIMENT TRANSPORT IN THE CAMBORIU ESTUARY - BRAZIL: PRE JETTY CONDITIONS

Estuarine hydrodynamics is a key factor in the definition of the filtering capacity of an estuary and results from the interaction of the processes that control the inlet morphodynamics and those that are acting in the mixing of the water in the estuary. The hydrodynamics and suspended sediment transport in the Cambori estuary were assessed by two field campaigns conducted in 1998 that covered both neap and spring tide conditions. The period measured represents the estuarine hydrodynamics and sediment transport prior to the construction of the jetty in 2003 and provides important background information for the Cambori estuary. Each field campaign covered two complete tidal cycles with hourly measurements of currents, salinity, suspended sediment concentration and water level. Results show that the Cambori estuary is partially mixed with the vertical structure varying as a function of the tidal range and tidal phase. The dynamic estuarine structure can be balanced between the stabilizing effects generated by the vertical density gradient, which produces buoyancy and stratification flows, and the turbulent effects generated by the vertical velocity gradient that generates vertical mixing. The main sediment source for the water column are the bottom sediments, periodically resuspended by the tidal currents. The advective salt and suspended sediment transport was different between neap and spring tides, being more complex at spring tide. The river discharge term was important under both tidal conditions. The tidal correlation term was also important, being dominant in the suspended sediment transport during the spring tide. The gravitational circulation and Stokes drift played a secondary role in the estuarine transport processes.

JUVENILE RECRUITMENT, EARLY GROWTH, AND MORPHOLOGICAL VARIATION IN THE ENDANGERED SANTA CATALINA ISLAND RATTLESNAKE, CROTALUS CATALINENSIS

Life-history information constitutes the raw data for building population models used in species conservation. We provide life-history data for the endangered Santa Catalina Island Rattlesnake, Crotalus catalinensis. We use data from 277 observations of C. catalinensis made between 2002 and 2011 on the island. Mean snout-vent length (SVL) of adult C. catalinensis was 643 mm for males and 631 mm for females; the difference was not significant. The degree of sexual size dimorphism (SSD; using SVL) was -0.02. However, sexes were dimorphic in total length ( SVL + tail length), relative tail length, and stoutness. Juvenile recruitment occurs during late-summer. In their first year of life, juveniles seem to grow at a rate of about 1.7 cm/mo. Females seem to become mature around 570 mm SVL, probably in the year when they become 2 y old. Scattered literature data corroborates the time of juvenile recruitment described herein. Growth in C. catalinensis seems to be slower than that of C. ruber, its sister taxa, but similar to other rattlesnakes.

Phylogeny and cryptic diversity in geckos (Phyllopezus; Phyllodactylidae; Gekkota) from South America's open biomes

The gecko genus Phyllopezus occurs across South America's open biomes: Cerrado, Seasonally Dry Tropical Forests (SDTF, including Caatinga), and Chaco. We generated a multi-gene dataset and estimated phylogenetic relationships among described Phyllopezus taxa and related species. We included exemplars from both described Phyllopezus pollicaris subspecies, P. p. pollicaris and P. p. przewalskii. Phylogenies from the concatenated data as well as species trees constructed from individual gene trees were largely congruent. All phylogeny reconstruction methods showed Bogertia lutzae as the sister species of Phyllopezus maranjonensis, rendering Phyllopezus paraphyletic. We synonymized the monotypic genus Bogertia with Phyllopezus to maintain a taxonomy that is isomorphic with phylogenetic history. We recovered multiple, deeply divergent, cryptic lineages within P. pollicaris. These cryptic lineages possessed mtDNA distances equivalent to distances among other gekkotan sister taxa. Described P. pollicaris subspecies are not reciprocally monophyletic and current subspecific taxonomy does not accurately reflect evolutionary relationships among cryptic lineages. We highlight the conservation significance of these results in light of the ongoing habitat loss in South America's open biomes. (C) 2011 Elsevier Inc. All rights reserved.

Water-in-Oil Micro-Emulsion Enhances the Secondary Structure of a Protein by Confinement

A scheme is presented in which an organic solvent environment in combination with surfactants is used to confine a natively unfolded protein inside an inverse microemulsion droplet. This type of confinement allows a study that provides unique insight into the dynamic structure of an unfolded, flexible protein which is still solvated and thus under near-physiological conditions. In a model system, the protein osteopontin (OPN) is used. It is a highly phosphorylated glycoprotein that is expressed in a wide range of cells and tissues for which limited structural analysis exists due to the high degree of flexibility and large number of post-translational modifications. OPN is implicated in tissue functions, such as inflammation and mineralisation. It also has a key function in tumour metastasis and progression. Circular dichroism measurements show that confinement enhances the secondary structural features of the protein. Small-angle X-ray scattering and dynamic light scattering show that OPN changes from being a flexible protein in aqueous solution to adopting a less flexible and more compact structure inside the microemulsion droplets. This novel approach for confining proteins while they are still hydrated may aid in studying the structure of a wide range of natively unfolded proteins.

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Species Distribution Modeling for Conservation Purposes

Species distribution models (SDMs) can be useful for different conservation purposes. We discuss the importance of fitting spatial scale and using current records and relevant predictors aiming conservation. We choose jaguar (Panthera onca) as a target species and Brazil and Atlantic Forest biome as study areas. We tested two different extents (continent and biome) and resolutions (similar to 4 Km and similar to 1 Km) in Maxent with 186 records and 11 predictors (bioclimatic, elevation, land-use and landscape structure). All models presented satisfactory AUC values (>0.70) and low omission errors (<23%). SDMs were scale-sensitive as the use of reduced extent implied in significant gains to model performance generating more constrained and real predictive distribution maps. Continental-scale models performed poorly in predicting potential current jaguar distribution, but they reached the historic distribution. Specificity increased significantly from coarse to finer-scale models due to the reduction of overprediction. The variability of environmental space (E-space) differed for most of climatic variables between continental and biome-scale and the representation of the E-space by predictors differed significantly (t = 2.42, g.I. = 9, P < 0.05). Refining spatial scale, incorporating landscape variables and improving the quality of biological data are essential for improving model prediction for conservation purposes.

A radioenzymatic assay to identify three groups of phospholipase A(2) in platelets

Phospholipases A(2) (PLA(2)) are key enzymes in membrane metabolism. The release of fatty acids and lysophospholipids by PLA(2) activates several intra-cellular second messenger cascades that regulate a wide variety of physiological responses. The aim of the present study is to describe a radioenzymatic assay to determine the activity of three main PLA(2) subtypes in platelets, namely extracellular calcium-dependent PLA(2) (sPLA(2)) and intracellular calcium-dependent (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)). The differentiation of these distinct PLA(2) subtypes was based on the enzyme substrate preference (arachdonic acid or palmitoyl acid) and calcium concentration. Our results indicate that this new assay is feasible, precise and specific to measure the activity of the aforementioned subtypes of PLA(2). Therefore, this protocol can be used to investigate modifications of PLA(2) homeostasis in distinct biological models addressing the pathophysiology of many medical and neuropsychiatric disorders such as schizophrenia and Alzheimer's disease. (C) 2012 Elsevier Ltd. All rights reserved.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86 % as Trichoderma asperellum, 33.33 % as Trichoderma harzianum, 14.29 % as Trichoderma tomentosum, 4.76 % as Trichoderma koningiopsis, and 4.76 % as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen. (C) 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Developmental and Epigenetic Anomalies in Cloned Cattle

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

Abstract Background Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. Methods Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. Results Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. Conclusion Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

BACKGROUND: Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. METHODS: Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. RESULTS: Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. CONCLUSION: Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.