The Importance of Microhabitat for Biodiversity Sampling

Responses to microhabitat are often neglected when ecologists sample animal indicator groups. Microhabitats may be particularly influential in non-passive biodiversity sampling methods, such as baited traps or light traps, and for certain taxonomic groups which respond to fine scale environmental variation, such as insects. Here we test the effects of microhabitat on measures of species diversity, guild structure and biomass of dung beetles, a widely used ecological indicator taxon. We demonstrate that choice of trap placement influences dung beetle functional guild structure and species diversity. We found that locally measured environmental variables were unable to fully explain trap-based differences in species diversity metrics or microhabitat specialism of functional guilds. To compare the effects of habitat degradation on biodiversity across multiple sites, sampling protocols must be standardized and scale-relevant. Our work highlights the importance of considering microhabitat scale responses of indicator taxa and designing robust sampling protocols which account for variation in microhabitats during trap placement. We suggest that this can be achieved either through standardization of microhabitat or through better efforts to record relevant environmental variables that can be incorporated into analyses to account for microhabitat effects. This is especially important when rapidly assessing the consequences of human activity on biodiversity loss and associated ecosystem function and services.     

Stabilization of firefly luciferase against thermal stress by osmolytes

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.     

PGE1 analog alprostadil induces VEGF and eNOS expression in endothelial cells

Endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor 1-alpha (HIF-1alpha) are important regulators of endothelial function, which plays a role in the pathophysiology of heart failure (HF). PGE1 analog treatment in patients with HF elicits beneficial hemodynamic effects, but the precise mechanisms have not been investigated. We have investigated the effects of the PGE1 analog alprostadil on eNOS, VEGF, and HIF-1alpha expression in human umbilical vein endothelial cells (HUVEC) using RT-PCR and immunoblotting under normoxic and hypoxic conditions. In addition, we studied protein expression by immunohistochemical staining in explanted hearts from patients with end-stage HF, treated or untreated with systemic alprostadil. Alprostadil causes an upregulation of eNOS and VEGF protein and mRNA expression in HUVEC and decreases HIF-1alpha. Hypoxia potently increased eNOS, VEGF, and HIF-1alpha synthesis. The alprostadil-induced upregulation of eNOS and VEGF was prevented by inhibition of MAPKs with PD-98056 or U-0126. Consistently, the expression of eNOS and VEGF was increased, and HIF-1alpha was reduced in failing hearts treated with alprostadil. The potent effects of alprostadil on endothelial VEGF and eNOS synthesis may be useful for patients with HF where endothelial dysfunction is involved in the disease process.     

Insight into the genetic variability analysis and relationships among some Aegilops and Triticum species,as genome progenitors of bread wheat,using SCoT markers

We assessed the molecular genetic diversity and relationships among some Aegilops and Triticum species using 15 start codon-targeted (SCoT) polymorphism markers. A total of 166 bands amplified, of which 164 (98.79%) were polymorphic. Analysis of molecular variance and inter-population differentiation (Gst) indicated high genetic variation within the studied populations. Our analyses revealed high genetic diversity in T. boeoticum, Ae. cylindrica, T. durum and Ae. umbellulata, low diversity in Ae. crassa, Ae. caudata and Ae. speltoides, and a close relationship among Ae. tauschii, T. aestivum, T. durum, T. urartu, and T. boeoticum. Cluster analysis indicated 180 individuals divided into 8 genome homogeneous clades and 11 sub-groups. T. aestivum and T. durum accessions were grouped together, and accessions with the C and U genomes were grouped into the same clade. Our results support the hypothesis that T. urartu and Ae. tauschii are two diploid ancestors of T. aestivum, and also that Ae. caudata and Ae. umbellulata are putative donors of C and U genomes for other Aegilops species that possess these genomes. Our results also revealed that the SCoT technique is informative and can be used to assess genetic relationships among wheat germplasm.     

Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gα_i and Gα_s families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.Signal transduction pathways are important for sensing and responding to different environmental stimuli in both lower and higher eukaryotes. The highly conserved heterotrimeric guanine nucleotide-binding proteins (G proteins) belong to a family of regulatory proteins that are crucial for the transduction of signals, which are perceived by a distinct family of cell surface receptors (4). Heterotrimeric G proteins contain three subunits (α, β, and γ) that are linked in the inactive state. Activation of a Gα subunit by a transmembrane receptor leads to exchange of bound GDP with GTP on the Gα subunit, resulting in dissociation of the Gα and the Gβγ dimeric subunits, which can now interact with downstream effectors that subsequently generate changes in cellular responses (for a review, see reference 10).Filamentous fungi have one Gβ- and usually three Gα-encoding genes that belong to three major groups. Encoded proteins in groups I and III are related to the mammalian Gα_i and Gα_s families, respectively, but group II fungal Gα proteins have no mammalian counterpart (1, 4, 14, 22, 33, 53). Interestingly, the corn smut fungus Ustilago maydis contains a unique fourth Gα-encoding gene, and Saccharomyces cerevisiae contains only two Gα proteins (10, 57). Irrespective of the observed numerical variation, Gα proteins regulate a variety of cellular and developmental responses (4). For plant-pathogenic fungi, Gβ-encoding genes have been characterized functionally (9, 14, 22, 27, 31, 48, 52). Apart from the fact that individual Gα-encoding genes and the Gβ-encoding gene have been demonstrated to regulate growth, reproduction, and virulence, comparative functional characterization of all Gα-encoding genes has been reported only for a few plant-pathogenic fungi, including Magnaporthe grisea, Cryphonectria parasitica, and U. maydis (5, 41, 57).Mycosphaerella graminicola (anamorph Septoria tritici) causes septoria tritici blotch disease in bread and durum wheat in areas with high rainfall during the growing season, particularly in Western Europe, where it is considered to be the most important wheat disease (30). It is a ubiquitous phytopathogen with a lifestyle completely different from that of the aforementioned plant-pathogenic fungi. It is a dimorphic pathogen, and therefore the transition from a yeast-like to a filamentous form is important for initiation of infection (45). M. graminicola does not form appressoria but penetrates the leaves through stomata without forming specific infection structures. Furthermore, as a hemibiotroph, it has a biotrophic phase of about 10 days that is followed by a rapid switch to necrotrophy. The necrotic foliar lesions bear anamorphic and teleomorphic fructifications. M. graminicola is the model fungus for the Mycosphaerellaceae and even for the order Dothideales, an extremely large and diverse class of fungi with over 1,000 named species, including major plant pathogens such as the banana leaf streak fungus Mycosphaerella fijiensis (12, 21). Large expressed sequence tag (EST) libraries and the recently released genome sequence have been instrumental for the identification and characterization of genes involved in the development and pathogenicity of M. graminicola (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html). Recently, we reported that genes encoding mitogen-activated protein kinases (MAPKs) (MgFus3, MgSlt2, and MgHog1) and the catalytic (MgTpk2) and regulatory (MgBcy1) subunits of protein kinase A (PKA) are essential pathogenicity factors and regulate specific steps during the infection process (8, 43-45). To extend our knowledge about the role of G proteins in the development and pathogenicity of M. graminicola, we functionally analyzed three Gα-encoding genes and one Gβ-encoding gene of M. graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Our results show the requirement of MgGpa1, MgGpa3, and MgGpb1 for pathogenicity, whereas the latter also negatively regulates cell fusion and anastomosis. Among the G protein-encoding genes characterized in this study, MgGpa3 and MgGpb1 positively regulate the cyclic AMP (cAMP) pathway. MgGpa1 seems to be dispensable for cAMP regulation, whereas MgGpa2 appears to be redundant, for none of the assays rendered altered phenotypes. Our results open new perspectives for studying the regulatory machinery of the cAMP pathway in M. graminicola and other plant-pathogenic fungi.