In vivo expression technology (IVET) selection of genes of Rhizobium leguminosarum biovar viciae A34 expressed in the rhizosphere

IVET was used to identify genes that are specifically expressed in the rhizosphere of the pea-nodulating bacterium Rhizobium leguminosarum A34. A library of R. leguminosarum A34 cloned in the integration vector pIE1, with inserts upstream of a promoter-less purN:gfp:gusA, was conjugated into purN host RU2249 and recombined into the genome. After removal of colonies that expressed the reporter genes of the vector under laboratory conditions, the library was inoculated into a nonsterile pea rhizosphere. The key result is that 29 rhizosphere-induced loci were identified. Sequence analysis of these clones showed that a wide variety of R. leguminosarum A34 genes are expressed specifically in the rhizosphere including those encoding proteins involved in environmental sensing, control of gene expression, metabolic reactions and membrane transport. These genes are likely to be important for survival and colonization of the pea rhizosphere.

Friends or relatives? Phylogenetics and species delimitation in the controversial European orchid Genus ophrys

Background and Aims Highly variable, yet possibly convergent, morphology and lack of sequence variation have severely hindered production of a robust phylogenetic framework for the genus Ophrys. The aim of this study is to produce this framework as a basis for more rigorous species delimitation and conservation recommendations. Methods Nuclear and plastid DNA sequencing and amplified fragment length polymorphism (AFLP) were performed on 85 accessions of Ophrys, spanning the full range of species aggregates currently recognized. Data were analysed using a combination of parsimony and Bayesian tree-building techniques and by principal coordinates analysis. Key Results Complementary phylogenetic analyses and ordinations using nuclear, plastid and AFLP datasets identify ten genetically distinct groups (six robust) within the genus that may in turn be grouped into three sections (treated as subgenera by some authors). Additionally, genetic evidence is provided for a close relationship between the O. tenthredinifera, O. bombyliflora and O. speculum groups. The combination of these analytical techniques provides new insights into Ophrys systematics, notably recognition of the novel O. umbilicata group. Conclusions Heterogeneous copies of the nuclear ITS region show that some putative Ophrys species arose through hybridization rather than divergent speciation. The supposedly highly specific pseudocopulatory pollination syndrome of Ophrys is demonstrably 'leaky', suggesting that the genus has been substantially over-divided at the species level.

Evidence of a possible role for Lys-gamma 3-MSH in the regulation of adipocyte function

Lys-gamma 3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma 3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma 3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma 3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma 3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma 3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyper-phosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma 3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.

The role of exotic plants in the invasion of Seychelles by the polyphagous insect Aleurodicus disperses: a phylogenetically controlled analysis

The accidental introduction of the spiralling whitefly, Aleurodicus dispersus Russell (Homoptera: Aleyrodidae) to Seychelles in late 2003 is exploited during early 2005 to study interactions between A. dispersus, native and exotic host plants and their associated arthropod fauna. The numbers of A. dispersus egg spirals and pupae, predator and herbivore taxa were recorded for eight related native/exotic pairs of host plants found on Mahe, the largest island in Seychelles. Our data revealed no significant difference in herbivore density (excluding A. dispersus) between related native and exotic plants, which suggests that the exotic plants do not benefit from 'enemy release'. There were also no differences in predator density, or combined species richness between native and exotic plants. Together these data suggest that 'biotic resistance' to invasion is also unlikely. Despite the apparent lack of differences in community structure significantly fewer A. dispersus egg spirals and pupae were found on the native plants than on the exotic plants. Additional data on A. dispersus density were collected on Cousin Island, a managed nature reserve in which exotic plants are carefully controlled. Significantly higher densities of A. dispersus were observed on Mahe, where exotic plants are abundant, than on Cousin. These data suggest that the rapid invasion of Seychelles by A. dispersus may largely be due to the high proportion of plant species that are both exotic and hosts of A. dispersus; no support was found for either the 'enemy release' or the 'biotic resistance' hypotheses.

Outlining eicosanoid biosynthesis in the crustacean Daphnia

Background: Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX) pathway; the lipoxygenase (LOX) pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Results: Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda) together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. Conclusion: We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.

Macroevolutionary patterns in the origin of mutualisms involving ants

Ants are a diverse and abundant insect group that form mutualistic associations with a number of different organisms from fungi to insects and plants. Here, we use a phylogenetic approach to identify ecological factors that explain macroevolutionary trends in the mutualism between ants and honeydew-producing Homoptera. We also consider association between ant-Homoptera, ant-fungi and ant-plant mutualisms. Homoptera-tending ants are more likely to be forest dwelling, polygynous, ecologically dominant and arboreal nesting with large colonies of 10(4)-10(5) individuals. Mutualistic ants (including those that garden fungi and inhabit ant-plants) are found in under half of the formicid subfamilies. At the genus level, however, we find a negative association between ant-Homoptera and ant-fungi mutualisms, whereas there is a positive association between ant-Homoptera and ant-plant mutualisms. We suggest that species can only specialize in multiple mutualisms simultaneously when there is no trade-off in requirements from the different partners and no redundancy of rewards.

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

The role of Wnt signalling in the development of somites and neural crest

The Wnt family of secreted signalling molecules controls a wide range of developmental processes in all metazoans. In this investigation we concentrate on the role that members of this family play during the development of (1) the somites and (2) the neural crest. (3) We also isolate a novel component of the Wnt signalling pathway called Naked cuticle and investigate the role that this protein may play in both of the previously mentioned developmental processes. (1) In higher vertebrates the paraxial mesoderm undergoes a mesenchymal-to-epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists which result in the segmental plate being unable to form somites. These results show that Wnt6, the only member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. (2) The neural crest is a population of multipotent progenitor cells that arise from the neural ectoderm in all vertebrate embryos and form a multitude of derivatives including the peripheral sensory neurons, the enteric nervous system, Schwann cells, pigment cells and parts of the craniofacial skeleton. The induction of the neural crest relies on an ectodermally derived signal, but the identity of the molecule performing this role in amniotes is not known. Here we show that Wnt6, a protein expressed in the ectoderm, induces neural crest production. (3) The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement (Pandur et al. 2002b). Recent work has identified the protein Naked cuticle as an intracellular switch promoting the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked cuticle-1 (cNkd1) and demonstrate that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly our study shows that the expression of cNkd1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Environmental variation at the onset of independent foraging affects full-grown body mass in the red fox

The period following the withdrawal of parental care has been highlighted as a key developmental period for juveniles. One reason for this is that juveniles cannot forage as competently as adults, potentially placing them at greater risk from environmentally-induced changes in food availability. However, no study has examined this topic. Using a long-term dataset on red foxes (Vulpes vulpes), we examined (i) dietary changes that occurred in the one-month period following the attainment of nutritional independence, (ii) diet composition in relation to climatic variation, and (iii) the effect of climatic variation on subsequent full-grown mass. Diet at nutritional independence contained increased quantities of easy-to-catch food items (earthworms and insects) when compared with pre-independence. Interannual variation in the volume of rainfall at nutritional independence was positively correlated to the proportion of earthworms in cub diet. Pre-independence cub mass and rainfall immediately following nutritional independence explained a significant proportion of variance in full-grown mass, with environmental variation affecting full-grown mass of the entire cohorts. Thus, weather-mediated availability of easy-to-catch food items at a key developmental stage has lifelong implications for the development of juvenile foxes by affecting full-grown mass, which in turn appears to be an important component of individual reproductive potential.

A comparison of the active surveillance of scrapie in the European Union

The abattoir and the fallen stock surveys constitute the active surveillance component aimed at improving the detection of scrapie across the European Union. Previous studies have suggested the occurrence of significant differences in the operation of the surveys across the EU. In the present study we assessed the standardisation of the surveys throughout time across the EU and identified clusters of countries with similar underlying characteristics allowing comparisons between them. In the absence of sufficient covariate information to explain the observed variability across countries, we modelled the unobserved heterogeneity by means of non-parametric distributions on the risk ratios of the fallen stock over the abattoir survey. More specifically, we used the profile likelihood method on 2003, 2004 and 2005 active surveillance data for 18 European countries on classical scrapie, and on 2004 and 2005 data for atypical scrapie separately. We extended our analyses to include the limited covariate information available, more specifically, the proportion of the adult sheep population sampled by the fallen stock survey every year. Our results show that the between-country heterogeneity dropped in 2004 and 2005 relative to that of 2003 for classical scrapie. As a consequence, the number of clusters in the last two years was also reduced indicating the gradual standardisation of the surveillance efforts across the EU. The crude analyses of the atypical data grouped all the countries in one cluster and showed non-significant gain in the detection of this type of scrapie by any of the two sources. The proportion of the population sampled by the fallen stock appeared significantly associated with our risk ratio for both types of scrapie, although in opposite directions: negative for classical and positive for atypical. The initial justification for the fallen stock, targeting a high-risk population to increase the likelihood of case finding, appears compromised for both types of scrapie in some countries.

Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish scyliorhinus canicula defines ancient and derived roles for fox genes in vertebrate development

In the human genome, members of the FoxC, FoxF, FoxL1, and FoxQ1 gene families are found in two paralagous clusters. Here we characterize all four gene families in the dogfish Seyliorhinus canicula, a member of the cartilaginous fish lineage that diverged before the radiation of osteichthyan vertebrates. We identify two FoxC genes, two FoxF genes, and single FoxQ1 and FoxL1 genes, demonstrating cluster duplication preceded the radiation of gnathostomes. The expression of all six genes was analyzed by in situ hybridization. The results show conserved expression of FoxL1, FoxF, and FoxC genes in different compartments of the mesoderm and of FoxQ1 in pharyngeal endoderm and its derivatives, confirming these as ancient sites of Fox gene expression, and also illustrate multiple cases of lineage-specific expression domains. Comparison to invertebrate chordates shows that the majority of conserved vertebrate expression domains mark tissues that are part of the primitive chordate body plan.

The use of ultraviolet-blocking films in insect pest management in the UK; effects on naturally occurring arthropod pest and natural enemy populations in a protected cucumber crop

Studies in polytunnels were conducted to investigate the effects of ultraviolet (UV)-blocking films on naturally occurring insect pests and their arthropod natural enemies on a cucumber crop. Within tunnels clad with Antibotrytis (blocks light < 400 nm) and UVI/EVA (UV transmitting), 5.8 and 23.4 times more aphids, respectively, were recorded on traps compared with those on traps within tunnels clad with XL 385 (blocks light < 385 nm). When all plants within the UVI/EVA tunnels had become heavily infested with aphids, half of the plants in XL 385 tunnels were uninfested. More Coleoptera and thrips (approximately two times) were recorded under the UVI/EVA film than under the UV-blocking films, but for other arthropod pests (e. g. whitefly, leafhoppers), clear conclusions could not be drawn as low numbers were recorded. Substantial numbers of chalcid parasitoids and syrphids were found under the UV-blocking films, but further research is needed to evaluate fully the effect of such films on biological control of aphids. Higher syrphid numbers and more aphid mummies were recorded under the UVI/EVA film, probably because of the higher numbers of aphids present in tunnels clad with this film. The potential that UV-blocking films have as an effective component of commercial Integrated Pest Management (IPM) systems, for protected horticultural crops, is discussed.

Are Cape floral clades the same age? Contemporaneous origins of two lineages in the genistoids s.l. (Fabaceae)

The hypothesis that the elements of the modern species-rich flora of the Cape Floristic Region (CFR), South Africa, originated more or less simultaneously at the Miocene/Pliocene boundary, in response to the development of a mediterranean climate, has been challenged by numerous molecular dating estimates of Cape floral clades. These studies reveal a more gradual emergence, with the oldest clades originating in the Eocene, but others appearing later, some as recently as the Pliocene. That there are factors which might affect the dates recovered, such as choice of calibration point, analysis method, sampling density and the delimitation of Cape floral clades, suggests a need for further critical evaluation of the age estimates presented to date. In this study, the dates of origin of two Cape floral clades (the legume Crotalarieae p.p. and Podalyrieae) are estimated, constrained by a shared calibration point in a single analysis using an rDNA ITS phylogeny in which 633 taxa are sampled. The results indicate that these two clades arose contemporaneously 44-46 mya, not at the Miocene/Pliocene boundary as had been previously supposed. The contemporaneous origin of these Cape floral clades suggests that additional more inclusive analyses are needed before rejecting the hypothesis that a. single environmental trigger explains the establishment of Cape floral clades. (c) 2007 Elsevier Inc. All rights reserved.

Diversification of a large genus in a continental biodiversity hotspot: temporal and spatial origin of Muraltia (Polygalaceae) in the Cape of South Africa

Phylogenetic relationships in the largely South African genus Muraltia (Polygalaceae) are assessed based on DNA sequence data (nuclear ribosomal ITS, plastid atpB-rbcL spacer, trnL intron, and trnL-F spacer) for 73 of the 117 currently recognized species in the genus. The previously recognised subgenus Muraltia is monophyletic, but the South African endemic genus Nylandtia is embedded in Muraltia subgenus Psiloclada. Subgenus Muraltia is found to be sister to subgenus Psiloclada. Estimates show the beginning of diversification of the two subgenera in the early Miocene (Psiloclada, 19.3+/-3.4 Ma; Muraltia, 21.0+/-3.5 Ma) pre-dating the establishment of the Benguela current (intermittent in the middle to late Oligocene and markedly intensifying in the late Miocene), and summer-dry climate in the Cape region. However, the later increase in species numbers is contemporaneous with these climatic phenomena. Results of dispersal-vicariance analyses indicate that major clades in Muraltia diversified from the southwestern and northwestern Cape, where most of the species are found today.