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Infection processes consist of a sequence of steps, each critical for the interaction between host and parasite. Studies of host-parasite interactions rarely take into account the fact that different steps might be influenced by different factors and might, therefore, make different contributions to shaping coevolution. We designed a new method using the Daphnia magna - Pasteuria ramosa system, one of the rare examples where coevolution has been documented, in order to resolve the steps of the infection and analyse the factors that influence each of them.
Using the transparent Daphnia hosts and fluorescently-labelled spores of the bacterium P. ramosa, we identified a sequence of infection steps: encounter between parasite and host; activation of parasite dormant spores; attachment of spores to the host; and parasite proliferation inside the host. The chances of encounter had been shown to depend on host genotype and environment. We tested the role of genetic and environmental factors in the newly described activation and attachment steps. Hosts of different genotypes, gender and species were all able to activate endospores of all parasite clones tested in different environments; suggesting that the activation cue is phylogenetically conserved. We next established that parasite attachment occurs onto the host oesophagus independently of host species, gender and environmental conditions. In contrast to spore activation, attachment depended strongly on the combination of host and parasite genotypes.
Our results show that different steps are influenced by different factors. Host-type-independent spore activation suggests that this step can be ruled out as a major factor in Daphnia-Pasteuria coevolution. On the other hand, we show that the attachment step is crucial for the pronounced genetic specificities of this system. We suggest that this one step can explain host population structure and could be a key force behind coevolutionary cycles. We discuss how different steps can explain different aspects of the coevolutionary dynamics of the system: the properties of the attachment step, explaining the rapid evolution of infectivity and the properties of later parasite proliferation explaining the evolution of virulence. Our study underlines the importance of resolving the infection process in order to better understand host-parasite interactions.
Host-parasite coevolution is the result of multiple adaptations and counter-adaptations evolving in concert within the constraints of a particular system. Hosts use diverse defence mechanisms that coevolve with the offensive mechanisms of the parasite. From phages to ectoparasites, the success of infection depends on a series of steps and for each of them, the hosts may have specific defence mechanisms [1,2]. The following steps may be distinguished, with greater or fewer steps potentially existing depending on the system and the level of resolution: The host encounter with the parasite is the first step. During this step, the host may exhibit particular behaviours in order to avoid the parasite [3] and there may be polymorphism for such behaviours within species [4]. Once the encounter has taken place, parasites with a dormant stage may need to be activated in order to terminate diapause and initiate the infection process - for example, by endospore germination [see, for example, [5]]. After the activation step, endoparasites need to enter the host tissues. For many parasites, including the one studied here, this occurs through the attachment of the parasites to the host tissues but hosts may evolve to prevent this attachment. For example, plants often have very specific mechanisms to prevent fungal pathogens from entering leaf tissue [6] and some species produce layers upon their epithelium - the first barrier against infection - to obstruct parasite penetration (for example, mucus in coral protection [7] or salivary mucins to preserve the oral cavity health [8]). After attachment and entering its host, the next step of infection is proliferation. To counteract parasite growth, the host adapts physiologically (for example, iron-withholding [9]) or actively defends itself with an immune response. In the final step of infection, the parasite releases transmission stages to infect other hosts.
It has been argued that the fact that infection trials often intermingle the effects of different infection steps strongly influences our interpretation of host-parasite interactions [1,10,11]. For example, if only one of the steps is specific, the entire infection process will be specific. The same is true for environmental effects and host genotype-parasite genotype interactions. Furthermore, even if each of the steps is under simple genetic control (one or few loci) the combination of all of them might behave as a quantitative genetic trait and become more difficult to investigate. Resolving the infection process into its component steps simplifies the complexity of the infection process and helps us to better understand host-parasite interactions. Evolutionary models of host-parasite interactions are usually based on relatively simple assumptions about the underlying genetics and the impact of the environment. They commonly consider binary (Yes/No) infection outcomes (for example, matching-allele matrix [12-14]), even though available experimental data suggests more quantitative outcomes when looking at host and parasite interactions [15-17]. Explicit analysis of individual steps of infection can help bring in line theoretical models and data concerning the entire infection.
As little is known about the degree of specificity of the individual steps, the specificity attributed to host-parasite interactions is usually the combined effect of all steps. Although it is reasonable to assume that different steps are under the control of different genes, and are influenced by the environment to different degrees, it is possible that a single component of the infection pathway may explain most of the observed variation in host-parasite interactions. This is particularly important because understanding variation in host susceptibility is central for controlling disease and understanding evolution. Here, we use the Daphnia-Pasteuria host-parasite system to investigate which step(s) best explain(s) the high degree of host genotype by parasite genotype interactions reported for this system [18-20]. We analyse the contribution of host and parasite genetics, host gender, host phylogeny and of the environment for the dynamics of host-parasite co-evolution.
Reproduction in planctonic crustacean Daphnia is primarily clonal, which is most suitable for dissociations of genetic and environmental effects of its interactions with parasites. Daphnia are frequently found to suffer from bacterial, fungal and microsporidial infections [21,22]; among them the Gram-positive bacterium Pasteuria ramosa [21-23]. P. ramosa produces endospores for transmission (Figure 1a and 1b[21]) that can remain dormant for decades [24]. Transmission is waterborne and endospores do not have flagella. The infection process is unknown but penetration of the host cuticula has been observed for the congeneric species P. penetrans, a parasite of root-knot nematodes [25]. Inside the host, P. ramosa proliferates in the haemocoel and musculature, castrates females and is transmitted horizontally after the release of endospores from the dead host [26,27]. The interaction of D. magna clones and P. ramosa clones has been shown to be specific [20]. Pasteuria was shown to impose strong selection on its host [28] and there is evidence for coevolution [29]. Furthermore, strong effects of the environment and genotype-environment interactions were reported for the overall infection process [30,31]. The goal of this study is to disentangle the different steps of the infection process and to analyse how they are shaped by host and parasite genetics, and the environment. We aim at finding the step that explains the greatest variance for the strong host-parasite interactions reported for the overall infection process.
Figure 1. Scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images of the activation and the attachment step of the infection process of Pasteuria ramosa in Daphnia magna. (a) SEM image of a resting stage of P. ramosa. (b) TEM image of resting stage before activation. The exosporium (ex) encloses the two peripheral fibres (pf) and the endospore (en). (c) SEM image of activated spores trapped by Daphnia phyllopods. (d) TEM image of activated spores in Daphnia oesophagus. Top left: the spore is in the process of activating and shedding the exosporium. Bottom right: the activated spore with its sombrero-like structure in cross-section. Spore coat (sc) surrounding the cortex (cx). (e) TEM image of peripheral fibres (pf) and its microfibres on the upper side (upf) and on the lower side (lpf). The upper side is more furnished in microfibres and is more likely to play a role in the attachment. (f) TEM image of Pasteuria attached to the Daphnia oesophagus wall (ew). The nomenclature were defined according to the nomenclature of Pasteuria penetrans in [58].
We consider the following steps of the infection process and will investigate in details the second and the third, previously undescribed: (i) encounter; (ii) activation (once in contact with Daphnia, parasite endospores need a signal to germinate); (iii) attachment (the parasite must attach to the host and cross the host epithelium); (iv) proliferation (parasite proliferation and spore production); and (v) termination (killing the host to release spores). Environmental and host clone effects have been shown for the encounter and the proliferation steps [4,30,32-35]. However, neither of them can explain the strong host genotype by parasite genotype interactions described for the overall infection process in this system. Here, we localize where the activation and attachment steps take place and test for the genetic and environmental factors that influence those steps.
Spore activation
We developed a new method that traces fluorescently-labelled spores of P. ramosa in the transparent Daphnia magna hosts in order to investigate the activation of parasite spores and the attachment of the parasite to the host. Within minutes of exposing the Daphnia host to the P. ramosa spores, we observed a characteristic change in spore morphology. Spores acquire a sombrero-like structure (Figure 1c and 1d) which corresponds to the shedding of the exosporium and the extension of the peripheral fibres. This morphology was never observed in spores not exposed to hosts. We call this morphological change in spore shape 'activation'. Activation was found to happen regardless of the host clone or Pasteuria clone used and was observed in both resistant and susceptible D. magna clones (Table 1).
Table 1. Results of infection trials, spore activation tests and attachment-tests.
Spore attachment
We used different combinations of hosts and parasite clones previously characterized to be resistant or susceptible to given Pasteuria clones [20]. We observed the fate of fluorescent spores of three parasite clones exposed to 14 D. magna host clones with the aim of identifying differences which correlate with the compatibility of a given host-parasite combination (Table 1). The parasites attach to the host oesophagus for all susceptible (compatible) host-parasite combinations, but they never do so for the resistant combinations (Table 1, Figures 1f and 2a). Thus, the result of this attachment-test was 100% consistent with the results of infection trials (Table 1). For susceptible combinations the host oesophagus was densely covered with spores forming a dense layer in the oesophagus, while there were no spores attached in resistant combinations. We never observed ambiguous cases (for example, only few spores attached). While spores in the mid and end gut moved with the flow of the food, those attached to the oesophagus were not disturbed by passing boluses of food, indicating that they strongly adhere. In resistant combinations spores were never seen attached to the oesophagus and all spores passed with the flow of the food through the gut (Figure 2b). Thus, spore attachment in the oesophagus was very specific to the D. magna and P. ramosa genotype and consistent with resistant/susceptibility status for each combination.
Figure 2. Fluorescently labelled parasite spores attach to the oesophagus of susceptible, but not resistant, Daphnia clones. (a) Picture of a susceptible Daphnia magna exposed to fluorescently labelled spores. The entire animal is shown. Parasites are attached on the epithelium of the oesophagus (arrow). Other labelled spores can be seen with the rest of the food in the end gut (arrowhead). (b) Picture of a resistant D. magna exposed to fluorescently labelled spores. The entire animal is shown. The oesophagus is free of parasite (arrow). Labelled spores can be seen with the rest of the food in the end gut (arrowhead). Note the autofluorescence of the mandibule. Extended focus images obtained by the camera Leica DFC 300FX and the program Leica Application Suite (Version 3.4.0, package 'Montage'). Intensity, contrast and sharpness were increased with the same strength.
Influence of gender and culture conditions
Activation of spores was observed in all treatments and in all host clone-Pasteuria clone combinations (Table 2). In contrast, the specificity revealed by the attachment-test was found to be independent of host gender, temperature and culture conditions (single versus crowded; high versus low food, Table 2).
Table 2. Influence of the environment and host gender on spore attachment, as determined by the attachment-test.
Spore activation and resistance of other Daphnia species
Spores were found to be activated after exposure to all Daphnia species tested (Table 3). We found that spores of the P. ramosa clone C19 were able to attach to the oesophagus and infect D. dolichocephala (Table 3) but did not attach to the oesophagus or infect D. arenata, D. galeata, D. barbata, D. similis or D. lumholtzi. We also tested other species for spore activation of P. ramosa. Upon exposure to Simocephalus vetulus (Daphniidae) spores were readily activated but did not attach to the oesophagus or infect any of the individuals tested. Upon exposure to mosquito larvae (Culex spp.), which are also filter-feeding but are not crustaceans, P. ramosa spores were neither activated nor attached to the host.
Table 3. Relationship between one Daphnia magna-derived clone of Pasteuria ramosa (clone C19) and several Daphnia species belonging to three different subgenera (D. magna belongs to the subgenus Ctenodaphnia).
The aim of the current study was to analyse two steps in the life cycle of a bacterial parasite, characterize the specificity of the interaction, with regard to genetic and environmental factors, and relate these findings to what is known about host-parasite coevolution in this system. We focused on the activation of the parasite's resting stages and on the attachment of the activated spores to the host tissue where it enters the host. Our study revealed that P. ramosa spores captured by the filter feeding Daphnia are indiscriminately activated by every Daphnia clone and Daphnia species tested (Tables 1 and 3). Furthermore, activation was not only found to be independent of the host genotype or species and host gender but also of the environmental conditions (namely, density, temperature and food conditions). The following step of the infection process, however, the attachment of the activated spore to the oesophagus wall of the host, depended strongly on the combination of the D. magna and parasite genotype, but not on the host's gender, nor the environmental conditions in which they were kept (Tables 1, 2, 3).
Previous studies with the Daphnia-Pasteuria system were not able to disentangle the activation, attachment and proliferation steps. Thus, variation in infection success as reported in earlier studies [19,32,35-40] may be explained by the combined effects of these steps. However, the binary polymorphism found in infection trials with high doses of single parasite clone [20] correlates perfectly with the results of our attachment-test (Table 1). This suggests that only Pasteuria clones able to attach to the oesophagus are able to infect the host. Ben-Ami et al. [38] proposed that D. magna might be either completely resistant or susceptible to P. ramosa depending on the genotype-genotype interaction. They called this the 'binary infection hypothesis'. Our data are consistent with this hypothesis and further pinpoint which specific step of the infection process is responsible for the high degree of specificity. For a given combination of host and parasite genotypes, the activated spores are either able to attach and then infect or they do not attach and do not infect. We did not see any evidence for a graded (quantitative) form of interaction.
Spore attachment is a key step in Daphnia-Pasteuria coevolution
The Daphnia-Pasteuria system has become one of the prime examples of antagonistic coevolution. Host and parasites show strong genetic effects for resistance, virulence and infectivity; genotype × genotype interactions have been reported within and across populations and selection acts rapidly in natural populations [18,19,40,41]. Our study suggests that the parasite-dependent [28] host population structure and the coevolution [29] described for this system are mainly driven by the properties of a unique step, the attachment step. First, this step revealed very strong host genotype by parasite genotype interactions (Table 1). Second, the attachment step is independent of the environmental conditions. Third, a recent study of D. magna - P. ramosa coevolution using resurrected host and parasite isolates from lake sediments showed a signal of fluctuating selection only for infectivity but not for parasite virulence [29]. Virulence (the parasite's effect on infected hosts) was also observed to evolve but at a slower rate [29]. The authors proposed that the difference between the evolution of virulence and infectivity resulted from different genes contributing to these traits. Here we give a mechanistic explanation for this finding. Infectivity depends on the attachment and, most likely, on the ligands present on the host and on the parasite. On the other hand, expression of virulence may depend on the host's immune response during the within-host proliferation step. It is likely that these processes are determined by different sets of genes.
The identification of the attachment step as the key step in the coevolutionary dynamics in this system will allow us to improve our understanding of the patterns of antagonistic coevolution. For example, evolutionary models studying the coevolution of the infectivity and the virulence steps [42] can fit our system in relation to the coevolution of the attachment and the proliferation steps. Those models typically characterize infection outcomes as binary (Yes/No), while empirical data suggest they are more quantitative [15-17]. We show that we can observe a binary outcome when individual steps of the infection process are considered. Furthermore, our method provides a fast and reliable way to test individuals and populations for their susceptibility to Pasteuria. Ongoing research in our group showed that up to 400 Daphnia individuals can be tested in a day (P Luijckx, in preparation). The assay we developed makes it possible to test for susceptibility without the potentially confounding effect of the within-host proliferation step in the infection trials.
From the environment to the host body cavity
The resting endospores of P. ramosa can remain dormant for decades under harsh environmental conditions [24,29]. Before attachment to the host, the spores need to be activated (Figure 1d). The filter-feeding Daphnia capture particles, including parasites, from the water and transport them on a mucus-layered pathway from the phyllopods to the mouth. During this process, the parasite's exosporium opens by an unknown trigger, releasing the activated spore form within less than 10 min (Figure 1). Despite the fact that spore activation is a necessary step for the infection, this step is entirely unspecific with regard to Daphnia species and clone, host gender and the environmental conditions (Tables 1, 2, 3). The signal that triggers spore activation may be related to chemical substances in the mucus of the filtering apparatus, but other factors (for example, mechanical) cannot be excluded.
Once the activated spore enters the oesophagus, it will attach to the oesophagus wall if the host and the parasite genotype are compatible. There it presumably penetrates the gut wall and enters the host's body cavity. A similar attachment process on the cuticula is also known from P. penetrans but, in this case, the parasite seems to be able to attach to any area of the nematode's body surface [25]. It has been proposed that the lower part of P. nishizawae attaches to the host because this part is densely covered by microfibres [43]. In contrast, in P. ramosa it is the upper part of the peripheral fibres (Figure 1e) that are most densely covered with a layer of microfibres. These fibres may be involved in the attachment (Figure 1f).
An endospore adhesin epitope, situated on the exosporium of P. ramosa, has been identified and it has been suggested that it may be a ligand responsible for the recognition and the binding onto the host [44]. However, according to our results, it is unlikely that this epitope is involved in the attachment because the exosporium of P. ramosa is removed during the activation step. A later study, analysing surface proteins of P. ramosa spores by two-dimensional gel electrophoresis, proposed that a collagen-like protein may be responsible for the binding onto the host but might suffer the same problem of the previous study [45]. We propose that later studies on candidate proteins responsible for the specific attachment to the host in this system should investigate the spores once activated.
The development of Pasteuria, from the moment they attach to the oesophagus until the vegetative stage can be detected in the hemolymph (about 8 days at 20°C [46]), is unknown. Also, the penetration mechanism is poorly described. Sayre and Wergin [25] show a transmission electron micrograph of P. penetrans with a structure they call a 'germ tube' crossing the host epithelium. Our hypothesis is that the endospore makes a hole across the host epithelium and injects its cortex into the host. As one response of Daphnia to wounding is an increase of Phenoloxidase (PO) activity [47], one might expect the penetration process to trigger an immune response but this remains an open question. However, resolving the infection process will allow the study of the immune response during the proliferation step without the confounding effect of genetic variation in the attachment step.
Environment effects and the proliferation step
We found that environmental effects do not influence the activation and attachment step (Table 2). Excluding these steps, we suggest that the proliferation step is the one responsible for the reported sensitivity of the overall infection process for environment effects [32,34]. The activation and the attachment steps seem independent of the host's immune system (defined as a system that is potentially able to kill parasites), while the proliferation step is likely to be governed by the host's immune system. The immune system may lead to variation between and within those Daphnia clones that allow Pasteuria attachment (and, thus, enable the parasite to enter the host), thereby contributing to local and temporal adaptation, maternal effects and induced resistance [29,34,48]. We suggest that future studies on host immunity should use only Pasteuria clones that can attach to a given clone of Daphnia so that all the variation observed is likely to originate from variation during the proliferation step. These factors highlight the importance of controlling the host and parasite genotypes and breaking down the infection process in order to understand the respective role of each step in host-parasite interactions.
Resolving the infection process leads to a better understanding of host-parasite interactions
Resolving the infection process in its sequential steps has been proposed in a number of theoretical models [10,11] but experimental data are scarce. Our approach is transferable to other host-parasite systems and our results suggest that this can provide important new insights into host-parasite interactions and their evolution. Increasing the degree of the resolution of the infection processes highlights a universe of possibilities of the different levels at which host and parasites interact. The different steps might differ in how they are influenced by the environment. They might also differ in which sets of genes regulate them. As it is probably the case for our study system, different steps of the infection process might follow distinct evolutionary dynamics and be explained by different model (for example, balancing selection, directional selection) [10,11]. However, because of the sequentiality of the steps, it is possible that the selection on one might depend on the selection on other steps. We propose that analysing infection as a succession of well characterized steps will help to reconcile the empirical data with predictions based on alternative coevolutionary models (for example, Red Queen and Selective Sweep models).
Spores of all P. ramosa clones tested, and which were isolated from natural D. magna populations, were activated by all D. magna clones as well as by six other Daphnia species (Table 3) and even a Cladoceran from a different genus, Simocephalus vetulus. Also, apart from the natural host, D. magna, D. dolichocephala, also became infected following attachment of the activated spores to the host oesophagus. This suggests that the triggers for spore activation and, to a lesser extent, for attachment are phylogenetically conserved. This may facilitate the host range evolution of the parasite. Indeed, despite its high specificity on the level of the host clone, P. ramosa infections have been reported in several species within the family Daphniidae [49]. It will be necessary to test more clones of different Daphnia species in order to determine their pattern of susceptibility and resistance to the parasite. Importantly, phylogenetically conserved steps of the infection process can be ruled out as major factors in coevolution, but are, perhaps, the most appropriate targets for vaccine and drug development. In fact, the genes involved in some infections steps have been worked out for some systems [50,51] and can be of use in biomedicine for diseases control [52,53].
Our study highlights the explanatory power of resolving the steps of the infection process in order to better understand host-parasite interactions and coevolution. Attachment appears to be the crucial step for the previously observed high specificity in the Daphnia-Pasteuria system and we speculate that it is the crucial step for coevolution as observed in this system [29]. Our results reveal that each step can involve different interactions between host, parasite and environment and that certain steps can be phylogenetically conserved. With this knowledge, it will be easier to apply simple models of host-parasite interactions to this system and identify the mechanistic basis of trade-offs, maternal effects, genotype × environment interactions and coevolution. The logic of this procedure can equally be applied to other host-parasite systems but also to study other types of biotic interactions.
Host and parasite
We used 14 isofemale lines (hereafter referred as clones) of D. magna and one clone each of six other Daphnia species (Tables 1 and 3). Unless otherwise stated, Daphnia clones were kept in standard medium (ADaM, [54] modified by using only 5% of the recommended Selenium dioxide concentration) at 20°C and fed with the chemostat cultured unicellular algae, Scenedesmus obliquus.
The parasites used were single genotypes of P. ramosa, C1, C14 and C19, characterized as clones in Luijckx et al. [20] and originated from D. magna populations in Moscow (Russia), Tvärminne (Finland) and Gaarzerfeld (Germany), respectively. Spore suspensions of Pasteuria were obtained by homogenizing infected D. magna in ADaM and quantifying spore density. The status of resistant or susceptible D. magna were defined previously [20]. The infection status of two further Finnish D. magna clones ('Kela-39-09' and 'Kela-18-10') exposed to Pasteuria clones were tested with the same protocol. All infections in these experiments were done with naïve individuals born to naïve mothers, kept under high food conditions. These conditions were applied because they are known to minimize the triggering of immune effect [34,35,55].
Fluorescence labelling of spores
Fluorescently labelled spores of P. ramosa were produced by homogenizing infected Daphnia in ADaM, followed by centrifugation at 10 000 g for 5 min at room temperature. The spore pellet was suspended in 0.5 mL of 0.1 M sodium bicarbonate (pH 9.1) containing 2.0 mg/mL of fluorescein-5(6)-isothiocyanate (F3651-100MG, Sigma-Aldrich, Miss, USA), a green fluorescent dye that stains proteins unspecifically [56]. Spores were incubated in the dark for 2 h at room temperature with occasional vortexing. The suspension was centrifuged at 10 000 g for 5 min and the supernatant removed. The spore pellet was suspended in distilled water and, again, subjected to centrifugation. This process was repeated until the supernatant was clear. Labelled spore suspensions can be stored at 4°C in the dark for several months.
The shape and location of the green labelled spores were examined in the transparent Daphnia using a microscope with fluorescent light (Leica DM 2500, at magnification 200 × and 400 ×) and filter cubes Leica B/G/R (bandpass filter excitation 420/30 nm; 495/15 nm; 570/20 nm - band pass filter suppression 465/20 nm; 530/30 nm; 640/40 nm). We increased the colour contrast by adding a red fluorescent dye to the medium in which the Daphnia were observed. This was done by preparing a solution of concentrated red dye (0.05 mL of DMSO with 0.0015 g of Tetramethylrhodamin-5-isothiocyanate; T0820-5MG by Sigma-Aldrich), which was homogenized in PBS to make the diluted dye (1 μL of concentrated solution with 10 mL of phosphate buffered saline). We added 1 μL of this solution to the Daphnia medium 10 min before observing the Daphnia. We obtained extended focus images using a camera Leica DFC 300FX and the program Leica Application Suite (Version 3.4.0, package 'Montage').
The separation of the different steps and their specificity
Adult Daphnia were put individually in 1 mL of medium in 24-well-plates and exposed for at least 1 h to around 17,000 labelled P. ramosa spores. Susceptible hosts exposed to labelled spores become infected, suggesting that the dye does little or no harm to the spores (data not shown).
Spore activation
Pilot trials revealed that the labelled spores remain in their typical spherical shape as long as they are not in contact with a host. Upon contact with the host phyllopods (swimming and respiratory appendages of branchiopod crustaceans), spores with a sombrero-like shape are observed (Figure 1c and 1d). We called this process 'spore activation'. We tested all combinations of 14 D. magna clones and three P. ramosa clones for spore activation (Table 1). The same was done for one clone each of six further Daphnia species but only in combination with one P. ramosa clone (Table 3). We used five replicates for each host-parasite combination [in total (14 × 3 × 5) + (6 × 1 × 5) = 190; details in Table 1].
Spore attachment
After exposure, Daphnia were placed on a microscopic slide and we examined the complete Daphnia body under a fluorescent microscope. The transparent body of Daphnia allowed us to determine in which body region activated fluorescent spores attach in the living animals. Once we determined the specific area, we tested resistant and susceptible Daphnia magna clones (five replicates of 14 clones, details in Table 1) for differences in attachment. The same was done with clones of other Daphnia species (five replicates of one clone per species; details in Table 3). In order to validate the assignment of individuals with apparently no spores attached to their oesophagus, we viewed the oesophagus of slightly squashed animals at 400 × magnification. For each experiment, the examiner was not informed whether the animals belonged to a susceptible or to a resistant clone. In order to confirm that the Daphnia ingested spores, the gut content was inspected for the presence of spores. All exposed animals had spores in the faeces. We call this procedure to test for spore attachment the 'attachment-test'.
Influence of gender and culture conditions
In order to see if the specificity pattern observed in the attachment-test was dependent on host sex or culture conditions, 10 host individuals of each sex were tested in each of six treatments. This was done with D. magna clones 'Kela-39-09' and 'Kela-18-10"'because these two Daphnia clones have the reverse pattern of infectivity to the two P. ramosa clones used and they are easily induced to produce male and female offspring in the laboratory. Daphnia were raised either at one of four temperatures (10°C, 15°C, 20°C, 25°C, with high food), two food levels (at 20°C, fed daily with 2.5 or 5 million algae) or two density levels (at 20°C, high food level, single Daphnia or Daphnia from crowded stock cultures; see Table 2). These conditions were chosen to represent various environments that are common in natural Daphnia populations. We did not employ a full factorial design, as our interest was not in establishing reaction norms but in testing for the influence of non-genetic conditions in general. Daphnia of both clones raised under these conditions were exposed to fluorescently labelled spores of P. ramosa C1 and C19. Given the very clear effects observed with the four combinations of hosts and parasites used and the range of conditions tested, we do not believe that other combinations would drastically change our results. However, we cannot exclude with certainty that some combinations might lead to a different result.
Resistance or susceptibility of other Daphnia species
One clone of each of six other Daphnia species (D. arenata, D. dolichocephala, D. galeata, D. barbata, D. similis and D. lumholtzi) were assayed for their propensity of oesophageal spore attachment using P. ramosa clone C19. For this assay, groups of five conspecific individuals were exposed to 200,000 P. ramosa spores of clone C19 in 20 mL medium. Four replicates per species were used. After 5 days we filled the jars to 100 mL and then changed the medium on a weekly basis. Animals were fed daily with 5 to 10 million algal cells per jar depending on the size of the Daphnia species. The infection status was investigated under a microscope with phase contrast (magnification 400 ×), at host death or 29 days after exposure.
Electron microscopy
In order to prepare Daphnia for transmission electron microscopy (TEM), infected individuals were fixed on ice in 4% glutaraldehyde buffer in Sorensen's phosphate buffer (0.1 M KH2PO4 and 0.1 M Na2HPO4) and kept in the dark for several hours. The animals were then rinsed five times on ice using the same buffer for a total of 5 min. Post-fixation was carried out with 1% OsO4 in Sorensen's phosphate buffer on ice. After post-fixation, the Daphnia were again washed in Sorensen's phosphate buffer on ice, dehydrated in a graded acetone series, and finally embedded in the epoxydic resin EPON.
Transversal and sagittal sections were made through the oesophagus. Semi-thin sections (diamond knife, 0.7-1 μm) were cut in order to approach the right spot on the resin block using a RMC MT 6000-XL (RMC Inc, AZ, USA) ultramicrotome. In order to identify regions of interest for TEM, the tissue was stained using Richardson's dye [57] and examined under a light microscope. In order to see parasite structures using TEM, 5-8 ultrathin sections (diamond knife, 60 nm) were cut after every 10 semi-thin sections. The ultra-thin sections were mounted on Formvar-coated copper grids and stained with uranyl acetate and lead citrate to enhance the contrast. Ultrathin sections were analysed using a FEI Morgagni™ transmission electron microscope at 80 kV equipped with a digital camera.
For scanning electron microscopy (SEM), D. magna were fixed in 3% glutaraldehyde buffer in 0.1 M phosphate buffer for 2 h at 20°C. Samples were washed twice in distilled water for 5 - 10 s, dehydrated in graded ethanol series and critical point dried overnight (16 h). The specimens were coated with gold (20 nm) and viewed using a Philips XL 30 ESEM under high volume conditions from 5 to 15 kv.
Authors' contributions
DD conceived and designed the study, performed the experiment, performed data analysis and drafted the manuscript. PL participated in the design of the study. FB performed the SEM. CL performed the TEM. DE conceived of the study, participated in its design and participated in drafting the manuscript. All authors read and approved the final manuscript.
We thank M. Ackermann and L. Du Pasquier for their thoughtful discussions; N. Boileau, J. Hottinger, M. Kredler and U. Stiefel for their laboratory assistance; and J. Andras, P. Beldade, S. Zweizig and the three anonymous reviewers for their comments on the manuscript.
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J Bacteriol 1977, 129:1091-1101. PubMed Abstract | PubMed Central Full Text
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Duncan AB, Little TJ: Parasite-driven genetic change in natural population of Daphnia.
Evolution 2007, 61:796-803. PubMed Abstract | Publisher Full Text
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Nature 2007, 450:870-U816. PubMed Abstract | Publisher Full Text
Mitchell SE, Rogers ES, Little TJ, Read AF: Host-parasite and genotype-by-environment interactions: Temperature modifies potential for selection by a sterilizing pathogen.
Evolution 2005, 59:70-80. PubMed Abstract
Vale PF, Little TJ: Measuring parasite fitness under genetic and thermal variation.
Heredity 2009, 103:102-109. PubMed Abstract | Publisher Full Text
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Ecology 2008, 89:313-318. PubMed Abstract | Publisher Full Text
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Am Naturalist 2010, 175:106-115. Publisher Full Text
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Little TJ, Carius HJ, Sakwinska O, Ebert D: Competitiveness and life-history characteristics of Daphnia with respect to susceptibility to a bacterial pathogen.
J Evolutionary Biol 2002, 15:796-802. Publisher Full Text
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Little TJ, O'Connor B, Colegrave N, Watt K, Read AF: Maternal transfer of strain-specific immunity in an invertebrate.
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Preston JF, Dickson DW, Maruniak JE, Nong G, Brito JA, Schmidt LM, Giblin-Davis RM: Pasteuria spp.: systematics and phylogeny of these bacterial parasites of phytopathogenic nematodes.
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Richardson KC, Jarett L, Finke EH: Embedding in epoxy resins for ultrathin sectioning in electron microscopy.
Chen ZX, Dickson DW, Freitas LG, Preston JF: Ultrastructure, morphology, and sporogenesis of Pasteuria penetrans.
Phytopathology 1997, 87:273-283. PubMed Abstract | Publisher Full Text


text:
RAF helicopters touch down at Trent Valley Academy in Gainsborough
Posted: September 01, 2012|
RAF helicopters touched down on a Gainsborough school's sports field to fulfil a promise to call in when they were passing.
Flight Lieutenant Jamie Anderson was at the controls of his Puma when it made a brief landing at Trent Valley Academy in Gainsborough.
The two-minute touchdown, during exercises from RAF Benson in Oxfordshire, was witnessed by only a handful of students and staff.
But the brief encounter came after 33 Squadron and the TVA history department honoured three of its First World War aviators.
Flight Lieutenant Anderson was one of four RAF Benson officers to attend a re-dedication ceremony in April, following the restoration of four damaged headstones in nearby General Cemetery.
Following research by TVA's head of history Peter Bradshaw, the memorials to the brave quartet, who lost their lives more than 90 years ago, defending the town from the Zeppelin menace, were restored to their full glory.
Argentinian Frank Benitz and Canadians Carey Pinnock, John Brophy and Arthur Menzies were all laid to rest in the cemetery on Cox's Hill after crashing while serving with 33 Squadron, based in Gainsborough during the Great War.
"I was really impressed that they came to both the ceremony and the school," said Mr Bradshaw. "And it was only at a day's notice that they contacted is to say they were going to be in the area.
"So we've had some frantic ringing round to get children in during the holidays to welcome them."
And the brief touchdown was a personal experience for 15-year-old George Forrest, who will be TVA's new head boy from September.
"The backdraft and the sound of the engines was amazing when they landed," said George.
"And it was even better because we knew at least one of the pilots. I met Flight Lieutenant Anderson when he came up to Gainsborough in April."
In charge of the aircraft was Squadron Leader Stewart Staudinger, who is in the RAF but originally from Canada.
He said: "We wanted to drop by and thank the people of Gainsborough for the tremendous work they have done in repairing the headstones of those brave men who paid the ultimate sacrifice.
"It is important to us on 33 Squadron that we maintain the community links with the people of Gainsborough."

subset: commoncrawl


text:
Hang tight!
Recipe by Cheryl
"This is a very good tasting cake, and easy to make."
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1 (3.4 ounce) package
instant pistachio pudding mix
1 (18.25 ounce) package
white cake mix
1/2 cup
vegetable oil
1 1/2 cups
1 1/2 cups
2 (1.5 ounce) envelopes
instant dessert topping
I have a couple of suggestions...not that the recipe isn't great they way it is..but I baked as it then sliced the cake in three layers - and iced in between the layers rather than only on the outside of the whole cake...although I had enough for both. Also, I dusted it with crushed pistachios. This cake is fabulous!!!!!
This is the recipe off of the Betty Crocker yellow cake mix box. It's for a yellow pound cake, but works well with any instant pudding flavor. I make this pistachio every year for my dad's birthday...but it's only 4 eggs and 1 cup of water (i use club soda instead)- it comes out moist every time. As for the frosting, I use 2 boxes of pistachio instant pudding with 1 pint of heavy cream (whipped) 1 cup of milk and add a little almond extract to it.
18 Ratings
Great Recipe! Just keep in mind that 1 box of vanilla pudding mix was left off the ingredient list(for the frosting)! Makes a beautiful Christmas cake with the white frosting and Christmas sprinkles. I have made this cake 3 times this Christmas season!
Okay I screwed up but it turned out great still! I've had requests to make it again!
I didn't read the directions well and put the milk in the cake on top of the water so it was quite moist!!! But it turned out great. Follow the real directions and I'm sure you'll like it. It takes some TLC but it works!
I am making one for Thanksgiving tomorrow by popular demand. Try it! I didn't think i'd like pistachio cake but it was wonderful!
i made the pudding mix like the recipe said and let it set up for a bit, then i folded in cool whip and let it set again for a bit. Then frosted the cake and refrigerated it. Patience paid off as it was the rave!
Plano, TX
There was no cake. It was like a piece of plastic, with all those eggs. Maybe my cake mix was on the small side? The cake dropped. I had made it for work for St. Patrick's Day, but it's going in the garborator. It was a disaster--something went wrong...I've triple checked the recipe. How did you guys get it to turn out??
Oh boy, what a disaster. I did as the recipe said, but it came out a cross between cake and custard. Maybe it has something to do with the order the ingredients are put in..or how long it's baked for. I also made 3 layers. Here's what I di...I could see this was going to be a flop as I watched it bake. So, I told my husband it was a new kind of desert called Pistachio jubilee. If he thought it was suppose to be a cake, he never would have tried it.! Well, he said it was good! I wouldn't make it again! Different,but good! Go figure!...
This is a great recipe. I also made a few minor changes. I use 24oz of cool whip, 1 cup heavy cream and a box of pudding for the icing and I cut into three slices and ice between each slice. Also I folded in the eggs one at a time to keep the cake from being too heavy or eggy tasting. This is hands down my families favorite cake for every occasion!
This is fantastic. Just as I remember having as a kid!!! My mom used to double the "icing" and mound most of it in the middle!! Fabulous!!
* Percent Daily Values are based on a 2,000 calorie diet.
Pistachio Cake I
Serving Size: 1/14 of a recipe
Servings Per Recipe: 14
Amount Per Serving
** Calories: 328
** Calories from Fat: 153
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text:
North Culinary Students Win Honors at MPTC
Eleven Sheboygan North High culinary arts students recently competed at the Moraine Park Technical College (MPTC) High School Culinary Olympics. Dia Xiong won a gold medal and scholarship in Layered Cake Decorating. Bronze medals were awarded to Hunter Lewis in Create A Plate; Mai Soua Chang in Layered Cake Decorating; and Katie Johnson, Chong Lee and Kiki Lo in Sandwich Construction Relay Team. An honorable mention (4th place) went to Rya Johnson in Layered Cake Decorating. Other students who also finished events successfully were David Callahan in Create A Plate and Derek Klockow, Kyle Mares and Adam Zitzer in Sandwich Construction Relay Team.
North Culinary Arts students won several honors on April 14 when they competed at the Moraine Park
Technical College (MPTC) High School Culinary Olympics.
Back Row: Derek Klockow, Kyle Mares, Adam Zitzer, Katie Johnson and David Callahan.
Front Row: Hunter Lewis, Kiki Lo, Rya Johnson, Mai Soua Chang, Dia Xion and Chong Lee.
Sheboygan Area School District Press Release
April 15, 2010


text:
Johnny Torque and Bobby Hart
Johnny opens wide so Bobby can fuck him deep! Hothouse says: Johnny Torque opens wide so Bobby Hart can fuck his face then stands up to fuck Bobby's ass. Bobby throws his legs over his head and opens his tight hole so Johnny can pound him hard. See Johnny Torque and Bobby Hart fucking here!


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Those of us who have had the great fortune of being initiated into a fraternity or sorority know that your letters stick with you for years to come. Just because you graduate and move on to other stages in life doesn't mean you're no longer a member of your beloved organization. At Greek Gear, you can outfit alumni with Greek gifts from head to toe. We also specialize in gifts for graduates and family members, including honorary four-legged members. Browse through this selection of stylish Greek alumni shirts, including ladies' and men's polos, T-shirts, sweatshirts, hoodies and hats specifically marked with your Greek letters and chapter. This impressive selection of alumni clothing guarantees that you'll find the right wearable item for your former sister or brother. The perfect gift for ex-students, Greek Gear's selection of alumni gifts would make any former fraternity guy or sorority girl happy. Whether it's mom, dad or a graduating member of your organization, these items are the most thoughtful thing you can give to a former member. Find gifts for the alums in your life who never want to forget the best years of their lives. Picture frames, stoles and tassels designed with your letters are a great way to show your loyalty to your organization even after you turn from active member to alum. Don't forget the four-legged friend who stayed by your side from rush to graduation. Dress Fido or Whiskers in Greek gear for dogs and cats with our variety of pet-friendly apparel, including bandanas, shirts, dog bowls, dog tags, collars and bowls marked with your fraternity or sorority letters. Dozens of other Greek items, from tote bags to flags to mugs to welcome mats, are available at Greek Gear and make great fraternity or sorority gifts. Remind graduating members that they're initiated for life with this selection of thoughtful gifts.


text:
Jason Gibbs, commissioner of the Department of Forests, Parks and Recreation, will step down May 14 so he can become a Republican candidate for Secretary of State.
Gibbs announced his intentions to his staff Thursday afternoon.
Gibbs served six years as spokesman for Gov. Jim Douglas before he was appointed commissioner in November 2008.
He has talked of running for secretary of state for several months. He said Thursday he wouldn't file to become a candidate, organize his campaign or begin to raise and spend money until he steps down as commissioner.
There are three other candidates who have already jumped into the secretary of state race because the seat will be open. The two Democrats are Charles Merriman and Jim Condos. The other Republican is Chris Roy.


text:
Click the button below or email firstname.lastname@example.com to contribute your experiences with this product. Although not necessary, it's interesting to have a first name, city, state and country (no email addresses will be used). Any information submitted becomes the property of Tennis Warehouse.
Please include any relevant information such as string type and tension, skill level and style of play.
Comments: Wow, what a pleasent stick! Control, spin -- it's all there. Due to the high stiffness rating, the comfort
is ok, no elbow problems. One of the best racquets for one handed backhands. I'm a bit surprised that it is not popular and that
TW didn't test this awesome stick. Strung with Alu Power Fluoro. I also tried the PS97, Ai 98, Radical Pro and this is the best choice
for me. Even the price is low.
From: Dexi, 3/15
Comments: Great 95 square inch players' racquet in the lighter side. My regular racquet is the old trusty Pro Staff
6.0 95, I found this one to be a great choice for those loking for a similar feeling yet lighter racquet, just need some changes in
terms of balance and weight, and it's good to go. Since it's a close to even balanced racquet, I like it with a leather grip, it takes
the racquet to something near 345 grams, with a more head-light balance. It's one of the best racquets that I have ever playtested
for my one handed backhand. Overall, a great stick for all-court attacking game. My only dislike is the oval beam (similar shape of
the 6.1), besides that, great stick from Dunlop.
From: Antonio, 1/14
Comments: This is basically what I've been looking for in a players' type racquet. Slightly less than 12 ounces, head
light with a tad more power than most players' racquets. I owned the 4D 200 Tour and it was great, but weighed in at 12.7
ounces. I went with the Prestige MP for 6 months and it is a fantastic racquet, a little dead in the upper hoop, but I wanted a little
more power. I would say this racquet is in between the IG Prestige MP and the IG Radical Pro line, kind of the best of what those
racquets offer. I have it strung at 56 lbs with RPM and might have to up the tension to 58 lbs to get where I want it. I demo'd
over the last 6 months, Volkl PB 10, Blade 98, PS 95, among others and this was my favorite.
From: Michael, 12/13
Comments: Prefer the Bio 200 to this one. This one is a bit too light, stiff and beam width is too wide for this type
of racquet. Prefer the plush feel of the Bio 200, but over time, I may get used to this one.
From: Tom, 11/13
Comments: Been a 200 fan since the Aerogel and the 4D was what I still used. Bought this one because I wanted a
stiffer and open string version of the 200 and the specs seemed to fall in place. I hit heavy topspin and have a one handed back
hand, and with the 4Ds I have lead tape at 9 and 3 strung up with poly at 55 lbs. The new version did feel stiffer and I did see an
improvement in spin, I felt like I lost some feel with the open string pattern and they changed the grips to more of a head shape it
seems. The racquet feels less stable and that was something I always loved about the 200 line, I've added weight to it and it feels
like I have that trust with my stability back. It does have a more modern feel but now that I have it set up the way I like I think I'll
feel more comfortable with the change.
From: Tri, 7/13
Comments: Update to my previous review. I made the switch to this frame the same week as a $15,000 pro event.
Scary, but went 3 rounds, losing a battle to a well-ranked ATP player. I am staying with this frame. I tried many strings, but the
Dunlop Explosive is my number one and number two is Dunlop Ice (but strung it way lower because it's very stiff).
From: RA, 7/13
Comments: Just an update to my previous review. This really plays different than the outgoing Bio 200 racquet. The
open string pattern (16 x 19) helps a ton on slice serves and the modern windshield wiper forehand. The old Dunlop 200 was
really more of a line drive racquet and one of the most arm friendly racquets ever made for a smaller head pattern of 95 sq inches.
This M 2.0 is definitely stiffer and just slightly more powerful.
From: Jon, 6/13
Comments: I really enjoy the 200 series and currently play with the Biomimetic 200. I saw the M2.0 had an open 16
x 19 string pattern and decided to buy it for more spin action. Well that is done, however the balance seems off, it swings not a
little but quite a bit heavier than my Biomimetic 200, feels like a a 200 Tour. I would advise this racquet for a stronger player.
From: Jon, 6/13
Comments: I had almost the exact same reaction as AH. Great feel, precision, power. I also made the switch.
From: SDB, 4/13
Comments: I was so impressed with this new M2.0. I always liked the Babolat APD and PD, but with a 1hbh, 100's
always seemed a bit 'out there.' This M2.0 (tried the F2.0 and it was too heavy and too oddly balanced) simply melted into my
has great feel and dwell time, and just bone-crushes the ball. Even my training coach said he'd never seen me hit better. Not
to get into each shot reviewed, simply that I had tried it all, literally, and made the switch.
From: AH, 4/13
Comments: This model is nothing like previous version's of the 200 series. Typically, the 200 series racquets were
flexy and feel oriented, this new incarnation is stiffer and has more inherent power and maybe a tad less control, reminds me of
old Wilson Hyper Pro Staff 5.0. It is taking me a little time to get used to the power, but Im starting to really enjoy the results!
From: Jeff, 3/13


text:
*image shown is 5 x 8 or larger rug
*smaller sizes will not show complete pattern, will be scaled down to fit.
Rating: 5.00
Read 1 Review >>
Name: Los Angeles Kings 1411 NHL Spirit Area Rugs
Manufacturer: NHL Rugs by Milliken
Collection: NHL Spirit
Material: Nylon
Construction: Machine Made
Colors: Blacks/Greys
Origin: USA
Description: Our NHL rugs are made of very soft, durable Nylon and have a non slip backing. Since our rugs are made to order they aren't sitting around collecting dust in a warehouse; they are brand new straight to you!! **RUGS ARE MADE TO ORDER AND SHIP WITHIN 7 to 10 BUSINESS DAYS ONCE ORDERED** (Los Angeles Kings 1411 NHL Spirit Blacks/Greys Machine Made Nylon Area Rug)
Other Related Patterns You Will


text:
Greg Olsen
Clifford Chance, London
Practice area M&A corporate, Antitrust competition, EU & trade
Sector Private equity, Mining & metals, Consumer goods & retail, Energy & resources, Banks, Insurance, Industrials
Tel +442070062327
Greg Olsen specialises in the application of EU and UK competition law to mergers, joint ventures and commercial arrangements as well as alleged behavioural violations such as cartels and abuse of market power.
Greg also advises on regulatory investigations in a diverse range of industries including financial services, insurance, media and consumer goods.
Relevant experience
- Advising Booker Group plc in obtaining Competition Commission clearance of its completed acquisition of Makro
- Advising NBCUniversal on the Competition Commission's Movies on Pay TV investigation
- Advising AB InBev in obtaining UK merger clearance for its US$52 billion acquisition of Anheuser-Busch
- Advising Barclays Bank PLC on the SME banking market study
Clifford Chance advises Citi on the sale of its consumer banking business in Greece
13 June 2014
Client Work
Clifford Chance advises Babcock International Group on its acquisition of Avincis Mission Critical Services
31 March 2014
Client Work
Clifford Chance advises GIC on the purchase of 50% interest in Broadgate
8 January 2014
Client Work
Career & qualifications
- University of Canterbury NZ (LLB) 1987
- University of Oxford, Keble College (B.C.L) 1990
- Winter Williams Commonwealth Scholar 1990
- Admitted as a solicitor in New Zealand 1990
- Admitted as a solicitor in England & Wales 2002
- Joined Clifford Chance 2007
Professional bodies
- Committee member and past Chair of the Law Society's Competition Law Section
- Trustee of the NZ-UK Link Foundation
Awards and citations
- Identified as a leading individual in Legal 500 UK 2013 (EU and competition)
- Ranked in Band 3 Competition/European Law in Chambers UK 2014
Clifford Chance publications and insights
20% of UK businesses have never heard of competition law
11 June 2015
Client Briefing
FCA to investigate competition in investment and corporate banking services
20 February 2015
Client Briefing
Too much of a good thing? Arguably. Article 9 commitments are being overused
21 October 2014
Client Briefing
European Commission reconsiders insurance sector exemption from antitrust laws
5 August 2014
Client Briefing
Financial Services and competition law : An overview of EU and national case law
28 July 2014
Client Briefing
Consumer Goods and Retail Industry Competition Bulletin July 2014
21 July 2014
Client Briefing
Financial Conduct Authority launches a review of competition in the wholesale financial sector
10 July 2014
Client Briefing
FCA updates retirement income market study terms of reference
11 June 2014
Client Briefing
Leasehold use restriction found to breach UK competition law
7 May 2014
Client Briefing
Upcoming EU restrictions on contractual auditor controls: implications for loan documentation
22 April 2014
Client Briefing
Clifford Chance partners explain changes to the UK competition regime
11 April 2014
Client Briefing
The New UK Competition Regime
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Business Lunch in Carrollwood
My husband is looking for a restaurant for a business lunch in Carrollwood. Friends have suggested Mimi's and Grillsmiths but I am looking for some other choices. Any ideas?
I am not a big fan of Mimi's. I really like Grillsmith, but have only been for dinner and/or drinks. There is a Bonefish in Carrollwood that is really good. There is another seafood place called Splash, but it's a little further north (not too far). And there is also a sort of high end place, but I forgot the name Grill 181 maybe. There is not much else other than these chains, but for chains I think they are halfway decent.
- The original comment has been removed


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Well put boryenka.
Someone underage sending someone else also underage photos of themselves should fall under PARENTS not GOVERNMENT.
This bill is a stupid waste of time.
Just add the emergency lanes!!
Well put mcn3ill
This sucks. Freak accidents happen. But we do need to keep drilling as long as we run on oil. Push for new technologies and we won't have to drill anymore.
Accident: An accident is a specific, identifiable, unexpected, unusual and unintended external action which occurs in a particular time and place, without apparent or deliberate cause but with marked effects.
There is no need for a Harahan PD, it's a waste of money. Get rid of it and let JP take over!
Time to put him (Leno) out to pasture...
Anyone sentenced to death should have their sentence carried out within 1 year. We need to stop wasting taxpayer $$ to house people that are going to die anyway.
I didn't know it was still on the air...
2nd that
So this guy comes out the gate slinging mud, what an idiot. I don't care what an candidate thinks about another candidate. Make the message worth the air time, tell me about YOU. I don't care who you are or what you are running for...trash another candidate = loose my vote.
That's horrible, might as well put up an 18 ft pink flamingo.
I guess the Legislature hasn't heard that we're in a recession.
It floods more in Jefferson Parish now than it did pre Katrina. WTF??? Forget another hurricane, if we get a tropical storm we're all fu$kd!
NOPD hire him.
They get killed for talking to the cops, sad but true.
The cheapest 1br around the area is at least $650 these days and thats for something small and crappy in a bad area.
Actually China is a 'Second World' country.


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We know how difficult it can be to choose a specialty when you don't know what it's like to work in particular fields. That's why we offer a unique Nursing Shadowing program that allows you to choose up to two sessions each series from 16 unique specialties.
Cardiac nursing is an incredibly intense environment; nurses are on the front-line of saving lives every day.
More information...
Each year over 87,000 adults visit BJH ER. No one shift is like the other; no one minute is like the next.
More information...
Shadowing a nurse at Barnes-Jewish Hospital is a great first step in choosing what specialty you want to work in upon graduation. This opportunity allows you to truly see what happens on a daily basis in a chosen specialty area before the application process begins.
Spending a few hours on a floor often eliminates misconceptions about a particular department and will better help you make an informed decision when considering where to begin your nursing career.
Learn more about our departments using the links on the right.
This is the most amazing program! I thought I wanted to go into ob/gyn, but after two sessions, I realised I really loved oncology. Who knew!?
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Make no mistake about it, what we are doing here is not, and has little hope of ever being, sustainable or self-sufficient. But that doesn't mean those aren't fundamental goals of the modern homesteader.
First of all, sustainability to me means not just for a community or a country, but for the world. Four people on 50 acres, even if it were a desirable prospect for the average person (and I know very few who even like to entertain the thought) could hardly happen in this country. In a country like India or China the proposition would be laughable. Few would applaud the latest self-sufficiency movement of urban homesteaders louder than I do, because I realize their goals have much more hope of being sustainable in our current global reality.
Because the nearest grocery store is 20 miles away we are more dependent on fossil fuels than city folks, and maybe even most suburban folks, too. But city and suburban life aren't for everyone, so I will continue to dream up some way that country life could ever again be considered remotely sustainable and somewhat self-sufficient.
I'm brand new at this, but like any industrious idealist, I have no doubt that we can make it happen, for us. I certainly don't have the answers to translate our inevitable success at homesteading, itself a loaded word as well, into a potential solution to fix any of the world's problems. Right now I'm just asking a lot of questions. And with every new question I look to those who have come before me. Sometimes they are old-timers, but other times they are the young, like Jenna Woginrich. In her new book Made from Scratch: Discovering the Pleasures of a Handmade Life, this 26 year old says, "The scary thing was that I was completely dependent of fossil fuels, and so was everyone I knew. My gas-heated apartment, my groceries from the supermarket, my station wagon parked outside-everything was part of the system. And if the system broke, I was going to be hungry, cold, and immobile. So I threw my hands in the air. I was done with Walmart and Wonder Bread. I wanted something real. I wanted something basic. I wanted a lifestyle that was no longer a part of the problem, or at the very least was constantly striving to be less involved in it." Thanks Jenna, my sentiments exactly.
The answers I'm continually seeking in this new lifestyle are sometimes practical, sometimes philosophical, and the words of all the others who are trying it their way too are always useful and inspiring to me.
What else I'm reading these days:
The Modern Homestead Manual: What it REALLY takes to Succeed Beyond the Sidewalks and Power Lines by Skip Thomsen and Cat Freshwater
The Self-Sufficient Life and How to Live It by John Seymour
VoluntarySimplicity: Toward a Way of Life That Is Outwardly Simple, Inwardly Rich by Duane Elgin
Complete Guide to Home Canning and Preserving by U.S. Department of Agriculture
Natural Beauty from the Garden by Janice Cox
The Woodlot Management Handbook by Stewart Hilts and Peter Mitchell
Lost Arts and Celebration of Culinary Traditions by Lynn Alley
Preserving Summer's Bounty A Rodale Garden Book
Storey's Basic Country Skills by John and Martha Storey
Texas Gardening the Natural Way by Howard Garrett


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SAN DIEGO -- The average couple, at age 65, is likely to need $261,000 to cover all their health care costs for the balance of their lives -- and those are just out-of-pocket costs, not those covered by Medicare. To make matters worse, health care costs overall are rising at about 5.8% a year.
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In Chechnya, troops killed a rebel leader and two Arabs serving with the rebel forces. Russia also announced that Chechnya would get a new flag, national anthem and coat of arms. Technically, Chechnya is a republic in the Russian federation. Russia is willing to grant more autonomy, but the current war is the result of Chechen Islamic fundamentalists crossing into adjacent Russian territory to establish an "Islamic Republic" in the Caucasus.
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Entertainment Weekly has the latest Breaking Dawn Part 1 posters and we are happy to report that they are just hot, and wolf-filled, enough to make us temporally stop dry heaving over that upcoming Breaking Dawn birth scene. We don't want to see an alien-looking Kristen Stewart giving birth to a monster slime baby via fang C-section! We don't wanna! Well, okay, we do wanna see that and we will see that, but before then we need a heavy dose of sexiness to balance it all out. Luckily these posters represent the the Twilight Saga we know and love it: smoldering with almost kisses, glowing with sunsets, and watched over by a pack of ravenous badass wolves, who just happen to be chillin' in the forest.
[Photo: EW]
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text: There's nothing quite like a World Cup to bring out a convoy of bandwagons. Suddenly, fans who wouldn't know a corner kick from a corner banquette get caught up in a drama and pageantry that is the world's greatest sporting event. And celebrities are no exception. On Instagram, national pride is everywhere—particularly among Brazilian supermodels—as stars sport their nations' uniforms and lead the cheering among their followers. Even Vice President Biden let his fan flag fly.


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Initiatives to help industry and government codify compatible cybersecurity requirements and capabilities are yielding some results, but acquisition experts say those plans have clouded federal cybersecurity acquisition efforts.
Participants in a May 22 panel, convened by the Coalition for Government Procurement in Tysons Corner, Va., to discuss how to navigate emerging cybersecurity acquisition rules, cited the National Institute of Standards and Technology's cyber framework and the joint "Improving Cybersecurity and Resilience through Acquisition" report issued by General Services Administration and the Pentagon, as two examples of useful programs.
But with sometimes-uneven acquisition and supply line protection rules across agencies, "the government isn't speaking with one voice" on cybersecurity acquisition, said Tom Barletta, a partner at Steptoe & Johnson who concentrates on federal acquisition law.
NIST's framework, meant to give critical infrastructure owners in 16 industries -- including banks, power and water utilities, and communications companies -- a more uniform approach to their cybersecurity practices was spurred by a February executive order. GSA's and the Defense Department's acquisition plans were submitted to the president as the order was rolled out.
David Bodenheimer, a partner at Crowell & Moring, said that as those rules and plans sink in, federal acquisition of cybersecurity IT can require expert navigation using the Federal Acquisition Regulation, Defense Federal Acquisition Regulation Supplement, the GSA Manual and other sources. "There are 12, 15, 20 different places to look" for guidance, he said.
Federal agencies are trying to simplify those rules for future contracts, said Richard Blake, business management specialist with GSA's Federal Acquisition Service Enterprise GWAC Center, particularly under the Alliant II and Alliant II Small Business government wide acquisition contracts. Those next-generation contracting vehicles look to provide comprehensive packages of IT solutions through customizable hardware, software and services.
Blake said his agency is looking for input from the private sector on how best to harmonize cybersecurity requirements in government contracts and the supply chain.
Emile Monette, senior advisor for cybersecurity at GSA's Office of Mission Assurance, said the GSA/DoD report "represents the best thoughts from government and the private sector" on how to proceed with cybersecurity-sensitive acquisitions. Given the sensitive nature of cybersecurity technology, Monette said, some vendors might be reluctant to provide official comments on the subject. He said he understood those concerns and urged companies to send their comments to him directly if they were concerned about their sensitivity.
Given that cyber threats are ever-changing, federal acquisition rules and regulations will continue to adapt to them, complicating and slowing the acquisition process, according to Don Johnson, who works in the Office of the Assistant Secretary of Defense for Networks and Information Integration.
That thought was echoed by Roger Waldron, president of the Coalition for Government Procurement.
"This struggle will continue as the threat evolves," Waldron told FCW in an interview at the conference. CGP will continue its efforts to keep members up to date on regulations, Waldron said, but he urged federal contracting officers and program managers to maintain flexibility in their processes to accommodate that threat evolution.
About the Author
Mark Rockwell is a staff writer covering acquisition, procurement and homeland security. Contact him at firstname.lastname@example.com or follow him on Twitter at @MRockwell4.
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Leidos, Accenture and Cerner pull off an unexpected win of the multi-billion-dollar Defense Healthcare Management System Modernization contract, beating out the presumptive health-records leader.
Millions of background-check records were compromised, OPM now says. Here's the jaw-dropping range of personal data that was exposed.
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Agency details results of an "interagency forensic investigation," plans for assisting affected individuals.
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Three-time Federal 100 winner is leaving government in July.
Members of Congress heaped hard questions on federal IT leaders at a June 16 hearing and suggested strongly that somebody needs to be fired.
Senate bill carves out exemption for supercomputing and other lab IT; an earlier version of the measure would have exempted the entire Department of Energy.
OPM is partnering with CSID to try to manage the fallout from a massive breach of some 4 million federal personnel records.
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Updated: 08/28/2014 6:00 PM
Created: 08/28/2014 4:34 PM WNYT.com
By: WNYT Staff
ALBANY - A symbol of urban blight in Albany will soon come down.
Habitat for Humanity and its partner, Housing Visions, have announced the demolition of a stretch of buildings on Sheridan Avenue.
The homes were once featured on Jeopardy as the definition of urban blight.
They're being torn down to make way for affordable housing.
"This is actually phase one of a three phase project that will be going on over the next several years. We are currently building ten homes up the block on Sheridan Avenue. Ten habitat homes and ten Touhey Foundation homes. So they will be brand new homes for our lower income families in the area," says Sara Rising of Habitat for Humanity.
Habitat says the first two homes on Sheridan will be completed by the end of September.


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The Sun's entry into the career area of your chart will boost your ambitions but you will do better in the long-term if you are realistic about what you can hope to achieve. Want more, but know your limits too.
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January 19, 2013 | 5:00am
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How it Works
Here is a quick overview of the consigning process with Just Between Friends.
1. Create a JBF Account to get a Consignor Number.
2. Register with a local sales event where you'd like to sell your items.
3. Prepare your merchandise, tag it and then bring it to the sale.
4. After the sale, you pick up any unsold items you do not wish to donate.
5. About 2 weeks later, you receive your consignor check in the mail!
Here's more detailed information on the process for you:
Anyone with children's and maternity items that are new or gently-used may register to sell as a JBF consignor. Feel free to go ahead and register now.
Many who start out as shoppers with JBF become consignors. Why? Because you can sell items you no longer need and then shop the sale for the items you do need! Plus, it lets you recoup some of the investment made purchasing your items.
Once you register with JBF, you will receive your Univeral Consignor Number which can be used to sell items at any JBF sales event. You will need to register for each sale with which you would like to consign.
Be sure to read through all email communications and your local JBF event website which is full of tips, dates/times, ideas and other important information about the sale. (NOTE: Typically, your local JBF hosts a Spring/Summer sale and a Fall/Winter sale each year. Some host a toy sale once a year, too).
After receiving your JBF Consignor number, you can log into your My JBF Profile to access the FREE tagging site. There you can create and print out your sales tags. You are then on your way! We have a Pricing Guide to help you determine the best price for your items. Then it's time to get busy preparing your items for the sale!
Prior to each sale, you will drop-off your items during the posted "DROP OFF" time. We encourage you to separate all clothing by gender and size before you arrive as this makes drop-off easier…and faster!
When you get to drop off, check in with the event coordinator. You will receive a consignor presale pass and, if applicable, an earlier Team Member presale pass. Next, you will bring your items to the "Inspection Area." While your clothing is being inspected, you can start placing all other items on the sales floor.
After inspection, you will hang your approved garments on the appropriate racks, typically by gender and size, before you leave.
You are now done with check in!
You will want to use your presale pass to shop the bargains yourself.
Once the sale concludes, items will be sorted for pick up. You can choose to donate unsold items to a local JBF charity or you can pick up unsold items. Then, about two weeks later, you will get your check (less a consignor fee which varies by sale)!
Please note that each sales event may have other specifics regarding the preparation of merchandise and drop off. Check your local sale website for further details.


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Related //
Galactic Spy
Become the galaxy's greatest spy – spot hidden characters, items, and other surprises in a fun new puzzle game.
LEGO Star Wars: Quest for R2-D2
Help find R2-D2 and hidden blueprints to build a powerful secret starship!
Star Wars Soundboard
Man the control panel of the incredible galaxy of Star Wars sounds, from classic quotes to sound effects!


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- ‹
- 12 of 45
- ›
- Start Over
After: Dramatic Wall Colour
You don't have to change much for a fresh look.
The original pale mustard-coloured tub and white walls were painted in a black-blue shade for dramatic effect. The medicine cabinet, pendant light and towel rack are the room's only new additions.
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Wenger said Arsenal had little choice but to let van Persie leave after the player engineered a move to one of their biggest rivals.
"We wanted to avoid it [the transfer] at all costs", Wenger told Telefoot. "They have the advantage of having played against him and so they knew his class.
"He's an outstanding player in every sense."


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Spanish Synagogue (Prague)
|Spanish Synagogue
Španělská synagoga
|Basic information
|Prague, Czech Republic
|Geographic coordinates
|50°05′25″N 14°25′15″E / 50.090278°N 14.420833°ECoordinates: 50°05′25″N 14°25′15″E / 50.090278°N 14.420833°E
|Reform Judaism
|Architectural description
|Architectural type
The Spanish Synagogue (Hebrew: בית הכנסת הספרדי, Czech: Španělská synagoga, German: Spanische Synagoge) is a Moorish Revival synagogue built in Prague in 1868 to the design of Vojtěch Ignátz Ullmann. The facade copies the form of the Leopoldstädter Tempel, built in Vienna, Austria, in 1853, a tripartite facade with a tall central section flanked by lower wings on each side. As in Vienna, the central section is topped by a pair of domed turrets.
The synagogue is most remarkable for the elaborate style of the interior, every surface is covered by elaborate Islamic-style polychrome and gilded patterns, some painted and some carved or molded.
During the Second World War, the Germans used the building as a repository for property taken from the Jews. The building underwent a restoration in the late 1990s.[1]
The building is owned by the Jewish Museum of Prague, and is used as a museum and concert hall.
Despite its name, the synagogue was never used by a Spanish or Sephardic congregation: it was in fact an early Reform temple. There are two theories to account for the synagogue's name.
- The Moorish architectural style may have been found reminiscent of the Golden age of Jewish culture in Spain.
- The synagogue was on the site of the city's most ancient synagogue, which may originally have been used by Byzantine Jews. Some awareness of this fact may have given rise to the legend of a historic Sephardic community.
See also[edit]
- Spanish Synagogue, Venice
External links[edit]
|Wikimedia Commons has media related to Spanish Synagogue in Prague.
|This article about a synagogue or other Jewish place of worship in the Czech Republic is a stub. You can help Wikipedia by expanding it.


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The University of Chicago relies on a service called Shibboleth to provide login authentication to University and external web services.
Shibboleth allows users with a valid CNetID to access multiple web applications with a single sign-on. Once a user logs in to one resource, they are granted access to multiple web applications, for which they have appropriate permissions, without being prompted to log in at each. When Shibboleth is utilized to control access to external web services, the system will evaluate the user's credentials and determine if access may be granted to the secure site. Whether or not the user may be granted access to secure external services with their CNetID depends on their affiliation with the University, various permissions associated with their account, and the requirements of the external service.
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Alumni account holders may contact email@example.com or call 1-877-292-3945 between 9 AM and 3 PM CST with any questions.


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Video:Repair Brick Wall Mortarwith Steve Crider
Though brick is low-maintenance, the elements and time can wear down brick mortar. Learn how to fix your home's brick walls with a technique called tuck pointing.See Transcript
Transcript:Repair Brick Wall MortarHi I'm Steve Crider for About.com Home. You know if you live in a brick house, chances are that sooner or later you're going to find out that brick is low-maintenance, not no-maintenance.
Brick Wall MortarOver time, the mortar starts to come out of these brick joints and they have to be refilled in a process called tuck pointing. Once upon a time you had to mix your own mortar. Now it comes in these handy tubes.
Prep the Brick WallFirst clean out the old joint with a brush and make sure it's clean and dry. Next, load up the tube in the caulk gun and go to work. Make sure that joint gets good and full. You'll also need one of these- this is called a jointer. Ask for it at your local home store. They'll know exactly what you are talking about.
Tuck Point the Brick WallNext, pull the tool along that joint and get a nice concave finish. Once it's dry, it will a lot like this.
Thanks for watching. To learn more visit us on the Web at homegarden.about.com. One down, and a lot to go!
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