Quirks of dye nomenclature. 8. Methylene blue,azure and violet

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.     

Bacterial networking

A report of the ESF-EMBO Symposium Bacterial Networks (BacNet08), Sant Feliu de Guixols, Spain, 13-18 September 2008.     

Interactions in the uptake of amino acids, ammonium and nitrate ions in the Arctic salt-marsh grass, Puccinellia phryganodes

The uptake of amino acids and inorganic nitrogen by roots of Puccinellia phryganodes was examined to assess the potential contribution of soluble organic nitrogen to plant nitrogen uptake in Arctic coastal marshes, where free amino acids constitute a substantial fraction of the soil‐soluble N pool. Short‐term excised root uptake experiments were performed using tillers grown hydroponically under controlled conditions in the field. The percentage reductions in ammonium uptake at moderate salinity (150 mm NaCl) compared with uptake at low salinity (50 mm NaCl) were double those of glycine, but glycine uptake was more adversely affected than ammonium uptake by low temperatures. Glycine uptake was higher at pH 5·7 than at pH 7·0 or 8·2. The glycine uptake was up‐regulated in response to glycine, whereas ammonium uptake was up‐regulated in response to ammonium starvation. Nitrate uptake was strongly down‐regulated when tillers were grown on either ammonium or glycine. In contrast to N‐starved roots, which absorbed ammonium ions more rapidly than glycine, the roots grown on glycine, ammonium and nitrate and not N‐starved prior to uptake absorbed glycine as rapidly as ammonium and nitrate ions combined. Overall, the results indicate that amino acids are probably an important source of nitrogen for P. phryganodes in Arctic coastal marshes.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Genetic diversity in the Eurasian otter, Lutra lutra, in Scotland. Evidence from microsatellite polymorphism

The relationship between microsatellite diversity and geographical fragmentation and isolation was studied in Scottish populations of the Eurasian otter, Lutra lutra. The geographic range of the study encompassed isolated archipelagos, islands adjacent to the Scottish mainland and both fragmented and continuous mainland populations. Tissue samples of 496 individuals from across Scotland were assayed for polymorphism at ten microsatellites. The isolation of populations on Shetland, and to a lesser degree on Orkney, was associated with reduced levels of microsatellite diversity. Most of the remaining island and fragmented mainland populations contained levels of microsatellite diversity similar to the high levels observed in die continuous mainland populations. Unexpectedly, both island and continuous mainland populations showed similar rates of departures from mutation-drift equilibrium. Such departures could have arisen from a variety of local demographic processes besides population bottlenecks. Gene flow appeared to be a major factor maintaining microsatellite diversity in all of these populations except the one on Shedand.     

Centrosome defects cause microcephaly by activating the 53BP1‐USP28‐TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53‐mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53‐mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non‐centrosomal protein SMC5 is also TP53‐dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.     

Reinforcing the native Otter Lutra lutra population in East Anglia: an analysis of the behaviour and range development of the first release group

The British population of the Eurasian Otter Lutra lutra has shown a serious decline since 1957–1958. In England the surviving population is broken into small fragments which may not be viable. One way to strengthen the population to enable it to recover would be to insert breeding units into the gaps. A practical methodology using captive-bred Otters in groups of three is described. This was tested successfully in 1983–1985 in East Anglia, with the first two release groups now breeding. The male in the 1983 group was tracked using a radio harness to be sure of survival and to monitor behaviour and use the environment. Data were obtained on die development of a home range in the new environment. Also, useful information was gathered on swimming and hunting speeds, time of emergence, distance travelled each night, activity periods and time spent in each habitat. This behaviour is compared to that of previously tracked wild Otters in Scotland. The very close similarity of many features is encouraging and results may be taken as indicative of the behaviour of wild Otters in lowland Britain.     

Carbon isotope discrimination in irrigated and droughted potato (Solanum tuberosum L.)

Carbon isotope discrimination (Δ) was measured in irrigated and droughted potato. Under irrigation, Δ in leaflets at given nodes increased (P < 0.001) between 21 and 63 d after emergence (DAE), which was attributed to increasing stomatal conductance (g_s) during leaf expansion. The effect of leaf position on Δ was non-significant in mature leaves. Under drought, Δ decreased (P < 0.001) in successive leaves up the stem, reflecting changes in g_s and water stress. At each node Δ remained constant or decreased, suggesting that effects of water stress were greater than changes with leaf expansion. There were significant differences in Δ between cultivars in both treatments, and in the progressive decrease in Δ up the stem under drought. Differences in Δ between cultivars were consistent with differences in stomatal control of leaf water status following water stress. Values for Δ in tubers were consistently lower than in stem and leaf, and decreased more rapidly. Differences in Δ between cultivars did not reflect dry matter production in either treatment, and differences in water use were non-significant between cultivars under drought. So, plants can achieve similar dry matter production through different growth strategies when irrigated or droughted, and Δ does not provide a simple, indirect method of selecting for dry matter production under water stress.     

Selective breeding of entomopathogenic nematodes for enhanced attraction to a root signal did not reduce their establishment or persistence after field release

We recently showed that the efficacy of an entomopathogenic nematode (EPN) as a biological control agent against a root pest could be enhanced through artificial selection. The EPN Heterorhabditis bacteriophora was selected for higher responsiveness towards (E)-β-caryophyllene (EβC), a sesquiterpene that is emitted by maize roots in response to feeding damage by the western corn rootworm (WCR). EβC is normally only weakly attractive to H. bacteriophora, which is one of the most infectious nematodes against WCR. By selecting H. bacteriophora to move more readily along a EβC gradient we obtained a strain that was almost twice more efficient in controlling WCR population in fields planted with an EβC-producing maize variety. However, artificial selection for one trait may come at a cost for other important traits such as infectiousness, establishment and/or persistence in the field. Indeed, infectiousness was slightly but significantly reduced in the selected strain. Yet, this apparent cost was largely compensated for by the higher responsiveness to the root signal. Here we show that the selection process had no negative effect on establishment and persistence of field-released EPN. This knowledge, combined with the previously reported results, attest to the feasibility of manipulating key traits to improve the efficacy of beneficial organisms.Key words: entomopathogenic nematodes, tritrophic interactions, artificial selection, biological control, Diabrotica virgifera virgifera, western corn rootworm, persistence, establishmentDiabrotica virgifera virgifera LeConte (Chrysomelidae: Coleptera, western corn rootworm, WCR) is a major well established pest of maize in the American Corn Belt and more recently also in Europe.¹ The larval stages of this beetle can cause significant damages to maize roots, leading to reduction of plant growth, deficiencies in nutrient and water uptake, lodging, increased susceptibility to water stress and reduced grain yield.² This combination of factors result in an estimated loss of one billion US dollars per year in the USA.³ The pest has been introduced in Europe in the early ''90s,⁴ and it is expected that at full establishment the costs resulting from WCR damages will be half a billion Euros.⁵ Several strategies are available to control this soil-dwelling pest, including crop rotation, pesticides and transgenic Bt maize, but WCR can readily evolve resistance to each of these methods.^6–8 This is why efforts have been invested in biological control alternatives.Entomopathogenic nematodes (EPN) show great promise as biological agents against WCR.⁹ Root-produced volatiles appear to play an important role in the recruitment of EPN^10–13 and one such volatile, (E)-β-caryophyllene (EβC), has recently been identified for maize roots¹⁴ and was found to be an ideal below-ground alarm signal.¹⁵ EPN efficacy can be improved by exploiting the ability of WCR-damaged maize roots to emit the attractant.¹⁴ Further studies have shown the importance of choosing the right species of nematodes.¹⁶ Among the EPN species tested against WCR, Heterorhabditis bacteriophora has proven to be one of the most virulent nematodes,¹⁷ but it barely responds to EβC.¹⁶ We therefore recently selected H. bacteriophora for higher responsiveness to EβC.¹⁸ In the field, the selected strain exhibited better abilities to control WCR larvae, but logically only in maize plots with plants that emitted EβC. However, previous studies have shown that enhancing beneficial traits through selective breeding can incur costs and negatively alter other traits in the selected strain.¹⁹ For EPN such trade-offs after selective breeding have also been reported, for instance resulting in reduced storage stability²⁰ or a lower capacity to kill their hosts.²¹ After selection for enhanced responsiveness to EβC response, we observed a small, but significant negative effect on infectiousness of the selected strains. However, this drawback was readily outweighed by the improved ability to locate hosts in the field.¹⁸Not only infectiousness is a crucial trait for the successful use of EPN in biological control: establishment and persistence in the field are of decisive importance as well. These traits vary with EPN species and are determined by biotic factors such as pathogens and predators²² or abiotic factors such as soil type,²³ humidity,²⁴ temperature²⁵ or pH.²⁴ But the main factor that is thought to determine long-term persistence in the field is the presence of available host insects.²⁵ In field trials in Hungary, three EPN species, H. bacteriophora, H. megidis and Steinernema feltiae, were released to test their control potential against WCR. They all persisted at least as long WCR were present in soil, during the same year.²⁶ There was no significant difference between the three species in the establishment or persistence. Yet, independent of timing of application, EPN populations dramatically decreased within five months after application. The authors²⁶ propose that this short persistence is due to the absence of suitable alternative hosts in intensively cultivated crop fields in Europe.To determine if the selection for enhanced responsiveness to EβC went at a cost for establishment and persistence we compared these key traits for the original and the EβC-selected stains. Using a metal auger (2 cm diam.; 20 cm high), 310 soil samples were dug out either two days (establishment) or 28 days (persistence) after EPN application. The soil was placed in plastic boxes (4.5 cm diam.; 60 cm high) and as previously described²⁶ Tenebrio molitor (Coleoptera: Tenebrionidae) larva was placed as bait in the boxes. Presence/absence of EPN was evaluated by visually checking T. molitor larvae for EPN infection. Soil samples from areas where no EPN were applied served as controls. No significant differences were found between the original and selected strain of H. bacteriophora strain (factor “strain”), neither in establishment after two days nor in persistence after 28 days (factor “time”) (Fig. 1, two-way ANOVA, Ftime_1,35 = 2.937, p = 0.097; Fstrain_2,35 = 10.359, p < 0.001; Ftime × strain_2,35 = 1.202, p = 0.315, statistical differences within factors were calculated using a Bonferoni post-hoc test). Hence, the selection of H. bacteriophora for a better response to EβC had no consequence for how the nematodes settled in the experimental fields. Future efforts to improve the effectiveness H. bacteriophora against WCR might also include selection for increased persistence in soil. This would allow lower application rates and could provide growers with an affordable and efficient control strategy against this voracious pest.Open in a separate windowFigure 1Establishment and persistence of the original and a selected strain of H. bacteriophora. The selected strain (squares) established and persisted as well as the original strain (diamonds). The triangles represent control samples from plots where no nematodes were released. Establishment (after two days) and persistence (after 28 days) was equal for both strains. Moreover, the number of soil samples containing EPN after 28 days was not significantly lower than after 2 days, independently of treatment. A few nematodes were detected in the control samples but again no differences over time were detected. Error bars indicate the SEM. Different lower-case letters indicate statistical differences within establishment (after 2 days) or persistence (after 28 days) (p <0.05).So far, manipulation of tritrophic systems in order to improve biological control has been largely theoretical.^27–29 We show here that for EPN this approach is realistic and that their responsiveness to root-produced foraging signals can be enhanced without significant costs for other relevant traits. It has also been shown that the emissions of the signals by the plants can be enhanced.³⁰ Combining these strategies opens new perspectives for the development of ecologically sound strategies in pest management.