Replacement of a native freshwater macroinvertebrate species by an invader: implications for biological water quality monitoring

The rate of freshwater invasions may be increasing, and macroinvertebrate invaders can have significant impacts on native macroinvertebrate assemblage structure through biotic interactions. More pollution-tolerant invaders can often replace native species. We examined implications of a species replacement for accurate biological monitoring of river systems using biotic indices. Our study uses Northern Ireland and the Isle of Man as examples of countries that possess river networks with many riverine macroinvertebrate assemblages subject to invasion. The introduced amphipod crustacean Gammarus pulex has replaced the native species G. duebeni celticus in many rivers in N. Ireland and the Isle of Man. Extensive seasonal data sets (119 sites) from three river networks, Lough Neagh and the Lagan in N. Ireland, and island-wide in the Isle of Man, were used to investigate the assumed equivalence of the native and invader in biotic indices concerned with the water quality monitoring system. Based on the derivation of the Biological Monitoring Working Party (BMWP) score, the Average Score Per Taxon (ASPT), as an example of a commonly used biotic index of water quality, we found index scores were lower in G. pulex sites compared to G. d. celticus-only sites. This indicated that assemblages were dominated by taxa more tolerant of organic pollution in the invader sites and more sensitive in the native sites. Inclusion of the invader in generation of the ASPT index, overinflated the ASPT values obtained compared to those with the native’s inclusion. This questions the accuracy of the ASPT and similar indices in rivers where the invader had replaced the native. We argue that with invasion pressures increasing, the validity of water quality indices such as the BMWP/ASPT needs to be re-examined in catchments where invaders have replaced natives. Indices such as the BMWP/ASPT are based on family level taxa and are inevitably coarse in their resolution given the wide range of water qualities tolerated by different genera within families. We argue that this resolution is even more compromised by the presence of very pollution-tolerant invaders, who may have replaced natives in disturbed or degraded river systems. The whole structure of water quality indices such as the BMWP/ASPT may need revising to take into account the presence of invasive species within monitored assemblages.     

Electrochemical and optical sensing of reactive oxygen species: pathway to an integrated intracellular and extracellular measurement platform

A comprehensive understanding of ROS (reactive oxygen species)-dependent cellular interaction requires the previously unmet ability to simultaneously monitor the intra- and extra-cellular environments. The present review assesses the potential of novel electrochemical and fluorescent-based nanosensor approaches to address the limitations of existing techniques for ROS analysis. Data generated by these new approaches have already contributed significantly to current understanding of the roles that these species play in various in vitro scenarios. However, integration of these novel approaches has the potential to offer, for the first time, the unparalleled ability to measure simultaneously and in real-time ROS flux in both the intra- and extra-cellular environments.     

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.     

Bioinformatic prediction and confirmation of beta-adducin as a novel substrate of glycogen synthase kinase 3

It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein β-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), β-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of β-adducin by GSK3 promotes efficient neurite outgrowth in neurons.     

Differential physico-chemical tolerances of amphipod species revealed by field transplantations

Physico-chemical regimes of river systems are major determinants of the distributions and relative abundances of macroinvertebrate taxa. Other factors, however, such as biotic interactions, may co-vary with changes in physico-chemistry and concomitant changes in community composition. Thus, direct cause and effect relationships may not always be established from field surveys. Equally, however, laboratory studies may suffer from lack of realism in extrapolation to the field. Here, we use balanced field transplantation experiments to elucidate the role of physico-chemical regime in determining the generally mutually exclusive distributions of two amphipod taxa, Gammarus (two species) and Crangonyx pseudogracilis. Within two river systems in Ireland, the former species dominate stretches of well oxygenated, high-quality water, whereas the latter dominates stretches of poorly oxygenated, low-quality water. G. pulex and G. duebeni celticus did not survive in bioassay tubes in areas dominated by C. pseudogracilis, which itself survived in tubes in such areas. However, both C. pseudogracilis and Gammarus spp. survived equally well in tubes in areas dominated by Gammarus spp. Physico-chemical regime thus limits the movement of Gammarus spp. into C. pseudogracilis areas, but some other factor excludes C. pseudogracilis from Gammarus spp. areas. Since previous laboratory experiments showed high predation rates of Gammarus spp. on C. pseudogracilis, we propose that predation by the former causes exclusion of the latter. Hence, presumed effects of physico-chemical regime on macroinvertebrate presence/abundance may often require experimental field testing and appreciation of alternative explanations. Received: 14 June 1999 / Accepted: 20 January 2000     

Relation of structural features to pyrrolic metabolites in livers of rats given pyrrolizidine alkaloids and derivatives

Levels of pyrrolic metabolites have been measured in the livers of rats given some pyrrolizidine alkaloids and semisynthetic derivatives. Structural and chemical features favouring the formation of such metabolites have been defined. The most important of these were: steric hindrance or chemical properties giving resistance to ester hydrolysis; lipophilic character, allowing access to hepatic microsomal enzymes; a conformation favoring microsomal oxidation of the pyrroline ring in preference to N-oxidation. In addition, the presence of ester groups gave the resulting pyrroles high chemical reactivity, leading to tissue binding.Amounts of pyrroles bound to liver were very low when animals were given either highly water soluble pyrrolizidine derivatives, including non-esterified bases or more-lipophilic esters if these were easily hydrolysed. Compounds prone to hydrolysis gave increased pyrrole levels in rats pretreated to deplete their esterase activity. Whereas heliotridine-based alkaloids usually give more pyrrole than similar retronecine esters, heliotridine ditiglate gave less pyrrole than retronecine ditiglate because the former was more open to hydrolytic attack.Among the carboxylic diesters, the cyclic retronecine diesters, in which the pyrrolizidine nucleus is more exposed to oxidative metabolism, gave the highest pyrrole levels in rats.Liver pyrrole measurements are useful for studying relationships between molecular structure, metabolism and toxicity of pyrrolizidine derivatives. They can be used for screening alkaloids for potential toxicity and for assessing dose levels suitable for toxicity tests when limited material is available.     

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.     

AutoCSA, an algorithm for high throughput DNA sequence variant detection in cancer genomes

The undertaking of large-scale DNA sequencing screens for somatic variants in human cancers requires accurate and rapid processing of traces for variants. Due to their often aneuploid nature and admixed normal tissue, heterozygous variants found in primary cancers are often subtle and difficult to detect. To address these issues, we have developed a mutation detection algorithm, AutoCSA, specifically optimized for the high throughput screening of cancer samples. Availability: http://www.sanger.ac.uk/genetics/CGP/Software/AutoCSA.     

CNP/cGMP signaling regulates axon branching and growth by modulating microtubule polymerization

The peptide hormone CNP has recently been found to positively regulate axon branching and growth via activation of cGMP signaling in embryonic dorsal root ganglion (DRG) neurons, but the cellular mechanisms mediating the regulation of these developmental processes have not been established. In this study, we provide evidence linking CNP/cGMP signaling to microtubule dynamics via the microtubule regulator CRMP2. First, phosphorylation of CRMP2 can be suppressed by cGMP activation in embryonic DRG neurons, and non‐phosphorylated CRMP2 promotes axon branching and growth. In addition, real time analysis of growing microtubule ends indicates a similar correlation of CRMP2 phosphorylation and its activity in promoting microtubule polymerization rates and durations in both COS cells and DRG neuron growth cones. Moreover, direct activation of cGMP signaling leads to increased assembly of dynamic microtubules in DRG growth cones. Finally, low doses of a microtubule depolymerization drug nocodazole block CNP/cGMP‐dependent axon branching and growth. Taken together, our results support a critical role of microtubule dynamics in mediating CNP/cGMP regulation of axonal development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 673–687, 2013     

Interactions between imidacloprid and Metarhizium brunneum on adult Asian longhorned beetles (Anoplophora glabripennis (Motschulsky)) (Coleoptera: Cerambycidae)

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), a longhorned beetle species native to Asia, has been introduced into several North American and European cities. Currently eradication and preventive measures are limited to identifying and destroying infested trees and protecting uninfested trees with trunk or soil-injections of the systemic insecticide imidacloprid. Because entomopathogenic fungi like Metarhizium brunneum Petch have been identified as virulent against these beetles we conducted several tests to determine the compatibility of the two agents in combination. Radial hyphal growth and the sporulation capacity of M. brunneum on Sabouraud dextrose agar with yeast were not significantly affected by the presence of imidacloprid. In a 2 × 3 factorial experiment investigating interactions between exposure to imidacloprid and M. brunneum we observed no effect of imidacloprid alone on beetle survival when beetles were given a single dose of 10 or 100 ppm compared to control insects. We observed a significant effect of exposure to M. brunneum, and a significant interaction between imidacloprid and M. brunneum representing a synergistic effect of dual treatment. Beetles exposed to the fungus alone lived significantly longer compared to insects treated with a single dose of 100 ppm imidacloprid (9.5 vs. 6.5 d). Consumption of striped maple twigs by beetles exposed to imidacloprid, across concentrations, was reduced 48% compared to control insects, where as consumption by M. brunneum-exposed beetles was reduced by 16% over the first 6-days of the test period. Beetles fed 100 ppm imidacloprid consumed 32% less over the first 3 d compared to beetles not exposed to imidacloprid and thereafter consumed as much as beetles not fed 100 ppm imidacloprid. M. brunneum-exposed beetles consumed significantly less food than control insects throughout the test period, and beetles treated with imidacloprid produced significantly fewer conidia compared to beetles not treated with imidacloprid.