Reproductive performance of asexual clones of the peach-potato aphid, ( Myzus persicae, Homoptera: Aphididae), colonising Scotland in relation to host plant and field ecology.

The population of peach-potato aphid, Myzus persicae (Sulzer), in Scotland comprises large numbers of a few superclones with much smaller numbers of other clones, and the reason for their differential success has yet to be elucidated. In the current study, the reproduction of lineages derived from these clones was measured by counting the numbers of offspring produced by a one-day-old nymph after 15 days. This was measured on four plant species, including local agricultural hosts and at two different temperatures (14 and 18 degrees C). There were significant differences in clonal lineage reproduction on different hosts and at different temperatures and amongst clonal lineages on the same hosts at the same temperature. Lineages of local insecticide sensitive clones did not have the best reproductive potential; instead, a recently introduced clonal lineage carrying MACE insecticide resistance was the best reproducer. The clonal lineage with the lowest reproductive potential also carried insecticide resistance, but this was kdr. A lineage from a local insecticide-sensitive clone was the least affected by reduced temperature. There was evidence of host plant specialisation in some of the clonal lineages.

Concerted and predictable acquisition of glial fibrillary acidic protein filaments by a cohort of astroblasts in culture.

Primary cultures of the 21 day foetal rat brain contain a cohort of astroblasts that concertedly acquire glial fibrillary acidic protein (GFAP) filaments between the 16th and 18th hour after plating. This burst of cytoskeletal differentiation is not observed in cultures initiated from the 18 day foetal brain and is not effected by the addition of cytosine arabinoside, an inhibitor of glial cell proliferation.

Acquisition of glial fibrillary acidic protein-containing fibres by astroglial cells in primary culture is orchestrated by a communicable factor.

In a previous study we discovered that primary cultures initiated from the whole brain of 21-day foetal rats contained astroblasts that concertedly acquired glial fibrillary acidic protein (GFAP) fibres. The mechanism of this burst of cytoskeletal differentiation could not be investigated in these cultures because it occurred too quickly (completed within 2 h). We report that cultures initiated from the region of the third ventricle display an extended burst of GFAP acquisition whose rate could be markedly reduced by medium changing. Temporary medium deprivation or the addition of cytosine arabinoside to the growth medium had no effect. Our findings suggest that an as yet uncharacterised communicable factor is involved in the orchestration of cytoskeletal differentiation in culture. This factor may be responsible for synchronising the appearance of GFAP-positive cells in the periventricular regions of the foetal brain.

Intraspecific Variation in Ribosomal DNA in Populations of the Potato Cyst Nematode Globodera pallida.

The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.

Innovative solutions to novel drug development in mental health.

There are many new advances in neuroscience and mental health which should lead to a greater understanding of the neurobiological dysfunction in neuropsychiatric disorders and new developments for early, effective treatments. To do this, a biomarker approach combining genetic, neuroimaging, cognitive and other biological measures is needed. The aim of this article is to highlight novel approaches for pharmacological and non-pharmacological treatment development. This article suggests approaches that can be taken in the future including novel mechanisms with preliminary clinical validation to provide a toolbox for mechanistic studies and also examples of translation and back-translation. The review also emphasizes the need for clinician-scientists to be trained in a novel way in order to equip them with the conceptual and experimental techniques required, and emphasizes the need for private-public partnership and pre-competitive knowledge exchange. This should lead the way for important new holistic treatment developments to improve cognition, functional outcome and well-being of people with neuropsychiatric disorders.

Clonal turnover of MACE-carrying peach-potato aphids (Myzus persicae (Sulzer), Homoptera: Aphididae) colonizing Scotland.

Peach-potato aphids, Myzus persicae (Sulzer), collected in Scotland in the years 1995 and 2002-2004 were characterized using four microsatellite loci and three insecticide resistance mechanisms. From 868 samples, 14 multilocus genotypes were defined (designated clones A-N). Five of these (denoted A, B, H, M and N) carried modified acetylcholinesterase (MACE) resistance, the most recent resistance mechanism to have evolved in M. persicae. The current paper shows that the continued presence of MACE aphids is due to turnover, as clones A and B were replaced in field samples by clones H, M and N in later seasons. Thus, insecticide-resistant populations in Scotland can be attributed to multiple waves of rapid clone colonisations and not to the continued presence of stable resistant clones or mutation or sexual recombination in local populations. The MACE clones carried varying levels of the other insecticide resistance mechanisms, kdr and esterase. The presence of these mechanisms could alter the clones success in the field depending on insecticide spraying (positive selection) and resistance fitness costs (negative selection).

Microsatellite marker analysis of peach-potato aphids (Myzus persicae, Homoptera: Aphididae) from Scottish suction traps.

The peach-potato aphid Myzus persicae (Sulzer) is an important vector of plant viruses. A network of suction traps collects aerial samples of this aphid in order to monitor and help predict its spatial distribution and likely impact on virus transmission in crops. A suction trap catch is thought to be a good representation of the total aphid pool. Sensitive molecular markers have been developed that determine the genetic composition of the M. persicae population. In Scotland, UK, these were applied to field collections revealing a limited number of clones. Molecular markers are less successful when applied to specimens that have been preserved in an ethanol-based trap fluid designed to preserve morphology. An assessment of different DNA extraction and PCR techniques is presented and the most efficient are used to analyse M. persicae specimens caught in the Dundee suction trap in 2001, a year when exceptionally high numbers were caught. The results reveal that the majority of the M. persicae caught belonged to two highly insecticide resistant clones. In addition, it was possible to compare the relative frequencies of genotypes caught in the trap with those collected at insecticide treated and untreated field sites in the vicinity. These results indicate that, in addition to suction trap data, the ability to sample field sites provides valuable early warning data which have implications for pest control and virus management strategies.

Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of Wolbachia.

Wolbachia are maternally inherited bacteria responsible for altering host reproduction. The two main groups found in insects, A and B, are based on molecular characterization using ribosomal, ftsZ, wsp (Wolbachia surface protein) or groE genes. We have used the wsp and ftsZ genes to study Wolbachia in byturid beetles. Byturus affinis contained a single copy of the ftsZ gene which grouped with A ftsZ sequences and a single copy of the wsp gene which grouped with B wsp sequences. This suggests that genetic exchange between A and B groups has occurred in the Wolbachia of this beetle. FtsZ and wsp sequences that were identical or nearly identical to those of B. affinis were found in B. tomentosus, suggesting that it also contains the same recombinant Wolbachia genotype. Most other byturids had more than one wsp sequence with at least one from the A and B groups, suggesting multiple copies of bacterial genes or multiple infections. B. ochraceus and B. unicolor both had four distinct wsp gene sequences. All the byturids had a closely related A wsp sequence and most a closely related B wsp sequence. Therefore, there appears to be an association between specific A and B wsp types.

The attack of the clones: tracking the movement of insecticide-resistant peach-potato aphids Myzus persicae (Hemiptera: Aphididae).

Myzus persicae (Sulzer) collected in Scotland were characterized for four microsatellite loci, intergenic spacer fingerprints and the resistance mechanisms modified acetylcholinesterase (MACE), overproduced carboxylesterase and knockdown resistance (kdr). Microsatellite polymorphisms were used to define a limited number of clones that were either fully susceptible to insecticides or possessed characteristic combinations of resistance mechanisms. Within these clones, intergenic spacer fingerprints could either be very consistent or variable, with the latter indicating ongoing evolution within lineages, most likely derived from the same zygote. Two clones (termed A and B) possessed all three resistance mechanisms and predominated at sites treated with insecticides. Their appearance on seed potatoes and oilseed rape in Scotland in 2001 coincided with extensive insecticide use and severe control failures. Clones C, I and J, with no or fewer resistance mechanisms, were found in samples from 1995 and were dominant at untreated sites in 2001. A comparison of Scottish collections with those from other UK and non-UK sites provides insight into the likely origins, distribution and dynamics of M. persicae clones in a region where asexual (anholocyclic) reproduction predominates, but is vulnerable to migration by novel genotypes from areas of Europe where sexual (holocyclic) reproduction occurs.

Analysis of eriophyid mite rDNA internal transcribed spacer sequences reveals variable simple sequence repeats.

Ribosomal DNA internal transcribed spacers of the eriophyid mites Cecidophyopsis ribis, C. selachodon, C. spicata, C. alpina, C. aurea, C. grossulariae and Phylocoptes gracillis were amplified using PCR, cloned and sequenced. Sequences for the ITS1 of Cecidophyopsids were 92-99% homologous. Cecidophyopsis inter-specific differences were found in seventeen simple sequence repeats (vSSRs), fourteen point mutations and two indels. No intra-specific variation in vSSRs was detected. A hypothetical structure for ITS1 was obtained and vSSRs were mapped onto this. Changes in vSSRs were compensated for by changes in complementary vSSRs or through multiple point mutations. A comparison with vSSRs of other arthropods suggested that the levels of intra-specific variation in Cecidophyopsis mites was less than in organisms which do not use arrhentoky for male determination.

A study of variation in rDNA ITS regions shows that two haplotypes coexist within a single aphid genome.

We report variation in the rDNA internal transcribed spacers (ITSs) of aphid species, the first for these insects. Variation at 6 sites within ITS1 sequences of the green peach aphid, Myzus persicae, identified two haplotypes coexisting within the same individuals, indicating that molecular drive has not homogenised different copies of rDNA. During this study, we found that PCR can cause a precise 58-bp loss in the amplified copies of an ITS haplotype (type 1). This occurs in all detectable copies under routine PCR conditions, at different annealing temperatures and with Pfu and Taq polymerases. In addition, "hot-start" PCR exclusively copied a different, rare haplotype (type 2). These observations have important considerations for using PCR, as large deletions in PCR products may not reflect real deletions in the genome, and changes in PCR conditions may be needed to copy cryptic haplotypes.

Glial fibrillary acidic protein in the cytoskeletal and soluble protein fractions of the developing rat brain.

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of clonal diversity of the peach-potato aphid, Myzus persicae (Sulzer), in Scotland, UK and evidence for the existence of a predominant clone.

Clones of the peach-potato aphid, Myzus persicae (Sulzer), mostly from Scotland, UK were examined using an rDNA fingerprinting technique. Eighty patterns (genotypes) were found amongst the 276 clones. A large number of clones (30%) from all sample areas in Scotland exhibited the same simple pattern, suggesting the presence of a single M. persicae clone. There was no difference in genotype distributions between M. persicae collected from brassica or potato crops, suggesting that host-adapted genotypes have no advantage in the field. Different fingerprints were randomly distributed in the environment, although clones taken from the same leaf were more often the same fingerprint. Highly distinctive fingerprints, which were more widely distributed, suggest that this technique could be used to follow individual clones. In addition to the common clonal type, multiple fingerprint bands were found over successive years, implying that, in Scotland, local overwintering asexual populations are the most common source of M. persicae in the following year.

Phylogeny of raspberry beetles and other Byturidae (Coleoptera).

Nuclear ribosomal ITS2 and mitochondrial cytochrome oxidase DNA sequences were analysed from the Byturidae (Coleoptera), which includes the raspberry beetles. The secondary structure of ITS2 was plotted and interspecific changes analysed. Evidence for selection on simple sequence repeats within the ITS2 was found. Phylogenetic trees based on the mitochondrial and ribosomal sequences were compared. They were in parity, indicating they reflect the true evolutionary histories of these insects. There was no evidence for hybridization in the populations surveyed, but there was evidence that the American raspberry beetle, Byturus unicolor, is divided into at least three distinct groupings. Despite sharing a related host, the raspberry pests from America were not the most related to the European raspberry beetle. Instead, links between Byturus affinis from Japan and the American raspberry beetle suggest that this lineage originated in Asia and colonized the Western USA.

Molecular evidence for multiple infections of a new subgroup of Wolbachia in the European raspberry beetle Byturus tomentosus.

Wolbachia, a group of maternally inherited intracellular parasitic bacteria, alter host reproduction, including the induction of thelytokous parthenogenesis, feminization of genetic males, son killing and, most commonly, the induction of cytoplasmic incompatibility (CI), in a diverse array of arthropods. CI can result in infertility and has attracted attention because of its potential in biological control and as an agent in speciation. Although there has been some analysis of overall infection rates in arthropods and within individual insect orders, there has been little exploration of within-species variation. In this study, primers specific for the ftsZ gene of Wolbachia were used to amplify it from different geographical samples of the European raspberry beetle (Byturus tomentosus), confirming the presence of Wolbachia. More than 99% of UK individuals were found to be infected with Wolbachia and 97% of these B. tomentosus beetles harboured multiple infections. Preliminary analysis of B. tomentosus beetles from continental European populations revealed a lower level of infection (24%) than those from the UK. Phylogenetic analysis using the ftsZ DNA sequences places Wolbachia from B. tomentosus into a new clade (Abt) within the A division, with some revisions to the existing Wolbachia phylogeny.

Gall mite molecular phylogeny and its relationship to the evolution of plant host specificity.

The phylogenetic relationships of all seven known species of Cecidophyopsis mites (Acari: Eriophyidae) with Ribes hosts have been inferred from ribosomal DNA sequences. This analysis found groups of closely related mites. The five gall-forming species, four of which are monophagous and one which has two hosts, were found in two groups. Another group consisted of the two non gall-forming species, one of which has two hosts, while the other is monophagous. The molecular phylogeny of their known Ribes host plants was calculated using the equivalent ribosomal regions as the mites. The structure of the two trees (mites vs hosts) was clearly different, implying that mite speciation did not closely follow speciation events in the plant hosts. Instead, the three groups of Ribes-infesting Cecidophyopsis mites have derived from a common galling ancestor millions of years ago. Each mite group has recently diversified onto different primary hosts. One group of mites has also lost the galling habit. The results have implications for host range changes and the durability of mite-resistance genes in cultivated Ribes.

Lactate utilization by isolated cells from early neonatal rat brain.

The utilization of lactate, glucose, 3-hydroxybutyrate, and glutamine has been studied in isolated brain cells from early newborn rats. Isolated brain cells actively utilized these substrates, showing saturation at concentrations near physiological levels during the perinatal period. The rate of lactate utilization was 2.5-fold greater than that observed for glucose, 3-hydroxybutyrate, or glutamine, suggesting that lactate is the main metabolic substrate for the brain immediately after birth. The apparent Km for glucose utilization suggested that this process is limited by the activity of hexokinase. However, lactate, 3-hydroxybutyrate, and glutamine utilization seems to be limited by their transport through the plasma membrane. The presence of fatty acid-free bovine serum albumin (BSA) in the incubation medium significantly increased the rate of lipogenesis from lactate or 3-hydroxybutyrate, although this was balanced by the decrease in their rates of oxidation in the same circumstances. BSA did not affect the rate of glucose utilization. The effect of BSA was due not to the removal of free fatty acid, but possibly to the binding of long-chain acyl-CoA, resulting in the disinhibition of acetyl-CoA carboxylase and citrate carrier.

Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria.

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.

Molecular analysis of ribosomal DNA from the aphid Amphorophora idaei and an associated fungal organism.

The ribosomal DNA cistron from the large raspberry aphid, Amphorophora idaei (Hemiptera: Aphididae), has been mapped by restriction analysis. The results showed that the map of A. idaei was similar to those of the previously characterized aphids Schizaphis graminum and Acyrthosiphon pisum. An extra Bgl II site was found in some of the ribosomal DNA intergenic spacer repeats in A. idaei. Using in-situ hybridization to aphid mitotic chromosomes it was demonstrated that probes derived from this region mapped to the pair of X chromosomes and it was therefore aphid in origin. Polymerase chain reaction using conserved rDNA primers also detected significant amounts of a fungal genome in the DNA samples. Microscopic investigation showed that the external surface of A. idaei harboured fungal propagules, hyphae and yeast-like organisms.

Ribosomal spacer length variability in the large raspberry aphid, Amphorophora idaei (Aphidinae: macrosiphini).

The large raspberry aphid, Amphorophora idaei, has several biotypes described by their abilities to overcome plant resistance genes. Bioassays of field populations showed a strong shift towards A1 resistance-breaking biotypes since the 1960s. RFLP analysis of the rDNA cistron was used to study variation found within and between standard clones of three A. idaei biotypes and twenty-nine field populations collected over 3 years. Probing genomic DNA with the ribosomal DNA probe pBG 35 produced consistent differences in RFLPs between standard clones of biotypes. However, analysis of field populations gave more complex RFLP patterns that were not biotype-specific, unlike characteristic intergenic spacer (IGS) patterns reported for Schizaphis graminum biotypes. All but one sample collected from separate fields showed considerable genetic diversity within populations, attributed to alate migrations of parthenogenetic females in summer and males in autumn.