We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5 capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5 ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.

The peach potato aphid, Myzus persicae is a globally distributed crop pest with a host range of over 400 species including many economically important crop plants. The intensive use of insecticides to control this species over many years has led to populations that are now resistant to several classes of insecticide. Work spanning over 40 years has shown that M. persicae has a remarkable ability to evolve mechanisms that avoid or overcome the toxic effect of insecticides with at least seven independent mechanisms of resistance described in this species to date. The array of novel resistance mechanisms, including several 'first examples', that have evolved in this species represents an important case study for the evolution of insecticide resistance and also rapid adaptive change in insects more generally. In this review we summarise the biochemical and molecular mechanisms underlying resistance in M. persicae and the insights study of this topic has provided on how resistance evolves, the selectivity of insecticides, and the link between resistance and host plant adaptation.

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2–16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

BackgroundMyzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids.ResultsBioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid) to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR). Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpβ1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T).ConclusionPrevious studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also provides further validation of exisiting models of neonicotinoid binding and selectivity for insect nAChRs.

We discuss methods using stimuli to manipulate behavior of a pest for the purpose of protecting a valued resource. The methods are divided into two categories: those that manipulate behavior over a long distance, e.g. volatile chemicals, visual, and auditory stimuli, and those that manipulate behavior at a short distance (< 1 cm), e.g. involatile chemicals. Particular emphasis is placed on methods that have been developed through studies of pest behavior and on combining stimuli to increase efficacy. Future prospects for behavioral manipulation methods in pest management are discussed.

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

The peach^potato aphid Myzus persicae (Sulzer) can resist a wide range of insecticides, but until recently (1990) the only mechanism identi¢ed was the increased production of carboxylesterases (E4 or FE4), which cause enhanced degradation and sequestration of insecticidal esters. We have now identi¢ed two forms of target-site resistance involving changes in the acetylcholinesterase (AChE) and sodium channel (kdr) genes. Biochemical and DNA diagnostic methods can be used to identify all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. Ampli¢ed genes underlie the increased production of esterases, but their expression is modulated by DNA methylation. Ampli¢cation of the E4 gene is in strong linkage disequilibrium with the kdr mechanism. This may re£ect strong insecticidal selection favouring aphids with multiple mechanisms, tight chromosomal linkage and/or the prominence of parthenogenesis in many M. persicae populations. The decreased ¢tness of resistant aphids under winter conditions may be a consequence of the altered sodium-channel gene a¡ecting behaviour and/or the perception of external stimuli.

The voltage-gated sodium channel is the primary target site of DDT and pyrethroid insecticides, and point mutations in the domain II region of the channel protein have been implicated in the knockdown resistant (kdr ) phenotype of several insect species. Here, we report that one of these mutations, a leucine-to-phenylalanine replacement in transmembrane segment IIS6, is also found in certain insecticide-resistant clones of the peach-potato aphid, Myzus persicae. The mutation was present in four clones with amplified E4 esterase genes, but was absent from both susceptible clones and those with amplified FE4 genes. The inferred presence of kdr-type resistance in the four E4 clones was subsequently confirmed by bioassays that showed this to be the primary mechanism of resistance to deltamethrin and DDT, although the esterase-based mechanism also contributes to the overall level of deltamethrin resistance. The kdr mutation on its own conferred 35-fold resistance to deltamethrin and this was enhanced up to 540-fold when it was present in a high (E4) esterase background. The esterase (FE4) mechanism was far less effective without the kdr mutation, conferring just 3-4-fold resistance to deltamethrin. These findings, and the linkage disequilibrium of the kdr mutation within clones overproducing the E4 esterase, have important implications for the evolution of resistance in this insect and for the use of pyrethroid sprays in the management of M. persicae populations in the field.