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Host cell and virus gene expression were measured five days after per os inoculation of 3rd instar lightbrown apple moth (LBAM) larvae with the Epiphyas postvittana nucleopolyhedrovirus (EppoNPV). Microarray analysis identified 84 insect genes that were up-regulated and 18 genes that were down-regulated in virus-infected larvae compared with uninfected larvae. From the 134 viral open reading frames represented on the microarray, 81 genes showed strong expression. Of the 38 functionally identifiable regulated insect genes, 23 coded for proteins that have roles in one of five processes; regulation of transcription and translation, induction of apoptosis, and maintenance of both juvenility and actin cytoskeletal integrity. Of the 34 functionally identifiable viral genes that were most strongly expressed, 12 had functions associated with these five processes, as did a further seven viral genes which were expressed at slightly lower levels. A survey of the LBAM-expressed sequence tag library identified further genes involved in these processes. In total, 135 insect genes and 38 viral genes were analysed by quantitative polymerase chain reaction. Twenty-one insect genes were strongly up-regulated and 31 genes strongly down-regulated. All 38 viral genes examined were highly expressed. These data suggest that induction of apoptosis and regulation of juvenility are the major 'battlegrounds' between virus and insect, with the majority of changes observed representing viral control of insect gene expression. Transcription and translational effects seem to be exerted largely through modulation of mRNA and protein degradation. Examples of attempts by the insect to repel the infection via changes in gene expression within these same processes were, however, also noted. The data also showed the extent to which viral transcription dominated in the infected insects at five days post inoculation.

The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.

The nucleopolyhedrovirus EppoNPV can control larvae of the leafroller Epiphyas postvittana in the field but has varying effects on New Zealands other leafroller pests An understanding of the infection process at the genetic/molecular level within the leafroller complex may lead to strategies to enhance the effectiveness of the virus Microarray technology was used to examine the effects of viral infection on gene expression levels in E postvittana gut tissue The results show that a 5 day infection with EppoNPV caused a significant downregulation of several E postvittana genes many of which were consistent with a response to injury or stress or with the triggering of apoptosis (such as components of the oxidative phosphorylation pathway) The majority of the expressed viral sequences detected are structural genes and genes associated with late gene expression These data provide a basis from which to compare EppoNPV infection in other less susceptible leafroller species

International audience; Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments. (C) 2010 Elsevier Ltd. All rights reserved.

While searching for ‘organ-specific’ genes in pea ( Pisum sativum L.) we have isolated a gene (designated PsMT A ) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMT A transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.

An extensin gene, designated extB , was isolated from an oilseed rape ( Brassica napus L.) genomic library by screening with an extensin cDNA species expressed abundantly in roots. The gene is a member of a large multigene family. Although expression of sequences homologous to the extB coding region was detected in roots and (at a lower level) in stems, no mRNA species could be detected with an extB -specific probe, isolated from a region encoding the predicted leader sequence of the extensin protein. The complete nucleotide sequence of the extB gene and its flanking regions was determined. The gene encodes a protein homologous to the extensin found in tomato stem, and notable for a regular repetitive structure and a high proportion of tyrosine residues.

A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1,300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.

A pea genomic library was screened with a pea seed lectin cDNA clone, a selected clone sub-cloned and the DNA sequence determined. We have designated this gene LecA; like other lectin genes LecA contains no introns. The coding sequence of this gene is identical to that of the cDNA used to select it, pLGA.10 (1) and that of the published cDNA sequence (2). Although these cDNAa differ in their sites of polyadenylation there is only one (simple) polyadenylation signal. At the 5' end of the gene there are three in frame ATG start codons. The first of these is followed by an in frame stop codon and both the sequence surrounding thio and the third ATG are poor matches to the functional consensus (3). The second agrees well and is therefore used. Multiple start codons have been noted in lectin genes from Phaseolus vulgaris (4). LecA contains a TATA box (5) 65 bases 5' to the start codon which lies within a 22bp region duplicated between this and the start codon; such repeats also occur in Phaseolus lectin genes (4). No other consensus seqquences can be recognised in the further 5' flanking sequence and comparison with lectin genes from related species (4, 6, 7) failed to reveal regions of significant homology. When this gene was used to probe Southern blots of restriction digests of pea genomic DNA at high stringency of hybridisation, to determine the copy number of LecA, all digests gave a result indicating the presence of a single gene.

SUMMARYDouble-stranded RNA (dsRNA) has been found to interfere with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is effective in reducing gene expression in the adult honey bee (Apis mellifera). A 1175 bp fragment of the hypopharyngeal amylase gene from the honey bee was cloned into a modified pGEM-5Zf(+) vector. The gene fragment was inserted between two opposing T7 promotors, thus enabling expression of dsRNA specific for the amylase gene when the plasmid was grown in an Escherichia coli strain expressing T7 RNA polymerase. This dsRNA was introduced into the haemolymph of forager honey bees by intra-abdominal injection and the effects on amylase activity in the hypopharyngeal glands of those bees examined. Compared to controls, injection of amylase gene dsRNA produced a significant decrease in amylase activity of 27.5% 3 days post-injection (P = 0.001). An experiment using radiolabelling to follow the fate of the dsRNA, revealed that most of t...