Changes in gene expression in the permissive larval host lightbrown apple moth (Epiphyas postvittana, Tortricidae) in response to EppoNPV (Baculoviridae) infection.

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.

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica).

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and alpha-amylase were still detected in the midgut of diseased larvae.

Serratia entomophila inoculation causes a defect in exocytosis in Costelytra zealandica larvae.

Rapid elimination of midgut luminal proteinase activity and gut clearance are the two major symptoms of amber disease in Costelytra zealandica larvae because of the three-subunit protein toxin complex produced in Serratia entomophila and Serratia proteamaculans. Quantitative PCR analysis of mRNA from the major serine proteinase gene families showed that loss of proteinase activity did not result from transcriptional downregulation. Unexpectedly, protein levels and rates of protein synthesis increased, rather than decreased, in the midgut of diseased insects. Proteomic analysis of midgut tissues showed marked differences between healthy and diseased midguts. Large increases in soluble forms of both actin and tubulin were identified from 2D-gels, together with concurrent decreases in the levels of polymeric actin-associated proteins: actin depolymerizing factor and cyclophilin. These results suggest that the Serratia toxin acts to cause degradation of the cytoskeletal network and prevent secretion of midgut gut digestive proteinases as both the actin cytoskeleton and microtubules are involved in exocytosis. Proteinases synthesized in the diseased midgut must be rapidly degraded because they do not accumulate in an inactive form.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

The secondary structure of Nosema apis large subunit ribosomal RNA.

The microsporidia are a group of obligate intracellular eukaryotic parasites, that lack mitochondria. Their ribosomes show several prokaryote-like features. This paper presents the secondary structure of the large subunit ribosomal RNA (LSU rRNA) of the microsporidium Nosema apis. With its 2481 bases, it is the shortest known non-mitochondrial LSU rRNA. The seemingly prokaryote-like features of the molecule cannot be used as evidence for the ancient origin of the microsporidia. The reduction in size can be attributed to changes in the regions of the LSU rRNA that are known to show great variability in length and sequence within the eukaryotes. The lack of fragmentation commonly seen in other eukaryotes may also be a derived feature.

Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae).

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 ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of a large tandem repeat unit size.

The ribosomal RNA (rRNA) gene region of the microsporidium, Nosema apis, has been examined. A new method for extracting microsporidian genomic DNA from infected host tissue is described. Complete DNA sequence data are presented for the small subunit gene (1242 bp), the internal transcribed space (33 bp), and the large subunit gene (2481 bp to a putative termination point). This is the first time that the complete large subunit rRNA gene has been published for any microsporidian species. DNA sequence is also presented for the regions flanking the 5' end of the small subunit gene and the 3' end of the large subunit gene. The intergenic spacer is shown to be heterogeneous, showing variation in sequence and restriction sites rather than length and containing sequence repeats, which are a characteristic feature of intergenic spacers. The rRNA gene region of N. apis is shown to occur in a head-to-tail, tandemly repeated manner, as in other eukaryotes. This repeat unit is shown to be approximately 18 kb in length. The nucleotide sequence presented has been submitted to the Genbank database under the accession number U97150.

Effects of time, temperature, and honey on Nosema apis (Microsporidia: Nosematidae), a parasite of the honeybee, Apis mellifera (Hymenoptera: Apidae).

Newly emerged adult bees were fed with Nosema apis spores subjected to various treatments, and their longevity, proportions of bees infected, and spores per bee recorded. Spores lost viability after 1, 3, or 6 months in active manuka or multifloral honey, after 3 days in multifloral honey, and after 21 days in water or sugar syrup at 33 degrees C. Air-dried spores lost viability after 3 or 5 days at 40 degrees, 45 degrees, or 49 degrees C. Increasing numbers of bees became infected with increasing doses of spores, regardless of their subsequent food (active manuka honey, thyme honey, or sugar syrup). Final spore loads were similar among bees receiving the same food, regardless of dose. Bees fed with either honey had lighter infections than those fed with syrup, but this may have been due to reductions in their longevity. Bees fed with manuka honey were significantly shorter lived, whether infected or not.

In vivo Responses of Honey Bee Midgut Proteases to Two Protease Inhibitors from Potato.

Potato protease inhibitors, POT-1 and POT-2, were fed to newly emerged adult honey bees in cages at different doses in either sugar syrup (0.2 or 0.01% w:v) or pollen food (1 or 0.2% w:w). In vivo activities of three digestive endopeptidases (trypsin, chymotrypsin and elastase) and one exopeptidase (leucine aminopeptidase; LAP) were measured after 3 or 8days' exposure of bees to inhibitor. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase, which did not change. POT-2 significantly reduced the activity of all endopeptidases at both timepoints, regardless of the dose level or the medium in which the inhibitor was administered. POT-1 acted in a similar manner, except that 0.01% POT-1 in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment. The survival of bees fed POT-1 or POT-2 at 0.2% in pollen or 0.01% in syrup did not differ from the controls.

Prey-mediated effects of the protease inhibitor aprotinin on the predatory carabid beetle Nebria brevicollis.

To investigate the potential non-target impacts of transgenic pest-resistant plants, prey-mediated impacts of a protease inhibitor (PI) on the predatory carabid, Nebria brevicollis, were investigated. The PI used was aprotinin, a serine PI of mammalian origin with insecticidal properties when incorporated in artificial diet or expressed in transgenic plants. Field-collected N. brevicollis adults, kept at 23 degrees C, 16:8 L:D, were fed, over their pre-aestivation activity period of 24 days, with Helicoverpa armigera larvae reared on an artificial diet containing 0.5% (w:w, fresh mass) aprotinin. These larvae contained 22.62 &mgr;g aprotinin/g insect. Control prey was reared on diet without aprotinin. Beetle survival and body mass were unaffected by prey type. Beetles consuming PI-fed prey lost significantly more mass than the control beetles during two periods of mass loss, but gained significantly more mass during the final period of mass gain. This was not due to differences in amounts of prey supplied or consumed. The final mass gain coincided with increased consumption of PI-prey. Female beetles were significantly heavier than males, but we found no consistent gender-based differences in response to PI-prey. At the end of the experiment, body mass of all beetles was similar to field-collected ones (approximately 55 mg). All experimental beetles had significantly lower activities of digestive cysteine proteases and the serine proteases chymotrypsin and trypsin than field-collected ones. Beetles consuming PI-fed prey had significantly lower levels of trypsin and higher levels of chymotrypsin and elastase than the control beetles.