Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

Preliminary Screening of Potential Control Products against Drosophila suzukii.

The first recording of Drosophila suzukii in the UK occurred in the south of England during August 2012. Since then sticky traps have continued to record the presence of individuals. Several products (both chemical and biological) were investigated for their efficacy against different life-stages of the pest. Both direct and indirect exposure to control products was assessed. Spinosad, chlorantraniliprole and the experimental product, TA2674, showed excellent potential as control agents when used as either a pre- or post-dipping treatment for blueberries with mortalities of 100%, 93% and 98% mortality, respectively, being achieved following pre-treatment. Direct spray application of all products tested had limited impact upon adult flies. Highest mortality (68%) was achieved following direct application of TA2674. Entomopathogenic agents (nematodes and fungi) tested appeared to reduce fly population development (ranges of 34-44% mortality obtained) but would seem unable to eradicate outbreaks. The potential of the tested products to control D. suzukii is discussed.

Determining the source of house flies (Musca domestica) using stable isotope analysis.

BACKGROUND: Intensive livestock units frequently produce flies in large numbers that, on migration, cause nuisance to the occupants of neighbouring dwellings. The resolution of such problems is often reliant on the unequivocal identification of the origin of the flies, particularly when several potential sources exist. This study evaluated stable isotope analysis as a method for differentiating adult houseflies (Musca domestica) on the basis of their dietary history so as to determine their likely source. RESULTS: Flies were reared in the laboratory on several substrates, including chicken and cattle manure, laboratory diet and household vegetable waste. Different fly parts (wings, heads and legs) and whole flies were analysed immediately after eclosion and after 10 days. The δ(13) C and δ(15) N values for adults that had developed on each diet type were highly distinct. Both isotopic ratios altered markedly after maintaining the flies for 10 days on a diet of cane sugar solution. CONCLUSIONS: Stable isotope analysis readily differentiated flies that had developed on a range of substrates. The technique, therefore, shows potential to be employed to determine the likely source of various nuisance insects, and to contribute to the abatement of such problems.

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome.

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.

First report of cyromazine resistance in a population of UK house fly (Musca domestica) associated with intensive livestock production.

BACKGROUND: House fly control in livestock-rearing facilities is heavily reliant on the use of the larvicide cyromazine. While extensive use of this compound has led to the development of resistance in several countries, no elevated tolerance has so far been reported from the United Kingdom. RESULTS: Tolerance to cyromazine in larvae derived from a field strain collected at an intensive pig unit was significantly elevated over that of insects taken from a susceptible laboratory strain. Resistance factors (RFs) of 2.9 and 2.4 were returned for assays initiated with eggs and neonate larvae respectively. The RF for field strain larvae exposed from neonate increased significantly to 3.9 and 5.6 following rounds of selection at 1.0 and then 1.5 mg kg(-1) cyromazine. CONCLUSION: Low-level resistance to cyromazine in UK house flies is reported here for the first time. The geographic extent of this resistance is unknown but, if widespread, may lead to control failures in the future, and indicates that careful stewardship of this compound in the United Kingdom is now required.

Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders.

BACKGROUND: The toxicity of a fusion protein, ButalT/GNA, comprising a venom toxin (ButaIT) derived from the red scorpion, Mesobuthus tamulus (F.), and Galanthus nivalis agglutinin (GNA), was evaluated under laboratory conditions against several pest insects. Insecticidal activity was compared with SFI1/GNA, a fusion comprising a venom toxin (SFI1) derived from the European spider Segestria florentina (Rossi) and GNA, which has been previously demonstrated to be effective against lepidopteran and hemipteran pests, and to GNA itself. RESULTS: Injection assays demonstrated that both fusion proteins were toxic to lepidopteran larvae, dipteran adults, coleopteran adults and larvae and dictyopteran nymphs. ButalT/GNA was more toxic than SFI1/GNA in all cases. GNA itself made a minor contribution to toxicity. Oral toxicity of ButalT/GNA towards lepidopteran pests was confirmed against neonate Spodoptera littoralis (Boisd.), where incorporation at 2% dietary protein resulted in 50% mortality and > 85% reduction in growth compared with controls. ButaIT/GNA was orally toxic to Musca domestica L. adults, causing 75% mortality at 1 mg mL(-1) in aqueous diets and, at 2 mg g(-1) it was orally toxic to Tribolium castaneum (Herbst.), causing 60% mortality and a 90% reduction in growth. CONCLUSIONS: Toxicity of the ButaIT/GNA recombinant fusion protein towards a range of insect pests from different orders was demonstrated by injection bioassays. Feeding bioassays demonstrated the potential use of the ButaIT/GNA fusion protein as an orally active insecticide against lepidopteran, dipteran and coleopteran pests. These experiments provide further evidence that the development of fusion protein technology for the generation of new, biorational, anti-insect molecules holds significant promise.

Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): basis of insecticidal activity.

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae).

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae).

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.

Insecticidal spider venom toxin fused to snowdrop lectin is toxic to the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) and the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The SFI1/GNA fusion protein, comprising of snowdrop lectin (Galanthus nivalis agglutinin, GNA) fused to an insecticidal spider venom neurotoxin (Segestria florentina toxin 1, SFI1) was tested for toxicity against the rice brown planthopper Nilaparvata lugens (Stål) and the peach-potato aphid Myzus persicae (Sulzer) by incorporation into artificial diets. Significant effects on the mortality of N. lugens were observed, with 100% of the insects fed on the SFI1/GNA fusion protein diet dead by day 7. The survival of the aphid M. persicae was also reduced when fed on the SFI1/GNA fusion protein. After 14 days, only 49% of the aphids that were fed on the fusion protein were still alive compared with approximately 90% of the aphids fed on the control diet or on diet containing GNA only. The SFI1/GNA fusion protein also slowed the development of M. persicae, and the reproductive capacity of the aphids fed on the SFI1/GNA fusion protein was severely reduced. The ability of GNA to act as a carrier protein, and deliver the SFI1 neurotoxin to the haemolymph of N. lugens, following oral ingestion, was investigated. The successful delivery of intact SFI1/GNA fusion protein to the haemolymph of these insects was shown by western blotting. Haemolymph taken from the insects that were fed on the fusion protein contained two GNA-immunoreactive proteins of molecular weights corresponding to GNA and to the SFI1/GNA fusion protein.

The pathogenicity of Vairimorpha necatrix (Microspora: Microsporidia) against the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae) and its potential use for the control of lepidopteran glasshouse pests.

A droplet feeding technique was used to feed known amounts of Vairimorpha necatrix (Kramer) spores to larvae of the tomato moth, Lacanobia oleracea (L) in order to assess the susceptibility of this lepidopteran pest to the pathogen. All first- to fourth-instar larvae died as a result of ingesting 1000 or more V necatrix spores. Two forms of death were observed, which were dependent on the dose and the age of the insect when treated. For first-instar larvae, rapid death (within 6days of dosing) occurred after ingestion of 2000 spores, whereas lower doses resulted in a proportion of larvae dying from chronic infection (microsporidiosis). For more advanced stages, increasing spore doses were required to give rapid death, such that a dose of 200,000 spores was needed to give 80% mortality within 6 days for third-instar larvae. Rapid death was not observed in fourth- to sixth-instar larvae. In all cases successful pupation and adult emergence were much reduced compared with non-infected larvae. Suspensions of V necatrix were sprayed on to tomato (Lycopersicon esculentum Mill) plants maintained in small glasshouses prior to infestation of the plants with L oleracea larvae. The numbers and biomass of pest larvae retrieved from the plants sprayed with V necatrix were significantly reduced by up to 40% and 70%, respectively, compared with plants sprayed with water (control). Similarly, plants sprayed with V necatrix showed a reduction in damage of up to 45% compared with the control plants.