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A transcriptome was constructed by pyrosequencing on the GS-FLX sequencer (Roche), for the whole body of the combined nymph and adult green stink bug, Acrosternum hilare (Say), collected from sites in North Carolina and Virginia, USA. The cDNA library for the combined sample was sequenced on one-half plate. The total output from the reads was 40, 500, 000 bps (base pairs) of data. The whole body library produced 162, 000 reads which were assembled using Roche software (Genome Sequencer FLX System GS De Novo Assembler, version 2.3, October 2009) into 1,598 contigs (contiguous sequences) with the assembly cutoff set at 400 bp, and into 2,534 contigs with the assembly cutoff set at 150 bp. BLASTp was used to compare contigs to the Uniprot database (January 3, 2010) to determine putative sequence identity. Of these contigs, 974 had an expect value of 1e-05 or lower. Of these, 876 had an assigned Uniprot GO (Gene Ontology) ID and were mapped and annotated. Wego software was used to translate the Uniprot GO ID for these contigs into GO Level 2 functional categories. Analysis of this recently-sequenced green stink bug transcriptome is aimed at the discovery of new insecticide targets for applications in plant, genetic pest management. [ABSTRACT FROM AUTHOR]

Transgenic cotton varieties that express Bacillus thuringiensis (Bt) toxins have been adapted readily because they reduce the need of insecticide application, while their environmental impacts are minimal. Recently, pyramided cotton varieties that express two different Bt toxins have been introduced; these varieties may reduce the risk of resistance development in lepidopteran pests populations. However, resistance monitoring in Bt cotton fields are necessary and mandated. Based on a previously described feeding disruption test (FDT) from our laboratory, the objective of this project was to develop a bioassay that uses Bt toxin obtained directly from Bollgard II plant material. The technology under development should be applicable to other pyramided and single gene commercial plants. The assay hardware is a novel 16 well plastic plate with each well of the plate containing a hydrateable, artificial diet containing leaf extract from Bollgard II. Dose-response studies were conducted using a laboratory susceptible strain of the tobacco budworm, Heliothis virescens, where different concentrations of leaf extract were used to hydrate a standard meal pad in each well. From these studies, a diagnostic dose was determined of 1.3 µg cotton / meal pad, which essentially stopped the production of fecal pellets by neonates of the budworm after a 24 h incubation. This diagnostic dose was further evaluated using three different populations of H. virescens assayed directly from the field from central North Carolina, USA. The diagnostic dose was also evaluated with the Bt resistant, laboratory reared H. virescens strains, YHD2 and CxC. The bioassay developed is a practical, reliable monitoring tool that can be used to monitor Bt susceptibility as part of an integrated resistance program in pyramided cotton fields. [ABSTRACT FROM AUTHOR]

With the eradication of the boll weevil, the development of narrow-spectrum insecticides for whitefly control, and the widespread adoption of Bt cotton in the last decade, several species of plant bugs (Miridae) and stink bugs (Pentatomidae) have become major pests of cotton. Reports of tarnished plant bug Lygus lineolaris (Palisot de Beauvois) resistance to several classes of cotton insecticides, and of variable insecticide susceptibilities of stink bug species that damage cotton, highlight the need for assays to monitor resistance. Assays in current use have several limitations. Topical assays used for plant bugs and stink bugs are limited to testing insecticides with contact activity. Except in the case of neonicotinoids, vial assays in current use for plant bugs require the addition of plant material as a food source and are limited to contact insecticides. Vial tests for stink bugs are limited to testing insecticides with contact activity. All of the topical and vial assays for these sucking pests rely on a mortality endpoint which is often not easy to read (requires probing the insect and/or discriminating between knockdown versus death) and un-necessarily extends the time to the assay endpoint. Feeding disruption test (FDT) bioassays circumvent these limitations. In FDT assays, insects feed on insecticide in rehydrateable artificial diet mealpads containing a blue indicator dye to mark feeding on the artificial diet. The assay endpoint is the dose-dependent amount of blue feces produced, usually within 24 hours. The objective of the work described in this paper was to determine the feasibility of developing FDT assays for plant bugs and stink bugs. Lab-strain adult tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), fed different concentrations of β-cyfluthrin and thiamethoxam in rehydrateable NI diet mealpads containing food-grade blue dye in FDT plates showed a dose-response in production of dyed feces for both insecticides. Lab-strain adult brown stink bugs, Euchistus servus (Say), fed different concentrations of thiamethoxam in nectar also showed a mortality dose response. Brown stink bugs fed NI diet containing food-grade blue dye produced dyed feces; this approach should also be applicable to resitance monitoring as was the case for the plant bug. [ABSTRACT FROM AUTHOR]

Lab-strain tobacco budworm (Heliothis virescens) moths were fed different concentrations of permethrin and spinosyn in 10% sucrose containing 0.2 mg/ml trypan blue dye to obtain dose-mortality, dose-nectar-ingestion, and dose-fecal production responses. Percent mortality increased with increasing concentration of permethrin. Ingestion (g of treatment solution per moth) declined with increasing concentration of permethrin. Trends in percent mortality and ingestion for permethrin were consistent with prior results reported for spinosyn (van Kretschmar et al., 2007). Blue-dyed fecal production declined with increasing concentration of both insecticides. These results demonstrate the feasibility of using dyed sucrose solution in an adult-based feeding disruption test to monitor Lepidoptera for development of resistance to chemical insecticides. [ABSTRACT FROM AUTHOR]

Lab-strain tobacco budworm (Heliothis virescens) moths were fed different concentrations of permethrin and spinosyn in 10% sucrose containing 0.2 mg/ml trypan blue dye to obtain dose-mortality, dose-nectar-ingestion, and dose-fecal production responses. Percent mortality increased with increasing concentration of permethrin. Ingestion (g of treatment solution per moth) declined with increasing concentration of permethrin. Trends in percent mortality and ingestion for permethrin were consistent with prior results reported for spinosyn (van Kretschmar et al., 2007). Blue-dyed fecal production declined with increasing concentration of both insecticides. These results demonstrate the feasibility of using dyed sucrose solution in an adult-based feeding disruption test to monitor Lepidoptera for development of resistance to chemical insecticides. [ABSTRACT FROM AUTHOR]

Lab-strain tobacco budworm (Heliothis virescens) moths were fed different concentrations of spinosyn and permethrin in 10% sucrose to obtain dose-mortality responses. Percent mortality increased with increasing concentration of both insecticides. Ingestion (g of treatment solution per moth) declined with increasing concentration of both insecticides. When trypan blue dye was added to an insecticide solution fed to moths, it provided a dramatic visual marker of disrupted fecal production. These results demonstrate the feasibility of an adult-based feeding disruption test to monitor Lepidoptera for development of resistance to chemical insecticides. [ABSTRACT FROM AUTHOR]

Feeding disruption tests (FDTs) were developed for monitoring insect resistance to three Bt proteins--Cry1Ab, Cry1Ac and Cry1F. The tests were designed to minimize false positives and negatives for resistance. The assays rely on a diagnostic dose of Bt toxin in a hydrateable meal pad containing a blue indicator dye. Insects resistant to Bt are able to consume the artificial diet and produce blue feces on a white background. Fecal production was inversely correlated with Bt concentration in the hydrated meal pad. The diagnostic dose for resistance determined by FDT for Cry1Ab against Heliothis virescens (Hv) was 15 micrograms a.i./ml hydration solution and for Helicoverpa zea (Hz) was 500 micrograms per ml; Cry1Ac: Hv=10, Hz=40; and Cry1F: Hv=20, Hz=200. The assays were successfully tested against Bt resistant tobacco budworms (YHD2 strain) and on H. virescens collected as eggs from tobacco in North Carolina. The kits were also evaluated by cooperators (results reported). FDT kits provide an easy-to-use (off-the-shelf) standard assay method for monitoring insect resistance to Bt and chemical insecticides. The technology has other applications in high-throughput insecticide screening, routine insect rearing and the evaluation of the insecticidal activity of Bt transgenic crops. [ABSTRACT FROM AUTHOR]

Feeding disruption tests (FDTs) were developed for monitoring insect resistance to three Bt proteins--Cry1Ab, Cry1Ac and Cry1F. The tests were designed to minimize false positives and negatives for resistance. The assays rely on a diagnostic dose of Bt toxin in a hydrateable meal pad containing a blue indicator dye. Insects resistant to Bt are able to consume the artificial diet and produce blue feces on a white background. Fecal production was inversely correlated with Bt concentration in the hydrated meal pad. The diagnostic dose for resistance determined by FDT for Cry1Ab against Heliothis virescens (Hv) was 15 micrograms a.i./ml hydration solution and for Helicoverpa zea (Hz) was 500 micrograms per ml; Cry1Ac: Hv=10, Hz=40; and Cry1F: Hv=20, Hz=200. The assays were successfully tested against Bt resistant tobacco budworms (YHD2 strain) and on H. virescens collected as eggs from tobacco in North Carolina. The kits were also evaluated by cooperators (results reported). FDT kits provide an easy-to-use (off-the-shelf) standard assay method for monitoring insect resistance to Bt and chemical insecticides. The technology has other applications in high-throughput insecticide screening, routine insect rearing and the evaluation of the insecticidal activity of Bt transgenic crops. [ABSTRACT FROM AUTHOR]